Solutions for 7.

012 Quiz III

11/23/09
Class Ave = 64/98 Standard Dev = 12.8 Approximate grade A B C D F Range 80 - 98 65 - 79 50 - 64 35 49 0 34 % 12% 36% 39% 12% 1%

Question 1 (16 points) The chromosomal position of the gene that encodes the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is diagrammed below, as is the location of two single nucleotide polymorphisms (SNPs). CFTR gene

SNP 2

SNP 3

a) Explain the advantage of using close, tightly linked SNPs to follow the inheritance of cystic fibrosis (CF) in this family. Using SNPs that are very close to the CFTR gene will decrease the chance that there will be a recombination between the SNPs and the CFTR gene. The pedigree shows the inheritance of CF. The individuals that are shaded show the disease phenotype, all affected individuals are indicated. Also listed are the alleles of SNP 2 for some individuals. *Assume complete penetrance of the CF phenotype.
1
CG

2
CC The two letters identify the two alleles of the SNP. For example, T C indicates that on one of the chromosomes you would find a T (a T/A base pair), on the other chromosome you would find a C (a C/G base pair).

3
GC

4
CC

5
CG

7
CG

8
CG

9
TC

10

11
C C

12
GT

13
GG

14
CT

CC

15

16
C G

17

b) Given this pedigree, answer the following questions. Assume no recombination between the CFTR gene and SNP 2. Individuals 5 and 6 have an affected child. What is the genotype for individual 6 with respect to Cystic Fibrosis locus? Choose from: +/+ +/ / Cant tell

Individuals 5 and 6 have an affected child. What is the genotype for individual 6 at SNP 2? Choose from: C G C C C T G G G T Cant tell

Assume that individual 17 is not a carrier of the disease allele. What is the chance that the offspring of individuals 16 and 17 is a carrier of the disease allele? Explain how you determined this using both the pedigree and the SNP information. From individual #15, we know that both mutant alleles of the CFTR gene are linked to the C version of the SNP in both #8 and #9. Thus we know that #16 is a carrier. Therefore, there is a 50% chance that any offspring will receive this allele and be a carrier.

Question 2

(30 points)

Above is a schematic of a protein kinase cascade. The cellular responses indicated alter gene expression, and result in cell division. a) Assume that the normal cells undergo cell division only in the presence of growth factor. Put an X next to any of the following mutations that could allow unregulated cell division. _____ A mutation such that the receptors lack the cytoplasmic domain __X__A mutation such that the receptors dimerize without growth factor _____ A mutation such that the receptors cannot be phosphorylated _____ A mutation such that the receptors cannot bind growth factor __X__ A mutation such that Ras fails to hydrolyze GTP __X__ A mutation such that MEK is constitutively phosphorylated b) Knowing that MAPK is a kinase, propose a mechanism for how MAPK could result in altered gene expression. MAPK phosphorylates a transcription factor that, once phosphorylated binds to specific DNA regions. The binding of the transcription factor to DAN increases expression of the genes specifically needed to stimulate cell division. c) You have two identical cell cultures. You treat one with growth factor but leave the other untreated. After 12 hours, you plan to compare the variation of RNA expression of the two cultures using DNA microarray technology. Limit your answers below to the space provided. Briefly describe what a DNA microarray is. Do not describe how it is made, but rather what features of a DNA microarray make it useful for the proposed experiment. A DNA microarray is a collection of specific DNA probes. Each DNA probe represents unique sequence. To be useful for measuring variation of RNA expression, each probe would represent DNA that is a transcribed region of a gene, and the collection of all of the probes should represent all of the genes in the organism. Briefly describe how a DNA microarray could be used to allow you to compare the gene expression profile of the two cultures. RNA from each sample is isolated, fluorescently labeled, and hybridized to the microarray. If more of a specific mRNA molecule is present in the sample, more molecules will hybridize to a specific probe. Thus more fluorescence will be associated with this specific probe, and the difference in fluorescence for each probe of the array can be detected.

Question 2, continued d) Consult the diagram on the previous page Briefly describe what is different between a ribosome that is producing the Raf protein and a ribosome that is producing the receptor protein. The ribosome producing the Raf protein will be free in the cytoplasm, whereas the ribosome producing the receptor protein will be associated with the endoplasmic reticulum. What is different about the first few amino acids of the nascent growth factor polypeptide as compared to the first few amino acids of the nascent Raf polypeptide that allows the proper localization of these two proteins? The first few amino acids of the nascent growth factor polypeptide will act as a signal sequence. This allows the SRP to bind and appropriately localize this protein. The first few amino acids of the nascent Raf polypeptide will not have a signal sequence.

e) You discover a cell that, under restrictive conditions, fails to secrete proteins and fails to localize proteins to the membrane. This cell is homozygous for a mutation in a single gene. Based on the inability to localize both secreted and membrane proteins and your knowledge of the secretion pathway, what gene would you predict is mutated in this cell? The gene encoding the SRP, signal recognition particle. Assume that this mutant cell can be infected with a single (+) stranded RNA virus. This virus is an enveloped virus that uses virally encoded glycoproteins to infect target cells. Assume that viral particles can be released from the cell under restrictive conditions. Will the viral particles released under restrictive conditions be infectious? Explain your answer. No. because all viral proteins are made using the host machinery, this cell is unable to localize the virally encoded glycoproteins to the plasma membrane membrane. Thus, the viral particles released from this cell will not have the virally encoded glycoproteins embedded in their envelopes. Without these glycoproteins, to specifically bind to a host cell protein they virus cannot enter the cell.

Question 3 (30 points) In neurons, different membrane pumps or channels are responsible for establishing the resting membrane potential and generating the action potential. a) Describe what is meant by the term resting membrane potential. The difference in charge across the membrane of a cell due to distribution of ions. b) List the two pumps or channels that are primarily responsible for establishing the resting membrane potential. Where appropriate, include the type of channel (i.e., ligand-gated, voltage-gated, etc.). For each list the ion(s) that flow through the channel, and the direction the ion or ions move to establish the resting membrane potential. Pump or channel Ion(s) moved Direction of ion(s) Into or out of cell (include type, if appropriate) Na+ out Na+/K+ pump (ATPase) K+ in Resting K+ channel K+ out

c) List the two pumps or channels that are primarily responsible for generating the action potential. Where appropriate, include the type (i.e., ligand-gated, voltage-gated, etc.). For each list the ion(s) that flow through the channel, and the direction the ion or ions move to produce the action potential. Pump or channel Ion(s) moved Direction of ion(s) Into or out of cell (include type, if appropriate) Na+ in Voltage-gated Na+ channel Voltage-gated K+ channel K+ out

Question 3, continued d) Funnel spiders produce atracotoxins, which open voltage-gated Na+ channels, and greatly slow their inactivation. Which of the action potentials shown below (1, 2 or 3) would be recorded in a single neuron after the neuron was treated with atracotoxin? Note that the normal action potential in this neuron is shown by the dashed line. Action potential 3. In terms of ion flow, explain why your chosen action potential differs in the shape as compared to the normal action potential. The repolarization of the membrane from +50 mV back to the resting potential of -70 mV is the result of two actions, the timed inactivation of the voltage-gated Na+ channel and the opening of the voltage-gated K+ channel. Slowing the inactivation of the voltage-gated Na+ channel will mean that Na+ is still able to move into the cell through the voltage-gated Na+ channel as K+ moves out of the cell through the voltagegated K+ channel. This lengthens the time it takes to return the membrane to -70mV.

+100mV

Action potential 1: Greater amplitude than normal

+50mV

+50mV

Action potential 2: failure to repolarize

-70mV +50mV

-70mV

Action potential 3: slower repolarization

-70mV

e) Widow spiders produce latrotoxins, which induce a large Ca2+ influx into the presynaptic terminal but do not alter Ca2+ influx into the postsynaptic cell. You have an excitatory presynaptic and postsynaptic pair of neurons that you treat with latrotoxin. Describe how the latrotoxin induced Ca2+ influx into the presynaptic terminal changes the response of each neuron below. presynaptic cell: An increase in Ca2+ influx into the presynaptic terminal will allow the presynaptic cell to release additional neurotransmitter. postsynaptic cell: Because the presynaptic cell is releasing additional neurotransmitter, the post-synaptic cell is more likely to generate an action potential.

Question 3, continued To further study synapses, you place Aplysia neurons in culture to create the arrangement shown below. The response of a postsynaptic neuron to input from the presynaptic neurons (neurons A, B, and C) can be recorded by placing a small recording electrode into the postsynaptic neuron.
Neuron A Recording electrode Neuron B

Neuron C

Neuron A makes an inhibitory synapse. Neurons B and C make excitatory synapses.

f) Neuron A makes an inhibitory synapse. What effect would release of neurotransmitter from neuron A have on the membrane potential of the post-synaptic neuron? Neuron A makes an inhibitory synapse, so release of neurotransmitter from neuron A hyperpolarizse the membrane of the post-synaptic cell. g) Neurons B and C make excitatory synapses. At the start of the experiment, both neurons B and C must be stimulated to generate an action potential in the postsynaptic cell. However, after much stimulation over a period of days, you discover that Neuron B alone now triggers an action potential in the postsynaptic cell. Note that a genetic mutation has not occurred in either cell. Your UROP advisor asks you to propose a hypothesis to explain what might have occurred in neuron B that is consistent with this observation. There may be several plausible hypotheses, but describe just one. Neuron B may produce additional synaptic vesicles that allow additional neurotransmitter release when Neuron B fires an action potential. Neuron B may make additional synapses with the post-synaptic cell that would allow additional neurotransmitter release. Neuron B may slow the reuptake of neurotransmitter that would allow longer exposure to the released neurotransmitter. Your UROP advisor asks you to propose a hypothesis to explain what might have occurred in the postsynaptic cell that could explain this observation. There may be several plausible hypotheses, but describe just one. The post-synaptic neuron may make additional receptor and/or ion channels that would allow a greater response to the released neurotransmitter. The post-synaptic neuron may make additional synapses with Neuron B that would allow additional neurotransmitter to be released to the post-synaptic cell. h) To study the synapses between nerves and muscles, you look for Aplysia mutants that do not move properly. Two different types of screens were used. Screen 1 looked for mutants that showed rigid paralysis, and Screen 2 looked for mutants that showed flaccid paralysis. Which one of the two screens could identify an animal with a loss of the acetylcholine receptor? Why? Screen 2. If an animal lacking the acetylcholine receptors, the muscle cells would be not bind to acetylcholine and thus could not contract. Which one of the two screens could identify an animal with a loss of acetylcholine esterase? Why? Screen 1. If an animal lacking acetylcholine esterase, acetylcholine released to the muscle cells would be present for much londer and cause the muscle to continue to contract.

Question 4 (22 points) Cells can become transformed (i.e., become capable of growing and dividing inappropriately) because of mutations in genes such as proto-oncogenes and tumor suppressor genes. a) How is the function of the protein encoded by an oncogene different from that of a proto-oncogene? The function of the protein encoded by a proto-oncogene is to promote cell division when the appropriate signals are present. The protein encoded by an oncogene promotes cell division even when inappropriate.

Five cells are described below. Cell Affected Normal function of protein Gene 1 2 3 Gene M Gene N Gene O Protein M activates the retinoblastoma protein, pRB. Protein N is a transcription factor that increases expression of a proto-oncogene Protein O prevents the cell from entering the cell cycle Protein P initiates a signal cascade that stimulates cell division Protein Q is a growth factor receptor

Allele1 A mutation that gives a constitutively active protein M A mutation such that protein N can no longer bind to DNA A nonsense mutation that replaces the fourth codon with a stop codon A mutation that gives a constitutively active protein P

Allele 2 A deletion of the entire gene Wild-type A deletion of the entire gene A deletion of the entire gene A mutation that activates the receptor in the absence of growth factor

Gene P

Gene Q

A mutation that prevents receptor localization

b) Identify the normal version of Gene M, N, O, P and Q as a either a tumor suppressor gene or a proto-oncogene and give the phenotype of the cell that carries the two alleles described above. Choose transformed (i.e., has characteristics of a cancer cell) or NOT transformed. Cell Affected Gene Gene M: Gene N Gene O: Gene P: Gene Q: Normal Gene functions as tumor suppressor gene or proto-oncogene tumor suppressor gene proto-oncogene tumor suppressor gene proto-oncogene proto-oncogene Phenotype of cell with alleles 1 and 2 above

1 2 3 4 5

NOT transformed NOT transformed transformed transformed transformed