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IMMUNOGENETICS AND
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MOLECULAR BIOLOGY
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Answers
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Explanations
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PGI Supplement November 2017

1. Which of the following DNA polymerase used in PCR has DNA polymerase in PCR www.bioline.com
proofreading activity? •• VELOCITY DNA- have proofreading 3’-5’ exonuclease activity
a. Pfu polymerase •• RANGER DNA- have proofreading 3’-5’ exonuclease activity
b. Taq polymerase •• VELOCITY DNA- proofreading 3’-5’ exonuclease activity
c. Bestaq DNA Polymerase
2. Which of the following motif is/are involved in specific
d. Pwo polymerase
protein-DNA interactions?
e. Tth polymerase
a. Helix-turn-helix
b. Zinc finger
“Taq polymerase remains to be the most ubiquitously used
c. Helix-loop-helix
polymerase for PCR. This enzyme has a high rate of dNTP
d. Leucine zipper
incorporation but does not have any proof-reading activity. This
means that Taq is prone to introducing base-pair errors during PCR
Several Motifs Compose the DNA Binding Domains of
amplification. If the PCR product is to be used for functional protein
Regulatory Transcription Harper 30th/444-45; Satyanarayan 4th/573-74
analysis, then this can be problematic”-www.sciencedirect.com
•• The specificity involved in the control of transcription requires
Alternative Polymerases and PCR Modifications that regulatory proteins bind with high affinity and specificity
Since the introduction of Taq polymerase, a variety of other

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•• to the correct region of DNA.
thermostable polymerases from other thermophilic organisms •• Four unique motifs—the helix-turn-helix, the zinc finger,
have also been used in PCR. Helix-loop-helix motif and the leucine zipper— account for

•• Pfu polymerase from Pyrococcus furiosus is used when
increased accuracy is desired because it possesses proofreading
ability—unlike Taq polymerase.
Tli polymerase isolated from Thermococcus litoralis (Vent™
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many of these specific protein-DNA interactions.
•• The first motif described was the helix-turn-helix.
Table 1. (Harper 30th/444): Examples of Transcription Factors That

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DNA polymerase) also has 3′ to 5′ proofreading exonuclease activity Contain Various DNA Binding Motifs
to create accurate copies- www.sciencedirect.com
Binding Motif Organism Regulatory Protein
“Both Bestaq DNA Polymerase and Kodaq DNA Polymerase tN
have excellent proofreading ability and processivity, with a processing Helix-turn-helix E coli lac repressor, CAP
speed of 3-4kb and 1kb per minute, respectively”-www.abmgood.com Phage lcl, cro, and 434 repressors
“Tth polymerase is derived from Thermus thermophilus, a Mammals Homeobox proteins Pit-1, Oct1,
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Oct2
thermophilic thermal vent bacterium (25). Tth DNA polymerase
functions optimally at 75°C with high processivity, but lacks Zinc finger E coli Gene 32 protein
proofreading 3’-5’ exonuclease activity”-www.abmgood.com Yeast Gal4
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“Pwo DNA polymerase is derived from the ultra-thermophilic Drosophila Serendipity, hunchback
Xenopus TFIIIA
archaeon Pyrococcus woesei found in deep marine environments
Mammals Steroid receptor family, Sp1
(24). Pwo polymerase functions optimally at 100-103°C, and displays
Leucine zipper Yeast GCN4
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high proofreading 3’-5’ exonuclease activity, giving it 18-fold higher

fidelity than Taq polymerase”-www.abmgood.com Mammals C/EBP, fos, Jun, Fra-1, CRE binding
protein (CREB), c-myc, n-myc, l-myc
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KOD DNA polymerase is a recombinant form of DNA

polymerase derived from the thermophilic solfatara bacterium
Thermococcus kodakaraensis KOD1 type strain. KOD DNA 3. Which of the following protein (s) is/are required for DNA
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polymerase functions optimally at 85°C and displays 3’-5’ exonuclease strand separation?
proofreading activity, producing blunt-ended DNA products- www. a. DNA Helicase
abmgood.com b. Single-stranded DNA-binding (SSB) proteins
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“Bacillus stearothermophilus is a thermophilic species c. Ligase

of bacteria common to soil, hot spring, and ocean sediment
environments. Bst DNA polymerase isolated from this species
displays helicase-like activity to unwind DNA strands in addition to
its polymerase activity. While Bst polymerase has 5’-3’ exonuclease
activity, it cannot proofread as it lacks 3’-5’ exonuclease activity”-
www.abmgood.com
“Bsu DNA polymerase is isolated from the mesophilic soil
bacterium Bacillus subtilis. Bsu DNA polymerase operates optimally
at 25-30° and denatures at 75°C, making it unsuitable for PCR. Bsu
polymerase cannot proofread as it lacks 3’-5’ exonuclease activity”-
www.abmgood.com
d. DNA polymerase
e. DnaA protein
Formation of the Replication Fork Lippincott 6th/399-400
•• Proteins required for DNA strand separation: Initiation
of DNA replication requires the recognition of the origin of
replication by a group of proteins that form the prepriming
complex. These proteins are responsible for maintaining the
separation of the parental strands, and for unwinding the
double helix ahead of the advancing replication fork. These
proteins include the following:

Answer
1. a. Pfu polymerase; c. Bestaq DNA Polymerase; d. Pwo polymerase [Ref: Lippincott 6th/482; Vasudevan 5th/458]
2. a. Helix-turn-helix; b. Zinc finger; c. Helix-loop-helix; d. Leucine zipper [Ref: Harper 30th/444-45;Satyanarayan 4th/573-74]
3. a. DNA Helicase; b. Single-stranded DNA-binding (SSB) proteins; e. DnaA protein
 [Ref: Harper 30th/453; Lippincott 6th/399-400;Satyanarayan 4th/525]

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 Immunogenetics and Molecular Biology

 DnaA protein: DnaA protein binds to specific nucleotide Enzyme Reaction Primary Use
sequences at the origin of replication, causing short,
tandemly arranged (one after the other) AT-rich regions in DNase I Under appropriate Nick translation; mapping
conditions, produces of hypersensitive sites;
the origin to melt. Melting is ATP-dependent, and results in
single-stranded nicks mapping protein-DNA
strand separation with the formation of localized regions in DNA interactions
of ssDNA.
 DNA helicases: These enzymes bind to ssDNA near the Reverse Synthesizes DNA from Synthesis of cDNA from
replication fork, and then move into the neighboring transcriptase RNA template mRNA; RNA (5' end)
mapping studies
double-stranded region, forcing the strands apart—in
effect, unwinding the double helix. Helicases require energy RNAse H Degrades the RNA Synthesis of cDNA from
provided by ATP [Note: DnaB is the principal helicase of portion of a DNA-RNA mRNA
replication in E. coli. Its binding to DNA requires DnaC.] hybrid
 Single-stranded DNA-binding (SSB) proteins: These Terminal Adds nucleotides to Homopolymer tailing
proteins bind to the ssDNA generated by helicases . They transferase the 3' ends of DNA
bind cooperatively—that is, the binding of one molecule of CRISPER-Cas9 RNA-targeted DNA- Genome editing and
SSB protein makes it easier for additional molecules of SSB directed nuclease modulation of gene

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protein to bind tightly to the DNA strand. The SSB proteins expression
are not enzymes, but rather serve to shift the equilibrium
between dsDNA and ssDNA in the direction of the single-

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4. Component (s) involved in RNA-induced silencing
stranded forms. These proteins not only keep the two complex (RISC):
strands of DNA separated in the area of the replication a. miRNa
origin, thus providing the single-stranded template required b. Drosha

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by polymerases, but also protect the DNA from nucleases c. Dicer
that degrade ssDNA. d. Pasha
•• Solving the problem of supercoils: To solve this problem, there tN e. Slicer
is a group of enzymes called DNA topoisomerases, which are
responsible for removing supercoils in the helix. “Degradation of homologous RNA in RNA interference is carried
 Type I DNA topoisomerases: These enzymes reversibly out by functional RNA-induced silencing complex (RISC). RISC
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cut one strand of the double helix. They have both nuclease contains Dicer, Argonaute protein, siRNA and other components”-
(strand-cutting) and ligase (strand-resealing) activities. www.ncbi.nlm.nih.gov
 Type II DNA topoisomerases: These enzymes bind tightly “The Argonaute family of proteins, specifically Argonaute 2
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to the DNA double helix and make transient breaks in both
strands. Type II DNA topoisomerases are also required
in both prokaryotes and eukaryotes for the separation of
interlocked molecules of DNA following chromosomal
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(also known as Ago2, eIF2C2 or Slicer) are essential components of
RISC, which are involved in mRNA cleavage”-www.abcam.com
“MicroRNAs (miRNAs) associate with Argonaute (Ago),

GW182, and FXR1 proteins to form RNA-induced silencing

replication. DNA gyrase, a Type II topoisomerase found in complexes (RISCs). RISCs represent a critical checkpoint in the
bacteria and plants, has the unusual property of being able
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regulation and bioavailability of miRNAs”-www.jneurosci.org

to introduce negative supercoils into relaxed circular DNA “The RNA-induced silencing complex, or RISC, is a
using energy from the hydrolysis of ATP. multiprotein complex, specifically a ribonucleoprotein, which
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Table 2. (Harper 30th/453): Some of the Enzymes Used in Recombi- incorporates one strand of a single-stranded RNA (ssRNA) fragment,
nant DNA Research such as microRNA (miRNA), or double stranded small interfering
RNA (siRNA). The single strand acts as a template for RISC to
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Enzyme Reaction Primary Use recognize complementary messenger RNA (mRNA) transcript.
DNA ligase Catalyzes bonds Joining of DNA molecules Once found, one of the proteins in RISC, called Argonaute, activates
between DNA and cleaves the mRNA. This process is called RNA interference
molecules (RNAi) and it is found in many eukaryotes; it is a key process in gene
silencing and defense against viral infections”-wiki
DNA Synthesizes double- Synthesis of double-
“The cytoplasmic RNA-induced silencing complex (RISC)
polymerase I stranded DNA from stranded cDNA; nick
single-stranded DNA translation; generation contains dsRNA binding proteins, including protein kinase RNA
of blunt ends from sticky activator (PACT), transactivation response RNA binding protein
ends (TRBP), and Dicer, that process pre-microRNAs into mature
microRNAs (miRNAs) that target specific mRNA species for
Thermostable Synthesize DNA at Polymerase chain reaction
DNA elevated temperatures (DNA synthesis) regulation. There is increasing evidence for important functional
polymerases (60°-C-80°C) interactions between the miRNA and nuclear receptor (NR) signaling

Answer
4. a. miRNa; c. Dicer; e. Slicer [Ref: Harper 30th/409-10; Lippincott 6th/459]

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PGI Supplement November 2017

networks, with recent data showing that estrogen, acting through the inhibition of translation by targeting the 5′-methyl cap binding
estrogen receptor, can modulate initial aspects of nuclear miRNA translation factor eIF4 or the ribosome directly. Recent data
processing. Here, we show that the cytoplasmic RISC proteins PACT, suggest that regulatory miRNA-encoding genes may be linked,
TRBP, and Dicer are steroid receptor RNA activator (SRA) binding and hence co-evolve with their target genes.
NR coregulators that target steroid-responsive promoters and
regulate NR activity and downstream gene expression. Furthermore,
each of the RISC proteins, together with Argonaute 2, associates with
SRA and specific pre-microRNAs in both the nucleus and cytoplasm,
providing evidence for links between NR-mediated transcription and
some of the factors involved in miRNA processing”-www.ncbi. nlm.
nih.gov
“DGCR8/Pasha is an essential cofactor for Drosha, a nuclear
RNase III that cleaves the local hairpin structures embedded in
long primary microRNA transcripts (pri-miRNAs) in eukaryotes.
DsRNA-binding proteins that contain dsRNA-binding domains
(dsRBDs) serve diverse roles during RNA metabolism. A recently

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emerging theme in RNA silencing pathways is that multidomain
RNase III proteins, such as Drosha and Dicer, necessitate dsRNA-
binding proteins for miRNA processing and/or RISC formation. For

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example, Dcr-1 in Drosophila interacts with Loquacious/R3D1 that
contains three dsRBDs and is required for miRNA processing, and
possibly also for RISC assembly”- www.ncbi. nlm. nih. gov

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The Microprocessor complex is a protein complex involved in
the early stages of processing microRNA (miRNA) in animal cells.
The complex is minimally composed of the ribonuclease enzyme tN
Drosha and the RNA-binding protein DGCR8 (also known as
Pasha) and cleaves primary miRNA substrates to pre-miRNA in the
cell nucleus-wikipedia
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Micro-RNAs Are Derived from Large Primary Transcripts
Through Specific Nucleolytic Processing Harper 30th/409
•• The majority of miRNAs are transcribed by RNA pol II into
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primary transcripts termed pri-miRNAs. pri-miRNAs

are 5′-capped and 3′-polyadenylated. pri-miRNAs are
synthesized from transcription units encoding one or several
distinct miRNAs; these transcription units are either located
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independently in the genome or within the intronic DNA

of other genes. Given this organization miRNA-encoding
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genes must therefore minimally possess a distinct promoter,

coding region and polyadenylation/termination signals. pri-
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miRNAs have extensive 2° structure, and this intramolecular

structure is maintained following processing by the Drosha-
DGCR8 nuclease; the portion containing the RNA hairpin is
preserved, transported through the nuclear pore via the action
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Fig. 1 (Lippincott 6th/459): RNA interference or RNAi by cleavage of

of exportin 5, and once in the cytoplasm, further processed by target mRNA. [Note: RNAi can also result from inhibition of target
the heterodimeric dicer nuclease-TRBP complex to a 21 or 22- mRNA translation.]
mer.
•• Ultimately, one of the two strands is selected for loading into the 5. In which of the following autosomal non-disjunction is/are
RNA-induced silencing complex (RISC), which is composed seen:
of one of four Argonaute proteins (Ago 1→4), to form a a. Patau syndrome
mature, functional 21-22 nt single stranded miRNA. siRNAs b. Edward syndrome
are produced similarly. Once in the RISC complex, miRNAs c. Klinefelter syndrome
can modulate mRNA function by one of three mechanisms: d. Cri-du-chat syndrome
(a) promoting mRNA degradation directly; (b) stimulating e. William syndrome
CCR4/NOT complex- mediated poly A tail degradation; or (c) •• Klinefelter syndrome involves sex chromosome non-disjunction
(so not autosomal)
Answer
5. a. Patau syndrome; b. Edward syndrome [Ref: Robbins 9th (SAE)/100,163,165; Harrison 19th/439,83e-6,2350]

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 Immunogenetics and Molecular Biology

Williams syndrome and Cri-du-chat syndrome are chromosome
deletion syndromes
“Nondisjunction is the failure of homologous chromosomes or
sister chromatids to separate properly during cell division. There
are three forms of nondisjunction: failure of a pair of homologous
chromosomes to separate in meiosis I, failure of sister chromatids to
separate during meiosis II, and failure of sister chromatids to separate
during mitosis. Nondisjunction results in daughter cells with abnormal
chromosome numbers (aneuploidy)”-www.medicinenet.com
Consequences Nondisjunction www.angelfire.com,wiki; Robbins 9th
(SAE)/100,163,165 ;Harrison 19th/439, 83e-6, 2350
•• Monosomy-Turner syndrome (X monosomy) (45, X0)
•• Autosomal trisomy: Down syndrome (trisomy 21), Edwards
syndrome (trisomy 18) and Patau syndrome (trisomy 13)
•• Sex chromosome aneuploidy: Klinefelter syndrome (47, XXY),
increase) are associated with Huntington’s chorea (CAG), myotonic
dystrophy (CTG), spinobulbar muscular atrophy (CAG), and
Kennedy disease (CAG)”- Harper 30th/378
Nucleotide Repeat Expansion Disorders Harrison 19th/440
•• Several diseases are associated with an increase in the number
of nucleotide repeats above a certain threshold
•• The repeats are sometimes located within the coding region
of the genes, as in Huntington’s disease or the X-linked form
of spinal and bulbar muscular atrophy (SBMA; Kennedy’s
syndrome). In other instances, the repeats probably alter gene
regulatory sequences.
•• If an expansion is present, the DNA fragment is unstable and
tends to expand further during cell division. The length of
the nucleotide repeat often correlates with the severity of the
disease. When repeat length increases from one generation to
the next, disease manifestations may worsen or be observed at

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XYY Male (47, XYY) and Trisomy X (47, XXX)
an earlier age; this phenomenon is referred to as anticipation.
•• Uniparental disomy (Prader-Willi syndrome and Angelman
syndrome) Table 3 (Harriosn 19th/440): Selected Trinucleotide Repeat Disorders

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•• Mosaicism syndromes (Pallister-Killian syndrome and Hy-
pomelanosis of Ito) Disease Repeat Inheritance Gene Product
•• Mosaicism in malignant transformation (Human X-chromosomal CAG XR Androgen receptor
retinoblastoma) spinobulbar

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“Klinefelter syndrome: The classic form of KS (47, XXY) muscular atrophy
occurs after meiotic nondisjunction of the sex chromosomes (SBMA)
during gametogenesis (40% during spermatogenesis, 60% during tN Fragile X-syndrome CGG XR FMR-1 protein
oogenesis)”- Harrison 19th/2350 (FRAXA)
“Humans have 22 homologous pairs of autosomes and one pair Fragile X-syndrome GCC XR FMR-2 protein
of sex chromosomes. The main difference between autosomes and (FRAXE)
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sex chromosomes is that autosomes are involved in determining Dystrophia CTG AD, variable Myotonin protein
the somatic characters of an individual and sex chromosomes are myotonica (DM) penetrance kinase
involved in determining the sex and the sex-related hormonal Huntington's CAG AD Huntingtin
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traits”-www.pediaa.com disease (HD)

“The first chromosome deletion syndromes were diagnosed Spinocerebellar CAG AD Ataxin 1
clinically and were subsequently demonstrated to be caused by ataxia type 1 (SCA1)
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a chromosome deletion on cytogenetic analysis. Examples of

Spinocerebellar CAG AD Ataxin 2
these disorders include the Wolf-Hirschhorn syndrome, which ataxia type 2 (SCA2)
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is associated with deletions of a small region of the short arm of

Spinocerebellar CAG AD Ataxin 3
chromosome 4 (4p); the cri-du-chat syndrome, associated with ataxia type 3
deletion of a small region of the short arm of chromosome 5 (5p); (SCA3); Machado-
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Williams syndrome, which is associated with interstitial deletions Joseph disease

of the long arm of chromosome 7 (7q11. 23); and the DiGeorge/ (MD)
velocardiofacial syndromes, associated with interstitial deletions of Spinocerebellar CAG AD Alpha 1A voltage-
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the long arm of chromosome 22 (22q11. 2)”- Harrison 19th/83e-6 ataxia type 6 (SCA6, dependent L-type
6. Microsatellite instability disorder (s) is/are: CACNAIA) calcium chan-nel
a. Myotonic dystrophy Spinocerebellar CAG AD Ataxin 7
b. Huntington chorea ataxia type 7 (SCA7)
c. Ataxia telangiectasia Spinocerebellar CAG AD Protein
d. Spinocerebellar ataxia ataxia type 12 phosphatase 2A
e. Xeroderma pigmentosa (SCA12)
Dentorubral CAG AD Atrophin 1
“Trinucleotide sequences that increase in number pallidoluysian
(microsatellite instability) can cause disease. The unstable (CGG) atrophy (DRPLA)
n repeat sequence is associated with the fragile X syndrome. Other
Friedreich ataxia GAA AR Frataxin
trinucleotide repeats that undergo dynamic mutation (usually an (FRDA1)
Answer
6. a. Myotonic dystrophy; b. Huntington chorea; d. Spinocerebellar ataxia [Ref: Harrison 19th/440; Harper 30th/378; Lippincott 6th/ ]

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PGI Supplement November 2017
“Roughly 100 syndromes of familial cancer have been reported,
although many are rare. The majority are inherited as autosomal
dominant traits, although some of those associated with DNA repair
abnormalities (xeroderma pigmentosum, Fanconi’s anemia, ataxia
telangiectasia) are autosomal recessive”-Harrison 19th/101e-4
7. Which of the following is/are part of 30S initiation complex:
a. ATP
b. GTP
c. Initiation factor
d. Elongation factor
e. mRNA
“30S initiation complex (30SIC) contains mRNA, fMet-tRNAfMet
and initiation factors IF1 and GTP-bound IF2”- www.nature.com
“In bacteria, during the initiation phase of protein synthesis
three initiation factors (IFs: IF1, IF2 and IF3), the messenger RNA
(mRNA), and initiator tRNA (fMet–tRNA) assemble in a 30S pre-
initiation complex (preIC) in which proper codon–anticodon base
pairing has not yet taken place”-academic. oup.com
Initiation of translation in prokaryotes Satyanarayan 4th/557
•• The 30S ribosomal subunit is bound to initiation factor 3 (IF-
3) and attached to ternary complex of IF-2, formyl met-tRNA
and GTP
•• Another initiation factor namely IF-1 also participates in the
formation of preinitiation complex
•• A 50S ribosome unit is now bound with the 30S unit to produce
70S initiation complex
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Fig. 2: Initiation complex formation in bacteria
Answer
7. b. GTP; c. Initiation factor; e. mRNA 
8. c. May result in 100 abnormal cells
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8. Single mutation in DNA of somatic cell of multicellular
organism results in:
a. No effect on organism
b. Every cell in the entire organism will be affected
c. May result in 100 abnormal cells
d. Can be transmitted to progeny
•• As tissue (collection of cells) can be formed from mutant
somatic cells, so option c (100 abnormal cells) may be true
In multicellular organisms, mutations can be classed as either
somatic or germline: ib. bioninja.com.au
•• Somatic mutations: occur in a single body cell and cannot be
inherited (only tissues derived from mutated cell are affected)
•• Germline mutations: occur in gametes and can be passed onto
offspring (every cell in the entire organism will be affected)
Mutation Harrison 19th/432
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•• Some mutations may be lethal, others are less deleterious, and
some may confer an evolutionary advantage.
•• Mutations can occur in the germline (sperm or oocytes); these
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can be transmitted to progeny. Alternatively, mutations can
occur during embryogenesis or in somatic tissues.
•• Mutations that occur during development lead to mosaicism,
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a situation in which tissues are composed of cells with different
genetic constitutions.
•• If the germline is mosaic, a mutation can be transmitted to some
progeny but not others, which sometimes leads to confusion in
assessing the pattern of inheritance.
•• Somatic mutations that do not affect cell survival can sometimes
be detected because of variable phenotypic effects in tissues
(e.g., pigmented lesions in McCune-Albright syndrome).
•• Other somatic mutations are associated with neoplasia because
they confer a growth advantage to cells. Epigenetic events may
also influence gene expression or facilitate genetic damage.
With the exception of triplet nucleotide repeats, which can
expand, mutations are usually stable.
Mutations and Evolution www.thoughtco.com
Sometimes, during replication, mistakes can be made, and these
mutations can change the DNA in the cells of the body. However, if
there is a mutation in a somatic cell, it most likely will not contribute
to the evolution of the species.
Since somatic cells are in no way involved in the process of
sexual reproduction, any changes in the DNA of somatic cells will
not get passed down to the offspring of the mutated parent.
Since the offspring will not receive the changed DNA and any
new traits the parent may have will not be passed down, mutations
in the DNA of somatic cells will not cause evolution.
If there happens to be a mutation in a gamete, that can drive
evolution. Mistakes can happen during meiosis that can either change
the DNA in the haploid cells, or create a chromosome mutation
which can add or delete portions of DNA on various chromosomes.
If one of the offspring is created from a gamete that has a mutation in
it, then that offspring will have different traits that may or may not be
favorable for the environment
[Ref: Satyanarayan 4th/557]
[Ref: Harrison 19th/432; ib.bioninja.com.au]
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