Biology 200

Sample Midterm Exam

Total time: 90 min

Section:

Family Name: Given Name: Student #:

(please print)

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 8 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.

Please write your name and student number on

both the first page AND page 8 of this exam.
Your grade will be written on page 8.
Question 1 (5 marks)
When doing research, scientists will often image the same cell using multiple different
microscopy techniques. In this case, the scientists were studying chloroplasts.

A. What type of microscopy was used to generate each of these two images? How do you
know? (3 marks)
Type of microscopy How do you know?
Image 1:
due to the low resolution and the scale range of 50um
brightfield which fits and how it does not show details of the
organelle

Image 2:
due to the higher resolution and scale of 5um which fits
TEM for electron microscopy and its a plane of section image
and it shows details as you can see the mitochondria

B. The cell in Image 2 is one of the cells seen in Image 1. In image 2, only 6 chloroplasts can be
seen, however in Image 1 there is not a single cell visible with only 6 chloroplasts. Explain how
this is possible. (1 mark)
This is possible because image 1 is brightfield so the magnification is lower to 50um compared to image two with TEM
with a magnification of 5um so in image 1 you are not able to see the details of each cell so you can not see each
individual chloroplast as to image 2 where TEM allows you to see the details of organelles and it is a plane of section, so
you are able to see in the cell to count the cholorplast
C. State one advantage of using the type of microscopy used to generate image 1 over
image 2. (1 mark)

With image 1 the cells can be alive where in image 2 the cells have to be dead, so brightfield in image 1 can be
useful if you want to see an organism and keep it alive for you experiments
Question 2 (8 marks)
For each of the experimental observations below, please state what it tells us about cellular
structure and/or function, and explain why.

A. Nucleoli become heavily radiolabeled when radioactive ribonucleotides are provided to the cell.

The nucleoi contains DNA and RNA which are made of nucleotides, specifically RNA is made in the nucleoulus
inside the nucleus which contain ribonucleotides for transcription and creating ribosome subunits, so if these are
tagged, the nucleoi will have the tagged ribonulceotides inside so it will also be radiolabelled.

Radioactive ribonucleotides move into the nucleus (0.5 marks), are incorporated to rRNA (1 mark) as
it is being transcribed in the nucleolus [ribosomal subunits assembly in nucleolus] (0.5 mark).

B. If the NLS from a nuclear protein is experimentally added to hexokinase (a cytoplasmic

protein), the altered hexokinase is detected in the nucleus.

IT IS SUFFICIENT

This tells us that the NLS from a nuclear protein has the function of entering the cell because hexokinase which
regularly found in the cytoplasm was instead found in the nucleus, so NLS can bind to the NIR which both can connect
with the fibrils and alternate the NPC and get access into the nucleus.

C. If mammalian cells are treated with hormones and then imaged using TEM, the results
show increases in the amount of euchromatin relative to heterochromatin in the nucleus.

That tell us that the hormones cause the DNA to decondense which is more euchromatin than heterochromatin and
the genes within the region would be actively transcribed and on the TEM image there would be seen more light
stained regions than dark which indicates a decrease in heterochromatin which are mostly darkly stained.

D. If cells are treated with SDS (a strong detergent) membrane proteins can be separated
form phospholipids.

A strong detergent breaks the non-covalent bonding in the membrane proteins, which include the hydorgen bonding,
which help seperate the hydrophilic ends of the the phospholipids so the membrane proteins can sperate and since the
interactions are degraded in the phospholipid bilayer.

SDS is structurally amphipathic and can form detergent micelles. Detergent miscelles can separate
membrane proteins from membrane phospholipids due to their long hydrophobic tails which associate
with the hydrophobic regions of membrane proteins and the fatty acid tails of phospholipids which
causes the membrane to disassemble.
Question 3 (8 marks)
This is the hydropathy plot of a new membrane protein of the plasma membrane of a
eukaryotic organism.

Further biochemical analysis indicated that this protein is a glycoprotein with a polysaccharide
chain located at its N-terminal end.

A. Predict how the protein will be arranged in the membrane. Draw a diagram and indicate
the plasma membrane, cytosol, extracellular space, N and C termini of the protein. (4
marks)

B. (4 marks) You want to examine a second membrane protein using trypsin digestion followed
by SDS-PAGE. This integral protein is a single membrane-spanning alpha-helix, with its N-
terminus on the extracellular side of the membrane. Predict the results that you would expect to
see in this experiment by drawing protein bands in the gel image for the treatments indicated
below. As an example, Lane 1 has been drawn for you.
Lane 1 (shown): Intact cells, no trypsin treatment
Lane 2: Intact cells treated only with a
solution of high salt concentration
Lane 3: Brief detergent treatment first, no trypsin
treatment
Lane 4: Trypsin treatment of intact cells 1 2 3 4 5
Lane 5: Brief detergent treatment first, followed by
trypsin treatment
 Question 4 (7 marks)

RasGTPase (Ras) is a lipid-linked, plasma membrane-associated protein involved in cell signalling.

Goodwin et al. tagged Ras with GFP and expressed it in cells to examine its mobility by
Fluorescence Recovery After Photobleaching (FRAP). They compared the mobility of Ras in the
presence (+) or absence (-) of a drug called methyl-β-cyclodextrin (MβCD) that removes cholesterol
from the membrane.

Data adapted from Goodwin et.al. 2005. Biophysical Journal. Vol. 89:2 1398-1410

A. Compare the FRAP recovery curves in the upper and lower sets of images. Make sure to
indicate which one is the control (& how you know it's the control) and how fluorescence
recovery is influenced by the test condition. (2 marks)
In the upper set of images that have the absense of MBCD, there is a little recovery seen at 80s as the bleach slowly fades
due to the fluidity of the membrane but in the lower set of images with the presence of MBCD, the membrane is seen to be
more fluid as at 80s the bleach seems to gone and it was fully recovered from the bleach as the band of bleach is no longer
visible. The control would be with MBCD because then we are able to compare the effects of MBCD if there are any.
Fluorescence recovery is influenced by the test conditions of the prescence and absence of MBCD, because it effects how
fluid the membrane will be, as the drug removes cholesterol of the membrane which chnages how fluid it is.
B. Based on the data, draw what you would expect the fluorescence recovery curves for each
condition to look like? Draw and label both curves on the axis below, as well as the x and y
axis. (2 marks)

brightness

time
C. Based on this data, explain what you can conclude about how the mobility of Ras within
the plasma membrane may be regulated. (1 mark)

The mobility of RAS within the plasma membrane is regulated by chloesterol content, which where in a high temperature
envionment, it would cause the membrane to be less fluid due to the van der waal interactions and in a lipid linked, plasma
membrane associated protein which makes it conected to the cell cortex and makes the membrane less fluid as well.

D. Predict how the mobility of Ras might differ in an artificial lipid bilayer (made entirely
of phospholipids) compared to a cellular plasma membrane? Explain. (2 marks)

It would be different by being more fluid because with a artificial lipid bilayer with only phophlipids, there would be less
connections with the cell cortex making it more fluid compared to a regular plasma membrane with proteins that are
embedded in the membrane which decreases fluidity.
 Question 5 (10 marks)
**Note: This question appeared on the 2020WT1 midterm.**
T53BP1 is a protein that plays an important role in repair of the DNA double strand breaks and must
therefore enter the cell nucleus. Two different sequences of the primary structure of T53BP1 have
been proposed to be NLSs. To determine whether these sequences serve as NLSs, two mutants of
T53BP1 were created as indicated in the diagram below. Next, T53BP1 and its mutants were fused to
GFP (green fluorescent protein) and expressed in cells. The images shown in panels A, B and C are
the result of this analysis.

A. Where is the fluorescent protein in each panel? (3 marks.)

Panel A Panel B Panel C

inside the nucleus surrounding the nucleus envelope in the

inside the nucleus
cytoplasm

B. When you compare all panels together, what can you conclude about sequence 1 and sequence 2
of T53BP1? Explain. (2 marks)
Sequence 2 is needed to enter the nucleus whereas sequence 1 is not necessary to enter the nucleus because as seen in
image B, where the mutant only includes the second sequence, it was still able to enter the nucleus wheras in image c, with
only the first sequence present, the nucleus was just surrounding the nucleus envelope, that means sequence was able to
get the NLS to the NIR but it was to able to bind to the cystic fibrils to get through the NPC.
Sequence 1 is not an NLS because when its basic amino acids are mutated to alanines the protein is still transported to nucleus
Sequence 2 is an NLS because when its basic amino acids are mutated to alanines the protein does not enter the nucleus.
C. As shown in the diagram below, T53BP1 contains a serine close to sequence 2 that can be
phosphorylated. To determine the possible role of phosphorylation in the nuclear import of T53BP1,
scientists studied the localization of GFP-tagged T53BP1 in conditions that inhibit or promote
phosphorylation of proteins. Their results are shown below. When you compare both panels, what
can you infer about the effect of phosphorylation on the nuclear import of T53BP1? Justify your
answer using the data provided and your knowledge of the nuclear import mechanism. Please write in
point form. (4 marks)

The effect phosphorylation on the nuclear import of

T53BP1 that stops it from entering the nucleus.
This is due to the fact that sequence two is basic
so it is positively charges whereas phosphorylation
is negative charge, which weakens the positive
charge. So then it inhibits the function of T53BP1
of connectining witth the fibrils to get through the
NPC as seen in image E where the fluorescence is
only seen surrounding the nucleus in the cytoplasm
whereas in image D, it is seen inside the nucleus.
Question 6 Synthesis Question (9 marks)

One of the course level goals of BIOL200 is that you are able to recognize that “all aspects of
protein’s function are ultimately encoded in its primary amino acid sequence.”

AP-1 is a transcript factor complex that functions as a heterodimer. A heterodimer refers to a protein
complex that is made up of two polypeptide chains with different compositions. AP-1 is composed of
two different proteins, c-Jun and c-Fos. The AP-1 heterodimer forms in the nucleus where it can
activate a wide variety of genes involved with cell growth.

How should the amino acids in the AP-transcription factor be arranged in the c-Jun and c-Fos
polypeptides, in order for this transcription factor to: (1) fold, (2) assemble properly, and carry out its
function? What kind of non-covalent interactions would be involved in each case? Please write your
answers to both questions in the table below.

Folding (3 marks)

When folding, it is stabilized by the interactions of the r-group and groups of the
backbone. These r-groups are probably electrically charged since they are smaller
and in the nucleus. Also it interacts with DNA which is negatively charged.

Assembly (3 marks)

They are probably hydrophilic, non-polar bonds created

Function(s) (4 marks)

the functions could probably be activated or repressor transcription of a certain gene that is
involved in cell growth because since it is in the nucleus and transcript factor complex. The
interactions involved would be covalent and ionic since it binds to the DNA sequence that is
negatively charged.
Family name: Given name: Student #:

For Marking Purposes do not write in this table.

BIOL 200 Midterm Grades

Question 1 2 3 4 5 6 TOTAL
Student
Score
Maximum
Score 5 8 8 7 10 9 47

THIS SPACE WILL NOT BE MARKED – USE IT FOR ROUGH

NOTES