Solution Key- 7.

013 EXAM 2 (4 / 8 / 15)

Question 1 (22 points)
a) Shown below is a segment of replicating DNA in the skin cells of a mouse. The direction of
movement of the replication fork and the alignment of a
3
primer is shown.
Primer
i. Label the 5 and 3 ends of the replicating DNA
5 Top Replication strands by filling in the shaded boxes. (2pts)
fork
Bottom ii. Which strand (top or bottom) is the template for
lagging strand synthesis? (3pts)
5

b) You isolate skin cells from a normal healthy mouse and culture them in two plates (1 & 2). You add
Drug 1 to Plate 1 and Drug 2 to Plate 2.

Drug 1 is 2- chloroethyl nitrosouria (CNU) that ACTIVATES DNA methylase

Drug 2 is valproic acid that decreases the affinity (binding strength) of histones for DNA.
Which treatment (choose from Drug 1 or Drug 2) will promote transcription in skin cells? Explain
why you selected this drug over the other.
Condensed packing of chromatin is promoted by the binding of histones to DNA and also by the
methylation of bases. If the chromatin is NOT condensed, the base sequences are exposed and
available for the transcription factors and other proteins to bind and initiate transcription. Drug 2
promotes transcription by promoting the chromatin to move from the condensed state to an open/ loose
state thus exposing these base sequences (4pts with 2 for explain)

d) The Retinoblastoma protein (Rb) is a transcription factor. This protein is active when unphosphorylated
in the G1 phase of cell cycle and prevents entry of the cell into S phase. The CyclinD-Cdk4 complex
phosphorylates and inactivates Rb allowing the cell to enter the S phase and complete cell cycle. The CyclinD-
Cdk4 complex is inhibited by p21 protein as shown below. Note: A T sign in the schematic represents inhibition.

CyclinD- i. What will be the ploidy of a mutant somatic

Cdk4 Rb S-Phase cell that has a constitutively active Rb (n/
complex 2n/ 4n)? (2pts)

ii. Which protein is expressed at maximal levels only at the G1/S

p21 checkpoint of cell cycle (CyclinD/ Cdk4/ p21/ Rb)?

Each cyclin is expressed and active only at specific cell cycle checkpoint. (3pts)

iii. Would a p21 temperature sensitive (inactivating) mutant, grown at the non- permissive
temperature (42oC), divide less rapidly, more rapidly or about the same compared to the cells
grown at the permissive temperature (37oC)? Explain your choice.
Inactivation of p21, promotes the formation ofCyclinD- Cdk4 complex, which inhibit Rb thus allowing the
cell to move from G1->S phase of the cell cycle (4pts, 2pts for explain)

e) Answer the following questions using RNA polymerase/ DNA polymerase/telomerase as the
options.
i. Which of the above enzymes carries its own primer? Telomerase (2pts)
ii. Is active in normal non- dividing adult cells? RNA polymerase (2pts)

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Question 2 (20 points)
The following is the DNA sequence for the transcription initiation region of Cyclin D gene in mice.
Note: Part of the promoter region is boxed. Transcription begins at and includes the bold & underlined A/T
base pair.
#1 #2 #3
5---TGGACTGCTATAATAGCAGTTCAGCTGAGATGACGAAGATTTGGCCAGGGTTCTTAAAGT--3 TOP
3---ACCTGACGATATTATCGTCAAGTCGACTCTACTGCTTCTAAACCGGTCCCAAGAATTTCA--5BOTTOM

a) Write the first 6 nucleotides of the nascent (newly synthesized) Cyclin D mRNA.
5AGCUGA3(3pts)

b) Write the first 3 amino acids of the newly synthesized Cyclin D protein.

N-Met-Thr-Lys-C (3pts)

c) You create homozygous mutants 1, 2 and 3 by substituting the G/C base pairs (bold and shaded) at
positions 1, 2 and3 by an A/T base pair as shown below.

G A (TOP)
C T (BOTTOM)

i. Which mutant (1/ 2/ 3) synthesizes full length Cyclin D protein but at a reduced amount?
Explain why you selected this mutant.
Mutations in the regulatory regions such as promoter sequence (as in Mutant #1) can influence the
amount without altering the sequence of the gene product. (3pts, with 1pt for explain)

ii. Of the remaining two mutants, identify the one that has a silent mutation. Explain why you
selected this mutant.
Mutant #2, Both 5AAA3 and 5AAG3 codons code for Lys making this a silent mutation (3pts, 2 for
explain)

d) You identify a disorder in mice in which the Cyclin D gene is NOT translated. Further analyses
reveal that the Cyclin D gene in affected and normal mice has the IDENTICAL gene sequence. Circle
the option(s), from below that could explain why Cyclin D is NOT translated in the cells of the affected
mice.
1. The cellular Capping enzyme which attaches the 5 methyl guanosine Cap is inactive.
2. The ribosome- binding site in the mature mRNA transcript corresponding to CyclinD gene is mutated.
3. The affected mouse inherits only the maternally imprinted copy of Cyclin D gene.
4. Splice donor and splice acceptor sites of the introns within Cyclin D gene are mutated.
Explain why you selected this option(s).
As per option #1, if the mRNA transcript is not capped it will have a reduced stability and will be
degraded before it can be translated. As per option #2, if the gene is imprinted, it is methylated and so
silenced. As the gene is not transcribed, there will be no translation. (5pts, 2pts for selection and 3
for explain)

e) Sometimes a gene may encode two different proteins in two different cell types. Provide one
explanation that supports this statement.
Alternative splicing, overlapping frames, protein modifications, alternative promoters may result in
different mature mRNA transcripts each of which can produce a specific protein (3pts)

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Question 3 (23 points)
Page 7 (which you can detach) has a schematic of Wnt secretory protein ligand mediated cell
signaling that triggers Cyclin D gene expression, which is critical for cell cycle.

a) Consider the following mutations in different components of the signaling pathway.

Mutant #1: Frizzled receptor lacks its ligand- binding domain (3pts)
Mutant #2: Gsk3 lacks its kinase domain (3pts)
Mutant #3: Cyclin D gene has a constitutively active promoter (3pts)

Complete the table for each of the following homozygous mutations in the presence of wnt ligand.

Mutants Dsh active Gsk3 catenin CyclinD Cell proliferation

(Yes/ No)? active (Yes/ stablized (Yes/ expressed (Yes/ (Yes/ No)?
No)? No)? No)?

#1 No Yes No No No

#2 Yes No Yes Yes Yes

#3 Yes No Yes Yes Yes

b) You identify a mutant cell, which produces misfolded - catenin.

i. Propose a mechanism that would help the misfolded protein to fold correctly.
This is done with chaperones, which are involved in proper folding of the proteins and also prevent the
misfolded proteins from aggregating. (2pts)

ii. Circle the correct option and explain why you selected this option. Proteasome mediated
degradation of misfolded proteins is an example of a (hydrolysis/ dehydration) reaction.
The proteasome degrades the protein, by breaking the peptide bonds that lead to the primary structure
together, through a hydrolysis reaction (3pts)

iii. From the options below, circle one that serves as monomer of wnt protein ligand. (1pt)

Amino acid Nucleotide Phospholipid Monosaccharide

c) Of the components listed in the signaling pathway (wnt / Frizzled/ Dsh/ Gsk3 / - catenin/
cyclinD), list two whose translation requires BOTH the cytoplasmic ribosomes and also the ribosomes
on the membrane of endoplasmic reticulum. Explain why you selected this option.
Wnt is a ligand being made and secreted by some other cell and frizzled is a cell membrane receptor
protein. So their translation starts in the cytosol using the cytosolic ribosomes but is completed on the
ER membrane using the ER ribosomes. (4pts, 1 for ach and 2 for explanation)

d) Circle the correct option. The signal sequence located at the N- terminus of Wnt ligand corresponds
to the base sequence at the (3 or 5) end of the corresponding mRNA, which is complementary to the
bases at the (3 or 5) of the DNA strand that has been used as a template for transcription.
(4pts or 2pts for each)

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Question 4 (23 points)
a) You use Cyclin D mRNA and an oligo dT (5TTTTTTTTT3) primer to prepare Cyclin D cDNA (first
strand).
i. What feature does the mature mRNA have that allows you to use this primer?
It base pairs with the 3poly-A tail (2pts)

ii. Where does the primer bind on the mRNA (close to the 5end/ close to the 3/in the middle
of mRNA)? (2pts)

CyclinD double-stranded cDNA has recognition sites for restriction enzymes X and Y as shown below.
You want to clone part of the cDNA into the following plasmid that has recognition sites for restriction
enzyme B, Y and A as shown. Note: A slash (/) represents the recognition and cut site for each restriction
enzyme. X Y X Bacterial
promoter
5 3
3 2kb 1kb
5
B
Size: 5Kb lacZ

Direction of transcription A
0.5kb
Bacterial
B A X Y Ori
5T/AATTA3 5GAATT/C3 5CAATT/G3 5GCCTT/C3 Y
3ATTAA/T5 3C/TTAAG5 3G/TTAAC5 3C/GGAAG5 Ampr
R
Note: Amp represents the ampicillin resistance gene. The lacZ gene
encodes an enzyme that converts white X-gal substrate into blue color
product
b) You cut the CyclinD cDNA with restriction enzyme X.

i. With which restriction enzyme (B/ A/ Y) would you cut the plasmid in order to insert CyclinD
cDNA into the plasmid? (2pts)

ii. Write the resulting 6- base pair sequences at the points of ligation of the plasmid and CyclinD
cDNA. (3pts) 5CAATTC3
5GAATTG3 CyclinD cDNA
3CTTAAC5 3GTTAAG5

c) You transform E. coli with the ligation mix in order to amplify your clones.
i. What medium would you use to select transformed E. coli cells? Medium with ampicillin (2pts)

ii. How would you screen for transformed E. coli cells that contain the recombinant plasmid?
What medium would help you do so?
Grow the transformed bacterial cells grow on medium with X- gal. Those transformed with recombinant
plasmids will have a disrupted LacZ gene. They will not convert X-gal to blue color and will appear as
white colonies. In comparison, those transformed with self-ligated plasmids (or those without an insert)
will have intact LacZ gene and will form blue colonies. (3pts)

d) You purify recombinant plasmids from two E. coli colonies (Colony 1 that has plasmid 1 and Colony
2 that has plasmid 2). You digest both plasmids with Restriction enzyme Y and resolve the digested
fragments by DNA gel electrophoresis. You observe the following DNA fragments on the gel.

Recombinant plasmid 1 from Colony 1 gives fragments of 1.5kb and 6.5 kb.
Recombinant plasmid 2 from Colony 2 gives fragments of 5.5kb and 2.5kb.

Which colony (1 or 2) can produce CyclinD RNA? Explain why you selected this colony.
Based on the size of the fragments the CyclinD cDNA in colony 1 is inserted in the right orientation with
respect to the promoter. So it will be transcribed to give cyclinD RNA (3pts)

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Question 4 continued
e) The following plasmid sequence flanks the CyclinD cDNA after cloning. (2pts or 1 each)
5TGCATCAGCTATTACC CyclinD GCACTTCCCGGGTAGG3Top strand
3ACGTAGTCGATAATGG cDNA CGTGAAGGGCCCATCC5Bottom strand
From the choices below, circle the primers they could use to PCR amplify CyclinD cDNA gene.

#1: 5TGCATCAGC3 #2: 5CCTACCCGG3 #3: 5GCTGATGCA3 #4: 5GCACTTCCC3

1 2 3
f) The first three amino acids of the wild type CyclinD sequence are N- Met -Trp -Gly ----C. A
CyclinD mutant that is associated with failure to undergo cell division yields dideoxy sequencing
fragments of the following lengths and ends corresponding to
5 A the first three codons. Explain what is wrong with the mutant
T that prevents it from undergoing cell division.
G The coding region of CyclinD mutant shows an insertion of A
T
as the 5th base making the 2nd codon a stop codon. (4pts)
A
G
G
G
G
3

Question 5 (12 points)

You are studying the following pedigree for anosmia (loss of ability to smell) that is associated with too
little cell division in the cells that become olfactory (smell) sensor cells. Assume that anosmia is caused
by mutation in Cyclin D gene. Please note: All affected individuals are shaded. Individuals 3, 8 and 10
marrying into the family do not have the disease associated alleles of the Cyclin D gene. Also listed are
the SNPs for some individuals in the pedigree.

a) You use SNP analysis to explore the mode of

inheritance of anosmia. Assume complete linkage
of CyclinD gene and the given SNP. What is the
mode of inheritence of anosmia?
(A, C) (A, A) Autosomal dominant (3pts)

b) What is the specific SNP(s) that is associated

with this disease?
A SNP (3pts)

c) Using somatic cell gene therapy you successfully alleviate the symptoms of anosmia in Individual
4. If she also has a daughter with Individual #3 what is the probability of her daughter having anosmia?
The ratio of the daughter being healthy or having anosmia is 1: 1. So the probability of her having the
disease is 50%. (3pts)

d) In all individuals, olfactory sensor cells undergo programmed cell death due to activation of
caspases. SC79 is a drug that activates the BCL-2 protein, where BCL-2 inhibits apoptosis. Would you
expect patients treated with SC79 to show improvement of their symptoms? Explain.
Yes, since this treatment will inhibit programed cell death or apoptosis. (3pts)

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 Wnt Signaling pathway for Question 3. You can detach this page.

Frizzled
receptor
Wnt ligand

Cell cycle CELL MEMBRANE

Dsh
CyclinD gene transcription
-catenin
NUCLEUS
GSK3

CYTOPLASM

-catenin

Wnt ligand binds to and activates the frizzled receptor in the membrane of target cell.
Active frizzled binds to and activates disheveled (Dsh).
Active Dsh phosphorylates and inhibits GSK3, a kinase.
When unphosphorylated and active, GSK3 phosphorylates the transcription factor -catenin
(cat). The phosphorylated - catenin is unstable and gets degraded.
Wnt signaling promotes -catenin stability and translocation to the nucleus.
-catenin promotes the transcription of Cyclin D gene thus promoting cell proliferation.