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F.y.B.Sc. Biotechnology Semester I Examination
paper: Basic BiotechnoloryII Molecular Biology
ModelAnswers
Q.1 Do as directed (Any
t5) (15 marks- one mark each)
1. F plasmid.
2. a. Insertional Inactivation of a marker
gene,
b. Visual screening methods
c. Plaque phenotlpe
3. Streptococcus pneumonia
-
smooth and rough sffain
4. Transposition involves integration of a copy of a defined segment
of a DNA from
another locus, either in the same or different molecule of DNA.
5. Mutation- sudden change in the heritable material.
6' Mutagenesis- the process of change in genetic material naturally or on induction
with a mutagen.
7. E.
coli or Bacillus subtilis
8 Ethidium b'romide, proflavin (any one)
9 False
10. True
11. Multiple Cloning Site
12. Semiconservative
13. Primer
14.
DNA Polymerase -
15.
3'-
I
5' exonuclease activity
16. Leading
17. Primase
18. Cyclin dependent kinases (Cdks)
19. G$cosylase
20. Thymine dimers
Q 2 a Explain how Meselson and Stahl proved semiconseryative
mode of DNA
replication (8 marks)
watson and crick proposed the semiconservative
model in which each progeny
molecule retains one of the parental st
Cs.
Five years later, Matthew Meselson and
Frank Stahl obtained experimental evidence
that semiconservative replication was
correct.
Organism used - Escherichia coli.
E'coli was grown for several generations in a
minimal medium n which the only N
source was l5NH4Cl.
As a result, all the bacteria's cellular N-containing
compound contained 15N instead
ofN14.
l5N labeled bacteria was transferred to a medium containing
nitrogen in the
normal 14N form and allowed to reproduce
in the new conditions for several
Then the
generations.
Throughout the period of growth in the r4N
medium, sample of E.coli were take4
lysed to release the cellular contents and cellular
DNA wasw analysed in cesium
chloride density gradients.
After one generation ----all the DNA had density
intermediate between that of totally
15N DNA and totally 14N DNA. After 2 genaations,
half the DNA wasof
intermediate and half of the density of DNA
containing entirely r4N. thus, the
observation was exactly what semiconserva ive
model predicted.
If the conservative model of DNA replication had
been correct, after one generation -- 2 bands of DNA" one in the heavy density position
of the gradient and other in the
light density position would be seen and in the
subsequent generations the amount of
DNA in the light density position would increase with
eaci generation. But the fact
that intermediate density DNA was found
ruled out the conservative model.
According
to the dispersive model after one
generation
all DNA would be of
intermediate density but after the 2"d generation,
the DNA molecules would be found
at one band located halfivay between the intermediate
density and light density band
positioned and with subsequent there would
continue to be one band and it would
become lighter in density with each generation.
Such a slow shift in DNA density was not seen
in Meselson
therefore dispersive model ruled out.
-
stahl experiment and
This is how Meselson and Stahl proved semiconservative
mode of replication.
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Q 2 b differentiate between the reading and lagging
strands formed during DNA
replication (7 marks)
To maintain the 5'- 3'polarity of DNA
synthesis and one overall direction of
the
replication fork movement, the direction of
DNA synthesis is different on the two
template strands.
Leading strand: As the replication fork migrates,
synthesis of one
v'v new
Dlrcu\r (the
'vvv strand
\
continuous.
leading strand) is
The template strand copied is in
3'_ 5, direction.
The leading strand synthesized is in the same
direction as that of the fork movement.
Lagging strand: synthesis of lagging strand
is discontinuous because the helix must
untwist to expose a new segment of 3'- 5'template
so that the new strand can be
made in the 5' - 3, direction.
Thus the DNA on the lagging strand is formed
in fragments called okazaki fragments.
The template strand copied is in
3'_ 5, direction.
The lagging strand is spthesized
in the opposite direction of the replication
fork
movement.
OR
Q 2c Elaborate on the different DNA polymerases in Eukaryotic
DNA replication
(8 marks)
Replication Enzymes and proteins:
Five different DNA polymertase have been
identified in mammalian cells, designated
as o (alpha), B @eta), y (gamma) and (epsilon).
e
The o, p, o and e polymerases af,e
located in the nucleus, while the y polymerase
is found in the mitochondria.
DNA polymerase o, 6 are the two enzymes responsible
for nuclear DNA replication.
They function essentially like the E.cori DNA polymerase
III.
DNA polymerase p serves a DNA repair function,
and DNA polymerase y catalyses
mitochondrial DNA rep lic action.
Both Nuclear replication polymerase a and
6 can use RNA primers for the initiation
DNA synthesis; D can also use a DNA primer.
The mitochondrial polymerase y uses an
RNA polymer.
uniquely among the DNA Polymerase, the polymerase
c
also has primase activity; a
separate enzwe is used for the synthesizing
a primer in DNA synthesis catalyzed.by
the other DNA polymerase.
It is not known whether or not these enzymes have
5, to 3, exonucrease activity.
only 6, e and y have been shown to have proofreading
(3, to 5, exonuclease) activity.
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Q2
d With
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neat labelled diagram illustrate the Holliiday model
of
recombination (7 marks)
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Q 3 a Explain the term mutation and discuss the various
tlpes of mutations
(8 marks)
Can be explained in form of a table
Mutation-
2 Marks
Tlpes (6 marks) - somatic, gerrn line, spontaneous,
Induced, point, Gross, forward,
Backward, lethal, nutritional etc.
Q 3 b Discuss Nucleotide Excision repair (7 Marks)
Can be explained only diagrammatically
U*ABC
endonuclease complex
is a
speciar class
of
endonucrease enzyme
involved in DNA repair of prokaryotes. The name
is derived from the root term
ultraviolet radiation, since the level of most of
these enzymes will lre elevated in
the bacterial cells when the cells are exposed
to UV light.
The U*ABC complex of E. cori consists of four enz)nnes.
They are named as
urzrA, UwB, uwc and uvrD (uwD is also called
as DNA hericase Irt).
Among these uw erzymes, uvrB and uwc are
the actual e,ndonucleases which
cut the phosphodiester backbone of the DNA.
UWABC complex is arso called as DNA excinuclease.
A;n excinucrease is a
special type of endo-nucrease enzyme which
cut two phosplodiester bonds at a
time simultaneously, which is different from conventional
endonur:leases since
usual endonucleases cut only one phosphodiester
bond at a tims.
NER mechanism also requires DNA pollnnerase
I enzyme wtrich wil do the gap
filling activity after the exinuclease removes the damaged
basesr.
NER also requires DNA ligase enzyme for the
final nick joining.
The sequential action of these enz)rynes such as
uwA, uwEt, uwc, uwD, then
DNA polymerase I and DNA ligase are essential for
the orchestr.tion of NER
machinery in the bacterial cells.
NI'CLEOTIDE EXCISION REPAI* IN PROI(ARYOTES
IIrrrABC ENDONITCLEASS [eXrNUCf-n.eslrl Er{ZyMEBY
[fV radiatio.rr irrcltr ces
DNA darnages
{JrrrABC extrnrrclease find.s darnage.
crrts DNA strand.
L2 or 13 nucLeotid.es are rerrro.rre*I
DIYA pol5rrnerasre f fil.ts in ttre
resulting gap
DNA ligase :sea,l.s ttre DlgA str:and
tllu$tratlan: @ JotlaD Jarnastad/Tho F?or.a, Swed/sar
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Q 3 c Explain the various types of point mutations (g Marks)
Base pair substitution
-
Transition, transversion
Base Pair addition/ deletion
- Frame shift
Resulting in phenotlpic changes
- Missence, Nonsense, silent, and Neutral.
Explanation in a table form should also be
considered can be explained with or
without example
Q 3 d Discuss the mechanism of mutation induced by Base
anarog (7 marks)
Base analog
-
chemical mutagen, analogous to nitrogen bases
of DNA example
5 BU- 5 Bromo uracil (2 Marks) Mechanism
5 marks (
with or without diagram)
Normal form of 5 BU and rare form of 5 BU
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Q4 A. How wourd you
marks)
use pBR
322 as a vector to introduce a gene of choice (g
pBF- 322 is an anificial cloning plasmid
vector, B Boliver, R _ Rodriguez. It has
4364 kbases' It has 2 selectable markers tetracycline
and ampicillin resistant gene.
Has unique single restriction site for 12 different
-
-
restriction endonucleases within the
marker genes' cut vector with PstI, place the insert
in the amp gene and make it non
functional' Bacteria with such a cloned vector
cannot grow in presence of amp but can
grow in presence of tetra. This is insertional
inactivation. cells are first grown ln
media containing tetra and a replica is made
on a plate containing amp in the medium.
which grow on master plate but fail to grow on the
replica plate contain the
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qeslred
msert.
48' Explain Isoration of genomic DNA by
a flow diagram (7 marks)
Protocol of genomic DNA from any material
can be considered bacterial DNA flow
sheet is given as an example examiners need
not restrict only to it. Expected is various
solution used in extraction and isolation protocol
of genomic
DNA.
Q4 c. Explain the different types of Restriction Endonucleases (g
marks)
Tlpe I - has 3 subunits - i. Restrictive subunit ii.
Modification subunit iii. specificity
subunit - specifies recognition site. Requir e Mg2+, _
S adenosyl methoinine and ATp
as cofactors. The recognition site is 15bp long
and cleavage site is l000bp away from
the 5' end ofrecognition site.
Tlpe II
- require Mg2+ as cofactor. They have separate activities for reshiction and
modification' They cut within the restriciion site
at a specific place. The restriction
site exhibits a twofold symmetry around
a given point. The restriction sites
palindromes.
Enzymes cut to give staggered or
blunt ends.
Tpe III - two subunits,
are
one for recognition and modification and
the other
for
cleavage. ATP is source of energy and
Mg2+ as cofactor. They have symmetrical
recognition sites and cut DNA at non palindromic
sequences, 25_27 bp away from
recognition site.
Q4 D. Explain with an example plant vectors (7 Marla)
Plant virus vectors are used for transfer
of genes. The DNA insert linked with viral
DNA replicates and expresses in the host cell
along with viral genes. These are
transmitted systemically in the plant
but the DNA never integrates into the plant
DNA' To act as vectors they should i. carry
extra NA ii. have broad host range iii.
be
easily transmitted.
Then write on any one
-
l.caulimoviruses - rinear open circle ds
DNA . Spreads rapidry throughout the plant
and found in high copy number. Replicates
through .*"rr" transcription. Two
genomic regions of the virus oRF
I and oRF vlr are non essential and can be
replaced by a foreign DNA.
2' Geminiviruses -
ss DNA. Their genome consists of
two elements - an origin of
replication and replication protein, which
are included in the vector. The coat protein
sequence is replaced by the desired
or reporter gene. They are used to insert
foreign
genes in protoplasts.
3' Tobacco mosaic viruses (TIW) linear
RNA genome. The vector is constructed by
ne into an intact viral genome which rapidly
spreads through the
with insert replicate only in protoplasts inoculated
leaves or
Q 5. Write short notes on any three (5 marks each)
a
S
'
'
.
15 marks
Early models of DNA Replication
emiconse.ative, Disp ersive and conservative
models
conservative. Replicalion produces one helix
made entirely of old DNA and one
helix made entirely of newDNA
semi-conservative. Replication produces two
helices that contain one old and one
new DNA strand.
Dispersive
b. Genetic Engineering in any one prokaryote _
Most common- E. coli
Methods
l'
-
Transformation
entry of naked DNA through the cell
membrane into the
recipient E. cori cell. The cell has to
be competent to take up the foreign
DNA.
Not all exogenous DNA survive in the recipient
celr, they are wrnerable to
2.
e specific transduction
only specific regions
recipient, either the gal or bio gene. The
3.
4'
-
conjugation transrer or genetic
-",:1tJ"Tf:*':'l:j,t::i',',.* or Hfr) to the
recipient cell (F-) through direct physical
contact.
Transposition - involves integration of
a copy of a defured segment of a DNA
from another locus, either in the same
or different molecule of DNA. these mobile
transposable elements carry one or
more genes that provide added qualities
to the
cell that carries it, for eg, resistance to drugs,
toxins; encode toxins.
c Photoreactivation
-
Repair ofI-IV induced thymine dimers
In prokaryotes
Also called as Light repair
Induction by visible light,
Role of photolyase enzyme
Splitting of bonds between the dimers
Can be explained diagrammatically.
d
-
W
radiations as physical mutagens
Non -ionizing radiations
Induction ofthymine dimers
Also C^C C^T
Atrecting formation of covalent bonds between
bases
Forming gaps in DNA, no replication
Bulging ofDNA
A,$Nitn$(p)Fr
e.
pUC vector-
Developed by Messing. has colEl
origin gene, amp resistance gene
and engineered
version of lac z gene which contain
*ut ipt, restriction sites within the first part
of
the coding region. This is calred
the MCS - multiple .toning site.
If
targetDNA is
inserted into any one of these
sites the lac z gine is inactivated.
Hence
such a
recombinant vector will form
white colony on a suitable plate as
compared
to non
recombinant vector.
cells canying just the vector are capable
of producing the hybrid beta galactosidase.
so when grown in presence of IprG (inducer)
and x-Gat ,,rb.,.ur", the enzyme
acts
on substrate to produce a blue product.
cells carrying the vector with insert are
not
able to produce the hybrid beta galactosidase.
Hence the substrate is not acted
upon
and the colonies remain white in
colour. This plasmid vector is designed
for blue
white
screening.