2.

40 Analysis of DNA
The techniques of DNA analysis have undergone a revolution in the past two decades. This
revolution has enabled the complete sequencing of several bacterial genomes and detailed
investigations into the regulation of growth and differentiation. These tools will play an
increasingly important role in medical care (diagnosis) and the legal system (DNA fingerprinting) in
the future.

2.41 DNA Sequencing

The most commonly used DNA sequencing method today is Sanger or dideoxy sequencing. Four
reaction mixtures are set up with all dNTPs, a DNA polymerase, radioactive dATP (to label the
DNA), a oligonucleotide primer, and the DNA to be sequenced. A small amount of one dideoxy
nucleotide triphosphate (ddNTP) is included into each reaction mixture to produce a ddATP,
ddTTP, ddGTP, and a ddCTP reaction. Then the mixture is incubated at 40 degrees C for a few
minutes and the samples are then analyzed by gel electrophoresis. Because the dideoxy nucleotide
lacks a 3’-OH group, it halts DNA synthesis whenever it is incorporated into the DNA. This
produces a series of DNA fragments that end with the ddNTP that was added to that specific
reaction. When all four reaction mixtures are electrophoresed, it is possible to determine the
sequence of 100-400 basepairs of the target DNA by ‘reading up the gel’. By independently running
a separate set of reactions with a primer specific for the complimentary strand, one could identify
any errors in sequencing made with the original primer.
2.42 Restriction Endonucleases
Restriction enzymes recognize short palindromic sequences, often 4-6 basepairs in length, of double
stranded DNA and cleave a specific phosphodiester bond on each strand of the helix. The DNA
fragments produced by restriction enzyme digestion can then be analyzed by electrophoresis, used in
other molecular biology applications, and used as genetic markers. These enzymes have been
isolated from bacteria that employ these enzymes to combat bacteriophages. The target sequences
of the restriction enzymes that a bacterium possess are methylated, preventing the enzyme from
recognizing the target. Invading bacteriophage DNA will not have the target sites methylated, so the
DNA will be cleaved into small fragments and later degraded to prevent infection. Below is an
example of EcoRI, a restriction enzyme with a recognition sequence of GAATTC cutting a DNA
molecule into two smaller fragments.
2.43 Polymerase Chain Reaction
The polymerase chain reaction (PCR) is an in vitro DNA amplification technique that employs a
thermostable DNA polymerase to replicate 100-3000 basepair segment of DNA that is flanked by
known sequences. A short stretch of the flanking sequence from each side of the target DNA is
used to make an oligonucleotide primer for DNA synthesis. The target DNA, the two primers, the
thermostable polymerase, and all four nucleotides are mixed in an appropriate buffer. This reaction
mixture is then placed in a thermocycler that brings the sample to 94 degrees C to denature the
strands, cools to 55 degrees C to allow the primers to anneal, and then heats to 72 degrees C to
synthesize the new strand. The denature-anneal-synthesize cycle is repeated 25 or more times to
amplify the target DNA by approximately 1 million fold. The resulting PCR product can then be
analyzed by gel electrophoresis and is suitable for other molecular biology applications such as
sequencing.
2.44 Hybridization
Hybridization is a technique that used the basepairing characteristics of DNA to identify target
sequences in a nucleic acid sample. A labeled oligonucleotide probe is synthesized to be
complimentary to the target sequence and is allowed to react with the denatured nucleic acid
sample. This probe will basepair with high specificity with the target sequence, and will identify the
presence or absence of the target sequence genome sized samples.
2.45 Plasmids
Plasmids are extrachromosomal DNA that can be maintained in prokaryotes and eukaryotes such as
yeast. These autonomously replicating plasmids first gained attention as mediators of drug
resistance in bacteria but have become essential molecular biology tools. Plasmids serve as a
vehicle to carry and replicate DNA sequences that can be used for sequencing, genetic mapping,
recombinant protein production, and numerous other applications. Each plasmid must contain an
origin of replication, a selectable marker such as an antibiotic resistance gene, and a multiple
cloning site (MCS) that contains unique restriction sites within the plasmid. In the example shown
below, the plasmid has two antibiotic resistance genes (tetracycline and ampicillin), one of which
contains the MCS within the gene. The plasmid is cut in the MCS with the restriction enzyme
EcoRI, which opens the plasmid. The foreign DNA is then cloned into the plasmid and the DNA is
used to transform host bacteria. Transformed bacteria are selected by their ability to grow in the
presence of ampicillin. If a ampicillin resistant colony cannot grow in the presence of tetracycline,
it is a recombinant plasmid. Plasmids that reannealed upon themselves would be identical to the
original plasmid and would be ampicillin and tetracycline resistant. The recombinant colonies could
then be transferred to media containing ampicillin and grown in large quantities to prepare for
further analysis.
2.7 Cell Cycle
Cells reproduce by replicating their contents and then dividing in two. Multicellular organisms are
generated by complex sequences of cellular division, periodically interrupted by sexual cell fusion.
In the duplication process only DNA must be replicated exactly and divided precisely between the
two daughter cells. The process of cell division is broken up into six distinct processes.

2.71 Prophase
In the cell cycle the transition from the G2 phase to the M phase is not a well defined process. The
chromatin slowly condenses into well defined chromosomes, which is duplicated in the S phase.
During the prophase the nucleoli disappear. The condensed and replicated chromosomes are made
up of two chromatids. On each chromatid is the specific DNA sequence known as a centromere. At
the end of the prophase process, the cytoplasmic microtubules which are part of the interphase
cytoskeleton disassemble and the mitotic bipolar spindle begins to form. The spindle consists of
microtubules and associated proteins attached to two separate poles. The spindle initially assembles
outside of the nucleus.
2.72 Prometaphase
Prometaphase starts abruptly with the nuclear envelope disintegrating into membrane vesicles which
are indistinguishable from endoplasmic reticulum. The mitotic spindle, which has been outside of
the nucleus, now enters the nuclear region. On each centromere specialized protein complexes
called kinetochore mature and attach to the spindle microtubules. The microtubules that are
attached to the kinetochore protein complexes are called kinetochore microtubules. The remaining
microtubules are polar microtubules while the microtubules which are outside of the spindle are
called astral microtubules. From each sister chromatids in each chromosome the kinetochore
microtubules extend in opposite directions, eventually causing the chromosomes to align in the
center of the spindle poles.

2.73 Metaphase
The chromosomes aligned in the center of the spindle poles by the tension applied to them by the
kinetochore microtubules. Each chromosome is held in place by the corresponding kinetochore
microtubule from each spindle pole.
2.74 Anaphase
This phase is triggered by a signal from the cell which causes the two attached microtubules on each
chromosome to separate. The chromatids are separated and pulled to each spindle pole. The
movement towards the spindle pole by the chromatids is accomplished by Anaphase A and
Anaphase B. In Anaphase A the chromatids are pulled to the spindle pole by the shortening of the
kinetochore microtubules. During Anaphase B the polar microtubules elongate which cause the two
spindle poles to separate from each other.

2.75 Telophase
During Telophase the chromatids reach the spindle poles. The kinetochore microtubules then
disappear, while the polar microtubules continue to elongate separating the spindle poles further.
During the elongation process a new nuclear envelope forms around the group of daughter
chromosomes. The chromosomes begin to expand from their condensed state. Once the nuclear
envelope is formed the nucleoli begin to reappear.
2.76 Cytokinesis
The Cytoplasm is divided by the process known as cleavage which begins during the anaphase.
During cleavage the cell membrane in the middle of the cell begins to draw together. This causes a
indention in the cell at the center separating the two new nuclei. The indention deepens until it
encounters the remains of the mitotic spindle. The remains of the mitotic spindle narrows and then
finally breaks leaving two separated cells.

6.0 Drugs
Drugs are chemical agents that are capable of interacting with cellular components to produce a
desired effect such as microbe killing. Examples of several broad classes of drugs that have known
mechanisms of action are presented here.

6.10 Antibiotics
Antibiotics are macromolecules that are produced by microorganisms that suppress the growth of
competing species or synthetic molecules that kill or inhibit microorganisms. The availability of
antibiotics for over 50 years has led to a great reduction in the number of deaths due to infectious
disease. The development of resistance to antibiotics has hampered the ability to effectively treat all
infections, but newer drugs and the use of multiple antibiotics have the capability of treating
infections from drug resistant microbes.
6.10.1 Aminoglycosides
Aminoglycosides are bactericidal antibiotics that halt translation by binding to the 30S ribosomal
subunit and inhibit the initiation step. If an aminoglycoside binds to a ribosome that has began
elongation, it will cause misreading of the remaining mRNA. These antibiotics are effective against
many gram negative organisms, and resistance arises from plasmids carrying
aminoglycoside-modifying enzymes. Since plasmids can rapidly spread through a microbial
population through conjugation and transformation, this represents a major public health concern.
Examples of aminoglycosides include streptomycin, kanamycin, gentamicin, neomycin, and
netilmicin. Streptomycin is illustrated below.

6.10.2 Tetracyclines
Tetracyclines have a broad spectrum of activity against gram negative and gram positive organisms.
It is also effective against both anaerobes and aerobes, with resistance developing from plasmid
encoded enzymes. These antibiotics are bacteriostatic and act by inhibiting translation by
competing with aminoacyl-tRNAs for the A site on the ribosome. Examples of tetracyclines include
tetracycline, demeclocycline, minocycline, and doxycycline. The structure of tetracycline is shown
below.
6.10.3 Chloramphenicol
Chloramphenicol is a potent antibiotic that inhibits translation in both bacterial and mammalian
cells. This drug binds to the 50S ribosomal subunit and inhibits the peptidyl transferase step in
protein synthesis. This drug is bacteriostatic and has a wide range of activity against many aerobic
and anaerobic organisms. Resistance usually develops through the acquisition of a plasmid
encoding for chloramphenicol acetyl transferase which inactivates the drug. Chloramphenicol is the
only drug of its class, and its use is limited because it is also capable of inhibiting mammalian
translation.

6.10.4 Quinolones
Quinolone antibiotics inhibit bacterial DNA gyrase, which is required to maintain a proper degree of
supercoiling in the DNA. These antibiotics bind to the gyrase and inhibit the strand nicking and
strand closing activities of the enzyme. Inhibition of DNA gyrase suppresses replication and
transcription. This is fatal to bacterial cells. These antibiotics have a broad spectrum of action
against aerobic bacteria, but resistance develops during prolonged administration due to mutations
within the bacterial DNA gyrase gene. Examples of quinolones include nalidixic acid, norfloxacin,
cinoxacin, ciprofloxacin, ofloxacin, sparfloxacin, lomefloxacin, fleroxacin, pefloxacin, and
amifloxacin.
6.10.4 Macrolides
Macrolides are bacteriostatic against gram positive bacteria. They bind to the 50S ribosomal
subunit and inhibit the translocation step and causing premature termination during transcription.
Resistance develops through mutation in the genes for the 50S ribosomal subunit. Examples of
macrolides include erythromycin, clarithromycin, and azithromycin. The structure of erythromycin
is shown below.

6.11 Antiviral Agents

Antiviral agents are drugs that inhibit the production of new viral particles by interfering with
specific stages in the virus life cycle such as replication. Because the viral enzymes are often quite
different from human enzymes with the same function, drugs can be designed that will inhibit only
the viral enzymes.
6.11.1 Acyclovir
Acyclovir is a guanine nucleoside analog that interferes with the replication of the herpes virus
genome. This nucleoside is converted to a nucleotide and terminates DNA synthesis when
incorporated into DNA because it lacks a 3’ OH group. Resistance to acyclovir has developed in
some strains of herpesvirus due to mutations in the thymidine kinase gene. Herpes virus thymidine
kinase converts guanine nucleosides and acyclovir into nucleotides. The mutation to thymine kinase
abolished the ability of the enzyme to convert acyclovir to an acyclovir monophosphate. This drug
is effective against a range of DNA viruses including the herpes simplex viruses and varacella
zoster. The structure of acyclovir is shown below.

6.11.2 Zidovudine
Zidovudine, also known as AZT, is a thymidine nucleoside analog that inhibits retroviral
replication. This drug is phosphorylated by thymidine kinase to form a zidovudine monophosphate
which is converted to a zidovudine triphosphate. The zidovudine triphosphate can then be
incorporated into DNA, where it causes chain termination due to the lack of a 3’ OH group. This
drug also inhibits thymidylate kinase, which results in the depletion of the nucleotide pool in the
infected cell. Mutations in the viral reverse transcriptase can confer resistance to zidovudine.
6.12 Anticancer Drugs
Approximately 25% of the US population will be diagnosed with cancer in their lifetime, therefore
making cancer a very active area of research. Numerous drugs that attack cancer (and normal) cells
have been developed, mostly interfering with DNA and RNA synthesis. The major pitfall in this
approach is that all rapidly proliferating cells are affected, not just cancer cells.

6.12.1 Methotrexate
Methotrexate is a folic acid antimetabolite that acts by inhibiting dihydrofolate reductase, preventing
the formation of the coenzyme tetrahydrofolic acid. Deficiency of tetrahydrofolic acid inhibits one
carbon metabolism that is central to many cellular processes such as energy production, nucleotide
synthesis, and amino acid synthesis. The most pronounced inhibition of cellular metabolism occurs
during S-phase, making this drug useful against rapidly growing cancers such as lymphocytic
leukemia, breast cancer, and osteogenic carcinoma. Cancer cells can become resistant to
methotrexate by amplifying their dihydrofolate reductase genes or by reducing the expression of
membrane bound methotrexate transport proteins. Side effects include myelosuppression, rash,
alopecia, nausea, vomiting, and diarrhea. All of these effects are the result of the killing of rapidly
dividing cells such as those found in the intestinal tract.

6.12.2 6-Mercaptopurine
6-Mercaptopurine is a nucleoside analog that is converted to 6-mercaptopurine ribose phosphate
(6-MPRP). This nucleotide has a similar structure to AMP, and can therefore inhibit the committed
step of purine biosynthesis. Additionally 6-MPRP can be incorporated into DNA and RNA,
forming a strand of nucleic acid with an aberrant structure. 6-Mercaptopurine is used to maintain
remission for acute lymphoblastic leukemia. Resistance to 6-mercaptopurine is due to reduced
affinity by HGPRT for this substrate and increased metabolism of 6-mercaptopurine to thiouric acid.
Side effects include myelosuppression, rash, nausea, vomiting, hepatotoxicity, and diarrhea. All of
these effects are the result of the killing of rapidly dividing cells such as those found in the intestinal
tract.

6.12.3 Dactinomycin (actinomycin D)

Dactinomycin is an antibiotic that intercalates between guanine-cytosine basepairs in the DNA
helix, inhibiting RNA polymerase and DNA polymerase. DNA strand breaks can be introduced at
high concentrations. Dactinomycin is used to treat Wilm's tumor, choriocarcinoma, and soft tissue
sarcomas. Dactinomycin sensitizes tissues to radiation. Resistance is mediated by the expression of
P-glycoprotein, which causes increased efflux of dactinomycin. The major side effect from
dactinomycin use is immunosuppression due to bone marrow suppression. Nausea, vomiting, and
diarrhea can also occur.
6.12.4 Anthracycline Antibiotics
Anthracycline antibiotics (doxorubicin and daunorubicin) intercalate into DNA to inhibit DNA and
RNA synthesis, alter membrane signal transduction (phosphatidyl inositol pathway), and generate
oxygen radicals. These oxygen radicals are produced by the cytochrome p450 system in the liver
and are potent denaturing agents. Anthracycine antibiotics are broad spectrum anticancer agents
that are used to treat many carcinomas, sarcomas, and leukemias. Resistance to anthracycine
antibiotics is mediated by the expression of P-glycoprotein, which causes increased efflux of these
anticancer drugs. Side effects include cardiotoxicity, myelosuppression, rash, nausea, vomiting,
alopecia, and diarrhea.

6.12.5 Bleomycin
Bleomycin is a metal cheating glycopeptide that interacts with DNA to cause strand breakage after
oxidation from bleomycin-DNA-Fe(II) to bleomycin-Fe(III). This strand breakage can cause
chromosomal abnormalities and cell death. Bleomycin is used to treat testicular tumors and
squamous cell carcinomas. Resistance is mediated by uncharacterized detoxification enzymes and
increased drug efflux. Side effects include pulmonary toxicity, alopecia, and hyperpigmentation of
the hands. In contrast to many other anticancer drugs, bleomycin rarely causes myelosuppression.

6.12.6 Mechlorethamine
Mechlorethamine is a DNA-alkylating agent that is derived from mustard gas employed during
World War I. It crosslinks the N7 positions of guanine residues in DNA and can cause
depurination. This leads to an inhibition of DNA and RNA synthesis and can lead to strand breaks.
This anticancer agent is used to treat Hodgkin's disease and solid tumors. Resistance is mediated by
increased glutathione conjugation of the drug and decreased membrane permeability to the drug.
Side effects include nausea, vomiting, and pronounced immunosuppression.

6.12.7 Vinca alkaloids

Vinca alkaloids (vincristine and vinblastine) are derived from the periwinkle Vinca rosea. These
alkaloids block mitosis in metaphase by binding to tubulin, preventing the formation of
microtubules. Vinca alkaloids are effective against a wide range of rapidly growing cancers such as
leukemias, sarcomas, lymphomas, and Wilm's tumor. Resistance is mediated by P-glycoprotein,
which increases the efflux of vinca alkaloids from tumor cells. Side effects include cellulitis,
nausea, vomiting, diarrhea, neuropathy (vinblastine only), and alopecia.

6.12.8 Paclitaxel (Taxol)

Paclitaxel is a taxane derived from the bark of yew trees that binds to tubulin, promoting its
polymerization and stabilization. These highly stable microtubules prevent mitosis and eventually
cause cell death. Paclitaxel has activity against ovarian and breast cancers. Resistance is mediated
by the presence of high levels of P-glycoprotein which promotes the efflux of the drug, or by
mutations in tubulin genes that prevent the binding of paclitaxel. Side effects include
hypersensitivity, neutropenia, and alopecia.
6.12.9 Alpha Interferon
Alpha interferon is a cytokine produced using recombinant DNA techniques that stimulates tumor
killing by macrophages and natural killer cells. It is used to treat hairy cell leukemia, melanoma,
multiple myleoma, and squamous cell carcinoma. Side effects include allergic reactions to the drug,
leukopenia, and thrombocytopenia.

7.0 Human Diseases

Due to efforts of medical research, the biochemical mechanisms of many human diseases are
known. A selection of diseases with mechanisms related to other sections of this program are
detailed here.

7.01 Trisomy 21
Trisomy 21, or Down’s syndrome, affects about 1 child in 700 and is the result of chromosomal
nondisjunction during meiosis. The symptoms of this syndrome include mental retardation,
epicanthic folds, a higher frequency of congenital heart defects (40%), and occasionally a single
palmar crease. These defects are due to ‘extra’ copies of the genes found on chromosome 21, but
the exact mechanisms that produce the specific defects are not known.

7.01.1 Trisomy 18
Trisomy 18, or Edward's syndrome, affects about 1 child in 8000 and is the result of chromosomal
nondisjunction during meiosis. This syndrome consists of severe mental retardation, rocker bottom
feet, micrognathia, congenital heart defects, and a prominent occiput. Because of the number and
severity of the defects associated with trisomy 18, death usually occurs within the first year of life.

7.01.2 Trisomy 13
Trisomy 13, or Patau's syndrome, affects about 1 child in 6000 and is the result of chromosomal
nondisjunction during meiosis. This syndrome consists of severe mental retardation,
micropthalmial, microcephaly, cleft lip or palate, abnormal brain formation, congenital heart
defects, and polydactyly.
Because of the number and severity of the defects associated with trisomy 13, death usually occurs
within the first year of life.

7.01.3 Cri-du-chat Syndrome

Cri-du-chat syndrome is caused by the deletion of the short arm of chromosome 5, producing a 46
XX (or XY) 5p- karyotype. This syndrome consists of microcephaly, high pitched crying (cat like),
severe mental retardation, and congenital cardiac defects.

7.02 Klinefelter’s Syndrome

Klinefelter’s syndrome affects 1 male in 850 and is the result of a male child having more than one
X chromosome (47, XXY). This is associated with chromosomal nondisjuction during meiosis.
The typical features of Klinefelter’s syndrome appear at puberty, and they include small testes, long
arms and legs, reduced levels of plasma testosterone, and increased levels of estrogens.
Gynecomastia and sterility are common complications of this syndrome.

7.03 XYY Syndrome

Approximately 1 in 1000 males has XYY syndrome due to the presence of an extra Y chromosome
that resulted from chromosomal nondisjuction. Males with the XYY karyotype have higher serum
levels of testosterone, FSH, and LH. It is unclear at this time if these elevated serum hormone levels
contribute to any pathology.

7.04 Turner’s Syndrome

Turner’s syndrome affects 1 in 2000 females and is the result of monosomy for the X chromosome
(45, XO). Females with this disorder may be described as having a webbed neck and a shield
shaped chest in childhood, but the problem often manifests as a failure of feminization during
adolescence. This can be treated by hormone replacement.

7.05 Xeroderma Pigmentosum

Xeroderma Pigmentosum is an autosomal recessive disorder of DNA repair that results in excessive
freckling and skin ulceration following exposure to sunlight. Skin cancer is the eventual result of
exposure to sunlight for afflicted individuals. These serious consequences are a result in a mutation
that inactivates the gene for the excision repair exonuclease. This defect in excision repair leads to a
rapid build up of thymine dimers in the DNA of skin cells when exposed to ultraviolet light of any
source. Because these dimers cannot be repaired, severely irradiated cells will die (ulcerations) and
surviving cells will accumulate mutations (cancer).

7.06 Arsenic Poisoning

Arsenic is a toxic heavy metal that blocks ATP synthesis by competing for inorganic phosphate
binding sites on enzymes (arsenate ion) and by covalently binding to lipoic acid cofactors (arsenite
ion). The enzymes inhibited include pyruvate dehydrogenase and alpha-ketoglutarate
dehydrogenase. The inactivation of these key enzymes and the competition for inorganic phosphate
binding sites greatly inhibit the ability of cells to generate ATP and carry out other cellular
processes.

7.07 Fructose Intolerance

Fructose intolerance arises from a deficiency of a liver aldolase that splits fructose 1-phosphate into
dihydroxyacetone phosphate and glyceraldehyde. Without the aldolase, fructose 1-phosphate
accumulates because fructokinase continues to phosphorylate fructose to produce fructose
1-phosphate. The growing pool of fructose 1-phosphate, a product useless to cells with this
disorder, depletes the liver of ATP and inorganic phosphate. If ATP levels fall below a minimum
level, the cells will die, eventually causing hepatic failure. This disorder can be treated by avoiding
dietary fructose.
7.08 Pyruvate Dehydrogenase Deficiency
Pyruvate dehydrogenase deficiency is a rare disorder caused by mutations in one or more subunits of
the pyruvate dehydrogenase complex. This causes elevated serum levels of lactate, pyruvate, and
alanine. The elevated lactate levels cause acidosis, and can be fatal in severe cases. Mild cases can
be treated with ketogenic diets (high protein - lysine and leucine, low carbohydrate) and
dichloroacetate (an inhibitor of pyruvate dehydrogenase kinase which inactivates pyruvate
dehydrogenase).

7.09 Mitochondrial Toxins

Mitochondrial toxins block electron transport at various steps, resulting in the rapid depletion of
cellular ATP. Tissues with high metabolic rates such as the central nervous system, heart muscle,
and the liver are rapidly damaged.

Toxin Step Inhibited

Amytal Transfer of electrons from NADH dehydrogenase to ubiquinone
Rotenone Transfer of electrons from NADH dehydrogenase to ubiquinone
Antimycin A Transfer of electrons from BC1 complex to cytochrome C
Cyanide Transfer of electrons from Cytochrome Oxidase to oxygen
Carbon Monoxide Transfer of electrons from Cytochrome Oxidase to oxygen
Oligomycin Inhibits activity of F1F0 ATPase
2,4 DNP Makes membrane permeable to protons, abolishes proton gradient needed
to produce ATP

7.10 Type I Glycogen Storage Disease (Von Gierke's Disease)

Type I Glycogen Storage disease is the most common type of glycogen storage disease. It is caused
by a hepatic and renal deficiency of glucose 6-phosphatase, resulting in the inability of the liver to
export glucose that was stored as glycogen. This is an autosomal recessive disorder with a
frequency of 1 in 200,000 births that results in hypoglycemia, lactic acidosis, hyperlipidemia, and
hyperuricemia. The symptoms of this disease can be reduced by providing a steady supply of
carbohydrates throughout the day.

7.11 Type II Glycogen Storage Disease (Pompe's Disease)

Type II glycogen storage disease is caused by the lack of alpha-1,4 glucosidase which leads to the
accumulation of glycogen in tissues. Alpha-1,4 glucosidase degrades damaged glycogen granules,
so without this enzyme, lysosomes become overloaded with glycogen that is not useful to the cell.
As a consequence of this enzyme disorder, cardiac hypertrophy followed by death at an early age
develops.

7.12 Type III Glycogen Storage Disease (Cori's Disease)

Type III glycogen storage disease is caused by a deficiency of the debranching enzyme, causing the
accumulation of glycogen with only the outer branches removed. This results in hepatomegaly and
mild hypoglycemia.
7.13 Type V Glycogen Storage Disease (McArdle's Disease)
Type V glycogen storage disease is caused by a deficiency in muscle phosphorylase, and enzyme
that phosphorylates imported glucose so it is retained in muscle tissue. This disease is manifested
by painful muscle cramps and low exercise tolerance.

7.14 N-Acetylglutamate Synthetase Deficiency

N-Acetylglutamate Synthetase Deficiency causes hyperammonemia and hyperaminoacidemia in
newborns due to the lack of N-Acetylglutamate Synthase. A low protein diet and carbamoyl
glutamate (activates carbamoyl phosphate synthetase) will reduce the symptoms.

7.15 Carbamoyl Phosphate Synthetase Deficiency

Carbamoyl Phosphate Synthetase deficiency causes mental retardation secondary to
hyperammonemia. Because carbamoyl phosphate synthase is present in very low amounts,
ammonia accumulates to toxic levels unless treated. Treatment consists of administering benzoate,
phenylacetate, a low protein diet, and arginine. This course of treatment regenerates urea cycle
intermediates and stimulates the activity of the carbamoyl phosphate synthetase present.

7.16 Ornithine Transcarbamoylase Deficiency

Ornithine Transcarbamoylase deficiency is an x-linked deficiency in a urea cycle enzyme that results
in mental retardation if left untreated. Treatment includes the removal of ammonia from the blood
and a low protein diet, which can be successful in preventing mental retardation.

7.17 Argininosuccinate Synthetase Deficiency

Argininosuccinate synthetase deficiency causes the urea cycle to halt after forming citrulline.
Citrulline accumulates in the blood and urine. Therapy includes a low protein diet and fortified with
arginine.

7.18 Phenylketonuria (PKU)

Phenulketonuria is the most common hereditary metabolic disorder, and it results in mental
retardation if untreated early in life. Classical PKU is an autosomal recessive deficiency in
phenylalanine hydroxylase resulting in the accumulation of very high levels of phenylalanine in the
blood. The excess phenylalanine inhibits aromatic amino acid transport in the brain and is excreted
in the urine as pheylpyruvic acid. Phenylpyruvic acid is oxidized in the urinary tract to
phenylacetate, which imparts a 'mousy' odor to the urine. Treatment consists of restricting
phenylalanine intake until reaching adulthood. Additionally, tyrosine becomes an essential amino
acid. Several non-classical forms of PKU exist (such as biopterin synthesis deficiency), and the
treatment varies depending on the nature of the defect.
7.19 Alcaptonuria
Alcaptonuria is a deficiency in homogentisate oxidase, an enzyme necessary for tyrosine
metabolism. This results in the accumulation and urinary excretion of homogentisic acid. Upon
contact with air, urinary homogentisic acid oxidizes and polymerizes to form a dark pigment. This
pigment is also deposited in bones and other tissues, and can cause arthritis if deposited in joints.
There is no treatment for alcaptonuria.

7.20 Vitamin A Deficiency and Excess

Vitamin A (retinol) is an essential component of visual pigments. Deficiency of this fat soluble
vitamin manifests as night blindness and dry skin. Excess consumption of vitamin A can lead to
arthralgia, headache, fatigue, skin changes, and alopecia. Use of high levels of vitamin A (in
Retin-A for example) during pregnancy is associated with birth defects.

7.21 Vitamin B1 Deficiency

Vitamin B1 (thiamine) is a component of thiamine pyrophosphate, which is a cofactor used in the
oxidative decarboxylation of alpha-keto acids such as pyruvate and alpha-ketoglutarate. Thiamine
pyrophosphate is also a cofactor for transketolase. Deficiency of this water soluble vitamin causes
Beriberi and Wernicke-Korsakoff syndrome. Beriberi consists of cardiomyopathy, polyneuritis, and
edema. Wernicke-Korsakoff syndrome is characterized by psychosis, opthalmoplegia, and ataxia.
Progresses to include memory loss, confusion, and confabulation.

7.22 Niacin Deficiency

Niacin is a component of NAD+ and NADP+ that are cofactors in many biosynthetic and energy
producing reactions. Deficiency of this water soluble vitamin leads to Pellegra, which is
characterized by dementia, dermatitis, diarrhea, and beefy glossitis.

7.23 Vitamin B5 Deficiency

Vitamin B5 (pantothenate) is a component of CoA, fatty acid synthase, and a cofactor in acyl
transfer reactions. Deficiency in this water soluble vitamin leads to enteritis, alopecia, adrenal
insufficiency, and dermatitis.

7.24 Vitamin B6 Deficiency

Vitamin B6 (pyridoxine) is converted to pyridoxal phosphate, which is a cofactor in transamination,
transsulfuration, and decarboxylation reactions. Deficiency of this water soluble vitamin leads to
irritability and convulsions in severe cases.