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P2: REGULATION GENE EXPRESSION & GENETIC ENGINEERING / SEM-III / SIDDY UNIT-I: GENE REGULATION IN PROKARYOTES.

Negative & positive control of gene expression (Lac Operon). Dual Promoters (Gal Operon). Dual function of Repressor(Ara Operon). Transcriptional control by Attenuation (Trp Operon). Phase variation (salmonella flagellar protein synthesis). Sporulation gene Expression in Bacillus. Regulation of Gene Expression in Lambda Phase (Repressor & Cro). Plasmid Copy number Control.

UNIT II: GENE REGUALTION IN EUKARYOTES Complex of eukaryotic genome & changes in chromatin structure in active & inactive states. Levels of gene regulation in Eukaryotes. Tissue specific expression. Transcriptional control. Transcriptional factors & the role of promoters, enhancers, DNA binding proteins. Post translational control including RNA splicing, RNA editing, RNA transport & RNA stability. Translational Feedback. Regulation of Harmones. Phosphorylation & gene silencing. Gal Operon of yeast.

UNIT III:PRINCIPLES OF GENETIC ENGINEERING 1 Discovery & significance of Restriction modification system in Bacteria. Enzymes Used in Gene manipulations:

Polymerases (DNA pol-I, Klenow, T4 Dna polymerase, Poly(A) polymerase). Terminal deoxy nucleotidyl transferase. Reverse transcriptase. DNA ligases (E.coli & T4). Nucleases (Mung bean, S1 nuclease,Dnase1,Rnase A,35 exonuclease). Methylases (DAM & DCM). Restriction endonucleases-type I,II,III, isoschizomers, blunt end & staggered end cutters, 5 & 3 overhangs) Polynucleotide kinase & phosphatases (Calf intestinal & shrimp alkaline phosphatases).

DNA Sequencing methods: Maxam-gilbert chemical method. Sangers dideoxy chain termination method & Automated sequencing.

Chemical synthesis of oligodeoxynucleotides(solid phase synthesis).

UNIT IV: PRINCIPLES OF GENETIC ENGINEERING II Overview of cloning methods. Methods of isolation of DNA. ligation, introduction of r-DNA & selection of recombinants. Vectors: plasmids & viral vectors(Lambda & M13), phagemids, cosmids, YAC, BAC, PAC, & Shuttle vectors. Reporter genes (beta-Gal, CAT, GFP, GUS, luciferase). Secretion of recombinant proteins. Baculoviral expression. Fusion proteins. Substaractive cDNA cloning & 2hybrid system. Phase display.

Strategies for preparation of genomic cDNA libraries. Enriched & substarctive cDNA libraries. Design & preparation of probes for hybridization(radio active & non radiolabled probes). Complete genome sequencing projects(Human genome sequencing project )& microarray technology.

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