Biology 200

Midterm Exam – October 25th 2022

Total time: 90 min. 20% of Course Grade
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Instructions:
1. Use black or blue PEN only. Exams that are written in pencil, erasable ink, or that have
corrective tape on anywhere on the exam will not be eligible for re-grading. A regrade request
requires that the exam does not look modified in any way.
2. There are 6 questions on 9 pages. Answer all questions in this exam booklet.
3. You are allowed an 8.5x11 inch, double-sided, hand-written memory aid for this exam. All
memory aids that do not conform to these rules will be taken away.
4. You will have 90 minutes for this exam.

Instructions for Exam Regrades

The exams were carefully and consistently graded. However, if you feel that
an error was made in the grading of your exam, please follow these
instructions.

1. Carefully read this answer key, and compare to your answer.

2. You must submit a letter to your instructor that outlines why the regrading is
needed for each question. You must refer to the answer key in your
explanation. It is not sufficient to say “please regrade Question X.” Instead,
you have to explain why your answers should be given more points.

3. The whole exam will be regraded. Therefore, there is a possibility that your
exam grade may go up, down or stay the same. The new grade will be your
final grade – you do not get to keep your old grade.

Please do not submit any requests for regrading until 48 hours after you
receive your mark.

Your request must be received no later than November 22nd, at 11pm to be

considered.

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Question 1 (_____/ 7 marks)

A. What type of microscopy generated these images? Provide one reason to explain how you know.
(3 marks)

Image A Image B

Image A Image B:

Type of TEM (1 mark), EM (0.5 marks), SEM (0 Brightfield (1 mark); Light microscopy (0.5
microscopy marks) marks), fluorescence (0 marks)

Supporting High resolution Low resolution

basis Cross-section Cannot see internal membranes
Amount of detail (internal membranes, (0.5 marks for any of these)
thylakoids) (0.5 marks for any of these)

Instructor comment: these microscopy images are similar the ones shown in the Peer Tutor practice exam,
especially Image B.

B. What organelle is visible in both these micrographs? (1 mark)

Plastid, chloroplast (1 mark)

Instructor comment: cell membrane is not accepted, as the membrane in Image A would be the chloroplast
membrane, not just simply ‘cell membrane.’

C. Your research team would like to investigate how tobacco mosaic virus enters a plant cell. Which
of the two techniques above would be more appropriate for your investigations, and why? (2 marks)
TEM would be more appropriate for studying viruses (1 mark). Viruses are extremely small (0.5 marks), and
therefore only visible with the resolution of electron microscopy (0.5 marks).

(If students incorrectly identified the type of microscopy in Part A, then mark this question according to their
answer in A.)

Instructor comment: the main reason against brightfield: viruses too small to be seen in brightfield. Simply
stating high resolution is not sufficient for reasoning. This is covered in Tutorial 2 in detail, and also shown in
Unit 3 Topic 3.1.

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D. In general, what is an advantage to using the microscopy technique you did not choose in (C)? (1
marks)
Image B (Brightfield): Live imaging, more economical, quicker. (1 mark)

(If students picked the wrong microscopy for C, then D was marked in context of that. i.e. they can still get full
marks for D if the reasoning is correct, even if C is wrong.)

Question 2. (_____/6 marks)

The statements listed below are all FALSE. For each statement, identify the part(s) that is/are
false, and provide a brief rationale explaining why it is false in the space provided. (2 marks each).

A. The hydropathy plot to the right predicts a beta barrel.

Transmembrane beta barrels alternate between hydrophilic and
hydrophobic amino acids (1 mark); thus, a hydropathy plot will
not show any peaks for the transmembrane domains of a beta
barrel, but will instead show a line closer to ‘0. (1 mark)’

Instructor comment: This was covered in Unit 2, Topic 2.3 with

the VDAC example in-class. If you answered that the plot shows
an alpha-helix, you received part marks, however you still need
to indicate why a beta barrel was not represented by the
hydropathy plot shown.

B. The nucleolus is the location of ribosome assembly, thus also where translation of mRNA into
proteins takes place.
Only ribosome subunits are assembled at the nucleolus (1 mark). mRNA is exported into the cytosol for
translation (1 mark).

Instructor comment: This was covered in Unit 3 – Topic 3.1 (slide on how ‘Ribosomes are made of rRNA and
protein’). There are two incorrect parts – your answer needs to address both for full marks.

C. Heterochromatin appears dark and dense under transmission electron microscopy because of
the presence of high amounts of protein required for transcription of genes.
Heterochromatin appears dense because of its highly compacted state with many DNA packing proteins (1
mark). Genes within heterochromatin are not accessible for transcription (1 mark).

Instructor comment: This was covered in Unit 3 Topic 3.3 (slides on comparison between heterochromatin &
euchromatin). There are two incorrect parts – the dark/dense appear is due to DNA packing proteins (e.g.
histones, DNA scaffolding proteins), and that heterochromatin is NOT the site of transcription.

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Question 3. (_____/8 marks)

As a Pokemon master, you are interested in understanding the nuclear organization of a rodent-like
Pokemon species called Pikachu. As part of your studies with Professor Oak, you isolate and treat
the chromatin of Pikachu in order to run a DNA nuclease digestion. The schematic of the structure
of Pikachu chromatin is shown below. Note that it only shows part of the chromatin structure.

You decide to have the following treatments in your nuclease digestion experiment:
Lane 1 = Treatment A, no nuclease treatment
Lane 2 = Treatment B, brief (30 sec) nuclease digestion of naked DNA
Lane 3 = Treatment C, extended (30 min) nuclease digestion of chromatin
Lane 4 = Treatment D, brief (30 sec) nuclease digestion of chromatin.

A. What is the purpose of Lanes 1 & 2? (2 marks)

Lane 1 is a (negative) control to show what chromatin looks like without nuclease (or the effects of nuclease)
(1 mark)
Lane 2 is a (positive) control to show what chromatin looks like after being partially digested by nuclease (1
mark)
Note: if they say “baseline control” – max. 1 mark out of 2 marks.

B. Draw what you would expect to see in the four

lanes in the gel to the right. If the DNA band sizes
are known, then label your DNA bands with their
respective sizes for full marks. A size ladder is
included already on the right of your gel for
comparison. (6 marks)
Lane 1: large band near the top of the gel (0.5 marks)
Lane 2: smear ranging from top of the gel to the bottom of
the gel (0.5 marks)
Lane 3: one large band size 150bp (1 mark, with label for
full marks, only 0.5 marks if no size indicated)
Lane 4: banding pattern (at least 3 regularly repeating bands, 1
mark)
• Smallest band clearly labeled with size OR is
unambiguously draw RIGHT at 150-200bp (1
mark; -0.5 marks if two separate bands or just the
200 bp)
• Remaining bands 400, 600, 800 (2 marks)
-0.5 marks for not including labels

Instructor comment: The concepts in this question were covered in Tutorial 5 (PS 3.2.1) , and also in Problem
Set Question 3.2.2 that the Peer Tutors also covered during their review.

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Question 4 (_____/ 8 marks)
In breast cancer tumour cells, a transmembrane protein receptor called CCR5 is expressed. CCR5
was found to be involved in the metastasis (spread) of breast cancer cells. For metastasis to
happen, the cancer cells respond to a cell signaling event that results in the reorganization of CCR5
and other protein receptors.

A UV-activated compound called INA was thought to be a possible cancer treatment candidate. To
test this, Viard et al. (2009) treated breast cancer cells with INA, and then subjected the samples to
UV light, in order to see how INA affected CCR5 proteins.

A. INA was found to be a highly hydrophobic molecule. Why do you think this makes it an effective
molecule to interact with CCR5? (2 marks)
Because CCR5 is a transmembrane protein, we expect that there are portions of the CCR5 protein that has
hydrophobic R groups that allow it to be embedded in the lipid bilayer (1 mark). INA, being a hydrophobic
molecule, would be able to form van der Waals or hydrophobic interactions with those R groups (1 mark).

Instructor comment: The concept here is very similar to Problem Set 2.2.1 with the liposome-based drug
targeting question.

B. Viard and his colleagues investigated how UV-activated INA

(INA-UV) affected CCR5 proteins using FRAP. CCR5-GFP was
expressed in the breast cancer cells that were treated or not
treated with 20µM INA-UV. Fluorescence signal recovery after
photobleaching was measured and graphed, as seen to the
right.

Describe the data and explain what it is telling us about how INA-
UV affects CC5R in the membrane. (4 marks)
[control: description] The control with untreated breast cancer cells,
after 110 seconds post-bleach, had recovered 80% of its original
fluorescence of labeled CCR5 (1 mark). [INA-UV: description] The
treatment with 20µM of UV-activated INA only recovered ~20% of its
original fluorescence of labeled CCR5 (1 mark). To earn full marks,
students should mention the quantities described by the data points.
[control: explanation] This means that the unbleached, fluorescently-tagged CC5R in the control is able to
move freely within the plasma membrane (1 mark)
[INA-UV explanation] while the treatment with UV-INA severely limits the movement of CC5R within the
plasma membrane (1 mark)

C. Upon further investigation, INA was discovered to be able to form covalent bonds with
transmembrane proteins nearby when it is activated with UV light. Based on the information
provided about INA and metastasis, and the FRAP data above, explain whether or not UV-activated
INA can be used as effective drug treatment for breast cancer. (2 marks)
INA is can be used as an effective drug treatment, because we know from the question that breast
cancer cells needs to undergo reorganization of the receptor proteins in the plasma membrane as
part of its cell signaling response.
UV-INA can form strong covalent bonds to CC5R, which inhibits its ability to move freely within the
plasma membrane (0.5 marks), as seen in the loss of recovery in the FRAP experiment (0.5 marks).
The inability of CC5R to reorganize, then, will inhibit or decrease the ability of breast cancer to
spread (1 mark). Students here should focus on the mobility of the CCR5 protein for full marks, and
not the fluidity of the membrane—the FRAP data shows the mobility of CCR5.
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Question 5 (______/ 14 marks)
Interferon (IFN) is a regulator of immune response to viral infection. When you are infected by a
virus, like SARS-CoV-2 (SARS), IFN induces expression of genes that are involved in immune
response. SARS expresses a protein called Orf6, as a defense against this IFN-dependent immune
response. Here, Miorin et al. (2020) explore how this Orf6 protein acts in the cell.

To test the ability of SARS Orf6 protein to affect expression of genes that are normally induced by
IFN, Miorin et al. placed the IFN response element upstream of a luciferase gene expression
construct (Figure 1). They expressed this engineered gene in cells treated with IFN only, or with IFN
and SARS Orf 6, then measured the amount of luciferase activity that could be detected in the cells
(Figure 2).

Figure 1

Figure 2

Instructor comment: The question set-up for the Figures 1 & 2 above is similar to that presented in PS 3.3.7,
which had a walkthrough.

A. Describe the results of the luciferase assay, and use it to explain how SARS Orf6 must be
affecting expression of genes regulated by IFN. (2 marks)

IFN only: High luciferase activity (0.5) indicates that IFN has induced expression of this gene (0.5).
IFN + SARS-Orf6: Low/decrease in activity (0.5) suggests that Orf6 is able to suppress expression of
luciferase/IFN-regulated genes (0.5)

Instructor comment: common issue – students mixing up “luciferase activity” with the transcription of this
gene.

B. In response to SARS infection, IFN signaling activates the transcription factor STAT1. When
activated, STAT1 binds with other transcription factors to the IFN response element, and turns on
expression of genes involved in immune response. Miorin et al. looked at how IFN and Orf6 affect
localization of STAT1. Here, they looked at cells without IFN, with IFN, and IFN with SARS Orf6.
Treated cells were fixed and stained with fluorescently labelled
antibodies against STAT1.

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Describe each panel of this experiment and explain what it tells you about the role of IFN and SARS
Orf6 in the localization of STAT1. (5 marks)

• Without IFN: STAT1 is found mainly in the cytosol. (1 mark)

• With IFN: STAT1 is found mainly in the nucleus (1 mark) indicating that IFN causes it to be imported to
the nucleus (1 mark)
• IFN + Orf6: STAT1 is mainly in the cytosol (1 mark), indicating that it is the SARS Orf6 protein that
prevents IFN-response mediated import of STAT1 into the nucleus (1 mark)

C. Miorin et al. then investigated the localization of

SARS Orf6 itself. Here, they have labelled SARS-
infected cells with a red fluorescent antibody against
Nup98 (a protein of the nuclear pore complex), and a
green fluorescent antibody against Orf6. To the right are
images of the same cells, showing either Nup98
fluorescence or Orf6 fluorescence. Note: SARS did not
infect 100% of the cells in this sample.

Describe the results of this experiment, and explain what it tells you about the location of SARS
Orf6. (4 marks)

NUP98 fluorescence appears as a bright ring (1 mark), indicating the location of nuclear pore complexes within the
nuclear membrane (1 mark).
SARS Orf6 appears in the same location as NOP98 (1 mark), also suggesting that it is localizing to nuclear pore
complexes within the membrane (1 mark).

Note: ‘Bright ring’ is subjective. Any loose description of the picture was accepted. “Fluorescence in a circle around the
nucleus” was common.

Instructor comment: The question set up for the above is very similar to the ideas presented in PS 3.1.8.

D. Propose a model by which SARS Orf6 suppresses immune response, and support your model
with data from these three experiments. (3 marks)

Overall model: SARS Orf6 must be suppressing immune response by preventing passage of STAT1 into the nucleus,
so that it is not able to bind to genes turned on by IFN and induce expression of genes involved in immune response.

Experiment 2 shows us that STAT1 remains in the cytosol in IFN-treated cells that are also expressing Orf6
(or infected by SARS), therefore preventing STAT1 from entering nucleus in response to IFN (1 mark)
Experiment 3 shows that that ORF6 binds to the nuclear membrane—possibly to the nuclear pores,
therefore preventing passage of STAT1 through the pore (1 mark)
Experiment 1 shows expression of Orf6 prevents IFN-induced expression of luciferase. This means that
IFN-induced genes are not transcribed/turned on, which is expected if STAT1 cannot enter the nucleus (1
mark)

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Question 6 (_______/9 marks)
The CFTR protein is an integral membrane protein that forms a channel that transports chloride ions
across the plasma membrane. Cystic Fibrosis results from a mutation in this protein that causes it to
mis-fold.
Describe the importance of the amino acids found within CFTR’s primary sequence, and explain
how the non-covalent interactions they make (with each other and with molecules in their
environment) determine the folding of this structure, its location within the cell, and the function of
this protein.

Major ideas for Sections ‘Folding of the polypeptide into its final structure’ & ‘Location within the
Cell’

Students should discuss the location of the specific properties of amino acids, the molecule in the
environment, the type of non-covalent interaction between them, and how this allows the CFTR protein to
function as channel protein. (Note: a general description of folding protein was not sufficient for this question
– it must apply to the CFTR protein.) The following ideas below may be applied fluidly between these two
boxes, depending on how the students structures it.

• Needs to fold spontaneously so that it can function as a transmembrane protein in the plasma
membrane, such that hydrophilic amino acids are on the surfaces that are exposed to water (1 mark),
and hydrophobic amino acids would be embedded in the membrane or with each other in interior of the
protein (1 mark)

Instructor comment: a good number of students simply copied the answers from the practice midterms and stated that
the protein was able to spontaneous fold in an aqueous environment. This is not entirely correct as we stated that CFTR
is a membrane protein, and not a soluble protein like in the example midterms. Also, ‘outside’ and ‘inside’ was often
ambiguous, especially in context of a membrane protein.

Secondary Structure
• Beta-barrel formed via H-bonding between the backbone atoms
• Multiple-alpha helices via H-bonding between the backbone atoms (must be multiple alpha helices
– no marks given for single alpha helix)

Instructor comment: we looked at the mitochondrial porin VDAC as an example of how a beta barrel can form a channel,
and we also looked at the structure of multiple alpha helices to form a channel as well in Unit 2 – Topic 2.3

Tertiary Structure
• Hydrophilic aa likely found on the surface exposed to aqueous environment (extracellular space &
cytosol) and will make H-bonds with water
• Hydrophobic aa interact with hydrophobic fatty acids tails of membrane via van der
Waals/hydrophobic interactions (half marks for only saying membrane, and not tails)

Targeting signals
• The primary sequence of this protein must encode a targeting signal that directs it to the plasma
membrane (and not a generic description of a targeting signal)
• These amino acids are hydrophobic in nature (Unit 4 knowledge; not required but accepted)
• These amino acids must form noncovalent interactions with the receptor protein that directs them to
the membrane.

Instructor comment: Discussion of targeting signals must be specific for this channel protein. Any mention of
targeting signals for other cellular locations is incorrect.

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 Function of this protein
• The amino acids lining the channel/pore must therefore be positively charged residues (No marks
were given if negatively charged R-groups were chosen here) , to interact with negative Cl- ions via
ionic interactions. (All four parts must be present to 3 marks, -0.5 mark for any missing components)
• Note that saying simply polar/hydrophilic R-groups to interact with Cl- was not accurate

Instructor comment: In general, this last section on function was the most well-done of the three sections.

Family name: ________________ Given name: _________________ Student #: _____________

For marking purposes only. Do not write in this table.

BIOL 200 Midterm Grades

1 2 3 4 5 6 TOTAL

Question

Student
Score
Maximum
Score 7 6 8 8 14 9 52