PBC5065 Advanced Genetics – Moore Section 2009 Test 10/26/09Name_____________________________

Show your work! Partial credit may be given if your answer is incorrect but I can follow your logic.

The grandson of Victor Frankenstein, Frederick, is a skilled neurosurgeon and teacher who has spent his life living down the legend of his grandfather, even changing the pronunciation of his name. He works at Shands Hospital on the campus of the University of Florida. To his surprise, Frederick inherits his grandfather’s castle in Transylvania.

When he visits the castle, Frederick finds that it houses a servant, Igor, a housekeeper, Frau Blucher, and a lovely and mellow plant geneticist, named Inga, who works at close-by Transylvania University.

Frederick (let’s call him Fred for short) also discovers that the monster his grandfather made is still alive and living in the castle and Frederick names him Dickkopf. Fred’s grandfather’s laboratory and notes are also still there. But Fred decides he is not interested in studying reanimation. Instead what interests him is Dickkopf’s friend, Larry.

Fred has obtained grant funding from the International Society of Hair Restoration Surgery, The Trichological Society, and the Luxuriant, Flowing Hair Club for Scientists to study a rare condition in humans that causes long, thick hair to grow all over their bodies. Although the condition is rare, Frederick has identified a few families in which it occurs. The pedigrees of the families point to a single gene as the cause of the phenotype. Frederick is calling the putative gene Werewolf. And now here is Larry, who also shows the phenotype.

But before beginning to work with humans, Frederick decides to use a mouse model for initial experiments, because there are some excellent mouse mutations available. For example, he has a stock where the mice are hairless. And Igor, in one of his late-night experiments, irradiated some of the nude mice and one of them gave birth to a male mouse with very long silky hair. Igor thinks it would make a nice pet or a tasty snack, but Fred can see that the mouse has scientific potential if its phenotype is due to a simple genetic alteration. To determine whether this is the case, Fred mates the hairy male mouse (H) to two inbred nude (N) female mice, each of whom produce 10 offspring (see next page).

2) (2 pts) After seeing the results of the crosses, Fred hypothesizes that the phenotype of the long-haired male mouse (number 12 in the figure), designated H for hairy phenotype, and nicknamed Harry, does appear to be due to a simple genetic alteration. Assign allele designations for this putative gene, which we will call Wf for now (H and N are phenotypes, designated in case the photograph is not clear, Wf is the allele for hair production). What is the genotype of Harry at the Wf locus? __Something designating heterozygosity_________

What is the genotype of both mom mice (Numbers 11 and 13, with their phenotype designated N for nude)? ___Something designating homozygosity ________

In both cases you need to show both alleles.

Fred uses 200 markers to genotype all of the mice. The results for 3 markers are shown in the figure above. Note that in Marker B, which is an RFLP, one of Dad Number 12’s alleles can give a two-banded phenotype (which is determined by where the restriction enzyme cuts and where the probe used hybridizes). Also note that Marker C is a dominant marker, where the band is present or absent.

2) (4 pts) Which marker or markers appear to be linked to Wf? _A and B_ .

3) (4 pts) What is the phase of the alleles of Wf and Marker A in Harry? __Dominant Wf allele and upper band allele of Marker A, recessive Wf allele and lower band allele of Marker A__

What is the phase of the alleles of Wf and Marker B in Harry? (Tell me what allele designations you are using) Dominant Wf allele and lower double-banded allele of Marker B, recessive WF allele and intermediate band of Marker B__

This question gave a lot of people problems, but enough people got it correct that I don’t think the question was flawed. If you got 0 points, I couldn’t tell that you had any inkling

what phase was. If you got 1 or 2 you seemed to have some idea of what I was asking, but you were still somewhat off the mark. If you got 3 points, you seemed to me to have a pretty firm grasp on phase and alleles, but some important details were not present. If you were totally correct, you got 4. If you did not get full credit for this question, please take some time to look at why. This is a very fundamental genetic concept.

4) (4 pts) Circle the progeny mice that appear to be recombinant between Wf and any of the markers (indicate which marker).

Progeny 10 & 20 are recombinant for Marker AProgeny 20 is also recombinant for Marker B.

With this kind of segregating progeny, it is possible to test for linkage using either Chi Square analysis or LOD score analysis.

I meant this as a statement, but many of you took it as a question; no points for a comment here, whether you were correct or not.

5) Set up a Chi square analysis to test for linkage between Marker A and Wf in the progeny mice (i.e. show the observed and expected categories and numbers for each class and the degrees of freedom for the test). (3 pts) (Marker A alleles lettered from top A and B.)

Genotype Observed Expected (O – E)2/E9 5 3.2 16/51 5 3.2 16/51 5 3.2 16/59 5 3.2 16/5

Totals 20 20 12.6 = X2

Df = 3

I gave a lot of partial credit here, that I probably shouldn’t have. Note to self: put a chi square problem back in homework.

Do you expect these numbers to yield a significant or nonsignificant Chi square value? (2 pts)

Significant. I gave no credit for “Yes” or “Nonsignificant” without further explanation, partial credit if you said nonsignificant but justified your answer logically (about the only way you could do this is to have mistakes above).

6) Set up a LOD score analysis for linkage between Marker B and Wf, using a Θ value of 0.1, all of the progeny mice, and the formula of your choice. (3 pts)

I gave full credit for answers that showed total n of 20, so n – r = 19, r = 1, and one of the formulas I gave you, e.g.,

Z (0.1) = (20 – 1) log [2 (1 – 0.1)] + 1 log (0.2)= 19 (0.25) – 0.7=4.85 – 0.7= 4.15

7) (4 pts) Which marker appears to be most closely linked to Wf? _____B________ If you were wrong in question 4 but gave an answer here that makes sense with what you said in question 4, you got partial credit.

Would this be a good marker from which to initiate a chromosome walk to Wf? Why or why not? I gave one point of credit if you said yes, but justified why. I gave 2 pts for realizing that, based on the recombination frequency in this one small population, this would be a very long way to walk, and that closer markers are needed. I don’t know why so many people said yes after the calculation you did in your homework on bp versus cM. This is another concept that could be very useful to you in the future.

Fred examined a number of segregating molecular markers scattered throughout the genome using informative families from matings with Harry. Shown in the table below are some of the marker loci that mapped to chromosome 8.

8) If Marker B in the question above was really one of the markers in the table, which one would you hypothesize it would be? Why? (2 pts)

The answer I expected was D8S1733 because the recombination value you calculated should have been 5 cM and this marker locus showed the highest LOD value at that theta value (0.05) and the LOD was well above 3.0. I also gave full credit for D8S1771 if you showed the same logic, although the LOD was not as high, since there could be many reasons for this (differences in numbers sampled, etc). Also the trend was more clear for this marker. If you came up with a different locus and gave a good argument, I gave partial credit.

9) Which marker would you use to initiate a chromosome walk toward Wf? (2 pts)

I expected D8S560 because it had the highest LOD at 0.0. Again, partial credit for another answer that was convincingly justified.

10) List two markers that clearly do not show linkage with Wf. (2 pts)

Markers where the LOD was never above 3.0 in table: D8S511, 1827, 549, 1739

11) Do you think that this means that they have been mistakenly mapped to chromosome 8? (2 pts)

No, they could be too far away to detect linkage.

12) Igor loves to do cytogenetics, especially with chromosome paints (FISH) (he thinks the resulting preparations are pretty). He prepares a number of mouse cell lines for Fred to use in his studies of Wf expression, and several different kinds of FISH paint probes. Match the type of paint probe Igor prepared with the experiment Fred did with it (some experiments can be done using more than one kind of probe and in some cases it would be useful to use more than one kind of probe) (8 pts total):

A. A probe composed of repetitive DNA (e.g. SINEs and LINEs)B. FISH with a whole chromosome library, where each chromosome is differently coloredC. A probe specific for all of chromosome 8D. A probe for a small region around WfE. A probe for telomeres: (1 pt each)

___B____ Determining the chromosomes with which chromosome 8 most often interacts in an interphase nucleus

___C or E____ Studying Wf expression in a cell line trisomic for chromosome 8

___C, D____ Determining the location of Wf in the chromosome 8 interphase territory, using 2 probes

___C ____ Studying cell lines with duplications or deletions of chromosome 8 (assuming they are large enough), or translocations involving chromosome 8, but not inversions in chromosome 8, using mitotic metaphase preparations

___D____ Confirming where on chromosome 8 Wf is located, using mitotic metaphase preparations

___A_____ An experiment where all of the chromosomes are colored the same color

___E_____ An experiment where the ends of all of the mitotic chromosomes are studied

________ Study of a cell line where there is an inversion on one homologous chromosome 8, using 2 probes I messed up here and accepted anything. I should have had a centromere specific probe on your list. Then the answer would be E and the centromere probe.

Other answers are possible. I gave credit to anything I thought was plausible.

After a lot of linkage analysis, Fred identified a marker, Marker X, that appeared to be very tightly linked to Wf. Using a rat/ mouse somatic hybrid chromosome panel (where mouse chromosomes are eliminated), Fred was able to map Marker X to mouse chromosome 8. Fred also had access to a set of radiation hybrid cell lines for mouse (made by irradiating mouse chromosomes and then fusing them with hamster chromosomes). The part of the panel that includes mouse chromosome 8 is shown. The individual hybrid lines are indicated by their names across the top of the figure and numbers 1 to 30 across the bottom of the figure. The portion of mouse chromosome 8 that each hybrid cell line harbors is shown by the line running vertically between the name and number. Individual regions of chromosome 8 based on Giemsa banding are designated at the far left of the figure by numbers.

Fred extracts DNA from each of the hybrid cell lines and probes it with a probe made from Marker X. Individual hybrid lines 1, 2, 4, 6, 7, 8, 10 13, 15, 17, 19 21, 23, 25, 26, 27, 28, 29, and 30 contain mouse DNA that hybridizes with Marker X. 13) (2 pts) The bands depicted on the chromosome are based on the staining of

___euchromatin________ versus ____heterochromotin____________.

What is this representation called? _____ideogram________________________________

14) In which region is Marker X and so presumably Wf, located? (2 pts)

Band 36, hybrid 30 is diagnostic for this.

Thanks to the genetic resources available for mouse, Fred is able to order large insert clones that cover the portion of chromosome 8 that appears to contain Wf. The clones have already been put in order in the mouse genome, and some markers have been identified on each clone, so Fred could do more linkage mapping to move still closer to Wf. However, since he now has a relatively small part of the genome to search, he decides to search for candidate genes in the region. After still more experiments and examination of the available databases, he identifies three transcripts in the region that he believes might have potential to be Wf.

15) On the figure above, identify (Draw a line to an example or label it) (12 pts):

Markers/ genes used in first construction of the genetic map.Markers/ genes used for more fine mapping (mapping more close to Wf).Large inserts that could be used for walking tom the region of Wf.A candidate gene.An exon.An intron.

I was pretty liberal here but not totally. I wanted to see whether you understood physical mapping. Most of you did but some of you clearly didn’t.

One of the things Fred does next is prepare RNA from various mouse tissues and assemble them on northern blots, that he then hybridizes with probes from each individual interesting transcript (i.e. potential gene of interest, Wf). The blots are shown below.

Large chrom. interval

Small interval fine mapping

Large insert clones

Candidate genes with introns,exons

Some indication that the DNA sequence was contained in the candidate clones

16) Which candidate gene is most likely to be Wf? Why? (2 pts)

Gene 1, expressed only in skin.

17) What would you expect to see if you probed a zoo blot with Wf (or did a homology search at NCBI)? (2 pts)

Perhaps homologues in mammals with hair but not for instance in worms or birds or fish. If you said it would be expressed in every species without a convincing rationale, I only gave you partial credit.

Candidate gene 1 →

Candidate gene 2 →

Candidate gene 3 →

The differences in gene number in the three mice are an example of what phenomenon we discussed in class? (2 pts) I was hoping to see “copy number variation”, because I made a big deal about this in class. However, I accepted “aneuploidy”, which I would consider a subset of copy number variation, but correct for this specific example, and “duplication”, which is also true. The point of the example is that it is copy number, not copy arrangement, that leads to the mutant phenotype.

The mutant mouse displays what cytological aberration if all of the rest of his chromosomes are normal? (2 pts) Trisomy for chromosome 8.

Igor found another mutant mouse that has long hair (top mouse in figure) compared to his littermate (bottom mouse). Using a Wf probe and FISH technology, Moe examined the configuration of Wf in the mutant mouse and its parents. His results are shown below. The DNA represents mouse chromosome 8 and the arrows represent copies of Wf.

18) The naked mother and daughter display what cytological aberration? (2 pts) __Deletion, deficiency___

19) What has to happen, compared to the other mice in the pedigree, for the affected mice to be naked? (2 pts)

They have to have one (or fewer?) copies of the gene, no matter the configuration of the genes if there is more than one copy (again copy number variation).

In addition, Fred has located a new mouse genetic stock which is segregating for mice with no hair. Below, a litter of 4 mice and their parents are shown, along with Igor’s cytological analysis. The naked mice are represented by dark symbols, chromosome 8 is represented by the DNA, Wf copies are represented by arrows, and the X indicates that the gene region is missing, not just defective.

20) Fred and Igor have located Wf to mouse chromosome 8 but human chromosome 1. Why? (The figure below may help you answer this question.) (2 pts)

Human chromosome 2 has a homologous region with mouse chromosome 12 (probably because of translocations during evolution and speciation) and the gene is located in the homologous region.

Below are two depictions of the central dogma of genetics. Which one do you think most accurately reflects reality? ____B____ (2 pts)

A.

B.

21) If histones were depicted in Figure B, where would they be? (2 pts) In or associated with the nucleosomes

22) What is the significance of showing some of the DNA without histones? (2 pts)It is opened up for various cellular processes like transcription and gene expression.

23) Can pre-mRNA such as is shown in the figure give rise to more than one gene form? (2 pts) Yes

24) How does this occur? (2 pts) Alternate splicing, RNA editing, others

Fred continues to study Wf, aided by his assistant Inga. Inga’s specialty is studying gene expression. So she studies transcription and expression of Wf and some of her findings are depicted below (3 pts).

25) What do the green circles represent? Nucleosomes

26) Where in the figure would the histones be most methylated? Where the nucleosomes are closely compacted, for the most part. I also accepted “on the histone tails”.

27) What is represented by the other objects in the figure? Other factors that interact with DNA during various processes, e.g. transcription.

The story has a happy ending Fred was able to produce a monoclonal antibody expressing antisense-Wf. When the antibody was applied to mice, they failed to grow hair:

So, Fred tried the same therapy on Larry.

See the lower row of mice compared to their littermates.

Larry is a happy guy (the monster, however, misses his friend).