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MOLECULAR BIOLOGY
NUCLEOTIDES
Questions:
1. Give the sources of carbon and nitrogen of purine and pyrimidine ring. Pon
May 2005
2. Name 4 different types of neucleotides giving their biological importance.
Pon May 2004
3. What is the pathway breakdown of purine nucleotide? Pon May 2004
4. Salvage pathway.
5. Write a note on purine salvage pathway. Pon May 2010
6. What is gout? Enumerate the clinical findings in gout. What is the mode of
action of the drug allopurinol in this condition?
7. What is Lesch - Nyhan syndrome?
8. Lesch - Nyhan syndrome. Pon May 2001
9. Name the in born error of metabolism associated with hyperuricemia and
self-mutilation.
10.Orotic aciduria
CHEMISTRY OF NUCLEOTIDES
Name 4 different types of neucleotides giving their biological importance. Pon May
2004
1. Nucleotides are precursors of deoxy-ribonucleic acid (DNA) and ribonucleic acid (RNA).
Nucleotides are also components of important co-enzymes like NAD+ and FAD, and
metabolic regulators such as cAMP and cGMP.
2. A nucleotide is made up of 3 components:
1. Nitrogenous base, (a purine or a pyrimidine)
2. Pentose sugar, either ribose or deoxyribose;
3. Phosphate groups esterified to the sugar.
3. NUCLEOSIDE = nitrogenous base + pentose sugar
4. Nucleotide (Nucleoside monophosphate) = Nucleoside + Phosphate
5. Nucleic acids (DNA & RNA) = polymers of nucleotide
6. The purine bases: adenine and guanine; present in both RNA and DNA .
7. The pyrimidine bases: cytosine, thymine and uracil. Cytosine is present in both DNA
and RNA. Thymine is present in DNA and uracil in RNA.
8. DNA (Deoxy ribonucleotides): There are four different types of nucleotides found in
DNA, differing only in the nitrogenous base.
1. d-adenosine + Pi d-AMP (d-adenylic acid)
2. d-guanosine + Pi d-GMP (d-guanylic acid)
3. d-cytidine + Pi d-CMP (d-cytidylic acid)
4. d-thymidine + Pi d-TMP (d-thymidylic acid)
9. RNA (Ribonucleotides)
1. Adenosine + Pi Adenosine monophosphate (AMP) (Adenylic acid)
2. Guanosine + Pi Guanosine monophosphate (GMP) (Guanylic acid)
3. Cytidine + Pi Cytidine monophosphate (CMP) (Cytidylic acid)
4. Uridine + Pi Uridine monophosphate (UMP) (Uridylic acid)
5. Inosine + Pi Inosine monophosphate (IMP) (Inosinic acid)
10.Nucleotides - biological importance:
1. Nucleotides are essential for RNA and DNA production and, therefore, proteins
cannot be synthesized or cells proliferate. They precursors of ATP, GTP, UTP, CTP
and their derivatives.
2. Nucleotides also serve as carriers of activated intermediates in the synthesis of
UDP-glucose and CDP-choline.
3. Nucleotides play an important role as energy currency in the cell eg. ATP
4. Sulfate donor: Adenosine 3'-phosphate-5'-phosphosulfate

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5. The methyl group donor: S- adenosylmethionine.
6. They are structural components of several essential coenzymes, such as coenzyme
A, FAD, NAD+, and NADP+.
7. Second messengers: cyclic adenosine mono phosphate (cAMP) cyclic guanosine
monophosphate (cGMP)
8. Allosteric regulator: GTP
9. cGMP serves as a second messenger in response to nitric oxide (NO) during
relaxation of smooth muscle.
10. UDP-sugar derivatives participate in biosynthesis of glycogen and proteoglycans.
11. UDP-glucuronic acid forms the urinary glucuronide conjugates of bilirubin
12. CTP participates in biosynthesis of sphingomyelin
13. Synthetic analogs like 5-fluorouracil, and 6-mercaptopurine, for cancer treamnet.
14. The purine analog allopurinol, used in treatment of hyperuricemia and gout
15. Cytarabine is used in chemotherapy of cancer, and azathioprine is employed
during organ transplantation to suppress immunologic rejection
BIOSYNTHESIS OF PURINE NUCLEOTIDES
1. The contribution of different atoms are from different sources for the formation of
the purine ring:
1. N1 of purine is derived from amino group of aspartate.
2. C2 and Cs arise from formate of N10- formyl THF.
3. N3 and N9 are obtained from amide group of glutamine
4. C4, C5 and N7 are contributed by glycine.
5. C6directly comes from CO2.

C&N sources of purines

2. Two methods of synthesis:
1. The de novo synthesis: the purine ring is synthesized from different small
components. The pathway is expensive in terms of the use of high energy
phosphate bonds.
2. Salvage pathway: Purine is synthesized from intermediates in the degradative
pathway for nucleotides.
DE NOVO SYNTHESIS OF PURINE
There are 11 steps in the de novo synthesis pathway:
Step 0:

Preparatory Step: Phospho ribosyl purophosphate (PRPP) synthesis:

Ribose-5-phosphate + ATP ADP + Phospho ribosyl pyrophosphate (PRPP) (enzyme:

PRPP synthetase)
Step 1:
Glutamine + PRPP 5-phosphoribosylamine.(enzyme- glutamyl
amidotransferase)

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Step 2:
5-Phosphoribosylamine + glycine + ATP glycinamide ribotide (GAR).
(Synthetae)
Step 3:

Glycinamide ribotide + N10-Formyl tetrahydrofolate formyl glycinamide

ribosyl 5phosphate.(formyl transferase)

Step 4:

Formylglycinamide ribosyl 5- phosphate+ Glutamine formyl glycinamidine

ribonucleotide (FGAM) (synthetase)

Step 5:

Formyl glycinamidine ribonucleotide + ATP 5-aminoimidazole ribonucletide

(AIR) (synthetase)

Step 6:

5-aminoimidazole ribonucletide + CO2 5-amino- 4-carboxy-aminoimidazole ribonucleotide (ACAIR). (carboxylase)

Step 7:

5-amino- 4-carboxy-amino- imidazole ribonucleotide + aspartate + ATP Nsuccinyl-5-amino-imidazole carboxamide ribonucleotide. (SAICAR)
(synthetase)

Step 8:

N-succinyl-5-amino-imidazole carboxamide ribonucleotide 5aminoimidazole 4 carboxamide ribonucleotide (AICAR) + Fumarate

(Adenosuccinate lyase)

Step 9:

5-amino imidazole 4 carboxamide ribonucleotide (AICAR) + N 1O-Formyl

tetrahydrofolate formamino imidazole 4-carboxamide ribonucleotide
(formyl transferase)

Step 10: Formamino imidazole 4-carboxamide ribonucleotide inosine

monophosphate( lMP) + H2O (cyclohydrolase)
Step 11: IMP+ Aspartate+ GTP AMP + Fumarate

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1.

2.

3.
4.
5.
6.

SALVAGE PATHWAY
A salvage pathway is a pathway in which nucleotides (purine and pyrimidine) are
synthesized
from intermediates in the degradative pathway for nucleotides. The free purines
(adenine, guanine and hypoxanthine) are formed in the normal turnover of nucleic
acids (particularly RNA), and also obtained from the dietary sources. The purines can
be directly converted to the corresponding nucleotides.
On the contrary, in De Novo pathways, the bases of the nucleotides are made from
scratch by using simpler starting materials (including amino acids). For this process,
ATP hydrolysis is required.
Salvage pathways recycle already used bases by reattaching them to a ribose. The
salvage pathway economizes intracellular energy expenditure.
Salvage pathway is important in erythrocytes and brain where de novo synthesis is nor
operating.
PRPP is starting material in this pathway. Phosphoribosyl pyrophosphate (PRPP) is the
donor of ribose 5-phosphate in the salvage pathway.
The free purines are salvaged by two different enzymes; adenine phospho ribosyl
transferase (APRTase) and hypoxanthine guanine phosphoribosyl transferase
(HGPRTase).
Adenine phosphor ribosyl transferase
i. Adenine+PRPP > Adenosine monophosphate+ PPi
Hypoxanthine guanine phospho ribosyl transferase
ii. Gunine+ PRPP -> Guanosine mono phosphate +PPi

Hypoxanthine guanine phospho ribosyl transferase

iii. Hypoxanthine-guanine + PRPP > Inosine
monophosphate +PPi
7. Absence of enzymes of salvage pathway produces specific clinical syndromes. A defect
in the enzyme Hypoxanthine guanine phospho ribosyl transferase(HGPRT) causes
Lesch-Nyhan syndrome .
Regulation of Purine Synthesis:
1. The intracellular concentration of PRPP regulates purine synthesis to a large extent
2. If AMP and CMP are available in adequate amounts to meet the cellular requirements,
their synthesis is turned off at the amidotransferase reaction.
3. AMP inhibits adenylsuccinate synthetase while CMP inhibits IMP dehydrogenase
4. The formation of AMP from IMP requires GTP; similarly formation of GMP requires ATP.
Hence both GTP and ATP are made available in sufficient quantities.
Purine Synthesis Inhibitors:
1. Folic acid (THF) is essential for the synthesis of purine nucleotides (reactions 4 and 10).
Sulfonamides are the structural analogs of paraaminobenzoic acid (PABA). These sulfa
drugs can be used to inhibit the synthesis of folic acid by microorganisms. This
indirectly reduces the synthesis of purines and, therefore, the nucleic acids (DNA and
RNA). Sulfonamides have no influence on humans, since folic acid is not synthesized
and is supplied through diet.
2. The structural analogs of folic acid (e.g. methotrexate) are widely used to control
cancer. They inhibit the synthesis of purine nucleotides (reaction 4 and 10) and, thus,
nucleic acids. Both these reactions are concerned with the transfer of one-carbon
moiety (formyl group). Mercaptopurine inhibits the conversion of IMP to GMP and
AMP.These inhibitors also affect the proliferation of normally growing cells. This causes

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many side-effects including anemia, baldness, scaly skin etc.
3. Azaserine (diazo acetyl-L-Serine) is a glutamine antagonist and therefore inhibits
reactions involving glutamine.
4. Other synthetic nucleotide analogues used as anticancer agents are 6-thio guanine
and 8-aza guanine.
DEGRADATION OF PURINE NUCLEOTIDES

Steps
1.
2.
3.

of Purine breakdown
AMP is first deaminated to produce IMP by AMP deaminase.
IMP is converted to inosine and GMP to guanosine by the action of 5'-nucleotidase.
Purine nucleoside phosphorylase converts inosine to hypoxanthine and guanosine to
guanine.
4. Hypoxanthine is oxidized by xanthine oxidase to xanthine & Guanine is deaminated to
xanthine by guanase
5. The common product Xanthine is further oxidized by xanthine oxidase to uric acid, the
final product of human purine degradation.
6. Uric acid is excreted primarily in the urine. The end product of purine nucleotide
catabolism is uric acid (urate).

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Disorders of Purine Metabolism:

HYPERURICEMIA & GOUT
1. Uric acid is the end product of purine metabolism in humans. The normal concentration of
uric acid in the serum of adults is of 3-7 m/dl. In women, it is about 1 mg lower than in
men. The daily excretion of uric acid is 500-700 mg.
2. It is defined as serum uric acid concentration exceeding 7 mg/dl in male and 6 mg/dl in
female. The manifestations are due to the low solubility of uric acid in water.
3. Gout is a disorder characterized by high levels of uric acid. The hyperuricemia can lead to
the deposition of mono -sodium urate crystals in the joints, and an inflammatory response
to the crystals, causing first acute and then progressing to chronic gouty arthritis. uric acid
is deposited in cooler areas of the body to cause tophi. Thus tophi are seen in distal joints
of foot. Increased excretion of uric acid may cause deposition of uric acid crystals in the
urinary tract leading to calculi or stone formation with renal damage. Gout may be either
primary or secondary.
4. Primary gout: lt is an inborn error of metabolism and is related to increased synthesis of
purine nucleotides. The following are the important causes of primary gout:
a. Abnormal PRPP synthetase : lack of allosteric control
b. Abnormal 5-phosphoribosyl amido transferase : overproduction of purine nucleotides
due to lack of feedback control
c. Deficiency of enzymes of salvage pathway; increased availability of PRPP and
decreased purines; so feedback inhibition is lost.
d. Glucose 6-phosphatase deficiency: ln type I glycogen storage disease (von Gierke's),
glucose 6-phosphate cannot be converted to glucose due to the deficiency of glucose
6-phosphatase. This leads to the increased utilization of glucose 6-phosphate by
hexose monophosphate shunt (HMP shunt), resulting in elevated levels of ribose 5phosphate and PRPP and, ultimately, purine overproduction.
e. Elevation of glutathione reductase: Increased glutathione reductase generates more
NADP+ which is utilized by HMP shunt. This causes increased ribose S-phosphate and
PRPP synthesis
5. Secondary gout:
f. Secondary hyperuricemia is due to various diseases causing increased synthesis or
decreased excretion of uric acid.
g. Increased degradation of nucleic acids (hence more uric acid formation) is
observed in various cancers (leukemias, polycythemia, lymphomas, etc.) psoriasis
and also in increased tissue breakdown.
h. The disorders associated with impairment in renal function cause accumulation of
uric acid which may lead to gout.
6. Clinical features:
a. Uric acid pool is tremendously increased to 3,000 mg. or even more/ in patients
suffering from gout
b. Gouty attacks may be precipitated by high purine diet and increased intake of
alcohol.
c. Gouty arthritis affects the first metatarsophalangeal joint (big toe), but other joints
may also be affected. The joints are extremely painful. Synovial fluid will show
birefringent urate crystals.
d. In chronic cases, uric acid may get deposited around joints causing swelling (tophi)
composed of sodium urate.
e. In chronic gout, the deposition of urate crystals in renal medulla occurs which
progresses to urolithiasis and renal damage.
7. Treatment Policies in Gout
a. Reduce dietary purine intake and restrict alcohol.

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b. Increase renal excretion of urate by uricosuric drugs, which decrease the
reabsorption of uric acid from kidney tubules, e.g. probenecid.
c. Allopurinol:
i. It has similar structure like hypoxanthine. Allopurinol is an analogue of
hypoxanthine. Allopurinol is a competitive inhibitor of xanthine oxidase
thereby decreasing the formation of uric acid.
ii. The durg causes a rapid fall in serum uric acid level and an increase in
concentration of hypoxanthine and xanthine in blood. Both xanthine and
hypoxanthine are more soluble and so are excreted easily in urine.
iii. Xanthine oxidase converts allopurinol to alloxanthine. It is a more effective
inhibitor of xanthine oxidase. This is a good example of suicide
inhibition'.
iv. Dosage: Initially 100 to 200 mg daily. Maintenance: 200 to 600 mg daily. Not
recommended in children.
v. In addition to gout, the drug can be used in secondary hyperuricaemia.
vi. Allopurinol also has an inhibitory action on the enzyme tryptophan
pyrrolase.
d. Colchicine, an anti-inflammatory agent is very useful to arrest the arthritis in gout.
e. Urate oxidase: The drug can be used in lowering uric acid level by oxidising uric
acid.
Pseudogout:
The clinical manifestations of pseudogout are similar to gout. But this disorder is
caused by the deposition of calcium pyrophosphate crystals in joints. Serum uric acid
concentration is normal in pseudogout.
2. HGPRT deficiency: LESCH-NYHAN SYNDROME
a. It is an X-linked inherited disorder of purine metabolism. Incidence is 1:10,000
males.
b. It is due to a defect of hypoxanthine-guanine phospho ribosyl (HGPRTase). So, the
rate of salvage pathway is decreased resulting in accumulation of PRPP and
decreased level of inhibitory purine nucleotides.
Hypoxanthine guanine phospho ribosyl transferase
i. Gunine+ PRPP -> Guanosine mono phosphate
+PPi
Hypoxanthine guanine phospho ribosyl transferase
ii. Hypoxanthine-guanine + PRPP > Inosine
monophosphate +PPi
c. Mutations that leads to the defect include deletions, frameshift mutations, base
substitutions, and aberrant mRNA splicing.
d. The disease is characterized by self mutilation (including biting and head
banging), mental retardation, excessive uric acid and nephro-lithiasis. Gout
develops in later life. The neurological manifestations suggest that the brain is
dependent on the salvage pathway for the requirements of IMP and GMP.
e. Allopurinol treatment that helps to decrease uric acid production, has no effect on
the neurological manifestations in these patients
IMMUNODEFICIENCY DISORDERS OF PURINE METABOLISM:(Hypouricemia)
a. Adenosine Deaminase (ADA) Deficiency
1. Adenosine deaminase plays a role in the breakdown of purines. Lymphocytes
have the highest activity of this enzyme. A deficiency of ADA results in an
accumulation of adenosine, which is converted to its ribonucleotide or
deoxyribonucleotide. As dATP levels rise, ribonucleotide reductase is inhibited,
thus preventing the production of all deoxyribose-containing nucleotides

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Consequently, cells cannot make DNA and divide. This would lead decrease in T
& B lymphocytes and a combined immunodeficiency.
2. This autosomal recessive deficiency causes recurrent infection and early death.
Antibiotics and periodic injections of immunoglobulin will be life saving. Weekly
intramuscular injections of bovine ADA were found to be beneficial.
b. The deficiency of purine nucleotide phosphorylase: it is associated with impairment
of T-cell function but has no effect on B-cell function. lt is believed that dGTP inhibits the
development of normal T-cells.
DE NOVO SYNTHESIS OF PYRIMIDINE:
Step 1: Carbamoyl Phosphate Synthesis:
Glutamine transfers its amido nitrogen to CO2 to produce carbamoyl phosphate. This
reaction is ATP-dependent and is catalysed by cytosomal enzyme carbamoyl
phosphate synthetase ll (CPS Il).
Step 2: Rate Limiting Step:
Carbamoyl phosphate and aspartate combine to form carbamoyl aspartate. The
enzyme is aspartyl transcarbamoylase (ATC), which is allosterically regulated. The
atoms C2 and N3 are derived from carbamoyl phosphate and the rest are from
aspartate.
Step 3: Formation of Pyrimidine Ring:
The 3rd nitrogen and 4th carbon are joined by a covalent bond and carbamoyl
aspartate is cyclised. Dihydo orotic acid is produced. The enzyme is dihydro orotase
(DHOase)
Step 4: Oxidation
Hydrogen atoms are removed from C5 and C6 positions, so that orotic acid is
produced. Enzyme is dihydro orotate dehydrogenase (DHODH). It requires NAD as coenzyme.
Step 5: Formation of OMP
Ribose-5-phosphate is added to orotic acid, so as to produce orotidylic acid or orotidine
monophosphate (OMP). PRPP is the donor of ribose-5-P. The enzyme is orotate
phosphoribosyl
transferase (OPRTase).
Step 6: Decarboxylation
The C7 of OMP is removed as carbon dioxide, so that uridine monophosphate (UMP) is
produced. This is the first purine that is synthesized. The enzyme is OMPdecarboxylase
(OMPDC). 6-aza-uridine inhibits this step, and so used as an anticancer drug.
Step 7, Synthesis of Triphosphates
UMP is phosphorylated to form UDP (uridine diphosphate) with the help of ATP. The
enzyme is nucleoside monophosphate kinase (UMP kinase). Next, the UDP is
phosphorylated to UTP (uridine triphosphate) with the help of ATP. The enzyme is
nucleoside diphosphate kinase.
Step 8, Formation of CTP
UTP is converted to CTP by adding an amino group from glutamine catalysed by CTP
synthetase. It needs ATP
Salvage pathway of pyrimidines
1. The pyrimidines (like purines) can also serve as precursors in the salvage pathway.
This reaction is catalysed by pyrimidine phosphor ribosyltransferase which utilizes
PRPP as the source of ribose 5-phosphate
2. The salvage of pyrimidine nucleosides is the basis for using uridine in the treatment of
hereditary orotic aciduria.

335
3. Uridine phosphorylase adds ribose-1-phospate to the free base uracil, forming uridine
monophosphate. Uridine kinase then phosphorylates this nucleoside into its
diphosphate and triphosphate forms.

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4. Deoxythymidine phosphorylase adds deoxyribose-1-phosphate to thymine, forming
deoxythymidine monophosphate. Thymidine kinase can then phosphorylate this
compound to deoxythymidine diphosphate and triphosphate
Regulation of Pyrimidine Synthesis:
1. CPS II, ATC and DHOase are present as a multienzyme complex and referred to as
CAD'. OPRTase and OMP decarboxylase are also present as a single functional
complex. Because of this clustering of enzymes, the synthesis is well co-ordinated.
2. Aspartate transcarbamoylase (ATC) is allosterically inhibited by CTP.
3. CPS II (enzyme 1) is inhibited by UTP and activated by PRPP
4. OMP decarboxylase is inhibited by UMP.
Degradation of pyrimidine nucleotides:
1. Cytosine and Uracil are degraded in a similar way:
1. First pyrimidine nucletides are dephosphorylated to the nucleosides by 5nucleotidases. Pyrimidine nucleosides are then phosphorolysed into free pyrimidines
and pentose 1 phosphate with the help of nucleoside phosphorylases.
2. Cytosine will form uracil by deaminase.
3. Uracil, is then reduced to 5,6-dihydrouracil by dihydrouracil dehydrogenase using
NADPH.
4. 5,6-dihydrouracil is hydrolyzed by hydropyrimidine hydrase to produce ureidopropionic acid.
5. -ureidopropionase convert -ureidopropionic acid into CO2, NH3 and -alanine.
6. The -alanine can either be used in the synthesis of Anserine, carnosine or CoA or can
be oxidised to acetate, NH3 and CO2.
2. Thymine
1. Thymine released from thymidine or produced by the deamination of 5-methylcytosine
is reduced to dihydrothymine by an NADH dependent dehydrogenase
2. Next is the hydrase that brings about the hydrolysis of dihydrothymine to give ureidoisobutyric acid.
3. The -ureidoisobutyric acid is hydrolysed by -ureidoisobutyrase into CO2, NH3 and amino isobutyrate.
Disorders of Pyrimidine Metabolism:
Orotic Aciduria
i. The condition results from absence of either or both of the enzymes, OPRTase and OMP
decarboxylase. It is an autosomal recessive disease.
ii. There is retarded growth and megaloblastic anemia. The rapidly growing cells are
more affected and hence the anemia.
iii. Crystals are excreted in urine which may cause urinary tract obstruction. Due to lack of
feedback inhibition orotic acid production is excessive.
iv. The condition can be successfully treated by feeding cytidine or uridine. They may be
converted to UTP which can act as feedback inhibitor.
v. Orotic aciduria may also occur in ornithine transcarbamoylase deficiency (urea cycle
enzyme) as carbamoyl phosphate accumulates due to defective conversion to
citrulline.
vi. Allopurinol competes with orotic acid for the enzyme orotate phosphoribosyl
transferase, leading to orotic aciduria and orotidinuria.
Anticancer Agents Acting on Pyrimidines
i. Methotrexate inhibits dihydrofolate reductase and thereby reduces the regeneration of
THFA; it is a anticancer agent.
ii. 5-fluoro-uracil, 5-iodo uracil, 3-deoxy uridine, 6-aza uridine, 6-aza cytidine and 5-iodo2-deoxyuridine are anticancer drugs, which competitively inhibit thymidylate synthase.
iii. Cytosine arabinoside where ribose is replaced by arabinose is another anticancer
agent.

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DEOXYRIBO NUCLEIC ACID

REPLICATION OF DNA
1. Describe the mechanism of DNA replication. Add a note on DNA repair
mechanism. Aug 2007
2. Name the primer required for DNA polymerase III. Pon May 2003
3. Define the terms replication, transcription and translation. Describe the
steps involved in protein biosynthesis. March 2002; Oct 1998
4. What is semi-conservative replication? Explain with a simple diagram. Pon
Dec 2003
5. What is Okazaki fragments? Pon May 2006
REPLICATION:
1. During cell division, each daughter cell gets an exact copy of the genetic information
of the mother cell. This process of copying the DNA is known as DNA replication.
2. In the daughter cell, one strand is derived from the mother cell; while the other strand
is newly synthesized. This is called semiconservative type of DNA replication.
Steps:
1. Origin of replication (ori)
2. Unwinding of DNA
3. Formation of the replication fork
4. Initiation and chain elongation
5. Formation of replication bubbles and ligation of the newly synthesised DNA segments.
Origin of Replication (ori)
1. There are specific sites where replication starts. The origin of replication on the DNA
strand in bacteria is termed as ori. Corresponding areas in higher organisms are called
replicators which contain the base sequences called the origin replication element
(ORE). This area is recognized by specific proteins collectively called the origin
recognition complex (ORC).
2. The protein A or DnaA binds at specific sites of origin, and opens the duplex (double
strand)
Unwinding of DNA:
1. Relief of supercoil is done by a group of enzymes called topoisomerases.
2. Helicase separates the strands of DNA, without a cut, but using energy from hydrolysis
of ATP.
3. DNA Replisome:
1. DNA replication needs more than 20 enzymes and proteins, collectively called
DNA replicase system or replisomes. Eg.
i. Helicases move on both directions, separating the strands in advance of
the replication. This forms a replication bubble with two replication forks.
ii. Single stranded DNA binding proteins (SSB) stabilize the complex.
Replication Bubble:
1. The two complementary strands of DNA separate at the site of replication to form a
bubble. Multiple replication bubbles are formed in DNA molecules for a rapid
replication process.
2. The enzyme primase in association with single-stranded binding proteins forms a
complex called primosome, and produces RNA primers.
3. DNA polymerase enzyme synthesises a new complementary strand of DNA, by
incorporating dNMP sequentially, making use of single stranded DNA as template.
4. The synthesis of two new DNA strands, simultaneously, takes place in the opposite
direction---one is in a direction (5'3') towards the replication fork which is continuous,
the other in a direction (5'3') away from the replication fork which is discontinuous.
5. Deoxyribonucleotides are added one after another, to 3' end of the growing DNA chain.

339
A molecule of pyrophosphate (PPi) is removed with the addition of each nucleotide.
Each ribonucleoside monophosphate is added through formation of a 3' 5'
phosphodiester bond.
6. Because the DNA strands are antiparallel, the polymerase functions asymmetrically.
On the leading (forward) strand, the DNA is synthesized continuously. On the
lagging (retrograde) strand, the DNA is synthesized in short fragments, called
Okazaki fragments. These pieces are made in the normal 5'3' direction, and later
joined together.
7. An enzyme capable of polymerising DNA in 3 5 direction does not exist in any
organism, so that both of the newly replicated DNA strands cannot grow in the same
direction simultaneously
8. Okazaki pieces:
a. The small fragments of the discontinuously synthesized DNA are called Okazaki
pieces. These are produced on the lagging strand of the parent DNA. Okazaki
pieces are later joined to form a continuous strand of DNA. DNA polymerase I
and DNA ligase are responsible for this process.
b. Several Okazaki fragments (up to a thousand) must be sequentially synthesized
for each replication fork. To ensure that this happens, the helicase acts on the
lagging strand to unwind dsDNA in a 5' to 3' direction.
c. The helicase associates with the primase to afford the latter proper access to
the template. This allows the RNA primer to be made and, in turn, the
polymerase to begin replicating the DNA. This is an important reaction
sequence since DNA polymerases cannot initiate DNA synthesis de novo. The
mobile complex between helicase and primase has been called a primosome.
d. As the synthesis of an Okazaki fragment is completed and the polymerase is
released, a new primer has been synthesized. The same polymerase molecule
remains associated with the replication fork and proceeds to synthesize the
next Okazaki fragment.

9. The template DNA strand (the parent) determines the base sequence of the newly
synthesized complementary DNA.

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3 and 5 ends of DNA

The DNA Polymerase Complex
1. A number of different DNA polymerase molecules engage in DNA replication. These
share three important properties: (1) chain elongation, (2) processivity, and (3)
proofreading.
2. Chain elongation:
a. Under the influence of DNA polymerase, the 3' hydroxyl group of the end
nucleotide combines with the 5' phosphate group of the new deoxynucleotide.
The pyrophosphate is released from the deoxynucleoside tri phosphate.
b. This newly added nucleotide would now polymerise with another one, forming
the next phosphodiester bond. The base pairing rule is always observed.

3. Proceesivity: DNA polymerase III is a highly processive enzymethat is, it remains

bound to the template strand as it moves along, and does not diffuse away
4. Proof reading:
a. The nucleotide sequence of DNA will be replicated without errors. Misreading of
the template sequence could result in mutations.
a. DNA polymerase III has a proofreading activity through 3' 5' exonuclease.
b. DNA polymerase III checks if nucleotide is correctly matched to its
complementary base on the template. If it is not, the 3' 5' exonuclease
corrects the mistake. If an incorrect base has been added, the enzyme makes a
cut at the phosphodiester bond and releases the incorrect nucleotide.
c. The proofreading exonuclease activity requires movement in the 3' 5'
direction, not 5' 3' like the polymerase activity. This is because the excision
must be done in the reverse direction from that of synthesis.]
Condensation into Chromatin Structure
Newly synthesized DNA is rapidly arranged into nucleosomes. This is facilitated by histone
chaperone proteins and chromatin remodeling complexes.
Inhibitors of DNA replication:
1. Bacteria contain a specific type Il topoisomerase namely gyrase. Bacterial gyrase is
inhibited by the antibiotics ciprofloxacin, novobiocin and nalidixic acid. These are
widely used as antibacterial agents

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2. Compounds that inhibit human topoisomerases are used as anticancer agents e.g.
adriamycin, etoposide, doxorubicin. The nucleotide analogs that inhibit DNA replication
are also used as anticancer drugs e.g. 6-mercaptopurnie, 5-flu orour acil.
3. Methotrexate (inhibits dihydrofolate reductase) and S-fluorouracil (inhibits thymidylate
synthase) block nucleotide synthesis.
4. In recent years, topoisomerase inhibitors are being used. They block the unwinding of
parental DNA strands and prevent replication.
5 steps of replication:

DNA REPAIR
1. List various DNA repair mechanisms and give their biological importance. Oct 2003
Causes of errors in DNA:
1. Endogenous:
a. Replication errors:
i. Incorrect base-pairing
ii. Insertion of one to a few extra nucleotides
b. Reactive oxygen species produced from normal metabolic byproducts
2. Exogenous
a. Chemicals - nitrous acid; Cigarette smoke contains carcinogens such as the
aromatic polycyclic hydrocarbons
b. Radiationi. Ultraviolet light, can fuse two pyrimidines
ii. High energy ionizing radiation, can cause double-strand breaks
iii. Radiotherapy
c. Chemotherapy
d. Viruses
Effects of errors:
i. It is fortunate that a great majority of the mutations probably occur in the DNA that
does not encode proteins,a nd consequentlyw ill not have any serious impact on the
organism.
ii. Gene Mutation: Change in a single base pair in the human genome can cause a
serious disease e.g. sicklecell anemia.

342
iii. Uncontrolled cell divisions: Cancer
iv. Cell death
Types of DNA damages:
I. Single-base alteration
A. Depurination
B. Deamination of cytosine to uracil
C. Deamination of adenine to hypoxanthine
D. Alkylation of base
E. Insertion or deletion of nucleotide
F. Base-analog incorporation
II. Two-base alteration
A. UV lightinduced thymine-thymine (pyrimidine) dimer
B. Bifunctional alkylating agent cross-linkage
III. Chain breaks
A. Ionizing radiation
B. Radioactive disintegration of backbone element
C. Oxidative free radical formation
IV. Cross-linkage
A. Between bases in same or opposite strands
B. Between DNA and protein molecules (eg, histones)
Types of repair:
1. Mismatch repair
2. Base excision- repair
3. Nucleotide excisionrepair
4. Double-strand break repair
Mismatch repair:
1. This mechanism corrects a single mismatch base pair (eg, C to A rather than T to
A) or a short region of unpaired DNA.
2. The original template DNA contains methylated residues. The newly synthesised
strand will not have methylated bases. So enzymes can recognize the original DNA
strand. The mismatched base is idendified by an endonuclease that makes
defective strand removed. A small segment of DNA with correct sequences of
bases is synthesised by polymerase beta. The gap is filled by DNA ligase.
3. Faulty mismatch repair has been linked to hereditary nonpolyposis colon cancer.
Base Excision-Repair:
1. The bases cytosine, adenine and guanine can undergo spontaneous depurination to
form uracil, hypoxanthine and xanthine. These altered bases do not exist in the
normal DNA, and therefore need to be removed. This is carried out by base excision
repair.
2. A defective DNA in which cytosine is deaminated to uracil is acted upon by the
enzyme uracil DNA glycosylase. This results in the removal of the defective base
uracil.
3. An endonuclease cuts the backbone of DNA strand near the defect and removes a few
bases. The gap so created is filled up by the action of repair DNA polymerase and DNA
ligase.
Nucleotide excisionrepair:
1. The DNA damage due to ultraviolet light, ionizing radiation and other environmental
factors often results in the modification of certain bases, strand breaks, cross-linkags
etc.
2. A n excision nuclease (exinuclease) cuts the DNA on either side of the damaged DNA.
This defective piece is degraded. The gap created by the nucleotide excision is filled

343
up by DNA polymerase which gets ligated by DNA ligase
3. Xeroderma pigmentosum (XP) is a rare autosomal recessive disease. The affected
patients are photosensitive a nd susceptible to skin cancers. lt is now recognized that
X P is due to a defect in the nucleotide excision repair.
Double-strand break repair:
1. The defect occurs as a result of ionizing radiation or oxidative free radical generation.
This defect may lead to chromosomal translocation, broken chromosomes, and finally
celI death.
2. DSBs can be repaired by homologous recombination or non-homologous end joining.
3. In nonhomologous system, the ends of two DNA fragments are brought together by a
group of proteins that effect their religation. However, some DNA is lost in the process.
In homologous recombination repair, uses the enzymes that normally perform genetic
recombination between homologous chromosomes during meiosis.
DEFECTS IN DNA REPAIR AND CANCER:
1. Xeroderma Pigmentosum (XP): Defective NER mechanism; sensitivity to UV light; skin
cancers
2. Ataxia Telangiectasia (AT): defective ATM gene; sensitivity to UV light; lymphoreticular
neoplasms
3. Fanconi's Anemia: Defective genes are in chromosomes 20q and 9q. Defect in DNA
cross link repair; increased occurrence of cancer.
4. Bloom's Syndrome: Gene is in 15q. Defect in DNA ligase or Helicase; lymphoreticular
malignancies
5. Cockayne Syndrome: Defect in NER mechanism; transcription factor II H is defective;
stunted growth and mental retardation.
6. Hereditary Polyposis Colon Cancer (Lynch syndrome): Defective gene in chromosome
2. Defect in hMSH1 and 2 genes; mismatch repair is defective.
RIBONUCLEIC ACID (RNA)
1.
1.
2.
3.
4.

Explain the different types of RNA. And their functions. Pon Apr 2000
Messenger RNA-April 2000
Indicate two differences between the nascent and mature mRNA. Pon Dec 2003
Structure and functions of t RNA Oct 2000; April 2001, Pon may 2014
What is the structure of mRNA. Pon May 2003
Ribonucleic acid (RNA) is also a polymer of purine and pyrimidine nucleotides linked by
phosphodiester
bonds. RNAs are the working copies of the DNA. The RNAs are synthesized from DNA, and
are
primarily involved in the process of protein biosynthesis.
RNAs are of 5 types:
1. Messenger RNA (mRNA). The gene present in DNA is transcribed into mRNA. This
constitutes about 2-5% of total RNA in the cell. They are generally degraded quickly.
2. Ribosomal RNA (rRNA). This constitutes about 80% of all RNA in the cell. 28S, 18S and
5S are the major varieties. They are involved in the protein biosynthesis. They are very
stable.
3. Transfer RNA (tRNA). There are about 60 different species present. They constitute
about 15% of the total RNA in the cell. They are very stable.
4. Small RNA. They constitute about 1-2% of total RNA in the cell. There are about 30
different varieties. They are very stable.
5. Small Nuclear RNAs (SnRNAs) are a subgroup of small RNA.
Messenger RNA (mRNA):

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Structure:
1. The mRNA is a complementary copy of the template strand of the DNA.
2. The gene present in DNA is transcribed into mRNA; The m-RNA carries a specific
sequence of nucleotides in triplets called codons, responsible for the synthesis of a
specific protein molecule.
3. 2-5% of total RNA in the cell; degraded quickly; thymine is not present in RNA; instead
uracil will be incorporated.
4. The 5' terminal of mRNA is "capped" by a 7-methylguanosine triphosphate. The cap is
involved in the recognition of mRNA by the translation machinery, and also helps
stabilize the mRNA by preventing the attack of 5'-exonucleases.
5. The
other
end
of
mRNA
molecules,
the
3'-hydroxyl
terminal,
has
an
attached
polymer of adenylate residues.
This poly (A) "tail" maintains the
intracellular
stability
of
the
specific mRNA by preventing the
attack of 3'-exonucleases and also facilitates translation.
6. Eukaryotic mRNA contains a leader sequence at the 5-end, a coding region, and a
trailer sequence at the 3 -end.
Functions of mRNA:
1. It acts as a messenger of the information in the gene in DNA to the protein
synthesizing machinery in cytoplasm. It carries the message to be translated to a
protein.
2. The template strand of DNA is transcribed into a single stranded mRNA. This is
accomplished by the DNA dependent RNA polymerase.
Ribosomal RNA (rRNA).
1. This constitutes about 80% of all RNA in the cell. 28S, 18S and 5S are the major
varieties. They are involved in the protein biosynthesis. They are very stable.
2. A ribosome is a cytoplasmic nucleoprotein structure that acts as the machinery for the
synthesis of proteins from the mRNA templates. On the ribosomes, the mRNA and
tRNA molecules interact to translate into a specific protein molecule information
transcribed from the gene.
3. During periods of active protein synthesis, many ribosomes can be associated with any
mRNA molecule to form an assembly called the polysome
4. Ribosome contains two major nucleoprotein subunit- the 60S subunit & the 40S
subunit
5. The exact functions of the ribosomal RNA molecules in the ribosomal particle are not
fully understood, but they are necessary for ribosomal assembly and also play key
roles in the binding of mRNA to ribosomes and its translation. Recent studies indicate
that the large rRNA component performs the peptidyl transferase activity and thus is a
ribozyme.
Transfer RNA (tRNA).
1. There are about 60 different species and constitute about 15% of the total RNA in the
cell. They are very stable.
2. They transfer amino acids from cytoplasm to the ribosomal protein synthesizing
machinery.
3. The transfer RNAs contain extensive internal base pairing and acquire clover leaf like
structure. They also contain a significant proportion of unusual bases. These include
dihydrouracil (DHU), pseudouridine, and hypoxanthine. Moreover many bases are
methylated.
4. All tRNA molecules contain four main arms.

345

5.
Small
1.

2.

3.
4.

a. The acceptor arm: It is at the 3' End. It carries the amino acid. This area has 7
base pairs. The end sequence is CCA-3'.
b. Anticodon Arm: At the opposite side of the acceptor arm is the anticodon arm.
It recognizes the triplet nucleotide codon present in mRNA. The specificity of
tRNA resides in the anticodon site, which has base sequences complementary
to
that of
mRNA
codon.
c.
The D
arm or
DHU
region is
so named
due to
the
presence
of
a dihydro
uridine in
that area.
The DHU arm serves as the recognition site for the enzyme which adds the
amino acid.
d. Pseudouridine Arm of tRNA: The opposite arm is called pseudouridine arm,
as it contains a pseudouridine. It is denoted with the Greek alphabet which
is pronounced as psi. It is involved in binding tRNA to ribosomes.
e. About 75% of tRNA molecules possess a short extra or variable arm, about 3 to
5 base pairs long and they belong to Class1. The tRNA molecules belonging to
Class 2, have a long extra arm, 13 to 21 base pairs in length.
Function: The tRNA molecules serve as adapters for the translation of the information
in the sequence of nucleotides of the mRNA into specific amino acids.
RNA.
Most of these molecules are complexed with proteins to form ribonucleoproteins and
are distributed in the nucleus, the cytoplasm, or both. They constitute about 1-2% of
total RNA in the cell. There are about 30 different varieties.
Small Nuclear RNAs (snRNAs): snRNAs, a subset of the small RNAs, are
significantly involved in rRNA and mRNA processing and gene regulation. They are
involved in mRNA splicing and take part in the formation of spliceosomes. All of them
are located in the nucleus. They complex with specific proteins, to form small nuclear
ribonucleoprotein particles (SnRNPs). It is pronounced as "Snurps". Production of
autoantibodies against Snurps cause systemic lupus erythematosis (SLE), a fatal
autoimmune disease.
Micro-RNA (miRNA). They alter the function of mRNA. They are moderately stable.
miRNAs cause inhibition of gene expression by decreasing specific protein production.
Small Interfering RNAs (siRNAs): duplexes between siRNA and mRNA results
inreduced specific protein production. siRNAs are frequently used to decrease or
"knock-down" specific protein levels in experimental contexts in the laboratory.

TRANSCRIPTION
1. Define the terms replication, transcription and translation. Describe the steps
involved in protein biosynthesis. Pon March 2002; Oct 1998
2. Write in details about the initiation, elongation and termination of transcription.
Give an account of post transcriptional processing.(MGR U)

346
Introduction:
1. Central Dogma of Molecular Biology: It states that, in a cell, information flows
from the nuclear DNA to RNA to protein synthesis i.e. "DNA makes RNA makes protein"
2. Genome: The total DNA (genetic information) contained in an organism or a cell is
regarded as the genome. The genome is the storehouse of biological information. lt
includes the chromosomes in the nucleus and the DNA in mitochondria, and
chloroplasts. The study of the structure and function of genome is genomics.
3. The word gene refers to the functional unit of the DNA that can be transcribed.
4. Codons: The letters A, G, T, and C correspond to the nucleotides found in DNA. Within
the protein coding genes these nucleotides are organized into three-letter code words
called codons, and the collection of these codons makes up the genetic code. Codon
consists of a sequence of three nucleotides; i.e. it is a triplet code.
RNA POLYMERASES
There are several distinct RNA polymerases in eukaryotic cells.
1. RNA polymerase I synthesizes rRNAs involved in facilitating protein synthesis by
the ribosome.
2. RNA polymerase II is responsible for the synthesis of mRNA and miRNAs.
3. RNA polymerase III catalyzes the synthesis of tRNAs.
4. RNA polymerase with the subunit structure 2 is called the holoenzyme.
TRANSCRIPTION:
1. Transcription is a process in which ribonucleic acid (RNA) is synthesized from DNA. The
genetic master plan of an organism is contained in the sequence of
deoxyribonucleotides in its DNA. Ribonucleic acid (RNA) are the working copies of
the DNA.
2. Transcription produces messenger RNAs that are translated into sequences of amino
acids to produce polypeptide chains or proteins, and ribosomal RNAs, transfer RNAs,
and additional small RNA molecules etc.
3. One of the two strands of DNA serves as a template called non-coding strand or sense
strand and produces working copies of RNA molecules. The other DNA strand which
does not participate in transcription is referred to as coding strand or antisense strand.
TRANSCRIPTION PROCESS: Transcription involves three different stages-initiation,
elongation and termination
Initiation:
1. Transcription begins with the binding of the RNA polymerase to a gene in DNA. RNA
polymerases must be able to:
a. Recognize the start point for transcription of each gene and
b. Choose the appropriate strand of DNA to use as a template.
c. Control the frequency of transcription.
2. For above purposes, the gene contains a coding region and a regulatory region.
a. The coding region has the DNA sequence (gene) that is transcribed into RNA.
b. The coding region of DNA includes not only the gene for mRNA synthesis, but
also the initiator, promoter, and terminator regions as well as the introns and is
called a transcription unit.
c. The regulatory region consists of following two classes of sequences in DNA.
i. The promoter sequences controls the binding of RNA polymerase to
DNA and identifies the start point of transcription in the DNA. Promoter
sequences have two components.
1. The proximal component, called the TATA box which directs RNA
polymerase II to the correct site (fidelity)
2. Distal component called CAAT &GC boxes which specify the
frequency of initiation.
a. In eukaryotes, proteins known as general transcription factors (or basal

347
factors) bind to the TATA box and facilitate the binding of RNA polymerase II.
This binding process involves at least six basal transcription factors.
a. Another class of sequence elements can either increase or decrease the rate of
transcription initiation of eukaryotic genes. These elements are called either
enhancers or repressors, depending on how they effect RNA synthesis.
3. After TFIID binds to the TATA box and TBP, five more transcription factors and RNA
polymerase combine around the TATA box in a series of stages to form what is known as
the preinitiation complex.

Elongation Process of Transcription

1. Transcription, the synthesis of RNA from a DNA template, is carried out by RNA
Polymerases. Synthesis of the new RNA molecule occurs in the 5-to-3 direction. The
ribonucleoside triphosphates adenosine triphosphate (ATP), GTP, CTP, and UTP serve
as the precursors. Each nucleotide base pairs sequentially with the complementary
deoxyribonucleotide base on the DNA template. The polymerase forms an ester bond
between the -phosphate on the ribose 5 -hydroxyl of the nucleotide precursor and
the ribose 3-hydroxyl at the end of the growing RNA chain.
2. The RNAP moves along the DNA template. New nucleotides are incorporated in the
nascent mRNA, one by one, according to the base pairing rule.
3. A transcription bubble containing
RNAP, DNA and nascent RNA is formed.
RNAP has no nuclease activity; so
there is no proof reading.
Another
diffrence in RNA synthesis is that RNA
polymerase does not require a primer
4. As the RNAP moves on the DNA
template, the DNA helix unwinds
downstream
and
winds
at
the
upstream areas.

Transcription bubble
Termination of Transcription:
1. The elongation of the single-stranded RNA chain continues until a termination signal is
reached.
2. Termination includes removal of RNA polymerase from DNA and the release of the RNA
molecule
3. Termination is brought about by two means:
a. Specific sequences on the DNA molecule function as the signal for termination
of the transcription process. This allows the RNA to fold back on itself, forming

348
a loop. This structure is known as a hairpin. Additionally, just beyond the
hairpin, the RNA transcript contains a string of Us at the 3'-end. The bonding of
these Us to the complementary As of the DNA template is weak. This facilitates
the separation of the newly synthesized RNA from its DNA template
b. A termination protein called Rho(p) acts as ATPase and terminates transcription
and dissociates RNA and RNA polymerase from the DNA.
4. After termination of synthesis of the RNA, the enzyme separates from the DNA
template and probably dissociates to free core enzyme and free factor.
Primary transcript:
1. The product formed in transcription is called primary transcript.
2. The primary mRNA transcript produced by RNA polymerase ll is referred to as
heterogeneous nuclear RNA (hnRNA). They undergo post-transcriptional modifications,
(splicing, terminal additions, base modifications etc.) to produce functionally active
RNA molecules.
3. Differences between primary and mature mRNA:
Primary transcript
It is known as
heterogeneous nuclear RNA
(hnRNA). Present in nucleus
No cap at 5 terminal; 5terminal is a pyrimidine

poly-A tail is not present

Contains introns, or
intervening sequences
which do not code for
protein
coding sequences, the
exons are separated by non
coding introns

Mature mRNA
Mature mRNA; present in cytoplasm

The 5, end of mRNA is transcription is capped with

7- methylguanosine. This cap protects it from
degradation (by 5 exonucleases) during
elongation of the RNA chain.
added to mRNA. These tails help stabilize the
mRNA, facilitate its exit from the nucleus, and aid
in translation
removal of introns is promoted by small nuclear
ribonucleoprotein particles (snRNPs).

Introns are removed and the exons are joined

together

POST TRANSCRIPTIONAL MODIFICATIONS

1. Post transcriptional modification of RNA April 2000
2. Post transcriptional modification of mRNA. Pon may 2014
3. What is Post transcriptional modification? Explain with two examples. Pon
Nov 2005
4. What is Post translational modification? Give two examples. Pon May 2004
5. Name the Post translational modification.Pon May 2010
6. Give two examples of proteins undergoing Post translational modification. Give
the recations taking place. Pon May 2003
1. The RNAs produced during transcription are called primaryt ranscripts. They undergo many
alterations- terminal base additions, base modifications, splicing etc., which are
collectively referred to as post-transcriptional modifications. This process is required to
convert the RNAs into the active forms.
2. In mammalian system, it undergoes extensive processing to become the mature mRNA. This
mainly in the nucleoplasm. These modifications are:
a. 5' capping

349

3.

4.

5.

6.

7.
8.

9.

b. Poly-A tailing
c. Endonuclease cleavage
d. Methylation
e. Removal of introns
f. Splicing of exons (connect together).
In bacteria, mRNA is not changed; and translation of mRNA starts even before completion of
transcription. Post-transcriptional processing is not only form RNA but for tRNA and rRNA
as well.
The 5, capping : This is the first of the processing reactions. The 5, end of mRNA is
transcription capped with 7-methylguanosine by an unusual 5'-+5' triphosphate linkage.
S-Adenosylmethionine is the donor of methyl group. This cap protects it from degradation
(by 5 exonucleases) during elongation of the RNA chain. The cap also helps the
transcript bind to the ribosome during protein Synthesis.
Poly-A tail : A large number of eukaryotic mRNAs possess an adenine nucleotide chain at the
3'-end. This poly-A tail is not produced during transcription. lt is later added to mRNA.
These tails help stabilize the mRNA, facilitate its exit from the nucleus, and aid in
translation. After the mRNA enters the cytosol, the poly-A tail is gradually shortened.
Removal of introns:
a. Maturation of eukaryotic mRNA usually involves the removal of RNA sequences
(introns, or intervening sequences), which do not code for protein from the
primary transcript. The remaining coding sequences, the exons, are joined
together to form the mature mRNA. The process of removing introns and joining
exons is called splicing.
b. The molecular complex that accomplishes these tasks is known as the
spliceosome. The removal of introns is promoted by small nuclear
ribonucleoprotein particles (snRNPs). snRNPs (pronounced as snurps) in turn, are
formed by the association of small nuclear RNA (snRNA) with proteins.
c. Mutations at splice sites can lead to improper splicing and the production of
aberrant proteins. It is estimated that 15% of all genetic diseases are a result of
mutations that affect RNA splicing. E.g. thalassemia
d. Production of autoantibodies against Snurps cause systemic lupus
erythematosis (SLE), a fatal autoimmune disease.
e. Alternative splicing of mRNA molecules: The pre-mRNA molecules from some
genes can be spliced in alternative ways in different tissues. This produces
multiple variations of the mRNA and, therefore, of its protein product
Methylations: Methylations of N6 of adenine residue and 2'-hydroxyl group of ribose are
common. These are mainly done in the cytoplasm.
Modifications for other RNAs:
a. Ribosomal RNAs: The pre-rRNAs are cleaved by ribonucleases to yield
intermediate-sized pieces of rRNA, which are further processed (trimmed by
exonucleases and modified at some bases and riboses) to produce the required
RNA species.
b. Transfer RNA: tRNA are also made from longer precursor molecules that are
also modified. Many modifications of the standard bases A, U, G, and C, including
methylation, reduction, deamination, and rearranged glycosidic bonds take place.
Further modification of the tRNA molecules includes nucleotide alkylations and the
attachment of the characteristic CpCpAOH terminal at the 3' end of the molecule
by the enzyme nucleotidyl transferase.
Editing: Coding information can be changed at RNA level by RNA editing. An example is the
apolipoprotein B (apoB) gene and mRNA. CAA codon in the mRNA is edited to form UAA.
Instead of ApoB100, a different protein, apoB48 is the result of this editing
Reverse Transcriptase

350
1. The genetic materials of some animal and plant viruses are made up of RNA instead of
DNA. Retrovirus is a subgroup of RNA viruses. The human immunodeficiency virus
(HIV) causing AIDS is a retrovirus.
2. Here the RNA acts as a template. Based on this RNA, the enzyme, RNA dependent DNA
polymerase or reverse transcriptase will make a new DNA strand.
3. The new DNA acts as a template to produce double stranded DNA. Thus genetic
information is transferred from RNA to DNA a reverse process!
4. Some of the tumor viruses were also shown to possess reverse transcriptase. The
presence of the enzyme may be taken as an indication of a retrovirus infection.
Inhibitors of RNA Synthesis
1. Actinomycin D: Actinomycin D binds with DNA template strand and blocks the
movement of RNA polymerase. This was the very first antibiotic used for the treatment
of tumors.
2. Rifampin: lt is an antibiotic widely used for the treatment of tuberculosis and leprosy.
Rifampin binds with the p-subunit of prokaryotic RNA polymerase and inhibits its
activity.
3. a-Amanitin : lt is a toxin produced by mushroom, Amanita phalloides. This mushroom is
delicious in taste but poisonous due to the toxin o-amanitin which tightly binds with
RNA polymerase ll of eukaryotes and inhibits transcription

GENETIC CODE & CODONS

1. What is a codon? Pon May 2004
2. What is genetic code? Mention two important features of genetic code. What
are synonymous codons? Pon Dec 2003
3. What is genetic code? Explain its features. Pon apr 2000
CODONS
1. Central Dogma of Molecular Biology: The flow of genetic information is dependent on
the sequence of bases in DNA, its mRNA transcript and the sequence of amino acids in a

protein.
2. The genetic information for the synthesis of proteins is contained in DNA originally in the
codingstrand and as it is complementary to the template strand, and then the information
is transcribed to mRNA.
3. The three nucleotide (triplet) base sequences in mRNA act as code words for amino acids
in protein is called codon. The collection of codons is known as Genetic code and it is

arranged in a table.

351
4. Since there are four different bases, they can generate 64 (4 3) different codons. Their
nucleotide sequences are always written from the 5 end to the 3 end. There are 31 tRNA
species, carrying 20 amino acids, which translate 61 codons.
5. There are three codons which do not code for any particular amino acids. They are
nonsense codons, more correctly termed as punctuator codons or terminator codons.
They put "fullstop" to the protein synthesis. These three codons are UAA, UAG, and UGA.
6. Every amino acid except methionine is represented by several codons. Codons that
represent same amino acid are called as synonymous codons. In synonymous
codons, the base of the third position is insignificant, because the codons differing only
in the third base represent the same amino acid.
7. Several codons serve special functions.
a. The initiation codon (AUG) is the most common signal for the beginning of a
polypeptide in all cells.
b. The termination codons (UAA, UAG, and UGA), also called stop codons or nonsense
codons, normally signal the end of polypeptide synthesis and do not code for any
known amino acids.
c. Changing a single nucleotide base on the mRNA chain is called a point mutation.
GENETIC CODE
1. Protein synthesis is called translation because the language of the nucleotide
sequence on the mRNA is translated into the language of an amino acid sequence.
The process of translation requires a genetic code, through which the information
contained in the nucleic acid sequence is expressed to produce a specific sequence of
amino acids.
2. It consists of a specific sequence of nucleotides at a given position on a given
chromosome that codes for a specific protein (or, in some cases, an RNA molecule). A
gene has 'start' and 'stop' codons and other regulatory elements.
3. Within the protein coding genes the nucleotides are organized into three-letter code
words called codons, and the collection of these codons makes up the genetic code.
4. A triplet sequence of nucleotides on the mRNA is the codon for each amino acid. Since
there are four different bases, they can generate 64 (43) different codons or code
words by combinations. There are 31 tRNA species, carrying 20 amino acids, which
translate 61 codons.
Features of Genetic Code:
1. Triplet Codons: The codes are on the mRNA. Each codon is a consecutive sequence
of three bases on the mRNA, e.g. UUU codes for phenylalanine
2. Nonoverlapping: The codes are consecutive. Therefore the starting point is
extremely important. The codes are read one after another in a continuous manner,
e.g. AUG, CAU, GAU, GCA, etc.
3. Nonpunctuated: There is no punctuation between the codons. It is consecutive or
continuous.
4. Degenerate: 61 codes stand for the 20 amino acids. So one amino acid has more
than one codon. For example, serine has 6 codons; while glycine has 4 codons. This is
called degeneracy of the code. If the amino acid has more than one codon, the first
two bases in the codon will be the same, only the third one is different. This reduces
the effect of mutations.
5. Unambiguous or specificity: Though the codons are degenerate, they are
unambiguous. That is, one codon stands only for one amino acid.
6. Universal: The codons are the same for the same amino acid in all species; the same
for "Elephant and E.coli". The genetic code has been highly preserved during
evolution.
7. Wobbling Phenomenon: The reduced stringency between the third base of the

352
codon and the complementary nucleotide in the anticodon is called wobbling. The
pairing of codon and anticodon can wobble at the third letter. For example, GGU, GGC
and GGA are the codes for glycine; all three will pair with the anticodon CCI (I =
Inosinic acid) of glycine tRNA. The degeneracy of genetic code and wobbling
phenomenon together will reduce the effect of mutations.
8. Terminator Codons: There are three codons which do not code for any particular
amino acids. They are nonsense codons, more correctly termed as punctuator
codons or terminator codons. They put "fullstop" to the protein synthesis. These three
codons are UAA, UAG, and UGA. UGA is a stop codon; but in special circumstances, it
stands for seleno-cysteine (the "21st" aminoacid).
9. Initiator Codon: In most of the cases, AUG acts as the initiator codon. AUG also acts
as the codon for methionine. In a few proteins, GUG may be the initiator codon.
10. Mitochondria have different codes. The protein synthesising machinery of
mitochondria is distinct from that in the cytoplasm. There are only about 22 tRNAs in
mitochondria; but there are 31 tRNA species in cytoplasm.
TRANSLATION OR PROTEIN BIOSYNTHESIS
1. Define the terms replication, transcription and translation. Describe the steps
involved in protein biosynthesis. March 2002; Oct 1998
2. Post translational modifications
3. Explain Protein synthesis in detail. Add a note on drugs that inhibit protein
synthesis.
Translation:
1. The pathway of protein synthesis is called translation because the language of the
nucleotide sequence on the mRNA is translated into the language of an amino acid
sequence. The process of translation requires a genetic code, because the sequence of
amino acids in the protein is determined by the nucleotide base sequence in the DNA
which is transcribed to mRNA and translated into protein with the help of ribosomes.
2. There are wide variations in the cells which synthesize proteins. Liver cells produce
albumin and blood clotting factors for export into the blood for circulation. The Iiver
cells are rich in the protein biosynthetic machinery, and may be regarded as the
protein factory in the human body. Erythrocytes lack the machinery for translation, and
therefore cannot synthesize proteins.
3. The tRNAs act as adapter molecules between mRNA and the amino acids coded by it.
The nucleotides of codons have no affinity for amino acids. So the tRNA molecules act
as mediators between the mRNA and amino acids.
4. Translation is a cytoplasmic process. The mRNA is translated from 5' to 3' end. In the
polypeptide chain synthesized, the first amino acid is the amino terminal one. The
chain growth is from amino terminal to carboxyl terminal.

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Steps of protein synthesis:
1. Requirement of Components:
a. All the amino acids present in a protein must be present at the time of synthesis. If
one amino acid is missing translation stops.
b. One specific type of tRNA is required for each amino acid. Some amino acids may have
more than one specific tRNA molecule.
c. Aminoacyl-tRNA synthetases catalyze a two-step reaction that results in the
attachment of the carboxyl group of an amino acid to the 3'-end of its tRNA.
d. The specific mRNA is required as a template for the synthesis of the desired
polypeptide chain.
e. Ribosomes is part of the macromolecular complexes in which the synthesis of proteins
occurs.
f. Protein factors like initiation, elongation, and termination factors are required for
peptide synthesis. Some of these protein factors perform a catalytic function, whereas
others appear to stabilize the synthetic machinery.
g. ATP and GTP are required as sources of energy
Phases of translation:
i. Activation of amino acid
ii. Initiation
iii. Elongation
iv. Termination and
v. Post-translational processing.
1. Activation of amino acid:
Amino acids are activated and attached to tRNAs in a two step reaction. The amino
acid is first attached to the enzyme aminoacyl tRNA synthetases utilizing ATP to form
enzyme-AMP-amino acid complex. The amino acid is then transferred to the 3' end of
the tRNA to form aminoacvl tRNA
2. Initiation:
a. Initiation of protein synthesis requires that an mRNA binds to the ribosome.
Ribosomes are the centres or factories for protein synthesis. Initiation can be divided
into four steps:
A. Dissociation of the ribosome 80S into 60S and 40S subunits.
B. Formation of 43S preinitiation complex
C. Formation of initiation complex
D. Formation of 80S initiation complex.
b. Dissociation:
i. The 80S (s=sedimentation coefficient) ribosome dissociates to form 40S and
60S subunits. Two initiation factors, elF-3 (eukaryotic initiation factor) and elF1A binds to 40S subunit.
c. Preinitiation complex:
i. Binding of GTP with elf-2, (eukaryotic initiation factor 2) forms a binary complex,
which then binds to Met-RNA (methionyl RNA), and forms a ternary complex.
This complex attaches to 40S ribosomal subunit to form 43 S preinitiation
complex.
ii. Meanwhile, the cap at the 5-end of the mRNA binds to components of the eIF4
complex containing eIF4F, known as the cap-binding complex. elF- 4E, a
component of elF-4F is primarilyThis step involving responsible for the
recognition of mRNA cap. And this step is the rate-limiting in translation
d. Initiation complex:

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i.

m-RNA plus the cap-binding complex combines with the 43S preinitiation complex
to produce the 48S initiation complex.
ii. After formation of 48S initiation complex, it searches for precise initiation codon
AUG for methionine.
iii. Recognition of initiation codon: In eukaryotes, the first amino acid
incorporated is methionine (5-AUG codon). This is the initiation codon and its
recognition by ribosome is facilitated by a specific sequence of nucleotides. This
marker sequence for the identification of AUG is called as Kozak consensus
sequences.
iv. The 48S initiation complex now binds to 60S, which was free after ribosomal
dissociation, forms 80S initiation complex. This requires hydrolysis of GTP for
energy. This reaction then results in release of 48S initiation complex, which are
then recycled for next protein synthesis.

e. 80S initiation complex:

i. 80S initiation complex is now a complete assembly for protein synthesis and
contains one small and one large subunit
ii. It has three binding sites for tRNA, known as the P (peptidyl), A (aminoacyl),
and E (ejection) sites.
iii. The p site is having the initiator codon, the met-tRNAi and is ready for the
elongation cycle to commence. Both the A site (aminoacyl or acceptor site) and
E site (deacylated tRNA exit site) are free.

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3. Elongation: Elongation involves several steps catalyzed by proteins called elongation

factors (EFs). These steps are:
i.

b.

c.

d.

e.

Binding of aminoacyl-tRNA to the A site with proper codon recognition

ii. Peptide bond formation, and
iii. Translocation of the ribosome on the mRNA.
Binding of aminoacyl-tRNA:
i. When Met-tRNAi is bound to the P site, the mRNA codon in the A site
determines which aminoacyl-tRNA will bind to that site.
ii. EF1A forms a ternary (three) complex with GTP and the aminoacyl tRNA to enter
the A site with the release of EF1A GDP and phosphate.
iii. EF-1A, and GDP are recycled to bring another aminoacyl-tRNA.
Peptide bond formation:
i. The alpha amino group at the incoming amino acid in the "A" site forms a
peptide bond (CONH) with carboxyl group of the peptidyl tRNA occupying the
"P" site. This reaction is catalyzed by the enzyme peptidyl transferase.
ii. Now the growing peptide chain is occupying the "A" site and the tRNA in the P
site is no longer contains an amino acid or peptide.
i. This is an example of ribozyme where RNA acts as the enzyme.
ii. It is estimated that about six amino acids per second are incorporated during
the course of elongation in eukaryotes
Translocation:
i. After the peptide bond has been formed, the ribosome advances three
nucleotides toward the 3 end of the mRNA. This process is known as
translocation and requires the participation of eEF-2 and GTP hydrolysis to GDP.
ii. This causes movement of the uncharged tRNA into the ribosomal E site to be
released and movement of the peptidyl-tRNA into the P site.
Termination:
i. After successive addition of amino acids, ribosome reaches the terminator
codon sequence (UAA, UAG or UGA) on the mRNA. Since there is no tRNA

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bearing the corresponding anticodon sequence, the "A" site remains free.
ii. As the termination codon occupies the ribosomal A-site, the release factor
namely eRF recognizes the stop signal. eRF-GTP complex, in association with
the enzyme peptidyltransferase, cleaves the peptide bond between the
polypeptide and the tRNA occupying P-site. ln this reaction, a water molecule,
instead of an amino acid is added. This hydrolysis releases the protein and tRNA
from the P-site.
iii. The 80S ribosome dissociates to form 40S and 605 subunits which are recycled.
The mRNA is also released.
Polysomes:
a. Many ribosomes can translate the same mRNA molecule simultaneously.
Because of their relatively large size, the ribosome particles cannot attach to an
mRNA any closer than 35 nucleotides apart. Multiple ribosomes on the same
mRNA molecule form a polyribosome, or "polysome"
b. In an unrestricted system, the number of ribosomes attached to an mRNA (and
thus the size of polyribosomes) correlates positively with the length of the
mRNA molecule.
INHIBITORS OF PROTEIN SYNTHESIS
1. Majority of the antibiotics interfere with the bacterial protein synthesis and are harmless
to higher organisms.
2. Streptomycin : Initiation of protein synthesis is inhibited by streptomycin. lt causes
misreading of mRNA and interferes with the normal pairing between codons and
anticodons.
3. Tetracycline : lt inhibits the binding of aminoacyl tRNA to the ribosomal complex and can
also block eukaryotic protein synthesis. This, however, does not happen since eukaryotic
cell membrane is not permeable to this drug.
4. Puromycin: This has a structural resemblance to aminoacyl tRNA. Puromycin enters the A
site and gets incorporated into the growing peptide chain and causes its release. This
antibiotic prevents protein synthesis in both prokaryotes and eukaryotes.
5. Chloramphenicol : lt acts as a competitive inhibitor of the enzyme peptidyltransferase and
thus interferes with elongation of peptide chain.
6. Erythromycin : lt inhibits translocation by binding with 50S subunit of bacterial ribosome.
7. Diphtheria toxin : lt prevents translocation in eukaryotic protein synthesis by inactivating
elongation factor eEF2.
POST.TRANSLATIONAL MODIFICATIONS OF PROTEINS
The proteins synthesized in translation are, as such, not functional. Many changes take place
after the protein synthesis is completed. These modifications include
a. protein folding
b. trimming by proteolytic degradation,
c. intein splicing and covalent changes
1) Ghaperones and protein folding
a. When a protein is being synthesized, it may assume different three-dimensional
structures, out of which only one will have the biological activity. Abnormal folding
of proteins may lead to prion diseases.
b. The word "chaperone" means elderly lady in charge of unmarried girls on social
occasions. Chaperones attach to nascent polypeptide chains and prevent wrong
foldings; so that folding is allowed only in the correct direction. They help in the
assembly of tertiary and quaternary structure of proteins.
c. Examples of chaperones are a large family of proteins called heat shock proteins

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2)
3)

4)

5)

(HSPs). Any stress to the cell including heat, toxins, heavy metals, free radicals,
radiation, bacteria, etc. will cause increased production of HSPs, and hence they
are more correctly termed as stress proteins.
d. Chaperonopathies are disorders resulting from "sick chaperones". These diseases
progress with age.
Proteolytic cleavage: Modification of polypeptides by partial proteolysis, e.g. conversion
of pro-insulin to insulin.
lntein splicing: Inteins are intervening sequences in certain proteins. These are
comparable to introns in mRNAs. lnteins have to be removed, and exteins ligated in the
appropriate order for the protein to become active.
Modifications of amino acids
1. Gamma carboxylation of glutamic acid residues of prothrombin, under the influence
of vitamin K.
2. Hydroxylation of proline and lysine in collagen with the help of vitamin C
3. Phosphorylation of hydroxyl groups of serine, threonine or tyrosine by kinases, e.g.
glycogen phosphorylase.
4. Cotranslational glycosylation: Carbohydrates are attached to serine or threonine
residues through O-glycosidic linkages and to asparagine or glutamine residues
through N-glycosidic linkages.
Subunit aggregation
Examples are immunoglobulin, hemoglobin and maturation of collagen. Failure of posttranslational modification affects the normal function of many proteins. For example,
poor cross-linking of collagen in scurvy, since ascorbic acid is required for the
hydroxylation of proline and lysine.
PACKAGING OF DNA

1. Which protein is involved in DNA packing. Pon apr 2000

2. Role of histones in maintaining DNA integrity. Pon May 2009
1. DNA molecules require special packaging to enable them to reside within cells because
the molecules are so large.
2. DNA consists of a double helix, with the two strands of DNA wrapping around each other
to form a helical structure. To be compact, the DNA molecule coils about itself to form a
structure called a supercoil.
3. Histones: They are proteins containing unusually higher
concentration of basic amino acids. There are 5 classes; H1,
H2A,
H2B, H3 and H4. The H1 histone is loosely attached to the
DNA
4. Eukaryotic DNA binds to an equal weight of histones, which
are
small basic proteins containing large amounts of
arginine and lysine. The complex of DNA and proteins
is called chromatin and appears as beeds on a string.
5. The beads with DNA protruding from each end are
known as nucleosomes, and the beads themselves
are known as nucleosome cores.
6. Two molecules of each of four core histone classes (histones H2A, H2B, H3, and H4) form
the center of the core around which DNA are wound. The DNA joining the cores is
complexed with the fifth type of histone, H1.
7. Other types of proteins are also associated with DNA in the nucleus. These proteins are
called nonhistone chromosomal proteins.
NUCLEIC ACID METABOLISM

358
1. Discuss about nucleic acids under following headings: (MGR U)
a) Types b) Functions c) Components d) Char gaffs rule of DNA composition
e) Different forms of DNA double helix and f) Differences between DNA and
RNA.
2. Write on DNA polymerase. Pon May 2009
TYPES OF NUCLEIC ACIDS:
1. Nucleic acids are non-protein nitrogenous substances made up of a monomeric unit
called a nucleotide.
2. DNA is a polymer of deoxyribonucleotides and is found in chromosomes and
mitochondria.
3. Ribonucleic acid is a polymer of ribonucleotides. May be found in nucleus but mainly
occurs in cytoplasm. Types of RNA:
1) Transfer RNA (t-RNA)
2) Messenger RNA (m-RNA)
3) Ribosomal RNA (r-RNA)
Functions of Nucleic Acids:
1) DNA:
a. DNA is the chemical basis of heredity and nay be regarded as the reserve bank of
genetic information. DNA is exclusively responsible for maintaining the identity of
different species of organisms over millions of years. Further, every aspect of
cellular function is under the control of DNA.
b. The DNA is organized into genes, the fundamental units of genetic information. The
genes control the protein synthesis through the mediation of RNA, as shown below
DNA RNAProtein
c. The interrelationship of these three classes of biomolecules (DNA, RNA and proteins)
constitutes the central dogma of molecular biology or more commonly the central
dogma of life.
2) RNA:
a. Messenger RNA (mRNA). The gene present in DNA is transcribed into mRNA. The
mRNA is a complementary copy of the template strand of the DNA. The gene present
in DNA is transcribed into mRNA; The m-RNA carries a specific sequence of nucleotides
in triplets called codons, responsible for the synthesis of a specific protein molecule.
b. Ribosomal RNA (rRNA). This constitutes about 80% of all RNA in the cell. 28S,
18S and 5S are the major varieties. They are involved in the protein biosynthesis.
A ribosome is a cytoplasmic nucleoprotein structure that acts as the machinery for
the synthesis of proteins from the mRNA templates. On the ribosomes, the mRNA
and tRNA molecules interact to translate into a specific protein molecule
information transcribed from the gene. During periods of active protein synthesis,
many ribosomes can be associated with any mRNA molecule to form an assembly
called the polysome
c. Transfer RNA (tRNA). There are about 60 different species present. They
constitute about 15% of the total RNA in the cell. They are very stable.They
transfer amino acids from cytoplasm to the ribosomal protein synthesizing
machinery.
d. Small RNA.
i. Most of these molecules are complexed with proteins to form
ribonucleoproteins and are distributed in the nucleus, the cytoplasm, or
both. They constitute about 1-2% of total RNA in the cell. There are about
30 different varieties.
ii. Small Nuclear RNAs (snRNAs): snRNAs, a subset of the small RNAs, are

359
significantly involved in rRNA and mRNA processing and gene regulation.
They are involved in mRNA splicing and take part in the formation of
spliceosomes. All of them are located in the nucleus. They complex with
specific proteins, to form small nuclear ribonucleoprotein particles (SnRNPs).
It is pronounced as "Snurps". Production of autoantibodies against Snurps
cause systemic lupus erythematosis (SLE), a fatal autoimmune disease.
iii. Micro-RNA (miRNA). They alter the function of mRNA. They are
moderately stable. miRNAs cause inhibition of gene expression by
decreasing specific protein production.
iv. Small Interfering RNAs (siRNAs): duplexes between siRNA and mRNA
results inreduced specific protein production. siRNAs are frequently used to
decrease or "knock-down" specific protein levels in experimental contexts in
the laboratory.
Components of nucleic acids:
1) Nucleic acids are the polymers of nucleotides (polynucleotides) held by 3' and 5'
phosphate bridges
2) A nucleotide is made up of 3 components:
a. Nitrogenous base, (a purine or a pyrimidine)
b. Pentose sugar, either ribose or deoxyribose;
c. Phosphate groups esterified to the sugar.
3) When a base combines with a pentose sugar, a nucleoside is formed. When the
nucleoside is esterified to a phosphate group, it is called a nucleotide or nucleoside
mono-phosphate. The nucleic acids (DNA and RNA) are polymers of nucleoside
monophosphates.
4) There are four different types of nucleotides found in DNA, differing only in the
nitrogenous base.
5) The purine bases present in RNA and DNA are the same; adenine and guanine.
6) The pyrimidine bases present in nucleic acids are cytosine, thymine and uracil.
Cytosine is present in both DNA and RNA. Thymine is present in DNA and uracil in RNA.
Char gaffs rule of DNA composition:
1) Erwin Char gaff in late 1940s quantitatively analysed the DNA hydrolysates from
different species. He observed that DNA had equal numbers of adenine and thymine
residues (A = T) and equal numbers of guanine and cytosine residues (G = C). This is
known as Chargaff's rule of molar equivalence between the purines and pyrimidines in
DNA structure.
2) The significance of Char gaff's rule was that the double helical structure of DNA
derives its strength from Char gaff's rule.
3) Single-stranded DNA, and RNAs do not obey Char gaff's rule. However, doublestranded RNA which is the genetic material in certain viruses satisfies Chargaff's rule.
Different forms of DNA double helix :
1) The DNA is a right handed double helix. lt consists of two polydeoxyribonucleotide
chains (strands) twisted around each other on a common axis. The two strands are
antiparallel, i.e., one strand runs in the 5' to 3' direction while the other in 3' to 5'
direction.
2) The double helical structure of DNA exists in at least 6 different forms-A to E and Z.
Among these, B, A and Z forms are important
3) B form: Watson and Crick described the B form of DNA, a right-handed helix. Each
turn of the B-form has 10 base pairs spanning a distance of 3.4 nm. The width of the
double helix is 2 nm.
4) The A-form is also a right-handed helix. It contains 11 base pairs per turn. There is a
tilting of the base pairs by 2O0 away from the central axis.
5) The Z-form (Z-DNA) is a left-handed helix and contains 12 base pairs per turn. The

360
polynucleotide strands of DNA move in a somewhat 'zig zag' fashion, hence the name
Z-DNA.
6) It is believed that transition between different helical forms of DNA plays a significant
role in regulating gene expression.
Differences between DNA and RNA:
RNA
DNA
1)
Mainly seen in cytoplasm
Mostly inside nucleus
2)
Usually 100-5000 bases
Millions of base pairs
3)
Generally single stranded
Double stranded
4)
Sugar is ribose
Sugar is deoxyribose
5)
Purines: Adenine, Guanine
Adenine, Guanine
6)
Pyrimidines: Cytosine, Uracil
Cytosine, Thymine
7)
Char gaffs rule: Guanine content is not
Guanine is equal to cytosine
equal to cytosine and adenine is not equal
and adenine is equal to
to uracil
thymine
8)
Easily destroyed by alkali
Alkali resistant

The DNA Polymerase Complex

Write on DNA polymerase. Pon May 2009
1. Enzymes that catalyze the synthesis of DNA are known as DNA polymerases. All DNA
polymerases copy a DNA template strand in its 3-to-5 direction, producing a new
strand in the 5-to-3 direction
2. E.coli has three DNA polymerases: pol I, pol II, and pol III. Pol III is the major replicative
enzyme. But in eukaryotic cells, five key eukaryotic DNA polymerases have been
identified and designated by Greek letters: , , , , .
a. Polymerase alpha: It is the major enzyme responsible for chromosome
replication. This enzyme polymerises about 100 nucleotides per seconds. It has
4 subunits, one of which has primase activity.
b. Mammalian beta DNAP is a repair enzyme.
c. DNAP gamma is concerned with mitochondrial DNA synthesis.
d. Delta enzyme is used for both leading and lagging strand synthesis.
e. Epsilon is used for leading strand synthesis.
3. DNA polymerase cannot initiate the synthesis of new strands; it requires the presence
of a free 3-OH group to function. Therefore, a RNA primer is required to supply the
free 3-OH group.
4. DNA polymerase molecules engage in DNA replication through: (1) chain elongation,
(2) processivity, and (3) proofreading.
Chain elongation:
1. Under the influence of DNA polymerase III, the 3' hydroxyl group of the end
nucleotide combines with the 5' phosphate group of the new deoxynucleotide. The
pyrophosphate is released from the deoxynucleoside tri phosphate.
2. This newly added nucleotide would now polymerise with another one, forming the
next phosphodiester bond. The base pairing rule is always observed.
Proceesivity:

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DNA polymerase III is a highly processive enzymethat is, it remains bound to the
template strand as it moves along, and does not diffuse away.
Proof reading:
1. The nucleotide sequence of DNA will be replicated without errors. Misreading of the
template sequence could result in mutations.
2. DNA polymerase III has a proofreading activity through 3' 5' exonuclease.
3. DNA polymerase III checks if nucleotide is correctly matched to its complementary
base on the template. If it is not, the 3' 5' exonuclease corrects the mistake. If an
incorrect base has been added, the enzyme makes a cut at the phosphodiester bond
and releases the incorrect nucleotide.
4. The proofreading exonuclease activity requires movement in the 3' 5' direction, not 5'
3' like the polymerase activity. This is because the excision must be done in the
reverse direction from that of synthesis.
GENE EXPRESSION & ITS REGULATION
MUTATION
1. Point mutation. Pon may 2014
2. What is point mutation? Give two examples of abnormal hemoglobin due to point
muataion. Pon May 2006
3. What is point mutation? What are the changes in protein structure that can be
caused by point mutation? Pon Nov 2007
4. What is frame shift mutation? Pon May 2003
MUTATIONS: defined as a change in nucleotide sequence of DNA. This may be either gross,
or subtle with a change in one or a few nucleotides. Mutation may result in an abrupt or
spontaneous origin of new character. Statistically, out of every 106 cell divisions, one
mutation takes place.
Classification of Mutations
1. A point mutation is defined as change in a single nucleotide. All of them may lead to
mis-sense, nonsense or frameshift effects. The point mutation present in DNA is
transcribed and translated, so that the defective gene produces an abnormal protein.
This may be subclassified as
a. substitution,
b. deletion, and
c. insertion.
2. Substitution:
a. Replacement of a purine by another purine ( A to G or G to A) or pyrimidine by
pyrimidine (T to C or C to T) is called Transition mutation.
b. If a purine is changed to a pyrimidine (e.g. A to C) or a pyrimidine to a purine
(e.g. T to G), it is called a transversion.
3. Deletion:
a. Large gene deletions, e.g. alpha thalassemia (entire gene) or hemophilia

362
(partial)
b. Deletion of a codon, e.g. cystic fibrosis (one amino acid, 508th phenyl alanine is
missing in the CFTR protein.
c. Deletion of a single base, which will give rise to frameshift effect.
4. Insertion: Insertions or additions or expansions are subclassified into
a. Single base additions, leading to frameshift effect.
b. Trinucleotide expansions. In Huntington's chorea, CAG trinucleotides are
repeated 30 to 300 times. This leads to a polyglutamine repeat in the protein.
The severity of the disease is increased as the numbers of repeats are more.
c. Duplications. In Duchenne Muscular Dystrophy (DMD) gene is duplicated in the
disease.
Effects of Mutations
1. Silent Mutation: A point mutation may change the codon for one amino acid to a
synonym for the same amino acid. Then the mutation is silent and has no effect on the
phenotype. For example, CUA is mutated to CUC; both code for leucine, and so this
mutation has no effect.
2. Mis-sense but Acceptable Mutation: A change in amino acid may be produced in
the protein; but with no functional consequences. For example, in the normal
hemoglobin A molecule, the 67th amino acid in beta chain (HbA -67) is valine. The
codon in mRNA is GUU. If a point mutation changes it to GCU, the amino acid becomes
alanine; this is called Hb Sydney. This variant is functionally normal. A conserved
mutation occurs when the altered amino acid has the same properties of the original
one; e.g. Glutamic acid to Aspartic acid.
3. Mis-sense; Partially Acceptable Mutation: In this type, the amino acid substitution
affects the functional properties of the protein. HbS or sicklecell hemoglobin is
produced by a mutation of the beta chain in which the 6th position is changed to
valine, instead of the normal glutamate. Here, the normal codon GAG is changed to
GUG (transversion). HbS has abnormal electrophoretic mobility and subnormal
function, leading to sickle-cell anemia.
4. Mis-sense; Unacceptable Mutation: The single amino acid substitution alters the
properties of the protein to such an extent that it becomes nonfunctional and the
condition is incompatible with normal life. For example, HbM results from histidine to
tyrosine substitution (CAU to UAU) of the distal histidine residue of alpha chain. There
is met-hemoglobinemia.
5. Nonsense; Terminator Codon Mutation:
a. A tyrosine (codon, UAC) may be mutated to a termination codon (UAA or UAG).
This leads to premature termination of the protein, and so functional activity
may be destroyed, e.g. betathalassemia.
b. Or, a terminator codon is altered into a coding codon (UAA to CAA). This results
in elongation of the protein to produce "run on polypeptide" (Hb Constant
spring).
6. Frameshift Mutation: This is due to addition or deletion of bases. From that point
onwards, the reading frame shifts. A "garbled" (completely irrelevant) protein, with
altered amino acid sequence is produced. An example:
Normal
mRNA
AUG UCU UGC AAA......
Normal
protein
Met
Ser
Cys
Lys.......
Deleted U
mRNA
AUG CUU GCA AA.........
Garbled
protein
Met
Leu
Ala
.............
In this hypothetical example, deletion of one uracil changes all the triplet codons
thereafter. Therefore, a useless protein or no protein is produced. Frame shift mutation

363
can lead to thalassemia due to premature chain termination and polypeptide that are
non-functional.
7. Conditional Mutations: Most of the spontaneous mutations are conditional; they are
manifested only when circumstances are appropriate.
a. Bacteria acquire resistance, if treated with antibiotics for a long time. This is
explained by spontaneous conditional mutations. In the normal circumstances,
wild bacilli will grow. In the medium containing antibiotic, the resistant bacilli
are selected.
b. In a tuberculous patient, a lung cavity may harbor about 1012 bacilli. This may
contain about 106 mutations, out of which a few could be streptomycin
resistant. Therefore if only one drug is given, there will be overgrowth of drug
resistant bacilli. To avoid this, a combination of two antituberculous drugs is
given. So, drug-1-resistant mutants are killed by drug-2 and drug-2-resistant
mutants are removed by drug-1. The statistical probability of a single bacillus
acquiring resistance against both drugs is negligible.
8. Mutagens and Mutagenesis:
a. Any agent which will increase DNA damage or cell proliferation can cause
increased rate of mutations also. Such substances are called mutagens.
b. X-ray, gamma-ray, UV ray, acridine orange, etc. are well known mutagens.
c. The rate of mutation was proportional to the dose of irradiation.
d. Tatum (Nobel Prize, 1958) showed that a mutation of a single gene resulted
only in a single chemical reaction, which gave evidence to the concept of "one
gene, one enzyme".
Manifestations of Mutations
1. Lethal Mutations: The alteration is incompatible with life of the cell or the organism.
For example, mutation producing alpha-4 Hb is lethal, and so the embryo dies.
2. Silent Mutations: Alteration at an insignificant region of a protein may not have any
functional effect.
3. Beneficial Mutations: Although rare, beneficial spontaneous mutations are the basis of
evolution. Such beneficial mutants are artificially selected in agriculture. Normal maize
is deficient in tryptophan. Tryptophan-rich maize varieties are now available for
cultivation. Microorganisms often have antigenic mutation. These are beneficial to
micro-organisms (but of course, bad to human beings).
4. Carcinogenic Effect: The mutation may not be lethal, but may alter the regulatory
mechanisms. Such a mutation in a somatic cell may result in uncontrolled cell division
leading to cancer. Any substance causing increased rate of mutation can also increase
the probability of cancer. Thus all carcinogens are mutagens.
5. Ame's Test to Detect Mutagenicity
a. Special strains of Salmonella typhimurium (bacteria causing typhoid) are
selected. They have the mutated histidine gene. They will grow only when
histidine is provided in the culture medium. They are sensitive to mutagens
because they are defective in excision repair system for correcting DNA
damage.
b. The compound to be tested is mixed with bacteria and introduced into histidinedeficient medium. All bacteria will die, except those who have reverted back to
wild type and acquire the capacity to synthesize histidine. This is called reverse
mutation. The number of colonies will be proportional to the quantity of
mutagen.
6. Site-directed Mutagenesis
a. Michael Smith described this technique. An oligo-deoxyribonucleotide is
synthesized, whose sequence is complementary to a part of a known gene.
b. A specific deletion/ insertion is produced in the oligonucleotide. It is then

364
extended by DNAP. After replication, one strand is normal and the other strand
contains the mutation at the specific site. This allows study on the effect of that
particular mutation.

1.

2.
3.

4.

5.
6.

7.

8.

CELL CYCLE
Write a note on cell cycle. Pon Nov 2010
Somatic cells are generated by the division of existing cells. They duplicate their contents and
then divide to produce two identical daughter cells . This sequence of duplication is known
as the cell cycle
It refers to the events occurring during the period between two mitotic divisions.
The cell cycle may be broadly divided into three stages:
i. Interphase, mitosis, and cytokinesis.
ii. Interphase can be further subdivided into three phases called G1 phase , S
phase , and G2 phase.
iii. Mitosis (M) can be divided into five distinct phases called prophase ,
prometaphase , metaphase , anaphase , and telophase.
iv. The third stage, cytoplasmic division or cytokinesis, culminates with the
separation into two distinct daughter cells
RNA and protein synthesis also take place during G1 phase. In addition, organelles and
intracellular structures are duplicated and the cell grows during this phase. cells in G1
phase that are not committed to DNA synthesis are in a specialized resting state called G0
phase. In a normal cell population, most of the cells are in Go phase. General metabolic
events are taking place in Go phase.
Synthesis of nuclear DNA, also known as DNA replication , occurs during S phase
G2, is a time of preparation for the nuclear division of mitosis and phasei s characterizedb y
enlargement of cytoplasm and this is followed by the actual cell division that occurs in the
mitotic phase.
Cell Cycle regulators:
i. Cyclins: The cyclins, categorized as cyclins D, E, A, or B, are a family of cell
cycle regulatory proteins. Different cyclins are expressed to regulate specifi c
phases of the cell cycle.
ii. Cyclin-dependent kinases: cyclin-CDK complex catalyzes the phosphorylation
of substrate proteins on serine and threonine amino acid residues. Such
alteration of regulatory proteins allows for initiation of the next phase of the
cell cycle.
Checkpoint regulation:
i. Checkpoints placed at critical points in the cell cycle monitor the completion of
critical events and, if necessary, delay the progression to the next stage of the
cell cycle. They are key regulators that ensure that G1 is completed prior to
the start of S phase.
ii. Tumor suppressor proteins normally function to halt the cell cycle progression
within G1 when the cell should not continue past the restriction point
iii. p53: This tumor suppressor protein plays a major regulatory role in G1
iv. Retinoblastoma (RB) protein: This tumor suppressor functions to halt a cell in
the resting or G1 phase of the cell cycle.
Human papillomavirus binds with RB and p53 and inactivate them leading to unregulated cell
cycle progression and malignancy.
LACTOSE (LAC) OPERON
1. Analysis of Lactose Metabolism in E Coli Led to the Operon Hypothesis. Lac operon
explains regulation of gene expression.
2. The concept of operon was introduced by Jacob and Monod in 1961 based on their

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observations on the regulation of lactose metabolism in E. coli. This is popularly known
as lac operon.
3. The genes that encode proteins are called structural genes. In bacteria, the structural
genes that code for proteins involved in a particular metabolic pathway are grouped on
the chromosome along with the regulatory elements that determine the transcription
of these genes. These units are called operons. The genes in an operon are expressed
coordinately; that is, they are either all turned on or all turned off.
4. The lac operon consists of a regulatory gene (l; I for inhibition), operator gene (O) and
three structural genes (Z, Y, A). Besides these genes, there is a promoter site (P), next
to the operator gene, where the enzyme RNA polymerase binds. The structural genes
Z, Y and A respectively, code for the enzymes b-galactosidase, galactoside permease
and galactoside acetylase. b Galactosidase hydrolyses lactose to galactose and
glucose while permease is responsible for the transport of lactose into the cell.
5. A single polycistronic mRNA is produced that codes for all the proteins of the operon.
An mRNA coding for more than one protein is known as polycistronic mRNA. The
transcription of these genes start from a common promoter (P), located close to the Z
gene. The RNA polymerase binds to the promoter and transcribes these 3 structural
genes as a single mRNA.

6. The regulatory I gene in lac operon produces a repressor molecule. When there is
absence of lactose in E. coli, this molecule prevents the binding of the enzyme RNA
polymerase to the promoter site (P), thereby blocking the transcription of structural
genes (2, Y and A) . This is called repression of lac operon.
7. When lactose is present, it binds with repressor molecule and allows the genes to
prduce lactase enzyme. This is called depression of lac operon where lactose acts by
inactivating the repressor molecules.
8. When glucose is the only sugar available , the lac operon is repressed (turned off).
When only lactose is available, the lac operon is induced (maximally expressed or
turned on).
9. When E coli is exposed to both lactose and glucose as sources of carbon, the
organisms first metabolize the glucose and then temporarily stop growing until the
genes of the lac operon become induced to provide the ability to metabolize lactose as
a usable energy source.
10. This is explained by the formation of CAP-cAMP. The attachment of RNA polymerase to
the promoter site requires the presence of a cataholite gene activator protein (CAP)
bound to cyclic AMP. The presence of glucose lowers the intracellular concentration of
cAMP. Due to the diminished levels of cAMP, the formation of CAP-cAMP is low and the
hence the transcription are almost negligible in the presence of glucose. Thus, glucose
interferes with the expression of Iac operon by depleting cAMP levels.
11. CAP-cAMP acts as a positive regulator for the gene expression. lt is, therefore, evident
that lac operon is subjected to both positive (by repressor) and negative regulation.
12. Clinical Applications:
1. Lactase in human intestine is an inducible enzyme.
2. In humans, examples of derepression include induction of tryptophan pyrrolase,
and transaminases by glucocorticoids; as well as ALA synthase by barbiturates.

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RECOMBINANT DNA TECHNOLOG AND GENE THERAPY
Questions:
1.
Recombinant DNA-April 2000
2.
Describe in detail the steps involved in recombinant DNA technology.
3.
Describe the apllications of recombinant DNA technology. Pon Nov 2011 Pon
Nov 2010
4.
What is restriction endonucleases? Pon May 2003
5.
Use of plasmids in genetic engineering-April 2001
6.
What is the function of P53 gene? Pon May 2006
7.
What is the principle of gene therapy. Pon May 2006
8.
Chimeric DNA
9.
What is cloning? Mention the various types of cloning.
10.
Cyclic AMP
RECOMBINANT DNA TECHNOLOGY
1.
Recombinant DNA-April 2000
2.
Describe in detail the steps involved in recombinant DNA technology.
3.
Describe the apllications of recombinant DNA technology. Pon Nov 2011 Pon
Nov 2010
4.
What is restriction endonucleases? Pon May 2003
5.
Use of plasmids in genetic engineering-April 2001
6.
Chimeric DNA
7.
What is cloning? Mention the various types of cloning.
RECOMBINANT DNA TECHNOLOGY
1. When a gene of one species is transferred to another living organism, by artificial means,
it is called recombinant DNA technology. In common parlance, this is known as genetic
engineering.
2. Applications of Recombinant Technology:
a. By means of recombinant technology, hormones like growth hormone can be
produced in large scale.
b. Vaccines like Hepatitis B can be prepared in pure forms
c. Specific Probes for Diagnosis of Diseases: Specific probes are useful for:
i. Antenatal diagnosis of genetic diseases. e.g. cystic fibrosis, phenyl
ketonuria, etc.
ii. To identify viral particles or bacterial DNA in suspected blood and tissue
samples.
iii. To demonstrate virus integration in transformed cells.
iv. To detect activation of oncogenes in cancer.
v. To pinpoint the location of a gene in a chromosome.
vi. To identify mutations in genes: Sickle cell disease is an example of point
mutation
d. Gene Therapy: Normal genes could be introduced into the patient so that genetic
diseases can be cured.
e. Special techniques have led to remarkable advances in forensic medicine.
3. Principles of recombinant DNA technology:
i. Generation of DNA fragments and selection of the desired piece of DNA (e.g. a
human gene).
ii. Insertion of the selected DNA into a cloning vector (e.g. a plasmid) to create a
recombinant DNA or chimeric DNA (Chimera is a monster in Greek mythology that
has a lion's head, a goat's body and a serpent's tail. This may be comparable to
Narasimhain lndian mythology .
iii. Introduction of the recombinant vectors into host cells (e.g. bacteria).
iv. Multiplication and selection of clones containing the recombinant molecules.
v. Expression of the gene to produce the desired product.

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4. Aspects of Recombinant DNA technology:
i. Molecular tools of genetic engineering (Restriction endonucleases).
ii. Host cells-the factories of cloning.
iii. Vectors-the cloning vehicles.
iv. Methods of gene transfer.
v. Gene cloning strategies.
RESTRICTION ENDONUCLEASES: (Short notes)
1. These are enzymes that can cut DNA from any source at specific sites. They were first
discovered in E.coli restricting the replication of bacteriophages by cutting the viral
DNA. Thus, the enzymes that restrict the viral replication are known as restriction
enzymes or restriction endonucleases.
2. A restriction enzyme is named according to the organism from which it was isolated.
The first letter of the name is from the genus of the bacterium. The next two letters are
from the name of the species. An additional letter indicates the type or strain, and a
final number is appended to indicate the order in which the enzyme was discovered in
that particular organism.
i. Eco RI is isolated from Escherichia coli RY13 strain.
ii. HaeIII is the third restriction endonuclease isolated from the bacterium
Haemophilus aegyptius.
3. Restriction enzymes cleave dsDNA so as to produce a 3'-hydroxyl group on one end
and a 5'-phosphate group on the other. They can produce DNA fragments which have
single-stranded (ss) sequences with sticky ends or fragments that have blunt ends
that are double stranded.
4. A DNA sequence that is recognized and cut by a restriction enzyme is called a
restriction site and dsDNA fragments are of different sizes.
5. DNA ligases: DNA ligase covalently join sticky ends of a DNA produced by restriction
enzymes by forming a phosphodiester bond between the phosphate group of 5'-carbon
of one deoxyribose with the hydroxyl group of 3'-carbon of another deoxyribose.
Another ligase, encoded by bacteriophage T4, can covalently join blunt-ended
fragments.
HOST CELLS:
1. Escherichia coli was the first organism used in the DNA technology
2. B.subtilis is an alternative to E.coli
3. most commonly used eukaryotic organism is the yeast- Saccharomyces cerevisiae
4. mammalian cells (such as mouse cells) are also employed as hosts
VECTORS: Vectors are the DNA molecules, which can carry a foreign DNA fragment to be
cloned
1. Plasmids: Plasmids are extrachromosomal, doublestranded, circular, self-replicating
DNA molecules. Almost all the bacteria have plasmids. pBR322 of E.coli is the most
popular and widely used plasmid vector.
2. Bacteriophages are the viruses that replicate within the bacteria. Phage vectors can
accept short fragments of foreign DNA into their genomes.
3. Cosmids are the vectors that possess the characteristics of plasmid and
bacteriophage.
4. Human artificial chromosome: synthetically produced vector DNA, possessing the
characteristics of human chromosome.
5. Yeast artificial chromosomes: synthetic DNA that can accept large fragments of
foreign DNA.
6. Bacterial artificial chromosomes: is based on one F-plasmid which is larger than the
other plasmids used as cloning vectors
PROCEDURE OF DNA RECOMBINATION:
1. Preparation of Specific Human Gene: cDNA (complementary copy DNA) is prepared

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from mRNA using reverse transcriptase:
2. Preparation of Chimeric DNA Molecules: (Short notes)
a. Chimera is the Greek mythological monster with a lion's head, goat's body and
serpent's tail. A vector carrying a foreign DNA is called Chimeric DNA or
Hybrid DNA or Recombinant DNA.
b. Plasmid vector DNA is cut with a specific restriction endonuclease (RE). If EcoRI
is used, sticky ends are produced with TTAA sequence on one DNA strand, and
AATT sequence on the other strand.
c. The human DNA is also treated with the same RE, so that the same sequences
are generated on the sticky ends of the cut piece.
d. Then the vector DNA and human cut-piece DNA are incubated together so that
annealing takes place. The sticky ends of both vector and human DNA have
complementary sequences, and therefore they come into contact with each
other.
e. Then DNA ligase enzyme is added, which introduces phospho-diester linkages
between the vector and the insert molecules. Thus the chimeric DNA is finally
produced.
3. Cloning of Chimeric DNA
a. The next step is cloning. A clone is a large population of identical bacteria or
cells that arise from a common ancestor molecule.
b. Cloning allows the production of a large number of identical DNA molecules.
The hybrid molecules are amplified by the cloning technique.
4. Transfection of Vector into the Host
a. The process by which plasmid is introduced into the host is called transfection.
Host cells that contain recombinant DNA are called transformed cells if they
are bacteria, or transfected cells if they are eukaryotes.
b. Host E.coli cells and plasmid vectors are incubated in hypertonic medium
containing calcium for a few minutes. The plasmid is imbibed into the host cell.
Only 5% of bacterial colonies contain the desired vector. Therefore, we have to
select the desired colonies.
5. Plasmid Carries Antibiotic Resistance Genes: eg. Cmr (chloramphenicol resistance):
a. When the foreign DNA is inserted, the resistance against chloramphenicol is
lost.

369

b. This insertional inactivation of Cmr gene is the marker for hybrid DNA.
6. Expression Vectors
a. To produce the human proteins, E. coli carrying the vector with the insert is
allowed to grow, without any protein inhibitors.
b. Such a vector carrying the foreign gene, which is translated into a protein, is
called expression vector. The human proteins can be harvested from the
bacterial culture.
GENE CLONING
What is cloning? Mention the various types of cloning.
What is cloning? Mention the various types of cloning.Describe in detail the steps
involved in recombinant DNA technology.
1. Propagation of the host organism containing the recombinant DNA forms a set of
genetically identical organisms, or a clone. This process is hence known as DNA
cloning.
2. There are three different types of cloning:
a. Gene cloning, which creates copies of genes or segments of DNA

370
b. Reproductive cloning, which creates copies of whole animals
c. Therapeutic cloning, which creates embryonic stem cells.
3. There are two types of gene cloning:
a. In vivo, which involves the use of restriction enzymes and ligases using vectors
and cloning the fragments into host cells (as can be seen in the image above).
b. In vitro type which is using the polymerase chain reaction (PCR) method to
create copies of fragments of DNA.
4. Molecular cloning allows for the production of a large number of identical DNA
molecules, which can then be characterized or used for other purposes. This technique
is based on the fact that chimeric or hybrid DNA molecules can be constructed in
cloning vectorstypically bacterial plasmids, phages, or cosmidswhich then continue
to replicate in a host cell under their own control systems. In this way, the chimeric
DNA is amplified.
5. Steps of cloning:
a. Generation of desired DNA fragments.
b. Insertion of these fragments into a cloning vector.
c. Introduction of the vectors into host cells.
d. Selection or screening of the recipient ells for the recombinant DNA molecules
6. Cloning can start by selection of DNA fragment from genomic DNA or using mRNA. The
farmer is ideal but the later is easier.
7. Plasmid vector DNA and the selected human DNA are incubated together so that
annealing takes place forming Chimeric DNA within the plasmid vector.
8. Next step is transfection of Vector into the Host Cell eg. E.Coli
9. The host cells probagate in appropriate culture media and bacterial colonies containing
the desired vector are identified by the technique of insertional inactivation of
Antibiotic Resistance Genes in bacterial colonies.
10. A collection of these different recombinant clones is called a library. A variety of
molecules can be used to "probe" libraries in search of a specific gene or cDNA.
11. Blotting techniques are used for the specific identification of desired DNA or RNA
fragmenfs from thousands of molecules in library. Blotting refers to the process of
immobilization of sample nucleic acids on solid support (nitrocellulose or nylon
membranes) The most comonly used blotting techniques are:
a. Southern blotting (for DNA)
b. Northern blotting (for RNA)
c. Dot blotting (DNA/RNA)
12. Determination of nucleotide sequence in a DNA molecule is important to understand
the functions of genes, and basis of inherited disorders. Further, DNA cloning and gene
nianipulation invariably require knowledge of accurate nucleotide sequence. Manual &
Automated Techniques Are Available to Determine the Sequence of DNA
13. Applications of DNA cloning:
a. Cloning has led directly to the elucidation of the complete DNA sequence of the
genomes of a very large number of species
b. Many useful proteins are currently available as recombinant products. These
include
i. Recombinant factor VIII
ii. Tissue plasminogen activator, used to treat strokes
iii. Recombinant subunit vaccines, (e.g. Hepatitis B vaccine)
iv. Recombinant proteins as standard material for diagnostic laboratory
tests.
c. Cloned genes may be inserted into organisms, generating transgenic organisms
d. Cloning techniques are part of gene therapy
Use of plasmids in genetic engineering-April 2001
1. Most species of bacteria normally contain small, extrachromosomal DNA molecules

371
called plasmids.
2. Plasmid DNA undergoes replication that may or may not be synchronized to
chromosomal division. Plasmids may carry genes that convey antibiotic resistance to
the host bacterium, and may facilitate the transfer of genetic information from one
bacterium to another.
3. Plasmids can be readily isolated from bacterial cells, their circular DNA cleaved at
specific sites by restriction endonucleases.
4. The recombinant plasmid can be introduced into a bacterium, and large numbers of
copies of the plasmid produced. The bacteria are grown in the presence of antibiotics,
thus selecting for cells containing the hybrid plasmids, which provide antibiotic
resistance.
5. Plasmids have several properties that make them extremely useful as cloning vectors.
a. They exist as single or multiple copies within the bacterium and replicate
independently from the bacterial DNA.
b. The complete DNA sequence of many plasmids is known; hence, the precise
location of restriction enzyme cleavage sites for inserting the foreign DNA is
available.
c. Plasmids are smaller than the host chromosome and are therefore easily
separated from the latter
d. The desired plasmid-inserted DNA is readily removed by cutting the plasmid
with the restriction enzyme.
e. They bear a special region of DNA called an origin of replication. It confers
on the plasmid the property that It will replicate its own DNA as well as any
passenger DNA.
f. Plasmid DNAs replicate independently and they can be easily separated from
host bacteria.

6. Types of plasmids:
a. F plasmids (Sex plasmids): This plasmid
transfers a
replica of the plasmid from a donor (F+) cell to a recipient (F) cell without the F+ cell
losing its plasmid.
b. R plasmids, drug resistance plasmids: These plasmids carry genes conferring
resistance to one or more antibiotics and usually can transfer this resistance to an Rfree recipient cell.
c. Col plasmids or colicinogenic factor plasmids: They carry genes for the synthesis of a
protein known as colicins.
d. Ent plasmids: Plasmids called Ent are responsible for travellers diarrhoea and some
types of dysentery.
e. Plasmid vector PBR 322 has both tetracycline (tet) and ampicillin (amp) resistance
genes.
What is the principle of gene therapy. Pon May 2006
1. The ultimate cure for genetic diseases is to introduce normal genes into individuals
who have defective genes.
2. Gene therapy consists of intracellular delivery of genes to generate a therapeutic
effect by correcting an existing abnormality. Only somatic gene therapy, by inserting
the new gene into somatic cell of the patient is under trial. Germ cell gene therapy is

372
considered as unethical.
Principles of the Procedure
i. Isolate the healthy gene along with the sequence controlling its expression.
ii. Incorporate this gene into a carrier or vector as an expression cassette.
iii. Finally deliver the vector to the target cells.
4. Methods of Introduction of the Genes
a. Ex vivo strategy;
i. lsolate cells with eenetic defect from a patient.
ii. Crow the cells in culture.
iii. Introduce the therapeutic gene to correct gene defect.
iv. Select the genetically corrected cells (stablet ransformantsa) nd grow.
v. Transplant the modified cells to the patient.
vi. This technique is not associated with adverse immunological responses
after transplanting the cells
b. In situ strategy when the expression cassette is injected to the patient either
intravenously or directly to the tissue
c. In vivo strategy, where the vector is administered directly to the cell, e.g. CF (cystic
fibrosis) gene to the respiratory tract cells.
The Vectors:
1. Different vector (carrier) systems used for gene delivery are:
a. Viruses: Retroviruses, adenoviruses, adeno associated viral vectors and herpes
simplex viruses.
b. Non-virus systems include liposomes,
plasmids and physical methods.
2. Viruses:
a. The vectors frequently used in gene
therapy are viruses, particularly
retroviruses. RNA is the genetic
material in retroviruses.
b. The gag, pol, env genes are deleted
from the wild type retrovirus, rendering
it incapable of replication inside human
body. Then the human gene is inserted into the virus.
a. As the retrovirus enters the host cell, it synthesizes DNA from RNA (by reverse
transcription).
b. The normal human gene can now express .
3. Plasmid Liposome Complex: Liposomes are artificial lipid bilayers, which could be
incorporated with plasmids carryinthe normal human DNA. The complexes can enter
into the target cells by fusing with the plasma membrane. Liposomes can form
complexes spontaneously with DNA.
4. Gene Gun Method Tungsten particles are coated with plasmid DNA, and accelerated by
helium pressure discharge. This enables particles to penetrate the target tissues.
Success stories of gene therapy:
3.

Disease
Severe combined
immunodeficiency
Duchenne muscular dystrophy
Cystic fibrosis
Hemophilia A and B

Gene transferred by
Adenosine deaminase enzyme in chromosome
13 and 20 into (SCID) lymphocytes; by
retrovirus
Dystrophin gene on short arm of X
chromosome; by retrovirus
CFTR gene on chromosome 7 to bronchial
epithelium; adenovirus
genes for factor VIII and

373

Cancer

IX into fibroblasts; retrovirus

Activation of p53 (tumor suppressor gene) by
liposome

STEM CELLS THERAPY:

1. Stem cells are totipotential cells from embryonic tissue.
2. Stem cells have the ability to divide for an indefinite period. They can give rise to a
variety of specialized cell types.
3. Stem cells can be isolated from embryos, umbilical cord as well as any other adult
tissue.
4. Stem cells have the unique capacity to produce unaltered daughter cells (renewal) and
also to generate specialized cells (potency).
5. Stem cells may be capable of producing all types of cells of the organism (totipotent),
or able to generate cells of the three germ layers (pleuripotent), or able to produce
only closely related cell types (multipotent), or may produce only one cell type
(unipotent).
6. Stem cells may be embryonic (capable to differentiate) or adult type (limited capacity
to differentiate).
7. The pluripotency and self-renewal are critical for sustaining the lifelong functions of
organs. Active research is being done to utilize stem cells in the treatment of the
following diseases: Stroke, brain injury, Alzheimer's disease, Parkinsonism, wound
healing, myocardial infarction, muscular dystrophy, spinal cord injury, diabetes,
cancers. It has been suggested that tumor tissue contains a type of stem cell referred
to as a cancer stem cell.

1.
2.
3.
4.

POLYMERASE CHAIN REACTION (PCR)

Polymerase chain reaction. April 2001; April 2001; April 2001
Application of Polymerase chain reaction. Pon may 2014
What is Polymerase chain reaction? What are its apllications? Pon May 2004
What do you undestand by the term PCR? Explain its importance. Pon apr
2000
Outline the process of PCR. Whar are its applications? Pon May 2010
We need two primers for polymerase chain reaction. Justify. MGR 2011

5.
6.
PCR:
1. PCR is an in vitro DNA amplification procedure in which millions of copies of a
particular sequence of DNA can be produced within a few hours. Starting material
could be a single molecule of rRNA, mRNA of DNA.
2. PCR uses DNA polymerase to repetitively amplify targeted portions of DNA. Each cycle
of amplification doubles the amount of DNA in the sample, leading to an exponential
increase in DNA with repeated cycles of amplification.
3. The amplified DNA sequence can then be analyzed by gel electrophoresis, Southern
blotting, or direct sequence determination.
4. It can amplify the sequence, even when the targeted sequence makes up less than
one part in a million of the total initial sample. The method can be used to amplify DNA
sequences from any sourcebacterial, viral, plant, or animal.
The reaction cycle has the following steps:

374

1. Primer construction: (We need two primers for polymerase chain reaction.
Justify. MGR 2011)
a. It is necessary to know the nucleotide sequence of short segments on each side
of the target DNA. They are called flanking sequences and are used to construct
two, single-stranded oligonucleotides.
b. These two synthetic oligo nucleotides function as primers in PCR reactions since
there are two DNA strands that become templates for DNA polymerase to
selectively amplify the target DNA.
c. The selectivity of PCR results from the use of primers that
are complementary to the DNA. Through complementary base pairing, one
primer attaches to the top strand at one end of the DNA segment of interest,
and the other primer attaches to the bottom strand at the other end.
d. This would explain that we need two primers for PCR

2. Denaturation: DNA strands are separated by heating at 95C for 15 seconds to 2

minutes.
3. Annealing of primers to ssDNA: The primers are annealed by cooling to 50C for
0.5 to 2 minutes. The primers hybridise with their complementary single stranded DNA

375
produced in the first step.
4. Polymerization:
a. DNA polymerase and deoxyribonucleoside triphosphates (in excess) are added
to the mixture to initiate the synthesis of two new strands complementary to
the original DNA strands.
b. DNA polymerase adds nucleotides to the 3'-hydroxyl end of the primer, and
strand growth extends across the target DNA, making complementary copies of
the target.
c. At the completion of one cycle of replication, the reaction mixture is heated
again to separate the strands. Each strand binds a complementary primer, and
the cycle of chain extension is repeated.
d. DNA polymerase used for this reaction is derived from bacteria Thermus
acquaticus. This is a heat-stable DNA polymerase (Thermus aquaticus that
normally lives at high temperatures), the polymerase is not denatured and,
therefore, does not have to be added at each successive cycle.
5. Each newly synthesized strand can act as a template for the successive cycles. This
leads to an exponential increase in the amount of target DNA with each cycle hence
the name polymerase chain reaction.
6. Probes can be made during PCR by adding labeled nucleotides to the last few cycles.
After the amplification procedure, DNA hybridization technique or Southern blot
analysis with a suitable probe, shows the presence of the DNA in the sample tissue.
Clinical Applications of PCR
1. Diagnosis of bacterial and viral diseases:
a. PCR could detect even one TB bacillus present in the specimen. Any other
bacterial infection could also be detected similarly. The specific nucleotide
sequences of the bacilli are amplified by PCR and then detected by Southern
blot analysis.
b. If reverse PCR is done, living organisms could be detected. This technique is
widely used in the diagnosis of viral infections like Hepatitis C, Cytomegalo
virus and HIV.
c. PCR offers a rapid and sensitive method for detecting viral DNA sequences
even when only a small proportion of cells is harboring HIV virus.
2. Medicolegal cases:
a. PCR allows the DNA from a hair follicle or a blood cell to be analyzed. The
restriction analysis of DNA from the hair follicle from the crime scene is studied
after PCR amplification.
b. This pattern is then compared with that of various suspects; (DNA
fingerprinting). This helps to identify the criminal.
3. Diagnosis of genetic disorders:
a. The PCR technology has been widely used to amplify the gene segments that
contain known mutations for diagnosis of inherited diseases such as sickle cell
anemia, beta thalassemia, cystic fibrosis, etc.
b. PCR is especially useful for prenatal diagnosis of inherited diseases, where cells
obtained fromfetus by amniocentesis are very few.
4. Cancer detection:
a. PCR is widely used to monitor residual abnormal cells present in treated
patients.
b. Similarly identification of mutations in oncosuppressor genes such as p53,
retinoblastoma gene, etc. can help to identify individuals at high risk of cancer.
5. Fossil studies: DNA can be isolated and PCR amplified from fossils and is used to study
evolution by comparing the sequences in the extinct and living organisms.
PCR types:

376
1. Reverse Transcriptase PCR (RTPCR):
a. m-RNA, r-RNA can be the starting material in such types of PCR. This is widely
used in expression mapping, determining when and where certain genes are
expressed.
b. In ordinary PCR, DNA is detected; that DNA could be from a living or nonliving
organism. But in reverse PCR, mRNA is detected; that means, it is derived from
a living organism. Presence of HIV RNA in blood can be detected as early as 4
weeks after infection.
2. Nested PCR:
a. Nested PCR is intended to reduce the contamination in products due to the
amplification of unexpected primer binding sites.
b. Two sets of primers are used in two successive PCR runs, the second set
intended to amplify a secondary target within the first run product. This is very
successful, but requires more detailed knowledge of the sequences involved.
3. Real Time PCR
a. By this method, quantitation of the number of virus present in a sample can be
calculated., e.g.,viral load in HIV or HBV.
b. So, the treatment modalities can be planned and the response to treatment
could be assessed.
4. Quantitative PCR
a. Q-PCR (Quantitative PCR) is used to rapidly measure the quantity of PCR
product.
b. This is commonly used for the purpose of determining whether a sequence is
present or not, and if it is present the number of copies in the sample.
5. Quantitative Real time PCR (RQ-PCR)
This method is used to measure the amount of amplified product in real time.
6. RACE-PCR
a. RACE, or Rapid Amplification of cDNA Ends, is a technique used in molecular
biology to amplify the ends of messenger RNA (mRNA) transcripts.
7. Multiplex-PCR
By targeting multiple genes at once, additional information may be elicited from
a single test run that otherwise would require several times the reagents and
technician time to perform.
NUCLEIC ACID BLOTTING TECHNIOUES
Southern blotting- (MGR U)
Blotting techniques. (MGR U)
Explain the Western Blot Technique- Pon Nov 2010
What is the principle of Southern blotting ? Give two apllication. Pon Nov 2011
1. Blotting refers to the process of immobilization of sample nucleic acids on solid support
like nitrocellulose or nylon membranes. The blotted nucleic acids are then used as
targets in the hybridization experiments for their specific detection.
2. M. Southern developed the technique, which bears his name, for identifying DNA
sequences on gels. Molecular biologists decided to continue with this geographic
theme southern) to name two other additional techniques.
3. Southern blots are produced when DNA on a nitrocellulose blot of an electrophoretic
gel is hybridized with a DNA probe.
4. Northern blots are produced when RNA on a nitrocellulose blot is hybridized with a DNA
probe.
5. Western blot involves separating proteins by gel electrophoresis and probing with
labeled antibodies for specific proteins
Southern Blot Technique:
1. Southern blotting is a technique that can detect mutations in DNA. It combines the

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use of restriction enzymes, electrophoresis, and DNA probes.
2. It is based on the specific base pairing properties of complementary nucleic acid
strands. This is used to detect a specific segment of DNA in the whole genome.
3. The DNA is isolated from tissues. It is then fragmented by restriction enzymes. This
mixture is loaded into a well in an agarose or polyacrylamide gel and then subiected
to electrophoresis. DNA, being negatively charged migrates towards the anode. The
smaller DNA fragments move faster.
4. The separated DNA molecules are denatured by exposure to a mild alkali. The pieces
become single-stranded. This is then blotted (adsorbed) over to a nitrocellulose
membrane.
5. This results in an exact replica of the pattern of DNA fragments on the gel. The DNA
can be annealed to the paper on exposure to heat (80'C). The nitrocellulose or nylon
paper is then exposed to labeled cDNA probes. These probes hybridize with
complementary DNA molecules on the paper. The paper after thorough washing is
exposed to X-ray film to develop autoradiograph. This reveals specific bands
corresponding to the DNA fragments recognized by cDNA probe.

Applications of Southern blotting

1. lt is an invaluable method in gene analysis. Southern blotting can detect DNA
mutations such as the insertion or deletion of nucleotides. It can also detect point
mutations (replacement of one nucleotide by another,

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2. lmportant for the confirmation of DNA cloning results.
3. Forensically applied to detect minute quantities of DNA to identify parenthood and
thieves and rapists etc.
4. Highly useful for the determination of restriction fragment length polymorphism (RFLP)
associated with pathological conditions.
NORTHERN BLOTTING
1. Northern blotting is the technique for the specific identification of RNA molecules. The
procedure is similar to Southern blotting.
2. RNA molecules are subjected to electrophoresis, followed by blot transfer,
hybridization and autoradiography. RNA molecules do not easily bind to nitrocellulose
paper or nylon membranes.
3. Blot transfer of RNA molecules is carried out by using a chemically reactive
diazobenzyl oxymethyl (DBM) paper. The RNA can covalently bind to DBM paper.
4. Northern blotting is a technique for determining the number of genes (through mRNA)
present on a given DNA. But this is not really p racticable since each gene may give
rise to two or more RNA transcripts. Another drawback is the presence of exons and
introns.
DOT BLOfTING
1. Dot-blotting is a modification of Southern and Northern blotting
2. ln this approach, the nucleic acids (DNA or RNA) are directly spotted onto the filters,
and not subjected to electrophoresis. The hybridization procedure is the same as in
original blotting
techniques.
3. Dot-blotting technique is useful in obtaining quantitative data for the evaluation of
gene expression.
WESTERN BLOTTING
1. In this technique, proteins (not nucleic acids) are identified. The proteins are isolated
from the tissue and electrophoresis is done. The separated proteins are then
transferred on to a nitrocellulose membrane. After fixation, it is probed with radioactive
antibody and autoradiographed.
2. This technique is very useful to identify the specific protein in a tissue, thereby
showing the expression of a particular gene.
APOPTOSIS (S.N)
1. In the body, superfluous or unwanted cells are destroyed by a pathway called
apoptosis , or programmed cell death.
2. Apoptosis is a co-ordinated and internally programmed process that mediates the
death of cells in a variety of biologically significant situations.
3. Structural and Morphological Changes in Apoptosis:
a. There is shrinkage of cell volume, accompanied by dilatation of endoplasmic
reticulum and convolution of the plasma membrane.
b. The cells break up into membrane bound spherical bodies containing
structurally normal but compacted organelles.
c. Nucleus undergoes profound chromatin condensation.
d. Nucleolus falls apart, nuclear pores appear to aggregate in those areas of
membrane which are free from condensed chromatin.
4. Apoptosis differ from necrosis, the injured cells swell and their plasma membranes
rupture to release proinflammatory materials.
5. Fate of Apoptotic Cells:
a. Some of the apoptotic cells are phagocytosed.
b. Some lose contact with their neighbours and basement membrane and hence
float in adjacent spaces.

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c. There is no acute inflammation associated with apoptosis.
6. Biochemical and Molecular Changes in Apoptosis
a. Proteins and the nuclear lamins undergo proteolysis.
b. Activation of transglutaminase results in protein-protein cross-linking.
c. Nuclease activation cleaves oligonucleosomal fragments of chromatin.
d. On the cell membrane, phosphatidyl serine appears on the outer as well as the
inner surface, and previously hidden residues including charged amino sugars
become exposed on the cell surface.
e. Mitochondrial membrane potential alters sometime before the structural changes
begin.