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NANYANG TECHNOLOGICAL UNIVERSITY

2018 H3 MOLECULAR BIOLOGY

EXAMINATION PAPER MARK SCHEME

Section A: Answer ALL questions

1. (a) 5 marks

Protein folding:

The single chain of the protein will fold to maximize hydrogen bonding for secondary structures. [2]

Hydrophobic effect drives non-polar side residues to be buried in protein interior and expose polar side
residues at water surface. [3]

(b) 5 marks

Sickle cell anemia: a single point mutation in the gene of the beta-chain of hemoglobin, results in one of the
polar side residue changing to a non-polar side residue. [3]

This mutated beta-chain causes aggregation (polymerization) of hemoglobin by hydrophobic effect and van der
Waals attraction. [1] This causes deformation of the red blood cell structure. [1]

2. (a) 4 marks

Lactoferrin, ribonuclease, casein kinase [3]

Explanation: All three of these proteins have pI above pH 8, hence they are positive charged in pH 8 buffer In
a cation exchanger, the gel matrix is positive charged, so it will bind positive charged proteins. [1]

(b) 6 marks

1 – catalase. 2 – Myosin. 3 – Insulin. 4 – Ovalbumin. 5 – carbonic anhydrase. [5]

Explanation: In gel filtration chromatography, separation is by size. The larger the size (MW), the faster it
elutes from the column. [1]

3. (a) 3 marks

The three functions of adipose tissue are storage of energy in the form of fats, mechanical protection of
major organs, and insulation against heat loss from the skin. [3]

(b) 1 marks

At 24 h after exposure to leptin, the apoptotic index was 50% compared to the controls which were 30%. At 48
h after exposure to leptin, the apoptotic index was 60%. [1]

(c) 2 marks

After 9 h of exposure to leptin, Ang2 gene expression increased about seven times compared to controls at 0 h.
At 24 and 48 h, Ang2 gene expression increased thirty-three times and eighty-seven times compared to
controls at 0 h. [1]

After 9 h of exposure to leptin, VEGF gene expression increase twelve times compared to controls at 0 h. At 24
and 48 h, VEGF gene expression increased very little and twenty-five times compared to controls at 0 h
respectively. [1]
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(d) 4 marks

Leptin induces apoptosis of adipocytes in adipose tissue [1], together with increased gene expression of
Ang2 but with less increased gene expression of VEGF. [2] This may suggest a restructuring of the blood
vessel vasculature in adipose tissue. [1]

4. (a) 6 marks

Cells were fixed with formaldehyde, lyzed and sonicated. [1]

Lysates were incubated with non-immune IgG, anti-sp2 antibodies, anti-sp3 antibodies and anti-zf9 antibodies
for immunoprecipitation. [2]

The immune complex pellet was washed and reverse cross-linked by heating to 65oC. [1]

The DNA was extracted and samples were PCR-amplified using primers for the BS5-B region and the EP7-D
region of the HSP47 gene. [1]

PCR products resolved by DNA gel electrophoresis. [1]

(b) 4 marks

Sp2, sp3 and zf9 binds to BS5-B region of HSP47 gene. [1]
Sp2 and sp3, but not zf9, binds to EP7-D region of HSP47 gene. [1]
At least one of them is the transcription factor. [1]
The other two may be activators that bind to enhancers. [1]

5. (a) 3 marks

After 2 days in the presence of M-CSF, CREB-1 and CREM-1 protein expression was relatively low and was
only slightly increased by 3-fold and 6-fold respectively after 4 days. At day 6, CREB-1 and CREM-1 protein
expression significantly increased by 16-fold and 6-fold respectively compared to day 2. At day 8, CREB-1
and CREM-1 protein expression was 26-fold and 10-fold greater respectively compared to day 2. [2]

Protein expression of phosphorylated CREB-1 and phosphorylated CREM-1 only increased at day 4 by 6-
fold and 14-fold compared to day 2. Expression of both phosphorylated forms significantly increased by 33-
fold and 14-fold respectively at day 6 and continued to increased to 78-fold and 17-fold at day 8 compared to
day 2. [1]

(b) 3 marks

Overexpression of CREB-1 gene stmulated the activity of the METS promoter, macrosialin promoter and
SR-A promoter by 3-fold, 2-fold and 3-fold over mock samples respectively. [3]

(c) 4 marks

CREB-1 and CREM-1 are induced and phosphorylated during macrophage differentiation and this suggests that
the phosphorylated forms may be involved in intracellular signaling during macrophage differentiation. [2]

Overexpression of the CREB-1 gene increases the promoter activities of the METS, macrosialin and SR-A gene
and this suggests that these genes are upregulated during macrophage differentiation. [2]
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Section B. Answer only THREE (3) of the FOUR (4) questions

6. (a) 3 marks

Truncated target gene

Homologous site 1 NEOR Homologous site 2 Thymidine kinase

5’- -3’

5’-homologous site 1, homologous site2-3’. (Note: make sure answers have the 5’ and 3’ ends.) [1]
Neomycin resistant gene in the correct position. [1]
Thymidine kinase gene in the correct position. [1]

(b) 4 marks

Integration of targeting vector into mouse genome is by homologous recombination. [1]

The two homologous sites in the targeting vector line up opposite the same two homologous sites in the
mouse genome. [1]
DNA sequences that are between the two homologous sites in the targeting vector are exchanged for the
DNA sequences that are between the same two homologous sites in the mouse genome. [1]
This results in the mouse genome containing the strand of DNA sequences comprising the neomycin
resistant gene and the truncated target gene. [1]

(c) 3 marks

Positive selection is to keep/select those stem cells that have the targeting vector inserted into their genome by
homologous recombination and non-homologous recombination, as well as to eliminate stem cells that do not
have the targeting vector inserted into their genome either by homologous recombination or non-homologous
(random) recombination. [2]

Negative selection is to eliminate those stem cells that have the targeting vector in their genome inserted by
non-homologous recombination. [1]

7. (a) 4 marks

There are three polypeptide chains in C3c. [1]

The molecular weight of native C3c (indicated by absence of beta-mercaptoethanol) is 145 kDa. Full
digestion by maximum concentration of beta-mercaptoethanol yields 3 bands in the gel which are 75 kDa, 43
kDa and 27 kDa. These 3 bands add up to give a total of 145 kDa. [3]

(b) 1 mark

Disulphide bond holds the polypeptide chains with each other. [1]

(c) 2 marks

75 kDa 27 kDa 43 kDa

S S S S

If there are other illustrations, they are evaluated on a case-by-case basis.

In general, marks will be awarded for:

i. Disulphide bonds indicated. [1]

ii. Correctly drawn sizes and arrangement of polypeptide chains. [1]
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(d) 3 marks

Possible secondary structures: alpha-helices, beta-sheets, beta-turns, omega-loops. [Mention two of these
structures to give 1 mark.]

Explanation: C3c is a plasma protein that has to be soluble in blood; therefore its structure would most likely be
that of a globular protein. It functions very much like an antibody because it has to attach to microbes,
thus it would be more conducive to have a majority of alpha helices that are more flexible and less rigid than
beta-sheets to bind to microbe surface proteins. [2]

8. (a) 7 marks

The major groove of DNA is more conducive because:

- it is wider, deeper and longer which allows easier access by a DNA-binding protein. [3]

- there are more nucleotides in the length of the major groove that will potentially can form more hydrogen
bonds between the donor and acceptor atoms. [1]

- There is a larger surface area of the DNA helix to enable intercalation between the opposite DNA strands
particularly for the helix-turn-helix DNA binding motifs and leucine zipper binding motifs to intercalate
through their extended alpha-helice fingers. [3]

(b) 3 marks

Hydrogen bonding, hydrophobic attraction, van der Waals, dipole-dipole, ionic. [Mention three of these.]

9. (a) 1 mark

CFU-E (Colony forming unit-erythrocyte), or Hemocytoblast. [1]

(b) 4 marks

EPO production is affected by blood oxygen levels. [2]

In response to low blood oxygen levels, EPO production by the kidneys is increased. [2]

(c) 5 marks

EPO stimulates differentiation of CFU-E to become proerythroblast. [1]

Proerythroblast differentiates to early erythroblast where there is increased ribosome synthesis. [1]

The early erythroblast differentiates to late erythroblast in which there is increased hemoglobin accumulation.
[1]

The late erythroblast differentiates to the normoblast that will eject the nucleus to form the reticulocyte. [1]

The final stage is the differentiation of the reticulocyte to the erythrocyte. [1]
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Section C. Answer this question

10. (a) 3 marks

In an autoimmune disease, the body’s immune system comprising of white blood cells that cannot recognize
their own body cells. [1] They will attack its own body cells [1] to cause inflammation [1].

(b) 4 marks

Activated TLR4-/- and MyD88-/-mouse macrophages showed about a 60% decrease and 90% decrease in
p19 gene expression respectively compared to activated WT mouse macrophages. Unactivated macrophages
from WT, TLR4-/- and MyD88-/- mice were unaffected in their p19 gene expression. [2]

Unactivated macrophages from WT, TLR4-/- and MyD88-/- mice had no protein expression of p19. Only
activated WT macrophages had a 10-fold increase in protein expression of p19; activated TLR4-/- and
MyD88-/- mice macrophages showed no protein expression of p19. [2]

(c) 2 marks

Both LPS and PGN activated normal macophages showed a 10 to 12-fold increase in p19 promoter activity. [2]

(d) 4 marks

There was a 6-fold increase in P19 promoter activity observed in the -1178/+10 construct compared to the
controls which is the original -1280/+10 construct. [1]
Further deletions of the -1178/+10 construct that gave the -807/+10 and -413/+10 constructs resulted in the
promoter activity returning to the level of the controls. [1]
Further deletions of the -413/+10 construct that gave the -238/+10 and -168/+10 constructs resulted in almost
complete loss of promoter activity in both constructs which was ~10% of the level of the controls. [2]

(e) 3 marks

In the absence of NF-kB, one distinct band (radiolabeled oligonucleotide probe) at the end of the gel was
observed. [1]
In the presence of NF-kB, two slower migrating bands (closer to top of gel) and the band of the radiolabeled
probe were observed. [2]

(f) 4 marks

Stimulation of IL-23 p19 gene expression by LPS involves both TLR4 and MyD88 intracellular signaling
molecules. [2]
1.3 kb of the IL-23 p19 promoter is activated by LPS in which the sequence from -413 to -238 is sufficient for
promoter activation. [1]
NF-kB appears to be the transcription factor and that there are at least one or two NF-kB binding sites in the
p19 promoter sequence from -807 to +10. [1]

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