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7.013 Problem Set 6- 2015

Please print out this problem set and answer the questions on the printout.
th
Answers to this problem set are to be turned in at the box outside 68-120 by 10.50 AM, Friday May8 .

Question 1
Cancer is caused by accumulation of two or more mutations in the same cell that affects its proliferation
and survival.

a) Why does a persons chance of having cancer increase with age?

Cancer is a multi- step process that involves accumulation of many mutations in tumor suppressor
genes and/ or oncogenes in the same cell. These mutations accumulate over the life of a cell, either
spontaneously or by exposure to carcinogens, by replication mistakes or by chromosomal
translocations. Since the chances of accumulation of these mutations increase over time, the chances
of an individual having cancer also increases with age.

b) Assume that you have the ability to introduce normal copies of a gene into a transformed cell.

i. If the cell was transformed due to mutation of a tumor suppressor gene, would you expect that
adding a normal copy of the mutated gene would restore the cells normal phenotype (Yes/ No)?
Explain your choice.
Mutations in tumor suppressor genes involve a loss of both of the alleles of the gene. Hence
introduction of a normal copy of the mutated gene could restore the cells normal phenotype.

ii. If the cell was transformed due to an oncogenic mutation, would you expect that adding a normal
copy of the mutated gene would restore the cells normal phenotype (Yes/ No)? Explain your
choice.
Oncogenic mutations involve a gain of function of one of two alleles of the same gene, and create a
transformed phenotype that is dominant. Hence introduction of a normal copy of the gene would not
restore the cells normal phenotype.

c) Complete the table below for each of the following genes.

Gene Normal function of encoded Is this a proto- Would cancer cells

protein oncogene or a with this mutation be homozygous /
tumor suppressor have a dominant/ heterozygous for a
gene? recessive mutation in this gene?
phenotype
compared to wild
type?
c-fos Encodes a transcription factor Proto-oncogene Dominant Heterozygous enough
that increases expression of (but homozygous
growth promoting genes possible)
Src Encodes a non-recptor tyrosine Proto-oncogene Dominant Heterozygous enough
kinase that stimulates growth (but homozygous
signaling pathways possible)
DPC4 Encodes a relay protein that Tumor suppressor Recessive Homozygous loss-of-
inhibits growth-signaling gene function mutation
pathways

MDM2 Encodes a ubiquitin ligase that Tumor suppresor Recessive Homozygous loss-of
adds ubiquitin tags to the protein function mutation
misfolded proteins and marks
them for degradation.

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Question 1 continued
d) Cell lines are often used to test the oncogenic potential of viruses. If cancer is a multi-
step process, why can the introduction of a single active viral oncogene transform these cells?
Unlike the normal cells, the cell lines are immortal i.e. they can divide indefinitely and they are made so
through the accumulation of mutations. Therefore the introduction of a single viral oncogene into a cell
line essentially reflects the addition of one more mutation to a series of mutations that were preexisting
in the cell line. Hence the transformation of a cell line by adding a single active viral oncogene does not
contradict the statement that cancer is a multi- step process.

e) For each of the following statements regarding cancer, circle True or False.

i. Unregulated cell proliferation and enhanced cell death result in cancer (True/ False).

ii. A benign tumor represents localized, rapidly dividing cells (True/ False).

iii. Cancer cells lose contact inhibition and form foci in vitro (True/ False).

iv. Cancers are only familial or caused by viral infections (True/ False).

v. Cancer cells show decreased angiogenesis i.e. decreased blood vessels formation (True or False).

vi. Metastatic cancer cells can degrade the extracellular matrix proteins (True/ False).

vii. Some cancer causing agents are NOT carcinogenic in their original form but may be metabolized by liver
enzymes to carcinogenic forms (True/ False).

f) Radiation therapy can be used to treat tumors.

i. Briefly explain how radiation therapy works to treat a tumor.

Radiation therapy works by massively damaging the DNA of the rapidly dividing cancer cells. With
extensive DNA damage, cells will often initiate a pathway for apoptosis or programmed cell death.

ii. Chemotherapeutic drugs have a wide range of structures and functions, yet many elicit the same
side effects. Explain why the side effects are the same for a variety of different drugs.
Most of these chemotherapeutic agents target the cancer cells since they are actively dividing cells
compared to the normal cells. However, normal cells that are actively dividing, such as hair cells, blood
cells and gut cells, are also targeted by these treatments resulting in hair loss and nausea. Therefore,
they all result in very similar side-effects.

iii. Describe what is meant by the therapeutic window of a drug used in chemotherapy, and how it
relates to the side effects seen in a patient.
The therapeutic window is a measure of the difference between the concentration of a drug that is
required to kill the cancer cells (effective dose) and the concentration of the drug that affects normal
cells. A drug with a wider therapeutic window will have fewer side effects at the effective dose.

iv. Explain how use of the following drugs may prevent cancer cell growth and /or proliferation.

Drug Target of drug How is cancer cell growth and / or proliferation prevented?

Vincristine Microtubule inhibitor Prevents the formation of mitotic spindle and hence
inhibits cell proliferation
VEGF Inhibits blood vessel formation Prevents supply of nutrients and removal of waste thus
inhibitor contributive to cell death.

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Question 2
a) Please take a look at the following pedigree. Individuals with predisposition to retinoblastoma, a
pediatric retinal cancer, are shaded in the pedigree. This cancer is caused by the loss-of-function
mutation of Rb, a tumor suppressor gene that encodes the Rb protein. Individual marrying into the
family do not have any predisposition to retinoblastoma.

i. Give the mode of inheritance of

predisposition to retinoblastoma.
Autosomal dominant

(A, C) (A, A) ii.

Individual 1 despite the predisposition
surprisingly DOES NOT develop
retinoblastoma as a child. The probability
that she develops retinoblastoma later in life
is almost zero. Based on this information
which cell cycle stage (G0/ G1/ G2/ S/ M) are the retinal cells of adults in? Explain why you
selected this stage.
They are in G0 phase since they are not dividing anymore (and acquiring mutations). This means that
Individual 1 is an obligate carrier and the pedigree is incompletely penetrant.

WT MUTANT
iii. Using Rb for the allele regulating the wild-type phenotype and Rb for the allele
regulating the retinoblastoma phenotype, give the genotype of the retinal cells of Individual 4

WT MUTANT MUTANT MUTANT

During birth: Rb Rb Once she develops retinoblastoma: Rb Rb

iv. Assume that Individual 14, at the zygote (one- cell) stage, successfully receives a wild- type
copy of the Rb gene through homologous recombination. Give the genotype(s) of the

Retinal cells of Individual 14 during birth: Rb+Rb+

Gametes produced by Individual 14 as an adult: Rb+

If individual 14 fathers a daughter with a normal, healthy female, what is the probability that their
daughter will be predisposed to retinoblastoma? 0%

b) Human papilloma virus (HPV) infection can result in cervical, head and neck cancer. The E7
protein of HPV binds to the Rb protein, preventing it from binding to and inhibiting E2F. Based on this
observation would you classify E7 as an oncogene or a tumor suppressor gene? Explain why you
selected this option.
It is an oncogene since it binds to and inhibits Rb protein thus promoting cell cycle by the active E2F.
This essentially results in a gain-0f-function mutation that causes uncontrolled cell division.

c) Rous sarcoma virus (RSV) is a retrovirus. It is a modified form of Avian leucosis virus (ALV). In
addition to the genes found in ALV genome, the genome of RSV also has v-src oncogene that causes
cancer. Based on this information, circle True/ False for each of the following statements for RSV.
i. RSV has a DNA genome (True/ False).
ii. RSV needs reverse transcriptase enzyme to make the cDNA copy of its genome in the host cell (True/
False).
iii. RSV is a rapidly mutating virus (True/ False).
iv. RSV causes sarcoma by encoding a Src kinase that has lost its regulatory domain (True/ False).

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Question 3
Ames test is used to evaluate the mutagenic properties of test compounds. The test uses amino acid-
dependent strains of S. typhimurium bacteria. In the absence of an external histidine source, the cells
cannot grow to form colonies. Colony growth is resumed if a reversion of the mutation occurs, which
allows the production of histidine.

a) Most cancer causing agents are mutagens. Assume that you incubate the his- bacterial strain
separately with two known mutagens, compound 1 and compound 2. You observe the formation of
colonies on his- plates that contains the bacterial cells treated with compound 1. However, treatment
with compound 2 produces no colonies. Note: You may assume that these test compounds are used at
a concentration that is not lethal to bacterial cells.

i. How can you explain the results that you obtained for compound 2?
Compound 2 is not mutagenic in original/ native form. However once inside the body, it can be
metabolized to form(s), which can be mutagenic and hence carcinogenic i.e. compound 2 is
promutagen unlike compound 1, which is a mutagen.

ii. How could you modify the Ames test to prove that compound 2 is also a mutagen?
You should pretreat compound 2 with the liver extract that has the enzymes that can metabolize
compound 2 to metabolic forms that are mutagenic and hence carciogenic.

b) What is a non-mutagenic carcinogen? Give a specific example of a non-mutagenic carcinogen and

explain how this may cause cancer.
This is a carcinogen that causes cancer without directly causing mutations. For example, alcohol and
asbestos. Alcohol for example can damage the liver cells. When the liver cells divide to replace the
damaged cells they may acquire mutations that are cancer causing.

c) The following are three bacterial mutants that have different mutations in the DNA sequence that
encodes the C-terminus of an enzyme (200 amino acids long) that is required for Histidine biosynthesis
and that requires Gln197 for its function. The DNA sequence corresponding to the last five amino
acids of the wild- type and three different mutant versions (1/ 2/ 3) of this enzyme is included within the
sequence below. Note: Please refer to a codon chart from the previous problem.

Wild-type: 5-ATTGCCAAAGATTAGGATGATAAAT-3 You treat mutants 1, 2 & 3 separately with

3-TAACGGTTTCTAATCCTACTATTTA-5 two mutagens; mutagen C causes point
mutations in comparison to mutagen D that
5-ATTGCCGAAGATTAGGATGATAAAT-3 causes frame-shift mutations.
Mutant #1
3-TAACGGCTTCTAATCCTACTATTTA-5
i. Which bacterial mutant (choose from
Mutant #2 5-ATTGCCAGAGATTAGGATGATAAAT-3 1, 2 or 3) can revert to wild- type
3-TAACGGTCTCTAATCCTACTATTTA-5 following treatment with mutagen C?
Explain why you selected this option.
5-ATTGCAAAGATTAGGATGATAAAT-3 Mutant #1, shows a point mutation that
Mutant #3
3-TAACGTTTCTAATCCTACTATTTA-5 results in a missense mutation i.e. - 5-CAA-
3 (encoding Glu197) is changed to 5-CGA-3
(encoding Arg197). Mutagen C can revert
this point mutation.
ii. Which bacterial mutant (choose from 1, 2 or 3) can revert to wild- type following treatment with
mutagen D? Explain why you selected this option.
Mutant #3, shows a point mutation (deletion) that results in a frameshift altering the last five amino
acids of enzyme E1. Mutagen D causes insertions that can correct the reading frame.
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Question 4
C. elegans is a transparent nematode that lives in temperate soil and has a total of 1031 cells of which
302 are neurons. All the neuronal connections in C. elegans have been mapped and many complex
** defined.
* circuits have been

a) The following are two motifs/ circuits in C. elegans. Note: The -----------< represents activation and---
------------O represents inhibition.

i. Would you see an action potential generated in

NS 1 5 neurons 5, 6 and 7of motif 1? Why or why not?
2 4 Most likely yes, since neuron 4 when activated releases an
6
excitatory neurotransmitter (NT), which binds to specific
* ** receptors in the cell body or dendrites of neurons 5, 6 and 7
3 7
Motif- 1 to generate an action potential.

ii. How is the activity of neuron 1 in motif 2 being

1 2 regulated?
;
-4 It is regulated by the feedback inhibitory signals that are
-16 -16
eat +, eat +,
-4
eat eat-3 D 3D provided by neuron 3. This motif is an example of feedback
glc (AF +) (AF +); inhibitory loop.
Motif-2
R IA RIA
lc-3 be defined as the movement of organism toward or away from a temperature. The
b) Thermotaxis gmay
neuronal circuitry involved in thermotaxis is well defined in C. elegans. This worm can choose between
epistasis between glc-3 and
moving to warmer or cooler temperatures via thermotaxis.
C-defective mutations
To the left is the neuronal circuit for thermotaxis in C. elegans.
D As shown
Temperature
Temperature is sensed by AFD and AWC sensory
neurons, which secrete glutamate neurotransmitter (NT).
90 s AFD AWC All glutamate receptors are excitatory except for GLC-3
* Glutamate that is inhibitory.
*
* GLC-3
The thermal information from AFD and AWC is transmitted
AIY AIZ to AIY interneurons and then further down to AIZ and
**
RIA interneurons, which also secrete glutamate and
regulate further neuronal information processing.
RIA

Glutamate Glutamate signals from AFD inhibit the activity of AIY

through activation of GLC- 3 and induce eventual
migration to the colder side (blue).

By contrast, glutamate signals from AWC stimulate the

Colder side Warmer side activity of AIY and induce eventual migration to the
warmer side (red) (Ohnishi et al, 2011).
Behavioural output
thermotaxis assays of AWC-defective mutants and glc-3 mutants in
. Error bar indicates
i. s.e.m.
EAT-4Single
gene asterisk, doubleencodes
in C. elegans asterisk, aand NS that concentrates glutamate into synaptic vesicles.
protein
mer tests for a comparison of each genotype. (B, C) In vivo calcium
You create EAT-4 transgenic mutants that express EAT-4 only in AFD but not in AWC. Would
B) Ratio change of yc3.6 in AIY according to temperature change.
these mutants move toward the warmer or colder temperature? Explain.
the results shown in B; (B, C) n 22 or more animals. Error bar
The higher concentration of glutamate in AFD would result in inhibition of AIY causing the worm to
ectively, in SteelDwass tests for a comparison of each genotype.
circuit. AFD,move
AWC,toandtheRIA
colder side.
release glutamate in EAT-4-dependent
nals from AFD inhibit the activity of AIY through activation of GLC-
side (blue). By contrast, EAT-4-dependent glutamate signals from
er side (red). 5
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Question 4 continued
ii. How would the neuronal circuitry be affected in C. elegans if its AWC neurons were laser-
ablated (laser-killed)? Explain.
There would be no activation of AIY or any of its downstream neurons. So the worm will not be able to
move to the warmer side and will restrict to the colder temperature.

iii. Toward which temperature zone (warmer or colder) would the worms that have a
GLC-3 homozygous loss-of-function mutation (GLC-3-/ GLC-3-) move? Explain.
In the absence of GLC-3 receptors, AIY will not be inhibited by AFD. So it will constitutively activate the
downstream neurons and cause the worm to stay in the warmer temeperature.

Question 5
Optogenetics is a technique that uses light-gated ion channels found naturally in microorganisms. The
channel rhodopsin ChR1 opens in response to blue light, and allows the Na+ and Ca2+ ions to diffuse
across the plasma membrane. The halorhodopsi, NpHR opens in response to orange light and allows
Cl- ions to pass through.

a) When expressed in the axon of a neuron, and exposed to the appropriate light, which of these
channels (ChR1 or NpHR) would stimulate an action potential? Explain.
The activation of ChR1 will stimulate the action potential by allowing the diffusion of Na+ and Ca2+ ions
from the outside to the inside of the neuron thus depolariizing the membrane and bringing the
membrane potential closer to the threshold level. NpHR channels, once activated, will hyperpolarize the
membrane.

You are studying the circuitry involved in mammalian memory.

The circuit to the left is a memory circuit in the

mouse brain that includes the following brain
regions: Hippocampal neurons, prefrontal cortex
neurons (IL), prelimbic cortex neurons (PL),
basolateral amygdala neurons (BLA), central
amygdala neurons (CEA) and intercalated
neurons (ITC).

Hippocampal neurons and the PL neurons

synapse directly to the BLA neurons. Indirect
connections occur between the hippocampus and
ITC or CEA.

Activation of CEA neurons promotes memory. In

contrast, their inhibition suppresses memory.
Note: Arrows indicate excitatory axonal connections, T-bars inhibitory connections.

b) To probe the contributions of ITC and BLA neurons in the above circuit, you insert the ChR1 or
NpHR genes into either neuron type using a viral vector.

i. What would you do to open the ChR1 channel?

You would activate or open it by exposing it to the light in the blue range (470nM).

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Question 5 continued
ii. If ChR1 is inserted into ITC and opened, would memory be promoted or suppressed? Explain.
If the ITC neuron that is expressing ChR1 is subjected to light, the ChR1 channel will be activated/
open allowing the diffusion of Na+ and Ca2+ ions from the outside to the inside of the neuron thus
depolarizing the neuron and triggering an action potential. The neurotransmitter released by the axon
terminus of ILC will bind to and inhibit ( hyperpolarize) CEA thus suppressing memory.

iii. If ChR1 is inserted into BLA and opened, would memory be promoted or suppressed? Explain.
If the BLA neuron that is expressing ChR1 is subjected to light, the ChR1 channel will be activated/
open allowing the diffusion of Na+ and Ca2+ ions from the outside to the inside of the neuron thus
depolarizing the neuron and triggering an action potential. The neurotransmitter released by the axon
terminus of BLA will bind to and activate (depolarize) CEA thus promoting memory.

iv. If NpHR is inserted into BLA and opened, memory is strongly inhibited. Explain.
This would cause Cl- influx thus hyperpolarizing the membrane. In the absence of activation signal from
BLA and presence of inhibitory signal from ILA , CEA will be inhibited thus suppressing memory.

v. Using optogenetics, how would you figure out whether input (shown by the shaded box) from IL
neurons to ITC neurons is normally excitatory or inhibitory? Note: You can use NpHR and
ChR1 constructs inserted into IL axons, or you can suggest some other method.
You can insert the ChR1 Gene in IL neuron and activate or open the ChR1 channel. This should
activate ITC and subsequently inhibit CEA to suppress memory. You can insert the NpHR1 Gene in IL
neuron and activate or open the NpHR channel. This should inhibit ITC and subsequently activate CEA
to promote memory.

Question 6
a) The following schematic represents a lineage tree originating from one stem cell- type. Note: Each
letter and shade designates a particular cell- type. Each number represents a cell division.

a) Why is cell type A the only

E stem cell in this schematic?
1 3 Which of these cells (A-H) are
A
most likely terminally
2 F
differentiated cells?
A C This is the only cell type that
G can divide asymmetry to self-
B 4
regenerate and also give rise to
D all the cell types in the lineage
H
shown. This self-regenerating
LINEAGE ability and the ability to form
different cell types makes it a
stem cell.

b) The stem cell shown is bipotent/ multi-potent/ unipotent? Circle the best option and explain why
you selected this option.
It is multi-potent since it is giving rise to multiple cell types.

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Question 6 continued
Skeletal muscle is a dynamic tissue that is capable of mounting an orchestrated regenerative response
to physiological stimuli (extensive exercise) or severe injury as is shown below with Type A being the
stem cells.
c) Why is it disadvantageous for
the stem cells to divide
uncontrollably?
If these cells were always
dividing, they may acquire harmful
mutations, which would be
propagated to all the cells that will
arise from these cell type A in this
Bromouridine
lineage. More importantly, the
(BrdU) stained cells muscle would become misshapen
and not work.

d) Cell type A expresses CD56 (a cell surface protein) and Pax7 protein (a transcription factor). You
want to purify these stem cells using a flow cytometer and use them for muscle regeneration. Which
protein (CD56 or Pax7) would you use as a marker to purify the live stem cells from a mixed cell
population? Explain why you circled this protein.
You would use cell surface protein CD56 since you want to get live cells with which you can work. You
can add a fluorescence conjugated CD56 antibody, which will bind only toCD56 on the surface of the
SC and NOT any other cells in the population. These antibody labeled cells can be separated from the
remaining cell population through Flouorsecence activated cell sorter (FACS)

e) Recently, scientists inserted the extracellular matrix (ECM devoid of all cells) from pig muscle into
damaged human muscles to attempt regeneration. Explain why it is important to remove all cells
attached to pig ECM prior to inserting it in humans.
Since the pigs cells are foreign/ non-self cells (xenografts), they if not removed will be attacked by host
immune system resulting in life threatening graft rejection.

f) You decide to clone animals by somatic cell nuclear transfer (SCNT) by transferring the nucleus from
adult muscle cell into an enucleated egg. In a separate experiment, you first treat the muscle cell
genome with 5-aza-cytosine (5-azaC) a nucleotide that prevents DNA methylation. You insert the 5-
azaC treated nucleus into an enucleated egg. You observe an increase the efficiency of cloning. How
can you explain this?
The 5-azaC treatment de-methylates the DNA thus bringing the methylation pattern of the cell closer to
the embryonic state.

h) The following schematic shows zebrafish heart regeneration as described below.

After injury, expression and secretion of TGF

ligand begins in the wounded area.
Two cell types, fibroblasts and myofibroblasts
appear in the wounded area.
TGF ligand binds to and activates TGF cell
surface receptors, which activate SMAD3, a
transcription factor.
Active SMAD3 promotes proliferation of
myofibroblasts and their differentiation into new
cardiomyocytes.

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Question 6 continued
i. Do any cells described in the schematic above show stem cell properties? Explain your answer.
Myofibroblasts, since they self renew and also differentiate into cardiomyocytes,

ii. Where would you expect to see the localization of TGF receptor (choose from
cardiomyocytes, ECM, myofibroblasts, blastema)?

iii. Constitutive expression of active SMAD in a TGF receptor null mutant results in cardiomyocyte
regeneration after heart injury. Explain this result.
In the absence of TGF receptor, active SMAD substitutes for the normal signaling sequence that leads
to its activation and promotes myofibroblast proliferation and differentiation.

Question 7
In C. elegans the ectoderm gives rise to the epidermis (outer layer of the skin) and to all neural cell
types as shown below. Note: Each letter represents a particular cell type.

a) If you were to perform the lineage analysis by

Bromodeoxyuridine (BrdU) labeling

i. Which cell type would you label with BrdU to

trace the entire lineage shown in the
schematic?
You would BrdU label the T cells in the schematic above.

ii. Explain why you selected this option(s).

T cell divides assymetrically to generate T cells and
all the cells shown in the lineage.

b) What is the potency of the cell that you decided to BrdU label? Multipotent

c) Epidermal stem cells have been pivotal in treating burn victims. Briefly describe an experiment that
you can do to prove that a test cell is an epidermal stem cell.
You can adopt many approaches. You can transplant your test stem cells into adults i.e. remove
endogenous cells and replace them with test cells. You can see if your test cells produce the epidermal
cells. You can inject the labeled stem cells into a developing embryo and see what they become. You
can culture the stem cells in the presence of specific inducers and observe their fate or identify the cell
type they produce.

d) The skin has two layers: epidermis (outer) and dermis (inner or deeper layer) with the epidermal stem
cells (ESCs) located at the interface of epidermis and dermis. If the epidermis is removed from a patch
of skin, the epidermal stem cells, which are otherwise quiescent, start to proliferate. In contrast, if the
dermis is removed from a similar size patch, epidermal stem cell proliferation decreases relative to the
unperturbed skin.

i. What is the role of the epidermis in epidermal stem cell proliferation?

Epidermis contains the epidermal stem cells, that can divide to produce more epidermal stem cells and
also the new epidermal cells that replace the old worn out or the dead cells of the epidermis.

ii. What is the role of the dermis in epidermal stem cell proliferation?
The dermis that underlies the epidermis most likely secretes ligands or provides the appropriate
environment (niche) to the epidermal stem cells that allows them to divide into the cells of the
epidermis.

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Question 7 continued
iii. When the epidermal stem cells proliferate, where would you find transit amplifying cells and how
would these be affected by epidermis and dermis removal?
They would be present at the interface of dermis and epidermis. It is the epidermal stem cells that
divide to produce more stem cells and progenitor cells. The progenitor cells further divide to form the
transit epithelial cells. Removal of epidermis will cause these transit-amplifying cells to divide and
replace the lost epidermis. In contrast, if you remove the dermis, that provides the right environment or
niche for the division of these cells, then you will either observe production of no new epidermal cells to
replace the dead worn out cells or the epidermal cells produced will be abnormal.

e) Prospero is a transcription factor that localizes assymterically during the epidermal stem cells division
i.e. one daughter cell gets all the prospero and develops into epidermis whereas the other does not. In
the epidermal stem cells that express prospero- GFP fusion protein

i. List the subcellular organelle that would fluoresce green in epidermal stem cells? Explain your
choice.
Nucleus, where the transcription factors function to regulate gene expression.

ii. Classify Prospero as an inducer or determinant? Explain your choice.

Prospero is a determinant since it is expressed in epidermal stem cells and its asymmetric localization
during cell division influence the development of cell into epidermis.

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