Solution key- 7.

013 EXAM 3 (4/ 22/ 15)

Question 1 (28 points) (David & Eugene)
a) Gardasil is a preventive vaccine that was designed against the surface proteins of Human
Papillomavirus (HPV), a DNA virus that causes cervical cancer.

i. Circle the immune response(s) that is triggered following injection with Gardasil: Innate/
humoral or antibodies mediated/ cytotoxic T cell mediated immune response. Explain why you
selected this option.
Since the proteins used as vaccine are not present in humans they will be regarded as foreign resulting
in immune response. Innate immune response is nonspecific and triggered following infection with any
immunogen. Since the protein is not a live entity it will not infect a cell to trigger a TC cell mediated
immune response. Instead, the antigen presenting cells (APC) will collect and degrade the immunogen
and present it on their surface through MHC-II to activate TH and B cells resulting in humoral or
antibodies mediated immune response. (4pts, 1for each selection and 2 for explain)

ii. The immune response(s) that you selected involves the dendritic cells that act as the antigen
presenting cells (APC). Circle the class of cell surface molecules that allow the APCs to present
antigens on their surface: Surface antibodies/ T- cell receptors/ MHC-I/ MHC-II. (2pts)

iii. An individual develops a secondary immune response against HPV infections only after 3-4
booster doses of Gardasil. Briefly explain why is this so.
Booster doses allow the preferential clonal expansion of B cells that produce high affiinity antibodies
through affinity maturation. It also promotes the proliferation of memory B , T cells to create a reserve
that can combat the HPV infection. (2pts)

b) The following schematic represents a portion of an antibody heavy chain gene locus in humans.
Primer 1 Primer 1
5 3
3 1 2 3 4 1 2 3 1 2 3 4 C C C C C 5
Primer 2 Primer 2
Regions V D J C
Antibody heavy chain gene locus
i. You want to PCR amplify the antibody heavy chain gene locus from both a skin cell and from an
antibody producing B cell of the same person. Would the same set of primers (Primer 1 and 2)
amplify the antibody heavy chain locus from the two cell types? Why or why not?
Yes, since the primers are outside the antibody chain gene locus, which is not altered following somatic
recombination. (2pts)
ii. Would the antibody heavy chain gene locus amplified from the skin cells be longer than/ shorter
than/ of the same length than that amplified from the antibody producing B cells? Explain why
you selected this option.
This is what I was looking for based on the details covered in the lectures. The antibody heavy chain
gene locus will undergo VDJ somatic recombination only in the B cells (not in the skin cells) thus
selecting one V, one D and one J segment and removing the remaining segments in between. (2pts)

iii. Show the promoter(s) as arrow(s) and enhancer(s) (a base sequence far from the transcription
start site that promotes the expression of a gene) as a box(s) on the schematic of antibody
heavy chain gene locus above. (2pts, 1 for promoters and 1 for enhancer)

iv. Briefly explain how the location of enhancer(s) relative to the promoter(s) region of antibody
heavy chain gene regulates (promotes) antibody gene expression.
The antibody H and L chain locus are transcribed only once the somatic recombination has occurred
as a result of which the enhancer is in close proximity to the promoter of the selected V segment to
influence the transcription of rearranged gene. (2pts)

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Question 1 continued
c) Which of the following process(es) is directly affected in each of the mutant cells below?
Processes: Junctional diversity, somatic recombination, , class switching, complement activation,
affinity maturation, clonal expansion (5pts, 1 for each correct answer)

i. Mutant cells do not express the RAG1 and RAG2 genes: Somatic recombination

ii. Mutant cells lack terminal deoxynucleotidyl transferase enzyme (TdT): Junctional diversity

iii. Mutant cells lack the enzyme(s) responsible for hypermutation in the base sequence
corresponding to antigen binding regions (Fab) of the antibody: Affinity maturation & clonal
expansion

iv. Mutant cells produce only IgM antibodies (and NO IgG) prior to and after exposure to an
antigen: Class switching

d) The following sequence corresponds to the first 5 amino acids of L chain produced by a mature B
cell.
5-AATGCAAAAGATTAGG-3 TOP
3-TTACGTTTTCTAATCC-5 BOTTOM

i. Which strand serves as the template for transcription

(Top or Bottom)? (2pts)

ii. Give the 2nd amino acid of the L chain. Gln (1pt)

iii. Show the direction of transcription by an arrow. (1pt)

iv. You observe that activation induced deaminase

enzyme (AID) function results in the clonal deletion of
this B cell. Explain why is this so. Note: AID acts by converting some Cs to Us.
The AID will convert the shaded C to a U, which will be result in a premature stop codon. Hence the B
cell will not be producing any antibody and will be clonally deleted. (3pts)

Question 2 (12 points) (Dexter)

a) Multiple sclerosis (MS) is an autoimmune disorder in which the immune system of the patient attacks
and destroys the myelin sheath of a neuron.

i. Of what is myelin composed? It is a hydrophobic membrane layer comprised of 70% lipids and
30% proteins (2pts)
ii. What class of cells in the nervous system produces myelin? Glia cells (2pts)

iii. What function does myelin have? It provides insulation and prevents ions from leaking out
(2pts)
iv. What happens to action potentials in a neuron when the myelin sheath is destroyed?
This causes the ions to leak or diffuse particularly through the open channels instead of moving down
the length of the axon. This abrogates the speed of propagation (conductance) of the action potentials
along the length of the axon. (2pts)

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 Question 2 continued
b) Specific ion channels (open and gated) and pumps regulate the resting membrane potential and
action potential of a neuron.

i. You treat a neuron in vitro (in a plate) with a reversible ATP synthase inhibitor. Circle the
correct option and explain why you selected this option. This inhibitor would inhibit a pump or a
channel.
tics
It would inhibit the pump since it actively transports specific ions across the membrane w, which
requires the expenditure of energy. In comparison, channels allow the diffusion of specific ions, which is
energy independent. (2pts)
+
ii. A voltage-gated Na is a multimeric protein that opens or closes in response to voltage. Circle
the correct option(s). What level of protein structure (Primary/ Secondary/ Tertiary/
Quaternary) is NOT altered when this channel converts from closed to open state? (2pts)

Question 3 (25 points) (Gina & Nicole)

Wakefulness is promoted by the excitatory neurotransmitter hypocretin that is produced in a part
of the brain known as the hypothalamus and regulates neurons in the brain cortex and spinal cord.

a) Based on the above information, where would you expect to find receptors for hypocretin?
On the cell body or dendrites of neurons in the brain cortex and spinal cord (or just in brain cortex +
spinal cord). (2pts, 1 for each)

b) After treatment with hypocretin, the membrane potential of the post-synaptic cell is closer to/ further
from (circle one) threshold. Explain your choice.
Hypocretin is an excitatory neurotransmitter, which means it promotes an action potential in the post-
synaptic cell, which occurs when threshold potential is reached. It acts by making the inside of the cell
more positive compared to the outside thereby depolarizing the membrane and bringing the membrane
potential close to threshold. (3pts, 2 for explain)

c) The response of neurons to 50pg/l and 20pg/l hypocretin is as shown below.

i. What term is given to each of the spikes on each trace? Action potential (2pts)

ii. Indicate the concentration of hypocretin that led to Trace A and Trace B. Explain your choice.

voltage
voltage

time

Trace A ____20pg/l_(1pt)___ Trace B ___50pg/l__(1pt)___

The increase in concentration of the neurotransmitter influences the frequency of the action potential.
Since hypocretin is an excitatory neurotransmitter more of this will increase action potential frequency.
but the amplitude of action potential for a neuron remains unchanged. (2pts)

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Question 3 continued
iii. Tetrodotoxin binds to the voltage-gated Na+ channel and inhibits its function. If you treated
responsive neurons with tetrodotoxin plus hypocretin, draw the action potential from these
neurons on Graph A and provide an explanation for your drawing.
Hypocretin, being an excitatory
Normal neuron will cause local
Action Graph A
+50mV +50mV transient depolarization.
potential However, the voltage gated
+
Na channels, in the presence
of tetrodotoxin will fail to open.
0mV 0mV Hence there will be in
depolarization and hence no
action potential. Alternatively,
-55mV -55m V you can also say that it will be
completely flat as threshold
-60mV -60m V also depends on the VG Na+
channels (4pts with for for
explain)
Time Time

d) The neurons that produce hypocretin are stimulated to release this by glutamatergic neurons that
innervate the hypothalamus. The circuit that modulates hypocretin release looks like the one below.
What type of feedback loop is this?
Secretes Secretes Secretes
hypocretin glutamate hypocretin

Neuron 1 Neuron 2 Neuron 3 Spinal cord

neuron
Positive feedback loop: hypocretin secreted by neuron 1 promotes hypocretin secretion by
neuron 3 through activation of glutamate secreting neuron 2. (3pts)

e) Hypocretin receptors are G-protein coupled and lead to Ca2+ influx (entry) into the post-synaptic cell.
Glutamate receptors are ionotropic and lead to Na+ influx into the post-synaptic cell.

i. When the above circuit is stimulated with glutamate + hypocretin, which cell will show the most
rapid response? neuron 2/neuron 3/neuron 4? Explain your answer.
Neuron 3, as the glutamate R is ionotropic and would respond most rapidly. (3pts)

ii. After the circuit is established, over a few days, the amount of glutamate released from neuron
2 increases five-fold. Describe two changes in neuron 2 that would lead to the increase in
glutamate increase.
The affinity of the hypocretin receptors on the cell body of neuron 2 may have increased. Number of
hypocretin receptors on the cell body of neuron 2 may have increased. Neuron synthesizes more
glutamate, which is released following activation. The released glutamate may undergo reduced uptake
and / or reduced degradation. (2pts)

f) Narcolepsy is a disorder where someone affected will be unusually sleepy all the time. Based on the
information given above, suggest one gene(s) where loss-of -function mutations lead to
narcolepsy. Justify your answer.
Hypocretin or something that promotes the synthesis of hypocretin. Or,something that promotes the
synthesis of glutamate i.e. hypocretin receptor, glutamate receptor. The absence of this
neurotransmitter or inability to respond to the neurotransmitter will prevent the activation of neurons in
the brain and spinal cord resulting in nacolepsy. (2pts)

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Question 4 (12 points) (Faye)
Hypocretin neurons in the hypothalamus arise from early neural precursors in the forebrain and path
find (grow) towards the developing hypothalamus.

a) Using a stripe assay, and young hypocretin neurons (shown by circles in the schematic below),
draw how these would grow if Fibronectin is a short-range attractive guidance cue. Note: If there is
no growth indicate no growth. (3pts)

Fibronectin

Fibronectin

b) Cytochalasin inhibits actin polymerization. Would application of this inhibitor alter hypocretin neuron
growth in the stripe assay, and what part of the neuron would be affected? Explain.
In the absence of actin polymerization the protrusions will not form or degrade preventing the growth of
the growth cone. (3pts)

c) Two genes necessary for hypocretin growth into the hypothalamus are Integrin 1 and Robo. They
have the following activity.

Integrin 1 receptor binds the ECM molecule Fibronectin (an attractive guidance cue).
Integrin 1 is expressed on hypocretin neurons
Robo receptor binds the diffusible ligand Slit (a repulsive guidance cue)
Slit is expressed throughout the brain but not in the hypothalamus
Robo is expressed on hypocretin neurons

You construct homozygous loss-of-function (LOF) mutants in Integrin 1 and Robo. The comparison of
Robo and Integrin 1 LOF phenotypes is shown below.

hypothalamus*

hypocre(n**
neuron*growth*

hypocre(n**
forebrain* neuron*cell*
body*

wild*type* Robo*LOF* Integrin*1*LOF*

i. Explain the Robo LOF phenotype, using the above information and as diagrammed above.
In the absence of Robo receptors, the neurons will not be able to sense the repulsive cue provided by
the Slit. Hence they will grow randomly and towards attractive guidance cues such as fibronectin.
(3pts)

ii. Indicate how hypocretin neurons would grow in the double Robo-/- Integrin 1 -/- mutant.
They will not grow at all, as they need Integrin to grow at all. (3pts)

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Question 5 (23 points) (DS & Laura)
The spleen is a multifunctional organ. Along with blood vessels (arteries and veins), it has two regions:
the red pulp and the white pulp. Each includes multiple cell types.

The genes Pbx1, Nkx2-5, Tlx1 (which are transcription factors) and p15 (which is a an inhibitor of the
CDK2 cell cycle regulator) work together to promote spleen formation as described below.

Pbx1 gene expression is required for the

formation of a normal spleen.
Pbx 1 activates Nkx2-5 and Tlx1 expression
in future spleen cells.
Pbx 1 represses (inhibits) expression of the
CDK-inhibitor p15 to promote spleen
formation.
Nkx2-5 represses the transcription of p15
and S phase Cdk regulator.
Nkx2-5 and Tlx1 act cooperatively (together)
to activate transcription of effector genes in
future spleen cells.

a) On the schematic, show interactions between genes, using arrows for activation and T-bars for
repression. (4pts: 2 for correct arrows and 2 for correct inhibitions)

b) You use in-situ hybridization to determine TIx1 RNA

Pbx1 localization in developing spleen cells. In this technique
a labeled RNA probe is incubated with cells, excess
probe is washed out and the position of label is
p15 Nkx2-5 TIx1 examined.

i. The probe interacts with Tlx1 RNA by__base

pairing_ (2pts)
Norm al spleen

ii. The signal shows mature Tlx1 mRNA. This is mostly located in this subcellular region(s)
____Cytoplasm_____(2pts)

iii. Using immunocytochemistry, a labeled Tlx1 antibody locates the functional TIx1protein. This is
mostly found in the _____Nucleus______ (2pts)

c) The steps involved in the formation of the spleen are described and diagrammed below.

On Day 12 of embryonic development a group of cells (circles) begins to proliferate between two layers of
mesogastrium epithelium (shaded bars).
By Day 14 the future spleen cells are located on the left hand side.
At Day 16 future red pulp and future white pulp cells are intermingled.
By Day 18 red (black circles) and white pulp (white circles) cells form separate regions.
At Day 20 the spleen becomes functional with many cell types.

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Question 5 continued
The steps of the development of spleen are diagrammed below:
Future spleen
cells
Day 20,
functional
spleen with
multiple cell
types in red and
white pulp.

i. When are spleen cells differentiated? Explain.

They are differentiated at Day 20, since at the point you see multiple cell types in red and white pulp
that produce a functional spleen. (2pts, 1 for explain)

ii. E-cadherin protein is necessary for spleen formation. You make chimeric embryos, where E-
cadherin is deleted EITHER in the mesogastrium epithelium OR in the future spleen cells.

Loss of E-cadherin specifically in the spleen cells leads to intermingled red + white cells. What
process does E-cadherin promote here?
E-cadherin allows the red and white pulp spleen cells to sort out, via homotypic adhesion (students do
not need to use this term: like-like cell interaction or something along those lines is fine) E-cadherin
protein is localized to the plasma membrane. (2pts)

Loss of E-cadherin specifically in the mesogastrium leads to the tissue falling apart through a

epithelial-mesenchymal transition OR mesenchymal-epithelial transition. Circle one and
explain your choice.
The cells in the mesenchymal state do not make cell-cell junctions and hence do not sort. (2pts)

d) Loss of NKx2-5 function leads to absence of red pulp and an excess of white pulp. What is the role
of Nkx2-5 based on this result?
It is positive regulator of red pulp formation i.e. causes the cells to differentiate into red pulp cells. It
probably does so by playing a role in determination since it is a transcription factor. It could be a
negative regulator of white pulp formation i.e. prevents some cells from differentiating into white pulp
cells. Alternatively, it could also play no role in white pulp formation. (2pts)

e) Loss of function of p15, a CDK inhibitor (a cell cycle regulator) results in in an enlarged spleen.
Based on this information, circle all correct options. Thus p15 controls spleen determination/
differentiation/ cell proliferation/ cell survival. Explain your answer.
P15 being a cell cycle inhibitor must be influencing a proliferation, to regulate spleen formation. (1pt)

f) You find that TIx1 (a transcription factor required for spleen formation) is produced in the
mesogastrium epithelium surrounding the future spleen. Suggest a likely mechanism by which this
gene controls spleen formation.
Tlx1 most likely activates expression of a protein that is secreted from the mesogastrium, binds to
receptors on the future spleen cells to regulate spleen formation. (2pts)

g) What is the potency of the future spleen cells at Day 12? (uni-/ bi-/ multi-/ pluri-/ totipotent)?
Explain why you selected this option.
It forms multiple cell types of red and white pulp. (2pt)