MCBI (BIOL-UA 21) NAME NetID (e.g.

, ms4131)
Midterm 1
October 4, 2024

Write concisely and legibly in the spaces provided!!! You do not need any more room for your
answers than what is provided. Write your name and NetID on each page.

PART I (short answer, limited partial credit, 5 pts each):

1. There are several similarities between nucleic acid and protein polymers including their use of
non-covalent bonds.

a) What is the role of hydrogen bonds in maintaining DNA structure? [2 pts]

Keeps the 2 DNA strands together

b) Name one non-covalent interaction apart from hydrogen bonding that is important in
maintaining protein structure. [1 pt]

Electrostatic / ionic or Hydrophobic

c) To which end of a growing nucleic acid chain are new nucleotides added? Write 5’, 3’, or
either. [1 pt]

3’

d) To which end of a growing peptide chain are new amino acids added? Write C-terminal, N-
terminal, or either [1 pt]

C-terminal

2. The sequence below is from the template DNA strand.

a) What is the sequence of the RNA transcribed from this strand? [2.5 pts]

5’- A U G U U C A C C G C G C A U

b) What is the sequence of the resulting protein translated from this RNA? [2.5 pts]

Met-Phe-Thr-Ala-His

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MCBI (BIOL-UA 21) NAME NetID (e.g., ms4131)
Midterm 1
October 4, 2024

3. The image below shows an electron micrograph of part of a chromosome from a developing
egg, with a gene being transcribed.

a) What do the numbered arrows point to? Add the appropriate number in the relevant box.
(Note that some boxes do not have corresponding numbers; leave those blank) [4 pts]

Translation initiation site

Translation termination site

Transcription initiation site 2

Transcription termination site 1

Promoter 3

b) Given that this is a eukaryotic cell, are the black dots at the end of transcribed RNA
ribosomes? Write “yes” or “no” in the box. [1 pt]

no

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MCBI (BIOL-UA 21) NAME NetID (e.g., ms4131)
Midterm 1
October 4, 2024

4. You want to radioactively label a DNA fragment to use on a Northern blot. There are various
sites that you could use to cut the DNA fragment and then use DNA polymerase to fill in the
ends and incorporate a radioactive nucleotide.

a) Circle the enzyme(s) below that are suitable for this kind of filling reaction. [3 pts]
SfuI

b) For one of the enzymes above that is appropriate, write the name of one dNTP in the box
below that you would choose to be radioactively labeled to label this DNA fragment. [2 pts]

dCTP or dGTP

5. You are studying an organism with a single origin of replication and a genome of 3,600,000
bases organized as a circular chromosome.

a) How long will it take to replicate all of its DNA assuming that a replication fork moves at 1000
bases / second? Leaving your answer in unreduced form as a fraction is acceptable (but make
sure to include units). [3 pts]

1000 bases / second = 60,000 bases / minute = 3,600,000 bases / hour

But there are 2 forks per replication origin – therefore, 7.2M bases / hour
 30 minutes

b) DNA polymerase can only add a nucleotide to the end of an existing nucleic acid polymer.
This creates a problem for the initiation of DNA synthesis. As concisely as possible, explain how
the replication machinery solves this problem. [2 pts]

DNA polymerase needs a primer to begin polymerization

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MCBI (BIOL-UA 21) NAME NetID (e.g., ms4131)
Midterm 1
October 4, 2024

6. Complete this diagram of a eukaryotic mRNA below by adding the names of the missing
pieces in the boxes. [5 pts]

5’ UTR (or 5’ untranslated region)

Coding region
PolyA tail

7. This is the sequence of a small piece of DNA to be amplified by PCR

a) In the box below, write the sequence of a 5 base forward primer to amplify this entire
sequence [2 pts]

5’-G T A C T

b) In the box below, write the sequence of a 5 base reverse primer to amplify this entire
sequence [2 pts]

5’-T A A G G

c) In the box below, write how long the amplified PCR product will be, with units [1 pt]

15 bp

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MCBI (BIOL-UA 21) NAME NetID (e.g., ms4131)
Midterm 1
October 4, 2024

8. This diagram shows RNA polymerase during transcription.

a. Which arrow points to the 5ʹ end of the growing RNA molecule? (Write the letter in the box)
[1.5 pts]

b. Which arrow points to the 5ʹ end of the template strand? (Write the letter in the box) [1.5 pts]

c. Which direction is the RNA polymerase headed as it continues to elongate? Write “Left” or
“Right” in the box. [2 pts]

RIGHT

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MCBI (BIOL-UA 21) NAME NetID (e.g., ms4131)
Midterm 1
October 4, 2024

PART II (long answer, partial credit given so show your work, 12 pts each):

9. You identify a new toxin, Inh, that works by inhibiting transcription in eukaryotic cells by
binding to RNA Polymerase II.

The Kd of Inh and RNA polymerase II is 1.25 x 10^-7 M. A typical yeast cell has a volume of 4 x
10^-14 L and contains about 30,000 RNA Polymerase II molecules. Recall that Avogadro’s
number is ~6 x 10^23 molecules/mole.

a) What is the total RNA polymerase concentration inside a cell? Show your work. [4 pts]

(3 x 10^4 molecules / cell) * (1 mole / 6 x 10^23 molecules) * (1 cell / 4*10^-14 L) = 1/8 x

10^-5 moles/L = 1.25 x 10^-6 M

b) What concentration of Inh is needed inside the yeast cell in order for 80% of all RNA
Polymerase II molecules in the cell to be bound to Inh? Show your work [4 pts]

RNA Pol II Inh Complex

Initial 1.25 x 10^-6 M x 0
Change in Conc -y -y +y
Concentration at Eq 1.25 x 10^-6 M-y x-y y
y = [Inh-Pol] = 0.8*1.25 x 10^-6 M = 1 x 10^-6 M
Kd = 1.25*10^-7 M = (1.25 x 10^-6 M-y)*(x-y)/y = 2/8 * (x – 1 x 10^-6 M)
x = initial [Inh] = 5*10^-7 M + 1 x 10^-6 M

total Inh concentration inside cell = [Inh]+[Inh-Pol] = 1.5 x 10^-6M

c) At the concentration above, what proportion of all Inh in the cell is bound to RNA Polymerase
II? [2 pts]

[Inh-Pol]/( [Inh]+[Inh-Pol]) = 1 x 10^-6 M/1.5 x 10^-6M = 2/3

d) Your lab isolates a mutant version of RNA polymerase II, Pol*; the Kd of Inh and Pol* is 8 x
10^-7. At the concentration of Inh calculated in (b), is
the percent Pol* that is bound to Inh higher or lower LOWER
than 80%? [2 pts]

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MCBI (BIOL-UA 21) NAME NetID (e.g., ms4131)
Midterm 1
October 4, 2024

10. Below are written the recognition sites for a number of restriction enzymes that cut GC-rich
DNA sequences.

a) Find two pairs of enzymes that produce compatible ends from the list and write their names in
the boxes below: [4 pts]

Pair 1: EagI and NotI

Pair 2: NaeI and SmaI

You want to clone the 3kb fragment shown below on the left with ends that have been cut by
EcoRI into a 3kb plasmid vector (on the right) that has been cut with EcoRI at a site in its
polylinker.

b) You isolate a number of colonies from bacteria and want to determine if you have managed
to insert the fragment into the vector. Your labmate suggests that you digest the plasmids with
EcoRI. Why is this not a definitive test to see if the resulting plasmid contains the inserted
fragment? [1 pt]

The vector is the same size as the insert, so we would rely on band intensity to tell the
difference.

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MCBI (BIOL-UA 21) NAME NetID (e.g., ms4131)
Midterm 1
October 4, 2024

c) The fragment can insert in either orientation. Which single enzyme or combination of
enzymes would you use in a single reaction to simultaneously determine (i) whether the
plasmid has an insert; and (ii) which orientation the fragment has inserted. Write your answer in
the box on the left below, and then draw the outcomes of the reaction that you chose on the gel
below with the sizes of the fragments. Lane 1: Vector with no insert. Lanes 2 and 3: Vector with
insert in the 2 opposite orientations. Label the sizes of the bands at the side of the gel. [7 pts]

Enzyme(s):
BamHI + XbaI (gel on the left)
OR
XhoI + XbaI (gel on the right)

Lane 1 Lane 2 Lane 3

4500

3750

3000

1500

750

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MCBI (BIOL-UA 21) NAME NetID (e.g., ms4131)
Midterm 1
October 4, 2024

11. You are studying expression of the New1 gene in different tissues of mice. You use
Northern blotting. The first step is to run a gel that includes formamide, which is a Hydrogen
bond donor and acceptor.

a) Given that RNA is single-stranded, why is it important to include formamide in the gel? [1.5
pts]

Formamide removes / denatures RNA secondary structure so that RNA molecules run
according to their size not structure.

You isolate mRNA from the brain, the heart, the kidney and the lungs. You then probe 3
identical blots with probes complementary to exon 1 (left blot), exon 2 (middle blot), and exon 3
(right blot). These are the only exons in the New1 gene.

The results are shown below with mRNA from the brain in lane 1, from heart in lane 2, from
kidney in lane 3, and from lungs in lane 4.

b) What can you conclude about expression of New1 in the brain? [1.5 pts]

New1 is not expressed in the brain (or expressed at very low / undetectable levels)

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MCBI (BIOL-UA 21) NAME NetID (e.g., ms4131)
Midterm 1
October 4, 2024

c) What control could you perform to ensure that there is mRNA in the brain lane? [1.5 pts]

Probe with a sequence complementary to a gene known to be expressed in the brain

d) How many different transcriptional start sites are used in these different tissues? [1.5 pts]

e) Assuming that the New1 polyA tail is always 200 bases long, what is the length of each
exon? Hint: draw out the structure of each mRNA to help you answer this question. [4.5 pts]

Exon 1: 500bp/bases/nuc Exon 2: 300bp/bases/nuc Exon 3: 100bp/bases/nuc

)
f) What would you expect to see if you probed a Northern blot with a labeled 200 base probe
made of Ts? [1.5 pts]

A smear as this sequence can bind to all mRNAs with a polyA tail.

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MCBI (BIOL-UA 21) NAME NetID (e.g., ms4131)
Midterm 1
October 4, 2024

12. There are many parts of a gene that can become mutated to cause disease. Some
mutations are in the coding region of a gene and change the resulting protein. However, other
mutations are in non-coding parts of the gene, but still affect protein production.

You are interested in mutations that cause thalassemia, a type of anemia, that are in non-coding
regions of the Hemoglobin A1 (HbA1) gene. You decide to PCR amplify the HbA1 genomic
region from a number of thalassemia patients, and then sequence the PCR products to search
for mutations.

a) Which of the following are essential for a PCR reaction? In the box below, list all the letters
corresponding to essential components. [3 pts]

A. Forward and reverse primers

B. ATP
C. dATP
D. dCTP
E. dGTP
F. dTTP
G. Radioactive dATP A,C,D,E,F
H. DNA ligase

b) In one patient, you find that the TATAA sequence in the promoter of the HbA1 gene is
mutated to a sequence that no longer recruits TATA-binding protein. Give a brief explanation at
the molecular level of how this could affect Hemoglobin production and cause thalassemia [3
pts]

Reduced transcription of the HbA1 gene. Less transcription means less mRNA and less
protein

c) In a second patient, you find that the G U sequence at the start of the first intron of the HbA1
gene is mutated to different nucleotides. Give a brief explanation at the molecular level of how
this could affect Hemoglobin production and cause thalassemia. [3 pts]

No splicing / reduced splicing. Therefore the first intron is retained and this changes the
sequence of the mRNA to be translated / the resulting protein.

d) In a third patient, you find a single-nucleotide mutation in the Kozak sequence just upstream
of the AUG. Give a brief explanation at the molecular level of how this could affect Hemoglobin
production and cause thalassemia. [3 pts]

Reduce translation of mRNA, therefore less hemoglobin protein

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MCBI (BIOL-UA 21) NAME NetID (e.g., ms4131)
Midterm 1
October 4, 2024

13. You identify a cold-sensitive mutation in yeast that prevents DNA replication and therefore
stops cells dividing at the non-permissive temperature of 20°C, but it allows normal DNA
replication and cell division at the permissive temperature of 30°C. You want to use functional
complementation to identify the gene that has been mutated.

a) First, you need to make a cDNA library from normal yeast. Order the following steps in
making a cDNA library (write the step number, from 1 to 6, in the box next to each step). [4 pts]

5 Add linkers

6 Clone into shuttle vector Although the order listed here is the one
described in class, some cDNA libraries are
made using RNA to prime second strand
3 Add alkali to remove RNA synthesis; the RNA is nicked using RNAse.
Because of this, the order of #3 and #4 can be
interchangeable here, and either received credit.
1 Isolate mRNA from yeast

4 Second strand cDNA synthesis using DNA polymerase

2 Reverse transcribe using reverse transcriptase and oligo-dT

b) A shuttle vector contains DNA sequences that allow it to be selected for in E.coli and in yeast.

Give an example of a specific DNA sequence that allows cells containing a shuttle vector to be
selected for in E.coli. For the example you chose, what needs to be either present or absent in
the media (make it clear which) for this selection to work? [3 pts]

DNA: Ampicillin resistance. Add ampicillin to the media.

Partial credit: Antibiotic resistance (2)

c) Give an example of a specific DNA sequence that allows cells containing a shuttle vector to
be selected for in yeast. For the example you chose, what needs to be either present or absent
in the media (make it clear which) for this selection to work? [3 pts]

DNA: URA3 gene. Leave uracil out of the media.

d) For the functional complementation experiment, at what temperature

would you grow the yeast to select colonies in which a particular cDNA 20°C
complements the defect in DNA replication. [2 pts]

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MCBI (BIOL-UA 21) NAME NetID (e.g., ms4131)
Midterm 1
October 4, 2024

SCRAP PAPER

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MCBI (BIOL-UA 21) NAME NetID (e.g., ms4131)
Midterm 1
October 4, 2024

Genetic code

Amino Acid Table

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