NOTE: Some Questions May Have Multiple Correct Answers; As Long As Yours Is Reasonable

Bio154 Final-2006

Name: ______

I.D. Number:______

TA:______

Score:

1.______(out of 12) 2.______(out of 14)

3.______(out of 12) 4.______(out of 12)

5.______(out of 12) 6.______(out of 14)

7.______(out of 10) 8.______(out of 14)

Total:______(out of 100)

NOTE: Some questions may have multiple correct answers; as long as yours is reasonable, logic and consistent, you can get full credit. Some questions may be challenging but don’t panic; grading will be on a curve. Please try to be as concise as possible. Almost all questions should be answered by one or two sentences. Good luck.

Question 1.

A.) After a long day of studying, you decide to go outside for a walk one night. Shining your flashlight into the bushes outside your dorm, you see a silvery reflection out of the eyes of a stray cat. Thinking of photos you have seen, you realize that shining bright white lights (like camera flashes) into human eyes produces not a white/silver reflection, but a strong red reflection (the dreaded “red-eye”). Why have many nocturnal predators (like cats) changed the way light is absorbed by the retinal epithelium? (2 pts)

B.) Walking a little further from the dorm, you come upon another animal, a rare nocturnal salamander. Recognizing a cheap supply of experimental subjects, you take a bunch of salamanders into the lab and begin to examine their rod responses. You begin by recording the dark noise currents of single salamander rods, and compare the responses to those of single rods in a related, diurnal (day-active) salamander (traces from 3 rods each are shown below). You choose to focus on the discrete events only.

Nocturnal Diurnal

Summarize the differences between these two responses, and provide a plausible mechanism for each. (4 pts)

Give one advantage and one disadvantage that these adaptations confer upon the nocturnal salamander? (2 pts)

C.) Extending your studies deeper into the retina, you examine the responses of single, ON-center retinal ganglion cells to dim flashes, using light spots of different sizes.

Nocturnal Diurnal

How do the receptive fields of ganglion cells differ between the two salamander species? (1 pt) How does this difference make sense in terms of the single rod responses? (2 pts) What is the disadvantage of this strategy to the nocturnal salamander? (1 pt)

Question 2.

A. 1991 marks an important year in olfaction research. Using molecular biology techniques, Linda Buck and Richard Axel identified a large class of odorant receptors in rodents. Please give two pieces of evidence used by Buck and Axel to argue that are indeed odorant receptors (2 pts).

B. Having the large class of odorant receptors cloned allowed Buck and Axel to discover two fundamental organizational principles of the olfactory bulb and epithilium. Describe two principles and one method each that leads to the discovery of these principles (4 pts).

C. In 2006, Liberles and Buck found a new class of olfactory sensory neurons that express another class of 7-transmembrane G-protein coupled “trace amine-associated receptors” (TAARs). TAAR expressing sensory neurons are found to be distinct from those that express conventional odorant receptors. To study the function of TAARs, Liberles and Buck transfected individual TAARs into HEK293 cells, applied different compound to the transfected cells, and assay for activity of a transcription reporter SEAP (secreted alkaline phosphatase) (see Figure).

1.  The SEAP is under the control of a cAMP response element (CRE) control. Describe one possible signal transduction pathway between TAAR activation and the SEAP transcription (2 pts).

2.  From the data presented, draw one conclusion about the specificity of the TAAR receptors? (2 pts)

D. Given the cloned TAAR receptors, describe two experiments to examine how information is relayed from TAAR expressing neurons to their downstream neurons. In other words, how are the TAAR neurons represented in the cortex? (4 pts)

Question 3

While doing a forward genetic screen in Drosophila for new circadian mutants, you discover that one of your mutants is arrhythmic in constant darkness. After some diligent genetics, you map the mutation to a gene which has not been previously described. You decide to name your gene cadence (cad). Though nothing is known about this gene, sequence comparison with known genes reveals that it has a predicted protein dimerization domain, as well as a domain which may be involved with DNA binding. Moreover, you raise an antibody against the gene and discover that the immunostaining is usually nuclear.

A) One of the grad students in your lab who is working on cad tries to find out which DNA sequence the protein may bind. After some work, your student reports to you that cad is unable to bind DNA. This puts you in mind of other components of the circadian clock. Which gene that we have studied does this new gene resemble, and what mechanism of action does this suggest for cad? (2 pts)

B) You decide to test your theory using the new antibody you have had made against cad; you will stain fly tissue and observe localization and amount of cad. What experiment will you do, and what do you expect to see? (4 pts)

Being an astute Drosophila researcher, you wonder if your gene is important for other biological rhythms. After all, you are aware that other circadian genes are necessary for some aspects of wildtype courtship song. Moreover, you know that some fruitless mutants display altered courtship song, and that cad and fruitless are co-expressed in some neurons. You wonder if the two cad phenotypes, circadian and courtship song, are separable. That is, can a single fly display one phenotype while being wildtype with respect to the other?

C) Assuming that these phenotypes are separable, what two experiment(s) can you perform to demonstrate that they are? You have access to any fly stocks used in the paper from Manoli et al., as well as flies carrying UAS-cadence and UAS-cadenceIR (an RNAi directed against the cadence gene product). For any flies you are examining please describe their genotype with respect to fruitless, cadence, and any transgenes they may carry. Also, please predict whether these flies will take the same length of time or longer than wildtype to copulate, and whether they will remain rhythmic in constant darkness. (6 pts)

Question 4

The year is 2030. We have now discovered a set of drugs that will make stem cells differentiate into cells that look like neurons—they are able to extend axons and dendrites in vitro. Scientific researchers are just beginning to experiment with adding these stem cell derived neural precursor cells into the adult brain to treat degenerative disorders. As a medical researcher, you are particularly interested in using this type of therapy to treat Parkinson’s Disease.

A.) From what you learned about the pathology of the Parkinson’s disease, where in the brain would be the most obvious place to put these stem cell derived precusors to treat Parkinson’s Disease? (1pt)

B.) If you added these neural precursors into the adult brain and the patients don’t recover movement control, give two possibilities about what else might have gone wrong? Give one experiment to test whether this was a problem. Note: the precursor cells survive in the brain and do not cause cancer.

(4pts)

C.) It has recently been discovered that you can couple axon guidance molecules to small beads. These beads can be placed in the brain without causing physical damage. To repel axons of neural precursors away from a bead, what molecule should you attach to the bead? To attract neural precursors, what molecule should you attach?

Note: it has been demonstrated that these stem-cell derived neural precursors express Robo and Dcc, but not Unc5.

(3pts)

D.) Given the numbered regions above, provide one region where you would put repulsive beads in the brains of Parkinson’s patients? One region where you would put attractive beads?

(4 pts)

Repulsive beads—

Attractive beads—

Question 5

Sound pressure fluctuations result in displacement of a membrane in the auditory cochlea such that different frequencies are represented as shown in the figure below.

A) Is auditory representation of sound frequency DISCREET OR CONTINUOUS and is this representation more similar to VISION OR OLFACTION? (2 pts)

B) When adults who are congenitally or prelingually deaf receive cochlear implants, the results are disappointing. They never gain language competence and often request that the implant be removed. In contrast, early cochlear implantation in congenitally or prelingually deafened children can lead to nearly perfect acoustic communication and language competence. What may explain this phenomenon, based on our knowledge of the development of the visual system? (1 pt)

In congenitally deaf cats, the CNS is deprived of acoustic input because of degeneration of sensory cells before the onset of hearing. However, primary auditory afferents survive and can be stimulated electrically by means of cochlear implants. Cochlear implants when placed in both ears at the appropriate time is effective in allowing congenitally deaf kittens to respond to different frequencies of sound.

C. Design an experiment with an appropriate control to determine when, after birth, cochlear implants placed in both ears will be effective in congenitally deaf kittens. (2 pts.)

D. Name two ways you can map the receptive field properties of neurons in the auditory cortex of kittens. (2 pts) What changes would you expect in the auditory cortex map of congenitally deaf kittens that received cochlear implants in both ears at the appropriate time compared to congenitally deaf kittens who did not receive cochlear implants? (2 pts)

E. What do you think would happen to the auditory cortex map of congenitally deaf kittens if the cochlear implants in both ears allowed transmission of only one frequency? (3 pts)

Question 6.

The neuromuscular junctions in C. elegans utilize the same excitatory neurotransmitter-receptor system compare as their vertebrate counterpart. In order to understand the molecular mechanisms of synaptic transmission, aldicarb was used to perform a genetic screen. Aldicarb causes paralysis and eventually death.

A) Use one or two sentences to explain the action of Aldicarb (2 pts)

B) A forward genetic screen was performed to isolate mutants that are resistant to Aldicarb induced paralysis. A number of mutations were isolated through this screen. Among the genes that should be uncovered by this screen, list 4 genes that functions in the presynaptic terminal and use one sentence to support your prediction for each gene (4 pts).

C) You just found a new mutation in this screen and it appears to be dominant (heterozygous animals showed a resistant-to-paralysis phenotype). After months of hard work, you finally identified the mutation in a gene (you have just cloned it!). By comparing the primary sequence of the gene to homologs in other species, you predict that this gene encodes a potassium channel. What region of the protein is most indicative that this is a potassium channel (not other channels)? (2 points) Give a one-sentence explanation why this is? (2 pts). How does the mutation affect the function of this protein (2pts)

D) To study the function of this protein, you generate a knockout allele of this gene, this time the mutation appears to be recessive. Can you predict what kind of phenotype you will have on an Aldicarb assay and why? (2 pts)

Question 7.

In October of this year, Suo et al. came out with a publication that investigated the starvation response in C. elegans. They noticed an alteration in the worm’s behavior as well as a metabolic adaptation for survival. Their first key result was that starvation induced cAMP response element-binding protein (CRH-1) activation in a specific set of neurons called the SIA neurons (Fig 1. A&B).

Figure 1

A) What is the most likely genetic tool that the authors utilize here to image this activation in live animals? (Although tzls is the background of the animal, it will not help you with your answer). (1 pt)

The authors go on to find that if the animals are fed the bioamine octopamine (which is found endogenously in the animal) with their normal bacterial diet, the following results are seen (Fig 2. A&B). However, if they ablate the upstream neurons (MIA) and starve the worms, the effect of octopamine is still present (Fig 2. C&D).

Figure 2

B) What role do you hypothesize octopamine is playing in this system? (1 sentence, 1 pt)

You decide to continue this research and find two interesting mutants: ser-3 appears to contain a loss-of-function mutation (unfed worms do not exhibit starvation phenotype) and egl-30 appears to have a gain-of-function mutation (fed worms appear to be in starvation mode constitutively). You find that these two genes encode SIA neuron specific proteins that bind to one another in vitro by using coimmunoprecipitation and western blot assays. You want to further understand the function of these two genes in vivo so you take a pharmacological approach. You feed these mutant worms forskolin (a drug that activates adenylate cyclase) thus promoting the cellular levels cAMP. After forskolin exposure you measure CRH-1 activation (indicated by + or -). The following results are seen (Table 1).

Table

Food / Y / N / Y / N
Forskolin / N / N / Y / Y
WT / - / + / + / ++
ser-3lof / - / - / + / +
egl-30gof / + / ++ / ++ / +++
ser-3lof, egl-30gof / + / ++ / ++ / +++

C)  With these data, you can tell that ser-3 lies UPSTREAM / DOWNSTREAM of egl-30 (circle one). How do you know this? (4 points)

D)  Using your answer from part C, the co-IP data and western blot analyses, what proteins do you think ser-3 and egl-30 encode? (2 sentences, 2 points)

You find a new mutant, unc-13 (unfed worms fail to show a starvation phenotype). You accidentally drop some of these mutants on a bacterial plate that has been supplemented with octopamine. You’re curious and decide to look at these worms even though your PI tells you to just chuck them out. Surprisingly, these worms now have the starvation phenotype!

E) Why do you think this is? What kind of process could unc-13 be mediating? (2 sentences, 2 points)

Question 8

You have found a new exotic animal that has neurons with the following properties:

Vrest = 0 mV

[X+]out < [X+]in