Biochemistry 442 FINAL EXAM June 8, 2010

__________________________ name student number

You have just been recruited to the recently established Department of Molecule Neurobiology as an Assistant Professor. Your credentials include training in molecular biology and animal behavior. You understand that it is important to make some breakthrough discoveries if you hope to be promoted. You decide to identify and study the gene(s) involved in unusual behaviors of dogs. Your first goal is to discover the gene(s) responsible for fetching chasing a ball and returning it to you. Certain breeds of dogs, e.g. a Golden retriever, will naturally fetch, whereas other dog breeds, e.g. a French bulldog, and most other animals will not.

1. (4 points) For your first experiment, you breed a female, French bulldog with a male Golden retriever and get a litter of 9 pups (G1 generation). When they are old enough you test each one separately and find that they will all fetch a ball as well as their father. You then breed three of the female offspring with one of the male offspring and obtain 24 pups (G2 generation). This time when you test the pups you find that 6 of them (G2 6/24) will sometimes run after the ball, but they will not return it, whereas the other pups are good retrievers. F or f You also notice that the 6 Bulldog Retriever G1 G2 (6/24) pupsthat do not return ball to you, Fetch + +++ +++ + are also smaller than the other A pups and they are black rather than golden colored. The +++ + +++ +++ Black coat B breeding results are summarized in the diagram (+++ means that + +++ +++ + Growth C this feature is prominent):

(1 pt each part) Based on these results, you hypothesize that a transcription factor (TF) regulates fetching behavior, coat color and growth as shown in diagram. Moreover, the gene encoding the TF exists in two forms (F and f). Assume that the Golden retriever expresses form F. (a) What form (F or f) of the TF regulates growth? F (b) What form of the TF regulates black coat color? f (c) What form of TF is present in the G1 generation? Both F and f (d) What form of TF was inherited by the 6 pups in G2? f

2. (18 points, 2 pt each part) Based on your hypothesis and knowledge of coat color control in mice, you decide to set up a functional assay to clone the TF responsible for black coat color. You start by isolating skin cells. Then you make a cDNA library from mRNA isolated from the skin and insert the double-stranded form of the cDNAs into the polylinker of an expression vector. Then you transfect the cDNA library into the skin cells you isolated and look for cells that turn black. You find a few black cells among many thousands. You pick those black cells, isolate the DNA and PCR amplify the DNA insert in the expression vector. a) What gene is being regulated in this assay? Melancortin 1 receptor MC1R) gene b) What breed of dog would you use to isolate skin cells for this experiment? Golden retriever because they are not black c) What kind of skin cell would be ideal for this experiment? Melanocytes (or skin pigment cells) d) What dog breed would you use to make the skin cDNA library? French bulldog because they can make black pigment e) What enzyme would you need to make the first strand of cDNA? Reverse transciptase f) After you made double stranded DNA, what TWO enzymes would you use to insert the DNA into the expression vector? A restriction enzyme to open up the vector and a ligase to seal it

g) What DNA element would your vector need to allow production of functional mRNA? A promote/enhancer that works well in melanocytes. h) What primers would you use to amplify the DNA insert? You would use primers in the vector flanking the site of cDNA insertion i) Name an alternative strategy for cloning this TF. You could use a positional cloning strategy

3. (9 points; 3 pt each part) You sequence one of the cDNA clones that was amplified by PCR in the previous question and discover that it includes a 30amino-acid sequence motif that is repeated 6 times near the N-terminus of the protein. You also use that cDNA to make a probe that you then use to screen a cDNA library made from Golden retriever skin cells. You isolate some clones from the new library and sequence them as well. You notice that predicted N-terminal and C-terminal protein sequences are the same but there is an internal deletion of 2 of the 6 motifs found in the first clone that you sequenced. a) What is the protein motif that is repeated? It is a zinc finger

b) What is the function of that motif? DNA binding

c) What functional domain would you expect to find at the C-terminus of this protein? An activation domain

4. (9 points) You next use one of your cDNA clones (from #3 above) to make a probe for screening a genomic library made from French bulldog DNA. You identify one clone, sequence it and generate the gene map shown below. The gene contains 11 exons as shown by the boxes; the open reading frame is indicated in black boxes. The six small exons are each 90 bp long. A Southern blot of DNA from a Golden retriever (GR) was compared to that of French bulldog (FB) after digestion with Bgl 2 and probing with a full-length, bulldog cDNA probe. You also perform RT-PCR with skin RNA from the 2 dog breeds using the primers (labeled a to d) shown under the genomic map.
Bgl 2

a) (3 pt) Estimate the size of the open reading frame for the protein encoded by the gene shown. Each small exon is 90 bp; black area = 16 x 90 = 1440 nt divided by 3 nt/codon = 480 amino acids. b) (6 pt) Using the information in this figure, provide a molecular explanation for the difference in the Golden retriever and French bulldog genes and mRNA.

5 kb

Bgl 2 Bgl 2 1 kb

Bgl 2 9 kb

a GR
Kb 10

b FB Kb 1.5 1.0

c GR GR GR a,d b,c a,c

PCR primers d FB FB FB a,d b,c a,c

0.5 Southern blot (genomic DNA cDNA probe) PCR products (RT-PCR from mRNA)

The Golden retriever gene is missing the 4th and 5th zinc-finger encoding exons that lie in the 1 kb Bgl 2 fragment; hence the mRNA is 180 nt shorter than that of French bulldogs.

5. (6 points) One of the prominent differences between the Golden retriever and French bulldog is size. You suspect that the TF you are studying regulates dog size by binding to the enhancer of the growth hormone (GH) gene. The enhancer for the GH gene is located between -300 and -500 bp of the transcription start site. So, you amplify that piece of DNA by PCR and label the ends with 32P using T4 polynucleotide kinase. You then incubate this probe with nuclear extracts made from pituitary glands of Golden retrievers (GR) or French bulldogs (FB) with the results shown below (left panel): You then perform competition experiments with a huge excess of the competitor fragments A and B that span -450 to -420 and -400 to -370, respectively (right panel). a) (3 pt) The TF from which breed binds to the GH gene enhancer? GR b) (3 pt) Where does the TF bind to the GH gene promoter? Between -370 and -420 bp
Small -500 32P -450 A -420 -400 B -370 GR GR GR A B -300 P32

Extract: none GR none FB Competitor: Large

6. (8 points) Within the binding site for the TF you recognize the DNA sequence (top strand): 5 GGCGACGCCGGC 3. To see if this is the binding site, you make a set of competitors in which you change one base at a time to T (only top strand shown) and test them as competitors with the probe shown in Q.5; the results are shown as compete (+) or not (-):
competition 1. 5 TGCGACGCCGGC 3 2. 5 GTCGACGCCGGC 3 3. 5 GGTGACGCCGGC 3 + 4. 5 GGCTACGCCGGC 3 5. 5 GGCGTCGCCGGC 3 + 6. 5 GGCGATGCCGGC 3 7. 5 GGCGACTCCGGC 3 8. 5 GGCGACGTCGGC 3 + 9. 5 GGCGACGCTGGC 3 10 5 GGCGACGCCTGC 3 11. 5 GGCGACGCCGTC 3 12. 5 GGCGACGCCGGT 3 +

Underline the bases below that are critical for DNA binding by the TF

5 GGCGACGCCGGC 3

7. (6 points) You perform DNA binding experiments with the TF isolated from French bulldog and discover that its preferred binding sequence is that shown below:

5 GGCGACGCCGTTGTTGGC 3
Using all the information acquired so far, provide a molecular explanation for why one form of the TF promotes growth, whereas the other promotes black coat color. Be as precise as possible mention the DNA binding domains and its recognition sequences in your answer.
Each zinc finger of the TF-F (from GR) binds to 3 nt. The French bulldog TF-f has two extra zinc fingers (4th and 5 zinc-finger encoding exons) that each recognize the GTT sequence. Thus, the TF-F from Golden retrievers cannot recognize the cisacting element in the MC1R gene and the French bulldog TF-f cannot recognize the cis-acting element in the GH gene.

Now that you have some molecular insights, you can begin to tackle the problem of how this transcription factor might promote fetching behavior. You reason that fetching behavior is probably controlled by the nervous system and that transcription of proteins important for neurotransmitter synthesis or receptor for that neurotransmitter might be affected. 8. (4 points) As a first step in deciphering how TF controls behavior, you decide to determine where in brain it is expressed. a) What is the name of the technique that would allow you to determine which neuronal cells express the TF? In situ hybridization b) How would you make the probe that you would you need for this technique?
You could either use random priming of TF cDNA clone in the presence of radioactive dNTPS, or you could make a radioactive RNA probe (using radioactive NTPs) from the cDNA clone using a bacterial RNA polymerase that would give you RNA complementary to mRNA.

9. (4 points, 2 pt each part) The results of your experiment in Q. 8 reveal that a small population of neurons in the hypothalamus of Golden retrievers expresses the transcription factor and it does not appear to be expressed anywhere else in the brain. These are also the only cells that express neuropeptide W (NPW). Given this new information, you hypothesize that NPW is involved in the fetching behavior. a) If your hypothesis is correct, what would you predict for NPW expression in French bulldogs? NPW expression very low because the TF-f does not bind b) What quantitative technique would you use to examine the validity of your hypothesis? The best method would be quantitative PCR (qPCR), but maybe a Northern blot would be sensitive enough.

10. (14 points, 2 pt each part) Assuming that you obtained encouraging results from your previous experiment, you decide to make a dog that lacks the ability to express NPW. Fortunately, the lab next door has isolated Golden retriever ES cells and has mastered the techniques of generating puppies from them. You isolate the dog NPW gene, subclone it into a plasmid and generate a restriction map of the insert as shown NPW gene here. You also have a plasmid Nco1 Aat2 Xba1 Age1 Avr2 Sma1 Ava2 Xho1 Nco1 containing a selectable gene (NeoR, p = promoter region) cloned into a polylinker of another plasmid. Your job is NeoR p to make a targeting construct and a probe (to use for Southern blot analysis) from Sma1,Avr2,Nco1 Aat2,Sma1,Ava2 the available plasmids. a) If you wanted to replace exon 3 with the NeoR gene, what restriction enzyme(s) would you use? Avr2 and Ava2 b) If you decided to insert the NeoR gene into the 3rd exon, what restriction enzyme(s) would you use? Sma1 c) Would one of the two strategies (a or b) be better than the other for disrupting the expression of NPW gene? If so, why? They would be equivalent; the first
method removes the middle of the open reading frame, while the second method disrupts the open reading frame.

d) What restriction enzyme(s) would you use to isolate DNA for electroporation into dog ES cells? Think about the next question before answering. Xba1 and
Xho1 would be good choices. You cant use Nco1 or Aat2 conveniently because they would cut near the NeoR insertion, too.

e) What restriction enzyme(s) would you use to isolate a DNA fragment to make a probe for a Southern blot procedure to identify correctly targeted alleles? Draw probe on the map If you use Xba1 and Xho1 for the targeting vector, then you
could use Nco1 and Aat2 to isolate a fragment for a 5 probe, or XhoI and Nco1 to isolate a fragment for a 3 probe f) What restriction enzyme(s) would you use to digest the DNA from each of the ES cell colonies that you want to analyze for homologous recombination by Southern blot using the probe fragment mentioned in (e)? Nco1 would work well with either the 5 and 3 probes g) Would you expect the targeted allele to give a larger or smaller DNA fragment than the endogenous gene after digestion with the restriction enzyme(s) that you chose for part f?

Smaller, because there is an Nco1 site adjacent to the NeoR gene insert 6

11. (10 points) The targeted ES cells are transferred into blastocysts isolated from French bulldogs and allowed to develop. Some of the puppies that are born are black while others are yellow and black. You breed some of the yellow and black dogs with French bulldogs and obtain some dogs that are all yellow. Then you breed some of the yellow offspring with each other to obtain dogs that carry two targeted alleles of the NPW gene (they are homozygous). When you throw a ball, these homozygous dogs chase after the ball, but they never bring it back to you, unlike normal Golden retrievers. You hypothesize that activation (+) of dopamine (DA)-producing neurons by NPW is necessary to activate a reward (or gratification) pathway that reinforces returning the ball to the person that threw it as shown in the pathway below.

Sensory Input

NPW

DA

Reward or Gratification Pathway

a) (4 points) Explain in molecular terms how the TF mediates retrieving behavior of dogs. TF-F presumably binds to the promoter of the NPW gene to activate transcription of the neuropeptide W gene. Release of NPW in response to the ball activates dopamine (DA) neurons.

b) (4 points) Suppose you wanted to turn French bulldogs into retrievers. What transgenic experiment would you do based on the information obtained so far? You could make a transgenic dog with a construct that has the NPW promoter driving the expression of the golden retriever TF-F that would then recognize the bulldog NPW gene. Alternatively, you could use gene targeting to remove the two zinc-finger exons from the bulldog TF-f gene.

c) (2 points) You discover that other breeds of dogs also fail to retrieve, but they have the same transcription factor as Golden retrievers. Genetic defects in what other genes in the pathway shown above would be expected to selectively disrupt retrieving behavior. Mention TWO genes Defects in the NPW gene, a gene that processes the NPW precursor, or a gene for the NPW receptor would be reasonable candidates. Dogs that failed to make DA would have many other problems as well.

12. (4 points) If the NPW gene is X-linked, how would the chromatin associated with the NPW gene differ in males and females? Chromatin associated with the NPW gene from females would be inactive due to heterochromatin on one allele (histones on that allele would be hyoacetylated and methylated, there would be a histone variant, and the DNA would be methylated and condensed into a Barr body). The NPW gene on the other allele in females would be active euchromatin, just as in males.

13. (4 points) The NPW gene has a simple repeat sequence (TG)n in the 3 untranslated region of the last exon that differs in length (n) between dog breeds, each of which is a good retriever. How would you use that information to determine whether the NPW gene was imprinted? You would need to mate the two breeds with each other to generate hybrids carrying both alleles. Then you would isolate mRNA from the brains of the offspring and amplify the repeat region by RT-PCR using primers that flank the repeat. If both alleles are expressed (i.e. no imprinting), then both long and short forms of the PCR product would be observed, if there was imprinting then only one form would be expressed. (This is a sufficient answer) If there was imprinting, then only one length of PCR product would be observed in the offspring; the length of the PCR product would depend on whether the gene was paternally or maternally imprinted. For example, if breed A has the larger TG repeat and there is paternal imprinting, then offspring of breeding male A with female B, would only express mRNA with the shorter length.