Part I

Part 2. Multiple Choice (8 points each)

2. Progression of the cell cycle from G1 to S phase requires

a) phosphorylation of Rb by the G1 cyclin/CDK

b) ubiquitination of Sic by SCF

c) growth factor signaling

d) all of the above

3. The Retinoblastoma (Rb) protein

a) ubiquitinates Sic

b) binds to Bax

c) is activated by death receptors

d) phosphorylates MPF

e) inhibits cell cycle progression from G1 to S

4. LEAFY

a) promotes inflorescence identity in reproductive meristems

b) promotes flower identity in reproductive meristems

c) is a homeobox gene.

d) is an A-gene.

5. CLAVATA3 signaling from the tunica to the corpus

a) regulates shoot apical meristem size

b) promotes gibberellin sensitivity in the shoot apical meristem

c) produces an inhibitory field for organ initiation

d) inhibits the vegetative to reproductive transition

e) all of the above

Part 3. Short Answer ( points

6. (8 points)

Describe the Xist gene product. (What do you know about its biochemical function?) (4 points)

A mouse has the following genotype: One X has a normal Xist gene, and the X encodes the orange coat color. The other X has a defective Xist gene, and it encodes the white coat color. What color is the mouse? Explain your answer. (4 points)

7. Stem cell research (25 points)

You are a stem cell researcher. You believe that you have isolated a population of stem cells in mouse adipose (fat) tissue. Your stem cells carry a ubiquitously expressed GFP marker, and you want to assay the potency of these cells. This will be a time-consuming and expensive set of experiments. Before you get started, your advisor asks the following questions. Answer each question with complete sentences.

a) State the specific hypothesis will you test in your first experiment. (4 points)

b) Describe your experimental design as follows: 1) Describe any aspect of the host mice that is important to your experimental design. 2) How will you introduce the stem cells? 3) Which tissues or organs will you assay for incorporation of stem-cell derivatives? (6 points)

c) What criteria will you use to determine whether the transplanted stem cells have differentiated into new cell types? (We discussed 3 criteria in class.) How will you determine whether these criteria have been met? (15 points)

8. (7 points)

In your lab rotation, you are working with a cancer cell line. You prepare a batch of medium but mistakenly add an extra ingredient. Thinking it won’t matter, you transfer the cells to your medium but the cells all die. Initially you are dismayed but then realize you have potentially discovered a new cancer treatment!

a) First you need to know if the cells have undergone apoptosis or merely been poisoned. Name 2 features that would distinguish apoptotic cells from necrotic cells.

b) Having confirmed that the cells are indeed apoptotic, you next want to determine the mode of apoptosis. What are the 2 major pathways to apoptosis?

c) What experiment(s) that would allow you to determine which pathway was activated in response to your miracle ingredient.

9. (8 points)

You want to make a plant with a really big flower. You hypothesize that since the flower develops from a shoot apical meristem, that by increasing the size of the meristem, you will increase the size of the flower. Your approach is to overexpress the WUSCHEL gene using the strong 35S promoter in transgenic plants. About half your transgenic lines indeed have enlarged meristems but to your surprise, about half have small meristems that decrease in size as the plants grow until finally the meristem disappears. Explain your results considering both the function of the WUSCHEL gene and the apparently opposite effects of the transgene.

10. (15 points)

In the diagrams below, shade in the boxes for whorls where each of the A,B and C genes would be expressed in each of the given situations and give the identity of the organs that would form at each whorl.

a) A wild type flower

A
B
C
Whorl	1	2	3	4
Organ Identity

b) A loss-of-function mutant in the A-gene

A
B
C
Whorl	1	2	3	4
Organ Identity

c) A C-gene loss-of-function mutant with a B transgene controlled by an A-gene promoter (transgene: __A-promoter ; B-gene coding region )

A
B
C
Whorl	1	2	3	4
Organ Identity

11. (15 points)

A group of entrepreneurs are working on a business proposal for a new French fast food chain that will specialize in serving frog’s legs. To maximize profits, they plan to raise the frogs themselves. They realize they could make much more money if only each frog had more than 2 back legs and they hire you to work on this problem. Thinking back to your developmental biology course, you recall that FGFR2b is expressed in the ectoderm and is the receptor for FGF10 in the initiation of limb buds in chicks. If only you had the frog genes for these proteins, you could engineer designer frogs with extra legs! You isolate candidate genes and begin testing the hypothesis that you have the homologs.

Circle all the letters for experimental results that represent functional evidence (as opposed to correlative) supporting your hypothesis. Assume all experiments are properly controlled. Be careful—the potential investors see this as an important part of the proposal and have hired your old instructors as consultants to review the results!

a. The candidate frog proteins have 92% amino acid sequence identity to the chick FGF10 and 89% identity to chick FGFR2b.

b. A legless frog mutant has a premature stop codon in the gene encoding the frog FGF10-like protein.

c. The candidate proteins interact in the yeast 2-hybrid system.

d. Expression of a dominant-negative form of the FGFR2b candidate blocks leg formation in frogs.

e. A legless mutant phenotype is genetically linked to the gene encoding FGFR2b.

f. Antisense suppression of the gene encoding the frog FGFR2b blocks leg formation.

g. Injecting frogs with antibodies specific to your FGF10 protein blocks leg formation.

h. FRET analysis (fluorescence resonance energy transfer—a technique to demonstrate intimate contact between proteins) indicates the candidate proteins interact in vivo.

i. Expression of a constitutively active form of FGFR2b causes constitutive leg formation, even in the absence of FGF10.

k. The proteins are expressed in the same tissues and cells in frogs as in chicks.

l. A frog mutant has a premature stop codon in the FGFR2b gene and a legless phenotype is genetically linked to this locus.

m. The candidate FGF10 protein co-immunoprecipitates from frog extracts with FGFR2b antibodies.

n. Exogenous application of your candidate FGF10 protein induces ectopic leg formation.

o. A homeotic mutant with ectopic leg formation also shows ectopic expression of the FGFR2b candidate.

p. You have a strong intuitive feeling that they are the right genes.

q. A chimeric FGFR2b receptor containing the extracellular domain of PDGF receptor induces leg formation in response to exogenous PDGF.

12. (10 points)

a) Describe the factors involved in the regulation of the cell cycle following DNA damage; what are their biochemical functions and what are their regulatory relationships? (eg. kinase X phosphorylates and inhibits transcription factor Y which activates the expression of protein Z)

b) Describe the factors involved in the regulation of apoptosis following DNA damage; what are their biochemical functions and what are their regulatory relationships?