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Mt s vn ca sinh hc phn t V Th Hng Lan

NXB i hc quc gia H Ni 2007. 181 tr. T kho: ADN, GEN, genome, nhim sc th, ty th, lc tp, geomic, ADN, ti t hp, ngn hng cc ADNc, cDNA, ADN genome , phn ng PCR, k thut gen, phng php lai, Protein, tng hp protein, vn chuyn protein, tn hiu t bo, truyn tn hiu t bo, Th th tyrosine kinase, Protein G, sinh trng, pht trin, h gen lng bi, phi, chu trnh t vo, phn chia t bo. Ti liu trong Th vin in t H Khoa hc T nhin c th c s dng cho mc ch hc tp v nghin cu c nhn. Nghim cm mi hnh thc sao chp, in n phc v cc mc ch khc nu khng c s chp thun ca nh xut bn v tc gi.

Mc lc
LI NI U........................................................................................................................ 5
U

Chng 1 ADN V GEN ....................................................................................................................6

1.1 1.2
1.2.1. 1.2.2.

Khi nim v gen........................................................................................................ 6 Genome (h gen) ...................................................................................................... 10


Genome ca t bo prokaryot (t bo nhn s) ............................................................... 11 Genome ca t bo eukaryot (t bo nhn thc) ............................................................. 13

1.3
1.3.1. 1.3.2.

Cu trc si nhim sc trong t bo eukaryot .......................................................... 14


Histone trong cu trc nucleosome.................................................................................. 15 Methyl ho ADN ............................................................................................................. 17

1.4
1.4.1. 1.4.2. 1.4.3.

Cc gen trong genome eukaryot............................................................................... 18


Cc gen trong cng mt h gen ....................................................................................... 20 Gen lp i lp li lin tc................................................................................................. 21 Pseudogen (gen gi)......................................................................................................... 23

1.5
1.5.1. 1.5.2.

Thnh phn ADN lp li trong genome eukaryot .................................................... 23


ADN v tinh (satelitte DNA) v ADN tiu v tinh (minisatelitte DNA)......................... 23 Cc on ADN c kh nng di chuyn............................................................................ 24

1.6 1.7

Tng tc ca T-ADN vi genome thc vt............................................................ 29 ADN trong ty th v lc lp ..................................................................................... 32

1.7.1. 1.7.2.

ADN ty th ...................................................................................................................... 32 ADN lc lp..................................................................................................................... 33

1.8

Genomics.................................................................................................................. 33

1.8.1 So snh genome ............................................................................................................... 33 1.8.2 Genome ngi ................................................................................................................. 34 1.8.3 Nghin cu Genomics thc vt .................................................................................... 35 Chng 2 HOT NG CA GEN TRONG T BO ...............................................................38

2.1
2.1.1 2.1.2 2.1.3

Kim sot hot ng ca gen khi phin m ............................................................. 41


Kim sot khi u phin m .......................................................................................... 42 Kim sot kt thc phin m ........................................................................................... 50 Cc protein iu khin (regulatory proteins) ................................................................... 51

2.2
2.2.1 2.2.2 2.2.3 2.2.4 2.2.5

Kim sot sau phin m ........................................................................................... 53


Km hm dch m lin quan n cu trc vng 5'UTR ca phn t ARNm.................... 53 di ca ui polyA nh hng ti bn vng ca phn t ARNm......................... 54 bn vng ca ARNm ................................................................................................. 54 ARN anti-sense................................................................................................................ 55 Phn ng c sa ARNm - "RNA editing" ..................................................................... 56

2.3 2.4

Kim sot giai on dch m v sau dch m ........................................................ 57 Bin i phn t ARNm trong t bo eukaryot ....................................................... 59

2.4.1 Phn ng ct intron v ni exon ...................................................................................... 60 2.4.2 Cc intron c kh nng t ct ra khi phn t ARNm-Phn ng self-splicing ............... 62 2.4.3 Phn ng trans-splicing ni hai exon ca hai phn t ARNm......................................... 64 2.4.4 Cu trc chung ca phn t ARNm................................................................................. 64 Chng 3 K THUT ADN TI T HP....................................................................................66

3.1 3.2
3.2.1 3.2.2

Phn ct, phn ly ADN............................................................................................. 66 a cc on ADN vo vector ................................................................................ 67


Cc vector s dng trong k thut tch dng .................................................................. 68 a ADN vo vector ....................................................................................................... 70

3.3
3.3.1 3.3.2

Ngn hng ADN....................................................................................................... 72


Ngn hng cc ADNc (cDNA library) ............................................................................ 72 Ngn hng ADN genome (genomic DNA library).......................................................... 74

3.4
3.4.1 3.4.2 3.4.3 3.4.4

Sng lc mt dng t ngn hng ADN .................................................................... 76


Phng php sng lc chung ........................................................................................... 76 Phng php sng lc phn bit "differential screening"................................................ 77 Phng php i dc nhim sc th chromosome walking ........................................... 78 Nhy bc trn nhim sc th jumping on chromosome ............................................. 80

3.5
3.5.1 3.5.2 3.5.3 3.5.4

Cc phng php lai................................................................................................. 80


Phng php Southern blots............................................................................................ 81 Phng php northern blots............................................................................................. 82 K thut lai in-situ ........................................................................................................... 82 iu kin phn ng lai..................................................................................................... 82

3.6 3.7
3.7.1 3.7.2

RFLP trong nghin cu genome v lp bn gen ................................................. 83 Phn ng PCR (Polymerase Chain Reaction) .......................................................... 86
Cc yu t nh hng n phn ng PCR....................................................................... 87 Mt s dng ca phn ng PCR ...................................................................................... 88

3.8
3.8.1 3.8.2 3.8.3 3.8.4 3.8.5

K thut gen ............................................................................................................. 89


Nghin cu vai tr ca ADN iu khin, chc nng ca gen hoc protein..................... 89 Thay th hoc gy t bin gen ....................................................................................... 92 Gy mt hoc tng cng chc nng ca gen ................................................................. 93 Gen bo co reporter gene ........................................................................................... 96 Bin i genome thc vt ................................................................................................ 96

Chng 4 TNG HP V VN CHUYN PROTEIN................................................................98

4.1 4.2 4.3


4.3.1 4.3.2

Vai tr ca ARN vn chuyn (ARNt) trong tng hp protein ................................... 98 Tng hp protein b my Ribosome.................................................................... 100 Vn chuyn protein ................................................................................................. 102
Vn chuyn vo mng li ni cht .............................................................................. 103 Vn chuyn protein cu trc mng (membrane proteins).............................................. 105

4.4
4.4.1 4.4.2 4.4.3

Bin i sau dch m v kim tra cht lng protein trong khoang ER................... 108
To cu lin kt disulfide (S-S) v cun gp trong khoang ER..................................... 108 Hnh thnh cu trc multimer t cc chui peptide....................................................... 109 Qu trnh ng ho protein.......................................................................................... 109

4.5 4.6

Vn chuyn t mng li ni cht n Golgi v Lysosome.................................... 110 Vn chuyn t Golgi n b mt t bo: Con ng tit ngoi bo (exocytosis) .......... ................................................................................................................................ 110 Th th trn b mt t bo...................................................................................... 114 Th th ni vi protein G ....................................................................................... 117
Protein G........................................................................................................................ 117 Hot ho hoc c ch cAMPase thng qua protein G ................................................... 119

Chng 5 TRUYN TN HIU T BO .....................................................................................112

5.1 5.2
5.2.1 5.2.2

5.3 5.4
5.4.1 5.4.2

Protein kinase ph thuc cAMP (cAPK hoc kinase A)........................................ 121 Th th tyrosine kinase v cc protein Ras ............................................................ 124
Th th tyrosine kinase (RTKs)..................................................................................... 124 Protein Ras v chui cc phn ng truyn tn hiu hot ho bi th th tyrosine kinase ....................................................................................................................................... 127

5.5
5.5.1 5.5.2 5.5.3

Tn hiu th cp Ca+2 trong chui truyn tn hiu.................................................. 129


Inositol phospholipid ..................................................................................................... 130 Inositol triphosphate (IP3) v s vn chuyn Ca+2 ra khi ER ................................... 130 Calmodulin- protein to phc vi Ca+2 trong t bo................................................. 132

5.6 5.7
5.7.1 5.7.2 5.7.3

Khuch i cc tn hiu bn ngoi t bo............................................................... 133 Truyn tn hiu qua cc th th ni vi enzym trn b mt t bo ........................ 135
Th th guanylyl cyclase ............................................................................................... 135 Cc oncogene v tn hiu dn truyn t th th tyrosine kinase.................................... 136 Protein MAP kinase....................................................................................................... 136

5.8 6.1 6.2 6.3 6.4 6.5 6.6

Tyrosine kinase phi hp vi th th. Th th Tyrosine phosphatase................... 137 Nhng c tnh c bn ca chu trnh t bo........................................................... 139 Chu trnh t bo giai on pht trin phi sm ................................................... 143 Protein cyclin.......................................................................................................... 145 Nm men v h thng kim sot chu trnh t bo .................................................. 147 Kim sot phn bo ng vt .............................................................................. 150 Vai tr ca si vi ng tubulin trong phn bo........................................................ 152

Chng 6 CHU TRNH V PHN CHIA T BO....................................................................139

Chng 7 SINH TRNG V PHT TRIN ............................................................................154

7.1 Kim sot xc nh gii tnh .................................................................................. 155 7.2 Pht trin rui gim Drosophila .......................................................................... 158 7.3 Hot ng ca cc gen c ngun gc t m trong qu trnh hnh thnh trc u-ui v trc lng-bng................................................................................................................ 159
7.3.1. 7.3.2. 7.3.3. 7.3.4. Nhm gen quyt nh pht trin ca phn u v ngc u th (anterior-group genes) . 160 Nhm gen qui nh pht trin phn ui (posterior-group genes)................................. 162 Nhm gen qui nh pht trin trc lng-bng (dorsoventral-group genes) ................... 162 Nhm gen qui nh pht trin cc cu trc tn cng ca u th (terminal-group genes)164

7.4
7.3.5. 7.3.6. 7.3.7.

Hot ng ca cc gen trong h gen lng bi (phi) ........................................... 164


Cc gen to t "gap" .................................................................................................... 166 Cc gen cp t "pair-rule"............................................................................................ 166 Cc gen phn cc t..................................................................................................... 167

7.5

Cc gen chn lc .................................................................................................... 167

Li ni u
Vi mong mun chia s cng bn c mi quan tm v Sinh hc phn t, mt lnh vc ang c hc tp v nghin cu Vit Nam, chng ti xut bn cun sch "Mt s vn c bn ca Sinh hc phn t" nhm gii thiu nhng qu trnh quan trng xy ra trong t bo (trnh by trong chng 1, 2, 4, 5, 6 v chng 7) v mt s k thut c bn c s dng nghin cu nhng qu trnh (chng 3). Nhng qu trnh ny c nghin cu mc phn t phn no lm sng t s ging v khc nhau trong cu trc ca genome, cu trc ca mt gen gia t bo prokaryot v eukaryot (chng 1). Nhng cu trc lin quan n cc cch thc kim sot hot ng ca cc gen giai on phin m, sau phin m v dch m tng hp protein (chng 2). Qu trnh tng hp protein, nhng bin i cu trc protein v nhng cch thc nhn bit v vn chuyn protein c hiu n nhng v tr ch khc nhau trong t bo hoc tit ra bn ngoi c gii thiu trong chng 4. Ngoi ra, chc nng v hot tnh ca nhng protein tham gia qu trnh truyn tn hiu c trnh by trong chng 5; ca protein tham gia chu trnh t bo c trnh by trong chng 6 v nhng protein tham gia kim sot bit ho, pht trin, sinh trng v hnh thnh c th c gii thiu trong chng 7. c th hc c nhng kin thc chuyn su trong lnh vc sinh hc phn t, ti xin by t lng bit n su sc ti cc thy, cc c trong Khoa Sinh hc Trng i hc Tng hp H Ni (nay l Trng i hc Khoa hc T nhin, i hc Quc gia H Ni). ng thi ti xin chn thnh cm n Ph Gio s Trng Nam Hi v Gio s Nguyn Mng Hng c nhng nhn xt v gp qu bu cho cun sch. Ln u xut bn, chc chn cun sch cn c nhng thiu st, ti rt mong nhn c s ph bnh, gp ca bn c v ng nghip. Vi s cm n chn thnh! Tc gi

Chng 1 ADN V GEN


1.1 Khi nim v gen

Tri qua mt thi gian di, cc khi nim v nh ngha v gen dn dn c hnh thnh da vo kt qu th nghim, trc ht l cc th nghim di truyn c in. u tin, t php lai gia cc cy u c nhng tnh trng khc nhau v theo di s di truyn ca chng, Menden a ra kt lun mi tnh trng c quyt nh bi cc allen ca mt gen. Mt gen c th c nhiu allen. Mc biu hin ca tnh trng ph thuc vo s kt hp gia hai allen. n gin nht l mt gen c 2 allen (Aa). Khi tnh trng c th biu hin 3 mc khc nhau: tri (AA), bn tri (Aa), hoc ln (aa). Tip theo , vi mt lot cc th nghim tin hnh trn rui gim Drosophila, Morgan v cng s nhn thy mt s tnh trng c quyt nh khng phi do cc alen ca mt gen m do nhiu gen. iu quan trng hn na, da vo tn s trao i cho gia hai nhim sc th tng ng trong qu trnh phn bo gim nhim (meiosis), Morgan c th lp c bn di truyn (genetic map) cho php xc nh v tr ca gen trn nhim sc th. Hai gen cng gn nhau th tn s trao i cho gia chng cng nh. Trn thc t, bn di truyn cho bit v tr ca nhng gen lin quan n cc tnh trng hoc cc t bin m khong cch gia chng c tnh bng tn s trao i cho (cM). Tuy nhin, trao i cho khng xy ra nh nhau mi v tr trn si nhim sc th khin cho khong cch gia cc v tr trn bn di truyn khng phi lc no cng t l vi tn s trao i cho. Trc nm 1940, v tr cc gen trn nhim sc th c xem nh cc ht cm trong mt chui. Trao i cho c xem l ch xy ra gia cc gen m khng th xy ra trong mt gen. V vy, t kt qu th nghim, cc nh di truyn a ra 3 c tnh xc nh mt gen: 1. Gen qui nh mt tnh trng c th quan st c v chim mt v tr trn nhim sc th. 2. Gen c xem l n v di truyn nh nht c th b t bin. 3. Gen c xem l n v di truyn nh nht m trao i cho khng th xy ra trong mt gen. Trao i cho c thc hin gia cc gen tng ng. T nhng c tnh ny, r rng hai tnh trng khng ging nhau c th phn bit c th phi do t nht hai gen khc nhau qui nh. R rng, khi nim v gen ban u ny ch cho php xc nh mi tng quan theo kiu mt t bin - mt tnh trng - mt gen. Trong thc t, vic xc nh tn s trao i cho tm ra v tr mt gen gp rt nhiu kh khn do phi sng lc cc c th t bin t s lng c th rt ln cc th h con chu qua cc php lai khc nhau. Mt khc, bng phn tch trao i cho, v tr cc gen c th c xc nh trn bn di truyn nhng khng phn nh c chc nng ring bit ca chng. Nhc im ny c khc phc nh th nghim b tr chc nng (complementation tests). V d, khi kt hp cc t bo nm men Neurospora dng n bi b t bin c cng mt biu

hin l mt kh nng mc trn mi trng thiu histidine, cc nh di truyn nhn c mt s t bo lung bi c th phc hi kh nng sinh si trn mi trng khng c histidine. Kt qu php lai gia cc dng t bo t bin cho php xc nh tnh trng ny lin quan n hai gen khc nhau trong con ng sinh tng hp histidine. Da vo tn s trao i cho, cc gen ny c v tr phn b nhng im khc nhau trn bn di truyn. Nh vy, th nghim b tr chc nng cho php phn bit tng gen trong nhm gen cng qui nh mt tnh trng. Cc nghin cu tip theo do cc nh di truyn Clarence P. Oliver v Melvin M. Green thc hin trn rui gim pht hin thy trao i cho c th xy ra ngay trong mt gen. Ni cch khc, mt gen c th cha nhiu t bin khc nhau. Nh di truyn hc Seymour Benzer xc nh c 199 v tr t bin trn gen rIIA thc khun th T4. c bit nh vo vic khm ph ra cu trc ADN, Charles Yanofsky v cng s ln u tin a ra bng chng r rng v trao i cho xy ra gia cc nucleotide ca mt gen khi nghin cu gen m cho enzym tng hp tryptophan E.coli. Nh cc kt qu c bit quan trng trn m khi nim v gen c hon thin hn. Lc ny gen c xem l mt on nucleotide mang m di truyn cho cc acid amin ca mt si peptide. T khi nim ban u cho rng mi gen l mt ht cm ca mt chui (chui chnh l nhim sc th trong genome) v trao i cho cng nh t bin ch xy ra gia cc ht cm th cc nh di truyn hc tm c mi lin h tuyn tnh gia cc m di truyn b ba ca mt gen vi trt t acid amin trn si polypeptide.

Hnh 1.1: Hai protein c m bi mt on ADN duy nht do im bt u (hoc kt thc) qu trnh phin m tng hp ARNm xy ra cc v tr khc nhau ngay trn on ADN to ra cc si ARNm khc nhau (A) hoc do im khi u dch m tng hp protein phn b cc v tr khc nhau trn mt si ARNm (B).

Tuy nhin, khi nim gen nu trn khng th gii thch cho mt s hin tng nh sau: a/ Hin tng cc gen gi ln nhau (overlapping genes): trn mt on ADN hai gen khng nm k tip nhau m gen ny nm gi u ln gen kia. Nh th, phn ADN c 2 gen nm gi ln nhau cha m di truyn cho c hai gen. C th xy ra cc trng hp sau:

* Hai phn t ARNm c phin m t cc v tr bt u hoc kt thc khc nhau trn mt on ADN. Kt qu l hai phn t protein (c dch m t hai si ARNm) c cha mt on acid amin ging h nhau mc d hai protein cc chc nng khc nhau trong t bo (Hnh 1.1A). * Mt phn t ARNm c phin m t mt on ADN c th dng lm khun tng hp hai chui polypeptide khc nhau do im bt u dch m (start codon) phn b lch nhau (hin tng lch khung c). Hai protein ny c th khc nhau hon ton v trnh t acid amin v chc nng trong t bo (Hnh 1.1B). b/ Mt on ADN mang m di truyn ca 2 gen nn c phin m tng hp nn 2 loi ARNm khc nhau. iu ny xy ra khi m di truyn phn b theo cc khung c khc nhau ngay trn on ADN (Hnh 1.2). Do , hai protein c thnh phn acid amin v chc nng khc nhau hon ton c tng hp. Mt t bin xy ra ti mt v tr trn on ADN ny c th gy nh hng n mt hoc c hai gen. iu gy kh khn cho vic xc lp bn tnh trng.

Hnh 1.2: Hai gen m cho hai protein cng nm trn mt on ADN do m di truyn ca hai gen ny phn b theo cc khung c khc nhau

c/ i vi sinh vt eukaryot, mt gen thng bao gm cc on nucleotide cha m di truyn (exon) xen k vi cc on khng cha m (intron). Cc exon v intron u c phin m sang phn t ARN (gi l phn t tin thn ARN thng tin-ARNm). Sau , cc intron s b ct b i, cc exon c ni li vi nhau theo ng th t to ra phn t ARNm hon chnh. C th xy ra trng hp hoc l ch mt s intron hoc l tt c cc intron u b loi i khi phn t ARNm. Mt khc c th xy ra hoc tt c cc exon hoc ch mt s exon c ni vi nhau. Vic la chn intron ct s to ra cc phn t ARNm khc nhau mc d chng u xut pht t mt loi ARNm tin thn c phin m t mt khun ADN (Hnh 1.3). y l hin tng ct ni intron-exon lun phin (alternative splicing).

Hnh 1.3: Qu trnh la chn, ct cc intron (I) v ni cc exon (E) theo cc th t khc nhau to ra cc phn t ARNm ch ging nhau mt s exon (E1 v E3). Chng m cho hai chui polypeptide c chc nng khc nhau trong t bo.

d/ Gen m cho polyprotein: Polyprotein l sn phm u tin ca vic dch m t mt phn t ARNm, nhng sau phn t protein ny b ct ra thnh cc on peptide nh hn. Phn t polyprotein khng c hot tnh. Ch c cc on peptide mi c cc chc nng khc nhau. V d, cc hormon adrenocorticotropic (ACTH), lipotropic (LPHs), hormon kch hot melanocyte (MSHs) v enkephalin c to ra t mt phn t proopiomelanocortin ban u (Hnh 1.4). Nh vy trn thc t, mt on ADN sao chp ra mt loi ARNm nhng c nhiu loi protein c to thnh. e/ Mt s gen khng mang thng tin di truyn cho protein: Mt iu r rng rng cc phn t ARN ribosome (ARNr), ARN vn chuyn (ARNt) u c sao chp t ADN nhng chng khng c dch m. Ngoi ra, trong nhn t bo eukaryot cn tm thy cc phn t ARNsn kch thc nh (small nuclear RNA) m nhim rt nhiu chc nng khc nhau nh tham gia vo vic bin i phn t ARNm (ct intron v ni exon), kim tra li thng tin di truyn trn chng (c ch c sa ARNm), tc ng n bn vng ca ARNm trong t bo cht hoc tham gia vo c ch bt hot gen (ARNi-interference RNA tm dch l ARN nhiu). Do , cc on ADN m cho cc loi ARN ny phi c xc nh nh cc gen bi l t bin trn chng u c th lin quan n vic xut hin cc tnh trng l.

Hnh 1.4: Phn t proopiomelanocortin c phn ct to ra cc hormon MSH, LPH, CLIP v -endorphin c hot tnh.

T cc khi nim v gen c hnh thnh v thay i dn ph hp vi cc kt qu th nghim, sinh hc phn t ngy nay nh ngha mt gen nh sau: Gen l mt on ADN cn thit cho s tng hp mt polypeptide c hot tnh hoc mt phn t ARN cn thit cho hot ng ca t bo. Nh vy, mt gen khng phi ch bao gm vng cha m di truyn (codon region) m cn gm cc on ADN (cc vng ADN iu khin (regulatory elements)) cn thit cho vic phin m (Hnh 1.5). Mt khc, c nhng on ADN c cu trc hay trnh

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t nucleotide rt ging gen nhng chng khng c phin m hoc khng biu hin chc nng g nn chng khng th c xem l gen.

Hnh 1.5: Cu trc gen m cho protein t bo nhn thc (eukaryote gene). V tr nucleotide u tin c phin m sang phn t ARN c k hiu l +1. Nucleotide nm trc v tr +1 c k hiu 1 (khng c v tr 0). Cc nucleotide nm trc v tr ( +1) thuc vng promoter. Cc intron nm xen k cc exon. Intron b loi khi phn t ARNm bi phn ng ct ni intron-exon (spilicing). Chiu phin m c ch bng mi tn.

1.2

Genome (h gen)

Genome cha ton b thng tin di truyn lp trnh m bo hot ng sng cho t bo. a s genome vi khun phn b trn mt nhim sc th c kch thc nh v c dng vng khp kn. Ngc li, phn genome trong nhn t bo eukaryot thng rt ln v phn b trn cc nhim sc th dng thng. Thng tin di truyn khng ch nm trong trnh t nucleotide (genetic information) m ph thuc rt nhiu vo cu hnh khng gian ca nhim sc th (di truyn ngoi sinh- epigenetic information). Trnh t nucleotide ca ton b genome c xc nh i vi mt s sinh vt m hnh (model organisms) i din cho mi gii sinh vt nh vi khun E.coli, nm men, rui gim, giun trn, Arabidopsis v ngi. Bn m khong cch gia cc v tr c tnh bng n v nucleotide c xem l chnh xc nht. Bn ny c gi l bn vt l (physical map). Ngoi ra cn c mt s loi bn khc. V d, bn di truyn (genetic map) cho bit mi lin h v v tr ca cc nhm gen vi nhau hay ca cc ch th (markers) trn nhim sc th. Cc ch th ny c th l hnh thi (biu hin tnh trng), s a dng ca protein (protein polymorphisms), a dng di ca cc on gii hn (restriction fragment length polymorphisms-RFLPs), a dng di cc trnh t n gin (simple sequence length polymorphisms-SSLPs) v a dng cc on ADN c khuych i ngu nhin (randomly amplified polymorphic DNA-RAPD). Khonh cch gia cc v tr trn bn di truyn c tnh bng cM (centiMorgan) da vo tn s trao i cho. Hai v tr cng gn nhau th cng kh xy ra trao i cho gia chng trong phn bo gim nhim. Tuy nhin, trao i cho khng xy ra nh nhau mi v tr trn nhim sc th nn n v centiMorgan khng phn nh chnh xc khong cch gia cc v tr trn bn di truyn. Kt hp gia bn vt l v bn di truyn cho bit chnh xc khong cch gia cc gen (tnh trng), gia cc ch th phn t lin quan n nhng tnh trng cn nghin cu. Genome khng phi n thun l tp hp ca cc gen. Genome ca vi khun v sinh vt eukaryot bc thp thng khng ln v cc gen phn b st nhau. Hu ht cc gen ny ch c mt bn sao trong genome v rt t b gin on bi cc on ADN khng cha m di truyn (intron). Ngc li, thnh phn ADN cha cc gen ch chim mt t l rt nh so vi ton b genome trong t bo eukaryot bc cao. Cc gen trong t bo eukaryot bc cao thng cha nhiu intron v phn b xa nhau. T nhng nm 70, bng cc th nghim gy bo ho t bin, cc nh di truyn hc c th xc nh c s gen nm trn mt on nhim sc th.

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Ngy nay cc k thut phn tch ADN hin i (cc php lai Southern, northern, microarray...), cho php xc nh s gen hot ng trong mt t bo. V d, t bo nm men (sinh vt eukaryot bc thp) c khong 4000 gen hot ng, cn t bo ng vt c v khong 10.000 - 15.000 gen. Nh vy, nu di trung bnh ca mt gen khong 10000 bp th tng s chiu di cc gen hot ng trong mt t bo cng ch chim 1-2% genome. Hay ni cch khc ch mt phn rt nh genome mang thng tin di truyn cn thit cho hot ng sng ca t bo. So snh kch thc genome ca mt s loi gn nhau trong bc thang tin ho (tc l c phc tp loi tng t nh nhau) cng nh genome ca nhng loi cch xa nhau (tc l c tnh phc tp khc nhau) cho thy kch thc genome khng phi lun lun t l vi tnh phc tp ca loi. V d, genome ca ngi c kch thc khong 3,3x109 bp, trong khi genome cc loi lng c di tng t c 3,1x109 bp hoc ca thc vt c th t n 1011 bp. C l no loi lng c li c tnh phc tp nh c th chng ta? Mt khc, ngay trong cng mt loi chng ta cng nhn thy s mu thun v kch thc genome. V d, rui sng trong nh (Musca domestica) c genome c 8,6x108 bp, ln gp 6 ln kch thc genome rui gim (D.melanogaster) vi genome c 1,4x108 bp. Ngoi ra, kch thc genome ca cc qun th lng c thay i t 109 bp n 1011 bp (khc nhau gp 100 ln). V sao ngay trong cng mt loi kch thc genome li bin thin nhiu nh vy ? Kt qu bc u so snh genome gia cc loi sinh vt vi nhau cho php rt ra ba c im ni bt. Th nht, cc gen phn b khng theo qui lut trong genome. Th hai, kch thc genome thay i khng t l vi tnh phc tp ca loi v cui cng l s lng nhim sc th cng rt khc nhau ngay gia nhng loi rt gn nhau. Nu phn tch chi tit i vi mt gen nht nh th v tr cc intron, cc exon, cc on ADN iu khin hot ng ca gen vv... u l nhng yu t quan trng so snh tm ra mi quan h gia cc loi. Ngoi ra, tng s gen ni chung, s lng cc gen c nhiu bn sao trong genome, t l cc loi ADN lp li v thnh phn ca chng cng nh s di chuyn ca cc gen t genome ring bit ca cc bo quan (ty th, lc lp) sang genome trong nhn u chu nh hng ca thi gian, u phn nh qu trnh tin ho ca cc loi. Mt khc, c c s so snh chnh xc hn, ton din hn, cn xt n cu trc si chromatin, cu hnh khng gian ba chiu ca nhim sc th cng nh ca ton b genome trong nhn.

1.2.1. Genome ca t bo prokaryot (t bo nhn s)


Genome trong t bo prokaryot khng ln nn s lng genome ca cc loi vi khun c xc nh trnh t ngy cng nhiu. Nh cc thng tin d liu v cu trc h gen prokaryot, s phn b cc gen, cch thc kim sot hot ng cng nh chc nng ca chng ngy cng phong ph v tr nn r rng. Genome prokaryot c kch thc nh hn rt nhiu so vi genome eukaryot. Bn cnh nhim sc th cha phn ln thng tin di truyn, t bo prokaryot cn c nhiu loi plasmid. Trc y, plasmid c xem l nhng phn t ADN dng vng cha cc gen khng quan trng. V d, plasmid thng mang gen lin quan n tnh chng chu khng sinh. Do , t bo vn c th tn ti ngay khi thiu vng cc gen ny. Tuy nhin, khi nim plasmid c m rng ra khi thc nghim tm thy mt s t bo prokaryot c cha phn t ADN kch thc nh, dng thng v mang cc gen tng t nh plasmid dng vng. V vy, plasmid c hiu l nhng on ADN kch thc nh mang mt s gen khng quyt nh s sng cn ca t bo. Hn na, mt loi plasmid i khi c tm thy trong cc loi t bo prokaryot khc nhau. Mt khc, plasmid c kh nng bin np t loi t bo prokaryot ny sang loi khc. V vy, mc d c cha gen nhng plasmid dng nh khng c xem l mt phn ca genome.

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Hu ht genome prokaryot nh hn 5 Mb (5.000.000 nucleotide) v thng c phn b trn mt nhim sc th dng vng. Mt s t bo prokaryot c genome l phn t ADN dng thng. c bit, mt s genome prokaryot l phn t ARN hoc kt hp c hai loi ADN v ARN. Ngoi ra, genome prokaryot c th bao gm cc gen phn b trn cc on thng ADN hoc trn c hai loi phn t ADN dng thng v dng vng. V d, nhim sc th dng thng c pht hin ln u tin Borrelia burgdorferi vo nm 1989. Nhim sc th ny di 910 kb gm 853 gen. Bn cnh , t bo Borrelia burgdorferi cn c ti 17 plasmid dng vng v dng thng vi tng chiu di l 533 kb lin quan ti 430 gen. Hu ht cc gen phn b trn plasmid khng quan trng, ch c mt s t gen cn thit cho qu trnh tng hp purine v protein mng t bo. Do , trong tng s 17 plasmid, mt vi plasmid cha cc gen ny c xem l mt b phn ca genome trong t bo Borrelia burgdorferi. Nhng dn liu thc nghim nhn c khi phn tch genome v cc plasmid Borrelia burgdorferi gy tranh ci gia cc nh sinh hc khi so snh genome Borrelia burgdorferi vi Treponema pallium. Theo phn loi, y l hai loi vi khun c quan h gn gi nhau. Ging nh a s cc t bo prokaryot khc, genome loi th hai l mt phn t ADN dng vng c kch thc 1138 kb vi 1041 gen. iu th v l khng mt gen no Treponema pallium tng ng vi cc gen phn b trn plasmid ca loi th nht. Phi chng cc plasmid va c t nhin bin np vo Borrelia burgdorferi? Genome prokaryot khng ng gi trong cu trc nucleosome (nh genome eukaryot) v khng c bao bc bi mng nhn. Nhim sc th dng vng c cu trc khng gian ging nh nhng cnh hoa ca bng hoa, mi cnh l mt on ADN c cu trc siu xon (supercoil). Cc cnh khng u nh nhau v c nh vo li protein. Genome vi khun c khong 40-50 cnh. Cu trc kiu bng hoa ny c gi l nucleoid (Hnh 1.6). Cu trc nucleoid gip genome ch chim mt th tch rt nh trong t bo. Ngoi ra, cu trc khng gian ny ca nhim sc th c duy tr nh cc phn t ARN kch thc nh tng tc vi protein. Do , ngay khi b t gy, cu trc siu xon ca nhim sc th cng ch m ra mt cch cc b cnh b tn thng ch khng xy ra trn ton b genome. Hai enzym ADN gyrase v ADN topoisomerrase gi vai tr chnh cng phi hp vi phc protein khc lm nhim v ng gi ADN vi khun. Thc nghim pht hin c t nht 4 protein tham gia phc ny, trong protein HU c chc nng tng t nh histone t bo eukaryot. Mc d c cu trc rt khc vi histone nhng HU dng tetramer to thnh li c qun quanh bi on ADN khong 60 bp. Nh vy, protein HU c chc nng tng t histone trong vic qui nh nghim ngt cu trc khng gian ca si nhim sc th. Tuy nhin, chng ta cha xc nh c cc li ny c phn b u n hay ch tp trung ti "nh hoa" nucleoid.

Hnh 1.6: M hnh cu trc nucleoid E.coli gm 40-50 vng siu xon kt nh vi li protein v si ARN. Khi c t gy xy ra mt vng siu xon, nhim sc th ch m xon cc b vng ny (theo Snustad v Simmons, 2000).

13

Nm 1995, genome Haemophilus influenzae l genome u tin c xc nh ton b trnh t. n nm 1998 c hn 18 genome vi khun khc c c hon ton. Trong s ny, Mycoplasma genitalium c kch thc nh nht gm 580.070 bp v Mycobacterium tuberculosis c kch thc ln ti 4.411.529 bp. E.coli c nghin cu chi tit nht v c xem l i tng m hnh ca di truyn, ho sinh v sinh hc phn t. H gen E.coli gm 4.639.221 bp vi 4.288 trnh t c cc c tnh cu trc ca gen m cho protein (putative protein coding sequences). Mt phn ba s trnh t ny c xc nh l cc gen trong khi 38% cha bit c chc nng. Cc trnh t nucleotide c gi nh l gen nhng cha bit sn phm protein m chng m cho th c gi chung l khung c m (open reading frame-ORFs). Mt khung c m thng bt u bi b ba m di truyn cho methynonine (start codon) v kt thc bi mt trong s ba m dng tng hp protein (stop codon). Mc d c kch thc nh hn nhiu so vi genome eukaryot, nhng genome prokaryot c mt phn b cc gen cao hn, s on ADN khng cha m di truyn t hn. Ni cch khc, khong cch gia cc gen ngn hn. V d, khong cch trung bnh gia hai gen E.coli l 118 bp. Cc gen v ORFs chim 87,8%; cc gen m cho ARNs chim 0,8%; cn thnh phn ADN lp li khng cha gen ch chim c 0,7%. Mt khc hu ht cc gen prokaryot u tn ti n bn (single-copy gen) v cc gen khng c intron. Kt qu so snh trnh t nucleotide ca ton b genome E.coli vi cc trnh t ADN lu tr trong ngn hng d liu cho php pht hin 6 gen mi m cho ARNt, 12 gen lin quan n sinh tng hp v lp rp roi cng nh 2 gen m cho cc enzym tham gia vo con ng phn hy cc hp cht hu c vng. R rng vic so snh trnh t h gen gia E.coli v cc sinh vt prokaryot khc c bit c ngha trong vic xc nh cc gen mi cng nh chc nng ca chng. Ngoi ra, khi so snh s lng gen tham gia vo mt qu trnh sinh hc cc vi khun khc nhau cho php nh gi s lng gen ti thiu cn thit cho qu trnh . V d, qu trnh trao i cht lin quan n khong 243 gen E.coli, 112 gen Haemophilus influenzae nhng ch cn n 31 gen Mycoplasma genitalium. Hn na, vic so snh s lng gen phn b trong nhng genome c kch thc nh nht nh M.genitalium, M. pneumoniae cho php nh gi c s lng gen ti thiu cn thit duy tr s sng cho c th n gin nht. M.genitalium c 470 gen v M. pneumoniae c 679 gen. So snh cc gen v chc nng ca chng hai loi vi khun ny cho php c tnh s gen ti thiu cn c duy tr s sng l 256 gen. Tuy nhin nh k thut di truyn phn t gy t bin nh hng chnh xc tng gen, thc nghim tng dn s lng gen cn b t bin v nhn thy t nht cn c 300 gen m bo s sng cho vi sinh vt n gin nht. Ngoi ra, vic so snh cc gen ging v khc nhau gia cc vi khun c quan h gn gi trong tin ho c bit c ngha xc nh nhng gen ring bit ca tng loi, tc l nhng gen ch th dng phn bit loi ny vi loi kia. V d, trong s 470 gen c M.genitalium th 350 gen cng tn ti Bacillus subtilis. Nh vy ch c 120 gen to nn s khc bit gia hai loi vi khun ny. Tuy nhin, nhng nghin cu v cch thc hot ng ca cc gen ring bit ny, chc nng ca tng sn phm protein m gen m cho cng nh cc qu trnh ho sinh m chng tham gia cha a n kt lun r rng v vai tr ca 120 gen c th cho M.genitalium.

1.2.2. Genome ca t bo eukaryot (t bo nhn thc)


Genome ca t bo eukaryot bao gm cc nhim sc th phn b trong nhn v ADN phn b trong mt s bo quan nh lc lp, ty th. Tuy nhin, do hu ht s lng ADN cng

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nh cc gen tp trung ch yu trong nhn nn ADN (nhim sc th) phn b trong nhn c cc nh sinh hc quan tm rt nhiu. Cc nhim sc th l cc phn t ADN lin kt vi protein, dng thng. Khng c mi lin h rng buc no gia ba thng s sinh hc: s lng nhim sc th, kch thc genome v tnh phc tp ca loi. V d, nm men S.cerevisiae c xem l sinh vt eukaryot bc thp nhng li c s lng nhim sc th nhiu gp 4 ln rui gim D. melanogaster. Ngoi ra, kch thc genome k nhng ln gp 30 ln h gen ca ngi nhng s lng nhim sc th ch bng mt na. Hn na, mt s nhim sc th c kch thc rt nh (cc nhim sc th mini) nhng c mt phn b cc gen rt cao. V d, h gen ca g gm 39 nhim sc th, trong 6 nhim sc th bnh thng chim 66% ADN nhng ch c 25% cc gen phn b trn 6 nhim sc th . Ba mi ba nhim sc th cn li u l nhim sc th mini chim 1/3 ADN v c ti 75% cc gen. Nhng so snh l th ny cho thy s b him gia tin ho v cu trc genome trong cc sinh vt khc nhau m hin ti sinh hc cha gii thch c. Kch thc genome trong nhn eukaryot thay i t 12 Mb (nm men S.cerevisiae) n 120.000 Mb (thc vt F.assyriaca). Genome bao gm thnh phn ADN khng lp li v ADN lp li. Phn ln cc gen phn b trong thnh phn ADN khng lp li v s lng ca chng tng cng vi tnh phc tp ca loi. Tuy nhin, iu c bit lu l tnh phc tp khng ch ph thuc vo s lng gen m cn c xc nh bi thnh phn ADN lp li. V vy, khng phi lun lun tn ti mi tng quan t l thun gia kch thc genome v tnh phc tp ca loi. V d, kch thc genome ca ngi khong 109 bp trong khi genome mt s loi lng c hoc thc vt c th t n 1011 bp. Genome ca ngi c gii m hon ton (2001) bao gm cc thnh phn ADN c trnh by trn hnh 1.7.

Hnh 1.7: Cc loi ADN trong genome ngi (theo Brown, 2001).

1.3

Cu trc si nhim sc trong t bo eukaryot

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Trong nhn t bo eukaryot, ADN lin kt vi protein to ra cu trc gi l chromatin (si nhim sc). C th phn bit cc protein ny lm hai nhm chnh: histone v non-histone. Thnh phn protein non-histone thay i gia cc m, t chc, gia cc loi. Mi loi protein non-histone ch chim mt s lng rt nh so vi tng s protein non-histone hoc vi bt k loi protein histone no. Tuy nhin cc protein non-histone gi mt vai tr rt quan trng qui nh cu trc khng gian c th ca tng vng nhim sc th. Hot ng ca nhiu gen, c bit cc gen lin quan n pht trin phi, khng ch ph thuc vo trnh t nucleotide m cn ph thuc vo cu trc ca nhim sc th. Thng tin di truyn cha trong cu trc khng gian ca nhim sc th c gi l thng tin di truyn ngoi sinh (epigenetic information). S dng dung dch c lin kt ion yu, c th tch ra khi nhn t bo cc si nhim sc dng si n, ng knh khong 30 nm, gm cc ht nh ging nh chui ht cm (ng knh ht khong 10 nm). Cc ht nh ny c gi l nucleosome. Chng khng phn b ng u mi vng trn si nhim sc. Khi si ADN b ct bi nuclease, cc nucleosome tch ra ring bit. Mi nucleosome gm mt on ADN di 146 bp qun 2 vng quanh li protein cha 8 tiu phn ca 4 loi histone H2A, H2B, H3, H4. Phn u NH2 ca histone khng nm trong cu trc nucleosome m tn ti t do. on ADN nm gia hai nucleosome c gi l ADN ni (linker DNA). on ny c kch thc khong 50-70 bp. Protein histone H1 lin kt vi ADN linker nm gia 6 nucleosome v ng gi chng li thnh mt cu trc c bit gi l solenoid (ging nh mt bng hoa 6 cnh). Cc solenoid qun chng ln nhau thnh si xon. Nh cu trc c bit nn th tch ADN chim trong nhn gim i rt nhiu. Si ADN qun quanh li histone trong cu trc nucleosome v c ng gi trong cc solenoid c trt t rt cao. Mt iu th v c t ra l cc cu trc ny thay i nh th no khi mt on ADN c s dng phin m (to ARNm) hoc sa cha khi xy ra sai hng? Liu khi chng c b ph v tm thi nh trong qu trnh ti bn ADN hay khng? Cc phn t histone c gii phng hay vn lin kt vi ADN? Gii p nhng cu hi ny c nhiu kt qu khc nhau cho thy vic phin m khng nht thit yu cu ph v cu trc chromatin. Tuy nhin chc chn c xy ra nhng thay i trong cc tng tc protein-ADN, histone-histone, histone-non histone. iu c bit c ngha l vic thm bt cc nhm chc trn tng phn t protein tham gia lin kt to nucleosome khin cho cu trc nucleosome thay i. Li histone c th khng b phn r thnh cc tiu phn nhng b dch chuyn mt cch cc b trn si nhim sc bi cc protein iu bin chromatin (remodelling chromatin proteins). Cc protein ny gip cho vic tho g cc b si ADN khi cu trc nucleosome m khng nh hng n cc vng khc. ADN c gii phng ra khi nucleosome khng c ngha chng trng thi t do, v nh vy ADN rt d b ph hy bi cc tc nhn khc nhau trong t bo, c bit bi cc nuclease. Lc ADN thng lin kt vi cc protein c hiu (cc factor) cn thit cho s phin m hoc cc phc cn thit cho qu trnh sa cha ADN. Th nghim cho thy khi cc factor phin m tng tc vi ADN, chng c kh nng ngn cn histone lin kt vi ADN. iu ny c th l gii vic tn ti nhng vng tr vi nuclease nm xen cc v tr nhy cm trong mt gen. Khi protein bm vo ADN, ADN c bo v khi s phn ct ca enzym.

1.3.1. Histone trong cu trc nucleosome


Mi nucleosome c 146 bp ADN qun hai vng quanh li histone gm 8 tiu n v 2x[H3-H4] v 2x[H2A-H2B]. Cc histone ca li c cu trc tng t nh nhau, gm cc on peptide (domain) tn cng u NH2 (N-terminal), domain chung gip histone gp khc

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v phn tn cng COOH (C-terminal). Domain cn cho histone gp khc cn lin quan n tng tc gia cc histone v gia histone vi ADN. Cc nucleosome c gn vi nhau nh histone H1. Protein ny lin kt li histone vi ADN ni (ADN linker) nm gia nucleosome. di ca cc ADN linker khng c nh nh nhau. Histone H1 thit lp nn cu trc c trt t cao cho si nhim sc. Cc histone tham gia cu trc li u c phn u NH2 nm ngoi li, phn b t do theo cc hng khc nhau (Hnh 3-GT). Chiu di ca on phn b t do thay i t 16 n 44 acid amin (H3-44; H2B-32; H4-26 v H2A-16 acid amin). Cc on ny gi vai tr quan trng i vi s co c ca si nhim sc. Nghin cu ng hc qu trnh thay i cu hnh ca si nhim sc cho thy n c th tn ti ba dng: khng co c (unfolded), co c mc trung bnh (moderately folded) v co m c (extensively foded). on t do ca H3 v H4 cn thit si nhim sc c mc co c va phi. c bit on t do ca H3 khng th thay th c. di v v tr ra khi phn li ca on ny cn thit cho vic hnh thnh cu trc khng gian ca si nhim sc. Nh vy cng vi histone H1, cc on t do ca bn loi histone trong cu trc li u cn thit duy tr cu trc khng gian ba chiu cho nucleosome, duy tr trng thi co m c ca si nhim sc cng nh tng tc gia cc si nhim sc vi nhau. Ngoi ra, chng cn l cc v tr tng tc vi cc protein non-histone. Sau khi c tng hp, c bn loi histone u chu cc bin i nh ubiquitin ho, phosphoryl ho, glycosyl ho v c bit c ngha l qu trnh methyl ho v acetyl ho. Hu ht cc bin i ny xy ra vng N-terminal. Qu trnh phosphoryl ho v methyl ho c th tc ng qua li vi nhau, nh hng n s co c ca nhim sc th khi bc vo mitose. Ring trng hp ubiquitin ho xy ra phn ui C-terminal ca histone, gip cho cu trc nucleosome b ph v tm thi trong qu trnh ti bn hoc tng hp ARN. Nh vy, cc bin i ho hc ca histone tc ng n cu trc khng gian ca nucleosome v hot ng ca gen, trc ht qu trnh phin m. Cc histone trong cu trc li b acetyl ho ti cc acid amin lysine c hiu phn b phn N-terminal. Ngoi tr histone H2A, cc histone li khc thng c 4 n 5 v tr c gn nhm acetyl. Mt nucleosome c th c ti 26 v tr mang nhm acetyl. Acetyl ho histone c mt vai tr quan trng, quyt nh n cu trc cngi nhim sc. Nh si nhim sc khng co c, ADN c gii phng ra khi nucleosome, s tng tc gia cc nucleosome b ph hy, gy thay i lin kt gia cc domain N-terminal ca histone vi cc protein nonhistone, hoc lin kt gia cc protein vi ADN. Nhng bin i ny gp phn hot ho phn ng tng hp ARN. Ch cn 46% trong tng s 26 v tr c bit b acetyl ho cng ph v trt t cu trc ca si nhim sc v tng cng qu trnh sao chp ARN cc gen. Thng thng acetyl ho v kh acetyl histone lin quan n hot ha hay km hm hot ng ca gen. Mi loi histone c th c gn nhm acetyl nhng v tr c hiu bi cc enzym ring bit. iu gy ra nhng tc ng khc nhau n biu hin ca gen. Ngoi ra, qu trnh acetyl ho cn lm thay i cu trc ca phc iu bin chromatin (remodeling chromatin complexes). Phc ny c chc nng ph v tm thi cu trc li histone hay dch chuyn nucleosome trn si nhim sc. Chng thng tng tc vi vng N-terminal ca histone. Khi vng ny c mang nhm acetyl, phc iu bin chromatin c th lm cho cc histone H2A-H2B b di chuyn ra khi cu trc li nucleosome. Nh , cc promoter c bc l, cho php qu trnh tng hp ARNm c bt u. ng hc ca phn ng acetyl ho v kh acetyl rt linh ng, phc tp, ph thuc vo hot tnh ca cc enzym lin quan. Bin i thun nghch gia hai dng acetyl ho v kh acetyl ca histone ph thuc vo hai loi enzym histone acetyl transferase (HAT) v histone

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deacetylase (HDAC) cng vi cc protein ng hot ha (coactivator) vi HAT hoc ng c ch (corepressor) vi HDAC. R rng, hai qu trnh acetyl ho v kh acetyl c tc dng ngc nhau trong vic lm thay i cu trc si nhim sc v hot ng ca cc gen. Cc enzym deacetylase HDAC lm gim mc acetyl ho histone, dn n km hm qu trnh phin m. Ngc li, enzym acetyl transferase HAT tng cng acetyl ho kch thch qu trnh phin m. Mt khc, cnh tranh gia hai phn ng acetyl ho v kh acetyl gip si nhim sc thay i cu trc linh hot, p ng kp thi vi tng cng hoc km hm hot ng ca gen. ng vt c xng sng, bn loi histone H2A, H2B, H3 v H4 t thay i gia cc loi. Tuy nhin protein H1 gm mt s dng c k hiu t H1a n H1e, H1t v H5. V tr phn b ca cc loi histone H1 ny cha c xc nh r rng. Mt khc trong cc t bo tinh trng, histone c thay th bi protein protamine. Hn na, histone c tnh kim do cu trc bc I ca chng c khong 20-30% arginine v lysine. y l cc acid amin tch in dng (+). Nh vy thay i in tch ca histone lin quan cht ch n kh nng tng tc vi ADN v bn vng ca tng tc v acid nucleic c in tch m quyt nh bi nhm phosphate.

1.3.2. Methyl ho ADN


Bn thn ADN cng chu cc bin i do gn thm cc nhm chc khc nhau. V d, hin tng methyl ho cytosine hoc adenine. Nhng thay i ny c tnh c th cho tng vng nhim sc th, tc ng n cu trc khng gian ca si nhim sc v tham gia kim sot hot ng ca cc gen. Nhng c th ring ca tng vng nhim sc th c di truyn cho th h sau. Sai lch trong cu trc khng gian ca si nhim sc c th lm xut hin tnh trng mi ngay khi trnh t nucleotide khng sai hng. Do , phn t ADN c cha hai dng thng tin: thng tin di truyn (genetic information) quyt nh bi trnh t nucleotide v thng tin ngoi sinh (epigenetic information) quyt nh bi tnh phc tp v cu hnh khng gian ca genome. Hin tng methyl ho xy ra vi c ADN prokaryot v eukaryot. S methyl ho ADN prokaryot c xem nh l mt c ch bo v h gen, trong khi eukaryot methyl ho ng vai tr quan trng trong dng thng tin th hai. chnh l mt trong cc c ch kim sot hin tng nh du DNA (DNA imprinting), tc l tnh trng ca gen c biu hin ph thuc vo ngun gc di truyn t b hay m. Cn lu DNA imprinting hon ton khc vi di truyn theo gii tnh. Hin tng nh du ADN s c xem xt chi tit phn sau. Methyl ho ADN c ngha c bit i vi hot ng ca gen eukaryot, nht l cc gen trong qu trnh hnh thnh pht trin c th. Phn ng methyl ho xy ra nhng v tr c hiu. Khong 2-7% ADN t bo ng vt b methyl ho. Hu ht nhm methyl c tm thy cytosine (C) phn b trong cp nucleotide CpG. T l cytosine b methyl ho thay i rt khc nhau gia cc loi. Hu nh khng pht hin c methyl-cytosine nm men S.cerevisiae. Khong 10% cytosine b methyl ho ng vt c xng sng v 30% thc vt. Ch n nm cui cng ca thp k 20 mi khng nh c c hin tng methyl ho ADN Drosophila. Tuy nhin ch c khong 0,4% ton b h gen rui gim b methyl ho. Hn na cytosine gn gc methyl nm trong cu trc CpT v CpA ch khng phi trong trt t CpG (nh i vi ng vt bc cao). c bit thc vt bc cao, hin phn ng methyl ho c th xy ra vi cytosine trong mi cu trc CpG, CpNpG v CpNpN, trong N = A, T hoc C.

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Khi a cc gen b methyl ho hoc b kh methyl vo genome t bo nhn (th nghim chuyn gen) th ch nhng gen khng c nhm methyl mi hot ng. Mt khc, vng ADN khng c nhm methyl thng trng vi vng c cc v tr nhy cm ADNase. Thc nghim nhn thy rt nhiu gen khi ang phin m tng hp ARN u khng c nhm methyl vng cha promoter v exon th nht (u 5'), mc d cc exon tip sau v pha u 3' c cha nhm ny. R rng methyl ho c tc dng ngn cn gen hot ng. Ngc li, nu kh nhm ny th gen li c hot ho. Do phn bit vi CpG b methyl ho, cc cp CpG khng c gc methyl, lp i lp li nhiu ln pha trc u 5' ca gen khong 1-2 kb c gi l cm CpG (CpG island). Khong 56% cc gen trong genome ngi c phn b gn vi cm CpG. Nhng gen hot ng trong mi loi t bo (housekeeping genes) u c cm CpG khng b methyl ho. Tuy nhin, i vi cc gen c hiu (ch hot ng trong t chc chuyn bit) th phn ng methyl ho cm CpG ca chng c kim sot cht ch. Cm ny khng b methyl ho trong t bo cn n sn phm ca gen nhng li b gn gc methyl trong nhng t bo m gen khng biu hin. Nh vy mt gen hot ng, ngoi vic xut hin cc v tr nhy cm vi nuclease gn promoter, ADN vng cha gen cn b kh nhm methyl. Khi a ADN b methyl ho vo t bo, n tip tc b methyl ha khng ngng qua mi ln nhn i ADN. Ngc li, nu a ADN khng c nhm methyl vo t bo, chng khng b methyl ho sau mi ln ti bn. Phn ng gn nhm methyl vo cytosine c xc tc bi cc enzym methyltransferase. C th phn bit cc enzym ny thnh 2 nhm. Nhm th nht lm nhim v duy tr gc methyl nhng v tr cytosine trn si ADN va c tng hp trong qu trnh ti bn ADN. Vic gn gc methyl mi ny da vo nhm mCpG trn si khun. Chng c gi chung l cc enzym duy tr nhm methyl (maintenance methyltransferase). Nhm th hai gm cc enzym xc tc phn ng gn gc methyl vo v tr cytosine trn phn t ADN m v tr ny trc khng c nhm methyl. V d, gn nhm methyl vo cm CpG promoter khi cn km hm hot ng ca gen. Ngoi ra, qu trnh methyl ho cytosine trong trt t CpNpG hoc CpNpN i hi protein v cc phn t ARN kch thc ngn (20-25 nucleotide) nhn bit nhng cytosine . Nh , cu trc si nhim sc cng nh hot ng ca gen b thay i. Enzym demethylase c th m nhn phn ng kh nhm methyl. Tuy nhin, enzym ny cha c tm thy trong t bo ng vt. Kt qu nghin cu gn y (20022005) cho thy mt s enzym tham gia sa cha ADN c lin quan n vic loi b cytosine mang nhm methyl. Lc , on ADN cha mC b loi i v thay th bi cytosine khng mang nhm methyl.

1.4

Cc gen trong genome eukaryot

Mt trong nhng sai khc c bn trong cu trc gen gia sinh vt prokaryot v eukaryot l hin tng gen b gin on (interupted gene). Hin tng ny c khm ph ln u tin nm 1977 v c tm thy ph bin mi sinh vt eukaryot. K l l hin tng ny cng c pht hin mt s thc khun th (bacteriophage). Khi so snh trnh t nucleotide trn mt gen vi phn t ARNm c phin m t gen , cc nh khoa hc pht hin thy gen c cha nhng on khng mang m di truyn. Nhng on ny khng tm thy trong phn t ARNm c s dng lm khun tng hp protein. Chng c gi l cc intron. Nh vy bn cnh vic cha nhng on mang m di truyn (gi l exon), a s cc gen eukaryot cn cha cc intron. Mc d khng cha m di truyn v b ct i khi phn t ARNm, t bin xy ra intron c th ngn cn phn ng ni cc exon vi nhau, do to nn phn t ARNm sai hng khng s dng c dch m tng hp protein.

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Khi phn t ARN c phin m t mt gen, n phi tri qua qu trnh loi b cc intron, ni cc exon vi nhau (phn ng splicing). Phn ng ct ni ny xy ra vi cc loi ARNm, ARNr v ARNt. to ra phn t ARN hon thin, vic ct intron, ni cc exon tun theo nhng qui lut nghim ngt v chnh xc m bo th t ca chng. iu th v l cc exon ca mt phn t ARNm c ni vi nhau. Him trng hp ni cc exon ca cc phn t ARNm khc nhau. Do cc intron khng mang m di truyn nn t bin xy ra trn chng thng khng c biu hin cu trc ca chui polypeptide. Tuy nhin cc t bin c th nh hng n phn ng splicing khi chng xy ra cc v tr cn thit ct ni intronexon. iu ng lu vi ADN ca ty th v lc lp, intron ca gen ny c th l exon ca gen khc v sn phm protein ca hai gen c chc nng hon ton c lp. Ngoi ra, mt s gen c phin m to ra ARNm nhng chng khng c dch m. Nhng phn t ARNm ny vn tri qua phn ng ct ni intron-exon to ra cc on ARN ngn. Chng tip tc c phn hu thnh cc phn t ARN kch thc nh 22-25 nucleotides (miRNAs: micro RNAs). Cc phn t miRNAs tham gia vo nhiu qu trnh kim sot hot ng ca mt s gen trong genome, ch yu qu trnh sau phin m. Trong c ch kim sot ny, miRNAs lm nhim v nhn bit ARNm ca mt s gen khc phn hy cc ARNm ny. y l mt c ch kim sot hot ng ca gen sau phin m c pht hin vo nhng nm cui thp k 20. sinh vt bc cao, cc gen m cho protein hay cc ARNt, ARNr hu nh u b gin on. di trung bnh ca exon khong 200 bp trong khi ca intron c th ln hn 10 kb hoc thm ch t ti 50-60 kb. Ngoi ra, hin tng cc gen nm gi ln nhau (overlapping genes) rt him xy ra ADN nm trong nhn t bo eukaryot. Hin tng ny hay gp trong genome prokaryot v vi gen phn b trong cc bo quan ca t bo eukaryot. Hn na, mt gen ny c th nm trong mt gen khc, tc l gen th hai c phn b trong intron ca gen th nht. V d, in hnh cho trng hp gen trong gen (genes-within-genes) genome ca ngi l gen m cho neutrofibromatosis loi I. Intron 27 ca gen ny c cha 3 gen khc, mi gen u c exon v intron ring ca mnh (Hnh 1.8).

Hnh 1.8: Cu trc gen trong gen intron 27 ca gen m cho neurofibromatosis. Intron 27 c cha 3 gen nh OGMP, EV12B v EV12A. Mi gen ny u c intron (I) v exon (phn sm mu).

C th phn loi cc gen ty theo cu trc ca gen hoc theo chc nng ca cc sn phm do chng m ho. Genome n bi cc c th a bo c khong 1/4 n 1/2 s gen m cho protein l cc gen n l (single copy gene), khng tn ti bn sao th hai. S gen cn li thng tn ti hai hoc nhiu bn sao trong genome. Cc bn sao ca mt gen khng bt buc phi ging nhau hon ton do trong qu trnh tin ho chng chu nhng t bin nh thm, mt, thay th hoc chuyn on cc nucleotide. Cc gen hnh thnh t mt gen t tin c xp vo mt h gen (family genes). Cc gen trong cng mt h c th tp trung thnh mt nhm (trn mt nhim sc th) hoc phn tn trong genome (trn cc nhim sc th khc nhau). Sn phm ca cc thnh vin trong mt h c chc nng ging ht nhau hoc c lin quan n nhau mc d cc gen ny thng hot ng nhng thi im nht nh v trong cc loi t bo bit ho khc nhau. V d, vic tng hp cc protein globin (c m bi cc gen trong cng mt h gen) xy ra nhng giai on nht nh trong qu trnh pht trin phi

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thai v c th trng thnh. Ngoi ra cn c nhng trnh t nucleotide ging vi mt gen bit nhng trnh t khng c phin m hoc khng c dch m. Chng c gi l gi gen (pseudogen). Mt s gen gm nhiu bn sao ging ht nhau lp i lp li lin tc trn mt vng nhim sc th (tandem repeat genes). V d, gen m cho ARNr, ARNt, histone vv... Nh vy, cc gen eukaryot c th phn thnh cc loi chnh nh sau: gen n l, cc gen thuc mt h gen, gen lp i lp li lin tc v cc pseudogen.

1.4.1. Cc gen trong cng mt h gen


Cho n nay, hu ht cc gen m cho protein c nghin cu sinh vt eukaryot u khng phi l nhng gen n l. Khong mt na cc gen bit trong genome ng vt c xng sng u c cc bn sao ging ht hoc tng t (s bn copy c th t 2 n 20). Hin tng tn ti nhiu bn sao ging hoc tng t ca mt gen c th gy ra do sai lch trong trao i cho gia hai nhim sc th tng ng trong phn bo gim nhim (meiosis). iu lm cho mt nhim sc th c s lng bn copy tng ln trong khi nhim sc th kia c s lng gim i (Hnh 1.9).

Hnh 1.9: Sai lch trong trao i cho gia hai nhim sc th (mi nhim sc th c hai bn sao ca mt gen) khin mt nhim sc th ch mang mt bn sao trong khi nhim sc th th hai mang ba bn sao.

Sn phm ca cc thnh vin trong mt h gen c chc nng ging nhau nhng thng c s dng nhng thi im pht trin khc nhau hoc trong cc loi t bo bit ho khc nhau. Trnh t acid amin ca chng ch tng t m khng ging nhau hon ton. Khi mt thnh vin trong h gen b bt hot, thnh vin khc c th c hot ho thay th mc d bnh thng thnh vin th hai khng hot ng cng vi gen ban u. Cc gen globin l th d in hnh v mt h gen (Hnh 1.10). mi loi ng vt, cc gen ny c cu trc tng t do chng c cng ngun gc t mt gen t tin. T bo trong c th trng thnh c globin tn ti dng tetramer gm hai chui polypeptide v hai chui . Cc gen m cho cc chui ny nm trn hai nhim sc th khc nhau. Do hot ng ca chng phi c phi hp ng thi sao cho s lng hai loi polypeptide c to ra mt cch tng ng vi nhau v mt s lng. T bo mu ca phi cng cha globin dng tetramer nhng gm hai chui tng t v tng t . Cc gen m cho chui v chui tng t u thuc mt h gen trong khi cc gen m cho chui v chui tng t thuc h gen khc. Ngoi ra, trong mi h cn c cc pseudogen (gen gi) v mt s thnh vin khc m sn phm ca chng i khi vn c s dng.

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Hnh 1.10: H gen globin v ngi tp trung thnh cc nhm trn hai nhim sc th. Chng gm cc gen m cho globin v cc pseudogen (). Cc gen hot ng theo trnh t t tri sang phi ph hp vi qu trnh pht trin t phi n c th trng thnh.

H gen globin chim 28 kb trn nhim sc th 16, gm cc gen , 1, 2 v . Sn phm ca hai gen 1, 2 ging ht nhau. H gen globin chim 50 kb trn nhim sc th 11 gm 5 gen hot ng (, G, A, , ) v mt pseudogen . Sn phm ca hai gen ch khc nhau duy nht mt acid amin ti v tr 136 (Glicine v Alanine). Cc chui polypeptide lin kt vi nhau to ra cc dng globin khng ging nhau v c s dng nhng giai on pht trin khc nhau ca c th (Bng 1.1).
Bng 1.1: Cc dng globin thay i trong qu trnh pht trin ngi Giai on pht trin Hemoglobin M phi (8 tun) 22, 22, 12 Thai nhi (3-9 thng) 2 2 C th trng thnh (t khi sinh) 2 2 (~ 2%), 2 2 (~97%), 22 (~1%)

Bn cnh h gen m cho globin tp trung ti hai vng trn nhim sc th 11 v 16, h gen m cho aldolase c xem l v d in hnh v s phn b ri rc ca mt h gen trn cc nhim sc th khc nhau. H gen ny gm 5 gen thnh vin phn b trn 5 nhim sc th 3, 9, 10, 16 v 17. Mc d phn tn trong khp genome, cc gen ny c tng ng rt cao v trnh t nucleotide cng nh trnh t acid amin tung ng.

1.4.2. Gen lp i lp li lin tc


Thng thng cc thnh vin trong mt h gen khng ging nhau hon ton. S sai khc gia chng m bo tnh hot ng c lp ca tng gen v c duy tr qua chn lc. Tuy nhin cng c mt vi trng hp c bit, s lng cc thnh vin trong h rt ln v chng ging ht nhau, thng tp hp thnh cc nhm phn b trn cc nhim sc th khc nhau. Mi nhm c th bao gm t hai cho n hng trm gen, gen n ni tip gen kia. Vic lp i lp li lin tip cc bn sao ca mt gen trn mt on ADN (trn mt vng nhim sc th) c th nhm mc ch p ng nhanh, s lng rt ln sn phm ca gen khi t bo yu cu, v d nh cn p ng kp thi cc phn t ARNr cho giai on sinh trng nhanh (phi) hoc cc loi protein histone cho qu trnh ti bn ADN. Gen m cho ARNr: ARN ribosome chim 80-90% tng s ARN c trong t bo. S gen m cho chng thay i t 7 E.coli, 100-200 eukaryot bc thp n vi trm ng vt bc cao. Trong nhn t bo eukaryot, hu ht cc gen m cho ARNr tp trung thnh tng nhm chim mt vng trn nhim sc th (vng ADNr). ARNr gm cc loi chnh ARNr-5S, ARNr-5.8S, ARNr-18S v ARNr-28S (tng ng vi hai tiu phn nh v ln ca ribosome). Phn t ARNr-5S c m bi gen ring bit v c tng hp bi ARN polymerase III.

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Genome ca ngi c cha khong 2000 gen m cho ARNr 5S. Tt c cc gen ny u tp trung trn mt vng ca nhim sc th s 1. Ba loi ARNr 5.8S, 18S v 28S c tng hp t mt gen bi ARN polymerase I (Hnh 1.11). Mt phn t tin thn ARNr c phin m t gen, sau b ct bi cc ribonuclease to thnh cc phn t ARNr 18S, 5.8S v 28S. on nucleotide nm gia cc phn t ARN ny s b phn hy. Mi mt nhm gen m cho ARNr gm nhiu gen ging ht nhau, khong cch gia mi gen thay i tu theo loi, thm ch ngay trong cng mt loi. Genome ngi c khong 280 bn sao ca gen m cho ba loi ARNr, tp trung thnh 5 vng (mi vng c t 50 - 70 bn copy), phn b trn 5 nhim sc th 13, 14, 15, 21 v 22. ng vt c v, mi gen thng chim 13kb, nm cch nhau khong 30 kb. Khong cch ny c vai tr trong khi ng qu trnh tng hp ARNr hoc gip cho ARN polymerase d dng bm vo promoter.

Hnh 1.11: Mt n v phin m (mt gen m cho ARNr) mang thng tin di truyn cho cc phn t ARNr 18S, 5.8S v 28S Gen ny c lp i lp li lin tc. Khong cch gia cc gen thay i tu theo tng loi sinh vt.

Gen m cho protein histone: Protein histone tham gia lin kt vi ADN hnh thnh cu trc nucleosome. C bn loi histone khc nhau. Histone H2A, H2B, H3 v H4 tng tc vi nhau to cu trc li. Li ny c qun quanh bi on ADN 146 bp to thnh nucleosome. Histone H1 lin kt vi ADN linker nm gia cc nucleosome. Histone chim khong 0,5-1% tng s protein ca t bo eukaryot. Vic tng hp protein ny xy ra trong sut 1/3 chu k t bo ( pha S). Tuy nhin phn t ARNm histone c thi gian bn sng ngn (vi pht). C l v l do , c rt nhiu gen m cho histone (50-500) phn b thnh cc nhm trn nhim sc th. Chng nm ni tip nhau, mi nhm chim khong 5-6 kb ( ng vt c xng sng) (Hnh 1.12). Cng ging nh nhm gen m cho ARNr, khong cch gia cc gen trong cng mt nhm v gia cc nhm thay i gia cc loi, thm ch ngay trong tng c th. C th phn bit cc gen m cho histone thnh hai nhm. Nhm th nht gm cc gen m cho histone dng trong qu trnh ti bn ADN. Nhm gen ny khng c intron v phn t ARNm phin m t chng khng c ui polyA. y l iu khc bit vi cc ARNm eukaryot. Nhm gen th hai gm nhng gen m cho histone tham gia vo qu trnh bin i cu trc khng gian ca nhim sc th (lin quan n thng tin di truyn ngoi sinh). Cc gen thuc nhm ny c cha intron v phn t ARNm tng ng c gn ui polyA.

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Hnh 1.12: Bn phn b cc gen m cho histone Cu gai (A) v Rui gim (B). Mi nhm c lp i lp li trn mt vng nhim sc th. Chiu tng hp ARNm cho mi loi histone khng ging nhau (chiu mi tn) chng t ngay trong mt nhm, cc gen hot ng c lp nhau.

1.4.3. Pseudogen (gen gi)


Mi thnh vin trong mt h gen u c th hot ng ty thuc trng thi t bo. Tuy nhin c nhng thnh vin m khng bao gi pht hin c sn phm ca chng mc d chng ging ht hoc c trnh t nucleotide tng ng rt cao vi cc thnh vin khc. Nhng gen c gi l cc Pseudogen (tm dch l cc gen gi, thng k hiu l ). Pseudogen khng to c sn phm cui cng l protein, mc d chng c th c phin m tng hp ARNm. Cu trc pseudogen c th ch gm ton exon hoc gm cc exon v intron hoc c trnh t nucleotide ging ht hay tng t cc gen hot ng khc nhng khng c promoter. Thc nghim cho thy t bin xy ra cc pseudogen khin qu trnh phin m khng th khi ng c, hoc khin qu trnh tng hp ARNm dng khng ng ch, hoc ngn cn phn ng ct ni intron-exon to phn t ARNm. Thm ch ngay khi phn t ARNm c to ra, n cha cc tn hiu lm dng qu trnh tng hp protein sm hn cn thit. Hu ht cc h gen u c cc pseudogen, mc d vi s lng rt nh. Cc gen ny c th xut hin do sai lch trong trao i cho gia cc allen ca hai nhim sc th tng ng. Theo thi gian, cc t bin thm, bt, chuyn on hoc thay th nucleotide ngy cng tch t trn cc pseudogen. Ngoi ra khng th loi tr kh nng enzym reverse transcriptase tng hp phn t ADN trn khun mu cc ARNm v cc bn sao ADN ny c ghp vo genome. Do , pseudogen thng khng c promoter, khng cha intron, khng c cc on nucleotide 5 v 3 nm trc m khi u v nm sau m kt thc phn ng tng hp protein. Hai on trc v sau ny c gi l on khng dch m (5 and 3untranslated regions).

1.5

Thnh phn ADN lp li trong genome eukaryot

1.5.1. ADN v tinh (satelitte DNA) v ADN tiu v tinh (minisatelitte DNA)
Bn cnh cc h gen v cc gen lp i lp li lin tip, genome trong t bo eukaryot cn cha nhng vng ADN gm cc oligonucleotide (thng t 5, 10 n 150, 300 bp) c lp i lp li rt nhiu ln. iu to ra nhng c tnh vt l ring bit ca loi ADN ny. Da vo ngi ta c th phn on v tch chng ra khi ADN genome. Chng c gi l cc

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ADN v tinh (DNA satellite). T l ADN v tinh thay i gia cc loi chim t 10 n 30% h gen. Trong hu ht t bo ng vt c v, ADN v tinh thng tp trung xung quanh tm ng (centromere) v vng cui hai u nhim sc th (telomere). S phn b ca chng c vai tr nht nh trong qu trnh phn chia t bo v m bo di ca telomere qua cc ln ti bn ADN. Khi cc nhim sc th phn ly v hai cc trong phn bo, cc protein c hiu bm dnh vo nhng v tr c bit tm ng kim tra, iu khin s di chuyn . ADN v tinh gi vai tr ca nhng v tr c bit ny. Ni chung chng khng c phin m sang phn t ARN. Ngoi ra, ADN v tinh tm ng c nhn bn cui cng trong qu trnh ti bn nhim sc th. Rt c th hin tng lp i lp li ca mt loi ADN ti tm ng nhm ngn cn s xut hin tm ti bn ti v tr ny. cn trng ADN v tinh thng bao gm cc on nucleotide rt ngn (khong 5-15 bp), cn ng vt c v thnh phn ny a dng hn v thng phn b thnh tng nhm trn nhim sc th. ngi, c t nht hn 10 loi ADN v tinh. Mi loi c th chim ti 0,5-1% tng s genome, tng ng khong 107 bp. i vi tng c th ring bit, trong mi loi ADN v tinh, cc on oligonucleotide c th lp li hon ton chnh xc nh nhau hoc c th xy ra s thay th, loi b hay thm vo mt vi nucleotide. Tuy nhin nhng bin i ny ph thuc tng vng trn nhim sc th. Chc nng ca ADN v tinh phn b ri rc trong genome cha c sng t. Nhng nm cui ca thp k 20, sinh hc hin i chng minh c cc on lp li phn b gn hoc nm ngay trong gen c vai tr kim sot hot ng ca gen . Thng thng cc on ADN lp li khng c phin m. Chng b bt hot do cc cytosine v histone H3 b methyl ho lysine 9 nhng histone H4 b kh nhm acetyl. Khi cc oligonucleotide gm khong 25-50 bp c lp li nhiu ln chim mt on ADN t 1 n 5 kb, thm ch n 20 kb th chng c gi l ADN tiu v tinh (minisatellite DNA) hoc ADN lp li ngu nhin a hnh VNTR (variable number tandem repeat). Tng t nh ADN v tinh, vic tn ti ca ADN tiu v tinh c lin quan n cu trc nhim sc th bi v loi ADN ny thng bt gp telomere. Tuy nhin, chc nng ca ADN tiu v tinh phn b ri rc trong genome cha c lm sng t. Ngoi ra khi s nucleotide rt t (1-4 bp) c lp li nhiu ln thnh tng on khong 200 bp th chng c gi l ADN vi v tinh (microsatellite DNA). ADN vi v tinh thng bao gm 1 n 4 nucleotide lp li khong 10 n 20 ln. S lng loi ADN ny rt ln trong genome, v vy chng c dng lm ch th phn t trong vic xc nh v tr ca gen trn bn . V d, trong genome ngi, ADN vi v tinh CA (CACACA...) lp i lp li chim khong 0,5% (15Mb), trong khi s lp li ca mt nucleotide A (AAA...) cng chim n 0,3%. Mc d chc nng ca ADN vi v tinh cha c bit nhng chng c mt c mt ngha rt quan trng trong lp bn ton b genome. Trong mi mt qun th, cc ADN vi v tinh tng t nh nhau, tuy nhin s ln lp li cng nh nhng bin i trong mi loi ph thuc vo tng c th. Ni mt cch khc, mi loi tiu v tinh tn ti trong mi c th ca qun th, nhng s ln lp li cng nh cc bin i trong trnh t nucleotide li c trng cho tng c th. Tnh cht ny c p dng phn bit cc c th khc nhau v phn tch quan h huyt thng (k thut DNA-fingerpring...).

1.5.2. Cc on ADN c kh nng di chuyn

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Tn s trao i cho gia cc ADN tiu v tinh ln hn khong 10 ln so vi trao i cho xy ra gia cc on nhim sc th tng ng trong phn bo gim nhim. l mt trong nhng nguyn nhn to ra s khc bit gia genome ca cc c th trong mt loi. Ngoi ra s a dng ca genome cn do cc on ADN c kh nng di chuyn (thng c gi l transposon). Khi di chuyn, cc transposon gy ra vic sp xp, t chc li genome ca tng c th nh to cc on ADN mi hoc thay i chc nng hot ng ca cc on ADN v tr chng ghp vo v tch ra. Chng c th di chuyn ti v tr bt k v hon ton khng yu cu mi quan h no gia hai v tr mi v c. Khi tch ra khi v tr c, transposon c th mang theo cc on ADN ph cn, gy s mt on ti v tr c. Ngc li, khi ghp vo v tr mi, chng gy ra hin tng thm on hoc chuyn on v tr mi. Do , transposon ging nh cc vector chuyn ch ADN t ni ny sang ni khc trong mt genome hoc t genome ny sang genome khc. Ngoi ra, trao i cho gia cc transposon tng ng hai v tr khc nhau trn mt hoc trn hai nhim sc th cng to ra nhng bin i tng t. Nhng bin i dn n sp xp li genome, to tnh a dng gia chng v tnh c th ring ca tng c th. c bit, s thay i v tr ca cc transposon cn c th gy nh hng n hot ng ca cc gen phn b xung quanh ngay khi chng khng lm thay i trt t nucleotide nhng gen ny. Do hot ng ca cc gen lin quan n s di chuyn ca transposons (thng l cc gen nm trong transposon) c kim sot rt cht ch. C ch kim sot ch yu thng qua bin i cu trc khng gian vng nhim sc th cha transposon nh methyl ho ADN, methyl ho histone H3, deacetyl histone H4 vv.... Cch thc di chuyn v ghp vo genome ca cc on ADN c bit ny tun theo hai cch lin quan n dng trung gian ADN hoc ARN. Nhng on ADN no m s di chuyn ca chng gn lin vi dng trung gian ARN c gi l retroelement hoc ADN retrotransposon. Vic di chuyn ca retroelement xy ra tng t vi cch thc xm nhim ca virus m genome ca chng l phn t ARN (nhng virus ny c gi l retrovirus). Mt khi xm nhim vo t bo, ARN ca retrovirus c sao chp bi reverse transcriptase to ra ADN. Phn t ADN ny s c ghp vo genome ca t bo ch. Khi virus sinh si, phn ADN li c dng phin m to ra cc phn t ARN mi cn thit cho vic ng gi to virus mi. Trong s cc loi retroelement, cn lu n yu t ERVs (endogenous retrovirus) v cc retrotransposons. Chng u l nhng on ADN c kh nng di chuyn trong genome. Tuy nhin ERVs c chung mt c im l hai u c tn cng bi hai on nucleotide lp li vi kch thc ln (long terminal repeat-LTRs). LTRs gi vai tr quyt nh trong qu trnh di chuyn. Ngoi ra, retrotransposons bao gm cc yu t LINEs (Long Interspersed Nuclear Elements) hoc SINEs (Short Interspersed Nuclear Elements) l nhng on lp li di hoc ngn phn b ri rc trn cc nhim sc th. Yu t LINEs khng cha LTRs nhng c mang gen m cho reverse transcriptase trong khi SINEs khng c gen nhng c kh nng "vay mn" enzym ny do cc retroelements khc tng hp. Trong genome ca ngi, yu t LINE-1 c ti 3500 bn sao di nguyn vn 6,1 kb v hng trm nghn bn sao c kch thc ngn hn. Bn cnh trnh t Alu gm hng triu bn sao l v d in hnh ca yu t SINEs. Mc d phn t ARN c tng hp t Alu nhng sn phm protein khng c to thnh. D sao s tn ti ca cc ARN ny cng lm tng c hi gip Alu ghp vo genome. Cc transposon ADN c kh nng thay i v tr trong genome eukaryot khng qua dng trung gian ARN chim t l t hn so vi cc retroelement. V d, genome ngi, ch c

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khong 100 loi ADN transposon. Tuy nhin, ADN transposon c mt ngha c bit quan trng i vi s a dng ho genome. Mt s transposon c mt trong genome ca cc loi sinh vt khc nhau. V d, yu t mariner c chiu di 1250 bp c tm thy rui gim Drosophila cng nh rt nhiu ng vt khc, k c ngi. Phi chng cc transposon ny c thin chc t nhin trong tin ho l chuyn ch gen gia cc genome khc nhau? Cc transposon c chung c im l hai u tn cng ca mi transposon c cha hai on oligonucleotide lp li ngc chiu (inverted repeats). Cc transposon c th chia lm hai loi da vo kh nng di chuyn c lp hay phi ph thuc vo s c mt ca transposon khc. *Loi th nht gm cc on ADN c kh nng di chuyn c lp. Chng cha gen m cho cc protein iu khin qu trnh , v d enzym nhn bit hai u transposon ct chng ra khi v tr c v ghp vo v tr mi. Do , chng tch ra khi v tr c, ghp vo v tr mi hon ton c lp. Nh kh nng ny, chng to ra cc t bin khng bn vng. *Loi th hai gm cc transposon khng c kh nng t hot ng, tc l chng khng c kh nng di chuyn do khng cha gen m cho cc enzym cn thit. Vic di chuyn ca transposon loi ny ph thuc vo s c mt ca transposon c kh nng hot ng c lp (transposon nhm 1) cng nhm. Hai transposon c th xp vo cng nhm khi chng c cu trc tng ng vi nhau, c bit l cc on oligonucleotide phn b hai u transposon. y l v tr enzym nhn bit v ct ni transposon v tr c v mi. Khi cc transposon loi ny di chuyn, chng to ra nhng t bin bn vng nu nh trong th h ni tip chng phn ly c lp (phn ly theo nh lut Mendel) vi transposon c kh nng hot ng c lp cng nhm. Cc transposon n gin nht vi khun c gi l on gn IS (Insertion Sequences). Chng c th nm trn chromosome hoc trn cc plasmid. din t vic ghp ca IS vo v tr no , k hiu hai ln du hai chm c s dng (::). V d, :: IS1 m t transposon IS1 gn vo genome ca bacteriophage . Transposons vi khun khng gi mt chc nng no trong t bo. Trnh t nucleotide mt u IS thng lp li nhng ngc chiu so vi u kia. Hai trnh t hai u mt IS c gi l trnh t lp li ngc chiu (inverted repeat). V d, cu trc ca mt IS c trnh t nh sau: GGTAT-Xn-ATACC (trong n l s nucleotide nm gia hai u lp li ngc chiu). Do khi si p IS tch thnh hai si n th mi si ny c kh nng hnh thnh lin kt b sung ti hai u ca IS to cu trc dng vng (stem-loop) (Hnh 1.13).

Hnh 1.13: Cu trc dng vng c to ra do lin kt to cp b sung gia hai trnh t lp li ngc chiu ca mt IS trn mt si n ADN.

Ngoi cc IS, vi khun cn c cc on ADN c kh nng di chuyn vi kch thc di hn, gi l transposon Tn. Cc Tn thng phn b trn plasmid (phn t ADN dng vng, kch thc thng khng ln) v c kh nng ghp xen vo bt k v tr no trong genome. Chng thng mang thng tin di truyn m cho cc protein chng chu khng sinh.

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Gia IS v Tn c mi quan h v trnh t cc nucleotide. Cc Tn thng c gii hn hai u bi mt loi IS no .

Hnh 1.14: Cu trc ca transposon Tn-9.

Hnh 1.14 m t cu trc ca transposon Tn-9. Transposon ny mang hai gen; mt m cho tnh chng chu chloramphenicol (Rch) v gen kia m cho protein cn thit cho s di chuyn. Hai u ca Tn-9 c gii hn bi IS-1 m trnh t nucleotide ca IS ny sp xp theo cng mt chiu. Mt s transposon cha gen m cho cc enzym transposase lm nhim v nhn bit chui nucleotide lp li ngc chiu (inverted repeat) ct transposon. ADN ca v tr mi b ct sao cho mi si n lch nhau vi nucleotide (ct thnh u so le). Transposon ni vo cc u ct, to ra hai khong trng (gaps). Khong trng c sa cha theo nguyn tc to cp b sung. Do cc nucleotide ca u so le v tr mi c sao chp thnh hai bn, mi bn mt u v trnh t sp xp cc nucleotide ging nhau. V vy chng c gi l lp li cng chiu (direct repeat) (Hnh 1.15). Chiu di ca chng thng khong 7-9 bp. Da vo s c mt ca cc on cng chiu v ngc chiu c th xc nh c v tr transposon ghp vo hoc chuyn i.

Hnh 1.15 : Mt transposon c hai u tn cng gm 7 nucleotide (1234567) lp li ngc chiu, gn vo v tr c 5 nucleotide (ATGCA) trong genome. Sau khi ghp ni, on ngn ATGCA c lp li nhng sp xp theo cng mt chiu.

Qu trnh di chuyn ca mt transposon t v tr c (donor) sang v tr mi (recipient) xy ra theo hai c ch khc nhau: C ch sao y bn chnh (transposon c mt c hai v tr) v c ch tch ra khi v tr c di chuyn n v tr mi. Trong c ch th nht, trnh t nucleotide

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ca transposon c sao chp t v tr cho v c ghp vo v tr nhn. Nh vy mi ln di chuyn th s lng bn sao c tng ln. Qu trnh ny lin quan n hai loi enzym: transposase (tc ng vo hai u bn gc transposon) v resolvase (tc ng ln bn sao). Trong c ch th hai, mt transposon c th tch ra khi v tr c v ghp vo v tr mi. Nh vy s lng transposon khng thay i. Kiu di chuyn ny ch i hi enzym transposase. Khi transposon chuyn i, v tr c b gy. N c ni li nh c ch sa cha ADN trong t bo. sinh vt eukaryot, cc transposon cn c gi l yu t kim sot (controlling elements). Chng c nghin cu t nhng nm 1940. Tuy nhin c ch hot ng ca chng mc phn t ch mi c sng t trong nhng nm gn y. Cc nghin cu in hnh c tin hnh vi transposon ng v rui gim Drosophila. Transposons di chuyn, sp xp v khi ng cc gen nhng thi im c trng cho qu trnh sinh trng pht trin ca c th. Hai loi transposon Ac v Ds c nghin cu kh k ng. Chng cng thuc vo mt nhm transposon, u c hai trnh t lp li ngc chiu ging nhau. Di chuyn ca cc transposon Ds ph thuc vo s c mt ca Ac. Trnh t nucleotide ca Ac gm 4563 bp, c gii hn hai u bi 11 bp lp li ngc chiu, tip n 8 bp lp li cng chiu ca genome. Mi Ds u c on lp li ngc chiu ging nhau mc d chiu di ca chng thay i (Hnh 1.16).

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Hnh 1.16: Cu trc ca transposon Ac/Ds. Cc Ds c chiu di khc nhau (do Ac b t bin mt on) hoc c th cha on ADN hon ton khng tng ng vi Ac, hoc c th nm xen vo nhau. Tuy nhin tt c cc transposon ny u c gii hn bi 11 bp lp li ngc chiu.

Cc transposon ng thng ghp vo gn cc gen, lm ri lon hot ng ca chng dn n vic xut hin tnh trng mi nhng khng gy t bin cht. S di chuyn ca transposon ghp vo v tr allen ca mt gen bt k trn nhim sc th xy ra t bo soma s tc ng n biu hin ca allen trong qu trnh pht trin ca cy. Tri qua phn bo nguyn nhim (mitose), con chu ca t bo cha allen t bin s c biu hin tnh trng mi (thng quan st c hnh dng, mu sc ca ht ng). Thay i ny xy ra trong qu trnh pht trin soma c gi l "variegation" hay cn gi l hin tng mosaic (xut hin cc m). rui gim Drosophila melanogaster, yu t P c kh nng di chuyn c pht hin khi tin hnh lai gia con c dng P vi con ci dng M. Hu ht con lai b bt dc, nhim sc th b t gy, b t bin. Hin tng ri lon di truyn ny ch xy ra theo mt chiu, tc l php lai gia con ci dng P vi con c dng M vn to ra cc con lai bnh thng. Hin tng ny gy ra do genome ca cc c th thuc dng P c cha yu t di chuyn P. Yu t di nht gm c 2907 bp c cha gen m cho transposase. iu ng ch l mc d c chiu di khc nhau, cc yu t P u c mang cc trnh t nhn bit bi transposase. Quan st qun th rui gim trong thin nhin cho thy s lng P thay i t vi bn sao n 50 copy/genome. Hn na, nhng loi rui gim pht hin trc nm 1950 u khng c P trong genome. Phi chng P ch mi xut hin trong genome rui trong nhng nm cui th k 20. Liu s c mt ca chng c phi do virus xm nhim rui gim gy nn? Hin tng tng t cng c quan st thy vi khun b nhim thc khun th mang IS. Yu t IS xut hin trong genome vi khun thng qua qu trnh tip hp (transduction). C ch kim sot s di chuyn ca P ph thuc vo yu t tn ti trong t bo cht ca trng (di truyn theo m). Khi yu t ny c mt th chng km hm s di chuyn ca P. V vy, t bo trng ca con ci dng P th tinh vi con c dng M vn cho con lai bnh thng do yu t trong t bo trng ngn cn P chuyn ch. Tuy nhin, t bo trng dng M th tinh vi con c dng P cho php P di chuyn gy ra nhng ri lon bt thng trong cu trc genome. iu khin con lai b bt dc hoc xut hin cc tnh trng l.

1.6

Tng tc ca T-ADN vi genome thc vt

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S di chuyn ADN t genome vi khun sang genome thc vt c nghin cu kh k i vi tng tc gia Argobacterium tumefaciens hoc A.rhizogenes vi hu ht cc cy hai l mm. Hin tng di chuyn ADN ny gy nhng bin i v mt di truyn, biu hin vic xut hin cc nt sn trn thn cy hoc mc rt nhiu lng r ti ni b nhim vi khun. Bnh xut hin nt sn hoc mc nhiu r trn thn ch xy ra khi c mt Argobacteria. Tuy nhin sau bnh c duy tr khng ph thuc s tn ti ca vi khun. l do mt s gen vi khun c chuyn vo genome cy ch v hot ng gy bnh. Cc gen vi khun c kh nng di chuyn v hot ng trong t bo thc vt nm trn plasmid Ti (Tumor inducing) ca A.tumefaciens gy bnh nt sn hoc trn plasmid Ri (Root-hairs inducing) ca A.rhizogenes gy bnh mc lng r. Cng ging nh cc khi u ng vt, cc t bo thc vt c ADN vi khun ghp vo genome b chuyn sang trng thi mi, s pht trin v bit ho ca chng hon ton khc vi cc t bo bnh thng. l do hot ng ca cc gen vi khun (prokaryot) trong genome ca thc vt (eukaryot). Bnh thng nhng gen ny c mt trong genome vi khun nhng chng ch c bt m sau khi ghp vo genome thc vt. Qu trnh ny c tnh cht c hiu, tc l mt loi vi khun ch c kh nng gy nt sn trn mt s loi cy ch ny m khng tng tc c vi cc loi cy khc. Vic to nt sn hay thc cht qu trnh chuyn gen t vi khun sang genome thc vt dn n bin i trng thi sinh l ca t bo thc vt i hi cc iu kin sau: a/ Phi c hot ng ca cc gen trn 3 vng chvA, chvB, pscA nm trn nhim sc th ca vi khun khi ng vic bm dnh vi khun vo thn cy. b/ Plasmid Ti phi mang vng vir - ADN (nm ngoi on T-ADN). Vng ny mang cc gen cn thit cho vic tch v vn chuyn T-ADN t vi khun sang t bo thc vt. Vi khun xm nhim vo t bo cy ch ti v tr tn thng trn thn cy. Cy c vt thng do s h hng ngu nhin ca mng t bo thc vt hoc do vi khun tit ra hn hp nhng cht c m bi cc gen vir. Hot ng ca cc gen ny c hot ho bi hp cht phenolic ca cy (v d nh acetosyringone, catechol, cc dn xut ca chalcone...). Ngoi ra, s c mt cc monosaccharides nh glucose, arabinose trong mi trng cng khin cho nhm gen vir ca vi khun nhy cm hn vi cc hp cht phenolic do cy tit ra. Sn phm ca nhng gen trn vng vir cn lin quan ch yu n vic ct T-ADN ra khi plasmid v vn chuyn n vo t bo ch. Bng cc th nghim b sung chc nng (complementation test), thc nghim pht hin t nht c 21 polypeptide sn phm ca cc gen vir cng nh xc nh c chc nng ca hu ht cc protein ny trong qu trnh vn chuyn T-ADN. Protein VirA ng vai tr quan trng trong vic qui nh tnh c hiu gia cc loi cy ch vi Agrobacteria. Trong thc t, Agrobacteria khng c kh nng xm nhp vo cy mt l mm. C th protein VirA khng nhn bit c cc tn hiu do cy mt l mm tit ra. Protein VirC1 nhn bit v tng tc vi cc nucleotide nm u bn phi ca T-ADN. Mc d hai u T-ADN c trt t tng i ging nhau (ch sai khc nhau 2 nucleotide trong tng s 25 nucleotide cn thit cho s vn chuyn T-ADN) nhng cc nucleotide u bn phi gi vai tr quyt nh ct TADN ra khi plasmid. t bin u ny khin T-ADN khng c ct ra khi plasmid trong khi t bin u bn tri hon ton khng nh hng n qu trnh vn chuyn T-ADN t t bo vi khun vo trong nhn t bo cy ch. iu cho thy vic ct T-ADN c bt u pha bn phi v tin dn sang bn tri. iu c bit lu l ch c mt si n TADN c ct ra v vn chuyn sang t bo thc vt. Si n c gi l si T. u 5' ca si T tng ng vi u bn phi ca on T-ADN. Cc protein VirD1 v VirD2 lin quan n phn ng ct si T ra khi plasmid. Tip theo , protein VirE2 tng tc vi si T dc theo chiu di ca si. Protein VirD2 gi vai tr quan trng trong qu trnh vn chuyn.

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Cu trc VirD2 gm nhiu vng c hot tnh khc nhau, lin quan n cc chc nng nh ct, vn chuyn si T. Bn cnh vic tham gia phn ng ct si T ti u bn phi ca T-ADN, protein VirD2 cn lin kt vi u 5' ca si ny to thnh phc. Nh T-ADN c vn chuyn di dng phc ra khi t bo vi khun v xm nhp vo nhn t bo cy ch. Bng cc th nghim trn cy chuyn gen, ngi ta pht hin c protein VirD2 c mt trong nhn t bo thc vt. Vn chuyn si T ra khi t bo vi khun v ghp vo genome t bo cy ch l mt qu trnh phc tp, i hi s tham gia nhiu protein. Trong s , operon virB nm trn vng vir gi mt vai tr c bit. Operon ny di 9,5 kb m cho 11 proteins, a s l cc protein tit hoc phn b trn mng t bo. Chng bao gm ATPase (VirB11) v cc protein k nc to nn knh dn trn mng. Cc nh nghin cu cho rng mt trong cc protein c m bi operon ny phn b pha ngoi mng lm nhim v tng tc vi protein ca t bo thc vt, to knh dn a T-ADN vo nhn t bo cy ch. c/ Cc gen trn vng T-ADN c ghp vo genome t bo thc vt gy bin i trng thi cc t bo ny. T-ADN l mt on ADN c chiu di khong 23 kb (tu thuc vo tng loi A.tumefaciens) nm trn plasmid Ti. Hai u ca on ADN ny c cha 25 bp lp i lp li ging nhau hon ton ch sai khc nhau hai nucleotide (imperfect repeat sequence). Cc nucleotide u bn phi gi vai tr quan trng trong vic ct T-ADN. Cc nucleotide u bn tri ng vai tr trong vic ghp T-ADN vo genome cy ch. T-ADN gm hai nhm gen. Nhm th nht gm cc oncogen m c ch hot ng ca chng khc bit gia A.tumefaciens v A.rhizogenes. iu dn n s hnh thnh cc nt sn hoc bnh lng r. Trong trng hp xut hin nt sn, T-ADN mang ba oncogen m cho cc enzym tham gia vo phn ng tng hp cc hocmon sinh trng auxin v cytokinin. Ch khi T-ADN c ghp vo genome thc vt, cc oncogen nm trn T-ADN mi hot ng mt cch t ng. Do t bo cy ch no c T-ADN ghp vo h gen lp tc pht trin khng bnh thng do ri lon hocmon sinh trng m T-ADN m cho. Nt sn xut hin ti v tr cy b nhim A. tumefaciens l tp hp ca cc t bo bnh thng v t bo b bin i h gen. Trong trng hp vi bnh mc nhiu lng r, R-ADN ca A.rhizogenes c cha cc oncogen m sn phm ca chng lm thay i ngng nhy cm ca t bo thc vt i vi nng hocmon c mt trong mi trng. T gy ri lon s pht trin ca cc t bo c R-ADN ghp vo khin cho rt nhiu r xut hin ti v tr nhim. Nhm gen th hai c mt trn on T-ADN gm cc gen m cho cc enzym tham gia tng hp nhng phc cht dinh dng cn thit cho sinh trng v pht trin ca vi khun. Cc phc cht ny c gi chung l opines. Vi khun s dng opines nh ngun cacbon v nit. c bit, khi T-ADN mang gen m cho mt loi opine no th ngay trn plasmid Ti, nm ngoi on T-ADN, c cc gen tham gia qu trnh chuyn ho loi opine ny, gip cho vi khun sinh trng v pht trin. iu ng lu l opine c tng hp li tr thnh tn hiu kch thch hot ng ca operon cha cc gen ng ho opine nm trn plasmid Ti. V opine l protein c m bi cc gen vi khun, s c mt ca chng trong t bo thc vt c xem l ch th pht hin s chuyn ghp thnh cng ca T-ADN vo genome thc vt. T-ADN c vn chuyn vo trong nhn t bo ch v c ghp vo genome. C th xut hin nhiu bn sao ca T-ADN trong mt genome. Dng vng ca T-ADN i khi c tm thy trong t bo cy ch. y l dng trung gian hay ch l s lin kt ngu nhin gia hai u tri v phi ca T-ADN ang l vn cn lm sng t. Khi ghp vo genome vt

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ch, cc gen trn on T- ADN mi c hot ng. Nh vy iu ng ch l cc gen trn T-ADN (prokaryot) ch hot ng di s kim sot ca cc yu t phin m trong genome eukaryot. Ni chung, mt gen c iu khin bi mt promoter. Argobacterium c kh nng a cc gen l vo genome thc vt. V vy, chng c s dng nh cc vector chuyn ch gen mt khi cc gen gy nt sn trn T-ADN b thay th bi gen nghin cu. Ngoi ra, promoter ca cc gen trn T-ADN u l nhng promoter hot ng mnh trong t bo nhn. V vy chng c s dng lm promoter bo co hoc promoter iu khin gen l trong k thut chuyn gen. K thut ny c ng dng rt rng ri trong nng nghip. V d, a cc gen chng chu su bnh, gen chu c mi trng trng trt khc nghit... vo cc cy trng qu him hoc cho nng sut cao.

1.7

ADN trong ty th v lc lp

i vi t bo eukaryot, ADN khng ch phn b trong nhn m cn c mt ty th v lc lp. Hu ht phn t ADN trong cc bo quan ny dng mch vng. Tuy nhin cng c mt s trng hp ADN trong bo quan c th tn ti c hai dng mch vng v mch thng. V d, phn t ADN trong ty th ca Paramecium, Chlamydomonas v mt s loi nm men lun lun l si ADN mch thng. Mi t bo thng cha nhiu ty th hoc lc lp. Hn na, mi bo quan c th c nhiu phn t ADN. Do , s lng ADN ty th (ADNmt) hoc ADN lc lp (ADNcp) c th t n hng nghn bn sao trong mt t bo. V d mi t bo ngui c ti 8000 phn t ADNmt, trong mt ty th c khong 10 phn t. T bo trng ca ng vt c v c cha ti 108 bn sao ca ADNmt. Vi to Chlamydomonas cha khong 1000 phn t ADN lc lp trong mt t bo. Ngoi ra, kch thc phn t ADN bo quan khng t l vi tnh phc tp ca c th. Phn t ADNmt c th thay i rt rng t 16-17 kb ng vt c xng sng n 2500 kb mt s thc vt c hoa. Do kch thc nh hn nhiu so vi genome trong nhn nn ADN cc bo quan cha s lng gen t hn v cc gen phn b st nhau hn (khong cch gia hai gen rt nh, thm ch ch vi nucleotide). Phn t ADNmt hay ADNcp cha nhng gen m cho protein thc hin chc nng chuyn ho c th ca ty th hay lc lp nh cc protein tham gia chui h hp. Ngoi ra, ADN trong ty th v lc lp cn cha gen m cho ARNr, ARNt v protein ribosome dng ring cho bo quan.

1.7.1. ADN ty th
Trnh t nucleotide ca phn t ADNmt mt s sinh vt c xc nh. Kt qu ny gip chng ta hiu r hn cu trc v trt t sp xp cc gen trn phn t ADN ca bo quan. ng vt c xng sng, ADN ty th c kch thc nh gm cc gen khng c intron v hu nh khng c khong trng gia cc gen. V d, ADNmt ca ngi gm 16.659 bp tng ng vi 37 gen, trong 22 gen m cho cc phn t ARNt, 13 gen m cho cc polypeptide lin quan n phn ng oxy ho kh. nm men, ADNmt c kch thc ln hn so vi ng vt (78.000 bp) do mt s gen c intron v khong cch gia cc gen kh ln. ADNmt nm men c t nht 33 gen, trong s ny c 2 gen m cho ARNr, 23 gen m cho ARNt, 1 gen m cho protein ribosome v 7 gen m cho polypeptide tham gia phn ng oxy ho kh. c bit, ADNmt ca thc vt c kch thc ln nht v cu trc phc tp a dng nht. Trnh t ADNmt ca Marchantia polymorpha, thc vt nguyn thu khng c h mao dn, c xc nh hon ton. y l phn t mch vng c kch thuc 186 kb tng ng vi 94 khung c m (ORFs).

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Trong s 94 ORFs ny, thc nghim mi xc nh c mt s gen m s lng intron ca mt gen ln n 32. i vi thc vt c h mch, ADNmt cn ln hn nhiu. V d, ng hay da hu, ADNmt tng ng vi 570 kb v 300 kb. cc loi thc vt bc cao, cc gen c th phn b v tr khc nhau trn phn t ADNmt mc d sn phm ca gen c cng mt chc nng trong t bo.

1.7.2. ADN lc lp
Thc vt c ba loi lc lp khc nhau tu thuc vo hp cht m chng c nh tinh bt, cc sc t hoc cc cht bo. C ba loi ny u c cha phn t ADN (ADNcp) vi kch thc thay i t 85 n 292 kb to v 120 n 160 kb thc vt bc cao. c bit mt s thc vt nh to xanh Acetabularia, ADNcp ln n 2000 kb. Phn t ADNcp ca mt s thc vt c xc nh trnh t nucleotide. Lc lp thuc l Nicotiana tobacum c ADNcp gm 155.844 bp tng ng vi khong 150 gen. S lng phn t ADNcp trong mi t bo ph thuc vo s lc lp trong mt t bo v s ADNcp trong mi lc lp. V d, t bo to n bo Chlamydomonas reinhardtii ch c mt lc lp cha khong 100 phn t ADNcp. S gen phn b trn ADNcp bao gm gen m cho ARNr, ARNt, protein ribosome v mt s polypeptide tham gia phn ng quang hp, hp th nng lng nh sng mt tri.

1.8

Genomics

1.8.1 So snh genome


Da vo trnh t nucleotide ca mt s genome in hnh, cc nh sinh hc c th phn tch cu trc, hot ng v chc nng ca cc gen, lm sng t c vai tr ca ADN lp li, ADN nm gia cc gen, ADN khng cha m di truyn (cc vng 5 v 3 khng c dch m) v cc on intron ca tng gen vv... iu c bit c ngha l khi so snh cc genome vi nhau, chng ta c c nhng hiu bit tng quan v hot ng ca genome cc sinh vt khc nhau, mi quan h gia chng, s a dng sinh hc v mc tin ho. V d, ton b trnh t nucleotide ca genome Arabidopsis c xc nh cui nm 2000 nhm mc ch pht hin, phn lp cc gen quan trng ca cc cy nng nghip da vo s tng ng ca chng vi cc gen ca Arabidopsis. y l thc vt u tin c genome c xc nh ton b trnh t do kch thc genome tng i nh (130-140 Mbp, nh hn khong 200 ln so vi cc thc vt khc). B nhim sc th n bi ca Arabidopsis gm 5 nhim sc th. Ngoi ra, Arabidopsis c vng i ngn, d trng v c th mc quanh nm. Hnh dng cy nh chim rt t din tch nn hon ton thch hp vi iu kin nui trng trong phng th nghim. Trnh t nucleotide ca genome cc sinh vt m hnh c a vo cc loi ngn hng ADN khc nhau tu thuc vo mc ch nghin cu. Ba ngn hng d liu chnh hin nay lu tr hu ht cc thng tin v ADN l EMBL (thuc Vin Tin hc chu u- European Informatics Institude), GenBank (thuc Trung tm Cng ngh Sinh hc ca M-US National Centre for Biotechnology) v DDBS (thuc Ngn hng d liu ADN ca Nht-DNA Database of Japan). Bn cnh trnh t ton b h gen, cc loi ADN khc nh cDNA, ADN ch-ESTs (Expressed Sequence Tags) vv cng c lu gi phc v cho vic so snh, phn tch v xc nh chc nng ca genome, ca gen v sn phm (protein hoc ARN) tng ng.

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So snh genome gia cc loi sinh vt vi nhau cho php rt ra ba c im ni bt: Th nht l s lng nhim sc th rt khc nhau ngay gia nhng loi rt gn nhau. Th hai l cc gen thng phn b khng theo qui lut. Mt gen hoc mt h gm nhiu gen m cho sn phm cng chc nng c th phn b trn cc nhim sc th khc nhau, nm thnh nhm hoc ri rc trong genome. V d, s phn b ca gen m cho ARNr c trnh by trn hnh 1.17. Th ba l kch thc genome thay i khng hon ton t l vi tnh phc tp ca loi. Nhn chung, kch thc genome thng phn nh tnh phc tp ca loi. Tuy nhin, iu khng ng ngha gia vic tng s lng cc gen vi mc tin ho. Ch khi so snh trnh t ton b genome ca mt s sinh vt cng nh hot ng ca mt s gen quan trng trong sinh trng pht trin m cc nh sinh hc mi nhn thy tnh phc tp lin quan ch yu n vic tng s lng cc on ADN lp li. V d, genome ca mt s loi lng c hoc thc vt c kch thc khong 1011 bp, trong thnh phn ADN lp li chim hn 60-70%. Genome ca ngi nh hn, ch khong 3x109 bp. Chc chn rng ch ring kch thc genome khng th quyt nh tnh phc tp hay mc tin ho ca cc loi.

Hnh 1.17: Phn b ca ADNr tng ng vi ARNr 45S v ARNr 5S trong cc loi Triticeae

Bn cnh so snh tng th ton b genome gia cc loi, vic phn tch chi tit i vi mt gen nht nh cn lin quan n v tr cc intron, cc exon, cc on ADN iu khin hot ng ca gen. y l nhng yu t quan trng so snh tm ra mi quan h gia cc loi. Ngoi ra, tng s gen ni chung, s lng cc gen c nhiu bn sao trong genome, t l cc loi ADN lp li v thnh phn ca chng cng nh s di chuyn ca cc gen t ADN ring bit trong cc bo quan (ty th, lc lp) sang genome trong nhn u chu nh hng ca thi gian, tc l u phn nh qu trnh tin ho ca cc loi. Mt khc, c c s so snh chnh xc hn, ton din hn, cn xt n cu trc si nhim sc, cu hnh khng gian ba chiu ca nhim sc th cng nh ca ton b genome phn b trong nhn.

1.8.2 Genome ngi


D n xc nh trnh t genome ngi (h gen trong nhn) c cp n t nhng nm 1984-1988. D n c bt u vo u thp k 90 vi s tham gia ca hn 20 nhm nghin cu t cc nc M, Nht, c, Anh, Php v Trung quc do t chc quc t Genome Ngi (Human Genome Organization-HUGO) v cng ty t nhn Celera Inc. cng tin hnh c lp vi nhau. D n c trin khai vi ba buc c bn: th nht l lp bn ca tt c cc gen (khong 70.000 n 100.000 gen), tip n l xc nh bn vt l ca 24 nhim sc th mc chi tit nht (m cc k thut hin i c th p ng c) v cui cng l c trnh t nucleotide ca ton b genome. Genome ngi c xem gm c hai phn phn b trong nhn v trong ty th. Phn t ADN ty th c dng vng vi kch thc 16.569 bp. Kch thc ny qu nh, c th coi l

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khng ng k so vi genome trong nhn. Tuy nhin, do ty th khng c c ch sa cha ADN nn cc t bin (thm, mt hoc o on) thng c tch ly trong phn t ADN ca bo quan ny. Mt khc, mi t bo c khong 800 ty th, mi ty th c hn 10 phn t ADN. Cc phn t ny khng ging nhau do cha cc t bin to nn tnh a dng rt cao ca ADN ty th gia cc t bo ngay trong mt c th. Cui nm 2000, hn 96% trnh t nucleotide ca genome ngi c cng b. Genome ngi c kch thc khong 3,2 x106 kb, tc l 3,2 Gb (Gigabase-n v ln nht dng o chiu di trn bn vt l). Trong khong 2,95 Gb l vng cht nhim sc (euchromatin). Ch c 1,1 n 1,4% cha gen m cho khong 30.000-40.000 protein, trong ch mi xc nh c 1/3, cn li l cc protein d on (predicted protein). Genome ngi c ti 1,4 triu ch th SNPs. Thnh phn ADN lp li (SINEs, LINEs, LTRs v transposon) chim gn mt na genome (~43%). Tuy nhin hu ht cc transposon v LTRs u trng thi khng hot ng.

1.8.3 Nghin cu Genomics thc vt


S lng cc gen v nhng thng tin v genome ca rt nhiu loi sinh vt ni chung cng nh ca thc vt ni ring lu tr trong ngn hng gen tng nhanh khng ngng. Nhng s liu ny rt hu ch trong nh hng nghin cu nhm la chn c phng php thch hp. Chng ta cng nhau tm hiu xem cc nh sinh hc s l nhng thng tin v genome, c bit l ca cy m hnh Arabidopsis nh th no trong hng nghin cu genomics i vi thc vt. Trnh t nucleotide ca genome Arabidopsis c xc nh hon ton vo cui nm 2000. Loi thc vt ny rt gn vi cc cy h ci (ci bp, sp l, xu ho, c ci vv...). y l nhng loi rau xanh rt ph bin trong i sng con ngi. Mt trong nhiu l do khin cho genome Arabidopsis c chn xc nh ton b trnh t l do genome c kch thc tng i nh (130 Mb) v cha t ADN lp li. Bn cnh , d n c trnh t genome cy la c thc thi. Genome ca la ln gp 3,5 ln so vi Arabidopsis nhng cng ch bng 20% genome ca ng v 3% ca la m. iu may mn l cu trc genome ca cc cy lng thc (la, la m, la min, k, ng ...) kh ging nhau. S khc nhau v kch thc genome ch yu do thnh phn ADN lp li. Khng phi genome cng ln th s lng gen khc nhau cng nhiu. Do , trt t cc gen, chc nng ca chng cng nh cu trc genome ca Arabidopsis v la rt cn thit phn lp v iu khin cc gen quan trng cc cy c quan h gn gi vi chng. Nhng kt qu ny c bit cn thit cho chn lc, to mi ngun ging cy nng nghip c tnh trng mong mun. Cng vi genome ca Arabidopsis v la, hn 127.000 cc trnh t biu hin hay cn gi l ADN ch- ESTs c ngun gc t thc vt c lu tr trong ngn hng d liu cho php so snh cc gen gia cc loi thc vt vi nhau. Kt qu cho thy hu ht cc gen bit thc vt bc cao u c mc tng ng rt ln. Do , t trnh t nucleotide c th suy on c chc nng ca gen v ngc li. Khi bit mt gen bt k mt loi c th phn lp c gen c chc nng tng t loi khc. V d, cc nh sinh hc tin hnh so snh mc ging nhau ca 64 protein c chn ngu nhin t la v Arabidopsis. Chc nng ca cc protein ny cng nh trnh t nucleotide ca gen m cho chng bit. trnh hin tng so snh cc thnh vin trong cng mt h gen, cc gen c chn u l nhng gen ch c mt bn sao trong genome. Kt qu cho thy s gen c tng ng 70-80% chim t l cao nht. iu ny c ngha, mc d la v Arabidopsis khng c quan h gn gi vi nhau

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nhng khng v th m cc gen ca chng phi khc nhau. Ngc li, a s cc gen c cu trc v chc nng ging nhau u tn ti c hai loi ny (Hnh 1.18).

Hnh 1.18: So snh mc tng ng protein gia Arabidopsis v la.

Thc vt c hoa c xem l xut hin v tri qua tin ho t khong 150 triu nm. Bn cnh nhng nt tng ng v mt di truyn, cc c im v sinh trng pht trin rt phong ph, a dng gia cc loi v ngay trong cng mt loi. Tuy nhin, kt qu so snh genome ca la v Arabidopsis (khng gn nhau) cho thy s khc bit gia chng khng phi do s gen c hiu cho tng loi m ph thuc rt nhiu n cch thc kim sot hot ng ca mt gen hoc cc thnh vin trong cng mt h gen. Cc kt qu nghin cu khc cng a ra nhn xt s a dng gia thc vt ch yu do thay i trnh t nucleotide cc vng ADN iu khin (cis-regulatory sequence) v cc loi ADN lp li. i vi cc ging trong cng mt loi, thay i rt nh trong cu trc ca mt gen hoc cch thc kim sot biu hin ca n thng dn n nhng s khc bit rt r v tnh trng. iu ny c minh ha bi v d in hnh vi cc gen lin quan n bin i cc acid bo. Thc vt bc cao c hn 200 loi acid bo khc nhau, thng c d tr trong ht. Ch xt n cu trc bc mt, cc acid bo ny khc nhau bi s lin kt i, lin kt ba, s nhm OH, epoxi vv... Tuy nhin, nhng sai khc u c to ra bi mt s enzym thuc h gen ch khng phi do 200 gen khc nhau. Chng ta bit t mt gen, c th c nhiu phn t ARNm khc nhau nh c ch ct ni exon-intron lun phin (alternative splicing). Ch cn thay i 4 acid amin khin cho dasaterase chuyn thnh hydroxylase. R rng vic hnh thnh cc hp cht ho hc mi khng nht thit cn phi xut hin gen mi m cho chng. Ngoi ra, chng ta bit cc thnh vin trong h gen m cho cytochrome P450s m nhn mt lot chc nng khc nhau lin quan n sinh tng hp polysaccharide, kinase, phosphatase, cc factor iu khin gen ... Nh vy, s dng c hiu qu cc d liu trong ngn hng, trc tin cn xc nh mt chc nng c th cn nghin cu trong s cc chc nng m mt gen n bn hoc cc gen trong mt h gen m nhn. Khai thc thng tin trong ngn hng d liu to nn mt hng mi, c gi l phylogenomics (tm dch l pht sinh genome) trong nghin cu pht sinh chng loi. Cc nh sinh hc da vo cc thng tin bit phn tch ngun gc ca mt gen cn quan tm, phn tch nguyn nhn ca s a dng bin i v trnh t nucleotide hoc acid amin (sn phm protein) ca chnh gen tn ti trong cc sinh vt khc nhau. Hn na, c th tin hnh phn tch mc tng ng v ADN hay sn phm protein ca gen vi cc gen khc. T y c th xc nh chc nng c th ca gen ny. Nhng kt qu t c trong nghin cu phylo-genomics cho thy khong 54% cc gen thc vt bc cao c th phn thnh cc nhm c chc nng khc nhau. Khong 13% cc gen ca Arabidopsis lin quan n cc yu t iu khin gen v con ng truyn tn hiu. Phn

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tch 389 gen bit hoc ORFs nm trn mt on 1,9 Mb trong genome Arabidopsis cng c th xp chng vo cc nhm c chc nng khc nhau lin quan n cc con ng chuyn ho, n tng hp nng lng, truyn tn hiu, sinh trng v phn chia t bo. Tuy nhin, mc d c th bit sn phm ca gen nhng vai tr c th ca n trong sinh trng pht trin i vi tng c th li rt kh xc nh r rng. khc phc c yu im , cn s h tr mt thit ca cc k thut khc nh by gen, t bin di truyn... V d khi a mt on ADN c trnh t nucleotide bit vo genome c th to ra cc dng t bin khc nhau. Cn c vo trnh t nucleotide hai bn v tr ghp (xc nh nh phn ng invert-PCR hoc TAIL-PCR) c th xc nh c gen b bt hot. Da vo biu hin ca tnh trng mi, c th bit c ng hc biu hin ca gen cc thi im v giai on khc nhau. Mt kh khn na m phylo-genomics gp phi l s lp li ca cc gen xy ra kh ph bin trong genome. Genome Arabidopsis c nhiu gen lp li nm st nhau, do khi gy t bin bi cc tc nhn ho l, hu nh khng th to c dng t bin kp nh trao i cho. iu ny rt cn bit khi xc nh t bin lin quan n mt hay nhiu gen cng nh hn ch hin tng cc gen cng chc nng b tr ln nhau khi mt gen b bt hot. Chnh k thut s dng COs (phc ADN/ARN) phn no khc phc c kh khn ny. Phc c thit k c mang m dng tng hp protein gy t bin im on gen bo th trong mi thnh vin ca h gen (hoc cc gen lp li). Ngoi ra, phng php s dng si p ARN gy bt hot (gene silencing) cng t ra c nhiu u im trong vic km hm hot ng ca gen. Hn na, khi b nhim virus, thc vt c kh nng nhn bit v km hm s nhn bn ca ARN virus (virus-induced gene silencing). Nu nh b nhim bi virus ti t hp c mang gen l tng ng vi gen ca chnh thc vt, cy ch s tng cng bt hot lun c gen trong genome ca mnh.

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Chng 2 HOT NG CA GEN TRONG T BO


Gen mang thng tin di truyn m cho sn phm cn thit cho s sng ca t bo. Mt gen c xem l hot ng khi thng tin c s dng to ra sn phm cui cng l protein hay phn t ARN. iu ng ghi nh l thng tin ch c phin m v dch m theo mt chiu t ADN sang ARN n protein. Khng xy ra hin tng thng tin truyn t ADN sang thng protein hoc t protein ngc tr li acid nucleic. Vic trao i thng tin gia cc dng acid nucleic (gia ADN v ARN) c th xy ra thun nghch. V d, ADN ARN (phin m xui t ADN sang ARN), ARN ADN (phin m ngc t ARN sang ADN - reverse transcription), ADN ADN (ti bn ADN), ARN ARN (nhn bn ARN bi ARN polymerase da trn khun ARN). Hot ng ca gen m cho protein gm hai giai on chnh l tng hp ARNm v tng hp protein. Xen gia chng gm nhiu giai on trung gian c nhim v sng lc v iu bin thng tin t gen n sn phm cui cng. u tin, mt gen bt u hot ng khi cu trc ca n trong nhim sc th c thay i (kh methyl cytosine, bin i mt s lin kt histone, tr nn nhy cm vi nuclease, c s sp xp li cc on ADN vv...). y l giai on thay i mc ADN. Tip l giai on tng hp ARNm (hnh thnh cc phc hot ho qu trnh phin m, kim tra vic bt u v kt thc tng hp ARNm vv...), giai on bin i phn t ARNm (RNA processing-ch xy ra t bo eukaryot), vn chuyn ARNm ra ngoi t bo cht (RNA export), kim tra thng tin di truyn trn phn t ARNm (editing RNAs) v quyt nh tnh bn vng ca n (RNA turnover). Cui cng l tng hp protein (protein synthesis), bin i chng thnh cc sn phm c hot tnh (protein modification), vn chuyn v phn b protein n cc v tr khc nhau (protein targeting) cng nh qui nh thi gian tn ti ca chng trong t bo (protein turnover). Bt k giai on no cng c kim sot cht ch bi nhiu c ch khc nhau. Tt c u nhm mc ch m bo mt cch nghim ngt, chnh xc s lng sn phm ca tng gen theo ng yu cu ca t bo. Trong mi t bo, cc c ch iu khin c th xy ra chung vi nhiu gen (global regulation) hay ring bit cho tng gen (specific regulation); c th l tch cc (positive control) hay tiu cc (negative control), c th l t iu bin (autoregulation) hoc kim sot phn hi (feedback regulation). S phi hp iu ho gia cc c ch thng khc nhau cc t bo prokaryot v eukaryot (ph thuc vo cu trc t bo khng nhn hoc c nhn, cu trc khng gian ca nhim sc th, cu trc cc gen thnh operon, v tr ca gen trong genome, cu trc cc intron trong mt gen vv...). Chng ta bit t chng 1, ADN trong t bo eukaryot c ng gi trong cu trc nucleosome. Cu trc s b thay i cc b ti vng ADN chun b c phin m. V d, ti vng chun b phin m, mt s cytosine u 5 ca gen b kh nhm methyl, cc acid amin lysine ca histone H3 c s thay i mc methyl ho, histone H4 b acetyl ho vv... Nhng thay i khin mt promoter tr nn nhy cm vi DNase. V vy, promoter c xem l ang tn ti trng thi sn sng hot ng khi n khng b methyl ho v d dng b phn ct bi DNase. Chng ta nhn thy cu trc khng gian, thng qua cu trc nucleosome,

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tr thnh yu t u tin kim sot mt gen chuyn t trng thi bt hot (silent state) sang trng thi sn sng cho khi ng phin m (active state). Tuy nhin, khi ng phin m c th xy ra c hay khng cn ph thuc vo nhiu yu t khc na. V d, khi ng cc gen chu s kim sot tch cc (positive control) cn ph thuc vo s c mt ca cc protein hot ho. Protein hot ho thng lin kt vi protein khc, gi l cht cm ng (inducer) to thnh phc. Phc ny (activator+inducer) c kh nng tng tc vi ADN hoc vi ARN polymerase. Nh , cu trc cc b ca ADN thay i, hoc i lc lin kt ca ARN polymerase vi promoter tng, to iu kin thun li khi ng phin m. Nh vy hot ng ca gen b kim sot theo c ch tch cc s tng hoc gim ph thuc vo s c mt hoc thiu vng cc activator v inducer (v d in hnh l hot ng ca operon ara). Ngc li, c ch kim sot tiu cc lin quan n protein c ch (repressor). Repressor cng thng lin kt vi protein khc, gi l corepressor to phc. Phc ny tng tc vi ADN hoc vi ARN polymerase ngn cn khi ng phin m. Do , hot ng ca gen b kim sot theo c ch tiu cc s c bt m hoc ng ph thuc vo s thiu vng repressor hoc c mt ca repressor v corepressor (v d in hnh l hot ng ca operon lac). C hai c ch kim sot tiu cc v tch cc c th kt hp cng nhau iu bin biu hin ca gen. Cc protein hot ho v c ch c th cnh tranh tng tc iu bin mt cch linh ng biu hin ca gen. Mt s gen thng biu hin lin tc nhng ch mc ti thiu trong t bo. Cc gen ny d dng tng cng hot ng khi xut hin cc yu t hot ho nh activator v inducer. V d, hot ng ca mt s gen m cho enzym tng mnh khi xut hin c cht. Nhng gen ny c gi l "inducible gene" (gen c th hot ho). Ngc li, mt s gen thng biu hin mc mnh. Chng d dng b km hm khi xut hin protein c ch-repressor. V d, hot ng ca nhng gen tham gia tng hp tryptophan trong t bo E.coli b gim khi b sung tryptophan vo mi trng. Nhng gen ny c gi l "repressible gene" (gen c th b km hm). y, chng ta hnh dung c s phi hp, s cnh tranh gia cc c ch kim sot nhm thay i mc biu hin ca gen mt cch rt linh ng. Cc c ch chung iu khin hot ng ca gen c nghin cu kh chi tit giai on bt u tng hp ARNm. Tuy nhin, chng ta nhn thy chng u c th thc thi i vi bt k giai on no trong sut qu trnh chuyn i thng tin t ADN n protein. Trong t bo eukaryot, cc gen chu iu khin theo c ch tch cc chim u th. Hot ng ca nhng gen ny thng kh xy ra nu nh thiu activator. Ngc li, trong t bo prokaryot, cc gen thng b kim sot bi cc protein c ch repressor. a s cc gen prokaryot hot ng mnh nn chng cn cc repressor km hm mc biu hin ca gen. Trong hu ht cc sinh vt, phn t ADN c chc nng quan trng nht l lu tr thng tin di truyn cn cc phn t ARN m nhim nhiu nhim v khc nhau trong t bo. S lng ARNm mang m di truyn tng hp protein chim t l rt t so vi tt c cc loi ARN khc c mt trong t bo (Hnh 2.1). Nhng phn t ARN ch c phin m nhng khng dch m (khng dng lm khun tng hp protein) c gi chung l ARN khng mang m (non coding ARNs). Thc cht y l nhng phn t ARN khng cha cc m b ba tng ng cho acid amin. Tuy nhin, thng tin di truyn trn nhng phn t ny cn thit cho rt nhiu phn ng chc nng ca t bo. Chng tham gia trc tip vo qu trnh ti bn ADN ( telomere), lm thay i trnh t nucleotide, thay i v tr cc on ADN trn nhim sc th cng nh thit lp cu trc khng

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gian ca ADN cc vng d nhim sc (heterochromatin formation). Do , ARN tc ng trc tip n phn t ADN mang m di truyn. Ngoi ra, ARN tham gia cc c ch kim sot qu trnh phin m (nh bt u s phin m), kim sot sau phin m nh ct ni intronexon (splicing), c sa ARNm (RNA editing), phn ct ARNm (posttranscriptional gene silencing), km hm hot ng ca cc gen nhy (transposon silencing) vv...

Hnh 2.1: Cc loi ARNs c mt trong t bo sinh vt. ARNsn (small nuclear RNA): ARN kch thc nh trong nhn. ARNsno (small nucleolar RNA): ARN kch thc nh trong hch nhn. ARNsc (small cytoplasmic RNA): ARN kch thc nh trong t bo cht. ARNtm (transfer-messenger RNA): ARN tng t ARNt c kh nng tng tc vi ARNm (theo Brown, 2001).

iu c bit cn lu i vi cc loi ARN c mt trong t bo (c gi chung l ARN tng s) l chng thay i v s lng v chng loi tu thuc vo s bit ho v trng thi ca t bo. S tng hp cc ARN mi lun xy ra cng vi s phn hu cc ARN c. Tuy nhin, s lng ca tng loi ARN cng nh s loi phn t ARN to ra thng khng ging vi s lng v s loi ARN b phn hu. iu ny th hin rt r vi cc loi ARNm. Trong qu trnh t bo pht trin, bit ho hoc tr li cc tn hiu t bn ngoi, mt s gen c th c bt m trong khi mt s khc b bt hot; mt s gen c th tng mc phin m trong khi cc gen khc gim. Tp hp tt c cc phn t ARNm c mt trong t bo c gi chung l transcriptome. Transcriptome s qui nh cc loi protein cn c trong t bo. Tp hp tt c cc protein s c gi chung l proteome. Nh vy, transcriptome s thay i trong qu trnh t bo sinh trng v bit ho, trong cc phn ng m t bo tr li tn hiu khc nhau t bn ngoi. S thay i ca transcriptome s tng ng vi thay i ca proteome. D bin i mt cch lin tc, nhng khng bao gi transcriptome ch gm cc phn t ARNm mi. Trong phn bo, t bo con u nhn mt phn transcriptome t t bo m ngay khi cc ARNm cha cn s dng dch m. V d, trong bo t vi khun hoc trong t bo trng, trong cc ht thc vt u cha transcriptome dng d tr cha c dch m sang proteome. Khi trng th tinh, khi ht ny mm... transcriptome s c gii m thnh proteome tng ng. Phn ng tng hp ARNm c thc hin nh enzym ARN polymerase da trn khun mu l si n ADN. Si n dng lm khun tng hp ARNm c gi l si khun (template strand). Trnh t nucleotide trn si ADN n b sung vi si khun ging ht phn t ARNm. Si c gi l si mang m di truyn (coding strand). Phn ng tng hp ARNm c th chia lm 4 giai on nh sau:

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1.

Nhn bit khun mu: Cc phc protein cn thit khi ng qu trnh tng hp ARNm ca mt gen s bm vo nhng trnh t c bit (c th phn b pha trc hoc pha sau, hoc ngay trong gen) v enzym ARN polymerase bm vo promoter. Si kp ADN m xon ti v tr promoter, gii phng si khun dng t do. ARN polymerase bt u phn ng tng hp ARN. u tin, enzym tng hp cc si ARN rt ngn ch khong 2-9 ribonucleotide. Sau , enzym mi tng hp phn t ARNm. Cc ribonucleotide ca si ARNm to lin kt b sung vi si khun. ARN polymerase chuyn ng dc theo phn t ADN v si ARNm di dn do cc nucleotide c thm vo u 3' ca si ARN. Lin kt ADN/ARN xy ra ti vng ADN m xon. Khi ARN polymerase chuyn ng qua, lin kt b ph v; hai si n ADN phc hi thnh dng kp, si ARNm tch ra di dng si n. ARN polymerase cng vi protein ph tr nhn bit c v tr c hiu kt thc phn ng tng hp ARN. ARN polymerase tch ra khi si ADN; si ARN tch ra khi phc enzym v ADN khun.

2.

3.

4.

2.1

Kim sot hot ng ca gen khi phin m

Trong t bo prokaryot, ch c mt loi ARN polymerase xc tc cho phn ng tng hp cc loi ARNr, ARNt v ARNm. Mt t bo E.coli c khong 7000 phn t ARN polymerase, trong khong 2000 n 5000 phn t lun trng thi hot ng. Phn t ARN polymerase ca t bo prokaryot gm hai phn: phn li tetramere (2') v yu t . Cu trc li khng thay i nhng c nhiu loi yu t . Phn li c kh nng tng hp ARN trn mi khun ADN, cn yu t cho php phn ng xy ra chnh xc ti promoter. vi khun, mt s gen nh cc gen m cho ARN ribosome (ARNr), ARN vn chuyn (ARNt), protein to b my ribosome, cc enzym ARN v ADN polymerase thng hot ng lin tc (constitutive expression). Chng c gi l cc gen housekeeping (tm dch l gen qun gia). Hu ht cc gen m cho protein trong t bo prokaryot c kh nng p ng rt nhanh nhng tc ng thay i ca mi trng bn ngoi. Hot ng ca mt gen thng c kim sot 5 mc k tip nhau: phin m to ARNm, bin i phn t ARNm (thay i cu trc, thay i nucleotide....), thi gian tn ti ca ARNm, dch m v cui cng l bin i sau dch m (bin i cu trc phn t protein chuyn t dng khng hot tnh sang c hot tnh). Tuy nhin, prokaryot, kim sot giai on u tin (giai on phin m) gi vai tr ch yu i vi hot ng ca gen. T bo prokaryot khng c nhn, do phn t ARNm va c phin m trn khun ADN va c s dng dch m tng hp protein. Nh vy, c hai qu trnh phin m v dch m xy ra ng thi. y l iu khc bit vi t bo eukaryot, ARNm c tng hp trong nhn, tri qua cc bin i, c vn chuyn ra ngoi t bo cht dng lm khun tng hp protein. Nh trnh by trong chng 1, cu trc ca mt gen gm hai phn chnh: phn mang cc m di truyn v phn cha cc nucleotide lin quan n chc nng iu khin phin m di truyn sang si ARN. V tr nucleotide u tin phin m sang ARN c k hiu l +1. Vi qui nh ny, cc nucleotide nm trong phn iu khin ( vng 5 pha trc v tr +1) s c k hiu l v tr (-). Promoter ca cc gen vi khun m cho protein thng bao gm cc nucleotide v tr -10 v -35. ARN polymerase nhn bit v tng tc vi v tr -35, sau v tr -10 b m xon

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bt u phin m. Kt qu nghin cu trnh t nucleotide ca hn 100 promoter vi khun cho php xc nh c tnh bo ton ca on nucleotide ti v tr -10 v -35 nh sau: (Cc ch s ch % xut hin ca base trong promoter) V tr -10: T80 A95 T45 A60 A50 T96 V tr -35: T82 T84 G78 A65 C54 A45

Hnh 2.2: Th nghim footprint" cho php pht hin v tr bm ca ARN polymerase trn mt on ADN. So snh hai kt qu in di cho thy enzym bm vo v tr -30 nn khng xut hin vch -30 (B).

iu nn lu l mt s gen khng c cu trc promoter in hnh hoc qu trnh phin m i hi mt s protein khc tham gia cng ARN polymerase. Ngoi ra, cc gen hot ng nhng iu kin khc nhau i hi ARN polymerase lin kt vi cc yu t khc nhau. V d, t bo vi khun ch c mt loi ARN polymerase phin m cho cc gen khc nhau. Khi nhit mi trng tng cao, ARN polymerase ca vi khun s ch lin kt vi yu t 54 nhn bit promoter ca cc gen hot ng khi nhit tng bt thng (cc gen chu sc nhit). T bo eukaryot c nhiu loi ARN polymerase, trong ARN polymerase III nhn bit promoter ca gen m cho cc phn t ARN vn chuyn (ARNt) v phn t 5S ARN ribosome (ARNr). Promoter ca nhng gen ny nm pha sau v tr +1 (tc l nm trong phn mang m di truyn). V tr promoter nm trn mt on ADN c th xc nh bng thc nghim thng qua phng php "footprints" (tm dch l in du vn) da vo nguyn tc: ARN polymerase bm vo promoter v bo v n khi tc dng phn hy ca DNase hoc cc tc nhn ho hc. Trong phng php ny, u tin ADN c x l vi tc nhn ho hc hoc vi DNase, sau c in di trn gen acrylamide. Vng promoter c bo v s xut hin nh khong trng (footprints) (Hnh 2.2).

2.1.1 Kim sot khi u phin m


Cc gen vi khun thng tp hp thnh nhm v c iu khin bi mt promoter. Cu trc ny gi l operon. Nh vy, phin m trn cc gen nm trong mt operon c kim tra, iu khin ng b theo nguyn tc: hoc l khng c gen no hoc l tt c cc gen cng c phin m sang ARNm. Phn t ARNm phin m t mt operon c gi l ARNm -

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polycistronic. Tuy nhin, thng tin di truyn ca mi gen nm trn phn t ARNm polycistronic c dch m mt cch hon ton c lp. Phn t ARNm - polycistronic mang cc v tr ribosome bm vo v tch ra tng ng vi tng gen nm trong operon. Tc tng hp cc protein trn mt phn t ARNm polycistronic hon ton khc nhau. Nh th, mc d cng c phin m nhng sn phm protein ca tng gen nm trong mt operon c tng hp mt cch c lp vi s lng khc nhau v ti thi im khc nhau. Qu trnh khi u phin m trong t bo prokaryot thng c kim sot ch yu bi hai c ch khc nhau: c ch tiu cc v c ch tch cc. Trong c ch th nht, hot ng ca gen b km hm do protein c ch tng tc vi v tr c hiu nm gn promoter. V tr ny c gi l operator (v tr c ch). Khi repressor bm vo operator, ARN polymerase cng nh cc protein hot ho khc khng th dch chuyn hoc tng tc vi si ADN khun. Do qu trnh phin m tng hp ARNm b km hm, s lng phn t ARNm b gim. Ngc vi c ch tiu cc, hot ng ca gen chu s kim sot theo c ch tch cc ch bt u khi c mt ca protein hot ho. Chng tng tc vi cc v tr c hiu vng ADN iu khin khin cho cu trc ADN ti promoter thay i. Nh ARN polymerase d dng nhn bit c cc v tr cn thit khi ng qu trnh tng hp ARNm. 2.1.1.1 C ch kim sot tiu cc Operon lac E.coli l operon u tin c pht hin v nghin cu kh chi tit t nm 1961 bi Jacob v Monod. Hot ng ca operon ny c kim sot theo c ch tiu cc. Operon lac gm 3 gen cu trc: lacZ, lacY v lacA. Sn phm ca cc gen ny gip t bo tip nhn v chuyn ha -galactosides. Gen lacY m cho enzym lm nhim v vn chuyn galactosides vo t bo; gen lacA m cho enzym chuyn nhm acetyl t acetyl-CoA vo galactosides v gen lacZ m cho enzym chuyn -galactosides thnh cc ng. Cu trc v c ch kim sot tiu cc i vi hot ng ca operon lac c trnh by trn hnh 2.3. Qu trnh phin m trn lac operon c kim sot bi protein c ch LacI . Protein ny l sn phm ca gen lacI nm cnh operon lac. Gen lacI c gi l gen iu khin v sn phm ca n lm nhim v kim sot hot ng ca gen (nhm gen) khc. Protein LacI ch c hot tnh khi tn ti dng tetramer. Khi tn ti dng tetramere, protein LacI c 2 v tr tng tc. V tr th nht tng tc vi on nucleotide thuc vng promoter ca operon lac. on ny chnh l operator (O). V tr th hai c th tng tc vi c cht -galactosides, hoc cc cht c cu trc ho hc tng t c cht, hoc mt s phn t c bit nh isopropyl-thiogalactoside (IPTG). Thc nghim nhn thy hot ng ca operon lac tng rt mnh khi c mt ca nhng cht c kh nng tng tc vi v tr th hai trn protein LacI. Cc cht c c tnh kch thch hot ng ca cc gen c gi chung l cht cm ng. Khi khng c cht cm ng trong mi trng, qu trnh phin m trn operon lac b km hm do protein LacI bm vo operator. Tuy nhin khi c mt cht cm ng, protein LacI tng tc vi cht cm ng nn b thay i cu trc khng gian. Do , mt khi tng tc vi cht cm ng th protein LacI khng cn kh nng bm vo v tr c ch na. Lc ny khi ng phin m trn operon lac khng cn b km hm nn s lung phn t ARNm tng nhanh.

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Hnh 2.3: Cu trc operon lac vi khun E.coli. Hot ng ca operon lac c kim sot theo c ch tiu cc. (A): Khi khng c cht kch thch, protein LacI bm vo operator ca operon lac ngn cn ARN polymerase bt u tng hp ARNm. (B): Khi c cht cm ng xut hin, cht cm ng tng tc vi LacI lm thay i cu trc ca protein c ch khin protein c ch khng bm c vo operator. ARN polymerase bm c vo vng promoter tng hp ARNm.

2.1.1.2 Kim sot theo c ch tch cc Hot ng ca operon ara trong genome vi khun li c kim sot theo c ch nguc vi c ch tiu cc. iu ny th hin ch operon ara ch hot ng khi c mt ca protein hot ho. Mt gen hoc operon hot ng mnh hn khi c mt ca protein hot ho c xem l chu s kim sot theo c ch tch cc. Operon ara gm c 3 gen: gen A m cho isomerase, gen B m cho kinase v gen D m cho epimerase (Hnh 2.4). Cc enzym ny cn thit cho phn ng chuyn ho arabinose thnh xylulose-5-phosphate. Khi mi trng c arabinose, hot ng ca operon ara tng mnh tng t nh lactose kch thch hot ng ca operon lac. Tuy nhin, khi xut hin c cht arabinose, hot ng ca operon ara cn i hi s c mt ca protein hot ha AraC. AraC c m bi gen araC (nm gn operon ara).

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Hnh 2.4: Hot ng ca operon ara c iu khin theo c ch tch cc bi protein hot ho AraC. (A): Khi mi trng khng c ng arabinose, AraC bm vo cc v tr O1, O2 v I1 to lin kt gia O2 v I1 gy un cong si ADN. Operon ara b km hm, lng ARNm to ra rt t. (B) : Khi c mt arabinose, phn t ng ny s tng tc vi protein hot ha AraC. Phc cht bm vo 4 v tr O1, O2, I1v I2. Tng tc ca phc cht gia O1v O2 kch thch hot ng ca gen araC. Ngoi ra, tng tc ca phc cht gia I1v I2 khin hot ng ca operon ara tng ln rt nhiu. (theo Lodish v cs., 2000).

Khi mi trng khng c arabinose, AraC bm vo 3 v tr O1, O2 v I1, nm pha trc st vi promoter ca operon ara. Hn na, cc phn t AraC bm vo hai v tr O2, I1 c th tng tc vi nhau (tng tc protein-protein) gy un cong si ADN. Lc ny operon ara khng hot ng. Khi mi trng c arabinose, protein hot ho AraC tng tc vi phn t ng, do b thay i cu hnh. Lc ny phc cht AraC v ng bm vo 4 v tr O1, O2 , I1 v I2. Tip , AraC hai v tr O1v O2 tng tc vi nhau lm cho hot ng ca gen araC tng ln. Mt khc, phc cht khi gn vo I1 v I2 gy hot ha operon ara. Qu trnh tng hp ARNm trn operon ny c khi ng. iu ng lu l protein AraC t iu khin hot ng ca chnh gen to ra n (gen araC) bng vic tng tc vi cc v tr O1 v O2. Khc vi protein c ch LacI ch c tc dng iu khin tiu cc i vi operon lac, protein hot ho AraC kim sot hot ng ca c operon ara v gen araC theo hai c ch ty thuc vo v tr lin kt ca n vi ADN. Trong khi protein AraC ch c kh nng kim sot theo c ch tch cc vi gen araC th vai tr ca chng i vi operon ara thay i ty thuc vo v tr tng tc vi ADN v tng tc gia chng vi nhau. Hai c ch tiu cc v tch cc xy ra hon ton i lp nhau. S khc bit gia chng c m t trn hnh 2.5: cc gen chu s iu khin theo c ch tiu cc b c ch khi c mt ca repressor. Ngc li, cc gen chu kim sot theo c ch tch cc ch hot ng khi c mt activator.

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Hnh 2.5: So snh hai c ch iu khin tiu cc (A) v iu khin tch cc (B). Mc d tham gia kim sot qu trnh tng hp ARNm theo chiu hng ngc nhau, hai c ch ny c th hot ng c lp hoc phi hp cng nhau iu bin mc biu hin ca gen mt cch linh ng (theo Lewin, 1993).

Cc protein c ch ngoi vic bm vo ADN ngn cn ARN polymerase khi ng vic phin m, chng cn c th tng tc vi nhau thc thi chc nng km hm mc ti a hoc chng bm vo ARNm ngn cn ribosome thc hin vic tng hp protein. Khi c nhiu repressor cng phi hp hot ng, chng c gi chung l cc co-repressor. Cn c vo mc hot ng khi c mt cc phn t protein iu khin bit c gen c kch thch hoc b km hm. Ngoi ra tng tc gia cc protein iu khin c th gy ng hoc m gen. V d, mt protein cm ng gy mt hot tnh ca protein c ch hoc phc hi hot tnh ca protein hot ho u dn n khi ng gen. Ngc li, biu hin ca gen b km hm khi cc co-repressor kch thch hot tnh ca protein c ch, hoc gy mt hot tnh protein hot ho. Nhng iu ny c trnh by trn hnh 2.6.

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Hnh 2.6: Cc c ch kim tra hot ng ca gen thay i mt cch linh hot nhm c ch hoc hot ho gen. Phi hp gia cc c ch ny m bo mc biu hin ca gen rt linh ng, p ng yu cu ca t bo (theo Lewin, 1993).

2.1.1.3 Kim sot theo c ch suy gim (attenuation control) Operon tryptophan gm 5 gen m cho cc enzym lin quan n vic tng hp tryptophan. Khi mi trng thiu acid amin ny, hot ng ca operon tng gp 10 ln. Nh vy, s c mt ca tryptophan trong mi trng km hm operon tryptophan. Tuy nhin, tryptophan khng phi l tc nhn gy c ch biu hin ca operon. Chng gi vai tr ca cht ng c ch khi tng tc vi repressor ngn cn hot ng ca operon tryptophan. Nh vy, operon ny c iu khin theo c ch tiu cc do chnh sn phm cui cng ca operon. C ch kim sot c bit ny c gi l c ch phn hi (feedback inhibition). Ngoi ra, operon tryptophan cn chu s kim sot ngay khi phin m bt u theo c ch suy gim trnh by trn hnh 2.7. Phn tch trnh t nucleotide trn phn t ARNm polycistronic ca operon tryptophan cho thy on nucleotide gm 160 base nm trc im dch m u tin (m cho Methyonine) ng vai tr iu khin qu trnh phin m to ARNm trn operon tryptophan. on ny c gi l on gy suy gim "attenuator" c cha 4 chui nucleotide ngn c trnh t tng ng nhng ngc chiu. Do 4 chui ny c th lin kt to cp vi nhau, to cu trc "hairpin" (gi l cp tc). Tuy nhin, do mc tng ng khc nhau nn khi xy ra lin kt to cp gia chng th tnh bn ca tng cp khc nhau. Tu thuc vo s c mt ca tryptophan trong mi trng m xy ra nhng kiu lin kt to cp gia hai chui to ra cc cu trc cp tc khc nhau. Nh , s tng hp cc enzym cn thit cho phn ng to ra tryptophan c kim sot ph hp vi s i hi ca t bo i vi loi acid amin ny.

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Hnh 2.7: Cu trc bc hai ca phn t ARNm phim m t operon tryptophan. Cu trc ny gi vai tr kim sot phin m suy gim khin phn t ARNm khng hon chnh (do phin m b kt thc sm). Tuy nhin kim sot phin m suy gim ph thuc vo s c mt ca Tryptophan trong mi trng. (A)- Khi mi trng tha Tryptophan. (B)- Mi trng thiu Tryptophan. (C)- Khng c s tng hp enzym.

Chui nucleotide 1 trn on suy gim "attenuator" c cha cc m di truyn ca tryptophan (Trp). Khi mi trng c nhiu Trp, cc ARNt vn chuyn Trp cho ribosome dch m trn chui 1 ny d dng tm thy Trp trong t bo. Do ribosome vt qua chui 1 nhanh v bao trm ln chui 2. Lc ny ch c chui 3 v 4 lin kt vi nhau to cu trc cp tc gy tn hiu ngng tng hp phn t ARNm. Lu rng vi khun qu trnh phin m bi ARN polymerase xy ra ng thi vi qu trnh dch m bi ribosome. Cu trc cp tc trn phn t ARNm l mt trong nhng yu cu cn c ARN polymerase nhn bit v tr dng qu trnh phin m. Ngc li, khi mi trng thiu Trp, cc ARNt kh tm thy Trp, do ribosome phi ch i lu chui 1. Lc ny chui 2 t do v lin kt vi chui 3. Tuy nhin, cu trc cp tc ny cn cch xa v tr bm ca ARN polymerase nn khng nh hung n vic tng hp ARNm. Nh phn t ARNm ca operon tryptophan c to ra mt cch trn vn. 2.1.1.4 Qu trnh d ho lin quan n iu khin tch cc Glucose l mt trong cc ng cn thit cho t bo vi khun pht trin. N c t bo s dng trc tip, nhanh chng v n gin nht khng yu cu tng hp mi bt k enzym no. Khi glucose cng vi mt s ng khc nh lactose, arabinose cng c mt trong mi trng, t bo ch s dng glucose v km hm hot ng ca cc operon lin quan n chuyn ha cc loi ng khc. Hin tng ny c gi l c ch qu trnh d ho (catabolite repression). Cc nh ho hc pht hin rng khi vi khun thiu glucose, chng s tng cng tng hp nucleotide cyclic adenosine-3,5'-monophosphate (cAMP). S lng cAMP tng ln ging nh tn hiu bo ng v nguy c chuyn ho. Hn na, khi b sung cAMP vo mi trng c cha glucose v cc ng khc th hot ng ca cc operon lin quan n chuyn ho cc ng ny khng b c ch m ngc li c hot ho. Nh vy cAMP ng vai tr quan trng bt m hot ng ca mt s gen ngay khi chng ang trng thi b km hm. Nghin cu cc t bo vi khun t bin khng c kh nng s dng bt c loi ng no ngoi glucose, cc nh khoa hc pht hin rng chng khng tng hp c cAMP trong iu kin mi trng thiu glucose. Ni cch khc, cc t bo ny chu t bin gen m cho enzym tng hp cAMP (adenylate cyclase). Ngoi ra mt s t bo khc c kh

49

nng tng hp cAMP nhng vn khng tng hp c enzym cn thit chuyn ho cc loi ng khc glucose. Nh vy phi tn ti mt protein tng tc vi cAMP hot ho cc operon. Thc nghim phn lp c protein ny v gi l protein hot ho qu trnh d ho (Catabolite Activator Protein- CAP). Trong th nghim tng hp ARNm invitro i vi operon lac, s lng ARNm tng rt nhiu khi thm cAMP v CAP vo phn ng. Nu ch thm mt trong hai cht , s lng ARNm ch chim 5% so vi khi c mt c hai. Nh vy ngoi vic b iu khin theo c ch tiu cc bi protein LacI, operon lac cn chu s kim sot theo c ch tch cc nh phc cht cAMP-CAP. Phc ny tng tc vi promoter gy un cong si ADN, bc l v tr bm cho ARN polymerase (Hnh 2.8). Khng phi ch tham gia kim sot hot ng ca operon lac, protein CAP cn ng vai tr protein hot ho trong qu trnh tng hp ARNm ca nhiu operon khc, v d nh vi operon ara, galactose... Nh vy operon ara cn hai protein hot ho khc nhau (AraC v CAP) t c mc hot ng cc i. Vi th d v operon ara, chng ta thy tnh phc tp an cho nhau ca cc c ch kim sot hot ng ca gen (operon).

Hnh 2.8: Kim tra hot ng ca operon lac bi hai protein LacI v CAP. (a) Nu mi trng khng c lactose, ARNm-lac khng c tng hp ngay khi c hoc khng c glucose v LacI bm vo operator. (b) Khi c lactose, LacI khng bm c vo operator nhng do c glucose nn cAMP khng c tng hp dn n phc cht cAMP-CAP khng c to thnh. Do hot

50

ng ca operon yu. (c) Khi mi trng khng c glucose nhng c lactose, operon c hot ho theo c hai c ch, do lng ARNm-lac c tng hp t cc i (theo Alberts v cs., 2002).

2.1.2 Kim sot kt thc phin m


Qu trnh tng hp ARNm c dng li khi ARN polymerase gp tn hiu kt thc (termination signals). Ti v tr c tn hiu kt thc, lin kt gia 3 yu t (si ADN khun, si ARNm v ARN polymerase) km bn vng. E.coli, tn hiu ny c th l cu trc c bit ca phn t ARN do trnh t nucleotide nm phn cui ca gen qui nh.

Hnh 2.9: Kt thc phn ng tng hp ARNm nh cu trc c bit "hairpin". Cu trc ny to ra do lin kt gia cc chui nucleotide lp li ngc chiu "inverted repeat" v c ni tip bi chui U. Enzym ARN polymerase b dng li v ri khi khun ti v tr c hiu ny. Phn t ARNm c tng hp hon chnh.

Phin m khong 50% cc gen vi khun thng kt thc v tr si khun ADN c on nucleotide lp li ngc chiu giu G v C. Cc nucleotide lp li ngc chiu c th lin kt vi nhau theo nguyn tc b sung to cu trc cp tc. Cu trc ny lm ARN polymerase dng hoc chuyn ng chm (thi gian dng khong 60 giy). Tuy nhin lm enzym tch ra khi khun ADN, tip theo cu trc cp tc l cc nucleotide A ni nhau lin tip. Lin kt A-U gia ARN v ADN l cc lin kt yu nn cn t nng lng b gy chng (Hnh 2.9). Bn cnh cu trc cp tc, phin m ca mt s phn t ARNm c kt thc vi s tham gia ca protein Rho c hot tnh helicase. Protein ny gip ARN polymerase kt thc tng hp ARN ti v tr chnh xc. Do cc phn t ARNm c kch thc nh nhau. Trong trng hp ny, v tr kt thc cng thng cha cu trc cp tc nhng sau khng c chui nucleotide A. C ch kt thc phin m bng protein Rho n nay cn cha c xc nh mt cch r rng. Tuy nhin, rt c th hot tnh helicase ca protein Rho ct t lin kt gia ADN/ARN khin ARN polymerase dng tng hp ARN (Hnh 2.10).

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Hnh 2.10: C ch hot ng ca protein Rho. Protein Rho nhn bit v tr c hiu trn ADN v lm ngng phn ng tng hp ARNm. Nh , cc phn t ARNm u c kch thc chnh xc nh nhau.

Tn hiu kt thc phin m c th b b qua mt khi c cc tn hiu chng kt thc (antitermination). Lc , ARN polymerase tip tc tng hp ko di si ARNm cho n khi gp tn hiu kt thc khc. y thc s l mt hnh thc kim sot bt m (c phin m) hoc ng (khng phin m na) i vi cc gen nm pha cui operon. Tn hiu chng phin m l protein (v d nh protein N thc khun th) c kh nng lin kt vi ARN polymerase, gip enzym vt qua tn hiu kt thc hoc ngn cn hot tnh ca protein Rho. Hin tng chng kt thc phin m c nghin cu kh chi tit i vi hot ng ca cc gen thc khun th (). Ngay sau khi va xm nhim vo t bo vi khun E.coli, genome thc khun th tn ti dng vng trn. Phin m cc gen ca phage c bt u ti hai promoter PL v PR (nm st cnh nhau) bi chnh ARN polymerase vi khun. Si ARNm phin m t PR c dch m sang protein Cro cn thit thc khun th xc nh hnh thc xm nhim sinh tan hay tim tan. Si ARNm phin m t PL c dch m sang protein N. y l protein chng kt thc phin m ti hai v tr kt thc nm ngay sau gen m cho Cro v N. Nh , ARN polymerase vi khun tip tc phin m cc gen k tip trn genome thc khun th.

2.1.3 Cc protein iu khin (regulatory proteins)


Cc protein iu khin tham gia kim sot hot ng ca gen. Nhng protein ny c th tng tc vi chnh ADN hoc tng tc vi cc enzym ARN polymerase hoc tng tc vi nhau. V vy, protein iu khin thng c nhiu domain qui nh chc nng khc nhau. Hu ht cc protein iu khin c nghin cu k c chc nng hot ho hoc c ch qu trnh phin m v sau phin m. Mt domain thng gm nhiu on peptide nm khng cnh

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nhau trn si polypeptide. Nh cu trc khng gian ca protein m nhng on ny c phn b cnh nhau to nn trung tm qui nh hot tnh c th. Mt protein c th c nhiu domain. V d, protein hot ho gen c th c cc domain khc nhau, mi domain c vai tr c hiu m bo tng tc vi ARN polymerase, vi ADN, hoc vi cc protein khc. Ngoi ra, mt s protein iu khin c tng hp di dng khng hot ng. V d, khi t bo vi khun mc trn mi trng thiu nit, cc operon ntr (nitrogen-regulated operons) c kch thch tng hp khong 20-25 proteins khc nhau. Nhng protein ny cho php t bo sng c trong iu kin thiu nit. Promoter ca cc operon ntr c nhn bit bi ARN polymerasse cha y t 54. Do vic tng hp yu t ny lin quan n hot ng ca cc operon ntr. Ngoi ra chng cn c iu khin bi protein NR1. Protein ny bm vo ADN nm trc v tr +1. Gen m cho NR1 hot ng lin tc ging nh gen lacI, do NR1 lun c mt trong t bo mi thi im. Tuy nhin khi mi trng nit, NR1 tn ti dng khng hot ng. Khi thiu nit, NR1 c hot ho. Qu trnh chuyn i c m t trn hnh 2.11.

Hnh 2.11: Chu trnh chuyn i gia hai dng hot ng v dng khng hot ng ca phn t NR1 nh NR2 kinase v P2-phosphatase. Trong t bo eukaryot, cc enzym kinase v phosphatase c vai tr rt quan trng trong qu trnh chuyn trng thi ca cc protein iu khin t dng khng hot ng sang dng hot ng v ngc li.

nghin cu tng tc gia protein v acid nucleic, ngoi phng php du vn "DNA footprint" kh ph bin, mt s k thut khc hay c s dng nh xc nh bng in di chm (gel retardation assay), test th kh nng bo v bin i (modification protection assay) ... * Phng php "DNA footprint" cho php xc nh chnh xc cc nucleotide c protein bo v che chn khi tc dng phn hy ca ADNase (Hnh 2.12). Nh khi in di trn gel acrylamide, cc on ADN tng ng vi v tr tng tc khng xut hin trn gel, to ra nhng vt trng "footprint". Phng php ny tng t nh k thut xc nh v tr tng tc ca ARN polymerase trn si ADN khun trong qu trnh tng hp ARN.

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Hnh 2.12: Phng php "DNA footprint" xc nh v tr tng tc ca protein vi ADN. Khi lin kt vi cc nucleotide, protein bo v cho chng khi tc dng ca nuclease. Do nuclease ch ct ADN cc im nm ngoi v tr lin kt, to ra cc on ADN khc nhau. Trn gel thu c vt trng "DNA footprint" tng ng vi v tr c bo v bi lin kt ADN-protein.

* Phng php xc nh bng in di chm (gel retardation assay) cho php xc nh phc ADN hoc ARN lin kt vi protein khi so snh tc di ng ca ADN hoc ARN t do vi phc ADN-protein hoc ARN-protein di tc dng ca in trng khng i. Do tng tc vi protein nn phc chuyn ng chm (v vy gi l gel retardation). Tip sau , protein lin kt vi ADN hoc ARN c th c xc nh bng cc khng th. Khi c mt khng th, tc di ng ca phc khng th- protein-ADN cng b chm hn. on nucleotide ti v tr tng tc cng c xc nh chnh xc khi s dng cc oligonucleotide cnh tranh (competitor oligonucleotide). Nu chui oligonucleotide cnh tranh c trnh t b sung vi cc base trn si ADN hoc ARN, chui s cnh tranh vi protein trong phn ng lin kt vi ADN hay ARN. Do , protein khng th lin kt vi ADN hoc ARN na. Trn gel khng quan st c nhng bng di ng chm (Hnh 2.13).

Hnh 2.13: Phng php xc nh bng in di chm cho php phn bit on ADN t do v phc ADN- protein. Khi c lin kt vi protein, bng ADN chuyn ng trn gel chm hn so vi bng ADN t do khng lin kt.

2.2

Kim sot sau phin m

2.2.1 Km hm dch m lin quan n cu trc vng 5'UTR ca phn t ARNm


Phn t ARNm c cc vng 5' khng dch m (5 UTR 5 Untranslated Region) nm pha trc m khi ng (AUG) v vng 3 khng dch m (3 UTR) nm sau m dng. u 5' khng dch m c th to cu trc cp tc ngn cn ribosome khi ng dch m. Ngoi ra, phn 5 UTR c th l v tr tng tc ca mt s protein repressor. Khi ribosome khng

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th dch chuyn trn si ARNm khin cho qu trnh dch m b km hm hon ton. R rng trong trng hp ny, ARNm vn c tng hp mt cch bnh thng nhng protein khng c to ra. C ch iu khin qu trnh dch m do protein tng tc vi u 5' ca ARNm c gi l "kim sot dch m tiu cc" (negative translational control). t bo eukaryot, kim sot dch m tiu cc c pht hin i vi phn ng tng hp ferritin - protein lm nhim v to phc vi cc nguyn t st. Khi mi trng khng c st, cc phn t ARNm ferritin b aconitase (protein repressor) bm vo u 5', do khng c protein ferritin c tng hp trong t bo. Khi c st trong mi trung, aconitase lin kt vi st v ri khi ARNm ferritin. Lc ny ferritin c tng hp vi s lng ln gp 100 ln so vi lc ban u.

2.2.2 di ca ui polyA nh hng ti bn vng ca phn t ARNm

Hnh 2.14: di ui polyA nh hng n bn vng v qu trnh dch m trn phn t ARNm. ui polyA c th c thm vo hoc ct ngn i, nhng di ca n khng c t hn 30 nucleotide A.

Phn ng gn ui polyA (~200 nucleotide A) vo cc phn t ARNm eukaryot xy ra trong nhn t bo. Mt khi chuyn ra ngoi t bo cht, ui ny b ct ngn dn. Tuy nhin trong thc t, khng pht hin c ui polyA no ngn hn 30 nucleotide A. Do ui polyA ngn nht phn t ARNm khng b phn hy c th l 30 nucleotide A. c bit, m bo bn vng ca ui, cc nucleotide A cn c thm vo. Ni cch khc, vic thay i di ui polyA c kim tra rt cht ch (Hnh 2.14).

2.2.3 bn vng ca ARNm


Trong t bo vi khun, cc phn t ARNm thng b phn hy rt nhanh. Thi gian bn hy ca chng thay i t 3-5 pht ngay khi c hoc khng c cc cht cm ng trong mi trng. Do khi t bo vi khun mc v phn chia trong khong thi gian 20-30 pht, cc ARNm cn c ti to mi 5-10 ln trong mi ln phn chia. Khi t bo yu cu mt s enzym, ARNm cn thit tng hp cc enzym c phin m nhanh chng. Khi t bo

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khng cn nhu cu, qu trnh phin m dng rt nhanh v cc phn t ARNm lp tc b phn hy. Enzym nuclease chu trch nhim phn hy ARNm n nay vn cha c xc nh r rng. i vi mt s ARNm polycistronic ca cc operon nh lac hoc trp, chng b phn ct bi endonuclease to ra cc monocistronic tng ng vi tng gen trong operon. Khong cch gia cc monocistronic khng cha m di truyn, do khng c che chn bi ribosome nn chng d b ct bi endonuclease. Cc ARNm prokaryot c tng hp cng nh b phn hy rt nhanh, do vi khun c th thch ng nhanh nhy vi nhng thay i ca mi trng. Ngc li, cc ARNm eukaryot kh bn vng. V d ARNm m cho -globin c thi gian bn hy khong hn 10h. Tuy nhin cng c nhng loi ARNm c thi gian bn hy ch 30 pht hoc t hn. Thng l nhng ARNm m cho cc protein lm nhim v iu khin hot ng ca gen. Cc phn t ARNm eukaryot km bn do chng mang cc on nucleotide c bit ti u 3' kch thch s phn hy ARNm. Thc nghim cho thy khi on nucleotide giu A v U vng 3' khng dch m ca phn t ARNm km bn c ghp vo mt s ARNm bn vng khc, th nhng phn t ny tr nn km bn do ui polyA b ct ngn nhanh. Nh vy on nucleotide giu AU kch thch s phn hy ARNm thng qua vic gim di ui polyA. Ngoi ra, mt s ARNm c cha v tr c hiu u 3' c nhn bit bi endonuclease.

2.2.4 ARN anti-sense


Thng thng, cc protein gi vai tr ch o kim sot qu trnh phin m cng nh dch m. Tuy nhin hot ng ca mt s gen li c iu khin bi cc ARNs. Cng ging nh cc protein iu khin, cc ARNs lm nhim v kim sot hot ng ca gen c tng hp mt cch c lp v tng tc vi cc v tr c hiu (cc on nucleotide). Xt trng hp c th v vai tr iu khin sau phin m ca ARNs i vi vic tng hp protein OmpF t bo vi khun E.coli. Protein OmpF nm pha ngoi mng t bo lm nhim v nhn bit nhng thay i v p sut thm thu trong mi trng. Khi p sut tng th gen iu khin micF hot ng mnh hn. Sn phm ca gen micF l phn t ARN 174 bases, c th to cp theo nguyn tc b sung vi v tr nhn bit ca ribosome trn phn t ARNm ca gen ompF. Nh vy phn t ARNm ca micF lm nhim v ngn cn vic tng hp protein OmpF. Cc phn t ARN c chc nng tng t micF c gi l ARN-antisense (Hnh 2.15).

Hnh 2.15: Phn t ARN-antisense-micF kim sot dch m ca phn t ARNm -ompF. Phn t p ARN/ARN to thnh khin ribosome khng tng hp c protein OmpF.

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2.2.5 Phn ng c sa ARNm - "RNA editing"


Phn ng c sa ARNm lm thay i trnh t nucleotide ca phn t ARNm. Phn ng ny xy ra trong t bo cht. Cc nucleotide ca mt s phn t ARNm b loi b hoc b thay th, mt s nucleotide khc c thm vo. Nh vy trnh t nucleotide trn phn t ARNm khc vi gen (ADN) m t phn t ARNm c phin m. Phn ng ny c pht hin u tin i vi ARNm m cho protein ty th ca trypanosome. Cc phn t ARNm ny mang thm mt vi nucleotide U khc vi trnh t trn gen ADN. Cc nghin cu tip theo cho thy phn t ARNm trong ty th ca t bo thc vt hu nh u tri qua phn ng "RNA editing". Tuy nhin i vi cc ARNm ny, ch c s thay th nucleotide C bng U m khng c hin tng thm vo hoc loi b cc nucleotide khc. Trnh t cc acid amin protein c th thay i n 10% hoc hn na so vi protein c m bi phn t ARNm cha tri qua phn ng ny.

Hnh 2.16: Thay i thng tin di truyn trn phn t ARNm ca gen apo-B bi c ch "ARN editing". Chiu di phn t ARNm khng i nhng nucleotide U thay th C to ra mt m dng tng hp protein.

ng vt, cc phn t ARNm t tri qua phn ng "RNA editing". Trng hp u tin c pht hin i vi gen apolipoprotein B (apo-B) di 4503 bp, l gen n bn trong genome ngi (Hnh 2.16). T gen ny hai loi phn t ARNm c tng hp. Chng ch khc nhau bi mt nucleotide C b thay th bi U. Do lm xut hin mt m dng phn ng tng hp protein. Protein ngn tng ng vi loi ARNm ny c trng lng phn t 250 KDal v ch tn ti trong rut. Phn t protein 512 KDal tng ng vi ARNm khng b c sa tn ti c gan v rut. Phn ng "RNA editing" c thc hin nh cc phn t ARN di khong 40 - 80 base. Cc ARN ny gi l ARN ph tr, k hiu l ARNg (guide RNA). Chng c tng hp c lp vi nhau. u 5' ca chng c th to lin kt vi ARNm cn sa i, cn u 3' mang ui polyU. Cc nucleotide U c chuyn trc tip t ui ny sang ARNm. Qu trnh c sa din ra t u 3' v tip tc v u 5' ca ARNm. C th c nhiu ARNg tham gia sa i mt phn t ARNm.

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2.3

Kim sot giai on dch m v sau dch m

Kt qu nghin cu hot ng ca operon lac v operon ara c xem l nhng th d kinh in cho cch thc kim sot tiu cc v tch cc giai on phin m. Ngoi ra, qu trnh phin m ca cc gen genome thc khun th Lamda sau khi xm nhim vo t bo E.coli minh ha r rng cho s phi hp gia hai c ch kim sot tiu cc v tch cc cng vi mt s c ch khc nh t iu khin, kim sot phn hi tiu cc... i vi t bo prokaryot, cc phn t ARNm polycistronic thng mang thng tin di truyn ca nhiu gen. Cc gen trn cng mt operon c phin m ng thi. Tuy nhin sn phm ca cc gen ny khng c tng hp vi s lng nh nhau. V d, khi mc trn mi trng ch cha lactose, mt t bo E.coli c khong 3000 phn t -galactosidase, 1500 phn t galactoside permease v 600 phn t -galactoside transacetylase. Nh vy operon lac cha 3 gen v sn phm ca tng gen l khc nhau, mc d ch c mt loi ARNm c tng hp. R rng rng, qu trnh dch m trn phn t ARNm polycistronic c kim sot cht ch. Qu trnh tng hp cc protein ribosome E.coli c xem l th d minh ha r rng cho c ch kim sot sau phin m (Hnh 2.17). Ribosome E.coli gm 3 phn t ARNr v 52 chui polypeptide. Chng c to ra mt cch ng b vi nhau, khng c s thiu hay tha bt c thnh phn no nhm trnh lng ph nng lng. Hu ht cc gen m cho protein ribosome nm thnh nhm trong cu trc operon. V d, operon cha gen m cho protein S10 (protein s 10 trong tiu n v nh ribosome) gm 11 gen, tt c cng m cho protein ribosome. Trong s , gen rplD m cho protein L4 tiu n v ln ribosome lm nhim v kim sot tiu cc qu trnh dch m trn ARNm polycistronic ca operon. Bnh thng, khi c ARNr t do trong t bo, L4 tng tc vi ARNr tham gia hnh thnh ribosome. Tuy nhin khi vng mt ARNr, L4 tng tc vi u 5' ca ARNm polycistronic v ngn cn phn ng tng hp protein. Ngn cn qu trnh dch m bi mt trong s cc protein c m di truyn trn phn t ARNm polycistronic xy ra kh ph bin t bo prokaryot. C ch ny c gi l iu khin tiu cc phn hi (negative autogenous regulation) hoc t iu khin tiu cc (negative self-regulation). Khi sn phm ca gen thuc cc thnh phn cu trc bo quan, c ch t iu khin thng xy ra bi cc phn t dng n phn (monomer) c mt t do trong t bo.

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Hnh 2.17: Cu trc v kim sot dch m trn operon m cho protein S10 E.coli. Operon ny gm c 11 gen m cho cc protein ribosome. Protein L4 ca operon lm nhim v kim sot dch m trn phn t ARNm polycistronic. Khi t bo c ARNr t do, 11 protein c tng hp ng b m bo cho qu trnh hnh thnh ribosome. Tuy nhin, khi thiu vng ARNr, L4 tng tc vi u 5' ca ARNm, ngn cn khng cho qu trnh dch m xy ra (theo Lodish v cs., 2000).

Hot ng ca mt s gen m cho cc enzym lin quan n mt chu trnh chuyn ha sinh tng hp trong t bo thng c kim sot bi sn phm cui cng. Mt khi sn phm cui cng c cung cp y th chng thng quay li c ch hot ng ca cc gen m cho cc protein hot ng trc trong chu trnh . y chnh l s km hm phn hi (feedback inhibition). Nh , sn phm cui cng lun c duy tr nng ti u m t bo yu cu. Trong c ch kim sot ny, sn phm cui cng thng c kh nng tng tc vi cc sn phm hot ng trc n. Nh vy, sn phm trung gian (thng l cc enzym) c cha ng thi hai v tr lin kt vi c cht v vi sn phm cui. Tng tc vi sn phm cui khin cho enzym b bin i cu hnh, do i lc lin kt ca enzym vi c cht b gim. Nh vy mc d sn phm trung gian c tng hp, nhng chng khng thc hin c chc nng ca mnh. Bn cnh c ch phn hi, hot ng ca mt s gen m cho enzym c kim sot theo con ng hot ho phn hi (feedback activation). Cu trc ca cc enzym thc hin chc nng ny thng c cc v tr tng tc vi vi c cht hoc mt s phn t trng lng nh. Ngoi ra, mt enzym cn c th tham gia c hai c ch iu khin c ch v hot ho phn hi. V d, enzym glutamine synthetase xc tc cho phn ng cui cng trong qu trnh sinh tng hp acid amin glutamine. Enzym ny c cu trc a phn (multimer) lin quan n c hai c ch kim sot sau dch m ca 16 sn phm trung gian trong qu trnh tng hp acid amin glutamine c t bo nhn s v t bo nhn chun. Glutamine synthetase thc hin chc nng iu khin thng qua vic bin i cu hnh khng gian ca cc sn phm khin chng khng c hot tnh. Trong t bo prokaryot, qu trnh phin m, dch m v phn hy ARNm thng bt u ngay khi ARNm cha c tng hp xong. Nh vy, ti bt k thi im no, mi phn t ARNm c th chu s kim sot ca c 3 qu trnh trn. C 3 qu trnh c th xy ra mt cch ng thi trn mt phn t ARNm. c bit tc di chuyn ca ribosome cng nh mc phn hy xy ra nhng vng c bit ca ARNm l khc nhau ngay trn mt phn t ARNm polycistronic. Ngoi ra, phn t ARNtm (transfer-messenger RNA) tng tc vi

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ARNm c kh nng nh du cc protein b tng hp sai bng vic gn on peptide ngn vo cc phn t ny sau chng b phn hy.

2.4

Bin i phn t ARNm trong t bo eukaryot

Trong t bo eukaryot, qu trnh phin m ph thuc vo tng loi promoter cng nh tng loi enzym ARN polymerase. Cc ARNr c tng hp nh ARN polymerase I, ARNm c tng hp bi ARN polymerase II v cc ARNt v 5S ARNr c phin m nh ARN polymerase III. Promoter cho cc ARN polymerase I v II thng nm trc vng cha m di truyn trong khi mt s promoter cho ARN polymerase III nm pha sau im bt u phin m (tc l promoter ca ARN polymerase III nm ngay trong vng cha m di truyn). Cng vi cc ARN polymerase, phn ng tng hp ARN trong t bo eukaryot ch thc hin c khi c s tham gia ca nhiu protein khc. Chng to thun li enzym khi ng phin m ti promoter ca cc gen eukaryot. y l iu khc bit gia qu trnh phin m t bo prokaryot v eukaryot. Promoter prokaryot thng km hm bi protein c ch, v th chng c xem l promoter mnh. Ngc li, promoter eukaryot thng i hi phi c cc protein hot ho; chng c xem l promoter yu.

Hnh 2.18: K thut xc nh v tr +1 s dng S1 nuclease. on ADN cha v tr +1 c lai vi phn t ARNm. Phn t lai dng kp ny c x l vi S1 nuclease phn hy phn nucleotide khng lai. Sau , phn t lai c in di cng vi mu xc nh trnh t ca on ADN ban u. Trn Hnh 2.12 cho thy nucleotide A chnh l v tr u tin c phin m sang phn t ARNm.

xc nh v tr +1, tc l nucleotide u tin c phin m sang phn t ARNm, chng ta c th p dng k thut "S1 nuclease mapping" (xc nh v tr +1 bi S1 nuclease). u tin, on ADN (sng lc t genome) c cha phn 5' ca gen c lai vi ARNm ca chnh gen . Tip theo, phn nucleotide ADN hoc ARNm khng lai vi nhau m dng si n s b phn hy bi S1 nuclease. Ch c phn t lai dng kp c in di trn gel acrylamide cng vi phn ng xc nh trnh t ca chnh on ADN ban u. Nh , v tr nucleotide u tin +1 c xc nh chnh xc (Hnh 2.18). Qu trnh tng hp ARNm eukaryot xy ra nh ARN polymerase II. Tuy nhin enzym ny khng t bt u phn ng m i hi s phi hp ca nhiu yu t phin m. Nh tng tc vi ARN polymerase II hoc vi ADN promoter hay cc v tr khc ngoi promoter,

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cc yu t ny gip enzym nhn bit d dng cc v tr bo ton trn promoter bt u qu trnh phin m. Cho n nay v tr dng chnh xc qa trnh tng hp ARNm trong t bo eukaryot vn cha c xc nh. Tuy nhin vi hu ht cc gen m cho protein, phn ng c dng li sau khi ARN polymerase II vt qua v tr c bit AATAAA (gi l v tr polyA) khong 0,5 n 2 kb. Trong th nghim invitro, vic loi b v tr polyA hoc gy cc t bin ti u khin cho phn t ARNm c tng hp rt di do enzym khng xc nh c v tr dng. C ch kim sot dng tng hp ARNm khi ARN polymerase II i qua v tr polyA vn cha sng t. Nhng kt qu thc nghim bc u cho thy y l v tr lin kt ca phc gm protein CPSF (Cleavage and Polyadenylation Specificity Factor) v protein CstF (Cleavage Stimulation Factor). Phn COOH ca tiu n v ln nht ca ARN polymerase II cn thit cho vic thm ui polyA. Do , vic kt thc phin m v thm ui c th xy ra ng thi trong nhn t bo eukaryot (Hnh 2.19). Sau khi c phin m, phn t tin thn ARNm eukaryot phi tri qua ba thay i c bn trc khi c dng lm khun tng hp protein. Trc ht ARNm c gn ui polyA vo u 3', gn "m" Gm7 vo u 5' ( v tr 2' ca ng ribose) v ct ni intronexon thnh phn t ARNm hon chnh. Phn ng methyl ho to cu trc m gip cho ribosome nhn bit c phn t ARNm v bm vo n. Cu trc m cn gip bo v u 5 ca ARNm khng b phn ct bi exonuclease.

Hnh 2.19: Phi hp gia v tr polyA (AATAAA) v phc protein trong qu trnh tng hp ARNm. Phc CPSF v CstF lin kt vi ARN polymerase II. n v tr polyA, factor b phn ly khi enzym khin enzym khng th tip tc tng hp ARNm. ng thi Poly(A) transferase thm ui polyA vo u 3 ca ARNm.

Sau khi c ui polyA v m Gm7, ARNm phi tri qua qu trnh ct b cc intron trc khi c vn chuyn ra ngoi t bo cht. Khong 30-40 nucleotide nm bin gii gia intron v exon rt quan trng phn ng ct intron xy ra chnh xc. t bin cc nucleotide nm trung tm intron khng nh hng n phn ng ny. c bit trong cu trc ca mi intron, hai nucleotide u tin GU u 5' v hai nucleotide AG tn cng u 3' gi vai tr quyt nh. Chng c bo ton mi intron v l v tr nhn bit ct intron ra khi phn t tin thn ARNm.

2.4.1 Phn ng ct intron v ni exon


Phn ng ct intron v ni hai exon vi nhau c thc hin theo cc bc c bn nh sau (Hnh 2.20):

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+ Ct ti bin gii exon-intron u 5' ca phn t tin thn ARNm, u 5' ca exon c gii phng. + Nucleotide 5'pG u intron va b ct lin kt vi 2'OH ca adenine nm cch u kia (pha 3') khong 25 base. Lin kt ny khin cho intron c cu trc vng. + Ct ti bin gii intron-exon u 3' ca phn t ARNm. Intron c loi ra v hai exon c ni vi nhau. Phn ng ct b intron i hi s tham gia ca mt s phn t snARNs kch thc nh (small nuclear RNA). Chng lin kt vi protein to thnh phc ribonucleoprotein (snRNPs). Mt snRNP thng cha mt phn t ARN v khong 10 protein khc nhau. Mt s protein c hiu cho tng loi snRNP, mt s khc chung cho cc snRNPs. Phn t snARN ca snRNP c kh nng to lin kt theo nguyn tc b sung vi cc on nucleotide vng bin gii 5' hoc 3' gia exon v intron. Mt s snARNs c th lin kt vi intron hoc vi cc snARN khc. Nhng lin kt ny gi vai tr quan trng trong phn ng loi b intron.

Hnh 2.20: Phn ng loi b intron ra khi phn t ARNm. Sau khi bin gii gia exon-intron u 5' b ct, cu trc vng ca intron c to ra do lin kt 5'-2' gia G ca u 5' vi A nm gn u 3' ca intron. Vic ct ti ni tip gip gia intron-exon u 3' cng nh vic ni hai exon vi nhau xy ra ng thi.

Thc nghim tm ra t nht c 6 phn t snARN k hiu t U1 n U6 tng ng vi 6 snRNPs. Phn t snARN U1 lin kt vi 15-20 nucleotide ti ni tip gip 5' exon-intron, snARN U2 lin kt vi khong 20 nucleotide ti on cha A (v tr to cu trc vng ca intron), cn snARN U5 lin kt vi cc nucleotide ti vng bin 3' intron-exon. Ngoi ra cc snARN U4, U6 c kh nng tng tc vi nhau v tng tc vi U2, U5. Mi tng tc gia cc snRNP u lm thay i cu trc khng gian ca phn t ARNm, mang cc u ni ca exon li gn nhau, to cu trc dng vng ca intron, xc tc cho phn ng ct b intron v ni exon vi nhau (Hnh 2.21).

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Hnh 2.21: Tng tc gia cc snRNP to spliceosome v hot ng ca chng trong phn ng loi intron, ni cc exon vi nhau.

Trong thc t, mi snRNP v cc protein tham gia phn ng ct b intron to nn mt cu trc c th quan st di knh hin vi in t gi l cc spliceosome. Chng c kch thc tng t nh ribosome, bao gm cc lin kt ARN-ARN, ARN-protein v protein-protein. Mt s protein ca spliceosome l cc enzym helicase phn gii ATP, gii phng nng lng lm thay i cu trc khng gian cn thit ca phn t ARNm.

2.4.2 Cc intron c kh nng t ct ra khi phn t ARNm-Phn ng selfsplicing


Trn y chng ta xt phn ng ct b intron, ni hai exon vi nhau to phn t ARNm hon chnh nh xc tc ca cc snRNPs v protein (trong cu trc spliceosome). Ngoi ra, mt s phn t tin thn ARNm ty th hoc lc lp c kh nng loi b cc intron m khng cn s tham gia ca cc snRNP hoc protein. Ni cch khc, cc intron ny c th t ct ra khi cc phn t tin thn ARNm v cc exon c ni vi nhau. Chng c chia lm hai nhm khc nhau, tu thuc vo cch thc xy ra phn ng.

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Hnh 2.22: Phn ng self-splicing ca cc intron nhm I. Phn ng ny ch i hi s tham gia ca GOH. Trong qu trnh t ct intron, phosphodiester c chuyn t phn t ng ny sang phn t ng khc (phn ng transesterification).

Intron nhm I: Phn ng ct intron nhm I ch yu cu phn t tin thn ARNm (c cha intron) v nucleotide G c nhm OH v tr 3'. S c mt ca guanosine ny dn n vic ct ti bin gii 5' exon-intron, ng thi G c gn vo u intron va c gii phng (bng vic chuyn lin kt phosphate t ng ny sang ng khc m khng nh hng n lin kt phosphodiester). u t do 3'OH ca exon s tng tc vi ni tip gip 3' intron-exon. Tip theo hai exon c ni vi nhau v intron tn ti dng cu trc vng. Cu trc ny sau s chuyn sang dng thng (Hnh 2.22). Intron nhm II: Cc intron thuc nhm ny cng c kh nng t ct ra khi phn t tin thn ARNm. Chng c cha GU u 5' v c ch t ct xy ra tng t phn ng i hi xc tc ca snRNPs (Hnh 2.23)

Hnh 2.23: Phn ng self-splicing ca cc intron nhm II. Nhm 2'OH ca A nm gn 3' intron-exon tng tc vi u 3' ca exon 1. Ni tip gip 5' exon-intron b b gy to cu trc vng cho intron. Tip theo phn ng ni hai exon vi nhau, intron c gii phng ra di dng vng.

Tuy nhin, trong phn ng t ct ny khng h c s tham gia ca bt k factor no ngoi bn thn phn t tin thn ARNm. iu ng lu , cc intron nhm II c cu trc khng gian rt phc tp. Cc on nucleotide trong intron c th tng tc to cp vi nhau. S thay i

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cu trc ny lm cho phn t ARNm c kh nng t xc tc cho phn ng ct b mt on nucleotide ca chnh n. Nh vy chnh cu trc khng gian phc tp ca intron nhm II ng vai tr ca cc snRNPs trong spliceosome.

2.4.3 Phn ng trans-splicing ni hai exon ca hai phn t ARNm


Phn ng ny khng i hi cc snARN hoc cc protein. Chnh phn t ARN cho exon th nht ng vai tr ca cc snRNP. Mt khc phn t ARN ny c kh nng nhn bit v tr 5' exon-intron phn t ARN th hai. Nh vy cc ARN tham gia phn ng trans-splicing t xc tc cho phn ng. Phn ng ny xy ra vi hu ht ARNm ca trypanosome, giun trn hoc lc lp trong t bo thc vt (Hnh 2.24).

Hnh 2.24: Phn ng trans-splicing to phn t ARNm m cho protein photosystem A (psaA). Genome ca lc lp dng vng, di khong 300 kb cha 3 exon ca ARNm ng vi protein ny. Exon1 c phin m ngc chiu vi exon2 v exon3. Ba exon ny c phin m mt cch c lp v c ni vi nhau thnh phn t ARNm hon chnh. Phn ng ny c th xy ra trong invitro.

2.4.4 Cu trc chung ca phn t ARNm


Ngoi phn ng ct intron ni exon, phn t ARNm eukaryot cn chu cc bin i u 5' v 3'. u 5' ca ARNm c 7-methylguanylate (m7G) lin kt vi nucleotide u tin ca ARNm. M m7G ny ng vai tr gip ribosome nhn bit ARNm trong qu trnh tng hp polypeptide. u 3' ca ARNm c ui polyA c xem l c vai tr trong vic vn chuyn ARNm t trong nhn ra ngoi t bo cht. Mt phn t ARNm eukaryot hon chnh ch cha exon, mang m Gm7 u 5', ui polyA u 3'. Phn t ARNm c cha vng 5' khng dch m (5 UTR) v vng 3' khng dch m (3UTR). Phn 5UTR nm trc m bt u tng hp peptide"start codon" cn phn 3UTR nm sau m dng tng hp protein "stop codon" (Hnh 2.25). Cc vng ny lin quan n kim sot tnh bn vng ca ARNm cng nh kim sot qu trnh dch m trn si ARNm. Vng 5' khng dch m thng cha cc on oligonucleotide c th tng tc vi cc protein c bit hoc c th to cu trc bc hai (to cc lin kt b sung gia chng). Nhng tng tc ngn cn ribosome di chuyn trn ARNm. Mt khc, vng 3' UTR ca mt s ARNm c cc v tr nhn bit bi endonuclease hoc cha cc on nucleotide c bit (giu AU) kch thch s phn hy ARNm.

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Hnh 2.25: Qu trnh phin m v dch m. Gen cha cc exon (E) v cc intron (I) c phin m sang phn t ARNm tin thn. Qu trnh bin i ARNm thnh phn t hon hnh s dng lm khun dch m trong qu trnh tng hp protein.

Phn t ARNm eukaryot kh bn vng. Thi gian bn sng cng nh kh nng dng lm khun dch m ph thuc phn no vo di ui polyA. Trong thc t, khng pht hin c phn t ARNm c ui polyA ngn hn 30A. di ca ui lin quan n tnh bn vng ca ARNm nn vic thm hoc bt cc nucleotide A vo ui c kim sot rt cht ch (cn lu ui polyA c gn vo ARNm ngay sau khi phin m trong nhn. Tuy nhin thay i di ca ui c th xy ra trong t bo cht). c bit trong qu trnh pht trin m phi, rt nhiu loi ARNm c phin m t cc gen ca m v c d tr trong t bo trng. Chng thng c ui polyA rt ngn (10-30 A) nn khng c dch m. Ch sau khi trng th tinh, cc ARNm ny c thm ui polyA v tr thnh khun tng hp protein.

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Chng 3 K THUT ADN TI T HP


Bt u t nhng nm 1970 tr i, cc phng php nghin cu, phn tch ADN v gen c pht trin mnh m. Nh nhng k thut hin i, gi chung l k thut tch dng v ti t hp, mt gen bt k c th c phn lp v c tch ra khi genome. Gen ny c bin i theo mun v c a tr li t bo nui cy. Tuy kh khn phc tp hn, gen mang t bin c a vo t bo sinh dc. Chng hot ng trong genome ca c th v c di truyn cho cc th h sau. K thut tch dng lin quan n vic to ra phn t ADN ti t hp t cc on ADN c ngun gc khc nhau. ct v ni cc on ADN vi nhau, hay ni cch khc c th thao tc vi cc on ADN, nht thit phi s dng cc enzym gii hn (restriction endonucleases) v ADN ligase. V d, mt on ADN c cha gen c ct ra t genome ngi v c ghp vo plasmid to thnh phn t ADN ti t hp. Phn t ny c a tr li t bo vi khun. Sau 24 gi nui cy, khong 1012 phn t ADN ging ht phn t ban u c to ra. Do , s lng on ADN mang gen ca ngi cng c nhn bn ln theo. Lc ny on ADN xem nh c tch dng v cc plasmid mang ADN l c gi l vector tch dng (cloning vector). Cc on ADN a vo vector c th xut pht t cc ngun khc nhau. Ngun s cp (primary source) c th l ADN tch chit trc tip t genome hoc ly t ngn hng ADNc. Ngun th cp (secondary source) thng l on ADN phn lp t ngun s cp c ct thnh cc on nh hn tip tc a vo cc loi vector khc (subcloning). Thng thng, tch c mt gen bt k t genome cn phi bit trc mt on ADN lin quan n gen (hoc nm gn n). on ny c s dng nh mi hay cn gi l u d (probe) tm ra gen cn nghin cu. Khi sn phm ca gen bit, c th xut pht t protein suy din trnh t nucleotide m cho n. l nhng thng tin u tin cn thit gip cho nghin cu ARNm v ADN (gen) tng ng vi protein. Vn t ra lm th no tch c mt gen ring bit ra khi genome? Lm th no xc nh c phn t ARNm tng ng vi mt gen? Khi c gen, chng ta c th tng hp c protein invitro v invivo hay khng? Mt khc chng ta c th iu khin hot ng ca gen sau khi thay i (gy t bin) trnh t nucleotide hay khng? Trong c th, a s cc gen hot ng mt cch chuyn bit, tc l chng thng biu hin mt s hoc thm ch trong mt loi t bo bit ho nhng thi im nht nh, vi mc biu hin khc nhau. Lm th no tm ra c cc gen v bit c chc nng ca chng? Ngoi ra c nhng cn bnh do t bin gen gy ra (bnh di truyn) nhng sn phm ca gen cha xc nh c. Lm th no phn lp c gen v tm ra nhng bin i mc ADN lin quan n bnh? vv... Tt c nhng vn nu trn v ang c sinh hc phn t gii quyt, c bit nh p dng cc k thut tch dng v ti t hp ADN.

3.1

Phn ct, phn ly ADN

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Ngy nay, chng ta c th d dng ct genome ca t bo eukaryot gm cc phn t ADN c kch thc rt ln (khong 106 n 1011 nucleotide) thnh cc on ADN c kch thc theo mun nh cc enzym gii hn (restriction enzymes hoc restriction endonucleases). V tr ct c th nm cch xa hoc ngay ti v tr nhn bit. Cc enzym nhn bit v ct ngay ti mt v tr c s dng nhiu trong k thut ADN ti t hp. Chng nhn bit cc v tr c hiu gm 4 n 8 nucleotide trn phn t ADN v ct cc phn t ny thnh cc on khc nhau. Hin nay cc hng thng mi gii thiu gn 600 enzym gii hn c sn xut theo cng ngh ADN ti t hp. Chng c tm ra v tch chit ch yu t vi khun.

Hnh 3.1: Phn t ADN b ct bi enzym gii hn. Cc enzym ny nhn bit trnh t sp xp ca 4 nucleotide v ct ADN cc v tr ny. Mt s enzym ct ADN thnh cc on c u bng (cc u "Blunt ends") nh HpaI. Ngoi ra cn c cc enzym ct ADN to ra cc u dnh hay cn gi l u so le (cc u "Cohensive ends") nh EcoRI.

Cc on ADN c kch thc khc nhau c phn ly bng phng php in di trn gel agarose (in di ngang) hoc gel polyacrylamide (in di ng). Phn t ADN tch in m nn chng chuyn ng t cc m sang cc dng trong in trng mt chiu. Cc phn t ADN c kch thc ln (~ 105-107 bp) c phn ly bng in di trn trng xung y "pulsed-field". Nhng on di t 200 bp n 10.000 bp c phn ly trn gel agarose hoc acrylamide vi nng khc nhau. Nng gel ln ph hp vi cc on ADN ngn. V d, nhng on ADN ngn 200 bp sai khc nhau khng qu 10 bp c th phn bit trn gel agarose 4%. Tuy nhin, nng agarose khng th tng cao do gel gin, d gy v c in tr ln. V vy, gel polyacrylamide c u th hn so vi gel agarose trong vic phn ly cc on ADN ngn, c s lng nucleotide chnh lch nhau t, mc d khu chun b v thi gian in di vi gel polyacrylamide lu hn. Gel polyacrylamide c kh nng phn gii nhng on ADN hn km nhau mt nucleotide (phng php xc nh trnh t nucleotide). Sau khi in di, cc on ADN trn gel c th quan st c di tia t ngoi khi chng c nhum vi ethidium bromide (EtBr) hoc c th quan st bng mt thng khi nhum bc hay c nh du bng cc cht pht mu khc nhau. Ngoi ra, ADN cn c pht hin bng vic gn phng x (P32, H3 hoc S35). Phng php nh du phng x rt nhy, cho php pht hin lng ADN rt nh (c ng).

3.2

a cc on ADN vo vector

Vector c s dng lu gi v chuyn ch ADN vo cc t bo nhn. Chng c th tn ti trong t bo vi sinh vt (v d plasmid trong t bo vi khun) hoc trong t bo eukaryot (vector YAC trong t bo nm men). Ngoi ra, vector c thit k c kh nng tn ti trong cc loi t bo prokaryot v eukaryot (vector con thoi). V vy, vic la chn v thit k vector ph thuc vo mc ch nghin cu.

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3.2.1 Cc vector s dng trong k thut tch dng


Tu thuc vo kch thc ca cc on ADN cn lu gi m chn cc vector khc nhau. Ph bin nht l cc plasmid, genome ca bacteriophage (gi tt l phage hay ), cosmid, cc vector BAC, YAC. Cc plasmid dng lm vector thng c kch thc nh, do chng khng mang c cc on ADN c kch thc ln (<7 kb). Tuy nhin vector nhn to YAC c th mang c on ADN c 1 Mb.

Hnh 3.2: Cc yu t c bn v cu trc v tr a im tch dng ca vector. V tr ct ca cc enzyme gii hn c k hiu bi cc mi tn. Cc enzyme ny c ngun gc t vi sinh vt. EcoRI-Escherichia coli RY13, BamHI-Bacillus amyloliquefaciens H, XbaI-Xanthomonas badrii, SalI-Streptomyces albus, PstI-Providencia stuartii, SphIStreptomyces phacochromogenes v HindIII-Haemophilus influenzae Rd.

Cu to ca vector s dng trong k thut tch dng thng c cha 3 yu t c bn: th nht, tm ti bn ADN (origin of replication) cho php vector ti bn trong t bo nhn. Th hai, ch th chn lc (selectable marker gene) hay cn gi l gen ch th, thng l gene qui nh kh nng chng chu khng sinh. Nh c gen ny m chng ta c th sng lc c nhng t bo mang phn t ADN ti t hp. Cui cng l v tr gn on ADN l vo (v tr a im tch dng-polycloning site) (Hnh 3.2). Plasmid l nhng phn t ADN dng vng, tn ti trong t bo vi khun v nm ngoi nhim sc th. Plasmid c kch thc thay i trong khong 1 kb n vi trm kb. Trong t nhin, plasmid c th ti bn ng thi vi nhim sc th hoc c kh nng ti bn c lp. Thng thng, cc plasmid ti bn c lp thng c kch thc nh v c s lng bn sao ln trong mt t bo vi khun. Plasmid thng mang nhng gen chng chu khng sinh, v vy nhng gen c s dng nh cc ch th (marker) chn lc t bo cha plasmid. Plasmid u tin c s dng trong k thut tch dng l pSC101 ch c mt gen ch th qui nh tnh chng chu tetracyline (tetr) v mt v tr EcoRI duy nht ghp ADN l vo. Ngy nay, c rt nhiu loi plasmid khc nhau mang hai hoc nhiu gen ch th v v tr a im tch dng gm nhiu v tr nhn bit ca cc enzyme gii hn. c bit, v tr a im ca plasmid thng c thit k nm trong mt gen ch th. Khi on ADN xen vo ti mt trong cc im ny, chng gy bt hot gen ch th. Do , t bo nhn plasmid gn ADN l s khng tng hp c sn phm ca gen ch th. S thiu vng sn phm ca gen ch th c th lin quan n s chuyn mu ca mi trng nui cy. V th, da vo mu sc, chng ta chn lc c t bo nhn plasmid gn ADN l. V d, cc plasmid pUC c v tr a im nm trong trnh t ca gen ch th m cho -galactosidase. Khi pUC c gn ADN l, enzym khng c tng hp. Nu chng ta cho vo mi trng nui cy c cht ca enzym, c cht

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khng b chuyn ho nn t bo nhn pUC gn ADN l s c mu trng. Ngc li, nhng t bo nhn pUC c gen ch th nguyn vn, chng s c mu xanh to ra do c cht b chuyn ho bi -galactosidase. Cc plasmid kch thc nh c s bn copy nhiu hay c s dng trong cc k thut ADN thng thung. Chng thch hp vi cc on ADN khng di qu 7 kb. Khi on ADN c kch thc ln hn, tri qua mt s ln nhn bn, on ny b ngn dn hoc b loi khi plasmid do t bo c xu hng o thi ADN khng cn thit. Bacteriophage (vector thc khun th): Hu ht cc vector thc khun th c thit k da vo nhim sc th ca thc khun th . Nhim sc th thc khun th gm 48.502 bp m cho 46 gen. Khi xm nhim vo t bo vi khun, nhim sc th tn ti dng vng do lin kt ca cc v tr cos hai u ca phn t ADN thng. ADN ca phage c "ng gi" trong v protein. Khi "ng gi", kch thc nhim sc th khng cn phi chnh xc m c th thay i nhng khng nhiu qu 5% hoc t hn 25%. S dng tnh cht t nhin ny, on ADN l c thm vo nhim sc th ca phage. Lc ny nhim sc th tr thnh vector. Cc vector phage mang ADN l c cho xm nhim t bo vi khun. Chng ti bn v gy trng thi sinh tan (ph hy cc t bo ch) v gii phng ra cc phage mi. Bng hnh thc , ADN l c nhn ln cng vi nhim sc th phage. Cu trc v chc nng ca tt c cc gen phn b trn nhim sc th c xc nh hon ton. Cc gen lin quan n trng thi tim tan chim khong 1/3 nhim sc th (khong 15 kb). Chng c th b loi i m khng lm nh hng n hot ng ca nhng gen gy trng thi sinh tan. V th chng c thay th bi ADN l. Do di ca ADN a vo nhim sc th c th t n 15 kb. Khi nhim vo t bo ch (t bo vi khun), cc vector phage mang ADN mt kh nng tn ti trng thi tim tan. Chng ti bn v gy trng thi sinh tan, gii phng ra nhiu th phage mi. Cosmid: Nhim sc th chuyn t dng thng sang dng vng nh cc v tr cos hai u ca s ADN thng. Do trong th nghim in-vitro, mt phn t ADN c kch thc thch hp mang v tr cos ny c th c "ng gi" trong v protein. Cosmid l cc plasmid mang cc v tr cos v c "ng gi" in-vitro ging nh thc khun th . Khi cc phage mang cosmid ny xm nhp vo t bo vi khun, cosmid s chuyn thnh dng vng v tn ti nh plasmid trong t bo nhn. Tuy nhin phage cha vector cosmid khng cn kh nng xm nhim t bo vi khun v khng mang nhng gen cn thit cho qu trnh ny. V vy, cosmid c a vo t bo ch nh cc phage ph tr. Cosmid c th mang cc ADN l di khong 35 n 45 kb. Ngoi ra, cn c loi vector Fosmid mang tm ti bn ca plasmid F (E.coli) v cc trnh t cos ca cosmid. Tuy nhin. do tnh cht ca tm ti bn nn fosmid c s lng bn sao t trong t bo E.coli. Vic s dng vector Fosmid gip hn ch phn no nhng sai st xy ra vi ADN l xy ra trong qu trnh vector ti bn. Vector bacteriophage P1: tng t nh vector thit k t nhim sc th lambda , vector P1 c thit k da vo vic loi b nhng on ADN khng cn thit trong nhim sc th thc khun th P1. Do genome P1 ln hn genome ca lamda , vector P1 c kh nng nhn on ADN l di ti 125 kb. Ngoi ra, khi kt hp cc c tnh ca vector P1 v vector BAC, loi vector PAC (P1-derived Artificial Chromosome) c thit k ph hp vi cc on ADN khong 300 kb. Vector BAC (Bacterial Artificial Chromosomes): c thit k nhn to c nhng u im ca vector YAC, tc l c kh nng cha cc on ADN kch thc ln c 105 bp. Tuy nhin, do khng phc tp nh YAC nn thao tc vi BAC c phn n gin hn. BAC c th

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ti bn d dng trong E.coli ging nh plasmid. Nhng nm cui thp k 20, vector BAC ang dn dn thay th YAC trong cc nghin cu vi on ADN kch thc ln nh vic xy dng ngn hng, xc nh bn ca ton b nhim sc th... Vector YAC (Yeast Artifical Chromosome): y l vector c thit k nhn to da vo cu trc ca nhim sc th nm men. Vector ny c cha gen ch th, v tr a im tch dng, trnh t nucleotide ca tm ti bn ARS (Autonomously Replicating Sequence), tm ng v telomere (trnh t u mt nhim sc th). Nh vy, thc cht YAC l nhim sc th mini ca t bo nm men. Thng thng, sn phm ca gen ch th trn vector YAC gip t bo nhn c kh nng b khuyt s thiu ht mt cht no trong mi trng. V d, vector YAC mang gen ch th URA+ gip t bo khuyt dng URA- mc c trn mi trng thiu uracil. Vector YAC c th mang ADN l di ti 1 Mb (106 bp).

3.2.2 a ADN vo vector


a mt on ADN vo vector, phn ng ni gia vector vi on ADN gi vai tr quyt nh. Hai u ni ca vector v ADN thng lin kt vi nhau theo nguyn tc to cp b sung. Mt s phng php hay s dng nh sau: * S dng cc homopolymer: Cc homopolymer c th lin kt vi nhau theo nguyn tc to cp b sung. Chng c ni vo cc u ca vector v on ADN. V d, oligo (polyT) c ni vo u 3' ca ADN l trong khi oligo (polyA) c ni vo u 3' ca vector. di cc oligo thng khong t 10 n 40 nucleotide. Enzym terminal transferase c kh nng thm mt loi nucleotide vo cc u ct ca vector hoc ADN. Sau c gn thm cc polyA, poly T, vector v on ADN l c ni vi nhau nh lin kt b sung gia A/T v nh ADN ligase (Hnh 3.3). * Ct ADN l v vector bng enzym gii hn to u bng (blunt ends). Chng c ni vi nhau bi T4 ADN ligase. Enzym ny c kh nng ni hai on ADN c u bng vi nhau. Phng php ny khng i hi cc nucleotide to cp b sung cc u ct. Tuy nhin, ADN l a vo vector c th khng ly ra nguyn vn do v tr nhn bit bi enzym gii hn ban u b thay i.

Hnh 3.3: Enzym terminal transferase thm cc ui homopolymer vo cc u ca vector v ADN l. Chng c ni vi nhau to phn t ti t hp ADN nh ligase.

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* Ct ADN v vector bi mt loi enzym gii hn to cc u so le (cohensive ends). Sau chng c ni vi nhau bi enzym ligase. u im ca phng php ny l dng chnh enzym gii hn ly li c on ADN t vector cha chng sau khi vector ny c nhn ln trong t bo. Tuy nhin trong phn ng ni cc u ct vi nhau, c th chnh hai u ca vector c ni li, hoc hai hay nhiu on ADN ni vi nhau trc khi ghp vo vector. trnh nhng nhc im , vector ( dng thng) hoc on ADN sau khi b ct bi enzym gii hn, c x l vi phosphatase loi nhm P ra khi u 5' (Hnh 3.4).

Hnh 3.4: S dng phosphatase kh nhm phosphat (P) u 5 ca vector trnh s to li dng vng ca vector khng cha ADN l.

* Phng php s dng linker: Mt s oligonucleotide c tng hp nhn to c cha v tr nhn bit ca cc enzym gii hn c gi l cc linker. on ADN l c u bng c th c ni vi linker trc khi a vo vector. Thng thng, enzym c v tr nhn bit trong linker s c s dng ct vector (Hnh 3.5). * Phng php s dng linker i: Phng php ny thng c s dng trong qu trnh xy dng ngn hng ADNc. Cc bc chi tit c m t trn hnh 3.7. Phn t ADNc c tng hp t khun ARNm v c ni vi linker S (c v tr nhn bit ca enzym S). u cong "loop" c to ra do tnh cht ca enzym phin m ngc reverse transcriptase s b ct bi S1 nuclease v c sa li thnh u bng nh ADN polymerase. Sau u bng ny c ni vi linker R (cha v tr nhn bit ca enzym gii hn R). on ADN tip tc c x l vi hai enzym S v R to ra cc u so le. Vector cng b ct bi hai enzym S v R. Bng k thut ny, c th iu khin c chiu ghp ADN vo vector.

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Hnh 3.5: S dng linker a ADN vo vector. Enzym T4 ADN ligase ni cc linker c cha v tr nhn bit ca enzym E1 vo hai u on ADN l. Sau dng chnh enzym ny ct ADN v ct vector. Ch c cc on ADN mang v tr nhn bit ca E1 c gn vo vector nh ADN ligase.

3.3

Ngn hng ADN

Genome (hay gn gi l h gen) t bo eukaryot c kch thc rt ln. iu khin chng ta gp nhiu kh khn trong thao tc thc nghim khi chng ta thng lm vic trn mt gen nht nh. V vy, chng ta c th tch chit ADN tng s, sau dng enzym gii hn ct thnh cc on v lu gi tt c cc on trong ngn hng. Khi cn nghin cu mt gen no , chng ta ch vic chn gen ny t ngn hng. Mt cch chi tit hn, chng ta c th xy dng ngn hng ca tng nhim sc th hoc ngn hng ch cung cp thng tin c mt trong cc si ARNm ca mt t chc t bo bit ho. Nh vy thng tin di truyn ca mt i tng sinh vt bt k c th c ct gi trong cc loi ngn hng khc nhau, tu thuc vo mc ch s dng.

3.3.1 Ngn hng cc ADNc (cDNA library)


Tp hp tt c cc phn t ARNm tch chit t mt t chc c th i din cho tt c cc gen hot ng trong t chc . Cc ARNm ch cha exon nn ngn hn nhiu so vi cc gen tng ng. V vy, thay cho vic thao tc trn gen (ADN), chng ta khai thc t ARNm mt s thng tin v cu trc, chc nng ca gen, ca sn phm protein m gen m cho. Tuy nhin, ARNm rt d bin tnh nn thao tc vi chng khng thun li. khc phc nhc im ny, chng ta s dng enzym phin m ngc reverse transcriptase sao chp chnh xc thng tin t si ARNm sang ADN. Enym ny tng hp si n ADN b sung da vo si khun ARNm. Si kp ARN/ADN c x l vi kim hoc ribonuclease H (phn hy si khun ARN) chuyn thnh si n ADNc (complementary DNA). Tip sau , ADN polymerase I tng hp si n ADNc th hai c c si i ADNc hon chnh. Phn t ADN b sung dng si i c gi l ADNc. Cc bc c th ca qu trnh to ADNc c m t trn hnh 3.6. Tp hp tt c cc ADNc tng ng vi mi ARNm ca mt loi m c a vo vector. Mi vector nhn mt phn t ADNc. Tp hp tt c cc vector cha ADNc c chuyn vo t bo nhn. Tp hp tt c cc t bo nhn cha y ADNc ca mt loi m

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to thnh ngn hng ADNc ca loi m . Lc ny mi ADNc trong ngn hng thng c gi l mt dng (mt clone). sng lc bt k mt clone ADNc t ngn hng, cn phi c mi (u d) c hiu i vi ADNc ny. V d, chng ta c th chn c ADNc ng vi mt on polypeptide nu chng ta bit trnh t acid amin. Da vo trnh t acid amin chng ta suy ra trnh t cc nucleotide tng ng da vo m di truyn b ba. on nucleotide ny c tng hp nhn to v s dng nh mi sng lc ADNc ng vi polypeptide . K thut sng lc s c m t chi tit phn sau.

Hnh 3.6: Tng hp ADNc t ARNm. Enzym reverse transcriptase tng hp si n ADNc trn khun ARNm (s dng mi oligoT to cp b sung vi ui polyA). Si n ARNm b phn hy trong iu kin kim hoc bi ribonuclease H. Si ADNc th hai c tng hp trn khun ADNc th nht nh ADN polymerase I. Phn t ADNc (dng si kp) c cc u bng c ni vi hai loi linker S v R. Do khi a vo vector, ADNc c gi ng chiu.

Cc vector nhn ADNc c a vo t bo nhn. Qu trnh ny c gi l bin np (transformation). T bo c kh nng nhn vector c gi l t bo kh bin (competent cells). T bo nhn vector c gi l th bin np (transformants). Tp hp tt c cc t bo cha vector to thnh mt ngn hng ADNc i din cho t chc m dung tch chit ARNm. i vi mt s phn t ARNm ch xut hin vi s lng khng ln, chng thng c lm giu trc khi tng hp ADNc. Phng php lai loi tr (subtractive hybridization) hay c s dng pht hin cc loi ARNm c hiu ch c tng hp trong mt loi m nht nh. Phng php ny ln u tin c ng dng xc nh ADNc tng ng vi cc protein th th (receptor) ch xut hin trn b mt t bo lympho T m khng c lympho B (Hnh 3.7).

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Hnh 3.7: Phng php lai loi tr c s dng lm sch nhng phn t ADNc tng ng vi ARNm ch xut hin t bo lympho T (khng c lympho B). Phng php ny cho php lm giu nhng phn t c hiu khc bit gia hai loi t bo.

u tin, ARNm tch t t bo lympho T c dng tng hp cc ADNc. Sau cc ADNc (vi s lng ln) c lai vi ARNm tch t t bo lympho B sao cho mi ADNc tng ng vi ARNm xut hin c hai loi t bo u b lai vi nhau; ch cn li ADNc tng ng vi ARNm ring bit ca lympho T. Sau phn ng lai, sn phm c i qua ct lc tch ring nhng phn t ADNc dng si n. Tip theo, chng c dng tng hp ADNc dng si kp v xy dng ngn hng ADNc. Ngn hnh ny c hiu cho ring loi t bo lympho T v ch cha thng tin dnh ring cho loi t bo ny. Nhng ngn hng ADNc chuyn bit nh th rt thun li khi cn xc nh hot ng khc nhau ca gen gia hai loi t bo. Cc ADNc c th c a vo vector c mang sn promoter, thng thng l nhng promoter mnh to ra s lng ARNm ln. Nhng vector ny c gi l vector biu hin gen (expression vector). Di s iu khin ca promoter, ADNc c phin m to ra ARNm. c im ca nhng si ARNm ny l khng cha intron. Do , khi a vector biu hin cha ADNc vo t bo prokaryot, protein ng vi ADNc c th c tng hp trong cc th bin np. Nh vy, khi a vector biu hin gen mang mt ADNc hon chnh vo t bo kh bin th sn phm tng ng ca ADNc c th c tng hp vi s lng ln trong mt thi gian ngn. Ngoi ra, vector biu hin c th mang on nucleotide m cho mt protein nh du, v d nh protein pht hunh quang (Green Fluoresence Protein- GFP). on nucleotide ny nm sau promoter v s ni vi ADNc. Nh vy, promoter s kim sot biu hin ca protein dung hp (fusion protein) gm on peptide nh du ni vi protein m bi ADNc. Nh s pht hunh quang ca GFP m c th bit c hm lng protein s phn b v chc nng ca n trong t bo sng.

3.3.2 Ngn hng ADN genome (genomic DNA library)


Mt phn t ADNc ch cha cc exon ca mt gen, trong khi gen ca t bo eukaryot thng gm cc intron. V vy, ngoi vic xy dng ngn hng ADNc (tng ng vi vng

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mang m di truyn), cc nh sinh hc cn xy dng ngn hng cha cc gen hon chnh. iu gip h nghin cu su hn hot ng ca cc gen, v d nh nghin cu vng ADN iu khin, cc ADN tng cng (enhancer), nghin cu vai tr ca cc intron i vi iu ho hot ng ca gen vv...

Hnh 3.8: Xy dng cc loi ngn hng ADN genome ca cng mt i tng. Cc clone nm gi ln nhau to iu kin thun li cho vic phn lp gen, xc nh bn genome. Cu trc mt contig gm cc dng tng ng vi nhng phn on nh ct ra t mt on ADN ln hn. Cc ngn hng di dng contig c bit cn thit cho vic xc nh gii m ton b genome.

a ton b cc gen ca genome vo mt ngn hng, trc ht genome phi b ct bi enzym gii hn thnh cc on ADN c kch thc thch hp vi loi vector s nhn chng. Vi mt genome c kch thc nht nh, thu c on ADN di cn chn enzym nhn bit s nucleotide ln (8 nucleotide). Ngc li, thu c cc on ADN ngn th nn chn enzym nhn bit s nucleotide t (4-6 nucleotide). S on ADN trong trng hp th nht s t hn so vi trng hp th hai. Ngoi ra, cn lu khng phi d dng nhn c mt gen nguyn vn nm gn trn mt on ADN. Trong thc t, mt gen b ct thnh cc on ADN do v tr nhn bit ca enzym nm ngay trong gen. hn ch kh nng , th thut ca phn ng ct ADN l cn phi khng ch thi gian cng nh nng ca enzym sao cho phn ng ct ch xy ra tng phn mi v tr nhn bit khng b ct ng thi. Do , phn ng ct xy ra c tnh ngu nhin vi tt c cc v tr nhn bit ca enzym. Nh mt gen c th c bo ton ngay khi n cha v tr ct trong gen. Sau phn ng, cc on ADN c a vo vector thch hp. Vector cng c ct bng chnh enzym s dng ct ADN genome. Tp hp ca mi vector cha ADN genome uc a vo t bo nhn v to thnh ngn hng ADN genome. Mi vector ca ngn hng c gi l mt dng (clone). i vi mt c th, s dng cc loi vector khc nhau, c th xy dng c cc ngn hng ADN genome cha cc on ADN di ngn khc nhau (Hnh 3.8). Tuy nhin cc ngn hng ny khng h c s khc bit v thng tin di truyn. Nh s dng ng thi nhiu loi ngn hng, vic lp bn di truyn, bn nhim sc th, nghin cu cu trc v chc nng mt gen tr nn d dng. Ngc li, mi c th c nhiu loi ngn hng ADNc c trng cho loi m t chc dng tch chit

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ARNm. Tt c cc ngn hng ADNc ch cha vng exon ca cc gen. Nh vy, s khc nhau ch yu gia hai loi ngn hng ADN genome v ADNc l thnh phn ADN. Bn cnh , s lng dng (clone) cng nh kch thc cc on ADN trong ngn hng ADNc nh hn nhiu so vi ngn hng ADN genome.

3.4

Sng lc mt dng t ngn hng ADN

m bo chn c mt dng cn nghin cu t ngn hng, chng ta cn tnh ton s dng cn thit ly ra t ngn hng dng vo vic sng lc. V d, genome rui gim c kch thc khong 105 kb c ct ra thnh cc on trung bnh 40 kb. Ngn hng cha cc on ADN ny cn c t nht N =11.513 dng m bo mt on ADN bt k s c mt trong ngn hng vi sc xut P = 99%. S N c tnh theo cng thc sau: N = ln (1-P)/ln (1-1/n) = ln (1-0,99)/ ln [1-(40/100 000)] Trong : n l s on ADN ct ra t genome. N l s dng cn ly t ngn hng m bo sng lc c mt dng vi xc sut P. Nh vy, sng lc mt dng t ngn hng genome rui gim, vic u tin l chng ta phi ly t ngn hng t nht l 11.513 dng (y l 11.513 khun lc hoc l 11.513 transformants nn d dng m c khi pha long nui cy trn mi trng). Sng lc mt dng trong s hng triu dng t ngn hng ADN ca sinh vt bc cao i hi nhiu thao tc v thi gian. Sng lc nhng gen m cho enzym (amylase, protease...) hay gen qui nh tnh chng chu (khng li khng sinh, c dc...) hoc gen khuyt dng c th thc hin d dng hn da vo hot tnh enzym, kh nng mc trn mi trng c c dc hoc b khuyt chc nng. Qu trnh sng lc nhng gen ny c gi chung l chn lc di truyn (genetic selection). Sng lc gen chng chu khng sinh c xem l trng hp in hnh ca chn lc di truyn. V d, gen khng li peniciline c sng lc d dng t ngn hng ADN genome ca Salmonella typhimurium khi cc vector ca ngn hng c bin np vo t bo nhn E.coli. Nhng t bo ny c tri trn mi trung c cha peniciline. Duy nht dng t bo nhn vector mang gen penr c kh nng to khun lc trn mi trng ny. Ngoi ra, cc gen tham gia mt s con ng chuyn ho trong t bo cng c th sng lc d dng theo cch thc chn lc di truyn. V d, mun chn c cc gen m cho enzyme tham gia sinh tng hp histidine S.cerevisiae th c th xy dng ngn hng ADN genome nm men gm cc vector ti t hp c bin np vo t bo E.coli khuyt dng khng c kh nng tng hp c acid amin ny. Qu trnh sng lc ch tuyn chn dng t bo to khun lc trn mi trung khng c histidine. Khi phng php chn lc di truyn khng thch hp trong tuyn chn, k thut lai acid nucleic da vo s to cp gia cc trnh t tng ng v k thut min dch da vo tng tc khng nguyn-khng th c mt vai tr quan trng sng lc c gen hoc ADNc ca gen cn nghin cu.

3.4.1 Phng php sng lc chung


Khi p dng k thut lai acid nucleic ly c mt dng (mt on ADN) t ngn hng (ADN genome hoc ADNc), nht thit phi c u d (probe) c hiu. l mt on ADN c trnh t nucleotide ging ht hoc c tng ng cao vi mt phn bt k ca on ADN cn tm. Nu cn sng lc mt gen m sn phm protein ca gen bit, u d c

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th c tng hp nhn to da vo trnh t acid amin ca protein. Do tnh cht thoi ho ca m di truyn nn chng ta khng nhn c trnh t nucleotide chnh xc. iu ny khng nh hng nhiu ti kt qu sng lc do php lai ch i hi tng ng nht nh gia u d v ADN cn tm.

Hnh 3.9: Sng lc qun lc mang dng cn tm t ngn hng. Mng cha cc qun lc c gi l cc replica. Chng c x l vi kim v nhit trc khi lai vi u d gn phng x hoc pht hunh quang. Sau khi lai, v tr qun lc lai vi u d c pht hin trn phim t ghi phng x hoc da vo tn hiu pht mu.

Phng php ph bin c p dng trc y sng lc (screening) mt dng t ngn hng c m t trn hnh 3.9. S lng N t bo vi khun (mang plasmid) hoc cc phage (mang vector hay cosmid) ca ngn hng c nui cy trn cc a petri, mc thnh cc qun lc. t ln trn b mt mi a mt mng sao cho cc qun lc c chuyn t a sang mng (thng l mng nilon c i lc lin kt cao vi ADN). X l mng vi kim ph v t bo, gy bin tnh ADN v c nh chng trn mng. Sau cc mng c vi u d c hiu. u d ny c nh du phng x hoc ho hc. u d s lai vi ADN c trnh t b sung vi chnh n. V tr ca qun lc mang dng lai s c pht hin v c tch ra khi cc qun lc khc. S dng u d nh du phng x cho php xc nh nhanh, nhy, chnh xc dng mong mun trong s hng triu dng. Qu trnh ny thng c lp li 3 ln vi cc iu kin lai thay i tng kh nng sng lc, loi tr cc dng lai nhiu do u d c kh nng lai vi c cc on ADN c trnh t tng ng vi u d. Ngoi ra nu khng th bit, c th s dng k thut min dch sng lc gen (ADNc) m cho khng nguyn. Lc cn s dng ngn hng ADNc cha cc vector biu hin gen (expression vector). Khng nguyn c tng hp t dng mang ADNc m cho khng nguyn . Khng th c nh du (bng phng x hoc ha hc) lm u d. u d lai vi khng nguyn v v tr dng t bo c pht hin. Mt khi dng ADNc c phn lp, c th s dng n lm u d sng lc tip gen tng ng trong ngn hng ADN genome. Hin nay, sng lc mt hay nhiu dng trong cng mt ln, k thut PCR hoc microarray c rt nhiu u im, cho php chn c cc dng mong mun mt cch nhanh chng v chnh xc.

3.4.2 Phng php sng lc phn bit "differential screening"

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Phng php sng lc phn bit ph hp vi mc ch phn lp cc ADNc xut pht t ARNm c hiu cho tng loi m hoc khi cc ARNm ch xut hin trong tng giai on pht trin c th. Phng php ny cho php xc nh c cc dng ging nhau trong hai hay nhiu loi ngn hng ADNc. ng thi, cc dng c hiu cho tng loi m cng c phn bit r rng. Cc bc c th ca phng php sng lc phn bit c m t trn hnh 3.10. Cc dng t bo ca mt ngn hng c tri trn a petri. Sau cc qun lc c chuyn sang hai mng. Mi mng c lai vi u d phng x khc nhau. u d th nht bao gm cc ADNc tng ng vi ARNm ca loi m A. u d th hai gm cc ADNc ng vi ARNm ca loi m B. So snh s c mt hoc m nht ca cc vt lai trn phim t ghi phng x c th xc nh c cc ARNm ch xut hin trong mt hoc trong c hai loi m.

Hnh 3.10: Phng php sng lc phn bit. Ln lt t hai mng ln trn mt mi a petri mang cc qun lc ca ngn hng ADNc sao cho v tr ca cc qun lc trn hai mng ging ht nhau. Sau mi mng c lai vi u d phng x khc nhau. u d th nht bao gm cc ADNc tng ng vi ARNm ca loi m A. u d th hai gm cc ADNc ng vi ARNm ca loi m B. So snh s c mt hoc m nht ca cc vt lai (trn phim t ghi phng x hoc mu pht ra) c th xc nh c cc ARNm ch xut hin trong mt hoc trong c hai loi m. Nh , phn lp c cc dng ADNc c hiu cn nghin cu.

3.4.3 Phng php i dc nhim sc th chromosome walking


Da vo kt qu phn tch cc t bin di truyn hoc cc php lai nucleotide, v tr mt gen c th c xc nh trn nhim sc th (bn nhim sc th), mc d sn phm ca gen ny cha bit. Vn t ra l cn phn lp v xc nh c chc nng ca gen m . Mt khi bit bn nhim sc th v mt on ADN nm cnh gen cn nghin cu, gen c th phn lp c t ngn hng ADN genome nh phng php "i dc nhim sc th" (Hnh 3.11). Phng php ny da vo qu trnh chn lc t ngn hng ADN genome cc dng cha cc on ADN nm gi ln nhau; phn tch dc theo chng n c gen cn nghin cu. Mc d trnh t cng nh sn phm ca gen cha bit nhng da vo cc on ADN bit phn b trn cng nhim sc th v khng xa vi gen cn nghin cu, chng ta c th tch c gen ny t ngn hng ADN genome.

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Hnh 3.11: Phng php i dc nhim sc th c s dng chn lc cc dng ADN nm gi ln nhau theo mt trt t nht nh. Cc dng ny bao ph mt vng nhim sc th cha gen cn nghin cu. bc nhanh, ngn hng genome cn cha cc dng c kch thc ln. Ch mt on ngn ADN u cc dng c dng lm u d. Cc u d ny khng ging nhau v khng cha ADN lp li.

u tin, on ADN bit c dng lm u d sng lc ngn hng ADN genome (Hnh 3.11). Mt u ca on ADN lai vi u d c ct ra v s dng lm u d th hai tip tc sng lc on ADN khc lai vi n. Kt qu thu c on ADN th hai nm gi ln on th nht. on th hai ADN c tinh sch v mt u ca n (nm v pha c gen cn tm) li c ct ra v dng lm u d tip theo sng lc ngn hng chn tip on ADN th ba nm gi ln on th hai. Qu trnh c lp i lp li nhiu ln cho n khi thu c on ADN cha gen cn tm. Vi phng php ny, chng ta c th i dc v hai pha trn nhim sc th xut pht t on ADN bit trc t ban u (on ADN dng lm u d th nht). Tuy nhin khi p dng phng php ny, cn lu n s tn ti ca cc trnh t nucleotide lp i lp li trong genome. Nu cc u d c cha on lp li th khng th tin c trn nhim sc th v u d s lai vi nhiu on ADN nm ri rc trong genome. Cc on ny u c cha trnh t lp li tng ng vi u d. Do kt qu sng lc thu c cc on ADN khng nm gi ln nhau. V vy, u d s dng i t on ny sang on khc nht thit phi l duy nht. Ngy nay k thut ny c thay th bng cc phng php khc nhanh v t cng sc hn nh PCR hoc potision cloning tm dch l tch dng theo v tr (da vo v tr trn bn ). Khi s dng k thut PCR tm ra cc on ADN nm gi ln nhau, cc cp mi c thit k da vo trnh t nucleotide ca on ADN bit ban u. Phn ng PCR c thc hin c lp vi cc vector ca ngn hng. Vector no cho sn phm PCR s cha on ADN nm gi ln on ADN bit. on ADN mi c phn lp v u cui ca n li c xc nh trnh t da vo thit k cc cp mi tip theo. Mc d kt hp vi phn ng PCR, trong thc t tin i trn nhim sc th thc hin rt chm v mt nhiu cng sc sp xp 15-20 on ADN vo mt contig (gm cc on ADN nm gi ln nhau - Hnh 3.8). Phng php tch dng theo v tr khc phc phn no nhng yu im trn. Nguyn tc ca phng php ny l xut pht t mt v tr c xc nh trn bn nhim sc th. V tr ch cch xa gen cn phn lp mt vi Mb. Nh vy

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tch dng theo v tr cn phi kt hp cc loi bn vi nhau nh bn v tr enzym gii hn, bn ch th ADN microsattelitte vv... tm ra v tr lm mc cng nh xc nh chnh xc khong cch n gen cn tm.

3.4.4 Nhy bc trn nhim sc th jumping on chromosome


Phng php i dc nhim sc th khng thch hp vi nhng on nhim sc th di hn 1000 kb. khc phc kh khn , hai u ca mt on ADN genome c kch thc rt ln c ni vi mt on ADN bit to thnh phn t dng vng khp kn (Hnh 3.12).

Hnh 3.12: Phng php nhy bc trn nhim sc th. ADN genome c ct bng enzym c t v tr nhn bit trong genome. Do thu c nhng on ADN rt ln. Phn ng ligase xy ra trong iu kin c li cho vic to phn t dng vng (nng vector>> nng ADN). Phn t ADN ti t hp mang hai on ADN nm hai u ca on ADN ln. Hai on ny rt xa nhau trong genome.

Tip theo phn t ny b ct bi enzym gii hn m on ADN bit khng cha v tr nhn bit ca enzym ny. Nh vy, hai u ca on ADN genome v on ADN bit cng nm trn mt phn t ADN mi. Phn t ny c a vo vector v hai u ca on ADN genome lc ny nm gn nhau trong cng mt vector (vi kch thc thch hp do vic trn enzym gii hn) mc d trong thc t chng rt xa nhau. Khi hai u ca mt on ADN rt ln nm trong cng mt vector, nu bit mt u ny th d dng tm c u kia m khng cn i dc nhim sc th (khng cn tm cc on nm gi ln nhau, bao ph ln c vng ADN ln). Ngn hng cha cc u ca nhng on ADN ln ct ra t genome cho php p dng k thut nhy bc, tin xa v nhanh trn nhim sc th.

3.5

Cc phng php lai

Nguyn tc ca phng php lai acid nucleic l da vo phn ng to ra phn t lai nh lin kt b sung gia hai si n ADN hoc hai si n ARN hay ADN/ARN. iu kin ca phn ng lai ph thuc ch yu vo nhit v nng mui trong m. Thng thng, si n lm u d c nh du bng phng x (P32) hoc bng cc cht pht hunh quang. Do , si ch lai vi u d c pht hin nh phim c nhy vi phng x hoc cc k thut nhum mu. Mc d gi thnh cao nhng m bo an ton cho ngi s dng nn cc cht nh du khng phng x ngy cng chim u th trong cc phng th nghim hin i.

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Phng php lai ADN vi u d c s dng ch yu phn bit mt on ADN trong s cc on khc m kch thc ca chng khng ln. V d, sau khi phn lp c mt on ADN (mt dng) t ngn hng, chng ta dng k thut lai nh v chnh xc gen phn b trn on ADN , xc nh cu trc ca gen (phn b ca exon, intron). Ngoi ra, lai ADN genome (ct bng cc enzym gii hn) vi u d c hiu ca mt gen cho php nh gi s bn copy ca gen trong genome. Php lai gia ARNm vi u d cho php xc nh kch thc phn t ARNm, nh lng s bn phin m (transcripts) v xc nh hot ng ca gen cc t chc bit ho.

3.5.1 Phng php Southern blots


Phng php lai Southern blots do Edward Southern pht minh nhm phn bit mt on ADN nht nh trong s cc on khc b ct bi enzym gii hn. C th gi on ADN cn tm l on ADN ch. Phng php ny i hi u d c hiu lai vi ADN ch. u d c th l ADN hoc ARN v c trnh t nucleotide tng ng cao vi ADN ch. u tin ADN b ct bi enzym gii hn thnh cc on c th phn ly trn gel agarose. Sau cc si kp ADN (trn gel) b bin tnh (do x l vi kim) v c chuyn sang mt mng (thng l mng nylon hoc nitrocellulosse). Bc chuyn ny c gi l k thut blotting. Blotting c th thc hin bng cch thm thu mao dn (i hi nhiu thi gian-thng tin hnh qua m) (Hnh 3.13A), hoc s dng my ht chn khng (ch ht khong vi gi). Sau mng cha ADN c x l nhit (thng 2h 80oC) hoc c chiu tia t ngoi (vi pht) c nh ADN trn mng. Mng ny c lai vi u d c hiu nh du, thng bng phng x P32 (hoc cc cht pht mu). V tr ca on ADN lai vi u d c xc nh nh phim hoc ho cht c bit (Hnh 3.13B). Phng php ny rt nhy cho php pht hin c on ADN xut hin mt ln (n bn) trong genome (~1/106) khi ch dng 5 g ADN genome.

Hnh 3.13: (A)-Blotting chuyn ADN t gel agarose sang mng bng phng php thm thu mao dn. Sau khi chuyn, mng cha ADN ging ht nh trn gel. Mng c x l nhit hoc chiu tia UV c nh ADN. (B)K thut Southern blot s dng pht hin on ADN mong mun.

Lai Southern c p dng pht hin s lng bn sao ca mt gen trong genome; pht hin thay i trt t sp xp ca cc on ADN, s c mt ca cc on ADN l, s thay

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i v tr ca cc transposon; phn bit mt gen trong h gen, xc nh cc intron, exon ca mt gen vv.... Khi s dng cc ch th phn t (marker ADN) khc nhau lm u d, chng ta c th phn bit c cc loi, cc loi ng hnh, cc c th di loi.

3.5.2 Phng php northern blots


Cc bc ca phng php ny tng t nh Southern blots nhng y ARNs c lai vi u d ADN hoc ARN. Phng php ny c p dng xc nh s lng cng nh kch thc cc phn t ARNm c trong t bo. u tin ARN tng s c tch chit khi t bo. Sau , ARN c chuyn sang mng v c lai vi u d c hiu. u d c th l ARN hoc ADNc. Kch thc cng nh s lng ARNm lai vi u d c pht hin da vo v tr ca bng lai v m nht ca n. Nh phng php ny, c th so snh s lng ARNm ca mt gen hot ng bnh thng v khi b t bin cng nh nghin cu mc hot ng ca mt gen cc t chc bit ho khc nhau hay chu cc tc ng bn ngoi. i vi nhng gen m s lng ARNm c tng hp t, trc khi tin hnh lai northern, c th lm giu ARNm bng vic tch ring chng ra khi cc loi ARN khc. Khi cho ARN tng s qua ct gn polyT, cc ARNm mang ui polyA s c gi li trn ct trong khi cc ARN khc b loi. Ra ct bng dung dch thch hp thu ARNm. Trong nhng nm u th k 21, k thut lai northern c s dng pht hin cc phn t ARNsn kch thc nh (vi chc nucleotide). Nhng phn t ny ng vai tr quan trng trong qu trnh hnh thnh cu trc d nhim sc (heterochromatin formation), trong kim sot bt hot gen (gene silencing). Tuy nhin, do kch thc rt ngn, nn cc ARNsn phi c tch khi cc loi ARN kch thc ln. Sau , ARNsn c phn ly trn gel poyacrylamide v c chuyn sang mng nh my. c bit u d sau khi nh du cng phi c phn ct thnh cc on ngn. Mi bc tip theo tng t nh vi lai Southern.

3.5.3 K thut lai in-situ


Pht trin t php lai Southern blots, k thut lai in situ s dng cc u d nh du (on nucleotide hoc khng th) lai vi ADN, ARN hoc vi protein ngay trong t bo m khng cn tch chit chng. Cc u d oligonucleotide thng c nh du bng phng php ho hc (cht pht hunh quang) thay cho vic nh du bng phng x. Chng c tng hp t cc nucleotide mang nhng lin kt ho hc c bit. Cc u d sau khi lai c pht hin nh cc protein khc nhn bit cc lin kt ho hc ny. S phn b cc phn t ARNm trong t bo c pht hin nh phn ng lai insitu. Khi nghin cu vi ARN, nhim sc th khng pH cao, do chng gi nguyn trng thi si i v khng lai c vi mi. Nh phng php ny m cc biu hin ca gen mc phin m c th quan st c trong t bo. V d, vi m phi rui gim Drosophila, cc t bo nhng v tr khc nhau trong qu trnh pht trin c th c pht hin nh da vo kiu cch hot ng ca cc gen lin quan.

3.5.4 iu kin phn ng lai


Khi thay i nhit hoc thnh phn, nng cc cht trong dung dch ca phn ng lai, tc l thay i tnh cht ch nghim ngt ca iu kin lai, kh nng lai gia cc si n ADN/ADN, ADN/ARN v ARN/ARN to thnh si kp c th thay i t mc trnh t nucleotide kh tng ng vi nhau n yu cu chng phi hon ton tuyt i to cp b

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sung. Do iu kin nghim ngt ca phn ng, c th xc nh c mt gen t bin trong s cc thnh vin ca mt h gen. Khi iu kin phn ng lai cho php lai gia cc si tng ng th c th pht hin cc gen trong mt h gen nu mt thnh vin trong mt h gen bit v c dng lm u d (Hnh 3.14).

Hnh 3.15: Thay i iu kin lai trong phn ng Southern s thu c cc kt qu khc nhau. Trong iu kin khc nghit, u d ch lai vi ng on to cp b sung chnh xc. Trong iu kin d dng hn, u d c th lai vi cc on tng ng.

Hn 3000 bnh di truyn ngi do t bin xy ra nhng gen ch tn ti mt bn sao duy nht trong genome (n bi). Nhng t bin ny thng trng thi ln v ch biu hin khi c hai allen ca gen u b t bin. V d, bnh thiu mu hng cu hnh li lim xy ra bi thay i ca mt nucleotide duy nht (GAG GTG) gen m cho chui globin. chn on thai nhi mang hai allen t bin ca gen ny (mt nhn t b, mt nhn t m), hai chui oligonucleotide (~20 base) c tng hp nhn to. Oligo th nht ng vi gen bnh thng, oligo th hai ng vi on gen mang t bin. Nh vy hai u d ny ch sai khc nhau mt nucleotide. Trong iu kin lai rt nghim ngt, cc oligo ch lai vi on nucleotide tng ng khi cc lin kt b sung chnh xc tuyt i. Do khi ADN tch ra t t bo ca bo thai (amniocentesis) lai vi u d l hai oligo trong php lai Southern, dng bnh thng v dng t bin ca gen m cho globin c th phn bit c. Thai nhi mang hai allen khim khuyt th ADN tch ra ch lai vi mt loi oligo tng ng vi on t bin. Trong qu trnh tin ho, mt gen mi c th c to ra do t hp nhiu phn khc nhau ca cc gen, hoc do thay i dn dn t mt gen ban u. Hu ht cc gen trong mt h gen c chc nng lin quan n nhau v thng phn b ri rc trong genome. Khi mt thnh vin trong h c phn lp, cc gen khc trong h c xc nh nh php lai Southern thc hin trong iu kin km cht ch (u d chnh l thnh vin bit). Lc cc si n ADN khng hon ton b sung vi u d vn lai c vi n (Hnh 3.14). Phng php lai ny c s dng rt hu ch trong nghin cu sinh hc phn t. V d, ton b cc gen m cho protein tng tc vi ADN lm nhim v iu khin hot ng ca gen trong pht trin m phi rui gim Drosophila c pht hin nh php lai iu kin khng ngt ngho. S dng cc on nucleotide ca cc gen c ngun gc t rui gim lm u d, cc h gen tng t genome ngi v chut c pht hin nh chnh k thut lai.

3.6

RFLP trong nghin cu genome v lp bn gen

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Bn genome c th c xc nh theo hai cch khc nhau: bn vt l v bn lin kt di truyn (physical map v genetic linkage map). n v o khong cch (gia cc gen) trn bn vt l c tnh bng s lng cc nucleotide. Ngc li, bn lin kt di truyn c xy dng da vo cch thc di truyn cc tnh trng. Bn lin kt di truyn da vo tn s cng di truyn ca hai hay nhiu tnh trng trong cc php lai. Cc tnh trng ny c s dng nh cc ch th (marker) di truyn. Mi mt marker l mt v tr trn bn . n v o khong cch gia 2 v tr trn bn di truyn l centiMorgan (tnh theo tn s trao i cho gia 2 v tr ). Khi c s thay i trn bn genome, nu s thay i xut hin rt t v t ngt th xem nh xy ra t bin. Khi thay i xy ra ph bin theo tin ho th c gi l a dng (polymorphism).

Hnh 3.16: Tnh a dng cc on ADN b ct bi enzym gii hn (A) Hu ht cc c th trong qun th c on nhim sc th b enzym gii hn E1 ct ti 3 v tr to hai on ADN. Mt trong hai on ny c pht hin nh phng php Southern blots s dng u d RFLP (B) Khi c s thay i ca nucleotide ti mt v tr ct, enzym khng nhn bit c v tr na, nn ch ct hai v tr kia to mt on ADN di (lc ny c xem nh mt RFLP) C) Khi xy ra t bin thm on, on ADN b ct di hn. Loi RFLP ny thng ph bin nhng vng nhim sc th cha cc on ngn nucleotide lp i lp li nh GTGTGT... S ln lp li thay i rt nhiu trong qun th. Ti mt vng nht nh, s ln lp li c trng cho tng c th. l cc minisatellite, microsatellite hoc cn gi l cc VNTR (variable number of tandem repeat) (theo Alberts & cs., 2002).

Thng thng, bn lin kt di truyn c xc nh nh biu hin ca cc t bin nh mu mt, nhm mu. Gn y, cc phng php ti t hp ADN s dng cc marker di truyn mi - cc on ADN c di khc nhau gia cc c th bnh thng, lp bn di truyn. c im ca cc on ADN ny l s sai khc gia chng khng h c biu hin tnh trng. Tuy nhin, thay i nh gia chng c th lm thay i v tr nhn bit ca enzym gii hn. V d, thay i mt nucleotide c th lm mt v tr nhn bit, do khi ct bi enzym th thu c cc on ADN di ngn khc nhau (Hnh 3.15). Tng t, s thm bt cc nucleotide vo mt vng ADN bt k c th khng gy ra thay i tnh trng nhng lm thay i kch thc ca cc on ADN b ct ra t vng . S sai khc ny c gi l tnh a dng ca cc on ADN b ct bi enzym gii hn, vit tt l RFLP (Restriction Fragment Length Polymorphism).

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Hnh 3.16: K thut Southern blots c s dng pht hin mt RFLP. S khc nhau ca mt vng trn nhim sc th s 3 di truyn t cha v m c th phn bit da vo kt qu ca php lai. RFLP c s dng rt hu hiu trong phn tch quan h huyt thng.

Khi mt RFLP rt ph bin trong qun th, s khc nhau gia hai c th c th xc nh c nh RFLP . Cc RFLP thng cha cc on ADN ngn, lp i lp li, c chiu di khc nhau do s ln lp li thay i (t 4 n 40 ln). S khc nhau v kch thc ca cc on ADN m mt c th c tha hng t cha v m c th pht hin d dng nh php lai Southern. u d lai phi to lin kt b xung vi mt chui nucleotide nm trong cc on ADN (Hnh 3.16). Khi hai marker di truyn nm trn hai nhim sc th khc nhau, xc sut mt c th nhn c hai marker ny ch chim 50% (khi hai nhim sc th cng i v mt cc trong phn bo gim nhim). Ngay vi hai marker nm hai pha ca mt nhim sc th (i din nhau qua tm ng), xc sut phn ly ca chng kh cao do xy ra trao i cho trong phn bo gim nhim. Cc marker cng gn nhau trn mt nhim sc th, xc sut chng cng c truyn cho mt c th con chu cng ln. Thng thng phn lp c gen lin quan n bnh di truyn, c th dng cc marker RFLP xc nh v tr ca gen da vo ph h ln gm nhiu ngi. Sng lc cc c th m RFLP v tnh trng uc th hin ng thi. Rt nhiu gen gy bnh ngi c tm ra bng phng php s dng RFLP, nh cc protein tng ng c phn tch r rng (Hnh 3.17).

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Hnh 3.17: S dng marker RFLP phn tch bn di truyn. Mt tnh trng c bit (bnh) ngi c di truyn sang th h sau cng vi marker RFLP. Nu i a s cc c th b bnh u c mang marker ny th gen gy bnh v marker RFLP c mi quan h lin kt nm gn nhau trn cng mt nhim sc th ( m bo tin cy, tnh cng di truyn phi c kim tra trn s lng ln c th). Cn lu n hin tng trao i cho trong phn bo gim nhim khi to t bo sinh dc (trng hoc tinh trng). Lc ny marker RFLP s phn ly khng cng nhim sc th vi gen gy bnh (trng hp s 6) (theo Alberts & cs., 2002).

Trn hnh 3.17 m t mt marker RFLP nm gn mt gen gy bnh. Khi marker nm cng gn gen th cng d pht hin c gen . pht hin mt gen bt k da vo RFLP th phi bit c v tr ca cc marker RFLP trn nhim sc th cha gen . V tr ca tt c cc marker RFLP (s lng n hng nghn) to thnh bn RFLP ca genome. Trn bn ny, khong cch gia hai marker khong 106 bp (tng ng vi ~0,1 cM). khong cch ny chng cng di truyn cho con chu vi xc sut 99%. Da vo bn RFLP, khi mt tnh trng cng c di truyn vi mt marker RFLP th v tr ca gen qui nh tnh trng c th xc nh c d dng. Nh , vic sng lc cc clone ADN tng ng t ngn hng genome v phn lp c gen lin quan n tnh trng tr nn thc thi.

3.7

Phn ng PCR (Polymerase Chain Reaction)

Mt on ADN bt k c th c nhn ln nhanh chng hng t ln m khng cn a vo t bo vi khun. iu xy ra trong phn ng tng hp in-vitro s dng ADN polymerase v cc oligonucleotide (cc mi-primers). chnh l kt qu tuyt vi ca phn ng PCR, tm dch l chui phn ng tng hp. K thut PCR c Kary Mullis tm ra gip cc nghin cu v ADN ni ring, v sinh hc phn t ni chung t c nhng thnh tu ng kinh ngc. Nh phn ng ny, mt on ADN mt vng bt k trong genome c khuch i ln rt nhiu ln khi trnh t nucleotide hai u on ADN bit. Da vo trnh t , cc cp oligonucleotide c tng hp nhn to, mi oligo to lin kt b sung vi tng si n. Chng c s dng lm mi tng hp ADN invitro nh enzym ADN polymerase. c bit phn ng PCR ch i hi mt lng ADN lm khun ban u rt nh (c 10-3 g). Mt phn ng PCR gm nhiu chu k (Hnh 3.18). Mi chu k phn ng i hi ba bc. u tin, ADN khun c x l nhit tch thnh hai si n. Bc tip theo, nhit

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gim xung cho php cc mi (s lng rt ln) lin kt to cp b sung vi hai si n ADN khun (Do s lng mi ln nn lin kt gia mi v ADN khun xy ra chim u th so vi lin kt phc hi gia hai si n). Trong bc th ba, hn hp ADN v mi c vi enzym Taq polymerase v bn loi deoxyribonucleotide triphosphate. on ADN nm gia hai mi c tng hp. Khi cc chu k c lp li, cc on ADN va c tng hp cc chu k trc tr thnh khun mu cho chu k sau. S lng phn t ADN to ra tng gp i sau mi chu k. Ch sau vi chu k u, sn phm to ra ch yu l cc on ADN c kch thc bng ng khong cch gia hai mi. S chu k lp li trong mi phn ng PCR thng l khng qu 60 v mi chu k thng ko di khong 5 pht.

Hnh 3.18: Phn ng PCR. Ba chu k u tin ca phn ng c m t trn hnh v. T hai si n ADN bin tnh, sau 3 chu k thu c 16 si n, trong 8 si c kch thc ng bng khong cch gia hai mi oligo. Sau mt s chu k, s lng si ADN c kch thc duy nht chim u th tuyt i trong phn ng.

N = (2n-2n).x n: S chu k; x: S si khun ban u ; 2n: S si c chiu di khng xc nh N: S si tng hp c trong phn ng.

3.7.1 Cc yu t nh hng n phn ng PCR


* Cc mi oligonucleotide: T l GC cc mi thng chim khong 40-75%. Khong cch gia hai mi thng khng di qu 3 kb. Cc mi khng c to cu trc bc hai do lin kt gia cc nucleotide ngay trong mt mi. di ca mi (s nucleotide) t 15 n khng qu 30. Nhng mi di hoc ngn hn nh hng n kh nng bt cp chnh xc vi ADN khun hoc nhn bn khng c hiu. ADN c tng hp khi u 3' ca mi lin kt vi khun mc d u 5' c th t do. Do u 5' ca mi c th c gn thm linker hoc on nucleotide iu khin (promoter). Nng mi khong 0,1 M n 1 M. Nng thng c xc nh da vo gi tr OD o 260 nm. * Lin kt gia mi v ADN khun: Mi gn vo si khun ADN nh lin kt to cp b sung gia chng. Lin kt ny ph thuc thi gian, nhit , nng mi cng nh nng si khun. Khi nng mi rt ln so vi nng khun, thi gian mi vi si khun khng cn thit qu lu. Nhit giai on ny ph thuc chiu di ca mi v hm lng GC. Khi oligo mi gm 12 n 15 base, nhit khong 40-45oC. Nhit 72oC thch hp cho phn ng tng hp ADN bng Taq polymerase (l ADN polymerase c hot tnh nhit cao). Lc ny cn lu cc mi ngn thng b bin tnh

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tch ra khi si khun. Ngoi ra, trong trng hp mi c tng hp xut pht t trnh t acid amin th lin kt gia mi vi ADN khun khng hon ton b sung do tnh cht thoi ho ca m di truyn. Do , mi loi ny d dng tch ra khi si khun nhit tng hp ADN 72oC. V vy trong trng hp ny, thi gian gn mi c th ko di hn so vi mi c trnh t chnh ly t on cn nhn bn v Taq polymerase c th tng hp ADN nhit gn mi. Sau mi tng nhit ln 72oC. R rng nhit v thi gian giai on gn mi c bit quan trng; nhit qu cao gy bin tnh, nhit qu thp to ra cc lin kt khng c hiu (mi c th bm vo v tr bt k c tng ng ch khng m bo chnh xc). Ngoi ra khi ADN khun chim t l rt nh (v d mt on ADN trong genome), thi gian cn thit mi tm ng v tr lin kt c hiu v to cp vi khun thng khong 2 pht trong mt s chu k u tin. iu ng lu khi ch vi nucleotide u 3' ca mi lin kt vi khun th Taq polymerase s ko di si mi thnh si ADN mi bt chp mi cha lin kt hon ton vi khun u 5'. * Nng mt s ion cng nh bn loi nucleotide dNTPs trong mi trng u nh hng n phn ng PCR. V d, ion Mg+2 kch thch phn ng tng hp ADN trong khi mun nhn c on ADN di cn loi b hon ton KCl khi mi trng. ADN khun c th l ADN genome tch ra t t bo, ADN t cc ngn hng genome hoc ADNc, cc ADN bt k; ARN tng s hoc ARNm. Nng ADN khun thng < ng vi ADNc, hoc <g vi ADN genome.

3.7.2 Mt s dng ca phn ng PCR


* PCR lng (Nested-PCR): Sn phm ADN ca phn ng PCR th nht c s dng lm khun cho phn ng PCR th hai vi khong cch (tnh bng bp) gia hai mi ln sau ngn hn ln trc. Cch tin hnh nh vy nhm lm tng tnh c hiu, chnh xc cho sn phm cn nhn bn. Cc on ADN khng c hiu c th c to ra trong ln PCR th nht nhng rt kh tr thnh khun trong ln PCR th hai do cc sn phm c hiu chim u th tuyt i. * PCR phc (Multiplex PCR): Phng php ny s dng nhiu cp mi trong mt phn ng PCR thu c cng mt lc cc bng c hiu cho tng cp. Nh gim c thi gian th nghim cng nh hn ch vic nhim mu do thao tc. V d, bnh teo c Duchenne (DMD) gy ra do thiu ht cc on ADN gen tng ng. Gen ny di khong 2000 kb gm 70 exon (kch thc trung bnh ca intron khong 35 kb). S mt on ch yu xy ra hai v tr trn gen (cc v tr nhy cm t bin). S dng cc cp mi c hiu ca tng exon trong cng mt phn ng PCR phc, c th pht hin c 80-90% bnh nhn teo c do mt on. * PCR o (Inverse-PCR): Dng phng php ny phn lp nhng phn nm trc hoc sau mt on ADN bit. T phn tch c chui nucleotide nm trc hoc sau ca mt gen. u tin ADN genome c ct bi enzym gii hn to ra cc on ADN khc nhau. Sau , cc on ADN c ni li thnh dng vng v s dng nh ADN khun cho phn ng PCR vi hai oligo xut pht t phn ADN bit. Phng php ny tng t nh phng php i trn nhim sc th. N c ng dng xc nh promoter hoc cc on nucleotide lm nhim v iu khin hot ng ca gen. * PCR ngc (Reversed-PCR): Phn ng ny nhm nghin cu s lng ARNm, lm giu cc ARNm him trong t bo. u tin ARN tng s hoc ARNm c dng lm khun tng hp ADNc nh enzym reverse transcriptase. Sau cc si n ADNc c tng hp thnh si kp nh Taq polymerase. ADNc dng si kp c s dng lm khun trong phn

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ng PCR. Cc oligo trong phn ng ny thng bt ngun t cc exon d dng phn bit cc on ADN cha intron to ra do nhim ADN genome vi cc on nhn ln t ARNm (ADNc). Ch cn 1 g ARN tng s (~106 t bo) nhn bn phn t ARNm him (1-10 copies/t bo). * PCR ti t hp: c p dng xy dng phn t ADN ti t hp, a vo hoc b i hoc thay th cc nucleotide trong mt on ADN. K thut ny c m t c th trong hnh 3.19.

Hnh 3.19: Kt hp cc phn ng PCR thay th cc nucleotide trong mt on ADN bit. Mi thit k mang cc nucleotide thay th. K thut ny cho php gy t bin ti v tr mong mun trn mt on ADN (gen).

* Nhn ngu nhin cc on ADN bng PCR (RAPD-PCR) (Random Amplified Polymorphic DNA-PCR): Da vo cc minisatellite hoc cc microsatellite (VNTRs) bit tm c cc on nucleotide nm trc v sau cc VNTR ny. Chng c dng lm mi trong phn ng PCR. Sn phm thu c l nhng bng ADN c kch thc khc nhau. Phn ng c th dng mt mi ring bit. Phng php ny hay c s dng xy dng cy phn loi.

3.8

K thut gen

Chng ta cng nhau thit k mt phn t ADN ti t hp. n gin nht khi phn t bao gm vector v mt on ADN l. Trn thc t, chng ta c th ghp hai hoc nhiu on ADN c ngun gc khc nhau (t cc loi khc nhau) hoc xut pht t cc gen khc nhau ca cng mt c th ri a chng vo cng mt vector. Bng cch chng ta c th thu c phn t ADN c chui nucleotide theo mun m khng nht thit phi tng hp chng bng con ng nhn to (Hnh 3. 20). Phn t ADN ti t hp gi vai tr c bit quan trng trong vic sn xut protein dung hp (fusion protein), bin i cu trc protein thng qua vic thay th trnh t gen, trong nghin cu cc domain, cc vng c chc nng khc nhau trn phn t protein vv...

3.8.1 Nghin cu vai tr ca ADN iu khin, chc nng ca gen hoc protein
Phin m t mt gen c kim sot bi vng ADN iu khin (promoter, enhancer ....) v ph thuc vo cc tn hiu trong tng loi t bo cc iu kin khc nhau. Cc on ADN tng cng (enhancer) c th phn b ngay trong gen (vng intron) hoc pha trc hoc sau gen hng nghn nucleotide v cng nhau phi hp hot ng. Vi mt gen bt k,

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cc on ADN iu khin c th xc nh c khi ghp on ADN nm trc hoc sau gen vi mt gen bit m cho mt protein c hiu. Gen bit c gi l gen bo co (reporter gene). Sn phm ca gen bo co c gi l protein bo co (reporter protein). Gen bo co hay c dng m cho sn phm l enzym hay cht pht mu. Do protein bo co d dng pht hin nh phn ng enzym hoc nhum t bo. Khi a phn t ADN ti t hp xy dng theo cch thc ny vo t bo, gen bo co c th ghp vo v tr bt k. Nu v tr nm sau mt promoter th chng ta c th xc nh c thi gian, mc hot ng cng nh cc c ch kim sot ca promoter thng qua hot ng ca gen bo co, tc l thng qua thi gian v mc biu hin ca protein bo co.

Hnh 3.20: Ni cc on ADN vi nhau bng phng php s dng ui 5'. Trong phn ng PCR khi u 3' bm vo ADN khun, Taq polymerase tng hp si ADN mi ngay khi u 5' cha to cp vi ADN khun. S dng tnh cht ny, mt chui oligonucleotide c cha v tr ct ca enzym gii hn E c thm vo u 5' ca mi, do mi sn phm PCR to ra u c v tr nhn bit ca enzym E. Ct sn phm PCR vi enzym ny to u dnh. Cc sn phm PCR c ni vi nhau nh AND ligase. Phng php ny c s dng a cc on ADN vo vector.

Khi cc phn t ARNm ca mt gen bit c tng hp rt t trong t bo, vic tch v tinh sch chng rt kh khn. khc phc nhc im , phn t ADN ti t hp c xy dng gm gen bit ni vi promoter virus. Cc promoter ny thng hot ng mnh. Promoter ny c nhn bit bi ARN polymerase ca virus. Phn t ADN ti t hp c vi enzym v 4 loi ribonucleoside triphosphate tng hp invitro cc phn t ARNm vi s lng mong mun. Nhng promoter bit oc dng nghin cu chc nng ca gen ghp vi n c gi chung l promoter bo co (reporter promoter). nghin cu chc nng, cu trc protein tng ng vi mt gen bit, phng php dng vector biu hin (expression vector) hay cn gi l phng php biu hin gen c p dng thu c s lng ln ARNm v protein trong t bo sng (Hnh 3.21). Vector ny mang promoter hot ng mnh nm gn v tr ghp gen. Khi a vector vo t bo sng (t bo vi khun, virus, nm men, t bo ng, thc vt), ARNm tng ng vi gen trong vector cng nh protein c tng hp vi s lng ln. trnh hin tng protein l gy nh hng ti s pht trin ca t bo, trong thc t thng s dng cc t bo nhy cm nhit . Phng php ny rt c hiu qu trong vic sn xut vaccine hoc bt k protein no vi s lng ln.

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Hnh 3.21: S lng ln protein c to ra nh tch dng (cloning) on ADN cha m di truyn vo vector hot ng. Vector ny mang promoter hot ng mnh, do s lng ARNm v protein tng ng c tng hp nhiu.

Protein thng c mt on peptide phn b phn u (NH2 hoc COOH) lm nhim v c nh protein v tr thch hp trong t bo hoc m bo bn vng ca n sau khi tng hp. nghin cu vai tr ca on ny, n thng c ni vi protein bo co b ct b phn tng ng. y l phn t protein hp nht hay cn gi l protein dung hp. K thut ti t hp ADN cho php xy dng phn t ADN gm nhng on nucleotide khc nhau ca mt gen ghp vi gen bo co. T sn phm protein dung hp cho php xc nh chc nng ca cc on quan trng trong chui polypeptide c lin quan n vn chuyn, c nh hoc lin quan n hot tnh ca protein.

Hnh 3.22: Hai k thut khc nhau cho php phn tch cu trc v chc nng ca protein X (A) Theo di chc nng mi xut hin do ghp protein bo co vi mt on ca protein X cn nghin cu (B) Nhng thay i do t bin c th so snh c gia protein bnh thng v protein t bin nh quan st "epitope" gn vo chng.

Khng phi mi phn quan trng ca mt protein u c th ghp c vi protein bo co. V d, vn chuyn protein t Golgi n lisosome, cc on peptide lin quan n tn hiu vn chuyn ca protein ny nm xa nhau trn phn t protein v phi c gp li theo

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mt cu trc c bit khin chng tr nn gn nhau. nghin cu vai tr ca cc on ny, k thut ch epitope "epitope tagging" c s dng. Ton b protein cn nghin cu c ghp vi mt on peptide ngn 8-12 acid amin. on ny pht hin d dng nh khng th. V tr, chc nng cng nh s di chuyn trong t bo ca protein lai c theo di nh khng th, do mi nh hng do thay th bt k acid amin no trong protein u c th xc nh c (Hnh 3.22).

3.8.2 Thay th hoc gy t bin gen


Genome trong t bo vi khun v mt s eukaryot bc thp thng tn ti dng n bi. Thc nghim cho thy i vi nhng c th ny, c th thay th mt gen bnh thng (ang hot ng) bng gen t bin nh hin tng trao i cho tng ng. Hin tng ny xy ra vi tn s khng qu nh (Hnh 3.23A).

Hnh 3.23: Thay th hoc b sung gen vo genome (A) i vi vi khun hoc cc c th n bo eukaryot bc thp, gen t bin thay th gen bnh thng nh trao i cho gia hai chui nucleotide tng ng (B) c th eukaryot bc cao, gen t bin thng c ghp vo nhim sc th, do genome mang c gen t bin v gen bnh thng.

Thng thng, cc gen t bin c chn sao cho sn phm ca chng nhy cm vi nhit , tc l gen ch hot ng mt nhit thch hp. Khi chuyn sang nhit cao hn hoc thp hn, chng s b c ch hon ton. Loi t bo nhy cm vi nhit c s dng rt rng ri trong nghin cu chc nng, vai tr ca protein. i vi sinh vt eukaryot bc cao nh ng vt c v, genome thng tn ti dng lng bi (cc cp nhim sc th tng ng). Qu trnh thay th gen xy ra rt him vi nhng l do cha c lm sng t. Tuy nhin khi a mt gen t bin vo t bo, gen ny thng c ghp vo mt v tr bt k trong genome. Do bn cnh gen bnh thng, mt bn sao ca gen cng tn ti trong genome nhng dng t bin (Hnh 3.23B). Thc nghim hay s dng phng php to si ARNm ngc ngha (ARNm-antisense) gy km hm hon ton hot ng ca gen bnh thng. Chng ta bit rng khi phin m, si kp ADN m xon, mt trong hai si n c dng lm khun tng hp ARNm. Nu gen a vo t bo c chn sao cho n c trnh t nucleotide chnh xc nh si n b sung vi si khun th vic sao chp thc hin trn gen s to ra nhng phn t ARN c kh nng lai vi phn t ARNm ca gen bnh thng. Do qu trnh tng hp protein b c ch hon ton (Hnh 3.24).

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Hnh 3.24: Bt hot gen bng phn t ARNm-antisense Phn t ARNm-antisense c trnh t nucleotide b sung vi phn t ARNm bnh thng (ARNm sense) Hai phn t ny lin kt to phn t dng kp, ngn cn qu trnh tng hp protein trn phn t ARNm sense.

Ngoi ra c th a cc phn t ADN hoc ARN ngn c tng hp nhn to (bng phng php ho hc hoc enzym) vo t bo sng to phn t lai vi gen hoc vi ARNm ca gen cn nghin cu. Do , qu trnh phin m hoc tng hp protein trn nhng ARNm ny b km hm hon ton. Tuy nhin trong thc t, vic lai gia ARNm-antisense v ARNm-sense khng phi lun lun thc hin c d dng. khc phc iu , cc nghin cu thng c tin hnh trn protein. Trong t bo, protein thng c hot tnh khi dng phc cht do lin kt gia hai hoc nhiu chui polypeptide. Nu nh protein gi vai tr quan trng trong hot ng sng ca t bo, vic gy mt hot tnh ca n s dn n t bo b cht. Do c th quan st c tnh trng th cc gen mang t bin ch hot ng trong nhng iu kin nht nh. V d, s dng cc t bo nhy cm nhit , khi chuyn chng sang nhit khng thch hp cc gen t bin mi hot ng (cng vi gen bnh thng) to phn t protein khng c hot tnh. Nh vai tr, chc nng ca protein c xc nh.

3.8.3 Gy mt hoc tng cng chc nng ca gen


Mt trong nhng phng php iu bin hot ng ca gen thng thng nht l p dng hin tng trao i cho gia hai trnh t tng ng gy bt hot. y l nguyn tc ca k thut loss of function (tm dch l gy mt chc nng). Chng ta bit y v chnh xc trnh t nucleotide ca gen. Do , ch cn thit k on ADN l c hai u tn cng l hai chui nucleotide ca gen. a on ADN vo t bo. Trao i cho xy ra gia hai u on ADN l v gen c mt trong genome khin cho trnh t nguyn vn ca gen b gin on bi ADN l. Lc gen b t bin nn khng th xy ra phn ng tng hp protein (Hnh 3.25). Thiu ht sn phm ca gen thng dn n s xut hin tnh trng mi.

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Hnh 3.25: Gy t bin bt hot gen bng trao i cho tng ng. Vector chuyn ch on ADN l c hai trnh t nucleotide ca gen bit. Trao i cho xy ra gia gen nguyn vn trong genome vi cc trnh t tng ng trn on ADN l. Lc gen b gin on bi ADN l v b bt hot.

K thut trn thc s n gin i vi c th n bo (nh vi sinh vt hoc nm men). Tuy nhin vi c th a bo, chng ta khng mong mun ch mt t bo n l m ton b cc t bo trong c th u mang gen t bin. tho mn yu cu , vector chuyn ch gen t bin c th c vi tim vo nhn ca trng va th tinh trc khi hai nhn n bi (ca tinh trng v trng) cha kt hp thnh nhn lng bi. Sau , trng th tinh c a tr li d con. Ngay trong giai on t bo trng bt u phn ct, ADN l c ghp vo nhim sc th ca genome lng bi theo c ch trao i cho tng ng. Mt t l rt nh cc t bo u tin ca phi c cha gen t bin. Cc t bo c th bit ho thnh dng t bo sinh dc. Ngoi ra c th tin hnh k thut gy t bin gen trc tip trn t bo mm phi (Embryonic Stem cell - ES cell). T bo ES mang gen t bin c cy tr li phi giai on sm. Cc t bo mm cha bit ho nn chng c tim nng pht trin thnh bt c loi t bo no trong qu trinh sinh trng pht trin, tc l chng c kh nng bit ho thnh dng t bo sinh dc. Nh vy, ng vt pht trin t phi b cy ES cha n tim nng mang dng t bo sinh dc c gen t bin. Nhng th nghim to ng vt chuyn gen thng c tin hnh trn chut nht. Chut sinh ra t phi cy ES hoc t trng b vi tim c gi l th h Go. Chng c s pha trn gia cc t bo bnh thng v cc t bo chuyn gen. Th h Go giao phi vi nhau s sinh ra chut thun chng (th h G1) i vi gen t bin. iu ny c ngha tt c t bo trong c th u mang gen bt hot. Chng c gi chung l chut knockout (tm dch l chut b nh bt gen). Tuy nhin, chut knock-out ch c ngha c bit quan trng khi nghin cu cc gen nu b bt hot cng khng gy cht.

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Hnh 3.26: K thut thay th gen theo hai bc u tin gen ch th (chng chu khng sinh) c ghp vo nhim sc th nh trao i cho tng ng. Chn lc cc t bo sng trong mi trng c khng sinh. Gen t bin hoc gen c promoter mnh thay th cho gen ch th nh trao i cho gia trnh t tng ng Sau cng l chn cc t bo nhy cm vi khng sinh cy vo m phi (theo Brown, 2000)

t bin lm mt chc nng ca gen da vo trao i cho gia cc trnh t tng ng, thng l trnh t vng cha m di truyn. Tuy nhin, c th tc ng n hot ng ca gen theo chiu hng ngc li, tc l tng mc biu hin ca gen. K thut ny c gi l gain of function (tm dch l tng cng chc nng) lin quan ch yu n vng ADN iu khin (promoter). Lc ny, vng ADN cha m di truyn (cn bo ton nguyn vn) c lp ghp vi promoter mnh v sau c a vo vector di dng nhiu bn sao. Vector c a vo t bo ES v chn lc sao cho mi t bo ES c th c ti 40-200 copy ca gen cn nghin cu. Hot ng qu mc bnh thng ca nhng bn sao ny thng gy ri lon cc qu trnh sinh hc trong t bo nn dn ti s biu hin tnh trng mi. Xc sut vector (ADN l) ghp vo genome nh trao i cho gia hai trnh t nucleotide tng ng thng rt nh. V vy chn lc c nhng t bo b mt hoc c tng cng chc nng, gen ch th (thng l nhng gen qui nh tnh chng chu khng sinh) c a vo genome cng vi gen nghin cu. C th tin hnh k thut chuyn gen theo hai bc (Hnh 3.26). Bc u tin l chuyn gen ch th v chn lc cc t bo c kh nng sng trong mi trng c khng sinh. Tip n l chuyn gen t bin hoc ghp gen

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vi promoter mnh. Lc ny cn chn lc cc t bo nhy cm vi khng sinh do gen nghin cu thay th cho gen ch th.

3.8.4 Gen bo co reporter gene


K thut lm mt hoc tng cng chc nng cho php nghin cu vai tr ca mt gen khi ch bit trnh t nucleotide. Ngoi ra, c th cn c vo thi im promoter c hot ho xc nh s hot ng theo thi gian v khng gian ca gen. T bit c tnh c hiu ca gen trong tng giai on sinh trng v bit ho t bo. Mt promoter c xem l hot ng khi xut hin cc phn t ARNm hoc sn phm protein tng ng vi vng m di truyn nm sau promoter. Do , promoter cn nghin cu thng c ghp vi vng cha m di truyn cho protein c kh nng pht hunh quang hoc tham gia phn ng to mu. Nh vy thi im cng nh v tr xut hin protein trong cc t chc m c th pht hin c mt cch d dng. Vng ADN tng ng vi m di truyn ca protein bit c gi l gen bo co. y, reporter gene c dch l gen bo co trnh s nhm ln vi khi nim gen chn lc hoc gen ch th (marker gene). Mt s gen bo co thng gp nh cc gen m cho -galactosidase (lacZ), -glucuronidase (uidA), luciferase (lux), Green Fluorescent Protein (GFP). Ngoi ra, c th ghp mt phn ca gen bo co vi vng cha m di truyn cho mt protein no (to nn protein ti t hp) nghin cu s phn b cng nh vai tr ca protein ny n hot ng sng trong t bo. Sn phm ca cc gen bo co nh lacZ hoc uidA ch quan st thy khi s l t bo hoc m chuyn gen vi mt s ho cht. Do , cc t bo u b cht. Tuy nhin, protein GFP s pht mu khi chiu nh sng xanh hoc tia t ngoi yu m khng cn bt c s h tr no khc. Do , c th pht hin t bo mang gen m cho GFP mt cch d dng m khng gy tn thng chng. Cn c vo thi im, v tr xut hin GFP cng nh cng pht sng c th theo di s tng hp, phn b v di chuyn ca protein ti t hp trong t bo hoc m sng. Bng cch gy t bin tng nucleotide ca gen m cho GFP, k thut ADN ti t hp to ra cc bin th khc nhau ca protein GFP pht ra cc mu khc nhau, vi cng mnh gp hng chc ln so vi cc dng GFP tn ti trong t nhin.

3.8.5 Bin i genome thc vt


Khi thc vt b tn thng, cc t bo bit ho bc vo phn chia lm tng s lng t bo. iu c bit l d bit ho, nhng t bo thc vt vn c kh nng phn chia to ra cc loi t bo khc nhau. Thm ch mt s loi cy, t bo bit ho vn c kh nng pht trin thnh cy hon chnh cho cc giao t. Tnh cht c bit ny ca t bo thc vt c ng dng ti to cy hon chnh t cc t bo nui cy. Khi nui cy nhng phn nh ca m thc vt trong mi trng y dinh dng v cc cht iu ho sinh trng, rt nhiu t bo b kch thch phn chia lin tc mt cch v t chc to ra s lng ln cc t bo khng bit ho gi l m so (callus). Khi iu chnh nng cc cht trong mi trng thch hp, ngn v r cy xut hin t callus. i vi nhiu loi thc vt, khi tch cc t bo callus ri nhau v nui cy trong mi trng v trng, t mt t bo ring l c th pht trin thnh cy con hon chnh. Bng cc k thut hin i, ADN l c a vo genome t bo thc vt. Nui cy cc t bo s nhn c cy chuyn gen hon chnh. Thc t thng s dng Agrobacteria nh l vector chuyn ch gen vo genome thc vt (Hnh 3.27).

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Hnh 3.27: S dng plasmid ti t hp vn chuyn gen vo genome t bo thc vt. on T-ADN ca plasmid Ti c bin i, b bt nhng gen nm gia hai u tri v phi, ng thi nhn thm gen cn chuyn. Khi Argobacterium xm nhim t bo thc vt, on T-ADN mang gen l c chuyn vo genome thc vt.

u vit ca thc vt chuyn gen to ra nhng bc tin nhanh trong chn ging, kt hp cc tnh trng di truyn c li ca cc loi khc nhau trong mt c th. Thc vt chuyn gen p ng nhu cu x hi v nng sut v hiu qu. Tuy nhin, tc ng ca thc vt chuyn gen i vi cn bng sinh thi, mi trng ang cn l vn tranh ci.

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Chng 4 TNG HP V VN CHUYN PROTEIN


Genome cha mi thng tin di truyn v lu tr cc chng trnh cn thit t bo sinh trng, duy tr v pht trin. Protein chim hn mt na trng lng kh ca t bo. S tn ti, i mi ca chng gi vai tr quyt nh nht duy tr s sng cng nh m bo sinh trng v pht trin ca c th. Ton b protein trong t bo c m bi cc gen thuc genome c gi chung l proteome. Trong mt t bo ng vt, s protein khc nhau dao ng trong khong 10.000-20.000 loi, tng ng vi chng 10 t (1010) phn t. S gen m cho proteome ch tng ng vi phn ADN mang m di truyn, do ch i din cho mt phn m khng phi ton b genome. Hn na, t mt gen c th c nhiu sn phm protein. Hot ng ca mt gen c nhn nhn nh mt qu trnh gm hai giai on: phin m v dch m. Trong cc chng trc, chng ta xt n cc bc chun b c hon thin trong nhn t bo v cc c ch kim sot giai on th nht to ra sn phm ARNm. Cc phn t ARNm c vn chuyn ra ngoi t bo cht dch m tng hp proteome. Qu trnh ny c thc hin bi b my Ribosome vi s tham gia ca ARNt. Sau khi c tng hp, protein b bin i trong qu trnh vn chuyn n nhng v tr khc nhau tu thuc vo chc nng ca chng. V d, cc enzym c vn chuyn v lysome, protein iu ho hot ng gen c a vo nhn vv... C th xem hon thin cu trc bc I (trt t acid amin) l bc cui cng lin quan n hot ng ca mt gen. Tuy nhin, hot tnh ca protein hon ton ph thuc vo cu trc khng gian bc hai, bc ba v bc bn. Cu trc bc hai lin quan n cu hnh khng gian cc b tng phn ca chui peptide. Cu trc bc ba phn nh cu hnh khng gian ba chiu ca ton b chui v cu trc bc bn c trng cho cu hnh khng gian khi cc chui polypeptide tng tc vi nhau to ra protein c hot tnh. Phn t protein c th chuyn t cu hnh khng gian ny sang cu hnh khng gian kia gn lin vi cc hot tnh khc nhau ca chng.
Bng 4.1: Cc phn t cu thnh nn Ribosome prokaryot v eukaryot Prokaryot Eukaryot Ribosome S lng S lng ARNr ARNr protein protein Tiu n v nh 16S 21 18S 33 Tiu n v ln 5S, 23S 31 5S; 5.8S; 28S 49

Qu trnh tng hp protein i hi s c mt ca 3 loi ARN. Ngoi s a dng ca ARNm, 3 n 5 loi phn t ARNr (ARN ribosome) khc nhau kt hp vi protein to thnh cu trc Ribosome (Bng 4.1). T bo c khong 35-60 phn t ARNt (ARN vn chuyn) gi chc nng adaptor chuyn i m di truyn trn ARNm sang acid amin. Mi acid amin c m bi ba nucleotide (m b ba). Mi m b ba trn ARNm c c bi i m (anticodon) nm trn phn t ARNt. Khi hai m ny khp nhau, ribosome chuyn mt acid amin t ARNt sang si peptide. Vic gn chnh xc acid amin vo ARNt c thc hin nh cc enzym aminoacyl-tRNA synthetase. C th c nhiu ribosome ng thi dch m trn mt si ARNm to nn cu trc polyribosome.

4.1

Vai tr ca ARN vn chuyn (ARNt) trong tng hp protein

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Phn t ARNt c kch thc nh (70 n 90 nucleotide), lm nhim v vn chuyn acid amin tng ng vi m di truyn trn si ARNm. Mt s phn t ARNt c cng kh nng vn chuyn mt acid amin. V vy, chuyn ch 21 acid amin, trong t bo prokaryot v eukaryot c 35 v 60 phn t ARNt khc nhau. Acid amin th 21 l selenocysteine. y l acid amin him gp, c cu trc tng t nh cysteine nhng selenium thay th cho sulfur. Hu ht cc acid amin ca protein c s bin i (thm, bt, thay th cc nhm chc...) sau khi chui polypeptide tng hp. Tuy nhin, selenocysteine c to ra nh bin i xy ra serine, sau c nhn bit bi ARNt c hiu ghp vo chui peptide. M di truyn ca selenocysteine l UGA, mc d y cng l m dng tng hp i vi hu ht cc protein. Cc phn t ARNm m cho cc protein c cha selenocysteine thng c cu trc khng gian c bit khin cho b my tng hp protein nhn bit m UGA khng phi l m dng. Phn t ARNt c phin m bi ARN polymerase I v c cha intron. iu ng lu , intron ca ARNt c kh nng t ct (intron thuc nhm I). Chc nng ca mt ARNt ph thuc rt nhiu vo cu trc khng gian ba chiu - tc l ph thuc vo tng tc to cp gia cc nucleotide b sung, hay thm ch khng b sung, to nn s gp khc chnh xc ca phn t. a s cc phn t ARNt thng c cu trc gm cc thy hnh "l nhp" v cc nucleotide thng b bin i (gn thm cc nhm chc) sau khi c phin m t gen. Chnh nhng bin i ny gip cho s hnh thnh lin kt gia cc nucleotide khng b sung c trng cho ARNt.

Hnh 4.1: Qu trnh gn acid amin vo ARNt thng qua 2 bc Th nht: acid amin c hot ho to phc trung gian acid aminAMP. Phc ny gi tng tc vi enzym chuyn sang bc gn acid amin vo ARNt C hai bc ny c xc tc bi enzym aminoacyl-tRNA synthetase.

Mi ARNt c hai v tr c hiu: pha u 5' c trnh t "anticodon" l v tr tng tc vi m b ba trn phn t ARNm, cn u 3 l v tr lin kt vi nhm carboxyl (COOH) ca acid amin tng ng vi m b ba . Lin kt ny c thc hin qua 2 bc nh xc tc ca enzym aminoacyl-ARNt synthetase (Hnh 4.1). Bc th nht l hot ho acid amin, bc tip theo l gn n vo ARNt c hiu cho acid amin . Phn t ARNt c th chn ng acid amin c hiu m n cn vn chuyn trong s cc acid amin khc cng tn ti trong t bo cht? l nh hot tnh ca aminoacyl-ARNt synthetase. Mt acid amin s tng ng vi mt enzym ring bit. Enzym xc tc cho phn ng hot ho acid amin bng vic s dng nng lng ca ATP to ra phc acid amin~AMP. Phc ny gn vi enzym cho n khi tm c ARNt c hiu. Enzym s chuyn acid amin t phc sang u 3' ca ARNt. Phn t ARNt mang acid amin i vo ribosome, i m trn ARNt tung tc vi m trn ARNm v acid amin c chuyn sang chui peptide. Bng con ng ho hc, c th thay

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th acid amin ng bng acid amin sai trong phc acid amin-ARNt. V d, cysteyl-ARNtCys (ARNt mang cysteine trng thi hot ho) c th thay th thnh alanyl-ARNtCys. Trong phn ng tng hp protein in vitro, alanine (acid amin sai) c chuyn t phc alanyl-ARNtCys vo chui peptide, mc d i m trn ARNtCys tng ng vi m b ba cho cysteine trn ARNm. iu cho thy tnh chnh xc ca qu trnh tng hp protein ph thuc vo cc phn t ARNt. Mc d khng tn ti m thoi ho cho methyonine nhng c hai loi ARNtMet. Trong t bo vi khun hoc trong cc bo quan nh ty th, lc lp, acid amin u tin ca chui polypeptide lun lun l methyonine b formyl ho ti nhm amin. Nh vy, initiator RNAtfMet nhn bit methyonine b formyl ho v a n vo v tr u tin ca chui peptide. Phn t RNAtMet bnh thng nhn bit cc m AUG cho methyonine nm trong chui. Tuy nhin, trong t bo eukaryot, methionine u tin khng b formyl ho. Do , hai loi ARNtMet ny ch khc nhau cu trc khng gian.

4.2

Tng hp protein b my Ribosome

Ribosome bao gm ARNr tng tc vi cc protein (Bng 4.1). Mt s protein ribosome gi vai tr to cu trc khung cho b my tng hp protein. Chnh cc ARNr quyt nh hot tnh ca cc phn ng xy ra trong ribosome, cn protein trong cu trc ribosome c tnh cht ph tr, thc y cc phn ng . Ribosome mang cc v tr tng tc c bit: ba v tr cho ARNt v mt v tr cho ARNm. V tr P (peptidyl-RNAt-binding site) lin kt vi mt phn t ARNt m phn t ny c gn vi chui polypeptide ang c tng hp. V tr A (amynoacyl-RNAt-binding site) l ni tng tc ca phn t ARNt th hai va mang mt acid amin vo. V tr E (exit site) l ni ARNt lm xong nhim v, chun b c gii phng khi ribosome. Cc ARNt ny ch c gi cht cc v tr P hoc A mt khi anticodon ca chng to cp b sung vi cc codon trn phn t ARNm. Qu trnh tng hp protein ribosome c chia lm 3 giai on: bt u tng hp chui peptide, ko di v kt thc chui peptide. u tin, tiu phn ribosome nh bm vo ARNm vi s h tr ca cc yu t khi ng (initiation factors-IFs). vi khun, tng tc gia tiu phn nh 30S ribosome/ARNm ph thuc vo s to cp b sung gia trnh t nucleotide gn u 3 ca 16S ARNr v trnh t phn b pha trc m b ba ca acid amin u tin (trnh t Shine-Dalgarno) trn ARNm. u 5' ca phn t ARNm c th mang nhiu m codon ca methyonine nm cnh nhau. Tuy nhin ch c mt trong s s l m bt u chui peptide. Chnh mt vi nucleotide nm ngay sau m ny s quyt nh s la chn chnh xc. on Shine-Dalgarno (UAAGGAGG) thung nm trc m u tin ng vi Methyonine khong 7 n 13 nucleotide. Trong t bo eukaryot, nh vo cu trc m 7mG u 5' ca phn t ARNm m tiu n v ribosome nh tm c phn t ARNm. Mt s protein lin kt vi m ny, tip n cc yu t khi ng lin kt vi nhng protein to thnh phc vi ARNm. Thng qua phc ny m tiu phn ribosome nh 40S tng tc vi ARNm v bt u d tm m AUG ca methyonine. Cc yu t khi ng s tch ra khi tiu phn nh tm thy m AUG v tiu phn nh sau s hp nht vi tiu phn ln ribosome to ra b my tng hp hon chnh. Phn t ARNt c bit (initiator tRNA) mang acid amin u tin (methyonine) l phn t duy nht c kh nng gn vo v tr P m khng i qua v tr A. Nh vy mi chui polypeptide u c bt u bng methyonine. Lc ny, m b ba cho acid amin th hai trn phn t ARNm nm vo v tr A sn sng lin kt vi i m trn ARNt mang acid amin th hai. Chui peptide c ko di (Hnh 4.2).

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Giai on ko di chui polypeptide v c bn l ging nhau t bo prokaryot v eukaryot. Giai on ny i hi s tham gia ca cc yu t ko di chui peptide (elongation factors - EFs). C th xem giai on ko di gm nhiu chu k lp li, mi chu k c ba bc c bn nh sau:

Hnh 4.2: Qu trnh tng hp protein. u tin ARNt mang Methyonine vo v tr P trn tiu n v nh Ribosome. Sau , tiu n v nh tng tc vi v dch chuyn trn si ARNm n v tr m AUG u tin. Tiu n v ln ribosome s to phc vi tiu n v nh to thnh b my ribosome hon chnh tng hp chui peptide.

Bc 1: Phc aminoacyl-ARNt tng tc vi v tr A. Ba nucleotide ca anticodon (trn ARNt) to cp b sung vi ba nucleotide ca codon trn phn t ARNm. Bc 2: u carboxyl ca chui polypeptide ang gn vi phn t ARNt v tr P c tch ra v chuyn sang to cu ni peptide vi acid amin lin kt vi phn t ARNt v tr A. y l phn ng ch cht c xc tc bi enzym peptidyl transferase vi s tham gia ca cc ARNr thuc tiu n v ln ca ribosome. Lc ny ARNt v tr P c gii phng ra dng t do, khng lin kt vi acid amin. Phn t ARNt ny c chuyn sang v tr E sau gii phng ra khi ribosome. Bc 3: Phc ARNt-chui polypeptide v tr A c chuyn sang v tr P. Ribosome dch chuyn i chnh xc mt codon dc theo phn t ARNm. Bc ny i hi s tham gia ca yu t EF-G c hot tnh thy phn GTP. Phn ng thm 1 acid amin vo chui peptide ch ht 0,05 giy E.coli. Do , chui peptide gm 300 acid amin c tng hp trong khong 15 giy. Qu trnh tng hp chui peptide kt thc khi mt trong 3 m dng (UAA, UAG, UGA) trn phn t ARNm c mt v tr A. Cc m ny c nhn bit bi cc yu t dng tng

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hp (RFs-release factor). Khi nhn bit m dng, cc RFs c mt v tr A s thay i hot tnh ca peptydyl transferase khin enzym thm phn t H2O vo u COOH ca chui peptide. Chui peptide tch khi phn t ARNt v tr P; si ARNm ri khi ribosome; hai tiu phn ribosome tch ri nhau. Mt si ARNm c th c dch m ng thi bi nhiu ribosome. V d, trn si ARNm m cho peptide fibroin (protein trong sa c trng lng phn t 200 KDa) c ti 50-80 ribosome ng thi dch m. Qu trnh tng hp protein tiu tn nng lng hn bt c phn ng sinh ho no xy ra trong t bo. t nht c bn phn t cao nng b phn hy to ra mt cu ni peptide: hai phn t dng gn acid amin vo ARNt, phn t th ba dng trong bc th nht v phn t th t dng trong bc th ba. Mt khc mt phn t ARNm c th mang thng tin cho ba khung c khc nhau (tng ng vi ba chui polypeptide khc nhau), cho nn vic chn chnh xc khung c khi bt u phn ng tng hp protein rt quan trng.

4.3

Vn chuyn protein

Phn ln protein t bo vi khun khng b bin i sau khi c tng hp. Chng thc hin ngay chc nng ca mnh bn trong hoc c tit ra bn ngoi t bo. T bo tit protein ra ngoi nhm p ng yu cu v cht dinh dng, truyn tin bo v hoc duy tr vt liu to cu trc bn ngoi mng t bo. Nhng protein c chc nng, hot tnh bn ngoi t bo c gi chung l protein tit. Cch thc a protein tit ra ngoi mng t bo c gi l cc con ng tit. Tu thuc cu trc mng m protein c vn chuyn qua mng theo nhng cch thc khc nhau. V d, t bo vi khun Gram dng, protein tit c vn chuyn vt qua mng t bo cht ra ngoi. Tuy nhin, t bo vi khun gram m, protein tit phi i qua mng trong, lp phn cch gia hai mng v cui cng i qua mng ngoi. Khng ging nh protein vi khun, hu ht protein ca t bo eukaryot sau khi c tng hp cn tri qua nhng bin i nh glycosyl ho, acetyl ho, gn thm cc nhm phospho, gc sulfat... trn con ng vn chuyn n ch. Nhng bin i ny c thc hin ch yu mng li ni cht v b my Golgi. Hu ht cc protein i vo mng li ni cht ER (Endoplasmic Reticulum) ngay khi chng ang c tng hp. V th trn b mt ca ER lun c nhiu ribosome bm vo khin cho b mt ER tr nn sn si khng trn. Do nhng vng ER c ribosome bm vo c gi l ER ht, cn vng khng c ribosome l ER trn. Vng ER trn cn c gi l vng ER chuyn tip (transitional ER) v y l ni hnh thnh cc ti vn chuyn protein v lipid i t ER n Golgi. Trong mt s loi t bo nh t bo gan, vng ER trn chim phn ln mng li ni cht tp trung cc enzym tham gia chuyn ho, tng hp lipoprotein v cc enzym xc tc phn ng kh c cc cht ho tan trong lipid. Chng ta cn lu rng ribosome tn ti t do trong t bo cht vn tham gia tng hp protein. Khng c s khc nhau gia cc ribosome t do v ribosome bm trn ER. Mi ribosome u bt u tng hp mt chui peptide bt k trong t bo cht. Chnh tn hiu vn chuyn n ER nm u NH2 chui peptide s a ribosome n b mt ER. Do nhiu ribosome cng tham gia tng hp peptide trn mt si ARNm nn si lun gn vi b mt ER. T mng li ER, cc protein i n Golgi v tip tc c a n cc v tr khc nhau nh cc bo quan hoc tit ra khi t bo (Hnh 4.3).

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Hnh 4.3: Cc con ng vn chuyn protein t ER n cc v tr ch khc nhau v t b mt t bo n mng li ni cht ER i qua cc bc trung gian ti ni bo sm v ti ni bo mun.

Trn ng vn chuyn, protein chu nhng bin i v cu trc khng gian v thnh phn ho hc tu thuc vo ch cn n. Cn ch rng cc protein c ch phn b trong nhn hoc ty th, lp th s c tng hp bi ribosome t do trong t bo cht. Nhng protein c tng hp trn vn v c cu trc khng gian hon chnh ngoi t bo cht, sau mi c vn chuyn n ch.

4.3.1 Vn chuyn vo mng li ni cht


Con ng vn chuyn protein t mng li ni cht ER sang Golgi v i v cc ch khc nhau (cc bo quan trong hoc bn ngoi t bo) c gi l con ng tit (secretory pathway). Bt chp v tr phn b cui cng khc nhau, cc protein i vo ER nh tn hiu dn nm u NH2. Tn hiu ny c gi l tn hiu bt u vn chuyn (start-transfer signal). Phn ng tng hp chui peptide c bt u nh cc ribosome t do trong t bo cht. Tuy nhin, khi u NH2 ca si peptide c cha tn hiu vn chuyn ra khi ribosome, tn hiu c nhn bit bi phc SRP (Signal Recognition Particle). Lin kt gia tn hiu v SRP lm dng tm thi phn ng tng hp protein, gip ribosome c thi gian n b mt ER. Chui peptide nm tip sau tn hiu vn chuyn c a vo khoang trong ca ER. Tn hiu vn chuyn l on peptide khong 30 acid amin trong c 6 n 12 acid amin k nc thng nm cnh nhau. on k nc gi vai tr quan trng i vi tng tc gia tn hiu bt u vn chuyn v th cm tng ng nm trn mng ER. Nu gy t bin loi b vi acid amin trong on k nc hoc thay th chng bng cc acid amin khc nhm gy bin i in tch th protein khng c vn chuyn vo trong ER. Vai tr ca tn hiu vn chuyn c minh chng bng th nghim tng hp protein in vitro vi s c mt ca microsome. Microsome l nhng phn ER to ra do ER b t gy khi t bo b nghin ng th. Cc phn ER nh c kh nng to thnh cc tiu phn dng trn (ng knh 100-200 nm). Khi khng c microsome, phn t protein tng hp invitro c on peptide mang tn hiu vn chuyn. Nu cho thm protease vo mi trng th protein b phn hy. Tuy nhin khi b sung microsome vo mi trng trc khi protein c bt u tng hp th bn trong microsome xut hin phn t protein khng cn mang tn hiu vn chuyn. Do , protein khng b phn hy nu b sung thm protease. Nh vy, tn hiu vn chuyn gip protein i qua mng ER vo khoang trong. Tn hiu ny b ct khi protein v b phn hy thnh cc acid amin bi peptidase v protease c trong khoang ER (Hnh 4.4).

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Hnh 4.4: Ribosome bm vo mng li ni cht (ER) nh th cm SRP nm trn mng Th cm tng tc vi phc SRP. Phc SRP gm 6 chui peptide v mt phn t ARN (c kh nng to cp vi ARNr). SRP c hai v tr lin kt vi tn hiu dn v Ribosome. SRP tn ti trong t bo cht lin kt vi on peptide (u NH2) do Ribosome tng hp. Sau khi SRP to phc vi Ribosome v protein, SRP tng tc vi th cm ca n trn mng ER (theo Alberts & cs., 2002).

K thut ADN ti t hp thit k cc phn t protein mang u NH2 bt k nhng m bo di v tnh k nc th chng u c th i qua mng ER vo khoang bn trong. Thc nghim xc nh c qu trnh vn chuyn vo khoang ER c bt u khi on peptide ra khi ribosome c chiu di khong 70 acid amin. Nh vy, qu trnh tng hp v vn chuyn vi hu ht cc protein c chiu di ln hn 100 acid amin s xy ra mt cch ng thi. u NH2 c a vo trong mng li ni cht ngay khi si peptide cha c tng hp xong. Phc SPR nhn bit mt cch c hiu tn hiu vn chuyn. Phc ny gm c 6 protein khc nhau v mt phn t ARN (~300 nucleotide) c trnh t b sung vi ARNr. Khi tn hiu vn chuyn xut hin v chui peptide ang tng hp c di nht nh th SPR tng tc vi tn hiu, vi ribosome v vi th th nm trn mng ER. Tng tc gip ribosome dng nh b dnh st trn b mt ER. Th th ca SRP c gi l "docking protein" gm hai chui polypeptide (nm trn mng ER) v (nm xuyn qua mng ER). Th nghim tng hp protein in vitro cho thy khi khng c microsome th phc SRP s gy ngng qu trnh tng hp protein khi u NH2 ra khi ribosome. Nh vy, SRP khng ch lm nhim v mang vc chui peptide ang c ribosome tng hp n b mt ER m cn c chc nng ngn cn qu trnh tng hp protein hon chnh nu nh thiu vng ER. Nh vy, phc SRP c ba domain qui nh ba chc nng khc nhau: tng tc vi tn hiu trn protein, tng tc vi docking protein v tng tc vi ribosome lm dng tm thi qu trnh tng hp protein. Ribosome tip tc tng hp protein sau khi SRP lin kt vi th th ca ring n trn mng ER. Sau , SRP tch khi ribosome v chui peptide c chuyn sang phc protein nm trong cu trc mng ER. Phc ny c gi l translocator gm 3-4 tiu phn khc nhau; mi tiu phn gm 3 protein nm xuyn qua mng ER. Thc cht translocator l phc to cu trc knh cho php polypeptide i xuyn qua mng. Bnh thng khi khng c chui polypeptide cn a vo ER th translocator s ng. Translocator nhn bit tn hiu vn chuyn trn chui peptide v s m ra cho php chui polypeptide c a dn vo trong khoang ER. Trong sut qu trnh a chui peptide vo ER, ribosome nm st translocator bt kn sao cho khng c bt c thnh phn no bn trong khoang ER, c bit l cc cation Ca+2, lt ra ngoi. Protein tham gia phc translocator phn b trn mng ER c xc nh

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bng th nghim tng hp protein in vitro (Hnh 4.5). Th nghim c tin hnh vi phn t ARNm c mang m di truyn cho lysin phn b on gia ca ARNm. M ny s c c bi phn t ARNt c bit lm nhim v vn chuyn lysin c gn thm cht nh du. Do , chui peptide c tng hp c mang lysin nh du. Khi chui c vn chuyn i qua translocator, cht nh du c kh nng tng tc vi mng ER nn chui peptide khng vo c trong khoang ER m b gi li translocator (do cht nh du tng tc vi mng ti v tr chui peptide i qua). Nh xc nh c cc protein tham gia cu trc phc translocator. Mt trong cc protein ca translocator l Sec61p. Protein ny ng knh khi khng c protein i qua. Khi c mt ribosome, Sec61p tng tc vi tiu phn ln ca ribosome, do si peptide c i qua trong khi ribosome bt cht knh khng cho cc phn t khc t do i qua.

Hnh 4.5: Cu trc ca translocon to knh dn cho php chui polypeptide c vn chuyn qua mng ER vo trong khoang khi ang c ribosome tng hp (theo Lodish v cs., 2000).

Khng phi mi protein vn chuyn vo ER trong khi ang c tng hp. Mt s protein, v d nh cc protein c khi lng phn t nh, c tng hp trong t bo cht. Sau , chng c a vo ER thng qua translocator. vi khun, protein SeA ATPase tham gia cu trc ca translocator. Protein ny phn b mt ngoi ER, tip xc vi t bo cht. Mi khi mt phn t ATP b phn hy, mt phn ca protein SecA s y si peptide va c tng hp vo l. i vi mt s protein kch thc nh, chng thng c tng hp trn vn bi cc ribosome phn b t do trong t bo cht. Khi , cc protein chaperon tn ti sn trong t bo cht s lin kt vi on peptide ra khi ribosome. Nh chui peptide khng c cu trc gp khc v c vn chuyn vo trong khoang ER. Nh vy, cc protein c cu trc khng gian khng th i qua translocator vo khoang trong ER. Vn chuyn phn t protein ang tng hp vo khoang ER gip protein khng trng thi t do trong t bo cht. Phi chng y l mt c ch bo m an ton cho t bo cht, ngn cn khng cho cc protein tit hoc cc enzym tn ti dng t do trong t bo cht, trnh ph hy cc thnh phn ni bo. Vi mt s t bo c bit lm nhim v tit hydrolase, t bo cht cha cc cht c ch hydrolase bo v t bo khng b phn hu bi cc enzym ny.

4.3.2 Vn chuyn protein cu trc mng (membrane proteins)

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Cc protein tham gia hnh thnh cu trc mng cht nguyn sinh hay mng cc bo quan c tng hp v vn chuyn ng thi vo ER. Tuy nhin, chng khng c gii phng vo khoang trong ER m vn gi li trn mng ER. Chiu phn cc ca nhng protein ny trn mng ER, tc l v tr cc u NH3+ v COO- trong khoang ER hay ngoi t bo cht, ging ht nh s phn cc ti v tr trn mng ch (mng ch c th l mng nguyn sinh cht hay mng ca cc bo quan). Trong qu trnh vn chuyn t ER n v tr ch, tnh phn cc ca protein mng khng thay i. Nh vy, s phn cc ca protein thuc cu trc mng c hnh thnh ngay khi chng ang c tng hp trn ER. Chng ta hy xt trng hp n gin nht l s vn chuyn ca protein mng m chng ch nm xuyn qua mng mt ln. Trong trng hp ny, mt s protein, v d nh glycophorin trn mng t bo hng cu hay th th ca insulin, c phn u COO- nm trong t bo cht cn phn u NH3+ nm pha ngoi mng. Tuy nhin, mt s protein khc, v d nh th th ca transferrin, phn cc ngc li; chng c u COO- nm pha ngoi mng cn phn u NH3+ nm pha trong t bo cht. i vi trng hp th nht, chui peptide c hai tn hiu c bit: tn hiu vn chuyn qua ER nh m t phn trn v tn hiu th hai gm khong 20-25 acid amin k nc to nn cu trc xon . Trong qu trnh chui peptide va ang tng hp va i vo khoang ER, tn hiu ER b ct khi chui peptide, gii phng u NH3+ vo trong khoang ER. Chui pepitde tip tc c a vo khoang cho n khi tn hiu th hai i qua translocator th b gi li. Do on pepitde nm tip sau tn hiu th hai (cha u COO-) khng c a vo khoang na. Tn hiu th hai b gi translocator cho n khi tng hp chui peptide kt thc. u COO- ca chui nm bn ngoi t bo cht. Sau , tn hiu th hai s di chuyn ra khi translocator (y l phc protein) v nm xen vo lp phospholipid ca mng ER. Lc ny chui peptide c c nh ti mng nh tn hiu th hai xuyn qua lp phospholipid. Tn hiu th hai ny c gi l tn hiu dng vn chuyn (stop-transfer signal). Tuy nhin n cn c chc nng gn gi chui peptide xuyn qua mng (membrane-anchor signal) (Hnh 4.6).

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Hnh 4.6: Vn chuyn protein mng Cc protein thuc cu trc mng c gi li mng ER nh tn hiu dng nm sau tn hiu vn chuyn (A), hoc nh mt tn hiu m nhn ng thi hai chc nng vn chuyn v dng vn chuyn (B v C). u NH3+ b a vo trong khoang ER (B) hay phn b pha ngoi t bo cht (C) ph thuc vo s phn cc ca cc acid amin nm pha trc v sau tn hiu.

i vi protein mng c u COO- nm bn ngoi mng v u NH3+ nm pha trong t bo cht, v d nh th th ca transferrin, chng c vn chuyn v c nh ti mng khi ang c tng hp. Tuy nhin, nhng protein ny ch c mt tn hiu, tn hiu ny ng thi m nhim c hai chc nng: chc nng vn chuyn ER v chc nng c nh ti ER. iu cn lu lc ny tn hiu khng phi l on peptide nm ngay pha u NH3+. Do , chui peptide nm pha trc tn hiu c tng hp t do trong t bo cht. Khi on tn hiu c tng hp i ra khi ribosome, n c nhn bit bi phc SPR c hiu v chui peptide c a n st mng ER nh m t mc 4.3.1. Tn hiu i vo translocator v on peptide nm pha sau tn hiu i vo trong khoang ER. Tn hiu c gi li ti translocator cho n khi tng hp chui peptide kt thc, phn u COO- c gii phng vo trong khoang. Sau , tn hiu s di chuyn ra khi phc protein ca translocator v xuyn vo lp phospholipid ca mng. Lc ny, chui peptide nm xuyn qua mng c u NH3+ nm ngoi t bo cht v u COO- phn b pha trong mng. Khi chui peptide l protein cu trc mng ca bo quan khc v ch xuyn qua mng mt ln th chnh on acid amin phn b qua mng c th m nhn ng thi cc chc nng vn chuyn, dng v gi chui trn mng ER. Gi s chui ny c u NH3+ phn b bn ngoi bo quan (tc l u NH3+ nm trong t bo cht) th u COO- s b a vo trong khoang ER v ti cha peptide ny hnh thnh t mng ER sao cho u NH3+ s nm pha ngoi ti tit. Sau , ti tit s i n b my Golgi v cui cng protein mng c a n v tr ch. Ti tit ch protein tit s tip hp vi mng bo quan v m bo gi ng chiu phn cc bn trong hay bn ngoi ca hai u NH3+ v COO-. Nu chui peptide l protein cu trc mng nguyn sinh cht v c u NH3+ phn b bn ngoi mng, th u NH3+ ca chui ny b a vo khoang trong ER. Sau , ti tit c hnh thnh c u NH3+ nm pha trong ti. R rng, s phn b ca cc u NH3+ v COO- trn mng ER c qui nh ph hp vi s phn b v tr ch cui cng (Hnh 4.6). Mt s protein thuc cu trc mng, v d nh protein to cu trc bm ion, knh vn chuyn ion, c th nm xuyn qua mng nhiu ln. Phn u NH3+ ca nhng protein ny thng cha hai tn hiu vn chuyn (start transfer signal) v tn hiu dng vn chuyn (stop transfer signal) ni tip nhau. Tip theo tn hiu dng u tin l cc tn hiu gi (membraneanchor signal) v tn hiu dng khc nm k tip nhau. S tn hiu tng ng vi s ln xuyn qua mng ca protein. Nhng tn hiu ny u l nhng on peptide c cu trc xon v k nc. i vi nhng protein c v tr phn b bn trong khoang ER, chng c gi li sau khi c a vo ER nh tn hiu c bit gm 4 acid amin KDEL (Lys-Asp-Glu-Leu). Sau khi c tng hp v vn chuyn vo khoang, chng c gi li nh tn hiu ny. Tn hiu c gi l tn hiu s hu "retention signal"; tn hiu ny thng nm u carboxyl ca phn t protein. Tn hiu KDEL c nhn bit bi th cm nm trn mng ER. i khi, chng c tm thy trn nang vn chuyn lm nhim v gi li cc protein mang tn hiu KDEL. C th xy ra trng hp v mt l do no m cc protein ny b ng gi nhm

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vo nang thay cho vic li trong khoang ER. Lc t bo sa cha sai st bng cch bt gi cc protein c tn hiu KDEL v a chng quay tr li ER. Mt trong nhng protein quan trng c gi li trong khoang ER l disulfide isomerase (PDI) c chc nng xc tc phn ng oxi ho nhm SH ca acid amin cystein to cu disulfide S-S. Hu ht cc cystein on protein phn b pha ngoi mng t bo u c lin kt cu disulfide. Tuy nhin, phn protein phn b trong t bo cht khng c cu disulfide. Bn cnh PDI, protein chaperon BiP cng c gi li trong khoang ER. Ging nh cc chaperon khc, BiP nhn bit nhng protein gp khc sai hoc nhng tiu phn t do ca mt phc protein. Nhng tiu phn ny v mt l do no cha c to thnh phc multimere. BiP s lin kt vi nhng protein sai hng v mt cu trc ny gi chng li trong ER. Ngoi ra, BiP cn c hot tnh phn hy ATP gip cho cc protein c th gp khc khi chng c vn chuyn vo ER.

4.4

Bin i sau dch m v kim tra cht lng protein trong khoang ER

Vo trong khoang ER, cc protein c gp khc to cu trc khng gian ring bit v chnh xc ca tng protein. Qu trnh ny lin quan n vic to cc cu ni disunfide, gp khc chui polypeptide v lp rp cc monomer vi nhau to phn t protein multimer. Ngoi ra, mt s proprotein c th cn c phn ct thnh cc chui nh ngay trong khoang ER. Tuy nhin qu trnh ny khng ph bin ER m xy ra ch yu trong Golgi. Mi phn t protein khng gp khc hoc c sai st trong vic gp khc hay b sai hng khi lp rp to multimer u b gi li trong ER. Thm ch v l do no m chng vn c chuyn t ER n Golgi th chng li b pht hin ti Golgi v a tr v ER. Cc protein sai hng s b y ra khi ER thng qua knh translocon v b phn hy trong t bo cht. Nhng protein b y ra ngoi t bo cht thng c gn vi ubiquinin v sau b phn hy bi protease. Cc protease thng phn b ngay pha ngoi mng ER sn sng thc hin nhim v ca mnh.

4.4.1 To cu lin kt disulfide (S-S) v cun gp trong khoang ER


Cc cu ni disunfide c vai tr quyt nh trong vic thit lp cu trc bc ba v bc bn ca phn t protein. c bit lin kt S-S gia hai cysteine gi vai tr quan trng m bo cu trc thch hp cng nh hot tnh ca cc enzym v hocmon. Do , bn trong khoang ER, phn ng to cu disunfide c thc hin trc khi hon thin cu trc khng gian cho protein. Cu disunfide c th c hnh thnh trn chui peptide ang vn chuyn vo khoang ER. Ngc li, cc protein c tng hp t do trong t bo cht hoc trn vng ER trn thng khng c cu disunfide. Chng phi ph thuc vo nhiu mi tng tc vi cc protein khc thit lp cu trc khng gian ca mnh. Enzym disulfide isomerase (PDI) phn b trong khoang ER v xc tc cho phn ng oxy ho nhm SH to cu disunfide gia hai cystein ca cng mt chui peptide hoc gia cc chui khc nhau. Mt protein c th c nhiu cu disulfide. V d, chui nh immunoglobin Ig c hai cu S-S. Khi chui ny ang c tng hp v vn chuyn vo khoang ER, cu S-S c thit lp cho hai cystein nm gn u NH2 ngay khi cystein th ba cha c gn vo chui Ig. iu m bo cho s to cu S-S chnh xc. Mt s cu disulfide c hnh thnh mt cch ngu nhin khin cho cu trc khng gian ca protein b sai lch. Trong trng hp ny, PDI s chnh sa li nhng cu S-S khng ng. Bn cnh phn ng to cu ni S-S, chui peptide c gp cun theo mt trt t c th ring ca peptide . Chnh cu S-S gip cho vic gp khc c chnh xc. Mt s protein khc ca ER nh cc enzym

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peptidyl-prolyl isomerase thc y s quay quanh cu lin kt peptidyl-prolyl ca on peptide cha gp khc, gip chng gp khc d dng hn.

4.4.2 Hnh thnh cu trc multimer t cc chui peptide


Cc protein c cu trc multimer c hnh thnh ngay trong khoang ER. Nghin cu s bin i cu hnh ca protein Hemagglutinin HA (protein cu trc v virus gy bnh cm) t dng monomer sang trimer xy ra bn trong ER ca t bo ch nhim virus l v d in hnh cho qu trnh hnh thnh cu trc khng gian thch hp i vi tng protein. Bng vic s dng cc khng th khc nhau, mi loi ch c kh nng tng tc c hiu vi HA dng monomer hoc dng trimer, ngi ta c th nhn bit c cu trc ca tng phn t HA trong khoang ER. Kt qu th nghim cho thy phn t HA chuyn t dng monomer sang trimer trong khong thi gian 7 pht. Ngay sau , cc cu disulfide trn mi si c to thnh v ba si tng tc vi nhau to phn t HA dng trimer hon chnh. Dng ny c vn chuyn tip n Golgi. Khi chui peptide mang cc t bin mt, thm hoc thay th acid amin, mi dng monomer cng nh trimer c cu trc khng thch hp u b gi li trong ER. Cc protein vo trong khoang cn c sa cha cu trc khng gian sai lch hoc cu trc polymer khng thch hp nh cc chaperone (BiP) v mt s enzym phn b trong khoang ER. Tng t nh trng hp HA, kt qu nghin cu protein 1-antiprotease do t bo gan tit vo mu nhm c ch hot tnh enzym elastase cho thy, khi 1-antiprotease c glutamate v tr 342 b thay th bng lysine th chui peptide ny khng c cu hnh thch hp. Do , chng b gi li trong ER m khng c tip tc vn chuyn tit ra ngoi. Lc t bo gan b trng phng ln bi mng li ER ca n cha y protein 1-antiprotease dng cu trc sai lch. S tch ly cc protein c cu trc khng ng s hot ho gen m cho nhng protein lm nhim v sa cha sai hng nh chaperon, peptidyl-prolyl isomerase v disunfide isomerase... Mt khi t bo gan khng cung cp 1-antiprotease, elastase vn c hot tnh. Enym ny s phn hy mng li hp th oxy phi. V vy, bnh nhn mc bnh di truyn Caucasian vi cc triu chng kh th, t chc phi b thoi ha bi elastase. Bnh di truyn ny do t bin lm cho 1-antiprotease khng c cu trc thch hp c tit ra ngoi. Sau khi c cu trc khng gian hon thin, protein c ng gi trong cc ti vn chuyn n Golgi. Mt s protein c th c gn thm cc nhm ng trc khi b a tip n Golgi. Tuy nhin phn ng ng ho tip tc xy ra Golgi.

4.4.3 Qu trnh ng ho protein


Sau khi vo trong ER, cc phn t protein b ng ho d dng phn loi trong qu trnh vn chuyn n tng ch. Chui oligosaccharide lin kt vi nhm amin ca acid amin asparagine thng qua N-acetylglucosamine (GlcNAc). Do lin kt vi nhm amin, chui oligo c gi l N-oligosaccharide. Chui N-oligosaccharide gm 14 gc ng (Glc)3(Man)9(GlcNAc)2, trong Glc-Glucose; Man-Mannose; GlcNAc-Gluco-N acetyl glucosamine. Acid amin Asparagine b ng ho thng c v tr Asparagine-XSerine/Threonine, trong X l acid amin bt k. Ba acid amin Asn-X-Ser/Thr v tr ng ho c gi l sequon. Khng phi mi sequon u c gn oligosaccharide. Do , cc acid amin bn cnh sequon hoc bn thn cu trc khng gian ca protein c lin quan n qu trnh ng ho ny. Ngoi ra, chui oligosaccharide c th lin kt vi nhm hydroxyl

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ca acid amin serine hoc threonine thng qua N-acetylgalactosamine (GalNac). Chui ny cng c th lin kt vi nhm hydroxyl ca acid amin hydroxylysine thng qua galactose. Do lin kt vi nhm hydroxyl nn chui ny c gi l O-oligosaccharide. Tuy nhin, rt t protein b ng ho nhm hydroxyl v phn ng ny, nu c, thng xy ra Golgi. Chui N-oligosaccharide gm 14 nhm ng c gi bn trong khoang ER nh lin kt vi dolichol. Khi phn t protein i vo khoang ER, c chui ng c chuyn sang protein. Phn ng c thc hin nh enzym oligosaccharyl transferase gn pha trong mng ER. Enzym chuyn oligosaccharide (Glc)3(Man)9(GlcNAc)2 sang chui peptide ngay khi ang c vn chuyn vo khoang. Tuy nhin ngay sau , glycoprotein li chu bin i tip. Chng b ct bt i ba gc ng (Glc)3 bi 3 enzym khc nhau. Lc oligosaccharide trn chui polypeptide ch cn li (Man)8(GlcNAc)2. Dng nh ba gc ng ny c vai tr nh du nhng chui N-oligo c hon chnh. Sau khi loi b 3 gc ng, phn t protein c chuyn sang cis-Golgi.

4.5

Vn chuyn t mng li ni cht n Golgi v Lysosome

Sau khi hon thin cu trc khng gian thch hp v tr thnh glycoprotein nh gn oligosaccharide vo asparagine sequon, cc protein trong khoang mng li ni cht ER s c vn chuyn n Golgi nh cc nang ti c ngun gc t mt s vng c bit trn mng ER. trong Golgi, chui oligosaccharide tip tc chu nhng bin i ty thuc vo tng loi protein. Ngoi ra, phn ng ng ho c th xy ra vi cc acid amin serine, threonine. C hai qu trnh ng ho v kh ng xy ra ER v Golgi ca t bo eukaryot m khng c trong t bo prokaryot. Rt c th s c mt ca oligosaccharide gip cho cc protein trnh c tc dng ca cc protease trong khi s kh cc ng khc nhau chun b cho protein c vn chuyn c chn lc v cc v tr ca ring mnh. T Golgi, protein c vn chuyn chn lc n cc ch khc nhau trong t bo nh mt s bo quan, mng t bo hoc c tit ra ngoi. V d, vn chuyn cc enzym v lysosome, cc enzym ny phi c phosphoryl ho ti phn t ng Mannose trong chui oligosaccharide. Nh , chng c "nh du" mannose 6 phosphate (M6P). V vy, cc enzym c phn bit vi cc protein khc v c nhn bit bi th th c hiu nm trn mng trans ca Golgi. Th th tng tc vi nhm M6P v "ng gi" cc enzym vo trong nang ti. Nang ny tch ra khi mng pha trans-Golgi ni c pH=7 m bo duy tr c tng tc gia th th vi M6P. Tip n nang cha enzym hnh thnh t trans-Golgi c dung hp vi nang mun (late endosome) c pH khong 5,5. trong nang mun, cc enzym tch khi th th, nhm phosphate b kh khi ng mannose nhm ngn cn th th tng tc tr li vi enzym. Cc enzym c t do trong nang mun v dung hp vo lysosome, gii phng cc enzym vo bn trong (ni c pH=5). S thiu vng mt vi enzym trong lysosome l nguyn nhn gy nn mt s bnh di truyn. Gen m cho cc enzym ny hon ton khng b t bin. Tuy nhin, enzym khng c mt lysosome l do thiu st trong qu trnh sng lc nhn din chng vn chuyn v lysosome. V d, nu khng c phosphoryl ho M6P th cc enzym s khng c a v lysosome mc d chng c mt trong Golgi.

4.6

Vn chuyn t Golgi n b mt t bo: Con ng tit ngoi bo (exocytosis)

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Trong t bo vi khun, cc protein phn b trn mng hoc cn phi tit ra ngoi thng c tng hp di dng preprotein vi tn hiu vn chuyn nm u NH2. Tn hiu ny c gi l "leader peptide", thung khng qu 25 acid amin. C rt nhiu phn t protein chaperone bm trn chui polypeptide ang c tng hp. Chaperon lm nhim v gi cho protein khng b gp khc ng thi tham gia vo vn chuyn, tit protein ra ngoi t bo. Leader peptide b ct khi protein khi n c vn chuyn n ch. i vi t bo eukaryot, cc th th, cc protein cu trc mng t bo, cc proteoglycan, glycoprotein cng nh mt s protein khc lm nhim v truyn tn hiu gia cc t bo c vn chuyn theo con ng tit ngoi bo (exocytosis). C hai con ng vn chuyn hay cn gi l hai con ng tit protein khc nhau: Vn chuyn lin tc (tit lin tc): ch yu xy ra vi cc protein cu trc. Vn chuyn chn lc: thng xy ra vi cc protein ho tan, mt s hormon, cc cht dn truyn thn kinh hoc cc enzym. Thng thng, cc protein ny ch c tng hp hoc gii phng ra khi cc nang tit khi t bo c yu cu.

i vi cc t bo bit ho lm nhim v tit, cc cht thng c tng hp v tch tr trong cc nang tit (secretory vesicle). l nhng nang c bit c bao ph lp clathrin pha ngoi. Cc protein cha trong nang tit c gi l protein tit. Nng protein tit trong nang rt m c (c th tng gp 200 ln so vi nng trong Golgi). Nh t bo c th tit ra mt s lng ln ngay khi c th yu cu. Mt nang tit chn lc c th cha nhiu loi protein tit vi nng khc nhau. Cc nang tit s dung hp vi mng t bo v nh cc cht tit ra ngoi. Bn cnh nang ph clathrin lm nhim v vn chuyn protein t trans-Golgi hoc t mng t bo n nang mun, thc nghim cn pht hin c t nht hai loi nang ph khc lm nhim v chuyn ch protein gia cc bo quan. Nang ph COPI vn chuyn protein gia cc np gp ca th Golgi theo hng ngc chiu t trans v cis v t cis-Golgi quay tr li mng li ni cht ER. Nang ph COPII vn chuyn protein t ER i cis -Golgi. Khi va tng hp, a s protein tit u dng khng c hot tnh do chng mang cc on peptide khng cn thit cc u COOH v NH2 (nn gi l cc pre-protein) hoc do chng c tng hp di dng polyprotein (gi chung l cc pro-protein), sau c phn ct to ra cc phn t c hot tnh. Chng ch c hot tnh khi c mt trong nang tit hay thm ch ch khi tit ra khi t bo. iu ny to iu kin thun li cho vn chuyn cc protein c kch thc rt ngn. dng polyprotein (cc on peptide ging ht nhau c lp i lp li lin tc) chng c nhn bit v ng gi d dng hn vo cc nang tit. Mt khc y c l cng l bin php bo m an ton cho t bo trnh tc dng ca cc enzym. Chng ch c hot tnh khi c ct gi trong nang hoc c tit ra khi t bo.

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Chng 5 TRUYN TN HIU T BO


c th a bo, cc tn hiu c truyn i gia cc t bo cng nh ngay trong mt t bo. Tn hiu truyn gia cc t bo cho php chng cng phi hp hot ng, gi cn bng cho c th thnh mt khi thng nht. Cc tn hiu gip tng t bo xc nh c v tr cng nh chc nng chuyn bit ca chng. iu m bo t bo ch bc vo phn chia khi cc t bo bn cnh cn n. V bt c l do g khin cho s phi hp hot ng gia chng b sai lch, t bo s phn chia khng tun theo s kim sot chung v iu c th dn n xut hin ung th git cht c th. Cc k thut hin i cho php nghin cu qu trnh truyn tn hiu trong mt t bo hoc gia chng vi nhau. Mi t bo eukaryot u c mt h thng truyn tn hiu phc tp nhng cc k hon ho, bao gm cc protein lm nhim v truyn v tr li tn hiu. c bit quan trng l cc th th phn b trn b mt mng t bo v bn trong t bo (trong t bo cht v nhn), cc protein kinase, cc phosphatase, protein Gs vv... t bo ng vt, tn hiu gi i di nhiu dng khc nhau nh cc phn t protein, on nh peptide, cc acid amin, cc nucleotide, cc dn xut ca axit bo, cc steroid, thm ch cc cht kh nh oxit nit, cc monoxit cacbon... Tn hiu ban u kch hot mt lot cc phn ng truyn tin ni tip hoc xy ra ng thi vi nhau. Mi tn hiu u tin c gi chung l ligand. Cc th th nhn bit ligand rt nhy cm, chng tng tc vi ligand nhng nng rt nh (thng < 10-8 M). Ngay sau khi tip nhn tn hiu, mt lot cc phn ng truyn tn hiu xy ra dn n thay i hot ng ca t bo nhm tr li tn hiu. Trong mt s trung hp khi th th nm bn trong t bo, cc ligand phi nh v c tnh k nc mi c kh nng khuch tn vt qua mng vo t bo cht v tng tc vi th th. Trong mi tng tc th th-ligand, vai tr ch yu ca ligand l lm thay i cu hnh ca th th thng qua lin kt. S thay i cu hnh ny ging nh tn hiu thng bo cho t bo cn c phn ng i vi ligand. Trong c th a bo, mt t bo bt k lun tip xc vi hng trm tn hiu khc nhau. Cc tn hiu ny c th l cht ho tan, hoc bm vo cht nn hoc bm trn b mt cc t bo ln cn. Chng c th kt hp vi nhau cng hot ng iu bin phn ng tr li tn hiu. Do mi t bo u c mt chng trnh ring bit chn lc cc tn hiu v phn ng p li chng. Cc t bo khc nhau s tr li khc nhau i vi cng mt tn hiu. T bo thng phi tr li cho tp hp ca nhiu ch khng phi tng tn hiu ring l. Cu tr li l kt qu ca mt chui cc phn ng lm thay i hot ng ca t bo; quyt nh t bo sng hay cht, bc vo phn chia hay tn ti trng thi tnh. c bit c nhng protein trong h thng truyn tn hiu c kh nng phn tch v x l tn hiu dn n nhng hiu ng sinh hc thch ng gip t bo gi c trng thi cn bng. iu ng lu l cc th th nhn bit hormon rt kh xc nh v tinh sch v nng ca chng rt nh trong t bo. S lng th th c hiu cho mt hormon thung dao ng t 10.000 n 20.000 phn t trn b mt t bo, tc l ch chim khong 10-6 tng s protein c trong t bo hoc c 10-4 tng s protein trn b mt mng. Trong thc t, s xut hin ca th th v hm lng ca chng c pht hin v xc nh nh th nghim chc nng da vo tnh cht lin kt c hiu gia chng vi cc hormon gn phng x hoc cht pht mu. Cc hormon ny vn gi c hot tnh tuy c nh du bng cc cch khc nhau.

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Cc th th hormon nm trn b mt thng c xc nh bng phng php nh du i lc "affinity labeling". Hormon gn phng x bm vo th th c x l bng cc cht ho hc to lin kt cng ho tr gia hormon v th th ca n. Nh , lin kt ny bn vng ngay khi c mt cc cht kh hoc cc cht gy bin tnh c s dng tch th th ra khi b mt t bo. Rt nhiu th th phn b trn b mt c tch ra khi mng t bo m vn gi c hot tnh nh sc k nh du i lc. Tuy nhin, c nhng trng hp khi s lng th th qu nh th khng th tinh sch bng phng php ny. K thut tch dng v ti t hp ADN cho php khc phc nhc im trn. Hnh 5.1 minh ha phng php tch dng biu hin (expression cloning), cho php thu c s lng ln th th ca hormon m khng cn phi tinh sch chng t protein tng s.

Hnh 5.1: Phng php tch dng biu hin xc nh cc ADNc him, m cho th th b mt t bo. ADNc a vo vector biu hin mang promoter hot ng mnh. Cc vector ti t hp c a vo dng t bo t bin khng tng hp c th th. Th th ch xut hin trn b mt t bo no nhn vector cha ADNc m cho th th v c kh nng lin kt vi ligand.

Trong phng php ny, hormon (ligand) ch bm vo t bo mang ADNc m cho th th. Hormon thng c nh du pht hunh quang, nh pht hin d dng. Trong c th, hu ht hormon l nhng phn t nh, c th ho tan trong lipid hoc trong nc. Mt s t hormon ho tan trong lipit c th th nm ngoi mng t bo. Rt nhiu hormon ho tan trong lipid c th th nm trong t bo cht. Chng phi khuch tn qua mng nguyn sinh cht tng tc vi th th. Ngc li, hu ht hormon ho tan trong nc c th th phn b trn mt ngoi ca t bo do hormon khng c th khuch tn qua mng t bo. Cc ligand do t bo tit ra c dn truyn i theo nhiu cch khc nhau. Tn hiu c th ch truyn n cc t bo nm ngay trong mt khu vc cng vi t bo gi tn hiu. Qu trnh ny c gi l paracrine (Hnh 5.2A). c im ca cch truyn ny l tn hiu khng c khuch tn i xa. Chng b phn hy bi cc enzym nm ngoi t bo hoc b gi li trong cht nn gia cc t bo.

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Hnh 5.2: Hai con ng truyn tn hiu thng qua cc phn t tit (A)- Truyn tn hiu paracrine (B)- Truyn tn hiu thng qua h mch dn. S khc nhau gia chng th hin tc truyn cng nh tnh c hiu ca t bo nhn.

Trong c th a bo, phi hp nhp nhng hot ng gia cc t bo nm cch xa nhau, c nhng t bo chuyn ho gi vai tr c bit trong vic truyn tn hiu i ton c th. l cc t bo thn kinh (cc nron) vi nhng axon. Khi tip nhn tn hiu t mi trng hoc t cc t bo thn kinh khc, nron gi cc xung in dc theo si axon n xi np nm tn cng si axon . Xi np nhn xung in v tit ra cc tn hiu ho hc c bit gi l cc cht dn truyn thn kinh (neurotransmiter). Cht ny c gi n t bo nhn rt nhanh v c hiu. Cch thc truyn tn hiu qua cc t bo thn kinh c cp k trong gio trnh thn kinh. Bn cnh t bo thn kinh, cn c cc t bo c bit lm nhim v gi cc tn hiu i kim sot hot ng ca c th, m bo s phi hp cn bng nhp nhng cho mt thc th thng nht. l cc t bo ni tit. Chng tit ra hormon gi vo mch mu ( ng vt) hoc vo nha cy ( thc vt), nh tn hiu c truyn i khp c th (Hnh 5.2B). Tuy nhin tn hiu do t bo tit gi vo h mch c truyn i chm hn so vi t bo thn kinh. Ngc li, cc hormon tit li c tc dng ngay khi nng ca chng rt thp (<108 M) trong khi cc cht dn truyn thn kinh c hot tnh nng khong 10-4 M. 5.1 Th th trn b mt t bo

C nhiu loi th th khc nhau. Trong chng ny chng ta ch n cc th th protein nm trn b mt t bo v cch thc chng tham gia chui cc phn ng truyn tn hiu. Hu ht ligand tng tc vi th th trn b mt t bo u l protein ho tan trong nc. Cc protein th th trn b mt c xp vo 4 loi da vo c ch truyn tn hiu: th th ni vi knh truyn ion, th th ni vi protein G, th th ni vi tyrosine kinase v th th ni vi enzym (Hnh 5.3). Tuy nhin, hot ng ca chng xy ra theo cng mt phng thc: Sau khi tip nhn tn hiu (bn ngoi mng t bo), th th truyn tn hiu cho cc cht trung gian. iu gy nn s thay i nng ca mt s cht truyn tn hiu trung gian (nu chng l enzym), hoc lm thay i cu trc ca cht truyn tn hiu (nu nh chng l tyrosine kinase), hoc lm thay i tnh thm ion ca mng t bo (nu chng thuc knh dn ion)... n lt mnh, cc cht truyn trung gian s lm thay i hot ng ca cc protein c hiu khc. Cui cng tn hiu c chuyn vo nhn, thng di dng cc yu t phin

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m, kim sot mc biu hin ca gen. Nh vy, t mt tn hiu ban u, mt lot thay i xy ra gip t bo tr li tn hiu nhanh, nhy v chnh xc. a) Th th ni vi knh ion: Cc tn hiu c nhn bit bi th th ni vi knh ion thng l cc cht dn truyn thn kinh. Do cc th th loi ny thng nm trn t bo thn kinh. Cht dn truyn thn kinh m hoc ng tm thi cc knh ion thng qua vic tng tc vi cc protein th th nm ngay trn knh ny.

Hnh 5.3: Bn loi th th nm trn b mt t bo nhn cc tn hiu t bn ngoi A- Th th ni vi knh ion thng phn b ngay trn knh B-Th th ni vi protein G hot ho protein ny (G+). G+ hot ho enzym xc tc phn ng to cht truyn trung gian C- Th th ni vi tyrosine kinase hot ho kinase. Enzym kinase phosphoryl ho tyrosine ca th th. Cht trung gian tng tc vi tyrosine b phosphoryl ho v tip tc truyn tn hiu D-Th th ni vi enzym xc tc cho phn ng truyn tn hiu trung gian (theo Alberts & cs., 2002).

b) Th th ni vi protein G: Sau khi nhn c tn hiu, cc th th loi ny s truyn tip tn hiu cho cc cht trung gian khc thng qua protein G (trimeric GTP binding regulatory protein). Cu trc v hot tnh ca protein G c nu trong phn 4.2. Mi th th ni vi protein G c cu trc kh ging nhau v chng u nm vt qua mng 7 ln. c) Th th ni vi enzym: Khi nhn tn hiu, cc th th loi ny hoc hot ng ging enzym hoc kt hp cng enzym. Hu ht chng ch vt qua mng t bo mt ln v c v tr lin kt vi tn hiu bn ngoi mng, cn v tr c hot tnh enzym nm bn trong mng. Cc th th loi ny khng ging nhau v ch yu l cc kinase hoc protein kt hp vi kinase. d) Th th ni vi tyrosine kinase: Tng tc gia th th vi ligand dn n vic to dimer ca th th. S thay i cu trc ca th th s hot ho tyrosine kinase nm trong t

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bo cht. Enzym ny gn gc phosphat vo tyrosine ca th th. Cht trung gian lin kt vi tyrosine c gc phospht v tip tc b phosphoryl ho bi cc kinase khc hoc bi chnh mnh. Nh tn hiu tip tc truyn i.

Hnh 5.4: Phi hp cc tn hiu trong con ung truyn tn hiu A - Hai tn hiu dn n phn ng phosphoryl ha protein X cc v tr khc nhau. Ch khi c hai v tr trn X c gn gc phosphate th X mi c hot tnh. Nh vy bt buc phi c ng thi hai tn hiu th protein X mi chuyn trng thi hot ng B - Hai tn hiu hot ho ng thi hai protein Y v Z to ra phc c hot tnh.

Chui cc phn ng phosphoryl ho bt u bng th th ni tyrosine kinase c thc hin ch yu bi cc kinase serine/threonine hoc tyrosine (Hnh 5.4). y l cc enzym c bit, xc tc cho phn ng gn nhm phosphate vo cc acid amin serine, threonine (t hn) hoc tyrosine phn b trn phn t protein. Cc nh nghin cu c tnh c khong 1% gen ca c th ngi m cho kinase. Mt t bo ng vt c v cha khong 100 loi enzym kinase khc nhau ch yu l serine/threonine kinase. Mc d s protein b phosphoryl ho bi tyrosine kinase ch chim 0,1% tng s protein trong t bo, tyrosine kinase ng vai tr then cht trong h thng truyn tn hiu. Tn hiu thu nhn bi hai loi th th ni vi protein G v ni tyrosine kinase thng c truyn qua mt s cht chuyn ti trung gian v cui cng l n cc yu t phin m. Cc yu t s iu bin mc biu hin ca gen sao cho t bo c phn ng tng thch vi tn hiu. Mt s protein c bit ho ch hot ng trong cc h thng truyn tn hiu. Chng thng tn ti hai trng thi: khng hot tnh (trc khi nhn tn hiu) v c hot tnh (sau khi nhn tn hiu). Khi nhn tn hiu chng chuyn sang trng thi hot ng. V d, phn ng phosphoryl ho v mc phosphoryl ho (nhn mt hoc nhiu nhm phosphate) xy ra vi tyrosine kinase lin quan mt thit n biu hin hot tnh ca protein ny. trng thi hot ng chng truyn tn hiu i bng cch gy phosphoryl ho cc protein tip theo, to nn chui cc phn ng phosphoryl ho. Sau chng tr v trng thi khng hot ng nh phn ng kh nhm phosphate. i vi, th th lin kt vi GTP, chng truyn tn hiu cho cc phn t trung gian v tr v dng khng c hot tnh khi lin kt vi GDP. Nh vy, chc nng nhn bit v truyn tn hiu ca c hai loi th th u ph thuc vo s thay i cu hnh ca chng thng qua phn ng phosphoryl ho hoc nh tng tc vi GTP/GDP (Hnh 5.5).

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Hnh 5.5: Hai c ch ch o truyn tn hiu trong t bo. i vi c hai trng hp, protein truyn tn hiu c hot ho bi phn ng phosphoryl ho (hoc kh gc phosphate). (A): Truyn tn hiu thng qua cc protein kinase. (B): Truyn tn hiu thng qua protein G.

5.2

Th th ni vi protein G

Hn 100 th th ni vi protein G c pht hin ng vt c v, phn ln nh k thut tch dng tng ng (homology cloning) da vo phn ng lai iu kin khng qu nghim ngt gia mi ADNc bit v ADN ca i tng cn nghin cu. Ngoi ra cc th th cn c tm thy nh k thut tch dng biu hin da vo cc tn hiu xut hin khi t bo b kch thch. Trong phng php ny, m c cha th th cn nghin cu c s dng tch ARNm v tng hp ADNc, xy dng ngn hng ADNc trong vector biu hin gen (vector c cha promoter). Sau , cc dng ca ngn hng c a vo trng ch Xenopus tng hp nn cc protein tng ng vi ADNc. Nhng protein l c gn vo mng t bo v c pht hin nh cc protein c nh du c kh nng lin kt vi chng.

5.2.1 Protein G
Protein G (trimeric GTP binding protein) c hot tnh phn hy GTP (Hnh 5.6). Chng lun phi hp hot ng cng vi cc th th nm trn b mt t bo to thnh mt cp truyn tn hiu. Tn hiu c truyn qua protein G vo trong t bo thng qua cc enzym hoc cc knh ion trn mng nguyn sinh cht. Protein G khc vi monomeric GTPase trong t bo eukaryot. Monomeric GTPase tham gia kim sot cc phn ng sinh hc v vn chuyn ti tit. Tuy nhin, protein G cng c hot tnh GTPase. N trng thi hot ng khi lin kt vi GTP nhng b mt hot tnh khi lin kt vi GDP. Khi cha c tn hiu, cc th th khng tng tc vi protein G. ng thi lc bn thn protein G trng thi lin kt vi GDP (tc l dng khng hot tnh). Khi th th nhn tn hiu, cu trc khng gian ca th th b thay i. Phc th th - ligand s tng tc vi protein G khin GDP b tch ra v GTP thay vo . Lp tc protein G tng tc vi cht trung gian tip theo (enzym hoc protein). Lin kt gia protein G v cht nhn tn hiu tip theo khin cho GTP b phn hy thnh GDP bi chnh hot tnh GTPase ca protein G. Phn ng phn hy GTP khin protein G tch ri khi cht trung gian. Protein G tr v trng thi ban u (mang GDP). Cht trung gian truyn tn hiu i.

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Hnh 5.6: M hnh hot ng ca protein G trong qu trnh truyn tn hiu Protein G duy tr trng thi hot ho mt khi ligand vn bm vo th th. V vy

Protein G gm ba chui polypeptide khc nhau: , v . Chui (s) c hot tnh GTPase. Chui ny s lin kt vi GTP v thy phn GTP gy hot ho adenylyl cyclase. Chui v to phc gi cho protein G bm trn b mt t bo bn pha t bo cht. Khi GDP lin kt vi chui , protein G khng c hot tnh. Khi GDP b thay th bi GTP, chui tch ra khi phc v lin kt vi adenylyl cyclase. Enzym ny c hot ho v n lt n xc tc cho phn ng to cAMP. Nh vy, protein G phn r thnh cc tiu n v v khi c hot ho. C th c nhiu loi th th ni protein G nhng tt c ch nhn bit mt loi tn hiu. Ngc li, c th c nhiu th th ni protein G, chng nhn bit cc tn hiu khc nhau. Hn na, t protein G, mi tn hiu c th c truyn theo con ng ring bit khin t bo c cu tr li c hiu vi tng loi tn hiu. Hu ht cc th th ni protein G hot ho cho mt chui cc phn ng lm thay i nng ca mt hoc vi phn t nh trong t bo. Nhng phn t ny lm nhim v trung chuyn v c gi chung l cc cht truyn tn hiu trung gian (hoc cn gi l cc tn hiu th cp). Hai trong s cc cht trung gian hay gp nht trong t bo l cAMP v ion Ca+2. Nng ca chng bin i do nhiu tn hiu khc nhau, trong hu ht l tn hiu nhn bit bi cc th th ni protein G. S d t bo tr li rt nhanh, nhy i vi s bin ng v liu lng ca cc tn hiu bn ngoi l do hot tnh thun nghch ca adenylyl cyclase. iu ny xy ra nh chui s ch tn ti trng thi hot ng trong thi gian rt ngn. Khi s lin kt vi adenylyl cyclase,

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hot tnh GTPase ca chui s c kch thch lm cho GTP b phn hy thnh GDP rt nhanh. Chui s tch khi adelylyl cyclase. Lc ny c s v adelylyl cyclase b mt hot tnh. Chui s lin kt tr li vi phc to thnh phn t protein G dng khng hot ng. Trong t bo ng vt, c t nht 6 loi ng phn adenylyl cyclase u phn b trn mng t bo phi hp vi cc th th nhn nhng tn hiu khc nhau. Vic chuyn i trng thi t hot ho sang khng c hot tnh ca nhng enzym ny u ph thuc vo protein G. c bit trn mng t bo thn kinh, enzym ny c hot ho bi ion Ca+2 nm trong phc vi calmodulin.

5.2.2 Hot ho hoc c ch cAMPase thng qua protein G


T nm 1959, phn t AMP dng vng k hiu l cAMP (cyclic adenosine-3',5'monophosphate cAMP) c xc nh nh l cht truyn tn hiu trung gian trong t bo. T n nay, thc nghim nhn thy cAMP tham gia vo nhiu qu trnh sinh ho khc nhau trong t bo prokaryot cng nh t bo ng vt. Nng ca cAMP trong t bo eukaryot nh hn 10-7 M. Khi b kch thch, nng ny tng gp 5 ln trong mt giy. iu i hi phn ng tng hp cAMP phi xy ra rt nhanh, ng thi phi c dng tc thi khi khng cn tn hiu kch thch. Phn t cAMP c tng hp t ATP nh enzym adenylyl cyclase (Hnh 5.7). Mt khc, cAMP b phn hy lin tc nh cAMP- phosphodiesterase. Nhiu tn hiu bn ngoi tham gia kim sot nng cAMP thng qua vic iu bin hot tnh ca adenylyl cyclase hn l thay i hot tnh ca cAMP-phosphodiesterase. Trong mt loi t bo nht nh, mi tn hiu gy hot ho adenylyl cyclase thng c chung hiu ng. V d, trong cc t bo m t nht c 4 loi hormon hot ho adenylyl cyclase cng dn n vic tng cng chuyn ho triglyceride (dng d tr ca cht bo) thnh acid bo. Cc th th cho nhng hormon ny hot ng khi lin kt vi protein G. C th mc bnh suy gim di truyn trong vic tng hp protein G thng ri lon chuyn ho, pht trin xng v pht trin tr no bt bnh thng. Th nghim minh ha r nht v s to cp cng hot ng gia th th v adenylyl cyclase l cc th th -adrenergic (lin quan n hot ng ca adrenaline v noradrenaline). Khi c th hot ng lin tc hoc chu s cng thng, cc m i hi b sung glucose v acid bo. Cc nguyn liu ny c t bo gan v cc t bo d tr cht bo (lipolysis) cung cp vo mu rt nhanh khi cc tn hiu adrenaline v noradrenaline tng tc vi th th -adrenergic nm trn b mt cc t bo ny. Ngoi ra, lin kt ca adrenaline vi th th -adrenergic cc t bo c tim lm tng tn s co bp tim, dn n tng lng mu i ti cc m. Lin kt gia adrenaline vi th th t bo c trn ca rut gy co gin c. Adrenaline cn c kh nng lin kt vi th th -adrenergic khin cc ng mch co tht li, ngn cn mu lu thng n cc vng ln cn (ni khng c tng tc gia adrenaline v th th). Nhng tc dng khc nhau ca hormon adrenaline u nhm mc ch cui cng l b sung nng lng cho cc c ng nhanh ca c trong nhng hot ng cng thng m c th ang chu ng.

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Hnh 5.7: Cu trc ca adenylyl cyclase gm 2 tiu phn phn b trong mng t bo, mi tiu phn gm 6 on helix. Hai trung tm hot ng ca enzym nm pha trong t bo cht s phn hy c cht khi enzym nhn tn hiu truyn t protein G.

Khi adrenaline lin kt vi th th -adrenergic, n hot ho adenylyl cyclase. Tuy nhin, khi adrenaline lin kt vi 2-adrenergic, n c ch enzym. iu cho thy cc protein G khc nhau c tc dng khc nhau (Hnh 5.8).

Hnh 5.8: Cc loi protein G tham gia hot ho (protein Gs) hoc c ch (protein Gi) enzym adenylyl cyclase. Cc tiu phn G l nh nhau trong cc loi protein ny. Chng ch khc nhau tiu phn G v th th m chng tng tc (theo Alberts & cs., 2002).

Adenylyl cyclase c th c hot ho hoc b c ch thng qua cc protein G khc nhau. Trong phn ng hot ho, th th -adrenergic cng to cp hot ng vi Gs. Trong phn ng c ch, th th to cp vi protein Gi. Protein Gi c cu trc tng t Gs ch khc tiu phn i. V d, tng tc gia adenosine v prostaglandin vi th th ca chng s kch hot protein Gi. Lc , tiu phn Gi tng tc vi GTP v tch khi protein Gi. Phc Gi-GTP c ch (ch khng phi l hot ho) adenylyl cyclase khin nng cAMP gim xung. Ngoi ra, khng phi ch ring Gi m c G cng c tc dng c ch adenylyl cyclase. Tiu phn Gi c ch theo con ng gin tip trong khi phc G c ch theo hai cch: hoc l phc G lin kt vi enzym hoc lin kt vi bt k tiu phn s t do c trong t bo, do hn ch chng hot ho enzym. Ngoi ra protein Gi cn kch thch vic m knh K+ trn mng t bo. Nh vy cc protein G rt linh hot trong vic truyn tn hiu, thc hin nhim v trung gian gia th th v cc tn hiu th cp trong t bo. cc th nghim tin hnh, hoc tiu phn G hoc phc G hoc c hai u tham gia phn ng. Tuy nhin, phc G lm tng hot tnh ca mt s dng adenylyl cyclase ch sau khi enzym c hot ho bi tiu phn Gs.

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5.3

Protein kinase ph thuc cAMP (cAPK hoc kinase A)

Trong cc t bo ng vt, chc nng ch yu ca cAMP l hot ho enzym kinase. Nhng enzym ny c gi chung l cAPK (cAMP-dependent protein kinase), hoc protein kinase A. Cc cAPK xc tc cho phn ng chuyn nhm phosphate tn cng t ATP sang acid amin serine hoc threonine ca protein c hiu. Khi b phosphoryl ho, hot tnh ca protein b thay i (t trng thi hot ng sang trng thi bt hot hoc ngc li). Protein kinase A c tm thy trong mi t bo ng vt v c xem nh lin quan n hu ht cc chc nng ca cAMP. C cht ca kinase A a dng, ph thuc vo tng loi t bo. iu gii thch v sao tc dng ca cAMP cng thay i theo tng loi t bo. trng thi khng hot ng, kinase A l phc tetramer gm hai tiu phn c hot tnh enzym v hai tiu phn kim sot vic lin kt vi cAMP. Lin kt gia hai tiu phn ny vi cAMP lm thay i cu trc khng gian ca hai tiu phn kia, khin chng tch khi phc h (4 tiu phn). Lc hai tiu phn c hot tnh enzym c hot ho tham gia xc tc cho phn ng phosphoryl ho protein c hiu (Hnh 5.9).

Hnh 5.9: Hot ho kinase A bi cAMP. Enzym kinase A c cu trc tetramer. Lin kt gia cAMP vi hai tiu phn iu khin lm thay i cu trc khng gian ca kinase A, khin hai tiu phn c hot tnh enzym tch ra v c hot ho. Mi tiu phn iu khin c hai v tr lin kt vi cAMP. Khi c hai v tr u lin kt vi cAMP th hai tiu phn kia mi tch ra v xc tc cho phn ng phosphoryl ho. C t nht hai loi kinase A trong t bo ng vt c v. Loi th nht tn ti ch yu trong t bo cht. Loi th hai, nh vo mng sinh cht, mng nhn v cc si vi ng (microtubulin) nh hai tiu phn iu khin. i vi c hai loi kinase A, khi cc tiu phn c hot tnh c hot ho, chng c th di chuyn vo nhn gy phosphoryl ho cc protein iu khin hot ng ca gen trong khi hai tiu phn kia vn tn ti trong t bo cht.

Phn ng phosphoryl ho xy ra nh vai tr trung gian ca cAMP c pht hin ln u tin khi nghin cu qu trnh trao i glycogen trong t bo c xng. Glycogen l dng d tr ca glucose. C hai qu trnh tng hp v phn hy glycogen c kim sot bi adrenaline. Khi ng vt b nh hoc gp bt k tn thng no, tuyn adrenaline tit hormon vo mu gy tn hiu bo ng n cc t chc khc trong c th. Adrenaline tun hon trong mu, kch thch cc t bo c phn gii glycogen thnh glucose, ng thi gy ngng qu trnh tng hp glycogen. Glucose b oxy ha to ATP cung cp cho c. Bng cch ny, adrenaline chun b cho t bo c tham gia hot ng cng thng. Hot tnh ca adrenaline c th hin khi n lin kt vi th th -adrenergic trn b mt t bo c, gy ra s tng nng cAMP trong t bo cht. Lc cAMP s hot ho kinase A v enzym ny phosphoryl ho hai enzym khc (Hnh 5.10).

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Hnh 5.10: Phn t cAMP kch thch chuyn ho glycogen thnh glucose trong t bo c. Nh lin kt vi cAMP, kinase A c hot ho, xc tc cho phn ng phosphoryl ho v dn n hot ho hai enzym phosphorylase kinase v glycogen phosphorylase. Nh , glycogen phn gii thnh glucose. Kinase A trc tip v gin tip lm tng qu trnh phosphoryl ho enzym glycogen synthase. Do b phosphoryl ho, glycogen synthase b mt hot tnh, dn n c ch tng hp glycogen (theo Alberts & cs., 2002).

Nh vy, s tng nng cAMP dn n mt lot phn ng va kch thch chuyn ho glycogen thnh glucose va c ch qu trnh chuyn glucose thnh glycogen m bo cho nng glucose t cc i trong t bo. Tng hp v phn hy glycogen ph thuc nng cAMP xy ra ch yu t bo gan v c, ni d tr glycogen. Tuy nhin, cAMP cn tham gia truyn tin khi c mt ca mt s hormon. V d, trong t bo to m, nng cAMP tng cao s kch hot lipase thng qua cAPK, do tng hp acid bo c tng cng. Mt cch tng t, trong t bo bung trng, cAMP tng khin cho cAPK kch thch phn ng tng hp 2 hormon estradiol v progesterone. Ngoi ra, cc cAPK trong t bo thn kinh tham gia iu bin hot ng ca cc knh ion lin quan n vic hnh thnh tr nh trong qu trnh pht trin v bit ho. Cc enzym cAPK thng c c nh ti mt vng trong t bo nh protein lin kt vi cAPK. Nhng protein ny thng c hai phn, mt phn lin kt vi cAPK, phn kia c nh li ti mt v tr tng t nh ci m neo gi cAPK khng khuch tn trong t bo cht. Trong mt s t bo ng vt, nng cAMP tng s kch thch hot ng ca mt s gen c bit. V d, trong t bo tit hormon somatostatin, nng cAMP t n mc nht nh s bt m gen m cho hormon ny. Qu trnh hot ho gen m cho somatostatin c thc hin nh sau: Gen c cha mt on ADN ngn, gi l vng CRE (cAMP response element) l v tr tng tc ca protein iu khin, gi l protein CREB (CRE-binding protein). Tuy nhin protein CREB dng b phosphoryl ho mi lin kt c vi CRE. Khi b phosphoryl ho bi kinase A, protein CREB lin kt vi vng CRE, hot ho gen m cho hormon. T v d c th ca kinase A, chng ta thy mt chui cc phn ng phosphoryl ho

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cng nh kh phosphoryl xy ra k tip nhau. Trong chui phn ng ny, kt qu ca mi mt phn ng l s hot ho hoc c ch protein c hiu nh sn phm ca phn ng xy ra trc . Chui cc phn ng dy chuyn thng c iu khin bi nng ca mt loi phn t ban u, v d bt u bi cAMP. Mt khc, cc phn ng c th xy ra thun nghch khi nng cAMP thay i. Hn na, chui cc phn ng cn cho php khuch i tn hiu nh b ban u ( nng rt nh). V d, nng ca adrenaline cn thit trong mu kch thch phn ng phn gii glycogen khong 10-10 M. Tng ng vi lng adrenaline rt t ny, nng cAMP khong 10-6 M. Sau ba phn ng lin tip xy ra, nng glucose tng 50% (Hnh 5.11). T l nng gia ba loi enzym kinase, glycogen phosphorylase kinase v glycogen phosphorylase t c sau chui phn ng l 1: 10 : 240. Nh vy tc dng hot ho ca adrenaline v cAMP c khuch i ln rt nhiu ln. Hot tnh ca cAMP ch c tc dng tm thi. T bo nht nh phi kh gc phosphate ca nhng protein b phosphoryl ho bi kinase A. Nhn chung, phn ng kh phosphate cho nhng protein b phosphoryl ho cc acid amin serine hoc threonine c xc tc nh cc enzym serine/threonine phosphatase. Cc enzym ny c xp vo 4 nhm: I; IIA; IIB v IIC. Enzym nhm I gi vai tr quan trng trong phn ng tr li i vi thay i nng cAMP. Enzym nhm IIA xc tc cho rt nhiu phn ng kh nhm phosphate do cc enzym kinase serine/threonine gy ra. N gi vai tr quan trng trong kim sot chu trnh t bo. Enzym nhm IIB c hot ho nh Ca+2 v xut hin nhiu trong no.

Hnh 5.11: Truyn v khuch i tn hiu t bn ngoi t bo qua th th ni protein G Phn t adrenaline tng tc vi th th lm nng cAMP tng v hot ho cc enzym trong chui cc phn ng dy truyn. Nh vy tn hiu c khuch i.

Hot tnh ca bt k protein no lin quan n phn ng phosphoryl ho u ph thuc vo s cn bng gia hot tnh ca kinase v phosphatase. Phosphatase I kh phosphate nhng protein b phosphoryl ho bi kinase A. V d, hot tnh ca CREB b c ch do enzym nhm I kh nhm phosphate, v vy hot ng ca gen iu khin bi CREB b ngng do CERB mt kh nng lin kt vi CRE. Trong cc t bo c b kch thch bi adrenaline, hot

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tnh ca phosphatase I b gim do protein c ch. Khi kinase A xc tc phn ng phosphoryl ho i vi protein c ch, protein ny lin kt vi phosphatase I v lm enzym mt hot tnh (Hnh 5.12).

Hnh 5.12: Vai tr iu khin ca phosphatase I trong phn ng to glycogen thng qua cAMP Phosphatase I b c ch bi cAMP (tri ngc vi phn ng phosphoryl ho c hot ho bi cAMP). cAMP hot ho kinase A (cAPK) gy phosphoryl ho protein c ch phosphatase. Nh , protein c ch c hot tnh v n lt n km hm hot ng ca phosphatase I.

5.4

Th th tyrosine kinase v cc protein Ras

Hu ht th th tyrosine kinase nhn bit cc factor tng trng v bit ho t bo. V d, th th ca factor tng trng xut pht t tiu cu (PDGF-platelet derived growth factor), t si nguyn bo fibroblast (FGFs-fibroblast growth factor), t t bo biu b (EGF-epidermal growth factor) vv... Tng tc gia tn hiu ban u (ligand) v th th tyrosine kinase (RTKs) dn n phn ng phosphoryl ho acid amin tyrosine v tip sau l mt lot cc phn ng phosphoryl ho khc trong chui truyn tn hiu. Chng ta cng xem xt cch thc hot ng ca nhng th th ny v nhng protein nhn tn hiu t chng tip tc truyn tin.

5.4.1 Th th tyrosine kinase (RTKs)


Tt c cc th th RTKs u gm hai phn. Phn th nht phn b trn b mt t bo lm nhim v tng tc c hiu vi tn hiu (ligand). Phn th hai nm trong t bo cht c hot tnh tyrosine kinase. Phn th hai ny xc tc phn ng phn hy ATP thnh ADP v gn gc phosphate vo tyrosine. Hai phn ca RTKs c ni vi nhau bi on peptide helix k nc. Cu trc ca mt s loi th th tyrosine kinase c trnh by trn hnh 5.13.

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Hnh 5.13: Cc th th tyrosine kinase Chc nng ca vng giu cystein v vng tng t immunoglobulin cha c xc nh. EGF yu t tng trng biu b (epidermal growth factor); NGF yu t tng trng thn kinh (nerve growth factor); FGF yu t tng trng nguyn bo (fibroblast growth factor).

Hu ht th th RTKs tn ti dng monomer khi cha c tn hiu. Tng tc vi ligand lm thay i cu trc ca th th khin chui n monomer RTKs chuyn sang cu trc dimer. Tip , cu trc dimer khin trung tm xc tc ca phn nm trong t bo cht chuyn sang trng thi c hot tnh kinase. Hot tnh kinase ca chui monomer ny s gn gc phosphate vo tyrosine ca chui monomer kia. Nh th, mi chui n RTK s ch c hot tnh kinase i vi tyrosine ca si n kia sau khi hai si tng tc vi nhau to cu trc dimer. y l phn ng t phosphoryl ho.

Hnh 5.14: hay i cu trc ca th th tyrosine kinase trc v sau khi tng tc vi tn hiu u tin (ligand). Th th (dng monomer) tng tc vi ligand s to dimer. Hot tnh kinase ca mi si monomer s phn hy ATP v gn gc phosphate vo tyrosine trn si monomer kia. y l phn ng t phosphoryl ho.

Yu t tng trng EGF (yu t kch thch cc t bo biu b cng nh mt s t bo khc bc vo phn chia) c nhn bit bi th th tyrosine kinase. Th th ca EGF nm xuyn

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qua mng. Phn th th nm ngoi t bo lin kt vi EGF nn cu trc khng gian ca phn nm ngoi t bo b thay i cho php chng to lin kt dimer. Chuyn i t monomer sang dimer hot ho phn c hot tnh tyrosine kinase nm pha trong t bo cht. Phn ng dimer ho l c ch chung hot ho th th tyrosine kinase (Hnh 5.14). Mt khi hot ho, th th xc tc phn ng t phosphoryl ho cho nhau. Gc phosphate c chuyn t ATP sang acid amin tyrosine c bit ca chnh th th hoc ca mt s protein khc thuc con ng dn truyn tn hiu. Ngoi ra, tn hiu c th dng dimer nh tn hiu nhn bit bi th th tng trng c ngun gc t tiu cu PDGF. Khi tn hiu lin kt vi th th, hai phn t th th tng tc vi nhau v gy phosphoryl ho ln nhau (Hnh 5.15).

Hnh 5.15: Yu t tng trng c ngun gc tiu cu (PDGF platelet drived growth factor) tn ti dng dimer gm hai v tr lin kt vi th th. Do tng tc ca yu t PDGF vi th th dn n vic to dimer gia hai th th vi nhau v khi ng qu trnh truyn tn hiu vo trong t bo. Mt s yu t tng trng khc cng hot ng dng dimer thc y dimer ho ca hai phn t th th tyrosine vi nhau.

C ch truyn tn hiu bt u t cc th th ca EGF, PDGF, FGF c nghin cu kh k. Trong tng trng hp, tyrosine ca th th b phosphoryl ho tr thnh v tr c i lc lin kt cao i vi cc protein lm nhim v truyn tn hiu. Lin kt gia mi protein vi cc v tr tyrosine (b phosphoryl ho) khc nhau trn th th ph thuc vo cc on polypeptide nm xung quanh v tr . Nh tng tc vi th th ti v tr c hiu, nhiu protein c kh nng t phosphoryl ho cc tyrosine ca ring mnh. Nh vy, nhng protein ny s c hot tnh kinase ch khi tng tc vi kinase b phosphoryl ho trc . R rng phn ng t phosphoryl ho tyrosine gi vai tr chnh trong con ng truyn tn hiu, chuyn cc thnh vin sang trng thi hot ng, m bo tn hiu c truyn vo su trong t bo. Cc th th tyrosine kinase khc nhau thng tng tc vi t hp cc protein khc nhau, do hot ho cc phn ng tr li khc nhau. Xt trng hp th th cho insulin v yu t tng trng tng t insulin IGF1(Insulin like Growth Factor-1). Cc th th ny dng tetramer, do khi lin kt vi tn hiu, cu trc ca th th b thay i nhng khng xy ra hin tng dimer ho. Mt khc khi c insulin bm vo, th th t phosphoryl ho ti vng c hot tnh kinase (phn nm pha trong mng t bo). Vng ny tip tc gy phosphoryl ho cc tyrosine ca protein khc. Cc tyrosine ny l v tr lin kt v hot ho cc protein trong con ng dn truyn tn hiu. Protein c tyrosine b phosphoryl ho c nhn bit bi cc protein khc c cha on peptide c hiu gi l vng SH2. Rt nhiu protein lin kt vi tyrosine c gn nhm

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phosphat u c cu trc kh bo ton ti hai vng gi l SH2 v SH3 (Src homology regions 2 & 3-tn gi bt ngun t cc vng 2 v 3 ca protein Src). Nhng protein c hai vng SH2 v SH3 cn c gi l protein tng hp (adaptor protein). Vng SH2 nhn bit cc tyrosine b phosphoryl ho, do nhng protein c cha vng ny u c kh nng lin kt vi th th tyrosine kinase hoc cc protein b phosphoryl ho tyrosine (Hnh 5.16). Vng SH3 ca protein tng hp tng tc vi cc on peptide giu proline. Nh vy, tng tc vng SH3 gy nn nhng thay i cu trc cn thit m bo cho tng tc c hiu gia vng SH2 v tyrosine c gn gc phosphat.

Hnh 5.16: Tng tc gia protein truyn tn hiu mang vng SH2, SH3 vi th th ca PDGF Th th c cha 5 acid amin tyrosine c kh nng t phosphoryl ho. y l v tr lin kt vi 3 protein c hiu (pha bn tri hnh v) c mang cc vng SH2 v SH3. Ngoi ra th th ca PDGF cn c hai tyrosine khc nm st pha trong mng nguyn sinh cht cng c kh nng t phosphoryl ho Chng tr thnh v tr tng tc vi tyrosine kinase khng phi l th th. Cc ch s ch v tr ca tyrosine trong chui peptide. Cc v tr lin kt c xc nh nh k thut ti t hp ADN khi gy t bin tng tyrosine.

5.4.2 Protein Ras v chui cc phn ng truyn tn hiu hot ho bi th th tyrosine kinase
Cc protein Ras nm trong nhm monomer GTPase; chng c hot tnh tng t tiu phn Gs ca protein G. Protein Ras tn ti hai trng thi khc nhau ph thuc vo cu trc khng gian: trng thi hot ng khi lin kt vi GTP v trng thi bt hot khi lin kt vi GDP. Protein Ras thy phn GTP chm hn t nht 100 ln so vi tiu phn Gs ca protein G. pha trong mng t bo, nng GTP lun cao hn nng GDP gp 10 ln. V vy, khi Ras lin kt vi GTP, n s duy tr trng thi hot ng nu nh khng c cc protein khc kim sot. Trong t bo c hai nhm protein truyn tn hiu kim sot hot ng ca Ras thng qua iu khin qu trnh chuyn trng thi ca protein ny gia dng hot ng v bt hot. c ch hot tnh ca Ras, cc protein kim sot s hot ho GTPase. Nhng protein ny c gi l cc protein GAPs (GTPase-activating protein). Hot ng ca GAPs dn n t l GTP bm vo Ras b phn hy tng, do Ras nhanh chng chuyn sang trng thi khng hot ng. Tuy nhin hot ng ca GAPs li b c ch bi cc protein gii phng guanine (guanine nucleotide releasing proteins GNRPs). GNRPs thc y phn ng thay th GDP bng GTP, do chng li hot ho Ras (Hnh 5.17).

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Hnh 5.17: Kim sot hot ng ca protein Ras Cc protein hot ho (GAPs) lm mt hot tnh ca Ras bng vic kch thch phn ng thy phn GTP lin kt vi Ras. Cc protein GNRPs hot ho Ras thng qua phn ng thay th GDP bng GTP. Do nng GTP trong t bo cht cao hn 10 ln nng GDP, Ras lun lun c xu th duy tr lin kt vi GTP. ng vt c v, hai loi protein GAPs c pht hin GAP v neurofibromin l p120 Hot ng ca chng ph thuc vo tng loi t bo nhm duy tr Ras trng thi khng hot ng.

Ras tham gia vo con ng truyn tn hiu t th th tyrosine kinase. Tuy nhin, Ras khng lin kt trc tip vi th th; Ras nhn tn hiu thng qua protein tng hp. Nhng th nghim nui cy t bo c b xung yu t EGF lm sng t hot ng ca Ras trong chui truyn tin t th th tyrosine kinase. u tin, yu t EGF c th th tyrosin kinase tip nhn. Tip n, cc tyrosine ca th th t phosphoryl ho. Nhng tyrosine ny s lin kt vi vng SH2 ca protein GRB2. n lt mnh, GRB2 lin kt vi protein Sos v hot ho protein ny. Chc nng ca Sos l thay th GDP ca Ras bng GTP. Cui cng Ras phn hy GTP truyn tn hiu i (Hnh 5.18).

Hnh 5.18 : Protein Ras tip nhn tn hiu t th th tyrosine kinase thng qua hai protein tung hp GRB2 v Sos. Vng SH2 ca GRB2 tng tc vi tyrosine b phosphyl ho ca th th. Vng SH3 ca GRB2 tng tc vi Sos. Sos hot ho Ras khin GDP b thay th bi GTP; GTP b phn hy tip tc truyn tn hiu.

Cng vi cc protein Rho v Rac, Ras tham gia truyn tn hiu t th th trn mt t bo n h thng mng li si actin. Chng phi hp hot ng cng vi cc protein Rab lin quan n iu khin vn chuyn cc nang trong t bo. Ging nh hu ht cc monomer GTPase, protein Ras c gi cht mt u vo pha trong mng sinh cht. Chc nng ca

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chng l truyn tn hiu t th th tyrosine kinase trn b mt t bo vo n nhn hot ho cc gen lin quan n tng trng hoc bit ho t bo. Khi hot ng ca Ras b c ch, t bo khng tr li vi cc kch thch ca bn ngoi. Tuy nhin khi protein Ras b t bin khin Ras lin tc tn ti trng thi c hot tnh hoc c tng hp qu nhiu s khin t bo tng trng v pht trin khng ngng, ngay khi khng c tn hiu bn ngoi. Trong thc t, cc protein Ras c pht hin khi chng hot ng qu mnh do gen m cho Ras b t bin. Lc t bo khng chu s kim sot ca qu trnh phn bo v bit ho, chng sinh si khng ngng dn n pht trin ung th. Khong 30% ung th ngi l do t bin xy ra gen ras. 5.5 Tn hiu th cp Ca+2 trong chui truyn tn hiu

Chng ta bit phn t protein G trung chuyn tn hiu t th th n adenylyl cyclase dn n thay i nng cAMP trong t bo. Chng ta s xt n vai tr khc ca protein G trong vic phi hp hot ng gia th th vi enzym phospholipase C. Enzym ny lm thay i nng ion Ca+2 trong t bo cht, khi ng chui cc phn ng truyn tn hiu khc nhau. Ion Ca+2 l tn hiu th cp c t bo s dng rng ri hn cAMP trong qu trnh truyn tn hiu.

Hnh 5.19: Kim sot nng Ca+2 trong t bo cht. S biu din mt s phng thc c bn m bo duy tr nng Ca+2 trong t bo cht nng thp. (A): Ca+2 c bm ra ngoi t bo bng bm trao i Na v Ca hoc +2 +2 bng bm Ca -ATPase. (B): Ca c bm t t bo cht vo mng li ni cht ER hoc vo ty th. Ngoi ra trong t bo cht cn tn ti cc phn t khc nhau lin kt vi Ca+2 lm gim nng Ca+2 trng thi t do. +2 +2 Ion Ca c bm vo ty th ch khi nng Ca t bo cht rt cao gy nguy him cho t bo.

Nng Ca+2 t do trong t bo cht ca bt k t bo no cng lun c kim sot cht ch khng qu <10-7 M, trong khi ngoi t bo v mng li ni cht ER nng Ca+2 cao hn rt nhiu (10-3 M). V vy, gradient nng ion Ca+2 lun tn ti theo chiu t ngoi vo trong t bo hoc t khoang trong ER ra ngoi t bo cht. Khi cha c tn hiu, t bo thc thi cc c ch kim sot m bo s chnh lch nng ny. Khi c tn hiu, knh truyn ion Ca+2 m ra, cc ion ny trn vo trong t bo cht lm nng Ca+2 tng vt ln gy hot ho cc protein lin quan n Ca+2. C nhiu c ch khc nhau duy tr nng ion Ca+2 mc rt thp trong t bo cht. Mi t bo eukaryot u c phc Ca+2 - ATPase trn mng ni cht, s dng nng lng do thy phn ATP bm Ca+2 ra khi t bo. Mt s t bo nh t bo c, t bo thn kinh cn c bm trao i Ca+2 v Na+. Bm ny ch bm Ca+2 ra khi t bo khi nng Ca+2 trong t bo vt qu 10 ln nng cho php. Ngoi ra trn mng ER cng c bm Ca+2. Khi t bo trng thi tnh, nng Ca+2 khong 10-7 M, cn khi t bo hot ng nng ny c

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th t n 5x10-6 M. Khi t bo tn thng khng c kh nng bm Ca+2, nng Ca+2 c th tng n mc gy nguy him cho t bo (10-5 M). Lc ny ngay c bm Ca+2 trn mng ty th cng hot ng, s dng nng lng ca cc phn ng truyn in t bm Ca+2 ra khi t bo cht. Cc con ng thi Ca+2 c m t trn hnh 5.19.

5.5.1 Inositol phospholipid


Rt nhiu hormon khi tng tc vi th th trn b mt t bo gy nn s tng nng Ca trong t bo cht, thm ch ngay c khi ion Ca+2 khng c mt trong mi trng xung quanh. cc t bo gan, t bo m v mt s t bo khc, thc nghim pht hin rng ion Ca+2 xut hin trong t bo cht do c gii phng ra t mng li ni cht ER hoc t mt s nang trong t bo ch khng phi t mi trng bn ngoi vo. Cu hi t ra l lm th no khi c tng tc gia hormon v th th trn b mt t bo, tn hiu c truyn n ER v t ion Ca+2 c bm ra t bo cht?
+2

Cho n u nm 1980, th nghim ch ra rng nng Ca+2 trong t bo cht tng ln lin quan n phn ng phosphoryl ho phn t phosphatidylinositol (PI) to nn inositol phospholipid. Phospholipid PI nm mng sinh cht v pha t bo cht. Hai dng inositol phospholipid hay gp nht trong h thng dn truyn tn hiu l PI 4-phosphate (PIP) v PI 4,5-bisphosphate (PIP2). Khi c tn hiu bn ngoi gi n, th th c hot ho. Thng qua protein G (gi l Gq), tn hiu gi n enzym phosphalipase C-. Enzym ny c hot ho, xc tc phn ng phn ct PIP2 thnh hai sn phm: 1,2-diacylglycerol (c gi li trn mng) v inositol 1,4,5-triphosphate (IP3) tan trong nc. C hai sn phm trn u tham gia vo hai nhnh truyn tn hiu khc nhau. Mc d con ng truyn tn hiu thng qua inositol phospholipid cha c nghin cu chi tit nh vi cAMP, t nht c khong hn 25 th th phn b trn b mt t bo ni vi protein Gq tham gia vo con ng truyn tn hiu thng qua inositol phospholipid.

5.5.2 Inositol triphosphate (IP3) v s vn chuyn Ca+2 ra khi ER


Inositol triphosphate IP3 l sn phm ca phn ng phn hy PIP2. y l nhng phn t kch thc nh tan trong nc v khuch tn rt nhanh vo t bo cht. , IP3 lin kt vi cc knh c bit trn mng ER (IP3-gated Ca+2). Knh ny c iu khin bi c ch phn hi tch cc. Khi Ca+2 ra khi knh, n tng tc tr li vi knh khin knh c m rng hn. Do s ion Ca+2 c gii phng ra khi ER tng ln rt nhanh. Ion Ca+2 c gii phng ra rt nhanh v t ngt theo kiu "tt c hoc khng c g" (all-or-none). Khi lng Ca+2 tng ln trong t bo cht, enzym c hiu gy phosphoryl ho IP3 cng c hot ho. Do mt s IP3 b phosphoryl ho to IP4. Cht ny gip cho t bo duy tr phn ng tr li tn hiu c lu v chm hn. ng thi IP4 kch thch vic tch ly Ca+2 trong t bo cht bng vic ly Ca+2 t ngoi vo. Nh vy, nng Ca+2 trong t bo cht c th tng bng hai con ng: chuyn Ca+2 t ER ra v ly Ca+2 t ngoi t bo vo. Sau khi tr li tn hiu bn ngoi, t bo phn ng li s tng nng Ca+2 theo hai c ch. C hai u nhm mc ch gim s lng ion Ca+2 trong t bo cht tr li nng ban u. Trong c ch th nht, IP3 nhanh chng b kh nhm phosphate (chuyn sang dng khng hot ng) bi enzym phosphatase c hiu. Do , qu trnh gii phng Ca+2 ra khi ER b ngng. Vi c ch th hai, ion Ca+2 nhanh chng c bm ra khi t bo bng bm Ca+2-ATPase.

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Cng vi IP3 lm thay i nng Ca+2 trong t bo cn c sn phm khc ca PIP2 l diacylglycerol. Mt trong nhng vai tr quan trng m diacylglycerol m nhim trong qu trnh truyn tn hiu l hot ho protein kinase serine/threonine. Enzym c hot ho bi diacylglycerol c gi chung l kinase C (hoc PKC) do hot tnh ca n ph thuc vo Ca+2. Khi nng Ca+2 trong t bo cht tng ln do tc dng ca IP3, protein kinase C bin i cu trc gip n di chuyn trong t bo cht n st mng sinh cht. n c hot ho bi ion Ca+2 v diacylglycerol. ng vt c v, c t nht 8 dng kinase C khc nhau. Chng gy phosphoryl ho protein ti cc acid amin serine hoc threonine c hiu. C cht ca kinase C rt khc nhau, ph thuc vo tng loi t bo. Nng kinase C cao nht c tm thy trong no, chng phosphoryl ho cc knh ion trong t bo thn kinh dn n thay i tnh cht ca mng sinh cht v kch thch mng ny.

Hnh 5.20: Hai nhnh truyn tn hiu ca inositol phospholipid Th th lin kt vi protein G (Gq) gy hot ho phospholipase C-. Enzym ny ct PIP2 to IP3 v diacylglycerol. Sn phm th hai li hot ho kinase C. Hai enzym phospholipase C- v kinase C tan trong nc. Khi di chuyn n st mt trong ca mng nguyn sinh cht, hai enzym ny mi bc l hot tnh ca mnh.

Trong rt nhiu t bo, hot ng ca kinase C lm tng qu trnh phin m ca mt s gen c bit. Cc gen c th c hot ho theo hai con ng khc nhau. Trong con ng th nht, kinase C hot ho cc protein kinase trong chui cc phn ng phosphoryl ho, dn n hot ho cc protein iu khin gen. Trong con ng th hai, hot ng ca kinase C gy phosphoryl ho protein c ch, nh cc protein iu khin c gii phng ra dng t do trong t bo cht, di chuyn vo nhn v bt m cc gen c hiu. Cc con ng truyn tn hiu ca inositol phospholipid c m t trn hnh 5.20. Mt s cht khi c b sung vo mi trng nui cy t bo c th khi ng cc con ng . V d, IP3 c hot tnh khi c mt ca phc Ca+2-inophore, cht ny cho php nng Ca+2 tng ln trong t bo cht. Ngoi ra phorbolester cng gy ra nhng tc dng tng t diacylglycerol. Cht ny lin kt vi kinase C v hot ho enzym. Trong cc t bo, cc c ch truyn tn hiu c kt hp cng nhau to ra phn ng ti u nht cho t bo. V d, kch thch cc t bo nui cy bc vo phn chia, c hai cht Ca+2-inophore v cht hot ho kinase C u c thm vo mi trng nui cy. Nu ch thm mt trong hai cht ny th t bo khng phn chia.

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5.5.3 Calmodulin- protein to phc vi Ca+2 trong t bo


Nng Ca+2 trong t bo cht lun phi nh hn 10-7 M v khng bao gi vt qu 5x10-6 M ngay khi cc knh dn truyn Ca+2 m. Chnh v vy tng cng kh nng kim sot nng Ca+2, trong t bo cht cn tn ti cc protein lin kt c hiu v c i lc cao vi Ca+2, m bo cho s tng nng Ca+2 ch xy ra trong khong thi gian ngn, chnh xc. Trong cc t bo eukaryot, protein tng tc c hiu vi Ca+2 hay gp nht l calmodulin. Mt t bo ng vt c khong 107 phn t calmodulin, tng ng vi 1% tng s protein c trong t bo. Calmodulin c nhiu chc nng khc nhau, c th lin kt vi Ca+2 hoc lm trung gian cho mt s phn ng sinh hc. Phn t calmodulin l mt chui polypeptid gm 150 acid amin c 4 v tr c hiu tng tc vi Ca+2. Cu trc khng gian ca Calmodulin thay i khi n lin kt vi Ca+2. Phc cht Ca+2/calmodulin khng c hot tnh enzym. Phc ny lm nhim v kim sot hot tnh ca protein lin kt vi chng. Thng thng phc Ca+2/calmodulin tng tc vi nhng protein gi vai tr vn chuyn cc cht qua mng. Khi nng Ca+2 trong t bo tng cao v bt k mt l do g th bm Ca+2 c kch thch bi phc Ca+2/calmodulin bm Ca+2 ra khi t bo. Bn cnh , hot tnh ca mt s kinase hon ton ph thuc vo phc Ca+2/calmodulin. V th hot ng chnh ca phc l hot ho nhng kinase c bit ny. Chng c gi chung l CaM-kinase (Ca+2/calmodulin-dependent protein kinase). Protein CaM-kinase xc tc cho phn ng phosphoryl ho acid amin serine/threonine ca mt s protein c hiu. Cng ging nh trng hp ca cAMP, s tng nng Ca+2 t do trong t bo cht ph thuc vo protein c hiu m nhng protein ny b kim sot bi CaMkinase. c bit, CaM-kinase II c mt trong mi t bo ng vt v nhiu nht cc t bo thn kinh. Ti CaM-kinase II chim 2% tng s protein tp trung cc synape. Khi synape hot ng, dng ion Ca+2 vt qua knh in th Ca+2 (volgate-gated Ca+2 channels) vo bn trong t bo, kch thch t bo tit ra cc cht dn truyn thn kinh. Dng ion Ca+2 ny s hot ho CaM-kinaseII. n lt mnh, CaM-kinaseII phosphoryl ho tyrosine hydroxylase khin enzym ny c hot tnh. y l enzym kim sot tng hp cc cht dn truyn thn kinh. Nh , qu trnh tit v tng hp cc cht dn truyn c kch thch khi t bo thn kinh trng thi hot ho do nng Ca+2 tng. c im ni bt ca cc CaM-kinase l chng c th hot ng nh cc phn t nh: chng chuyn sang trng thi hot ng khi c mt ca phc Ca+2/calmodulin v gi c hot tnh ngay khi nng Ca+2 gim. l do sau khi b hot ho bi phc Ca+2/calmodulin, CaM-kinase phosphoryl ho cc protein khc v c kh nng t phosphoryl ho chnh n. Nh vy CaM-kinase t hot ho mnh. trng thi t hot ho, enzym CaM-kinase gi c hot tnh ngay khi khng c Ca+2. Nh thi gian hot ng ca enzym c ko di cho n khi phosphatase p o khin enzym mt tc dng (Hnh 5.21).

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Hnh 5.21: Hot ng ca CaM-kinase. Phn t enzym gm 12 tiu n v. Khi khng c phc Ca+2/calmodulin, enzym khng c hot tnh do vng qui nh hot tnh lin kt vi vng c ch. Khi phc Ca+2/calmodulin tng tc vi enzym, cu trc khng gian ca enzym thay i, enzym t phosphoryl ho vng c ch, gii phng vng c hot +2 tnh. T phosphoryl ho cho php ko di hot tnh enzym theo hai cch: Th nht, gi phc Ca /calmodulin +2 khng tch ra khi enzym cho n khi nng Ca trong t bo gim v nng cho php (t nht trong 10 giy). Th hai, enzym c duy tr trng thi hot ho khng ph thuc nng Ca+2 ngay c khi phc +2 Ca /calmodulin tch ra khi enzym. Hot tnh duy tr cho n khi phn ng phosphatase p o c phn ng phosphoryl ho. Kinase CaMII ng vai tr quan trng trong hot ng thn kinh. Chut b t bin mt trong cc protein ca khu dn truyn tn hiu m t trn hnh u mt tr nh v v tr khng gian (theo Alberts & cs, 2002).

5.6

Khuch i cc tn hiu bn ngoi t bo

Thng thng khi nng tn hiu thay i, tr li ca t bo cng thay i theo mt cch gn nh tuyn tnh. V d, phn ng ca t bo vi s thay i nng hormon xy ra c tnh cht ng bin. l do mi th th ch tng tc vi mt phn t hormon v hot ng ca cc gen lin quan l c lp vi nhau. Khi nng hormon tng, nng phc hormon-th th cng tng theo mt cch tuyn tnh. Do , s phc cht bm vo ADN hot ho gen cng tng theo tng t. Kt qu l tr li ca t bo vi hormon tng t l thun hoc khng i. Tuy nhin, trong mt s trng hp, tr li ca t bo thng xy ra t ngt khi nng tn hiu tng dn. nng nh hn ngng nht nh, t bo khng pht hin c. Nhng khi nng vt qua ngng, phn ng c th t ngay gi tr cc i. V sao nng tn hiu tng dn dn nhng tr li ca t bo xy ra rt t ngt, gn ging nh cch thc "tt c hay khng c g"? Mt trong nhng yu cu phn ng tun theo cch thc trn l cn c hai hay nhiu phn t (hoc mt phc cht) tng tc vi cc i phn t kch thch phn ng tr li. V d, trong mt s phn ng tr li kch thch ca hormon steroid, khng phi ch mt phc th th -hormon m phi c hai phc tr ln cng tng tc vi ADN hot ho mt gen. Do khi nng hormon va tng, gen b hot ho rt t ngt v t ngng hot ng cc i. C ch hot ho kinase A v calmodulin xy ra mt cch tng t. Nhiu ion Ca+2 cng tng tc vi calmodulin gy thay i cu trc khng gian ca protein ny khin n b hot ho rt nhanh. Nh vy, khi nng Ca+2 t do tng 10 ln th hot tnh ca calmodulin tng 50 ln. Phn ng tr li ca t bo cng xy ra theo cch thc t ngt khi tn hiu hot ho enzym th nht, ng thi c ch enzym th hai, m hai enzym ny xc tc cho hai phn ng

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c chc nng ngc nhau ca cng mt qu trnh truyn tn hiu. V d, mt tn hiu ng thi lin quan n hai phn ng: phn ng th nht gy phosphoryl ho nhng phn ng th hai li kh gc phosphate. Chng hn nh trong t bo c xng, vic tng nng cAMP va hot ho phosphorylase kinase va c ch phosphatase. Do phn ng phn gii glycogen xy ra nhanh, t ngt so vi vic tng nng cht kch thch ban u. Vi mt s v d in hnh v cch thc truyn tn hiu, chng ta hnh dung c phn no h thng cc knh dn truyn an xen vo nhau, kim sot qua li i vi nhau. Cc th th ni vi protein G c th gy hot ho hoc gy mt hot tnh mt cch gin tip cc enzym bm trn mng sinh cht hoc trn cc knh dn truyn ion. Mt s th th khc m nhn chc nng hot ho hoc c ch adenylyl cyclase lm thay i nng ca cht dn truyn tn hiu trung gian cAMP trong t bo. Mt s th th lin quan n hot ho phospholipase C-. Enzym ny thu phn PIP2 (phosphatidylinositol bisphosphate) to ra hai cht truyn tn hiu trung gian l IP3 (inositol trisphosphate)v diacylglycerol. Vai tr ca IP3 lm tng nng Ca+2 trong t bo cht bng vic kch thch gii phng Ca+2 t ER. Cn diacylglycerol c gi li trn mng sinh cht lm nhim v hot ho kinase C. Cc enzym kinase A, kinase C v kinase CaM gy phosphoryl ho cc protein c hiu cc acid amin serine hoc threonine, lm thay i hot tnh ca cc protein ny. Mi loi t bo cha cc protein c hiu c trng cho loi t bo . Chng m bo cho t bo tr li chnh xc vi tng kch thch. Tn hiu thng c khuch i ln rt nhiu ln, do phn ng tr li ca t bo c th xy ra rt nhanh v t ngng cc i. Tuy nhin, phn ng kt thc rt nhanh ngay khi ngng kch thch bn ngoi. l do bn thn phn t protein G c kh nng t thy phn GTP cng nh phn t IP b kh gc phosphate bi phosphatase rt nhanh vv... Vic thay i trng thi lin tc ca cc tn hiu th cp trong t bo m bo nng ca chng p ng vi kch thch bn ngoi m khng cn phi tng hp mi. Khi mt tn hiu bn ngoi t bo tng tc vi th th, th th c th hot ho ng thi nhiu phn t protein Gs. Mt phn t Gs li c th hot ho nhiu phn t adenylyl cyclase. Mi phn t Gs c th tn ti vi giy trng thi hot ho trc khi n thy phn GTP tr v trng thi bt hot. Thi gian ny cng cho phn t adenylyl cyclase tng tc vi Gs ang trng thi hot ha v xc tc to ra s lng ln cAMP t ATP (Hnh 5.22). Mt c ch khuch i tn hiu tng t xy ra trong con ng truyn tn hiu inositolphospholipid. Nng tn hiu bn ngoi t bo c 10-10 M kch thch to ra nng 10-6 M ca cc tn hiu th cp nh cAMP hoc Ca+2. Cc tn hiu th cp ny c chc nng hot ha cc enzym c hiu hoc cc knh dn truyn ion. Khi t bo ch tip nhn mt phn t tn hiu bn ngoi, hng trm phn t khc trong t bo c th b thay i cu trc, hot tnh hoc nng ...

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Hnh 5.22: Chui phn ng khuch i tn hiu c kch thch bi mt tn hiu ban u. Bc khuch i u tin i hi phn t tn hiu phi tng tc vi th th trong thi gian phc ny hot ho cc phn t Gs. Trong mt s trng hp khc, ligand phi tch ra khi phc rt nhanh cho qu trnh khuch i bt u xy ra (theo Alberts & cs., 2002).

Vi bt k chui truyn v khuch i tn hiu xy ra nhanh v nhy th u phi m bo s quay v trng thi cn bng ca tng phn ng trong chui khi ngng tn hiu kch thch. V vy trong t bo lun tn ti c ch rt hiu lc m bo vic phn hu cAMP, gim nng Ca+2 trong t bo cht cng nh gy mt hot tnh ca nhng enzym hoc protein truyn tn hiu trung gian b hot ha trc . C ch ny khng ch ngng phn ng tr li tn hiu m cn phi lp li rt nhanh trng thi tnh ging nh trc khi b kch thch. Ni chung, khi phn ng tr li vi kch thch xy ra nhanh th c ch gip t bo quay li trng thi tnh cng c thc hin mt cch mau chng tng t. 5.7 Truyn tn hiu qua cc th th ni vi enzym trn b mt t bo

Cng ging nh cc th th ni vi protein G, th th ni vi enzym thng l cc protein xuyn mng (trans-membrane protein). Nhng th th ny c phn nhn bit tn hiu nm pha ngoi mng. Tuy nhin, phn nm pha trong mng khng lin kt vi protein G m n c hot tnh enzym hoc tng tc trc tip vi enzym. C 5 loi th th ni vi enzym: 1/ Th th guanylyl cyclase xc tc cho phn ng to cGMP trong t bo cht. 2/ Th th kinase tyrosine gy phosphoryl ho cc acid amin tyrosine c hiu ca cc protein dn truyn tn hiu trung gian. 3/ Th th phi hp cng kinase tyrosine. 4/ Th th phosphatase tyrosine chuyn nhm phosphate khi acid amin tyrosine ca protein c hiu. 5/ Th th serine/threonine kinase gy phosphoryl ho cc acid amin serine hoc threonine ca mt s protein. Chng ta ln lt xt tng loi th th trn.

5.7.1 Th th guanylyl cyclase


Cc th th guanylyl cyclase s dng cGMP nh tn hiu trung gian tng t nh cc th th ni vi protein G s dng cAMP. Tuy nhin, th th va nhn bit tn hiu ng thi ng

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vai tr ca enzym xc tc phn ng tng hp cGMP. V d in hnh cho hot ng ca th th loi ny l tng tc gia th th vi tn hiu ANPs (atrial natriuretic peptides) c tit ra t cc t bo c trong tm nh ca tim khi huyt p tng. Kt qu cui cng ca tng tc ny l ion Na+ v H2O c thi ra khi t bo c trn. Thnh mch mu gin ra, nh huyt p gim. Phn th th nm ngoi mng t bo c v tr lin kt vi ANPs cn phn nm trong c hot tnh guanylyl cyclase. Khi ANPs lin kt vi th th, tn hiu c truyn vo bn trong t bo v phn th th c hot tnh enzym xc tc phn ng tng hp cGMP. Cc phn t cGMP lp tc hot ho cc protein kinase ph thuc cGMP (gi chung l cc Gkinase). Bng cch ny tn hiu c truyn vo trong t bo. Trong thc t, cc tn hiu guanylyl cyclase t gp trong cc nghin cu hot ng v chc nng ca t bo.

5.7.2 Cc oncogene v tn hiu dn truyn t th th tyrosine kinase


ng vt bc cao, t bo ch bc vo phn chia khi chng c kch thch bi cc factor tng trng do cc t bo khc tit ra. Tuy nhin i vi cc t bo ung th, chng phn chia lin tc ngay khi khng c cc tn hiu bn ngoi. Nh vy, chng khng chu s kim sot chung ca qu trnh sinh trng. Mt trong nhng nguyn nhn dn n s mt kh nng kim sot l do t bin xy ra nhng gen m cho protein tham gia con ng dn truyn tn hiu (v d cc protein Ras, Src...). Nhng gen khi hot ng bt thng dn n ri lon qu trnh pht trin, gy ung th c gi l cc oncogen. Bt k mt t bin no lin quan n vic to ra sn phm protein c hot tnh khng bnh thng trong con ng dn truyn tn hiu u c th khi ng qu trnh hnh thnh khi u. T bo bnh thng tr thnh t bo ung th s phn chia lin tc m khng yu cu cc tn hiu bn ngoi gi n. V d, oncogen erbB m cho th th ca factor EGF c vng qui nh hot tnh tyrosine kinase lun lun trng thi hot ho. T bo mang oncogen ny s phn chia lin tc. Ni chung, s phn chia v t chc thng do t bo mang oncogen m cho protein iu khin m protein ny lun lun c hot tnh. Ngoi ra, mt s hormon kch thch t bo bc vo phn chia khi lin kt vi th th ni vi protein G hn l vi th th tyrosine kinase.

5.7.3 Protein MAP kinase


Qu trnh phosphoryl ho tyrosine Ras khin protein ny c hot ho rt nhanh. Tyrosine b kh gc phosphate nh enzym c hiu tyrosine phosphatase v Ras b mt hot tnh khi GTP (lin kt vi Ras) b phn hu thnh GDP. kch thch t bo sinh si hoc bit ho, nht thit cc tn hiu ban u phi to nn chui cc phn ng xy ra lin tip truyn tn hiu vo trong nhn. H thng truyn tn hiu lin quan n mt lot phn ng phosphoryl ho serine/threonine. Phn ng xy ra trc s hot ho phn ng sau v chng u xy ra lu hn phn ng phosphoryl ho tyrosine. Mt nhm protein serine/threonine kinase gm t nht 5 thnh vin ng vai tr c bit quan trng trong chui phn ng truyn tn hiu. l cc protein kinase kch thch qu trnh phn bo mitose, c gi l cc protein MAP (Mitose Activator Proteins) hay l MAP kinase. Hot tnh kinase ca MAP ch t cc i khi c hai acid amin threonine v tyrosine ca n cng b phosphoryl ho. Hai acid amin ny ch cch nhau bi 1 acid amin. Kinase xc tc cho c hai phn ng phosphoryl ho trn MAP kinase c gi l MAP kinase-kinase. Vic yu cu c hai acid amin cng c phosphoryl ho m bo cho MAP kinase lun c gi trng thi khng hot ng tr khi b hot ho bi chnh MAP kinase-kinase (lu rng MAP kinase khng c kh nng t phosphoryl ho m n c hot tnh kinase ch khi c hai

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acid amin b phosphoryl ho bi MAP kinase-kinase). Bn thn MAP kinase-kinase c hot tnh khi cc serine/threonine ca n c gn gc phosphate. Phn ng c xc tc bi MAP kinase-kinase-kinase. n lt mnh, MAP kinase-kinase-kinase c hot ho c l nh tng tc vi protein Ras (Hnh 5.23).

Hnh 5.23: Chui cc phn ng dy chuyn phosphoryl ho serine/threonine c hot ho bi protein Ras hoc kinase C. Tn hiu c nhn bit u tin nh th th tyrosine kinase v c truyn thng qua Ras. (MAP kinase-kinase-kinase cn c gi l Raf khi tn hiu truyn t th th ni protein G qua kinase C) (theo Alberts & cs., 2002).

Mt khi MAP kinase hot ng, n truyn tn hiu i nh phosphoryl ha cc protein khc nhau gm cc kinase hoc cc protein iu khin. Thng thng, qu trnh phin m ca mt s gen xy ra sau vi pht khi c mt cc factor tng trng. Phc hp cc protein gi vai tr quan trng trong hot ho cc gen khi chng lin kt vi ADN c hiu. Khi c hot ho, MAP kinase di chuyn t t bo cht vo nhn v phosphoryl ho cc protein ca phc hp . Lc phc mi c kh nng tng tc vi ADN v bt m cc gen. 5.8 Tyrosine kinase phi hp vi th th. Th th Tyrosine phosphatase

C nhiu th th trn b mt t bo, v d nh cc th th c hiu cho khng nguyn trn t bo lympho B v T, khng phi l protein ca knh ion cng khng phi l th th ni vi protein G. Hn na chng khng c vng mang hot tnh kinase. Chng hot ng nh phi hp vi cc tyrosine kinase. Khi th th lin kt vi ligand, lp tc tn hiu c truyn n tyrosine kinase v cc enzym ny s phosphoryl ho cc protein khc truyn tip tn hiu

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i. Nhng tyrosine kinase kt hp cng th th c nghin cu nhiu nht l nhm Src hoc Janus. Nhm tyrosine kinase Src gm t nht 8 protein khc nhau nhng chng u cha vng SH2, SH3 v chng u nm pha trong mng t bo cht. Chng c c nh vo mng nh tng tc vi chui lipid v tng tc vi th th. Mi protein trong nhm c th lin kt vi nhiu loi th th khc nhau. C th l th th tyrosine kinase hoc cc th th khng phi l tyrosine kinase. Do tn hiu bt u t cc th th ny c th thng qua Src c truyn i theo nhiu con ng khc nhau. Nh trn nu, tyrosine b phosphoryl ho lp tc li b kh gc phosphate rt nhanh nh tyrosine phosphatase. Cc enzym ny ch kh gc phosphate tyrosine ca mt s protein. Tnh c hiu m bo nng tyrosine mang gc phosphate trong t bo rt nh. Nh vy, cc enzym ny c vai tr rt quan trng trong cc con ng truyn tn hiu. Bn cnh , tyrosine phosphatase cn ng vai tr th th vi mt s ligand c bit. Mt v d in hnh cho th th tyrosine phosphatase l protein CD45 c tm thy trn b mt bch cu gi vai tr ch yu trong qu trnh hot ho cc lympho B v T bi cc khng nguyn. CD45 l mt glucoprotein nm xuyn qua mng, vng c hot tnh tyrosine phosphatase nm pha trong mng. Khi CD45 lin kt vi cc khng th (bn ngoi mng t bo), vng c hot tnh c hot ho, xc tc cho phn ng chuyn nhm phosphate khi tyrosine ca mt s protein c hiu, thng l cc kinase. Nh , nhng protein ny c hot tnh tip tc gy phosphoryl ho, truyn tn hiu vo trong t bo.

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Chng 6 CHU TRNH V PHN CHIA T BO


Hu ht cc t bo eukaryot u tri qua qu trnh phn chia m kt qu l cc nhim sc th c nhn i v phn ly v hai t bo con ging ht nhau. y l mt qu trnh gm nhiu giai on, tt c u c kim sot rt cht ch, m bo s pht trin chnh xc v ng b trong mt c th a bo hon chnh. Hu ht cc sai lch khng tun th s kim sot u dn n hin tng t bo cht hoc pht trin thnh ung th. S phi hp cc k thut sinh ho, di truyn v c bit l ADN ti t hp cho php nghin cu chu trnh t bo cc i tng sinh vt khc nhau v rt ra c nhng nt c trng chung cho mi t bo eukaryot. c bit kt qu nghin cu xc nh c cc phc protein lm nhim v kim sot hai giai on chnh ca chu trnh phn bo l ti bn ADN (pha S) v phn chia t bo (pha M). Trong chng ny, chng ta quan tm n cc th nghim, cc k thut c p dng nghin cu tm hiu bn cht v hot tnh ca cc phc protein. T chng ta hiu c c ch kim sot chung chu trnh phn bo t bo eukaryot.

6.1

Nhng c tnh c bn ca chu trnh t bo

Chu trnh t bo l qu trnh t bo sinh sn bng vic nhn i mi vt cht cha trong t bo v phn chia thnh hai t bo mi. i vi sinh vt n bo nh vi khun; nm men, mi chu trnh t bo u to thm c th mi. Sinh vt a bo i hi rt nhiu ln phn chia t bo to nn c th hon chnh. Ngoi ra, s phn bo vn lun xy ra trong c th trng thnh i mi hoc thay th cc t bo b tn thng hoc b cht (do tc ng bn ngoi hay do chnh chng trnh gy cht t bo c lp trnh sn trong b my di truyn). Mi mt giy, c th chng ta to ra hng triu t bo mi duy tr trng thi cn bng. V mt l do no , v d nh c th b chiu x mnh, phn chia t bo b ngng s dn n t vong trong vi ngy. Chu trnh t bo xy ra bt c sinh vt no cng i hi s nhn i nhim sc th v cc bo quan. Cc qu trnh xy ra trong nhn (nhn i ADN) v trong t bo cht (to cc bo quan mi) phi hp cht ch nhp nhng trong sut mt chu trnh. C ch no v yu t g quyt nh iu ? Trc y cc hin tng lin quan n chu trnh phn chia t bo nh nhn i ADN, di chuyn cc nhim sc th v hai cc phn bo c nghin cu kh k v mt t bo hc. Trong nhng nm gn y, cc nh khoa hc quan tm n h thng kim sot chu trnh t bo (cell-cycle control system). Kt qu phn tch cc protein tham gia h thng ny cho thy chng c bo ton trong qu trnh tin ho. Mt thnh phn ca h thng kim sot nm men c th thc hin chc nng ca mnh mt cch hon ho trong cc c th khc nh ngi v ngc li. Chng ta xem xt n h thng kim sot chu trnh t bo v s phi hp gia cc phn ng sinh hc thc hin s kim sot . Chu trnh t bo sinh vt eukaryot c xem gm 4 giai on hay cn gi 4 pha (Hnh 6.1). Ba giai on G1, S v G2 c gi chung l gian k (interphase). Trong pha G1 ngay

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sau pha M (mitose), t bo hon thin kch thc ring m bo mi iu kin cn thit tip tc pha S (nhn i ADN). pha G2 sau pha S, t bo kim tra qu trnh nhn i ADN, m bo qu trnh xy ra khng c bt k mt sai st no. Sau t bo mi chuyn sang pha M.

Hnh 6.1: Chu trnh t bo eukaryot gm 4 pha M, G1, S v G2. T bo pht trin trong giai on gian k (gm cc pha G1, S, G2) v phn chia trong pha M.

Chu trnh t bo xy ra nhanh hay chm ph thuc vo tng loi t bo ngay trong mt c th. ng vt c v, vi nhng m sinh trng nhanh, ton b chu trnh phn bo ch xy ra trong khong 12 n 24 gi. c bit chu trnh phn bo xy ra rt nhanh giai on pht trin phi sm (khong 8 n 60 pht). Lc hai pha G1 v G2 hu nh khng xy ra v ton b chu trnh t bo ch c hai pha S v M. Khi khng c nhu cu phn chia lin tc, t bo ngng li pha G1 v sau bc sang mt trng thi c bit gi l Go. pha Go, t bo gi nguyn trng thi trong vi ngy, vi tun, vi nm thm ch trong sut c thi gian sng. Chu trnh t bo c nhng im then cht - gi l cc im kim tra (checkpoints) quyt nh s chuyn tip gia cc pha. Ton b chu trnh c iu khin bi trung tm kim sot. Trung tm c nhim v x l tn hiu din bin trong tng pha gi v. Ti cc im kim tra, tn hiu phn hi t pha trc s c trung tm phn tch quyt nh t bo c chuyn sang pha tip theo hay khng. Chng ta cn phn bit s khc nhau gia cc thnh phn ch cht tham gia trung tm kim sot vi ton b nguyn liu cn thit cu thnh nn b my thc thi cc giai on c bn ca chu trnh. V d, cc phc ca trung tm kim sot c chc nng iu hnh din bin ca pha S s khc vi cc enzym, cc protein tham gia qu trnh ti bn ADN giai on ny. iu ny tng t nh trong mt doanh nghip c phn: hi ng qun tr kim sot chung hot ng ca ban gim c; ban gim c iu hnh c th cc mc tiu do hi ng qun tr ra. H thng kim sot bao gm mt s protein gi vai tr trng yu trong tng pha v phi hp gia cc pha, c bit cc im kim tra. Nhim v ca h thng kim sot l m bo tnh chnh xc ca qu trnh nhn i ADN, phn ly nhim sc th v hai cc v hnh thnh hon ho hai t bo con. Khi tip nhn cc tn hiu khng bnh thng (t cc din bin trong qu trnh phn bo hoc t mi trng bn ngoi), h thng kim sot c th iu khin chu k phn bo chm hoc dng li mt im kim tra. im dng trong pha G1 c gi l im bt u "Start" ( nm men) hoc n gin gi l im kim tra G1. Khi h thng kim sot vt qua im G1, chu trnh phn bo chuyn sang pha S. Ttng t nh pha G1, khi h thng vt qua im kim tra G2, chu trnh phn bo c php bc vo pha M (Hnh 6.2).

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Hnh 6.2: Cc tn hiu mi trng v tn hiu phn hi t cc din bin xy ra trong qu trnh phn chia cho php t bo vt qua cc im kim tra G1 v G2. Ti cc im G1 v G2, h thng kim sot phn tch cc iu kin cn v trc khi cho php t bo chuyn sang pha tip theo.

Cc th nghim u tin da vo hin tng dung hp cc t bo ng vt nui cy cho php xc nh cc yu t quan trng ca h thng kim sot. T bo nui cy cc pha khc nhau c th dung hp vi nhau to t bo mi. V d, t bo gian k c th dung hp vi t bo pha M. Lc trong t bo dung hp, nhim sc th t t bo gian k tr nn co c li, mng nhn bao bc xung quanh b v. Hin tng ny xy ra tng t nh nhn t bo chun b bc vo phn chia. Nh vy trong t bo cht c tn ti yu t gy ra nhng bin ng trn. Mt lot cc th nghim dung hp t bo cc pha khc nhau, kt hp vi nghin cu chu trnh phn bo nm men v k thut phn tch non bo trng ch pht hin ra cc phc protein ca h thng kim sot phn bo. H thng kim sot phn bo hot ng da vo phc d dimer (heterodimer) gm hai h protein gi vai tr ch o. l cc protein kinase ph thuc cyclin (cyclin-dependent protein kinase), vit tt l Cdk v nhm protein cyclin. Cdk hot ho chui cc phn ng nh kh nng phosphoryl ho protein c hiu ti cc acid amin serine/threonine. Tuy nhin kh nng ny ca Cdk c kim sot bi cyclin. Mi Cdk c th lin kt vi nhiu cyclin khc nhau to thnh phc Cdk-cyclin c hot tnh kinase c hiu, tc l quyt nh protein ring bit s b phosphoryl ho bi phc. R rng ba s kin: to phc Cdk-cyclin; hot ho phc v phn r phc l nhng bc chuyn rt quan trng iu khin ton b chu trnh phn bo. Cc kt qu nghin cu cho thy h thng kim sot gn lin vi s hnh thnh v hot ho phc Cdk-cyclin ba giai on: pha G1 (phc G1Cdk), pha S (phc SCdk) v pha M (phc MCdk). Khi t bo c kch thch chun b ti bn ADN th phc G1Cdk hot ng nhm hot ho cc yu t phin m (transcription factor) i vi nhng gen m cho cc enzym cn cho ti bn ADN v cc protein cn cho to phc SCdk. Khi mi c hnh thnh, cc phc SCdk c gi dng khng c hot tnh bi cc protein c ch. Ch n cui pha G1, protein c ch b phn hu, phc SCdk c gii phng hot ho t bo bc sang pha S. Mt cch tng t, phc MCdk c tng hp trong pha S v trong pha G2, nhng chng ch c hot tnh sau khi ton b nhim sc th ti bn trn vn. Cc phc MCdk thc y qu trnh co c ca nhim sc th, ph v mng nhn, xut hin tm ng, thoi v sc v sp xp cc nhim sc th mt phng xch o. Ch khi hot tnh ca phc MCdk b gim th nhim sc th mi gin ra, mng nhn xut hin xung quanh hai nhn con v t bo phn chia thnh hai t bo mi.

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Din bin mt chu trnh t bo c thc hin theo ng trt t t G1 sang pha S, n G2 v kt thc bi giai on phn bo Mitosis. Tin trnh ny ch xy ra theo mt chiu m khng th o ngc li. c th bc cao, yu t ngoi bo (mitogen) kch thch t bo bt u chu trnh phn chia bng vic tng hp v hot ho cc phc G1Cdk. Cc protein cyclin c th phn lm hai nhm chnh: cc cyclin lin kt vi Cdk trong pha G2 t bo bc vo mitose (nn gi l mitose cyclin) v cc cyclin lin kt vi Cdk trong pha G1 chu trnh chuyn sang pha S (nn cn gi l G1 cyclin) (Hnh 6.3). Nhng nghin cu u tin trn nm men cho thy mt nhm protein Cdk tham gia h thng kim sot chu trnh t bo c hai im kim tra G1 v G2. Tuy nhin ng vt c v, mi im kim tra c kim sot bi t nht mt nhm Cdk ring bit. Hot tnh ca Cdk gp phn quyt nh t bo tn ti mt pha nht nh hoc chuyn pha. V mt l do no m nhm Cdk ph trch mt im kim tra c hot ho th t bo bc sang pha tip theo bt chp cc nguyn liu cn thit (protein, ADN...) hon thin pha c y hay khng.

Hnh 6.3: Cc thnh phn c bn (Cdk v cc loi cyclin) ca h thng kim sot chu trnh phn bo. Hot tnh ca Cdk ph thuc vo s c mt ca cyclin (theo Alberts & cs., 2002).

t bo ng vt c v, hot ng ca h thng kim sot im G2 c nghin cu kh chi tit. Thc nghim nhn thy nng mitose cyclin tng dn trong pha G2. Cyclin ny lin kt vi Cdk to phc c gi l yu t khi ng phn bo MPF (Mitosis Promoting Factor). Tuy nhin, phc MPF dng khng hot tnh. Phc chuyn sang dng hot ng khi b phosphoryl ho ti mt s v tr c hiu. Mt khi xut hin phc MPF dng hot ng, nng MPF c hot tnh tng nhy vt. S thay i t ngt hm lng MPF lin quan n c ch iu khin phn hi tch cc (positive feedback mechanism). iu ny c ngha, s xut hin cc enzym kinase hoc phosphatase (c hot ho bi dng hot ng ca MPF) s thc y s chuyn trng thi ca MPF. Do , khi xut hin dng hot ng MPF th lng enzym c hot ho tng khin cho lng MPF chuyn t dng khng hot tnh sang

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dng hot ng cng tng theo. Ni cch khc, cng c nhiu MPF trng thi hot ng th lng enzym c hot tnh gy hot ho MPF cng tng. Khi nng MPF dng hot ng t n mt gi tr ngng, mt lot cc phn ng sinh hc xy ra khin t bo bc sang pha M. Khi chu trnh t bo bin gii gia metaphase v anaphase, MPF b mt hot tnh t ngt do mitose cyclin b kh gc phosphate. Lp tc t bo ra khi pha M. Nh vy, phn ng chuyn gia hai trng thi hot ng v khng hot ng ca phc cycline-Cdk trong h thng kim sot tng ng vi chuyn pha trong chu trnh t bo. C ch hot ng ca h thng kim sot ti im G1 cha c nghin cu k nh im G2. Tuy nhin, nhng nguyn tc chnh u c tun th c hai im ny. iu ng lu l cc phn ng xy ra sau cc im kim tra G1 (trong pha S) v G2 (trong pha M) hon ton khc nhau.

6.2

Chu trnh t bo giai on pht trin phi sm

bc vo phn chia, t bo phi pht trin t kch thc nht nh, s lng ADN cng nh mi thnh phn khc trong t bo cht phi c nhn i. Thi gian hon thin mi cng tc chun b ny lu hay nhanh ph thuc vo mi trng xung quanh. Do cc c ch kim sot chu trnh t bo phi m bo sao cho mi ch tiu cn thit c thc hin trn vn, chnh xc theo ng trt t trc khi t bo bc vo phn chia to thnh hai t bo con. Trng hp c bit t bo phn chia khng tri qua bn pha G1, S, G2 v M xy ra giai on pht trin phi sm. Lc t bo phn chia lin tc m khng tun th qui nh cht ch ca cc c ch kim sot. Trong giai on ny phn bo xy ra nhanh v hu nh khng dng li cc im kim tra G1 v G2. Thc cht chu trnh t bo lc ny ch thc hin nhim v c bn nht l nhn i genome v phn ly chng v hai t bo con. Chng ta hy xt xem yu t no gi vai tr quan trng trong h thng kim sot chu trnh t bo i vi pht trin phi cng nh hot ng ca chng lin quan nh th no n cc giai on c th ca phn bo. Cc kt qu t c kh chi tit khi nghin cu s pht trin ca trng ch sau th tinh. Trng ch l mt t bo khng l hnh cu, ng knh t ti vi mm v cha lng t bo cht nhiu gp 100.000 ln so vi cc t bo bnh thng trong c th. Non bo phi tri qua phn bo gim nhim pht trin thnh trng chn (trng c kh nng th tinh). Nh chng ta bit, non bo ch dng li pha G2 hay cn gi l prophase ca gim nhim (meiosis I). im dng ny nm trc pha M v tng ng vi im kim tra G2 trong chu trnh phn bo thng thng. pht trin thnh trng chn, hormon s tc ng vo non bo khin n vt qua im dng G2 bc vo phn chia (meiosis II). Lc trng chn (Hnh 6.4). Sau khi th tinh, t bo trng phn ct lin tc m khng pht trin v kch thc to hng nghn t bo mi ngy cng nh. Ln phn ct u tin ko di khong 90 pht, tip theo l 11 ln phn ct (30 pht /ln) to 4096 t bo mi (212) trong vng khong 7 gi. Trong mi ln phn ct, pha M chim 15 pht v pha S chim 15 pht, cn hai pha G1 v G2 khng xy ra. K thut phn tch non bo ch (xenopus oocyte assay technique) cho php ly t bo cht ca trng chn cha th tinh tim vo non bo. Lp tc non bo khng dng pha G2 m chuyn sang pha M v pht trin thnh trng chn. R rng khng cn kch thch bi hormon progesteron m vn to c trng chn. Nh vy, phn chia t bo trng ch trong giai on u tin c kim sot bi c ch ph thuc vo t bo cht. Phng php ny cho php xc nh c tc nhn tn ti trong t bo cht ca trng chn ng vai tr quyt nh t bo chuyn sang pha M. Tc nhn ny c gi l tc nhn kch chn do n quyt nh s thnh thc ca non bo thnh trng chn (maturation promoting factor).

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Hnh 6.4: S hot ho trng ch Xenopus. X l vi hormon progesteron khin non bo thc hin gim nhim th nht v pht trin thnh trng chn. Th tinh gip trng ny hon thnh ln gim nhim th hai v bc vo chu trnh phn chia u tin ca phi (theo Alberts & cs., 2002).

Nghin cu din bin xy ra t khi trng chn n th tinh v bc vo phn ct cho thy tc nhn kch chn c hot tnh mnh khi non bo chuyn t G2 sang metaphase (meiosis I) v t meiosis I sang meiosis II. Hot tnh ny gim ngay khi trng va th tinh nhng sau li tng mnh khi trng th tinh bc vo ln phn ct u tin, bt u qu trnh pht trin phi. Tri qua 12 ln phn ct lin tip, hot tnh ca tc nhn kch chn bin i theo chu k m cc i ng vi thi im t bo bc vo phn bo (Hnh 6.5).

Hnh 6.5: Hot tnh MPF dao ng theo gim nhim (meiosis) v nguyn nhim (mitosis) trong non bo v hp t bt u bc vo phn ct.

Vai tr ca tc nhn kch chn c pht hin u tin khi nghin cu qu trnh pht trin trng ch sau th tinh. Nhng th nghim tip sau tin hnh vi cc t bo phn chia nguyn nhim cc loi sinh vt khc nhau khng nh tm quan trng ca yu t tn ti trong t bo cht tham gia kim sot chu trnh t bo. V d, th nghim vi cc t bo ng vt nui cy b x l vi colchicine (cht c ch phn bo, ngn cn s hnh thnh cc si thoi v sc) cho thy t bo cht chit ra t nhng t bo b x l c tc dng lm cho non bo tr thnh trng chn. Nh vy, yu t thc y phn chia nguyn nhim (mitosis promoting factor-MPF) t bo soma nui cy cng c hot tnh tng t yu t khi ng chn (maturation promoting factor) t bo non. Ngoi ra, cng vi phn tch non bo, k thut dung hp t bo cho php mt t bo pha M c dung hp vi t bo giai on gian k. Nh nhn t bo giai on gian k c a vo t bo cht ca t bo khc pha M. Kt qu cho thy nhn bc vo giai on ti bn v t bo phn chia. Nh vy tc nhn tn ti trong t bo cht kch thch qu trnh phn bo. Ni cch khc th mitosis promoting factor c mt trong t bo cht c kh nng thc y t bo soma chuyn t giai on gian k sang pha M, tc l kch thch t bo bc vo phn chia nguyn nhim. Tp hp cc kt qu nghin cu cho php a n kt lun maturation promoting factor cng chnh l mitosis promoting factor. iu th v l c hai khi nim ny c cng cch vit tt l MPF.

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Hnh 6.6: Hai tiu phn hnh thnh nn yu t MPF: Cdk v cyclin.

Kt qu tinh snh v phn tch hot tnh cho thy MPF l phc c hot tnh kinase gy phosphoryl ho acid amin serine/threonine ca mt s protein c bit. Phc MPF gm hai tiu phn: tiu phn c hot tnh enzym chnh l protein kinase ph thuc cyclin v tiu phn mitose-cyclin kim sot hot tnh kinase (Hnh 6.6). Phc MPF c hot tnh cc i trong pha M v cc tiu gian k i vi mi t bo eukaryot t nm men n ng vt c v. V vy, iu ng quan tm trc ht l bit c thi im xut hin v vai tr c th ca mitose cyclin trong vic kim sot hot tnh ca phc MPF.

6.3

Protein cyclin

Protein cyclin lin quan n pha M trong qu trnh phn bo c tinh sch ln u tin t phi cu gai (sea urchin embryo). Trng cu gai va th tinh c vi acid amin nh du ng v phng x. Protein tng s c tch chit cc thi im cch u nhau (10 pht) tnh t khi bt u . in di ng thi cc mu c th pht hin c nhng protein mi v din bin nng ca chng trong giai on pht trin phi sm. Trong s nhng protein ny ch c mt protein c nng bin i theo chu trnh phn chia t bo: nng cc i khi bt u pha M v t ngt gim xung cc tiu trong giai on anaphase. Protein c bit ny c gi l cyclin B. K thut tch dng v ti t hp ADN phn lp c ADNc tng ng vi gen m cho cyclin B cu gai. on ADNc ny c dng lm u d lai Southern vi ngn hng ADNc ca trng ch. Nh phn lp c dng ADNc m cho cyclin B ca Xenopus. Khi a ADNc ny vo vector biu hin th thu c protein. a protein vo th to khng th. iu th v l khng th ny tng tc vi MPF tch chit t trng ch trong php lai Western. Nh vy, cyclin B chnh l tiu phn ca phc MPF. Vy th cyclinB kim sot hot tnh ca MPF nh th no? Thc nghim tin hnh tht trng ch lm hai phn ngay sau khi trng va th tinh cha bc vo ln phn ct th nht: mt phn c nhn v phn kia cha t bo cht. Phn c nhn s tip tc phn ct lin tc to ra cc t bo nh dn trong khi phn th hai cng tri qua cc bin ng c tnh chu k. iu ny cng c quan st thy rt r trng cu gai sau khi th tinh b ly nhn i nhng vn tip tc phn ct lin tip to cc t bo nh hn v tt c chng u khng c nhn. Nh vy, yu t thc y phn bo tn ti trong t bo cht v hot tnh ca n khng ph thuc vo nhn (ADN). Vy th yu t ny c d tr trong trng trc khi th tinh hay l c tng hp mi trong giai on pht trin phi sm? Phn tch dch chit t trng ch cha th tinh, thc nghim pht hin c protein nonhistone, histone, cc enzym lin quan n ti bn ADN v mi thnh phn cn thit tng hp protein nh ribosome, ARNm. V t bo c th phn chia khi khng c nhn, tc l t bo c th khng s dng n cc thnh phn cn thit cho ti bn ADN. Vy th t bo c s dng cc thnh phn lin quan n qu trnh tng hp protein khi phn chia hay khng?

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Th nghim nhim sc th ca tinh trng (n) pha gian k vi dch chit t trng ch cha th tinh cho php quan st mt lot din bin xy ra vi b nhim sc th n bi. u tin, mng nhn hnh thnh xung quanh nhim sc th to thnh nhn n bi. Tip n, nhim sc th co c li, mng nhn b v ra v ADN tinh trng ti bn thnh lng bi (2n). Sau , tt c cyclin B trong dch chit t ngt b phn hy tng t nh t bo giai on anaphase (mitose). Lc ny nhim sc th gin ra v mng nhn li xut hin. C qu trnh ny c lp li u n vi chu k khong 20 pht. Cui mi chu k, cyclin B b phn hy. Vo u chu k tip theo, lng cyclin B tng dn do c tng hp mi t ARNm tng ng d tr sn trong trng. Khi hm lng cyclin B t n gi tr cc i th nhim sc th bt u co c, mng nhn t gy vv... R rng t bo thc hin phn ng tng hp mi protein t ARNm cung cp cyclin B cn thit cho phn chia. S tng hm lng cyclin B t l vi s tng hot tnh ca MPF. Khi s dng cycloheximide, cht c ch qu trnh tng hp protein th cyclin B khng c to thnh. ng thi cng khng pht hin c hot tnh ca MPF v khng quan st c hin tng nhim sc th co c cng nh mng nhn b t gy. Ngoi ra, khi x l dch chit t trng cha th tinh vi RNase nng thp nhm phn hy ton b ARNm m khng lm nh hng n ARNr v ARNt (hai loi ARN ny ch b phn hy bi RNase nng cao), th mng nhn vn xut hin bao quanh nhim sc th tinh trng v ADN vn c ti bn. Tuy nhin, sau cc din bin tng t nh phn chia t bo khng lp li na. Nu b sung thm ARNm m cho cyclin B vo th cc chu k bin ng tun hon vi thi gian 20 pht li din ra. Kt qu ny chng t ch c mt loi ARNm duy nht c mt trong th nghim (ARNm cyclin B), c dng lm khun tng hp cyclin B. cui phn bo mitose, cyclin B b bin tnh thng qua ubiquitin, tc l cyclin B c mang on peptide tng tc vi ubiquitin to tn hiu nhn bit cho protease. Nu trong th nghim trn, b sung ARNm cyclin B c mang t bin phn m cho tn hiu nhn bit sao cho cyclin B khng b gn ubiqutin, th cyclinB khng b phn hy bi protease v quan st thy nhim sc th khng gin ra v mng nhn khng xut hin. R rng, cyclin B c tng hp mi (de novo) c vai tr quyt nh hot tnh ca MPF. Hm lng cyclin B nhiu hay t ko theo s tng hay gim hot tnh ca MPF. Nh vy cyclin B quyt nh hot tnh ca MPF v kim sot mi din bin xy ra ngay sau khi trng th tinh. Nhng kt qu thu c vi cc t bo ng vt khc tm c t nht ba loi cyclin c chc nng tng t cyclin B trng ch. Chng c gi chung l mitose cyclin. S tng hp cc cyclin ny cng quan trng nh s phn hy chng t bo bc vo hoc ra khi pha M. Thng thng, cyclin b phn hy t ngt bc chuyn tip gia metaphase v anaphase. Khi cyclin b t bin sao cho n vn cn kh nng kch thch hot ng ca MPF nhng khng b phn hy do thiu on peptide c hiu tung tc vi ubiquitin th t bo bc vo pha M nhng khng th chuyn t pha M sang G1. R rng, hot ho phc MPF bc vo pha M i hi tng hp cyclin nhng bt hot phc MPF bc sang pha G1 li yu cu phn hu cyclin. Cyclin l thnh phn quan trng ca MPF c mt trong mi t bo eukaryot. C nhiu loi protein cyclin c m bi cc gen trong cng mt h gen. iu khin t bo bc vo pha M, phc MPF gy ra nhiu thay i nh nhim sc th co c li, mng nhn b ph v v kt cu khung t bo phi c t chc li to thoi phn bo. Tt c nhng thay i ny ph thuc vo hot tnh kinase ca MPF gy phosphoryl ho mt s thnh phn c bn to nn cu trc t bo. V d, cu trc nhn b ph v khi protein lamin ca nhn b phn r. Phn ng ny c xc tc bi MPF gy phosphoryl ho serine ca lamina. Khi lamina khng c cc serine c bit ny (do gen m cho lamin b t bin), chng s khng b phn hu khi t bo mitose, mc d mng nhn t gy to ra cc nang v cc din bin trong pha M vn xy ra bnh thng.

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Protein histone H1 c chc nng "ng gi" ADN thnh cu trc nucleosome cng b phosphoryl ho bi MPF. C l phn ng ny dn n vic nhim sc th co m li. Ngoi ra MPF cn gy phosphoryl ho mt s protein tng tc vi si vi ng microtubule (dng to thoi phn bo). nm men, Cdk cn c gi l Cdc (cell-division-cycle). C hai thnh phn mitose cyclin v Cdc2 kinase ca MPF u c th lin kt vi tm ng. C th mitose cyclin nhn bit c cc thnh phn ca tm ng v gip cho Cdc2 lin kt ng v tr. Cyclin B v cc cyclin chc nng tng t nh cyclin B b phn hy phase mun anaphase khi cc nhim sc t tch nhau ra v c ko v hai cc. Pha u NH2 ca hu ht cyclin ny u c on peptide nhn bit bi ubiquitin. on ny c gi l hp phn hy (destruction box). Nng cyclin B gim khin t bo nhanh chng ra khi pha M.

6.4

Nm men v h thng kim sot chu trnh t bo

Nhng th nghim tin hnh trn trng ch lm sng t vai tr ca cyclin B trong phc MPF. Tuy nhin cyclin ch l tiu phn lm nhim v iu khin cn hot tnh ca phc do tiu phn protein kinase m nhim. Nhng th nghim nghin cu bn cht cc kinase v s thay i hot tnh ca chng trong chu trnh t bo, c bit pha S v M c tin hnh trn nm men ny chi S.cerevisiae v nm phn chia S.pombe. T bo S.cerevisiae dng hnh cu, sinh sn bng phng thc ny chi. T bo con hnh thnh nh hn t bo m, do chng phi tng trng t n kch thc nht nh trc khi tip tc phn chia. T bo S.pombe c dng hnh que vi chiu di tng dn trong qu trnh pht trin. Khi bc vo phn chia, t bo s phn chia lm hai phn c kch thc nh nhau. Dung hp t bo v phn tch non bo l cc k thut chnh c p dng nghin cu i vi trng ch, trong khi nghin cu nm men lin quan n nhng k thut di truyn nhm sng lc v phn tch cc dng t bin khng c kh nng vt qua cc im kim tra trong chu trnh t bo. Khc vi cc t bo giai on pht trin phi sm, t bo nm men cng ging nh a s cc t bo trong c th eukaryot u phi pht trin t kch thc nht nh mi bc vo phn bo mitose. Thi gian t bo tn ti giai on gian k chim phn ln chu trnh t bo. c bit hai pha G1 v G2, kim sot chu trnh t bo phi m bo ng b gia tc pht trin v thi gian thc hin mt chu trnh phn bo. H thng kim sot s kim tra kch thc t bo ti mt s v tr c bit (size check point) thng phn b hai pha G1 v G2. Chu trnh t bo s b gi li ti nhng im ny t bo t kch thc ti hn hoc genome c ti bn hon chnh. Kim tra im G1 quyt nh ADN c c ti bn hay khng. Tng t nh th, kim tra im G2 cho php t bo bc vo phn chia. Ty thuc vo tng loi t bo v iu kin mi trng m xu hng gi t bo im G1 chim u th hn im G2 hoc ngc li. C ch kim tra ti im G1 v im G2 c sng t nh p dng cc k thut di truyn phn t nm ny chi S.cerevisiae v nm phn chia S.pombe. nghin cu chu trnh t bo v cc c ch kim sot phn bo, nm men c chn lm i tng in hnh tm ra cc t bin gen m cho cc sn phm tham gia h thng kim sot cng nh b my phn bo. Khi mt gen thuc h thng kim sot phn bo b t bin, t bo khng th thc hin trn vn mt chu trnh phn bo nn s lng t bo khng tng. Do , c th chn v duy tr c cc t bo ny, cc t bin ch c biu hin trong nhng iu kin c bit. Hu ht l nhng t bin nhy cm nhit (temperature-sensitive mutant). Chng ch biu hin tnh trng t bin khi nhit cao (restrictive condition), cn nhit thp (permissive condition) th t bo vn phn chia v pht trin bnh thng.

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Ch yu cc nghin cu nm phn chia S.pombe tp trung vo hai dng t bin nhy cm nhit . Dng th nht gm nhng gen m khi b t bin s lm cho t bo dng li ti mt im trong chu trnh t bo. Chng c gi chung l cc gen cdc (cell-divisioncycle). Dng th hai gm nhng gen m khi b t bin s cho php t bo vt qua cc im kim tra kch thc bc vo phn bo. Nh vy t bo s phn chia ngay khi kch thc cn cha t yu cu bnh thng. Cc gen ny c gi chung l wee (theo ting Scotlen c ngha l nh). Hai nhm gen cdc v wee c chc nng tri ngc nhau: t bin gen cdc khin t bo khng qua c im kim tra trong khi t bin gen wee gip t bo vt qua mt cch d dng. V vy, cc gen cdc thc y t bo vt qua cc im kim tra cn cc gen wee km hm qu trnh ny. im kim tra kch thc t bo nm men nm trong pha G2 (im bc vo mitose) tng t nh im kim tra G2 ti MPF c hot ho trong chu trnh phn bo giai on pht trin sm ca phi Xenopus. nm men S. pombe, tiu phn c hot tnh kinase tng t nh Cdk ca MPF. Tiu phn ny c m bi gen cdc2. Protein Cdc2 gi vai tr quyt nh nht, thc y t bo nm men bc vo mitose. Khi Cdc2 b mt hot tnh th khng xy ra qu trnh phn bo. Tuy nhin, khi Cdc2 khng b kim sot bi cyclin khin n lun trng thi hot ng th t bo phn chia lin tc. Gen cdc2 c tch dng t S. pombe c kh nng phc hi kiu hnh hoang di (wild type) cho dng t bin cdc2- trong th nghim b sung chc nng. Hn na, cc gen tng ng vi cdc2 phn lp uc t mt s sinh vt eukaryot khc cng c kh nng phc hi t bo nm men b t bin cdc2-. Khi chuyn gen tng ng vi cdc2 phn lp t ngi vo t bo nm men b t bin gen cdc2- th cc t bo ny li sng st v phn chia nh t bo bnh thng. Nh vy hot ng ca cc yu t kim sot phn bo rt ging nhau gia nm men v ng vt c xng sng. R rng c nm men v ng vt c xng sng, vic t bo bc vo phn chia hon ton ph thuc vo cc kinase v cyclin. iu ny chng t c ch kim sot t bo bc vo pha M c bo ton trong tin ho i vi mi t bo eukaryot. Protein Cdc2 c hot tnh kinase. Hot ng ca protein Cdc2 c kim sot bi ba protein khc (m bi cc gen wee1, cdc13 v cdc25). Trong s , protein Cdc13 c tng ng cao vi mitose cyclin, v d nh cyclin B ca Xenopus. Thc nghim chng t rng Cdc2 v Cdc13 l hai tiu phn ca MPF nm men S.pombe. Phn tch cc t bin cdc- v wee- khc cho thy hot tnh ca phc MPF (Cdc2-Cdc13) S.pombe cn ph thuc vo mt s protein kinase khc. V d, khi Cdc2 tng tc vi Cdc13, Cdc2 tr thnh c cht cho hai protein kinase Wee1 v MO15. Hai protein ny iu khin hot tnh kinase ca chnh Cdc2. Kinase MO15 gn nhm phosphate vo threonine (acid amin 161) nm trn phn t Cdc2. Ch khi b phosphoryl ho v tr ny th phc MPF mi c hot tnh cc i. Ngc li, kinase Wee1 gn nhm phosphate vo tyrosine (acid amin 15) trn Cdc2 gy c ch hot tnh kinase ca Cdc2 v ngn cn phc Cdc2-Cdc13 chuyn sang trng thi hot ng, ngay khi v tr 161 b phosphoryl ho. iu ny c ngha khi b phosphoryl ho hai v tr 15 v 161 th phc MPF s lun trng thi khng c hot tnh. Chnh protein Cdc25 ( nm) lm nhim v kh gc phosphate tyrosine, gip cho phc c hot tnh thc y t bo bc vo phn chia. Nhng thay i cu hnh khng gian ca Cdc2 c m t trong Hnh 6.7.

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Hnh 6.7: Hot tnh ca MPF. Tong tc vi cyclin cho php Cdc2 b phosphoryl ho bi Wee 1 v MO15. MPF dng khng hot tnh khi b phosphoryl ho bi Wee1. MPF chuyn sang dng hot ng nh kinase Cdc25. n lt mnh, cc phn t MPF c hot tnh s gy hot ho Cdc25 v c ch Wee1, nh chng tng cng hot ng ca chnh mnh (theo Alberts & cs., 2002).

i vi nm ny chi S.cerevisiae, t bo s vt qua im kim tra G1 chuyn sang pha S mt khi tho mn ba iu kin: m bo kch thc t bo, mi trng c cc cht dinh dng v t bo c kh nng bc vo sinh sn hu tnh. Cc gen khi t bin lm t bo khng th bc sang pha S ngay khi c ba iu kin cng c gi l gen cdc. iu ng lu l cc gen khng t bin S. pombe c k hiu bng ch thng in nghing (cdc2) nhng S.cerevisiae th c vit bng ch hoa in nghing (CDC28). Gen CDC28 c tng ng rt cao vi cdc2. iu l th l cdc2 quyt nh t bo S.pombe vt qua im kim tra G2 chuyn sang pha M, th CDC28 quyt nh t bo S.cerevisiae vt qua im kim tra G1 (start) chuyn sang pha S. t bin cdc2- khin t bo S. pombe dng li G2 trong khi t bin CDC28 khin t bo S.cerevisiae dng li G1. Tuy nhin th nghim b sung chc nng (complementary test) cho thy sn phm ca cdc2 v CDC28 c th thay th cho nhau, tc l CDC28 gip t bo S.pombe mang gen t bin cdc2- phn chia bnh thng v ngc li. H thng kim sot trong hai loi t bo ny u ch c mt protein kinase ph thuc cyclin (cdc2 hoc CDC28). Mc d c th thay th cho nhau nhng mi kinase c tc ng khc nhau trong chu trnh phn bo i vi tng loi t bo nm men. i vi S.pombe, h thng kim sot phn bo bt u s dng kinase Cdc2 ti im G2 cn S.cerevisiae trung tm kim sot iu hnh Cdc28 ti im G1. Do khi chuyn cc t bo t bin nhy cm nhit t iu kin nhit cho php sang nhit cao, trung tm kim sot hai loi t bo nm men chu tc ng cc giai on khc nhau. V vy, s thiu ht kinase Cdc2 v Cdc28 c biu hin cc pha khc nhau trong chu trnh phn bo. Nh vy hai loi kinase ny u cn thit t bo bc vo pha S v pha M S.pombe hoc S.cerevisiae. c th iu khin chu trnh t bo c hai im kim tra G1 v G2, protein Cdc28 tng tc vi cc cyclin khc nhau. pha G1, Cdc28 tng tc vi ba loi cyclin (G1 cyclin), c gi l Cln1, Cln2, Cln3, to nn phc c chc nng kch thch nhim sc th nhn i (t bo bc vo pha S). Th nghim nh bt gen (gene knockout) cho thy S.cerevisiae vn c th mc trn mi trng giu dinh dng khi ch mang mt trong ba gen CLN. t bin CLN3 khin t bo tn ti pha G1 lu hn vi gi, tc l bc vo pha S chm hn so vi bnh thng. Nu c ba gen b t bin th t bo cht. giai on G2, Cdc28 lin kt vi cc mitose cyclin (Clb1, Clb2 ...) to phc MPF thc y t bo vo mitose. Cc phc ca Cdc28 vi mi loi cyclin u c kh nng phosphoryl ho cc protein khc nhau. Nh vy, hot tnh ring bit ca Cdc2 hai im kim tra ph thuc vo loi cyclin m n lin kt. Mt chu trnh phn bo gm bn giai on phi c hon thnh trc khi t bo bc sang chu trnh mi. Cc c ch kim sot phn hi (feedback control) c nhim v dng chu k phn bo mt s v tr t bo hon thnh giai on trc ri mi chuyn sang giai on sau. Khi s dng cc cht ho hc c ch nhn i ADN hoc khi gy tn thng ADN,

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c ch phn hi s ngn cn t bo phn chia. iu m bo an ton cho cc th h t bo tip theo nhn c thng tin di truyn chnh xc, nguyn vn. Thc nghim cho thy cc c ch phn hi tiu cc gi vai tr chnh so vi phn hi tch cc. iu ny c ngha tn hiu phn hi gy ngng h thng kim sot phn bo hn l chuyn t bo sang giai on mi.

6.5

Kim sot phn bo ng vt

Hu ht nhng nghin cu h thng kim sot phn bo ng vt c thc hin i vi t bo nui cy. Khi thiu cc factor kch thch sinh trng (mitogen), t bo dng li pha Go. Nu b sung cc factor ny vo mi trng, t bo s chuyn sang pha S sau khong 14-16h v hon thnh ti bn ADN sau 6-8h. Sau t bo s phn chia. Tng t nh nm men, chu trnh phn bo ng vt cng tn ti cc im kim sot G1, G2. Nu chuyn t mi trng y sang mi trng khng c cht kch thch khi t bo truc im kim tra G1 th chng khng phn chia. Tuy nhin nu vt qua im G1 th mc d tn ti trong mi trng thiu mitogen, t bo vn thc hin trn vn mt chu trnh phn bo. Ging nh nm men, chu trnh phn bo ca t bo ng vt bc cao cng c iu khin bi cc kinase to phc vi cyclin. Nng ca cc kinase c gi n nh nhng nng cc cyclin thay i rt phc tp cc im khc nhau trong chu trnh. Ngoi ra, khc vi nm men ch c mt loi Cdc, t bo ng vt c nhiu loi kinase ph thuc cyclin (cyclin dependent kinase Cdk), v d nh protein tng ng Cdk2 tham gia h thng kim sot phn bo. Trong khi phc gia Cdc2 v cyclin B thc y t bo ng vt c xng sng bc vo mitose th phc gia Cdk2 v cyclin E thc y t bo vt qua im kim tra G1 cn phc gia Cdk2 v cyclin A cn thit hot ho b my ti bn ADN (bc vo pha S). T bo nm men (c th n bo) ch cn mt loi kinase Cdk kim sot phn bo. Ngc li trong c th a bo, chu trnh phn bo i hi s kim sot rt cht ch nghim nght vi s tham gia ca nhiu loi Cdk (Cdk1, Cdk2, Cdk3...) v cyclin khc nhau (A, B, D, E...). Protein Cdk1 c phn lp u tin ngui, c tng ng cao vi Cdc2 S. pombe. Kinase Cdk1 c th b sung chc nng cho t bo t bin cdc2-. Tip n, protein Cdk2 ca ngi c tm thy nh kh nng phc hi chc nng cho Cdc28 ca S.cerevisiae. c th trng thnh, a s cc t bo khng phn chia v gi nguyn mt trng thi nht nh thc hin chc nng bit ho ca t chc m chng cu to nn. Nu nh iu kin cc cht dinh dng y c th thc y cc t bo vi khun, nm pht trin sinh sn, th trong c th a bo, khng phi ngun dinh dng m chnh cc tn hiu gi i gia cc t bo ng vai tr quan trng. Phn ln cc tn hiu ny l cc yu t kch thch tng trng (growth factor) c nhn bit bi cc th th nm trn b mt t bo. Nng ca chng rt nh ch khong 10-9 n 10-11 M. Tng tc c hiu gia tn hiu v th th c tc dng thc y t bo bc vo phn chia. Nh vy khc vi nm men c chu trnh phn bo c kim sot ch yu bi cc c ch phn hi tiu cc, phn chia t bo trong c th a bo c iu khin ch yu bi cc c ch kim sot tch cc. Vy th bn cht ca cc tn hiu iu khin tch cc l g? C ch no chng tc ng n pht trin v phn chia t bo? Yu t tng trng u tin c phn lp c ngun gc t tiu cu (cc mnh t bo tun hon trong mu gip mu ng cc ti vt thng). Chng c gi l PDGF (plateletderived growth factor). Khi mu ng, cc tiu cu tng tc vi nhau tit ra cc cht d tr trong cc nang tit ca chng, trong c PDGF. Factor ny ng vai tr quan trng kch thch t bo phn chia trong sut thi gian phc hi vt thng. PDGF ch l mt trong s khong 50 protein bit c hot tnh ca factor kch thch tng trng. Mi loi t bo bit ho thc hin cc chc nng chuyn bit s c cc th th

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c hiu vi cc factor khc nhau. Tuy nhin, PDGF c kh nng kch thch i vi nhiu loi t bo nh t bo thn kinh, t bo c trn, cc nguyn bo si (fibroblast) ... trong khi factor erythropoietin ch kch thch duy nht cc t bo tin hng cu bc vo phn chia. Khi factor kch thch tng trng tng tc vi th th nm trn b mt t bo th phn th th nm pha trong mng t bo s xc tc cho chui phn ng truyn tn hiu. Tnh phc tp ca cc phn ng dn truyn tn hiu trong t bo c th hin ch mt tn hiu trung gian c th khi ng cho nhiu phn ng xy ra ng thi. Mi phn ng ny li gy ra cc phn ng tip theo khc nhau. Nh vy, h thng dn truyn mt tn hiu ban u to ra nhiu tn hiu trung gian an xen nhau v c tc ng qua li vi nhau. Khi c mt factor kch thch tng trng, t bo chuyn t Go sang phn chia. C th sp sp nhng gen b hot ho bi mitogen thnh hai nhm. Nhm th nht gm cc gen tr li tn hiu sm (early-response genes). Chng c hot ho sau khi c tn hiu khong 15 pht v khng i hi s tng hp protein. l do cc factor phin m ca nhng gen ny tn ti sn trong t bo trng thi Go. Tuy nhin chng phi c bin i, v d nh b phosphoryl ho, chuyn sang dng c hot tnh. Mt s gen thuc nhm th nht m cho factor phin m i vi gen thuc nhm th hai. V vy, hot ng ca cc gen tr li chm (delayed-response genes) thuc nhm hai ph thuc vo qu trnh tng hp protein. Nhm th hai thng hot ho sau khi c tn hiu khong 1h. Nh vy, sn phm ca gen tr li sm hot ho gen tr li chm, trong c cc gen m cho protein tham gia h thng kim sot phn bo nh Cdk2, Cdk4, Cdk6 v cyclin D, cyclin E. Mt trong nhng factor phin m lin quan cht ch n giai on t bo chuyn t G1 sang pha S l E2Fs. E2F l factor phin m ca nhng gen m cho mt s Cdk, cyclin; cc protein tham gia tng hp deoxyribonucleotide v ti bn ADN. Hot tnh ca factor ny c kim sot nh tng tc vi cc protein Rb, p107 v p130. Hn na, khi tng tc vi Rb, E2F chuyn t dng c hot tnh kch thch sang dng c chc nng km hm phin m do (Rb - E2F) lin kt vi phc kh acetyl cho histone (kh nhm acetyl c lin quan n hin tng co c nhim sc th). V phn mnh, kh nng tng tc ca Rb ph thuc vo kinase. Mt khi Rb b phosphoryl ho, E2F c th tn ti dng t do thc hin chc nng khi ng phin m. Nh m t bo bc vo ti bn ADN. Trong mt s trng hp ADN b tn thng, t bo dng li cc pha G1 hoc G2. Dng li G1 nhm ngn cn vic ti bn ADN cha cc sai hng. Dng li G2 gip t bo sa cha cc t gy ADN trc khi phn ly v cc t bo con trong pha M. C ch dng t bo hai pha ny lin quan n protein p53 v mt s protein c ch khi u khc (tumorsupressor protein). Trong t bo bnh thng, protein p53 rt khng bn. V th lng p53 rt kh t n ngng khi ng phin m i vi mt s gen, v d nh gen m cho p21CIP. y l protein c kh nng tng tc v km hm hot tnh ca hu ht cc phc Cdk-cyclin. Do , khi hm lng protein p53 t, p21CIP khng c tng hp. Tuy nhin, khi ADN b tn thng, p53 tr nn bn vng (do tc ng ca c ch cha r). Lc gen m cho p21CIP c hot ho v sn phm ca gen ny km hm hot tnh ca cc phc Cdk-cyclin khin t bo b dng li cc im G1 hoc G2 cho n khi ADN c sa cha phc hi nguyn vn. Nu nh ADN b tn thng qu nng hoc cc c ch sa cha ADN khng khc phc c sai hng th p53 tham gia thc y t bo cht theo chng trnh c lp sn trong genome (cht theo chng trnh-apoptosis). Chng trnh t cht c bt m c tc dng loi tr t bo mang ADN tn thng bc vo phn chia, ngn cn vic khuch i cc sai

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hng. Nh vy, apoptosis lm gim thiu kh nng t bo mang ADN tn thng pht trin thnh ung th.

6.6

Vai tr ca si vi ng tubulin trong phn bo

Phn bo nguyn nhim c bt u bng vic nhim sc th co m c li trong nhn v thoi phn bo xut hin ngoi nhn (giai on prophase). Thoi phn bo bao gm ch yu cc si vi ng microtubule lm nhim v y trung th (centrosome) ra xa nhau v ko cc nhim sc th v hai cc. Mt lot din bin lin quan n vic hnh thnh v lin kt si thoi vi tm ng, ko nhim sc th v mt phng xch o v tip y chng v cc cc vv... ph thuc rt nhiu vo ng hc cc phn ng xy ra hai u si vi ng. Si vi ng microtubules c cu trc dng ng gm 13 si nh kt hp li, mi si c cu to t cc phn t tubulin. Mi phn t tubulin gm hai tiu phn (heterodimer) -tubulin v -tubulin. Do tnh a dng ca cc tiu n v v -tubulin nn c nhiu loi tubulin. Cc si vi ng rt mm do nhng bn, phn b thnh mng li trong t bo. Chng m nhn nhiu chc nng khc nhau nh gp phn to nn khung t bo; tham gia vn chuyn cc nang, cc phc protein v c bit trong qu trnh phn bo. Si vi ng c tnh phn cc ph thuc vo ng hc qu trnh polymer v kh polymer hai u ca si. Mt u ca si [gi l u (+)] c th di ra rt nhanh nh phn ng polymer ho vi tc gp ba ln u kia [gi l u (-)]. Tuy nhin si vi ng c kh nng ngn li t ngt do mt i cc phn t tubulin. Chnh hot tnh phn hy GTP ca tiu phn -tubulin quyt nh tnh khng bn ca si. Tng tc gia -tubulin vi GTP cn thit phn t tubulin dng d hp t gn c vo hai u ca si microtubules (qu trnh polymer ho), trong khi phn hy GTP cn thit ph v lin kt gia cc tubulin v tch chng ra khi si (kh polymer). Hn na, phn ng polymer gn phc tubulin-GTP vo cc u xy ra nhanh hn phn ng phn hy GTP GDP khi kh polymer. Ngoi ra i lc tng tc gia cc tubulin mang GTP ln hn gia tubulin mang GDP. Nh vy, chnh lch nng GTP v GDP cng nh tc polymer v kh polymer nh hng rt ln n ng hc ca si vi ng microtubules. Sau khi gn vo si microtubules, tiu n v -tubulin chu bin i acetyl ho cc acid amin lysine ng thi loi b tyrosine u carboxyl (post-translational modification). Cc bin i ny khng xy ra vi cc phn t tubulin t do cha polymer ho. Phn ng acetyl ho v kh tyrosine lin quan n kh nng lin kt ca microtubules vi mt s protein tng bn vng ca si cng nh to thun li cho tng tc vi cc thnh phn khc trong t bo. Nhng protein ny c gi chung l MAPs (Microtubule-Associated Proteins). C th phn loi cc MAPs thnh hai nhm ty thuc vo trng lng phn t. Nhm HMW (High Molecular Weight) v Tau proteins. Cu trc ca cc protein trong c hai nhm u gm hai domain; mt domain lin kt vi microtubules v domain kia tng tc vi cc thnh phn khc. Khi cc MAPs lin kt vi u si microtubules, chng s ngn cn qu trnh kh polymer, do gi cho si c bn vng. Ngoi ra cn phi lu n cc MAPs c kh nng s dng nng lng phn hy ATP di chuyn dc theo si microtubules. Nhng protein MAPs c bit c gi chung l microtubule motor. Phn bo nguyn nhim phi m bo tuyt i mi thnh phn c trong t bo c phn chia nh nhau v hai t bo con. Do cc bo quan nh ty th, lc lp v ngay c th Golgi, mng li ER khng c kh nng t hnh thnh t cc nguyn liu ring bit trong t bo cht, cho nn cc bo quan ny khng th xut hin trong cc t bo con nu nh chng

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khng c di truyn t t bo m di dng c bit. Trong mt t bo, a s bo quan thng tn ti hai hay nhiu copy. Do trong t bo con, cc bo quan c duy tr nh nhn i t mt bo quan ban u. c bit i vi Golgi v ER, cc t chc ny b t gy thnh cc phn nh trong phn bo v chng c ko v hai t bo con nh cc si vi ng ca thoi phn bo. Si vi ng tham gia qu trnh phn bo c chia lm ba loi. Si cc (polar microtubules) c cc u tn cng gp nhau mt phng xch o lm nhim v y cc cc xa nhau. Si lin kt vi kinetochore (kinetochore microtubules)- phc protein c bit tng tc vi tm ng, lm nhim v iu khin s chuyn ng ca nhim sc th trong phn bo. Loi th ba l si sao hay cn gi l si trung th astral microtubules do chng xut pht t trung th, hng ra mi pha v thng ngn hn hai loi si kia, gi vai tr tng cng lc y phn chia hai cc (Hnh 6.8). Ngay khi mng nhn cn nguyn vn trong giai on prophase, mt s si vi ng xut hin trung th v gp nhau ti mt phng xch o. Mt s protein microtubule motor lm nhim v lin kt cc si microtubules vi nhau ti u (+) ca si, to lc y v ko cc cc xa nhau. Cc si microtubules lin tc polymer ho di ra v ng thi b kh polymer ngn li. T l gia tc polymer v kh polymer khng c mt gi tr nht nh m thay i ph thuc vo tng giai on ca phn bo. C ch phn t ca ng hc bin i si microtubules cha r rng mc d c lin quan n MPF trong phn ng phosphoryl ho v kh phosphoryl mt s protein lin kt vi si microtubules.

Hnh 6.8: Ba loi si vi ng cu to nn thoi phn cc trong phn bo mitosis.

Bc vo phn bo nguyn nhim, mi si nhim sc th c nhn i to hai nhim sc t (sister chromatid) dnh nhau tm ng. Mi bn tm ng u c phc protein tng tc vi ADN vng to nn cu trc kinetochore. Chnh u (+) ca si vi ng s lin kt vi kinetochore thng qua cc protein "motor" iu khin s chuyn ng ca nhim sc th, y nhim sc t xa nhau v a chng v cc cc i din. Ngay khi tng tc vi kinetochore, u (+) ca microtubules vn tip tc polymer ho v depolymer (tc l vn di thm v ngn bt do thm vo v ng thi mt i cc tiu phn tubulin).

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Chng 7 SINH TRNG V PHT TRIN

Qu trnh sinh trng pht trin ng vt c th c xem nh bt u t s th tinh ca trng mc d trc mi bc chun b phi hon tt qu trnh c thc hin mt cch hon ho. V d, mt s gen ca m c phin m v dch m. Tuy nhin, nhng sn phm ny hon ton c d tr trong trng nh ngun dinh dng v tp hp cc yu t kim sot pht trin ca hp t ngay khi trng va th tinh. Sau khi th tinh, hp t tri qua cc giai on pht trin phi nang, phi d v hnh thnh cc b phn ca mt c th hon chnh. giai on phi nang, hu ht cc t bo cha bit ho. Tuy nhin, bc sang giai on phi d, phi thai bt u hnh thnh vi 3 lp t bo: ni b, trung b v ngoi b. T 3 lp t bo ny, cc t bo c s phn khc nhau, chng pht trin bit ho theo nhng chng trnh khc nhau to thnh cc b phn ring bit ca mt c th hon chnh. Tht th v khi t mt t bo trng ban u, mt c th a bo vi nhiu loi t bo bit ho khc nhau c hnh thnh. mc phn t, s khc bit gia cc loi t bo c xem nh ph thuc vo cc cch thc hot ng ca gen v tng tc gia cc sn phm ca chng. Hot ng ca gen lin quan ch yu n cc protein. Chng c th lm thay i cu trc ca on ADN cha promoter, khi ng qu trnh sao chp thng tin, iu khin hot ng ca cc enhancer hoc c ch cc factor phin m. Qu trnh iu khin bt m hoc ng cc gen do protein m nhim dn n vic hnh thnh cc b phn ca mt c th hon chnh c nghin cu kh k trn i tng rui gim D.melanogaster v giun trn C.elegans. Sau khi th tinh, mt lot din bin xy ra trong trng rui gim, bt u t s phn b khng ng nht ca cc cht trong t bo cht dn n mc biu hin khc nhau ca cc gen thay i theo thi gian v khng gian. iu khin cho cc phn cc b phn b bn trong trng c nhng c tnh ring bit. bt k giai on no trong qu trnh pht trin, vic ng hoc m mt gen u lin quan n hot ng ca cc gen khc trong giai on trc . Ni mt cch khc, mt protein iu khin ny s kim tra hot ng ca gen m cho protein iu khin khc. Nh vy, trong giai on m phi, hot ng ca gen xy ra mt thi im nht nh gi vai tr ch o trong bit ho pht trin. Nghin cu sinh trung pht trin ng vt thng da vo phn tch di truyn, i hi cc i tng tha mn nhng yu cu nh s lng c th nhiu, vng i ngn, d dng thu nhn c cc th t bin trong t nhin cng nh bng k thut chuyn gen vv... Rui gim Drosophila melanogaster v giun trn Caenorhabditis elegans p ng c nhng yu cu trn nn c s dng nh nhng sinh vt m hnh nghin cu v hot ng ca gen trong qu trnh pht trin, c bit giai on phi sm. H gen ca hai i tng m hnh ny khng ln (170.000 kb rui gim, 80.000 kb giun trn), to thun li cho vic phn lp cc gen lin quan. Mt khi bit trnh t nucleotide ca mt gen c hiu, hot ng ca gen ny theo thi gian v ph thuc vo t chc m chuyn ho c xc nh nh cc k thut lai acid nucleic hoc to khng th. Tt c nhng phng php sinh hc hin i gip chng ta hiu c thi im v v tr m cc gen bt u hot ng trong qu trnh pht trin phi. Ngoi ra, cc gen phn lp c t hai i tng m hnh ny c th c dng to ra cc c th chuyn gen. T , chc nng c th ca gen cng nh cc qu trnh sinh hc

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lin quan n sn phm ca gen c hiu mt cch y hn. C th ni nhng nghin cu trn giun trn v rui gim gip cc nh sinh hc c nhng hiu bit chi tit v tng th v cc cch thc kim sot sinh trng pht trin. Trong khi nhng gen c vai tr quyt nh qu trnh pht trin rui gim l nhng gen m cho protein tham gia vo bin i ARN th giun trn li l nhng gen m cho cc yu t iu khin phin m. Tuy nhin, cch thc kim sot xc nh gii tnh giun trn v rui gim ging nhau: gii tnh c xc nh ph thuc vo t l gia s lng nhim sc th gii tnh X v s lng nhim sc th thng. Khi t l ny bng hoc ln hn 1 ( 1), phi pht trin thnh con ci. Khi t l ny bng hoc nh hn 0,5 ( 0,5) th phi pht trin thnh con c. Giun trn C.elegans c cc nh di truyn hc quan tm rt mun (1960) so vi rui gim Drosophila (1909). Giun trn c th nui trong a petri vi ngun thc n chnh l vi khun E.coli. Trong iu kin mi trng dinh dng y , vng i ca giun trn ch trong khong 3 ngy. c bit, C.elegans l ng vt lng tnh; trng v tinh trng sinh ra t mt c th c kh nng t th tinh pht trin thnh mt c th mi. V th d dng chn lc c cc t bin ln dng ng hp t. Ngoi ra, c th giun trn trong sut nn c th nhn thy cc t bo v theo di c s phn ca chng trong qu trnh pht trin t trng th tinh. Ph h ca tt c cc t bo to ra trong sut qu trnh t trng th tinh n c th trng thnh c xc nh vi giun trn khin cho C.elegans tr nn m hnh l tng trong nghin cu sinh trng pht trin. Rui gim Drosophila c ton b chu trnh sng ch ko di 9 n 10 ngy. Trng rui gim sau th tinh phn chia rt nhanh to thnh cc t bo khng c mng. V vy, giai on u ngay sau th tinh c th xem trng l mt t bo c rt nhiu nhn ging nhau. Trng thi ny ca trng c gi l hn bo. Sau 9 ln phn chia nguyn nhim, tng s 512 nhn di chuyn trong t bo cht v pha mng t bo trng v tip tc phn chia thm 4 ln na. Tip n tt c cc nhn ny u c bao bc bi mng to thnh cc t bo ring bit. Nhng t bo ny nm st mng t bo trng (pha t bo cht) to thnh lp n bo (gi l lp b phi) trn b mt ca phi. Phi rui gim cn phi tri qua mt lot bin i hnh thi t u trng, nhng n c th trng thnh. 7.1 Kim sot xc nh gii tnh

giun trn C.elegans, giun c ch c mt nhim sc th X, giun ci c 2 nhim sc th XX. Cch thc xc nh gii tnh C.elegans lin quan t nht n sn phm ca 10 gen khc nhau. t bin cc gen ny gy nn nhng lch lc trong pht trin gii tnh. V d, t bin gy mt chc nng (loss-of-function) gen her-1 (hemaphrodite-1) khin phi XO (ch c mt nhim sc th X) pht trin thnh c th lng tnh. Trong phi XX, protein ny khng c tng hp. Tuy nhin, trong phi XO, protein HER-1 c chc nng ca protein iu khin (regulatory protein) gy km hm hot ng ca mt lot gen lin quan n pht trin gii tnh ci. Nh vy gen her-1 m cho protein cn thit phi XO pht trin thnh giun c. S lng nhim sc th X v s lng nhim sc th thng (k hiu l A) c mt trong hp t phi c xc nh chnh xc v t s X/A gia hai s lng quyt nh bit ho gii tnh ca phi. Vic xc nh chnh xc s lng nhim sc th gii tnh ph thuc vo cc yu t t s (numerator elements) v vic xc nh chnh xc s lng nhim sc th thng ph thuc cc yu t mu s (denominator elements). Mt s yu t t s c pht hin nhng s tn ti ca yu t mu s cha c chng minh bng thc nghim. rui gim Drosophila, tng t nh giun trn C.elegans, qu trnh xc nh gii tnh cng i hi phi xc nh t s gia s lng nhim sc th X v s lng nhim sc th

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thng (A). Thng tin t s X/A s chuyn i thnh cc tn hiu hot ho cc nhm gen khc nhau lin quan n hnhthnh cc biu hin c th ca tng gii. S lng nhim sc th X v s lng nhim sc th thng uc xc nh giai on rt sm ca phi. Trong giai on phi sm, mt s protein c m bi cc gen phn b trn nhim sc th X v mt s khc c m bi cc gen nm trn nhim sc th thng. Cc protein ca phi tng tc vi nhau v tng tc vi protein c d tr trong t bo cht ca trng. S lng protein do gen nm trn nhim sc th X trong phi XX s nhiu gp 2 ln so vi phi XY. Do , s chnh lch nng sn phm ca cc gen lin kt nhim sc th X so vi cc gen trn nhim sc th thung cho php m chnh xc s lng nhim sc th XX. Do c s lng nhiu hn nn protein ca gen trn nhim sc th X trong phi XX d dng tng tc vi protein d tr sn trong t bo cht ca trng. Tng tc ny s dn n khi ng nhng gen quyt nh pht trin gii tnh (Hnh 7.1). Nh vy, nhng protein m bi gen lin kt gii tnh m nhn chc nng ca yu t t s (numerator elements). Ngc li, nhng protein m bi gen trn nhim sc th thng m nhn chc nng ca yu t mu s. T s gia nng yu t t s v nng yu t mu s t l vi t s X/A. Do , gii tnh c quyt nh ph thuc vo chnh lch nng sn phm ca cc gen nm trn nhim sc th X vi sn phm cc gen trn nhim sc th thng. (Cn nhc li l cc gen ny hot ng rt sm trong giai on pht trin ca phi). Mt khi t s X/A c xc nh, gi tr ca t s s chuyn i thnh tn hiu iu khin hot ng ca gen Sxl (Sex-lethal gene) ti promoter PE. Gen Sxl nm trn nhim sc th X. Protein SXL ca gen Sxl ch c pht hin trong giai on sm phi XX. Sau , phin m t PE c thay th bi promoter PM. iu c bit th v l promoter PE cng hot ng phi XY. Tuy nhin, qu trnh bin i (ct ni exon-intron) ca phn t ARNm phin m t PM ch c thc hin hon ho khi c mt ca protein SXL (nhc li l protein ny c dch m t ARN ca promoter PE trong phi XX). Trong phi XY, c ch ct ni lun phin khi khng c mt protein SXL b lch lc lm xut hin m dng (stop codon) khng ng ch trn phn t ARNm. Nh vy, s vng mt ca protein SXL tng ng vi c 2 promoter khin cho phi pht trin thnh rui c.

Hnh 7.1: T s gia s lng nhim sc th gii tnh X v s lng nhim sc th thng quyt nh hot ng ca gen Sxl; protein SXL kim sot cc gen cn thit cho xc nh gii tnh ca phi Drosophila (theo Snustad, 2000).

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Trong phi XX, protein SXL m nhn chc nng ca protein kim sot theo c ch tch cc qu trnh bin i phn t tin thn ARNm. iu ny c ngha, s c mt ca SXL cho php s lng protein SXL tng ln nh ct ni lun phin chnh xc. Nh vy, protein SXL kim sot tch cc hot ng ca chnh gen Sxl. Ngoi ra, SXL cn kim sot sau phin m i vi ARNm ca gen tra (tranformer gene). Cc phn t ARNm tin thn phin m t gen tra c ct ni lun phin. Trong phi XY, ct ni lun phin ca tra- ARN to ra m dng ngay trong exon th hai. Trong phi XX, s c mt ca SXL gip tra-ARNm ct ni ng tip tc dch m to protein TRA. Nh vy, s c mt ca protein SXL cho php xut hin protein TRA trong phi XX.

Hnh 7.2: Kim sot xc nh gii tnh phi rui gim Drosophila. Protein SXL ca gen Sxl bt m chui cc bc kim sot hot ng ca cc gen xc nh gii tnh thng qua kim sot ct ni lun phin cc phn t tin thn ARNm (theo Snustad, 2000).

Protein TRA, n lt mnh, phi hp vi protein TRA2 (m bi gen nm trn nhim sc th thng) kim sot hot ng ca gen dsx (doublesex gene). Gen dsx m cho 2 loi protein DSX; chng c dch m t 2 loi phn t ARNm khc nhau to ra do ct ni lun phin t mt dsx-ARNm tin thn ban u. Trong phi XX c mt protein TRA, TRA tc ng n ct ni lun phin ca dsx-ARNm. Do , loi protein DSX ca phi XX c chc nng km hm hot ng ca nhng gen cn thit cho phi pht trin thnh con c. Trong phi XY khng c protein TRA, dsx-ARNm khc vi dsx-ARNm trong phi XX. Loi protein DSX c trong phi XY c chc nng km hm hot ng ca nhng gen cn thit cho phi pht trin thnh con ci. Nh vy, 2 protein DSX xut pht t mt gen dsx. Tuy nhin, mi loi DSX quyt nh con ng pht trin gii tnh duy nht cho phi. T s la chn ny, hng lot cc gen s biu hin khc nhau gia phi XX v phi XY trong qu trnh bit ho pht trin gii tnh (Hnh 7.2). Trong giai on sm ca pht trin phi, hot ng ca cc gen trn nhim sc th X s c kim sot theo cch thc iu tit xut liu (dosage compensation). V d, giun trn C.elegans, phc protein ch xut hin trong t bo phi c hai nhim sc th XX v tng tc vi chng km hm hot ng ca cc gen trn nhim sc th X. iu m bo iu tit c liu lng protein ca nhng gen ny trong t bo c 2 XX (giun ci) ging nh trong t bo ch c mt X (giun c). Ngc li giun trn, trong t bo rui c (XO) Drosophila c phc protein tng tc vi mt nhim sc th X tng liu xut hot ng ca nhng gen

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nm trn nhim sc th X. Nh vy trong t bo phi Drosophila c hai nhim sc th XX, khng xy ra hin tng bt hot mt nhim sc th X nh ng vt bc cao. Cc gen trn 2 nhim sc th XX u hot ng nhng mc biu hin c kim sot theo c ch km hm. Trong cc t bo phi Drosophila ch c mt nhim sc th X th hot ng ca cc gen trn nhim sc th ny c kch hot tng liu xut. iu m bo s lng protein trong phi XX tng t nh trong phi XO (Hnh 7.3). iu tit xut liu Drosophila i hi t nht sn phm ca 4 gen khc nhau, gi chung l nhng gen msl (male-specific lethal genes). Trong phi c, phc protein MSL bm trn nhim sc th X c xc nh nh k thut lai s dng khng khng th i vi MSL. t bin nhng gen ny khin phi c b cht do nhim sc th X duy nht ca phi khng c kch hot. Trong s 4 protein MSL, protein MLE (sn phm ca gen mle-maleless) c hot tnh helicase gy m xon chui kp ADN. iu ny gi nhim sc th X duy nht trong phi c m xon cc gen tng cng hot ng. iu c bit l phc MSL c lin kt vi ARN tng tc vi nhim sc th X. Hai gen roX1 v roX2 nm trn nhim sc th X (RNA on the chromosome X) phin m to roX1-ARN v roX2-ARN. Hai loi ARN ny khng c dch m mc d chng u tri qua bin i thm ui poly A v ct ni exonintron. Chng phn b trn nhim sc th X cng vi phc MSL. Hot ng ca 2 gen m cho roX-ARNs chu kim sot tiu cc ca protein SXL. Hai gen ny ch phin m khi gen sxl khng hot ng. Nh vy, cc phn t roX-ARNs ch xut hin trong phi XY. Khi phi XX c t bin trn c 2 allen ca gen sxl, phi khng pht trin thnh rui c nh d on m phi b cht. Tuy nhin, phi cht khng phi do ri lon trong qu trnh chuyn i gii tnh (do gen sxl t bin) m do ri lon gy ra trong iu tit liu xut ca nhng gen nm trn nhim sc th X trong phi XX. Trong phi ng hp t t bin Sxl, cc gen trn hai nhim sc th XX khng c km hm nn dn n s qu liu cc sn phm ca nhng gen . iu ny khin cho phi cht. Nh vy, bn cnh nhim v kim sot biu hin ca cc gen trong qu trnh xc nh gii tnh, protein SXL cn iu tit liu sut ca chnh cc gen nm trn nhim sc th X.

Hnh 7.3: C ch iu tit liu sut i vi hot ng ca cc gen nm trn nhim sc th X giun trn v rui gim. Hai cch thc iu tit xy ra ngc nhau i vi hai loi ng vt ny v khc vi cch thc bt hot mt nhim sc th X ng vt c v.

7.2

Pht trin rui gim Drosophila

rui gim Drosophila, trng c hnh thnh t non bo. Mt non bo c mi lin h vi 15 t bo nui (nurse cell) thng qua cc cu ni t bo cht (Hnh 7.4). Nh m cc cht dinh dng v ARNm tng hp trong t bo nui c vn chuyn sang non bo. Nhng nguyn liu ny cn thit cho non bo pht trin thnh trng cng nh i vi s pht trin ca m phi giai on u.

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Hnh 7.4: Cc t bo nui ni vi nhau v ni vi mt cc ca t bo trng thng qua cu ni t bo cht. Cc t bo nang l t bo soma trong khi t bo trng v cc t bo nui xut pht t t bo sinh dc.

Hu ht cc gen lin quan n qu trnh pht trin ca Drosophila c pht hin nh cc t bin gy cht giai on pht trin phi sm hoc khin cho mt b phn c th pht trin khng bnh thng. T cc kt qu nghin cu di truyn phn t, thc nghim phn loi cc t bin thnh ba nhm chnh tu theo hot ng ca chng cng nh chc nng sn phm tng ng. Mt s c im chung ca tng nhm t bin nh sau: 1. t bin xy ra vi cc gen c ngun gc t m (maternal genes). Cc gen ny hot ng trong giai on pht trin trng trong c th m. t bin trn nhng gen ny khng gy nh hng n i sng bnh thng ca rui m, nhng li c biu hin tnh trng th h con. Chng c th hot ng trc hoc sau khi trng chn. t bin cc gen to t (gaps genes). Cc gen ny hot ng sau khi trng th tinh. t bin trn chng lm thay i s lng hoc tnh phn cc ca cc t. t bin xy ra cc gen chn lc (selector genes) hay cn gi l gen homeotic (homeotic genes). Cc t bin ny khng lm thay i s lng, kch thc hoc tnh phn cc ca cc t. Cc gen homeotic qui nh nhng c tnh ring bit ca tng t. Do , nhng t bin xy ra lm cho mt phn ca c th mang nhng tnh trng ca phn khc. V d t ngc biu hin cc c tnh ca t bng; chn hoc cnh c th xut hin phn u vv...

2. 3.

Cc gen ca mi nhm hot ng ni tip nhau hon thin cc tnh cht ring c tnh c th ngy cng cao ca cc phn trong phi. Cc gen c ngun gc t m phn nh cc vng trong trng. Trong mi vng, sn phm ca cc gen c ngun gc t m phn b khc nhau. iu dn n s hot ho khc nhau ca cc gen phn t trong vng. Nh m cc t c hnh thnh. Trong mi t, cc gen homeotic s hot ng xc nh cc tnh cht c th ca t, tc l hnh thnh cc c quan c v tr phn b nht nh ti t . 7.3 Hot ng ca cc gen c ngun gc t m trong qu trnh hnh thnh trc uui v trc lng-bng

i vi ng vt, c th lun c hai trc nhm phn bit phn u vi phn ui v phn lng vi phn bng. C hai trc ny c hnh thnh rt sm, thm ch mt s loi c th c nh hnh ngay trc khi trng th tinh. Trn i tng rui gim Drosophila, qu trnh hnh thnh hai trc u-ui v lng-bng c nghin cu kh chi tit da vo cc loi t bin nh hng n giai on pht trin rt sm ca phi. Phn tch tng th cc t bin lin quan n giai on pht trin u tin ca phi rui gim cho php xc nh bn nhm gen c ngun gc t m tham gia kim sot qu trnh hnh

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thnh cc trc u ui, lng bng ca phi. hiu r mc phn t nhng c ch lin quan n sn phm ca gen c ngun gc t m, sinh hc hin i c bit l di truyn phn t tin hnh phn lp gen, xc nh hot ng ca gen mc tng hp ARNm, xc nh s phn b ca sn phm tng ng ti cc phn khc nhau phi cng nh tc ng ca sn phm n chc nng ca cc protein khc. Thc nghim nhn thy cc gen c ngun gc t m gi vai tr rt quan trng trong giai on pht trin phi sm. t bin xy ra nhng gen ny khng lm xut hin tnh trng mi i vi m m ch biu hin th h sau. Trng ca nhng con m mang t bin cc gen thng b hng, phi b cht trong qu trnh pht trin. Cc gen c ngun gc t m hot ng trc khi trng c th tinh. Sn phm ca chng c th c s dng ngay hoc c d tr trong trng di dng ARNm hoc protein. Ngay trong trng xut hin gradient ca cc protein hoc ARNm phn b theo hai trc u-ui v lng-bng. Cc gradient chu trch nhim hnh thnh trc u-ui xut hin trc, cn gradient chu trch nhim trc lng-bng xut hin chm hn. Cc gen c ngun gc t m c chia lm bn nhm khc nhau: nhm gen lng-bng (dorsoventral-group genes), nhm gen hai vng tn cng u v ui (terminal-group genes), nhm gen phn u bao gm u v ngc rui gim (anterior-group genes) v nhm gen phn ui (posterior-group genes). Mi nhm gm nhng gen cng quyt nh chung cho mt cch thc pht trin ca tng vng. Trong mi vng, cc din bin cc b xy ra u tin, c th ngoi hoc trong trng. iu dn n phn vng ca cc tn hiu trong trng. Tip n, s phn b cc b ca cc protein c th dn n hnh thnh mt cu trc ring bit ti vng . Ni mt cch khc th ti mi vng, nng protein c hiu t ngng nht nh s quyt nh s phn pht trin ca vng . Nhng protein ny c gi l morphogen (tm dch l protein m liu). giai on u tin pht trin phi, s hnh thnh vng u-ui v vng lng-bng c quyt nh bi s phn b cc b ca gn 30 morphogen. Thc nghim tm c hn 30 gen lin quan n sinh tng hp, vn chuyn v phn b cc morphogen. S phn vng xy ra hon ton c lp vi nhau. 7.3.1. Nhm gen quyt nh pht trin ca phn u v ngc u th (anterior-group genes) Khi chc thng mt l nh ti u trc ca trng (ni tip gip vi cc t bo nui) t bo cht ca phn thot ra ngoi, phi s pht trin khng c u. Nu ly t bo cht phn sau ca trng b sung vo phn u th phi pht trin s c hai ui (m khng c u). Nu ly t bo cht ca phn u tim vo v tr khc trn trng, ti cu trc u s hnh thnh. Cc kt qu ny chng t rng phn u u th c quyt nh bi cc cht nm ti mt cc ca trng. Di truyn phn t tm c nhng con m b t bin gen bicoid s trng pht trin thnh phi khng c u. Nu ly t bo cht cc trc ca trng bnh thng tim vo trng ca con m t bin th phi ca trng li pht trin c u. Nh vy chnh sn phm ca gen bicoid d tr cc trc ca trng gi vai tr kim tra qu trnh pht trin phn u u th. Kt qu php lai insitu cho thy ARNm ca gen bicoid c tng hp trong cc t bo nui v a vo t bo trng theo cu ni t bo cht. Chng c gi li cc trc do phn khng dch m u 3' tng tc vi cc thnh phn ca khung t bo ti cc . Cc phn t ARNm ny ch c s dng tng hp protein Bicoid sau khi trng th tinh. Nng protein Bicoid ln nht ti cc trc v gim dn v pha ui. Gradient nng thay i khi genome ca con m c ghp thm cc bn sao ca gen bicoid. Do s bn sao ca gen

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bicoid trong c th m tng, nng ARNm cng nh protein Bicoid trong trng cng tng. iu ny dn n phi pht trin c phn u rng ln t ra pha sau. Nh vy protein Bicoid l mt morphogen. Phn u ca phi c qui nh do s chnh lch nng ca morphogen ny. Protein Bicoid bm vo promoter ca mt s gen v hot ho chng. Mt trong nhng gen ny l gen hunchback. Hot ng ca hunchback ph thuc nng protein Bicoid. Gen hunchback ch bt m khi nng Bicoid t mt gi tr nht nh v hot ng mnh hay yu ph thuc vo s lng gen bicoid, tc s lng protein Bicoid c nhiu hay t. Promoter ca gen hunchback cha 3 v tr c i lc lin kt cao v 3 v tr c i lc yu i vi protein Bicoid. Rui gim chuyn gen mang gen bo co (reporter gene) gn vi promoter hunchback b loi b cc v tr c i lc lin kt khc nhau chng t mc tng tc ca Bicoid vi nhng v tr s quyt nh mc hot ng ca gen hunchback. Do , gradient nng Bicoid s ng vi mc biu hin khc nhau ca gen hunchback theo khng gian. Mt khc ngay cng mt nng Bicoid, do cc v tr vng ADN iu khin ca gen hunchback c i lc tng tc khc nhau vi Bicoid nn s cnh tranh lin kt cng nh hng n hot ng ca gen ny. Ni mt cch khc, s khc nhau v s lng morphogen tham gia khi ng cc gen c chuyn thnh s khc nhau v bn cht, cu trc t bo trong giai on pht trin phi (Hnh 7.6). iu ng lu l cc phn t ARNm-hunchback c to ra t hai ngun. Ngun th nht t qu trnh phin m trn gen hunchback c kim sot bi nng Bicoid. Cc phn t ARNm ny phn b cc b vng u ca phi. Ngun th hai c sn trong t bo cht ca trng, chng c vn chuyn t cc t bo nui sang t bo trng v phn b ng u trong t bo cht. Mc d cc ARNm- hunchback t ngun th hai c mt mi vng trong trng nhng protein tng ng Hunchback khng xut hin phn ui ca phi. l do s mt ca protein Nanos. Gen m cho protein ny c ngun gc t m, tc l ARNm- nanos c d tr sn trong trng. Cng ging nh hu ht ARNm ca cc gen c ngun gc t m khc, ARNm-nanos phn b cc b pha cui ca phi. Do , xut hin gradient protein Nanos theo chiu ngc li vi gradient protein Bicoid. Hn na, chc nng ca hai protein ny cng ngc nhau. Trong khi Bicoid hot ho th Nanos km hm hot ng ca gen hunchback. Protein Nanos ngn cn qu trnh dch m t cc phn t ARNm- hunchback c mt vng ui. Hai protein Bicoid v Nanos u tham gia kim sot hot ng ca hunchback. Tuy nhin cn lu , Bicoid l factor phin m trong khi Nanos tc ng n qu trnh dch m.

Hnh 7.6: Gradient nng ca hai protein Bicoid v Hunchback theo trc trc sau ca phi Drosophila. Protein Bicoid hot ho gen hunchback thng qua tng tc vi cc v tr c hiu nm trong vng iu khin ca gen hunchback. Cc tng tc ny c i lc khc nhau tng ng vi biu hin mc khc nhau ca hunchback.

Mt lot cc protein khc c tng hp t cc gen c ngun gc t m tham gia vo giai on pht trin u tin ca phi rui gim. V d, gen pumilio m cho protein c chc nng

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tng t Nanos, tc l km hm qu trnh tng hp Hunchback. Kh nng km hm ca Nanos v Pumilio ph thuc vo vng 3' khng dch m trn phn t ARNm- hunchback bao gm c ui polyA. Thc nghim quan st thy phi rui gim bnh thng, ui polyA c di thm trc khi phn t ARNm- hunchback c dch m. Protein Nanos kch thch phn ng ct ngn ui polyA khin vic dch m trn ARNm- hunchback b c ch. 7.3.2. Nhm gen qui nh pht trin phn ui (posterior-group genes) Kt qu cc th nghim di truyn cho thy t bin mt s gen c ngun gc t m dn n vic phi pht trin khng c phn ui v khng c cc t bo sinh dc. Tin hnh cc th nghim tng t nh lm vi cc trc ca trng (thay t bo cht cc sau ca trng bnh thng vo trng b t bin hoc tim t bo cht ny vo mt v tr bt k... ) u khi phc c cu trc phn ui ca u th. Thc nghim tm ra protein Nanos ng vai tr morphogen trong vic hnh thnh cu trc ui. Gen nanos hot ng trong t bo nui v ARNm ca n c vn chuyn qua cu ni t bo cht n cc sau ca trng. Chng c gi cc sau nh phn khng dch m u 3'. C l phn ny c nhn bit v tng tc vi sn phm ca mt gen hot ng trc nanos v cng phn b cc b cc sau. Qu trnh hnh thnh cc t bo sinh dc, cng vi cc gen cn thit cho vic thit lp cu trc phn ui cn i hi mt s gen c ngun gc t m m sn phm ca chng c tch ly ti cc sau ca trng. Gen oskar ng vai tr then cht trong vic hnh thnh t bo sinh dc. Khi gen ny b t bin, phi pht trin khng c t bo sinh dc. ARNm v protein Oskar tp trung cc sau. Chng xm nhp vo mt s t bo cc ny v quyt nh s phn ca chng pht trin thnh t bo sinh dc. a ARNm ca gen oskar vo cc u, cc t bo sinh dc xut hin cc ny. 7.3.3. Nhm gen qui nh pht trin trc lng-bng (dorsoventral-group genes) Sn phm ca nhm gen ny iu khin s hnh thnh trc lng-bng t khi trng th tinh n giai on xut hin cc t bo phi b (blastoderm). t bin bt k gen no trong nhm u dn n hin tng phi pht trin khng c cu trc bng. Ti v tr ca phn bng xut hin cu trc lng. Cc t bin kiu ny u c khc phc khi a t bo cht ca phi pht trin bnh thng vo phi pht trin t trng ca con m cha cc gen t bin thuc nhm trc lng bng. u tin cc nh di truyn dng tc nhn ho hc gy t bin tng nhim sc th ca rui gim. Rt nhiu gen mang t bin v tnh trng lin quan c pht hin. Trong s , gen dorsal c xem ng vai tr quyt nh n vic hnh thnh trc lng bng. Protein Dorsal c tng hp t m v d tr trong t bo cht ca trng. n giai on phi b, Dorsal di chuyn vo trong nhn ca nhng t bo phn b vng bng. Lc Dorsal bt m hot ng ca hai gen twist v snail, ng thi km hm gen zerknullt v decapentaplegic. Nh , cc t bo vng bng s bit ho thnh trung b. Din bin hot ng ca bn gen trn trong cc t bo phn b vng lng xy ra ngc li. Do protein Dorsal khng di chuyn vo nhn cc t bo phn b vng lng nn trong cc t bo ny hai gen zerknullt v decapentaplegic hot ng cn hai gen twist v snail b km hm hon ton. Cc t bo ny s bit ho thnh biu b. iu ng quan tm l v sao ch xy ra s di chuyn v tr ca Dorsal trong t bo vng bng? Tn hiu quyt nh s chuyn ch pht i t bn trong trng hay cc t bo nui? Thc nghim cho thy tng tc gia hai protein Toll v Spatzle nm trn b mt trng,

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ni tip gip vi vng bng ca phi l du hiu khi ng s di chuyn ca Dorsal. Protein Toll phn b ng u trn b mt pha trong mng nguyn sinh bao ph phi. Protein Spatzle phn b trong cc khoang gia mng nguyn sinh v mng non hong (Hnh 7.7). Protease m bi gen easter ch phn ct Spatzle phn b vng bng phi thnh polypeptide c kh nng tng tc vi Toll. Phc Toll- Spatzle c nhn bit bi Dorsal khin cho protein ny di chuyn vo nhn. V sao protease Easter ch c hot tnh vi Spatzle vng bng? Phi chng gen easter ch c hot ho vng ny hay bn thn sn phm ca gen c d tr v c hot ho mt cch cc b? Hot ng ca cc gen thuc nhm trc lng-bng lin quan n cc tn hiu c trao i gia t bo trng v cc t bo nui. Tng tc mc phn t gia cc tn hiu ny cha c hon ton sng t. Tuy nhin sau khi th tinh, kt qu tng tc gy nn s di chuyn ca nhn t bo trng t cc ui v pha vng lng ca phi. iu ny dn n s bit ho ca cc t bo nui vng lng v vng bng. Nh , protein Spatzle phn b vng bng c nhn bit v b phn ct bi protease Easter. Protein Toll c m bi gen toll ng vai tr quyt nh trong vic phn trc lng-bng. Mi t bin gen toll u gy ri lon trong qu trnh hnh thnh trc ny. Cc gen khc lin quan n qu trnh phn trc u ch tham gia iu ho hot ng ca protein Toll m khng mt gen no thay th c n trong vic khi ng qu trnh. iu ng lu l protein Toll phn b ng u trong t bo cht ca trng. Do protein Spatzle ch b phn ct phn di (pha bng) ca t bo trng nn cc tn hiu (cc peptide to ra t Spatzle) hoc b receptor Toll tng tc rt nhanh hoc khng c kh nng khuych tn nn chng ch hot ho c cc phn t Toll phn b phn di. Sau khi Toll tng tc vi Spatzle, mt lot cc phn ng truyn tn hiu xy ra trong trng: Toll Tube Pelle Cartus Dorsal. Chc nng ca protein Tube cha c xc nh trong khi Toll ng vai tr th cm cn Pelle c hot tnh kinase. Protein Toll hot ho gen tube; sn phm gen tube hot ho gen pelle. Protein Pelle c chc nng kinase phn hu phc cht Cartus-Dorsal, nh Dorsal c gii phng dng t do. T t bo cht, Dorsall di chuyn vo nhn, hot ho cc gen c lin quan n pht trin cu trc bng.

Hnh 7.7: Hnh nh ct ngang phi rui gim Drosophila cho bit v tr phn b v tng tc gia protein Toll v

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Spatzle khi u qu trnh hnh thnh trc lng-bng phi Drosophila. Tng tc ny xy ra cc b vng bng ca phi trong khoang gia mng nguyn sinh v mng non hong bao quanh phi. Kt thc ca qu trnh ny l protein Dorsal c di chuyn vo trong nhn gy hot ho hoc c ch cc gen twist, snail, zerknullt v decapentaplegic. Hot ng ca cc gen ny lin quan n s hnh thnh trc lng-bng (theo Snustad, 2000).

Protein Dorsal phn b ng u trong t bo cht ca trng. Sau khi cc nhn di chuyn n b mt phi to phi b th cc t bo nm pha lng c Dorsal phn b t bo cht cn cc t bo nm pha bng c Dorsal phn b trong nhn. Tuy nhin, u tin Dorsal khng di chuyn c vo nhn do tng tc vi Cartus. Ch khi nhn tn hiu t Toll, protein Pelle mi c hot tnh kinase gy phosphoryl ha Cartus khin Dorsal c gii phng ra dng t do. Lc Dorsal mi di chuyn vo nhn. Chu s kim sot ca Dorsal, cc gen twist v snail, zerknullt v decapentaplegic c hot ho hoc km hm tu thuc vo nng ca protein ny. Cc t bo pha bng c nng Dorsal trong nhn cao nht gy hot ho cc gen pht trin cu trc bng. Cc t bo pha lng c nng Dorsal trong nhn thp nht cho php cc gen pht trin cu trc lng hot ng (tc l cc gen ny b c ch nng cao ca Dorsal). vng gia, nng Dorsal cao gy c ch mt s gen nhng li qu thp bt m cc gen khc. iu dn n s pht trin c hiu ca ngoi b. 7.3.4. Nhm gen qui nh pht trin cc cu trc tn cng ca u th (terminal-group genes) Phn cu trc c bit nm tn cng cc u khng phn t ca phi (cu trc acron phn u, cu trc telson phn ui) c qui nh bi hot ng ca mt s gen. Hot ng ca nhm gen ny cng tng t nh nhm gen phn trc lng-bng. Sau khi trng th tinh, protein Torso c tng hp t ARNm d tr trong t bo trng. Protein ny nhn cc tn hiu cc cc ca phi v tr nn hot ho, m u cho cc phn ng truyn tn hiu khc. Cc protein Tarless, Huckebein c tng hp t cc ARNm d tr cc cc, chng tham gia iu khin bt m cc gen c lin quan n cu trc ca cc cc tn cng. Nh vy cc c ch hot ng khc nhau ca cc gen c ngun gc t m dn n hnh thnh cc trc lng-bng v u -ui ca phi. y l nhng bc u tin trong qu trnh pht trin m phi xc nh hng v cu trc khng gian ca phi. i vi trc u-ui, phn b cc b ca ARNm, cn i vi trc lng-bng phn b cc b ca protein, ng vai tr quyt nh. Cc ARNm v protein ny u l sn phm ca cc gen c ngun gc t m. Sau khi phn trc, hot ng tip theo ca cc gen thuc h gen lng bi (zygote) s quyt nh s hnh thnh cu trc t cng nh cc c tnh ring bit ca tng t. Cc gen c ngun gc t m c phin m t c th m v ARNm tng ng c d tr trong trng. Ngoi ra, chng cn c hot ho t h gen lng bi ca phi (c th con). V d, gen hunchback c ARNm xut pht t hai ngun. Sn phm ca gen c ngun gc t m cng vi mt s gen trong h gen lng bi tham gia kim sot cc gen phn t (gap genes). 7.4 Hot ng ca cc gen trong h gen lng bi (phi)

rui gim trng thnh, ngoi phn u, c th cu to gm 3 t ngc v 8 t bng. Mi t c th chia lm 2 tiu phn, gi l tiu phn trc v sau (a v p). giai on pht trin phi v u trng cng xy ra s to t. Thc nghim quan st c 14 t v nhn thy mi t phi s ng vi hai tiu phn ca 2 t c th trng thnh. Mi t phi gm tiu phn sau (p) ca t ny v tiu phn trc (a) ca t lin k c th trng thnh (Hnh 7.8). V vy cc t ca phi c gi l phn t. C th quan st c cc phn t

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ny sau khi phi pht trin c 6h. Tuy nhin, sau 9h th phn t bin mt. Bin gii gia hai phn t tr thnh phn gia ca t mi. Phn t th t s ng vi tiu phn P ca t ngc T1 v tiu phn A ca t ngc T2 (Hnh 7.8). Khi phi phn chia n ln th 11, cc gen ca h gen hp t (phi) bt u hot ng. Hot ng khc nhau ca cc gen ny gia cc vng hoc ngay trong mt vng khin cho cc sn phm ca chng cng phn b khc nhau. Cc gen phn t "gap genes" hot ng chia phi thnh nhng vng ln, trong xy ra s phi hp a dng ca cc protein nhm iu khin s bit ho ca cc vng. iu quyt nh s phn ca mi vng s pht trin thnh mt s t ring bit. Tuy nhin nh mnh ny ch c hon thin nh hot ng ca cc gen to cp t, cc gen phn cc t. u th rui gim cng nh c th trng thnh cu to gm cc t. Qu trnh hnh thnh cc t lin quan n hot ng ca khong 30 gen. t bin mt gen bt k u nh hng n s lng cc t hoc cu to ca tng t. Cc gen phn t hot ng sau cc gen c ngun gc t m. Cc gen ny u thuc h gen lng bi ca phi. Chng c chia lm 4 nhm: nhm gen to t, nhm gen cp t, nhm gen phn cc t v nhm gen chn lc. Vic phn nhm ph thuc vo biu hin tnh trng khi gen b t bin v thi im cng nh v tr m gen hot ng (Hnh 7.9). Chng thng bt m sau 2 gi trng c th tinh, trc khi hnh thnh cc t bo trong phi.

Hnh 7.8: V tr tng ng ca cc phn t xut hin trn phi sau 6h pht trin v cc t xut hin sau 9h. Ngoi phn u, cc t ngc gm T1-T3 v cc t bng gm A1-A8 (theo Lodish & cs., 2000).

Phn ln sn phm ca cc gen c ngun gc t m v ca mt s gen phn t ng vai tr yu t phin m i vi nhm gen phn t. Nhng yu t ny cng lc m nhn hai chc nng va hot ho mt s gen phn t ny ng thi va km hm mt s gen khc. Do nng ngng ca mt factor cng nh t l nht nh gia factor hot ho v factor c ch s quyt nh mc biu hin ca tng gen phn t. V d nng protein Hunchback, sn phm ca gen phn t u tin m cao th qu trnh phin m ca gen kruppel b km hm nhng nu nh di mt ngng nht nh th hot ng ca gen ny li c hot ho. Nh vy nng ngung ca Hunchback quyt nh ranh gii xut hin ca protein Kruppel. Gradient nng Hunchback gim dn t u n ui ca phi. Do , nng Kruppel s tng dn theo chiu . Mt cch tng t, Kruppel khi t n ngung no s quyt nh hot ng ca nhng gen phn t khc tip theo.

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Hnh 7.9: Vng hot ng ca cc gen hunchback v kruppel. Hai gen ny m cho protein iu khin. Chng hot ng cc phn t phn u v ngc (da vo s phn b ca ARNm ti nhng phn t ny trong phi). Tuy nhin, cc protein Hunchback v Kruppel li nh hng n bit ho pht trin ca nhng t nm sau cc t c hai gen ny phin m. S phi hp ca cc protein iu khin kim sot s bit ho khc nhau ca cc vng trong phi (theo Lodish & cs., 2000).

7.3.5. Cc gen to t "gap" t bin nhng gen ny gy thiu ht mt hoc mt s t nm cnh nhau. Cc gen hunchback, kruppel c xem l nhng gen to t do t bin nhng gen ny lm cho cc t phn ui khng c to thnh. t bin n l thng khng gy tc ng nghim trng n vic xut hin cc t phn ui nhng t bin c hai gen bicoid v kruppel khin cho phi khng c ton b cc t vng ui. 7.3.6. Cc gen cp t "pair-rule" t bin chng gy thiu ht cc t chn hoc l lm cho s t gim i mt na. Sn phm ca mi gen to cp t s xut hin trong 7 t chn hoc 7 t l. Trong mi t, gen uc kim sot bi cc yu t hot ha v km hm mt cch c lp vi nhau. Hu ht cc factor phin m ny l sn phm ca gen c ngun gc t m hoc gen to t hot ng trc . Hot ng ca gen eve to t chn s 2 c xem l v d v s phi hp kim sot ca cc factor m bi gen bicoid v cc gen to t hunchback, kruppel v giant. Cc protein ny tng tc vi nhng v tr c hiu nm trc promoter gen eve. Bicoid v Hunchback kch thch hot ng ca eve trong phm vi rng nhng Kruppel v Giant li km hm eve trong khong hp xung quanh ranh gii gia hai tiu phn trc v sau. Gradient nng mi factor iu khin ny bin i theo qui lut c th ring cho tng protein (Hnh 7.10). V vy, chng kim sot cht ch ranh gii gia cc tiu phn ca tng t.

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Hnh 7.10: Bin i nng protein Eve cng nh cc factor hot ho Bicoid, Hunchback v yu t km hm Giant trong qu trnh kim sot hot ng ca gen eve. Phi hp gia cc protein xc nh chnh xc ranh gii gia cc tiu phn trc v sau ca t th hai. Mt cch tng t, s xut hin ca cc t khc c quyt nh do t hp cc factor c m bi gen c ngun gc t m v gen phn t.

7.3.7. Cc gen phn cc t Gen phn cc t qui nh trt t cc vng ngay trong mt t. y l nhng gen m khi b t bin thng khng lm thay i s lng t nhng mt phn no ca t s b loi b v thay th bi phn cn li ca t v tr i xng (hnh nh gng). V d, t bin trn gen gooseberry khin cho tiu phn sau ca mi t khng c hnh thnh. Thay vo l cu trc ca tiu phn trc nhng sp xp theo kiu hnh nh gng. Hot ng ca cc gen phn t xy ra tng t nh cc gen c ngun gc t m, tc l sn phm ca gen ny s bt m cc gen tip theo hoc c ch cc gen hot ng trc . Nh vy, cc gen phn t hot ng theo khng gian v thi gian. Hot ng ca cc gen phn t s phn chia phi thnh nhng phn nh dn dc theo trc u-ui. Trong mi phn nh, hot ng ca cc gen chn lc s qui nh cc c tnh ring bit ca tng phn. 7.5 Cc gen chn lc

Cc gen lin quan n s hnh thnh cc t phi rui gim c iu khin theo cch thc sao cho sn phm ca mt s gen hot ng trc s quyt nh mc biu hin ca cc gen tip theo. Nh , tng nhm t bo s c chung s phn quyt nh s phn vng dc theo trc u ui. Tuy nhin, trong mi vng, iu g khin cc t bo bit ho khc nhau to nn cu trc c th ring ca vng ? Ni cch khc v sao mi c quan ch phn b ti mt vng nht nh trn c th? Liu chng ta c th gy nh hng n hot ng ca cc gen thay i v tr ca tng b phn c hay khng? V d, rui gim, c th gy t bin mt xut hin vng bng hoc chn xut hin vng u hay khng? Bng k thut gy t bin v sng lc cc c th c s sai lch v v tr ca mt b phn trn c th, di truyn hc phn t xc nh c nhm gen c bit gi chung l gen chn lc (selector genes). y l nhng gen hot ng lin tc t giai on pht trin phi n c th trng thnh vi chc nng m bo v duy tr tnh c th ca tng vng dc theo cc trc u ui, lng bng trong sut qu trnh sinh trng v pht trin. u tin nhng gen ny c gi l gen homeotic do t bin nhng gen ny gy nn s sai lch v tr ca mt s b phn trn c th. Tuy nhin, tng hp kt qu nghin cu trn nhng gen lin quan n s duy tr tnh bit ho c th ca t bo ti mi vng m chng c gi l gen chn lc. Khc vi cc nhm gen c ngun gc t m hay gen phn t ch hot ng nht thi, cc gen

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chn lc mt khi bt m th phi hot ng lin tc. Nh vy hot ng ca cc gen chn lc nhm xc nh cu trc ca cc phn c th khc nhau dc theo cc trc trong sut thi gian c th tn ti. Cc gen chn lc cng hot ng hoc hot ng sau cc gen phn cc t. t bin xy ra nhng gen ny lm cho mt phn ca c th biu hin cc tnh trng ca phn khc hoc cc b phn ca t ny li xut hin t khc. V d, chn mc u hoc xut hin hai cp cnh ging ht nhau. Cn lu rng khi cc gen chn lc b t bin hoc hot ng ca chng xy ra khng ng thi im hoc v tr khng thch hp, ch c v tr cc b phn b sai lch ch khng h to ra b phn mi. nh mnh pht trin ca cc t bo c qui nh bi cc gen hot ng trc cc gen chn lc, tuy nhin nh mnh ny s khng c thc hin trn vn khi hot ng ca cc gen chn lc xy ra khng ng v tr. Phn ln cc gen ny m cho cc protein iu khin (lin kt vi ADN ti cc enhancer hoc promoter). Chng lm nhim v bt m cc gen nhng mc khc nhau. Do , mt t bin xy ra i vi mt gen chn lc khng ch gy bt hot gen m cn thay i hot ng ca cc gen chn lc khc. Cho n nay, 8 gen chn lc c pht hin rui gim v chng c xp vo 2 nhm Bithorax (BX-C) v Antennapedia (ANT-C) cng phn b trn nhim sc th s 3, cch nhau khong 10 cM. Nhm Antennapedia c 5 gen (lab, pb, Dfd, Scr, Antp) kim tra s sai khc gia cc t u v t ngc (t phn t 1-5) cn nhm Bithorax c 3 gen (Ubx, abdA, AbdB) lm nhim v kim tra s khc nhau gia cc t bng v t ngc (phn t 5-14). t bin nhng gen ny thng biu hin tnh trng cu trc biu b, h c, thn kinh dc theo trc u ui. Tt c 8 gen u c tnh cht chung l vng khng cha m di truyn (intron) rt ln. Nhng vng ny c vai tr rt quan trng trong kim sot mc biu hin ca gen theo thi gian v khng gian dc trc u ui. Nhng gen chn lc c vai tr tng t BX-C v ANT-C c pht hin ng vt c v. Tuy nhin, chng phn b ri rc thnh bn nhm trn cc nhim sc th khc nhau. Tt c nhng gen ny ng vt u c gi chung l nhm gen Hox. Ring i vi rui gim th cc gen Hox (thuc hai nhm BX-C v ANT-X) cn c gi l gen Hom. Nhn chung cc gen Hox u c cng nhng c im sau: + Vng ADN iu khin ca gen Hox cha mt on ADN di khong 180 bp c tnh cht bo ton. on ny c gi l homeo box. Homeo box m cho on amino axit (khong 60 a.a) c kh nng tng tc vi ADN. on amino axit ny c gi l homeo domain. + Cc exon ca mt gen thng nm xa nhau, phn cch bi cc on ADN di khng cha m di truyn nhng ng vai tr iu khin hot ng ca gen. + Cc gen trong mi nhm nm dc nhim sc th theo mt th t nht nh, chnh xc nh trnh t hot ng ca chng dc theo chiu di c th t u n ui. Cc gen ny c hot ho ni tip nhau nh cc tn hiu truyn dc nhim sc th, ph hp vi ch dn v khong cch gia cc phn ca c th (tnh theo trc u-ui).

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Hnh 7.11: Cu trc ca cc gen chn lc thuc nhm BX-C. (A). Cc exon c k hiu bi cc en m. Cc intron kch thc ln ng vai tr rt quan trng trong kim sot hot ng ca gen theo khng gian v thi gian. (B). Mc biu hin (ARNm/protein) ca cc gen thuc nhm BX-C trong cc phn t 4 n 14. Cc vng m ch mc biu hin cao ca gen trong tng vng dc theo trc u ui (theo Alberts & cs., 2002).

Nghin cu chi tit vai tr ca cc gen Hox thuc nhm BX-C cho thy th t hot ng ca 3 gen tun theo trt t sp xp trn nhim sc th. Sn phm ca chng phn b dc theo trc u ui cng tun theo qui lut nghim ngt. Hon ton khng c s phn b ng u m tn ti s chnh lch nng protein ca 3 gen gia cc phn t (Hnh 7.11). Khi c 3 gen ca nhm BX-C b t bin th phi b cht. Phn tch lp biu b ca phi cht pht hin thy ton b cc phn t 5 n 13 u ging ht phn t 4. Ni cch khc, t bin ton b nhm BX-C khin cho cc phn t 5-13 khng c to thnh. Nh vy, nhm Bithorax ch c 3 gen nhng chu trch nhim v s sai khc gia 10 phn t. c th l kt qu ca phn ng bin i phn t ARNm (ct intron theo cc trt t khc nhau) dn n nhiu loi phn t ARNm c tng hp t mt gen. Mt khc rt nhiu t bin c tm thy phn b trn nhng on ADN khng cha m di truyn. Trt t cc t bin ny trn nhim sc th cng tng ng vi trnh t sp xp ca cc vng c th m chng c nh hng. iu ny cho thy s khc bit gia cc vng ca c th khng ch do sn phm ca cc gen Hox quyt nh m cn ph thuc cc on ADN khng cha m di truyn (cc intron). Ni cch khc, v tr ca t bo ch c ngha trong pht trin phi nh hot ng ca cc gen Hox cng nh cch thc iu khin hot ng ca chng. Hin nay vn ang c cc nh nghin cu sinh hc quan tm n chnh l xc nh c ch no gip cc gen Hox nh c v tr hot ng ca chng. C ch iu khin phn hi c th c lin quan. Thc nghim chng minh c sn phm ca mt s gen Hox hot ho phn ng tng hp ARNm trn gen ca chnh n. V d, gen dfd trong nhm Antennapedia c nhiu v tr lin kt vi chnh protein Dfd. S lin kt ny duy tr hot ng ca gen. Ngoi ra, nhm gen polycom gm cc gen cn thit km hm hot ng ca cc gen Hom. Bt k mt gen no trong nhm ny b t bin, cc gen Hom s hot ng, mi u mc bnh thng v ng v tr, nhng sau chng hot ng khng chu iu khin v hot ng trn mi vng ca phi. Protein Polycom bm vo chromatin ca gen Hom lm thay i cu trc chromatin. Do s bin dng chromatin c th gip cho gen Hom nh c v tr hot ng ca mnh.

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Dng on homeo box ca cc gen Hom rui gim lm mi cho cc php lai Southern vi ADN genome tch ra t cc loi ng vt khc nhau, kt qu cho thy cc gen Hox u tn ti trong genome ch, chut v ngi. c bit cc gen ny u hot ng trong qu trnh pht trin phi. Khi a cc gen hox b t bin ca chut vo phi rui gim, chng gy ra s pht trin ba bi ging nh khi cc gen Hom ca rui b t bin. Cc gen Hox ng vt cng u hot ng theo thi gian v khng gian tng ng vi trnh t sp xp ca chng trn nhim sc th v theo trt t nht nh t u n chn. Tuy nhin gia cc gen Hox v gen Hom c s khc nhau: Cc gen Hox thng nh khng qu di v s lng gen trong mi nhm nhiu hn. C th tn ti nhiu bn sao ca mt gen Hox hoc nhiu bn sao ca cc nhm gen trong genome, do kh pht hin c nhng t bin biu hin ra tnh trng. Cng vi cc gen BX-C v ANT-C, di truyn phn t pht hin hng lot cc gen homeotic khc c lin quan n s hnh thnh ca tng c quan ring bit phn b tng vng nht nh trn c th. V d in hnh l gen eyeless qui nh v tr ca mt rui gim. Sn phm ca gen ny c cha homeo domain. Protein m bi eyeless ng vai tr yu t phin m lin quan n biu hin ca rt nhiu gen khc qui nh s hnh thnh mt ng v tr. Thc nghim to ra rui gim chuyn gen mang gen eyeless chu s iu khin ca promoter l m promoter ny ch hot ng trong m khng lin quan n vic hnh thnh mt. Sn phm ca gen eyeless xut hin khng ng v tr khin cho mt c th xut hin chn, cnh hoc vng ngc. Nhng mt c bit ny c th nhn bit nh sng nh mt bnh thng khc. ng vt c v c cha gen tng ng vi gen eyeless ca rui gim. V d gen tng ng chut c gi l gen Small eye do t bin gen ny khin mt chut b nh li. Lp li th nghim chuyn gen Small eye ca chut vo rui gim (tng t th nghim nu trn) th rui gim chuyn gen cng c mt xut hin sai lch v tr. R rng gen Small eye chut l gen homeotic v m cho protein cha homeo domain. Gen Small eye cng c chc nng qui nh vic hnh thnh mt ng v tr. Tuy nhin, rui gim mang gen Small eye li c thm mt rui ch khng phi mt chut. Nh vy sn phm ca hai gen Small eye v gen eyeless c chc nng tng t nh nhau, tc l c kh nng iu khin mt lot gen khc ca rui gim dn n kt qu cui cng l mt xut hin v tr mi. V vy nht nh mt rui ch khng th l mt chut c hnh thnh. Gen tng ng vi hai gen Small eye v gen eyeless c pht hin ngi. t bin gen ny khin trng en ca mt b nh hoc mt hn. Kt qu nghin cu ca di truyn phn t trong nhng nm cui th k 20 trn i tng m hnh Drosophila lm sng t phn no qu trnh pht trin ca trng sau th tinh. Hot ng k tip nhau theo mt trt t nghim ngt ca cc gen c ngun gc t m, gen to t, gen chn lc quyt nh vic hnh thnh cc trc ca c th, s phn vng phn t, s bit ho ca cc t bo, s xut hin cc c quan trong tng vng... c bit s tng ng ca cc gen homeotic c tm thy rui gim cng nh ng vt c v v con ngi. Phi chng nhng gen ny u c chung ngun gc t mt gen ban u. Tri qua thi gian tin ho, chng vn phn no b tr cho nhau thc hin mt chc nng c th trong sinh trng v pht trin?

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GII NGHA THUT NG CHUYN DNG


activator: protein hot ho. Protein tng tc vi ADN lm tng tc khi ng phin m promoter. allele: Mt trong nhiu dng ca gen phn b trn nhim sc th tng ng. Mt gen c 2 hoc nhiu allen (a allen). allele-specific oilgonucleotide (ASO) hybridization: Lai xc nh mt trong hai dng si n ca mt phn t ADN kp bng oligonucleotide. allosteric transition: S thay i cu trc bc 3 hoc bc 4 ca protein do tng tc vi phn t trng lng nh. Tng tc ny lm thay i hot tnh ca protein. alpha () helix: Cu trc bc 2 ca phn t protein to nn do mt on peptide cun xon nh lin kt gia nhm carboxyl v amin. amphipathic: Phn t hoc cu trc bao gm hai phn k nc v a nc. annealing: gn mi. Tng tc ca on oligonucleotide vi khun ADN hoc ARN. antibody: Khng th. L phn t protein tng tc vi khng nguyn ti mt v tr c hiu v kch thch c th loi b khng nguyn bng cc c ch khc nhau. anticodon: i m. L trnh t 3 acid nucleic trn phn t ARNt to cp b sung vi mt m di truyn b ba trn phn t ARNm. Lin kt b xung cho php acid amin do ARNt vn chuyn c gn vo chui peptide ang c tng hp. antigen: Khng nguyn. Bt c phn t no khi xm nhp vo c th c kh nng kch thch s to ra khng th v tng tc c hiu vi khng th c gi l khng nguyn. antisense ARN: Phn t ARN i ngha. Phn t ny c trnh t b sung vi trnh t ARN khc, thng l ARNm hn ch s to ra ARN hoc ngn cn qu trnh dch m trn ARNm. apoptosis: cht theo chng trnh. Qu trnh gy cht t bo theo chng trnh c m trong h gen vi nhng biu hin hnh thi c th ring. autonomously replicating sequence (ARS): Tm ti bn ( nm men). Trnh t ADN trn nhim sc th m bo kh nng ti bn cho nhim sc th . bacterial artificial chromosome (BAC): Vector tch dng c kh nng nhn on ADN kch thc ln, c thit k da vo plasmid F ca vi khun E.coli. bacteriophage (phage): Virus c kh nng xm nhp vo t bo vi khun. basal promoter: tm promoter. V tr nm trong promoter eukaryot, ti phc khi ng phin m bt u hnh thnh. base exsision repair: sa cha loi base. C ch sa cha ADN trong base sai hng loi b v thay th mi. base pair: cp base. Hai nucleotide b sung vi nhau bi lin kt cu hydrogen. Khi c vit tt bp c ngha l n v chiu di ngn nht ca phn t ADN kp.

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blund end: u bng. u tn cng ca si kp ADN kt thc bng hai nucleotide lin kt b sung vi nhau. ADNc (complementary DNA): phn t ADN b sung c phin m t ARNm bng enzym phin m ngc. ADNc khng cha intron, ch gm exon so vi ADN (gen) trong h gen (genome). centromere: tm ng. L on nhim sc th ti hai nhim sc t dnh vo nhau trong phn bo nguyn nhim. Tm ng l v tr phc protein kinetochore bm vo v tng tc vi cc si thoi v sc ko nhim sc th v cc cc. chaperone: tn gi protein c kh nng hoc ngn cn s cun gp sai ca protein ch hoc to thun li cho protein ch gp cun chnh xc. chaperonin: phc protein tr gip vic gp khc to cu trc khng gian ca nhng protein khc. checkpoint: v tr (im) trong chu trnh t bo ti s chuyn gia 2 phase c quyt nh ph thuc vo s p ng y cc iu kin cn c cho phase tip theo. chimera: c th c cha hai hay nhiu loi t bo c ngun gc di truyn khc nhau. chromatin: nhim sc cht, bao gm ADN, histone v protein nonhistone phn b trong nhn t bo eukaryot. Trong giai on phn bo metaphase, cht nhim sc co c thnh nhim sc th. chromatin-remodeling: iu bin cht nhim sc. Qu trnh lm thay i cu trc v v tr ca nucleosome trn si nhim sc. chromosome walking: i trn nhim sc th. K thut cho php xc nh th t sp xp ca cc on ADN trn nhim sc th vi iu kin cc on ny phi nm gi ln nhau. cis-acting: tc ng ni sinh. Thng dng ch on ADN kim sot hot ng ca gen nm trn cng mt nhim sc th. t bo prokaryot, yu t tc ng ni sinh thng nm gn gen b kim sot. Tuy nhin, t bo eukaryot, cis-acting c th phn b rt xa gen. clone: thng ch mt nhm t bo cha cng mt loi phn t ADN ti t hp. cloning vector: phn t ADN c kh nng mang on ADN l v c kh nng ti bn trong t bo nhn phn t . competent: Thng dng cho cc t bo vi khun c kh nng nhn phn t ADN l. consensus sequence: trnh t bo th. on ADN c trnh t tng ng cao nhng khng ging nhau hon ton v qui nh mt chc nng c bit . constitutive heterochromatin: d nhim sc th bn. Vng nhim sc th c cu trc co c bn vng. constitutive gene: gen lun lun trng thi hot ng trong mi loi t bo ca c th. contig: tp hp ca nhng on ADN nm gi ln nhau. cotransformation: ng bin np. Hai hay nhiu gen cng nm trn mt phn t ADN c bin np vo t bo vi khun. crossing over: Trao i cho. Qu trnh trao i vt cht di truyn gia hai nhim sc th xy ra do t gy, trao i v ni li vi nhau. CpG island: o CpG. on ADN giu CG nm trc gen eukaryot. Hu ht cc gen hot ng u c o CpG khng b methyl ho cytosine.

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degenerate: Thoi ho. Thng dng ch trnh t nucleotide m cho cc acid amin khi nhng acid amin ny c m bi hai (hoc nhiu hn) m b ba. degradosome: Phc enzym c chc nng phn hy ARNm vi khun. delayed-onset mutation: t bin chm. Nhng t bin ny ch c nh hng giai on mun trong thi gian sng ca c th. de novo methylation: thm cc gc methyl vo cytosine m trc khng b methyl ho. DNA fingerprint: Cc bng lai nhn c t php lai ADN Southern blotting gia h gen ca i tng sinh vt vi u d c hiu. DNA photolyase: Enzym pht hin c vi khun v thc vt c kh nng s dng nng lng nh sng trong phn ng sa cha ADN. DNA shuffling: k thut pht trin da trn phn ng PCR to nn nhng phn t ADN ti t hp c chc nng sinh hc hon thin hn. V d nh to nn gen m cho protein c ph hot ng rng hn v c hot tnh cao hn enzym tn ti trong t nhin. domain shuffling: k thut to gen mi. K thut ny sp xp ni cc on ADN ca mt hoc nhiu gen vi nhau; mi on mang m di truyn ca mt vng chc nng ca mt hoc cc protein khc nhau. dosage compensation: hiu qu ca mt gen tng hoc gim ph thuc vo s lng bn sao ca gen trong genome. dot-blot hybridization: k thut lai gia u d ADN hoc ARN vi ADN hoc ARN vi nhng nng bit c nh trn mng. embryonic stem (ES) cell: t bo ton nng c ngun gc t phi thai. endocytosis: nhp ni bo. Qu trnh thu nhn vt cht t bn ngoi t bo nh nhng ti c ngun gc t mng t bo. endogenous retrovirus (ERV): h gen ca retrovirus dng hot ng hoc khng hot ng ghp xen trong nhim sc th ca t bo ch. enhancer: on tng cng, lm nhim v kim sot hot ng ca gen v c th phn b cch xa gen. on ny rt him h gen prokaryot. episome: plasmid c kh nng chn ghp vo nhim sc th ca t bo nhn. epitope: Quyt nh khng nguyn. L phn c bit thuc phn t khng nguyn tng tc vi khng th. euchromatin: cht nhim sc thc. L nhng on t co c ca cht nhim sc trong giai on gian k. Cht nhim sc thc cha hu ht cc gen trng thi phin m. exocytosis: Xut ngoi bo. Qu trnh tit xut cc cht ra bn ngoi t bo nh cc ti tit dung hp vi mng t bo. exon: L nhng phn ca mt gen c mt trong phn t ARN hon chnh. Nhng phn t ARN tn ti trong t bo cht, bao gm ARNm, ARNr v ARNt. expressed sequence tag (EST): trnh t xut pht t ADNc c dng xc nh nhanh cc gen trong h gen. expression cloning: K thut ADN ti t hp c s dng phn lp ADNc hoc on ADN ca h gen (genome) da vo cc c tnh chc nng ca protein c m bi

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ADNc hoc on ADN m khng cn phi tinh sch protein ny. K thut ADN ti t hp to ra s lng ln protein nh tch dng ADNc hay gen tng ng vi protein . expression vector: Nhng plasmid hoc genome virus chuyn ch gen hoc ADNc vo t bo nhn thch hp. Protein do ADNc hoc gen m cho c tng hp trong t bo nhn. facultative heterochromatin: vng d nhim sc khng bn. Cu trc d nhim sc thng xut hin mt s gen khng hot ng trong mt s t bo hoc mt s thi im ca chu trnh t bo. fluorescent in situ hybridization (FISH): k thut lai hunh quang gia cc du (marker) gn hunh quang v nhim sc th xc nh v tr ca ch th trn nhim sc th. F plasmid: yu t gii tnh F kim sot chuyn gen bng con ng tip hp gia vi khun. footprinting: du vn tay. K thut xc nh trnh t ADN hoc ARN tng tc vi protein. Phn t acid nucleic nh du (bng phng x hoc bng cht pht hunh quang) b ct vi nuclease khi thiu hoc khi c mt ca protein. Khi c mt protein, on nucleotide tng tc vi protein c bo v nn khng b ct bi nuclease. So snh s xut hin cc bng ct trong hai trng hp cho php xc nh c trnh t nucleotide tng tc vi protein. frameshifting: chuyn khung c. S di chuyn c kim sot ca ribosome t mt khung c ny sang khung c khc v tr phn b trong gen. fusion protein: protein dung hp. Protein to nn do s dung hp ca hai polypeptide hoc hai hay nhiu phn ca cc chui polyppetide c m bi cc gen khc nhau. G protein: Cc protein c cu trc trimer (gm 3 tiu n v) c kh nng lin kt v phn hy GTP. G protein tham gia con ng truyn tn hiu trong t bo. gain-of-function mutation: t bin to tnh trng mi. gel retardation analysis: k thut xc nh v tr tng tc ca protein vi ADN da vo di ng ca ADN v phc ADN-protein trn gel in di. gene: n v mang thng tin di truyn. mc phn t, gen l on ADN bao gm c exon, intron v on nucleotide lm nhim v kim sot phin m, cn thit to ra sn phm chc nng cho hot ng sng ca t bo. V vy, gen m cho protein v cc phn t ARN. gene control: Cc c ch kim sot hot ng ca gen, bao gm giai on phin m, sau phin m, dch m v sau dch m. gene convesion: Chuyn hon gen. Hin tng cc allen ca mt gen chuyn hon cho nhau trong qu trnh trao i cho phn bo gim nhim. gene expression: Biu hin ca gen. Qu trnh chuyn i thng tin di truyn t gen thnh tnh trng. genes-within-genes: gen trong gen. Khi nim dng ch hin tng gen th hai nm trong intron ca gen th nht. genetic code: M b ba nucleotide trn ADN hoc ARN m cho mt acid amin ca phn t protein. genome: H gen. Tng s thng tin di truyn (ADN hoc ARN) c mt trong t bo hoc c th. genomic DNA: H gen di dng ADN.

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genotype: Kiu gen. Khi nim dng cho allen ca mt gen hoc rng hn l ton b cc kt cu di truyn ca mt t bo hoc mt c th. germ cell: T bo mm (thng ch dng t bo sinh dc). y l loi t bo pht trin thnh giao t. germ line: Dng t bo mm. Khi nim ny cn c dng ch vt cht di truyn t th h trc sang th h sau thng qua giao t. growth factor: Phn t polypeptide (tn ti bn ngoi t bo) tng tc vi th th trn b mt t bo gy ra chui phn ng truyn tn hiu vo bn trong t bo, dn n s tng sinh, bit ho t bo. guide RNA: on ARN ngn tham gia c sa ARNm bng vic xc nh v tr trn ARNm ti mt hoc vi nucleotide c thm vo lm thay i trt t m di truyn trn ARNm so vi m trn ADN. hetechromatin: Vng nhim sc th ti nhim sc th co c v cc gen thng trng thi khng hot ng trong giai on gian k. heteroduplex: Si kp ADN c cha cc nucleotide khng to cp b sung vi nhau. heterokaryon: T bo c t hai nhn tr ln, to ra do dung hp ca hai hay nhiu t bo khc nhau. heterozygous: D hp t. T bo lng bi hoc c th c hai allen khc nhau ca mt gen. homeobox: Trnh t ADN bo th m cho mt on oligopeptide gi l homeodomain, ca protein kim sot phin m. on ny tng tc vi ADN gip protein thc hin chc nng ca chng. Gen m cho nhng protein c vng homeodomain c gi l homeotic gene. homeosis: S bin i mt b phn c th thnh b phn khc gy ra do t bin hoc do ri lon hot ng ca mt s gen quan trng cho bit ho pht trin phi thai. homology: S tng ng v trnh t nucleotide hoc acid amin hoc cu trc ca mt b phn biu hin cng chung ngun gc ban u. Cn lu , analogy cng phn nh s tng ng v cu trc hoc chc nng nhng khng c s lin quan trong tin ho. homozygous: ng hp t. T bo lng bi hoc c th c hai allen ca mt gen ging ht nhau. Hox complex: Nhm cc gen chn lc tng ng vi nhau. Cc gen ny quyt nh s hnh thnh cc b phn khc nhau ca c th. hybridization: S kt hp ca 2 si n nucleic b xung vi nhau to ra phn t kp. S kt hp c th xy ra gia 2 si n ADN, gia si n ADN v ARN hoc gia 2 si ARN. hybridoma: Dng t bo lai tn ti vnh cu v to ra khng th n dng. Dng t bo by c to ra nh s dung hp gia t bo lympho B bnh thng v t bo u ty. intron: Nhng phn ca si ARN u tin va c phin m t ADN s b loi b i trong qu trnh bin i phn t ARN thnh phn t c chc nng sinh hc. intein: on ngn trn si polypeptide b ct i sau khi dch m kt thc. y l mt cch thc bin i sau dch m c c phn t protein c hot tnh. isoform: Nhng protein c hot tnh ging nhau mc d chng c s khc nhau v trt t acid amin.

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karyotype: S lng, kch thc, hnh dng ca ton b cc nhim sc th trong t bo nhn thc giai on metaphase. kinase: Enzym chuyn nhm phosphate cui cng ca ATP sang c cht. Kinase ng vai tr c bit quan trng trong kim sot hot tnh ca cc protein trong t bo. kilobase pair: 1000 cp base (1000 bp). kinetochore: Cu trc protein ba lp phn b ti tm ng, ti si thoi v sc bm vo ko nhim sc t v cc cc. Kinetochore ng vai tr rt quan trng trong vic di chuyn nhim sc th v hai cc trong anaphase. knockin gene: K thut trong trnh t m ho ca gen ny b thay th bi trnh t ca gen khc. knockout gene: K thut gy bt hot gen mt cch chn lc bng cch thay th gen bnh thng ca c th bi gen t bin. label: K thut nh du mt phn t bng cht pht quang hoc bng ng v phng x. Nh c th xc nh v tr hoc tinh sch phn t hoc theo di qu trnh di chuyn ca n trong chui cc phn ng ho hc. leaky mutation: t bin tc ng mt phn n tnh trng. Ligand: kch th. Phn t thng c trng lng nh, tng tc c hiu vi th th hoc i phn t to nn phc. Phc ny khi ng chui cc phn ng khin t bo tr li li s c mt ca kch th. LINE: trnh t nucleotide lp li phn b ri rc trong h gen, thng c hot tnh transposase. linker DNA: ADN nm gia hai nucleosome, tng tc vi histone H1. locus: V tr ca gen trn nhim sc th. Mi allen ca mt gen u phn b ti mt locus. LTR element: on ADN lp li tn cng bi trnh t lp li di (long terminal repeats). maintenance methylation: methyl ho bo ton. Duy tr gc methyl trn si ADN va c ti bn da vo gc methyl sn c trn si khun. MAP kinase: Enzym c hot tnh chuyn gc phosphat t phn t ATP vo c cht (hot tnh kinase) trong chui phn ng t bo tr li kch thch i vi cc yu t kch thch sinh trng. mapping: Xc nh v tr nhn bit ca cc enzym gii hn (restriction map) hoc xc nh trt t sp xp ca cc gen (genetic mapping) hoc v tr chnh xc ca cc gen (physical mapping) trn nhim sc th. maternal-effect gene: gen c ngun gc t m. Nhng gen ny c pht hin rui gim Drosophila. ARNm ca nhng gen ny c tch ly trong trng v c dch m trong giai on pht trin sm ca phi. metastasis: Di cn. S di chuyn ca cc t bo c tnh t v tr pht sinh ban u n v tr mi v pht trin v t chc ti . microsatellite DNA: ADN vi v tinh. Trnh t gm 1-4 nucleotide lp li lin tc trn nhim sc th. ADN vi v tinh cn c gi l trnh t n gin lp li lin tc (STR-simple tandem repeats).

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minisatellite DNA: ADN tiu v tinh. Trnh t gm vi chc nucleotide lp li lin tc trn nhim sc th. Loi ADN ny cn c gi l trnh t thay i v lp li lin tc (VNTRvariable number of tandem repeats). mitosis-promoting factor (MPF): yu t khi ng phn bo. MPF l protein d lng cc gm cyclin v kinase ph thuc cyclin (Cdk), thc y s co c nhim sc th, ph v mng nhn, bt t bo bc vo phn chia. Mitosis-promoting factor cn c gi l maturation-promoting factor (yu t khi ng chn). mitogen: tc nhn kch thch t bo bc vo chu k phn chia. mobile DNA element: on ADN khng c mt v tr c nh trn nhim sc th trong t bo ca mi c th trong mt loi. monoclonal antibody: khng th n dng c to ra t cc t bo xut pht t mt t bo B ban u. Khng th n dng c tnh c hiu vi ch mt khng nguyn. morphogen: M lng. Phn t can thip n c tnh ca t bo trong pht trin bit ho (giai on phi thai) tu thuc vo nng ca phn t . motif: Thng dng ch cu trc khng gian cc b ca protein m cu trc lin quan n mt chc nng. motor protein: Nhm enzym phn hy ATP trt trn vi si myosin hoc trn vi ng dynein v kinesin. multigene family: nhm cc gen, nm cnh nhau hoc phn b ri rc trn nhim sc th, c trnh t nucleotide tng t vi nhau. mutagen: tc nhn (ho hoc l) gy t bin. nonhomologous end-joining (NHEJ): C ch sa cha phn t ADN khi c hai si n u b t gy tn thng. northern blotting: K thut xc nh phn t ARN c hiu t cc ARN khc bng vic lai u d nh du (labeled probe) vi cc ARN. nucleolus: hch nhn. Cu trc nm trong nhn t bo eukaryot ti ARNr c phin m v bin i kt hp vi protein to ra tiu phn ribosome. nucleosome: cu trc gm 146 bp ADN qun quanh li protein histone, to nn cu hnh khng gian ca si nhim sc. oligonucleotide-directed mutagenesis: t bin nh v. K thut gy t bin gen bng vic thay th mt (mt s) nucleotide ca gen bng nhng nucleotide theo mun, thng qua s dng oligo tng hp nhn to c cha sn cc nucleotide mong mun ny. oncogene: Gen gy ung th. Sn phm ca gen gy ung th c tc dng bin i t bo lnh thnh ung th. Hu ht cc gen gy ung th xut pht t cc gen tin ung th (protooncogene). Cc gen tin ung th m cho cc yu t kim sot tng sinh hoc phn chia t bo. Khi b t bin, phn ln gen tin ung th tr thnh gen gy ung th. operator: im kim sot tiu cc ca gen vi khun hoc virus im ny thng l trnh t ngn cc nucleotide phn b ngay trc vng m di truyn ca mt gen. y l v tr tng tc vi protein c ch ngn cn khi u phin m ca gen. operon: Tp hp cc gen nm cnh nhau c kim sot bi mt promoter, do operon c phin m to nn mt phn t polycistronic ARNm. Cu trc operon ch gp vi khun.

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origin of replication: V tr trn trnh t ADN ti qu trnh ti bn ADN c bt u khi ng. PEST sequences: trnh t acid amin gy nh hng n phn hy ca protein cha trnh t PEST . polymerase chain reaction (PCR): phn ng trng hp nhn bn ADN. K thut cho php nhn bn nhn bn c hiu mt on ADN trong s tp hp rt nhiu on khc nhau. phage (thc khun th phage): Virus xm nhim vi khun v c th gy cht t bo vi khun. phage display: k thut cho php xc nh cc protein c kh nng tng tc vi nhau. phenotype: kiu hnh (tnh trng). Cc c tnh c th quan st c ca t bo hoc c th tng ng vi mt kiu gen. pheromone: Tn hiu truyn tin, l nhng phn t kch thc nh pht i t mt c th lm thay i tp tnh hoc hot ng ca gen c th khc cng loi. pinocytosis: Nhp ti bo. Hin tng cc ti trong t bo cht thu nhn khng c hiu cc git lng t bn ngoi t bo vo. plaque assay: K thut cho php xc nh s lng cc th virus xm nhim trong mu vt bng vic nui cy cc nng pha long ca mu vt trn b mt t bo ch ca virus v tnh m cc vt tan do virus gy ra. plasmid: Phn t ADN kch thc nh, dng vng nm ngoi nhim sc th, c kh nng t ti bn trong t bo. Plasmid thng dng lm vector tch dng trong k thut ADN ti t hp. point mutation: t bin im. Thay th hoc thm bt mt nucleotide lm thay i hot ng ca gen mc phin m (nu t bin vng iu khin ca gen), hoc lm xut hin m dng tng hp (stop codon) hoc lm thay i khung c m (ORF) nu t bin nm vng mang m di truyn ca gen. polyadenylation: thm ui polyA vo u 3 ca phn t ARNm eukaryot. polyadenylation editing: thm ui polyA to nn m dng tng hp protein trn phn t ARNm trong ty th ng vt. y c xem l c ch c sa ARNm bng vic thm ui polyA vo u 3 ca phn t ARNm kt thc l U hoc UA. positional cloning: Xc nh v tr v phn lp gen t bin da vo lin kt di truyn v cc ch th phn t ADN. pre-ARNm: bn phin m ARNm s cp. Phin bn c bin i thnh ARNm dng lm khun dch m. primary transcript: bn phin m s cp. Bn phin m ny cn tri qua qu trnh bin i c chc nng hot tnh sinh hc c trng cho tng loi phn t ARN. primer: mi. on nucleotide ngn cha nhm OH t do u tn cng 3, c trnh t b sung vi si khun n v l ni bt u tng hp si mi. primosome: phc protein tham gia ti bn ADN. probe: u d. on ADN hoc ARN gn ng v phng x hoc cht pht quang, dng pht hin trnh t nucleotide tng ng vi on . u d thng dng trong cc php lai acid nucleic. promoter: im khi u phin m.

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proofreading: chnh sa. Khi nim ny hay dng trong ti bn ADN ch hot tnh exonuclease 3 5 ca ADN polymerase loi b cc nucleotide khng to cp vi khun nhng b gn nhm vo si ang tng hp. prophage: ch trng thi tim tan ca genome thc khun th khi genome ny ghp vo nhim sc th t bo ch. proteome: tp hp tt c cc protein c mt trong t bo. pseudogene: gen gi. Mt gen khng c chc nng sinh hc. RACE (Rapid Amplification of ADNc ends): K thut PCR nhn bn phn u ca phn t ADNc. Ras protein: Phn t monomer tng tc vi GTP, phn b trong t bo cht, tham gia vo con ng truyn tn hiu sau khi c hot ho bi lin kt gia kch th v th th trn b mt t bo. reading frame: khung c. Trnh t nucleotide gm bi s ca 3 nucleotide (m b ba) trn phn t ARNm, tnh t v tr bt u dch m (start codon) n v tr dng tng hp protein. Mt s phn t ARNm c th ng vi 2 khung c khc nhau. receptor: th th. Bt c phn t protein no tng tc c hiu vi kch th bn ngoi t bo v khi ng chui phn ng bn trong t bo tr li kch th. recombinant DNA: ADN ti t hp. Phn t ADN c to ra do s ghp ni ca cc on ADN c ngun gc khc nhau. K thut to phn t ADN ti t hp thng da vo vic ct ADN c ngun gc khc nhau bng enzym gii hn v ni chng bng enzym ligase. recombination: ti t hp. Qu trnh trao i cc on nucleotide gia hai phn t ADN hoc nhim sc th to ra mt t hp mi. repetitive DNA: trnh t ADN lp li nhiu hn hai ln trong mt gen hoc trong h gen (genome). replication origin: tm sao chp (tm ti bn): Trnh t nucleotide duy nht ti sao chp ADN c bt u. Trn mt nhim sc th eukaryot c nhiu tm sao chp nhng mt nhim sc th prokaryot ch c mt. replicon: vng ADN c sao chp t mt tm sao chp. reporter gene: gen bo co. Da vo tnh trng bit ca gen bo co phn tch cc on nucleotide kim sot hot ng ca gen ny. restriction enzyme: enzym gii hn. Chng nhn bit trnh t nucleotide c hiu v ct phn t ADN si kp thnh cc on. restriction fragment length polymorphism (RFLP): a dng cc on ADN c ct bi enzym gii hn do s a dng v v tr nhn bit ca enzym. retrotransposon: on ADN c kh nng di chuyn trong genome thng qua dng trung gian l ARN nh qu trnh phin m ngc. retrovirus: Virus xm nhim t bo eukaryot c genome l ARN. Ti bn genome ca retrovirus tri qua bc phin m ngc to ADN. ADN ny c ghp vo nhim sc th t bo ch. Ti , ADN c phin m to ARN genome v ARNm dch m tng hp protein ca virus. reverse transcriptase. Enzym phin m ngc c hot tnh tng hp si ADN t si n ARN. Rho-dependent terminator: V tr kt thc phin m trn ARNm prokaryot, ti s kt thc ph thuc vo protein Rho.

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ribozym: Phn t hoc mt on ARN c hot tnh xc tc phn ng. RNA-depedent RNA polymerase: enzym phin m tng hp si ARN trn khun ARN. RNA editing: c sa ARN. Trnh t nucleotide trn phn t ARNm b thay i. RNA splicing: ct intron, ni exon. satellite DNA: ADN v tinh. Nhng trnh t nucleotide lp li to thnh cc bng v tinh trong ly tm theo gradient. secretory vesicle: ti tit. Nhng bo quan nh cha cc cht c tit ra ngoi t bo. selfish DNA: DNA khng c bt c chc nng g trong t bo. sequence tagged site (STS): Trnh t nucleotide duy nht trong genome. shotgun approach: cch thc xc nh trnh t ca mt phn t ADN bng vic xc nh trnh t cc phn on khi phn t b ct t mt cch ngu nhin. silencer sequence: trnh t nucleotide khi ng s co c cc b si nhim sc th, ngn cn protein kim sot phin m tng tc vi vng iu khin ca gen nm cch vi trm bp. simple-sequence DNA: on nucleotide ngn lp li lin tip, thng phn b tm ng (centromere) v u mt nhim sc th (telomere). southern blotting: k thut xc nh phn t ADN t nhng on ADN khc bng vic lai u d nh du (labeled probe) vi nhng ADN . spliceosome: phc ribonucleoprotein bm vo phn t pre-ARNm thc hin phn ng ct ni intron-exon. stem cell: t bo gc. T bo c tim nng phn chia to thnh nhng dng t bo bit ho khc nhau trong sinh trng pht trin. TATA box: hp TATA. on nucleotide bo th promoter eukaryot ti phc khi ng phin m tng tc vi promoter hoc vi nhau. trans-acting: tc ng ngoi sinh. Thng dng ch sn phm ca mt gen khi sn phm c kh nng khuch tn trong t bo gy tc ng n hot ng ca mt s gen khc trn cng mt nhim sc th hoc trn cc nhim sc th. transcription unit: n v phin m. on ADN c gii hn bi im khi u v im kt thc phin m cho mt si ARN. Si ARN ny c th cn tri qua qu trnh bin i thnh cc phn t ARN chc nng. transfection: chuyn nhim. Qu trnh chuyn gen hot ng vo t bo nui cy nghin cu tc ng ca gen . transformation: bin np. Qu trnh a ADN l vo t bo nhm gy bin i h gen ca t bo mt cch bn vng v c truyn cho th h sau. transgene: Gen c tch dng v c chuyn vo h gen ca t bo ng thc vt, ti gen l tn ti bn vng v c di truyn cho th h tip sau. transgenic: ng thc vt chuyn gen. transposon: ADN di chuyn. on ADN c kh nng di chuyn trong h gen. vector (dng trong gii hn sinh hc phn t): tc nhn mang ADN chuyn vo t bo hoc c th.

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Western blotting: K thut cho php xc nh protein c hiu bng khng th nh du. wild type: dng bnh thng, tn ti trong t nhin khng mang bt k t bin. Khi nim ny c dng cho mt phn t, t bo hoc mt c th. i vi c th, c th gi l loi di.

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