MOL 214

Final Exam – Part A

You must also complete the
Module Exam - Part B

May 16, 2013

Your exam code number is:

Write this number on each

page of your exam.

DO NOT write everything you know about a topic, this will waste your time. If you
provide more than one answer for a question only your first answer will be graded.

If you need extra space, continue only on the back of the page that the question is
written on. Clearly label that you are using the back for your answer.

Remember to write legibly, if we can’t read it, we can’t grade it!

You have 3 hours to complete the exam.

“I pledge my honor that I have not violated the Honor Code during this

examination.”

Signature:

Printed Name:
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Multiple Choice 32 points (2 points each):

1. Which of the following statements is true?

a) Disulfide bonds are formed by the cross-linking of methionine residues.
b) Disulfide bonds are formed mainly in proteins that are retained within the
cytosol.
c) Disulfide bonds stabilize a protein’s final conformation.
d) Disulfide bonds are a special kind of hydrogen bond.

2. The phosphorylation of a protein is typically associated with a change in

activity, the assembly of a protein complex, or the triggering of a downstream
signaling cascade. The addition of ubiquitin, a small polypeptide, is another
type of covalent modification that can affect protein function. Ubiquitination
often results in ______________.
a) membrane association
b) protein degradation
c) protein secretion
d) nuclear translocation

3. Transcription in bacteria differs from transcription in a eukaryotic cell

because:

a) RNA polymerase (along with its sigma subunit) can initiate transcription on
its own
b) RNA polymerase (along with its sigma subunit) requires the general
transcription factors to assemble at the promoter before polymerase can
begin transcription
c) The sigma subunit must associate with the appropriate type of RNA
polymerase to produce mRNAs
d) RNA polymerase must be phosphorylated at its C-terminal tail for
transcription to proceed

4. The octameric histone core is composed of four different histone proteins,

assembled in a stepwise manner. Once the core octamer has been formed,
DNA wraps around it to form a nucleosome core particle. Which of the
following histone proteins does not form part of the octameric core?
a) H4
b) H2A
c) H3
d) H1

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5. Which of the following statements about RNA splicing is false?

a) Conventional introns are not found in bacterial genes.
b) For a gene to function properly, every exon must be removed from the
primary transcript in the same fashion on every mRNA molecule produced
from the same gene.
c) Small RNA molecules in the nucleus are part of the splicing machinery
necessary for the removal of introns.
d) Splicing occurs after the 5′ cap has been added to the end of the primary
transcript.

6. A strain of yeast translates mRNA into protein inaccurately. Individual

molecules of a particular protein isolated from this yeast have variations in the
first 11 amino acids compared with the sequence of the same protein isolated
from normal yeast cells, as listed below. What is the most likely cause of this
variation in protein sequence?

a) a mutation in the DNA coding for the protein

b) a mutation in the anticodon of the isoleucine tRNA (tRNAIle)
c) a mutation in the isoleucyl-tRNA synthetase that decreases its ability to
distinguish between different amino acids
d) a mutation in the isoleucyl-tRNA synthetase that decreases its ability to
distinguish between different tRNA molecules

7. Which of the following statements about the lac operon is false?

a) The LacI repressor binds DNA when lactose is present in the cell.
b) Even when the CAP activator is bound to DNA, if lactose is not
present, the lac operon will not be highly transcribed.
c) The CAP activator can only bind DNA when it is bound to cAMP.
d) The lac operon is highly expressed when lactose is present and
glucose is absent.

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8. A DNA library has been constructed by purifying chromosomal DNA from

mice, cutting the DNA with the restriction enzyme NotI, and inserting the
fragments into the NotI site of a plasmid vector. What information cannot be
obtained from this library?
a) gene regulatory sequences
b) intron sequences
c) sequences of the telomeres
d) amino acid sequences of proteins

9. In situ hybridization can be used to determine the _________________.

a) sequence of a cloned gene
b) distribution of proteins in tissues
c) size of a gene
d) distribution of a given type of mRNA in different tissues

10. Which of the following does NOT describe a mechanism by which enzymes
catalyze reactions?
a) The enzyme provides additional energy, making conditions more favorable
for the reaction.
b) The enzyme rearranges the charges in the substrate, making conditions
more favorable for the reaction.
c) The enzyme orients two substrates in a position favorable for the reaction.
d) The enzyme bends the substrate into a conformation that is more
favorable for the reaction.

11. Why did the U.S. withdraw its support for the Biological and Toxin Weapons
Convention?
a) Because the penalties for violating the convention were too strict.
b) Because it is too difficult to verify that countries are not violating the
convention.
c) Because the U.S. would have to contribute too many resources to verify
that countries are not violating the convention.
d) Because the penalties for violating the convention were not strict enough.

12. Which of the following does not occur during M phase in animal cells?
a) growth of the cell
b) condensation of chromosomes
c) breakdown of nuclear envelope
d) attachment of chromosomes to microtubules

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13. MPF activity was discovered when cytoplasm from a Xenopus M-phase cell
was injected into Xenopus oocytes, inducing the oocytes to form a mitotic
spindle. In a control experiment, Xenopus interphase cytoplasm was injected
into oocytes and shown not to induce the formation of a mitotic spindle. Which
of the following statements is not a legitimate conclusion from the control
experiment?
a) The piercing of the oocyte membrane by a needle is insufficient to
cause mitotic spindle formation.
b) An increased volume of cytoplasm is insufficient to cause mitotic
spindle formation.
c) Injection of extra RNA molecules is insufficient to cause mitotic
spindle formation.
d) Components of an interphase nucleus are insufficient to cause
mitotic spindle formation.

14. Which of the situations below will enhance microtubule shrinkage?

a) addition of a drug that enhances GTP hydrolysis by tubulin dimers
b) addition of a drug that inhibits GTP hydrolysis by tubulin dimers
c) addition of a drug that inhibits ATP hydrolysis by tubulin dimers
d) addition of a drug that increases the affinity of tubulin molecules
carrying GDP for other tubulin molecules

15. Which of the following statements is correct?

Kinesins and dyneins ____________________.
a) have tails that bind to the filaments
b) move along both microtubules and actin filaments
c) can move in opposite directions to each other
d) derive their energy from GTP hydrolysis

16. Which of the following statements is false?

a) In mammals, multiple CDKs and cyclins drive the cell cycle.
b) CKIs prevent premature progression through the cell cycle.
c) A gain-of-function mutation in one copy of an oncogene could lead
to cancer.
d) A loss-of-function mutation in one copy of a tumor suppressor gene
could lead to cancer.

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Short Answer: 88 points:

1. Order the DNA sequences listed below according to relative melting
temperatures (from lowest Tm to highest Tm). Assume that they all
begin as stable double-stranded DNA molecules. Explain your answer.
(2 points)
A. GGCGCACC
B. TATTGTCT
C. GACTCCTG
D. CTAACTGG
B, D, C, A (1 point)
Higher GC content means more Hydrogen bonds and more energy
needed to break the bonds. (1 point)

2. Answer the following questions about DNA replication.

A. On a DNA strand that is being synthesized, which end is growing—the 3′
end, the 5′ end, or both ends? Explain your answer. (2 points)
The 3’ end is growing (1 pt)
DNA polymerase can only add nucleotides to a 3’OH or DNA polymerase only
adds nucleotides in a 5’ to 3’ direction.

B. On a DNA strand that is being used as a template, where is the replication

occurring relative to the replication origin—3′ of the origin, 5′, or both?
Explain your answer. (2 points)

Both (1 pt)
Because of the bidirectional nature of replication (or one direction is
leading and the other is lagging (1 pt)

3. Most cells in the body of an adult human lack the telomerase enzyme
because its gene is turned off and is therefore not expressed. An important
step in the conversion of a normal cell into a cancer cell, which circumvents
normal growth control, is the resumption of telomerase expression. Explain
why telomerase might be necessary for the ability of cancer cells to divide
over and over again. (2 points)

In the absence of telomerase the lifespan of a cell and its progeny is limited
because with each round of replication the length of the telomeric DNA will
shrink. If telomerase is provided to cells they can divide indefinitely because
their telomeres will remain a constant length even after each division.

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4. In the absence of glucose, E. coli can grow by using the sugar arabinose. The
genes that encode proteins that allow E. coli to use arabinose, araA, araB,
and araD, are arranged in an operon and are controlled by the same
promoter. The araC gene is located elsewhere and encodes a transcriptional
regulator that binds adjacent to the promoter of the arabinose operon. AraC
also has a binding site for arabinose. To understand the regulatory properties
of the AraC protein, you engineer a mutant bacterium in which the araC gene
has been deleted and look at the effect of the presence or absence of the
AraC protein on the AraA enzyme.

A. If the AraC protein works as a gene repressor, would you expect araA
RNA levels to be high or low in the presence of arabinose in the araC-
mutant cells? Explain your answer (2 points)

High (1 point) because without the repressor araA would be

transcribed/expressed all the time.(1 point)

B. What about in the araC- mutant cells in the absence of arabinose? Explain
your answer. (2 points)

High (1 point) because without the repressor araA would be

transcribed/expressed all the time.(1 point)

C. Your findings from the experiment are summarized below. Do the results
in the table indicate that the AraC protein regulates arabinose metabolism by
acting as a gene repressor or a gene activator? Explain your answer. (3
points)

AraC is an activator (1 point). In the araC- cells the level of AraA RNA is
always low (with or without arabinose), which indicates that transcription
needs to be activated. In the araC+ cells in the presence of arabinose the
transcription of araA increases, further supporting that it is an activator.

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5. In class, we discussed the possibility that RNA was the original hereditary
molecule. Based ONLY on the methods used by Hershey and Chase, in
which the bacteriophage particles were labeled with P32 & S35, what results
would they have seen if RNA, not DNA, was the molecule that carried the
genetic information? (2 points)

They would have seen the same results because P32 labels both RNA and DNA

Briefly describe another classic experiment that was performed to show that
DNA, not RNA, is the molecule that carries the genetic information. (2 points)

The Avery, MacLeod and McCarty experiment (don’t need the names)
The use of DNase and RNase allowed them to distinguish between the two
nucleic acids using the S and R bacteria transformation experiment.

6. Briefly explain what is meant when we say that biological weapons are often
infectious but not contagious. Why might this be a desirable feature of a
biological weapon? (4 points)

It means that although the weaponized disease can infect people it comes
directly into contact with, it cannot spread from person to person. This
would help to prevent unintended targets from being infected.

7. What two factors limit the resolution of an image seen through a microscope?
(2 points)

The wavelength of light and the properties of the lens.

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8. The bacterium Bacillus subtilis sporulates (forms spores) under certain growth
conditions. You have obtained a mutant that no longer sporulates, even
when exposed to these conditions. You have managed to identify the gene
that is mutated, and have replaced it with the GFP gene to create a reporter
construct. You expose the bacteria to different conditions that result in
sporulation, and measure the fluorescence produced. The data is shown
below.

a. What do these results suggest about the expression of your protein of

interest? (2 points)
The protein of interest is expressed at equal levels in all conditions.

b. What conclusion can you make about how the protein of interest affects
sporulation? (2 points)

It must affect sporulation through some mechanism other than a change in

expression.

You have managed to make an antibody to your protein of interest, and you
run an SDS-PAGE gel and you perform a Western blot of unmutated cell
extracts in the same 3 conditions used above. The results are shown below.

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c. Based on the results of the western blot, suggest a hypothesis about how
your protein of interest affects sporulation. (2 points)

In conditions that promote sporulation, the protein of interest is phosphorylated

(other post translational modifications were also accepted for full credit).
No credit for cleavage or dissociation of a complex.

d. Suggest an experiment that you could perform to prove or disprove the

hypothesis you suggested in part c. (2 points)

Treat the cell extract with phosphatase and run a gel to see if the upper band
goes away.

9. Thymosin is a protein that can bind actin monomers. If you were to add a
drug that inhibits the ability of thymosin to bind actin monomers, what effect
would this have on actin polymerization? Explain your answer. (3 points)

Addition of a drug that keeps thymosin from binding actin monomer will
increase the rate of actin polymerization in the cell. In this case, free actin
monomers are bound by thymosin and are thereby prevented from adding to
the end of an actin filament

(Also accepted credit for decreased as long as the answer states that
thymosin helps create seeds.)

10. Mutations in p53 are associated with >50% of cancers. What kind of protein
is p53? What does p53 do in response to stress like DNA damage? (3 points)

p53 is a tumor suppressor. In response to stress, p53 can arrest the cell
cycle or induce apoptosis.

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11. Your labmate is trying to clone a gene into a

plasmid (pictured here). He has added EcoRV
sites to either end of the gene using PCR as
shown (the start codon of the gene is closest to
the side labeled “1”), digested, ligated the PCR
product to the vector, and transformed the
ligation into bacteria. He wants to ensure that
the gene is in the correct orientation relative to
the promoter. In his attempts to determine this,
he performed a plasmid prep and digested the
purified plasmid with 4 different restriction
enzymes: EcoRV, HaeIII, PvuII, and AluI.
Unfortunately, while he was incubating the tubes
in the water bath, the labels washed off, and he
doesn’t know which tube corresponds to which
enzyme. He runs the digests on an agarose gel
anyway, and asks for your help in understanding
the results.

Label the lanes shown below with the restriction enzyme used for the digestion.
(4 points)

HaeIII AluI PvuII EcoRV

You tell him that you can determine whether the gene is cloned into the
correct orientation using only one of the digests. Which one? (1 point)

PvuII

Based on his gel, is the gene in the correct orientation? (1 point)

Yes

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12. The E. coli MalE protein is synthesized in the cytoplasm and then secreted
across the bacterial inner membrane through the Sec channel. As part of
your thesis project, you are characterizing the E. coli mutant strain prlD21.
Pictured below are the results from a pulse-chase experiment tracking
secretion of MalE in wild-type and prlD21 mutant cells. After pulsing the cells
with radioactive (hot) amino acids, you add non-radioactive (cold) amino
acids, collect protein extracts (at 10 sec, 30 sec, 1 min, etc.), and analyze the
extracts by SDS-PAGE.

A. Explain the rationale behind a pulse-chase experiment. What are you

measuring? (2 points)
The rationale behind a pulse-chase experiment is that you can follow protein
made at a specific time as it matures or is secreted. This means that you can
tell the difference between 2 possible hypotheses: 1) that the protein takes
longer to reach certain areas and 2) that the protein is actually moving from
one area/organelle to another.

B. Why is there a size difference between pMalE and mMalE? (2 point)

There is a size difference because the signal sequence has been cleaved in
the mMalE

C. Which strain is able to export MalE most efficiently? Explain your

reasoning. (2 points)

Wildtype is able to export MalE more efficiently, because there is more

mMalE in the wildtype and/or very little pMalE

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13. You are investigating the synthesis and trafficking of phospholipids in your favorite
eukaryotic cell line. You have devised an experimental system in which you can
feed your cells radioactive phosphorus, then break open the cells, isolate the various
organelles by density centrifugation and measure the radioactivity in different
organelles and membranes. An experiment where you add radioactive phosphate at
time zero and then analyze the cells over time gives the following data (ER =
endoplasmic reticulum, MT = mitochondrion, PM = plasma membrane):
160

140

120
CPM / mg lipid

100 ER
Golgi
80
MT
60 PM

40

20

0
0 10 20 30 40 50

Time (min)

A. Provide two different explanations for the pathway of phospholipid synthesis

and trafficking that are consistent with these data. (4 points)

Phospholipids are synthesized in the ER, then trafficked to the Golgi, the
mitochondrion and the plasma membrane in a linear pathway. OR, some
variation of: phospholipids are synthesized in the ER then trafficked to the
different membranes independently at different rates

B. In an effort to resolve this issue, you conduct another experiment in which

you add a small amount of radioactive phosphate at time zero, then a much
larger amount of unlabeled phosphate five minutes later. This time, you get
data like this:
60

50
CPM / mg lipid

40
ER
Golgi
30
MT
PM
20

10

0
0 10 20 30 40 50

Time (min)

Based on this result, what is the pathway for phospholipid synthesis and
trafficking? (2 points)

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synthesized in ER  Golgi  mitochondrion (faster) and PM

(slower)
14. You are attempting to replicate the Brainbow experiment. To do this, you
have created your own Brainbow construct, which is similar to the original
Brainbow construct, and it is pictured below. The patterned triangles
represent lox sites in this diagram, and the fluorescent proteins are Green
Fluorescent Protein (GFP), Cyan Fluorescent Protein (CFP), Red Fluorescent
Protein (RFP), and Orange Fluorescent Protein (OFP).

If recombination in this construct occurred between the two lox sites

represented by striped triangles, what would the resulting construct look
like? Draw it here: (2 points)

What color would the neuron expressing the recombined construct be? (1
point)

Red

15. The phosphorylation status of CDK is important for regulating cell cycle
progression. Why are cdc25 mutants arrested in the cell cycle, while wee1
mutants enter the cell cycle prematurely? (3 points)

Normally, the Cdc25 phosphatase removes the inhibitory phosphate

from CDK leading to activation. The wee1 mutation is a gain-of-
function mutation that results in the Wee1 kinase adding the inhibitory
phosphate to CDK. In both cases, the cyclin/CDK complex is in the
inactive state.

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16. To characterize the cell cycle of a newly discovered strain of yeast, you have
generated a library of cold-sensitive mutants and have started to take
photographs of interesting mutants. You have checked to make sure that
each strain in the library carries only a single mutation.

A. Mutant A stops growing when it is moved to cold temperature.

Measurements indicated that the cells contained a G2 amount of DNA.
You took pictures of the Mutant A cells before and after shifting them into
the cold. Although the cells were initially at all stages of the cell cycle, the
cells arrested as either one or two large-budded cells (see diagram below)
after the shift. What is the explanation for the different ways that the cells
arrest? (3 points)

Small budded cells are before the “execution point” when the protein is
required. (1.5 pts) Larger budded cells are already past the point and
divide again. After cells traverse the execution point they arrest in
G2/M as large budded cells. (1.5 points)

B. Mutant B also stops growing when moved to the cold temperature, but
under the microscope it appears that half of the cells arrested at one point
in the cell cycle while the other half are arrested at a different point. How
could you explain this result? (2 points)
The mutated gene is acts at two different checkpoints. Since the
population of cells is asynchronous, individual cells in the population are at
different stages of the cell cycle and will arrest at whatever checkpoint
they hit first.

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Mystery of the TA eating in lab

We have tried all semester to remind students that food is not allowed in lab. Shockingly,
when the staff opened the lab last Monday morning they found a partially eaten pizza
and large drink sitting on the TA desk in lab. We are convinced that the students did not
bring the pizza to lab, and are equally convinced that one of the TAs left it there over the
weekend. Each of the TAs claims that the pizza is not theirs! Luckily, we were able to
isolate some DNA from a partially eaten slice of pizza and mRNA from some hair root
cells attached to a hair found on the pizza. Please help us identify who was eating in the
lab last weekend.

Below is a table for you to keep track of whom you eliminate as suspects.

Suspect Microarray SNP STR Sequencing

Colleen
Kenric
Amanda
Anne
Julia
Jean
Geoff
Leslie
Nick
Daniela

1. You decide to use microarray analysis to attempt to identify the culprit, because you
remember from class that this will allow you to examine many genes at once. You
are able to isolate RNA from the hair root cells attached to the hair found on the
pizza. And you collect a check cell sample from each of the TAs.
a. If you isolate total RNA from the cells, what property of mRNA could you use
to separate mRNA from the rest of the RNA? (1 point)

The poly A tail

b. Name and briefly describe the function of two other types of RNA (i.e.- not
mRNA) found in the cell. (2 points)

tRNA – used in translation, links the amino acids to the correct codon
rRNA – part of the ribosome
miRNA – regulate gene expression
snRNA – part of splicing complex

c. To perform the microarray experiment, you first make cDNA from the mRNA
from the cells of the culprit and the TAs. What is the name of the enzyme
you use? (1 point)
Reverse Transcriptase

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d. Before you even start to perform the microarray experiment you lab partner
insists that the experiment won’t work to identify the culprit. After thinking it
over you agree. Give one reason why the experiment as outlined above
would not be helpful to identify the culprit. (2 points)
Possible answers:
You are comparing expression of genes in two different cell types (hair vs.
cheek cell)
Levels of gene expression are likely to be similar in different people, so it will
hard to identify a match with the culprit
Microarrays only show relative trends or relative changes in expression

2. You decide to start over and use the DNA samples that you collected from the culprit
and the TAs. You decide to investigate a gene, Gene T, which has a single
nucleotide polymorphism (SNP) that creates a site recognized by the restriction
enzyme, PstI. Even though this SNP is relatively common, you hope it will help you
identify the culprit.

a. Below is the portion of Gene T that has the SNP. In some people, the circled
G:C basepair is changed to generate the PstI site. Based on what you learned
about the general characteristics of restriction enzyme recognition sites, what is
the basepair change that these people possess that generates a PstI site? (1
point) G to A and C to T

5’ – C T G C G G – 3’
3’ – G A C G C C – 5’
You use PCR to amplify a 500 bp region of gene T that includes this SNP at the
200bp position. You then digest the PCR products with the enzyme PstI. Below
are the results of this experiment.

b. Knowing that the culprit is heterozygous for the SNP in Gene T, whom can you
eliminate as a suspect based on these data? Why? (2 points)
Colleen, Kenric, Julia, Jean, Leslie -- -1 point if only Colleen and Julia are mentioned.

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3. You still have multiple suspects, so you decide to do another test. This time, you do
a PCR-based test for a STR (short terminal repeat). At chromosomal region Z, it is
common to find one or more copies of a 25 bp sequence.
The first step in doing PCR is designing primers. Below, the two lines represent the
two strands of DNA at chromosomal region Z. The thicker region represents region Z
and is the sequence you want to amplify. Using short arrows, draw on the diagram
below where you would like your primers to anneal. (3 points)
• label the 5’ and 3’ ends of both primers
• label the 5’ and 3’ ends of the template DNA

5’ 3’
3’ 5’
(25 bp)n
5’ 3’
3’ 5’

The length of region Z without an insert is 100 bp. Below are the results of your experiment.

Unfortunately, you ran the culprit’s sample on a separate gel and forgot to add a DNA
ladder to that gel. All you know for certain is that the culprit who ate the food in lab is
homozygous for the number of repeats for this STR at chromosomal region Z. Whom
can you eliminate as a suspect after performing this experiment? (2 points)

Kenric, Anne, Julia, Leslie, Daniela

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Finally, another friend from class suggests that you sequence the DNA to identify the
culprit. Since you don’t have enough money to sequence the entire genomes of all the
TAs, you choose one small region of DNA to sequence. Below is a gel that represents
the results from sequencing a portion of Gene X from the culprit:

ddATP ddCTP ddTTP ddGTP

+
a. Write the double-stranded DNA sequence for this region of Gene S as shown in
the sequencing gel above. Indicate the 5’ and 3’ ends of the DNA sequence. (2
points)

5’ AGCATCTAG 3’
3’ TCGTAGATC 5’

b. Is it possible that you sequenced the beginning of the protein-encoding region of

the gene? Justify your answer. (2 points)

Yes, because there is an ATG in the 5’ to 3’ direction

The sequencing results from three of the TAs: Amanda, Colleen and Leslie all
perfectly matched the culprit.
Based on all the results, which TA left the pizza in the lab? (1 point)

Amanda

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