name:______student ID:______Genetics L311 exam 3 April 8, 2016

Directions: Please read each question carefully. Answer questions as concisely as possible. Excessively long answers, particularly if they include any inaccuracies, may result in deduction of points. You may use the back of the pages as work sheets, but please write your answer in the space allotted. However, you must show all your work. Clearly define your genetic symbols. We will not make guesses as to what a particular symbol is intended to mean. Also, don’t assume that strains are true-breeding unless this is stated in the question. Finally, show all your work. Good luck.

page 2 ______(20 points possible)

page 3 ______(22 points possible)

page 4 ______(26 points possible)

page 5 ______(24 points possible)

page 6 ______(8 points possible)

total ______(of 100 points possible)

1 name:______student ID:______1. Short answers (2 points each, 20 points total) A. Usually sterile, monoploids are the rare products of defective chromosome segregation in which the individual has only one copy of each of the chromosomes.

B. Fusion of the long arms of two acrocentric or telocentric chromosomes produces a(n) Robertsonian translocation .

C. The ability of some phage to transfer only a specific portion of a bacterial chromosome between bacteria is called specialized transduction .

D. Expression of as few as four genes in differentiated cells can cause them to de- differentiate, producing induced pluripotent stem cells.

E. The mitochondria , major sites of ATP synthesis, are small cytoplasmic organelles that include their own genomes.

F. The situation where three, four or more sets of chomosomes are present is called polyploidy .

G. The general approach of moving from a cloned gene to identifying a mutation in that gene is called reverse genetics or knock out .

Please provide concise definitions of the following terms:

H. prophage: A phage genome integrated into the bacterial host chromosome.

I. merodiploid: A bacterial cell that has two copies of a portion of its bacterial chromosome. This occurs when a cell contains an F’ episome.

J. CRISPR/Cas9: A bacterial system that can be used either to produce a mutation in a specific gene or to correct a mutation that is already present.

2 name:______student ID:______2. Below is a pedigree of a genetic disorder. Individuals that marry into the pedigree do not have the mutation. A. What is the most likely mode of inheritance in this pedigree (3 points)? extranuclear

B. Will the child indicated by the question mark inherit the trait (3 points)? ~0

C. Give a brief description of this mode of inheritance (3 points). Produced by segregation of cytoplasmic organelle that includes its own genome, such as mitochondria. These pass from mom through the egg to offspring. D. Provide one example of a trait that we discussed in class (doesn’t have to be human, although it can be) that is inherited in this way (3 points). MERFF, male sterility in corn

3. In studying a particular animal, you find that one pair of the chromosomes appear a bit unusual. The relevant chromsomes are shown below. The normal chromosome is the one on top.

A. What is the name of the chromosomal alteration that generated the abnormal chromosomes (2 points)? deletion B. Show the chromosomes synapsed in meiosis. Please include the genes (4 points). The segment that is deleted (i.e. that includes E and F) will be looped out.

C. Would you expect to show any phenotypes? Why or why not (4 points). This is likely to result in a mutant phenotype because loss of a large segment of chromosome as shown here results in haploinsufficiency for multiple genes, usually producing phenotypes. We saw this with Cr-du-chat and Wolf-Hirshhorn.

3 name:______student ID:______4. You have identified mutations in a gene, denticle, that produces short dermal denticles in the skin of the rare freshwater shark. To understand its function, you decide to clone the gene. Interestingly, your good friend D. P. Diver has cloned the fin gene, which you find is tightly linked to denticle. A. Please name the method you would use to clone denticle (you do not need to provide the steps involved, 3 points). Positional cloning or chromosome walk

B. You are so intrigued by shark genetics that you purify a protein called Fng that seems to regulate tooth development. You determine the amino acid sequence of a portion of Fng protein. How would you clone the Fng gene? You do not need to give all the steps, just enough information to know the method you will use to find the clone (3 points). Produce degenerate primers using the amino acid sequence, make genomic or cDNA library, screen using colony lift with degenerate primers as probe.

C. Your research indicates that a mouse homolog to the shark denticle gene functions similarly, where it regulates hair growth. To further study denticle function, you decide to mutate the mouse denticle homolog. Assume that you have cloned the mouse denticle gene, please describe how you would generate homozygous mouse denticle mutants. Include a picture of the vector that you would use (6 points).

1. Infect ES cells from black mouse using vector shown above. 2. Select for recombinants in neomycin and ganciclovir. 3. Verify that recombinants had deletion within denticle by Southern or PCR. 4. Mix engineered ES cells with embryo derived from white mouse. 5. Implant into pseudopregnant mouse to produce chimeric mice. 6. Cross chimeric X white to find those chimeric mice that produce all black offspring. Use Southern or PCR to ID hets. 7. Cross hets to generate homozygotes.

D. You wish to know which tissues express the mouse denticle homolog. Again assuming that you have the cloned gene in hand, please briefly discuss the method that you would use to figure out which cell types express denticle. You need not include the steps (4 points). You would make a reporter transgene by fusing dent promoter or whole gene to an easily detected gene produce like gfp, luciferase or lacZ, and then produce a transgenic mouse. You could then determine which tissues express the reporter.

4 name:______student ID:______5. You have decided to get a jump on graduate school by cloning yourself and sending your clone off to grad school. A. Please describe briefly how you will go about cloning yourself (6 points). 1. Obtain human eggs, 2. Remove nucleus, 3. Fuse enucleated eggs with your somatic cells 4. Allow to develop B. At present cloning is fraught with difficulty. Please give two ways in which your attempt to clone yourself might go awry (I’m not looking for reasons why your clone might be different from yourself but rather potential problems with the process itself (4 points). Many of the embryos won’t “take” (i.e. it’s extremely inefficient) Many of those that do develop have problems that can lead to death, often around the time of birth. 6. While marooned on an uninhabited island, you discover an interesting species of sea slug. You observe two different strains: one with blue gills and another with red gills. You decide to cross true-breeding blue-gilled females with true-breeding red-gilled males. The resulting progeny are all blue-gilled. You then cross the F1’s, which results in all blue-gilled F2’s. A. What modes of inheritance are consistent with these results (6 points)? maternal effect or extranuclear B. After lighting a signal flare, you decide you still have time to cross the F2’s together before being rescued. You find that the resulting progeny are a mix of some blue-gilled and some red- gilled. Which of the modes of inheritance that you listed above best explains this new result (6 points)? maternal effect C. What is the phenotypic ratio observed in the F3 generation (4 points)? 3:1 blue:red D. After being picked up by the Coast Guard, you head straight to the lab and clone the gene responsible for the beetle’s wing color. You then sequence the DNA. The following autoradiogram shows the sequence of a small region of the gene. What is the sequence of this region? Be sure to indicate the 5’ and 3’ ends (2 points). 5’-GAACTATCTCGCATC-3’ 7. Gene therapy in its simplest form introduces a wild type gene into some of the somatic cells of an individual suffering from some genetic disorder. Introduction of the wild type gene involves inserting it into a vector and then using that to infect the relevant cell type. We discussed four different types of vector in class. Name any two and tell me one major advantage and disadvantage that it provides. (Note: If you can’t remember the viral names tell me the major advantage and disadvantage of two types for partial credit. 6 points) retrovirus: can produce insertional mutagenesis; provides a means of introducing gene of interest into a population of cells adenovirous: remains extrachromosomal thereby eliminating insertional mutagenesis, can accommodate relatively large genes; can provoke excessive immune response adeno-related virus: remains extrachromosomal, less likely to provoke immune response; but can only accommodate small DNA pieces limiting it’s utility to introduce larger genes lentivirus: provides a means of introducing gene of interest into a population of cells, less prone to insertional mutagenesis than other retroviruses; still can produce insertional mutation

5 name:______student ID:______8. In an interrupted mating experiments, you obtain the data shown below for the time of entry. jes 6 min kat 13 min pol 14 min kev 19 min sha 35 min mel 50 min

A. Draw a genetic map showing the location of and distances between each gene on the bacterial chromosome. The position of the Hfr is shown (4 points).

B. Two of the genes map very near one another (within a minute) and a third closely linked gene, jan, that is 6.5’ from jes. Time of entry mapping does not provide accurate positioning for genes that are close to one another. Briefly describe how you could more accurately determine the relative positions of these two genes, including the steps involved. Assume the genes are required to synthesize a compound of the same name (eg. the kat gene would be required to synthesize Kat, pol makes Pol, etc., 4 points). Pol and kat are within one minute of one another. To map them more accurately we would use a generalized transducing phage, allowing it to infect wild type cells. After we would isolate phage and allow them to infect pol- kat- jan- cells. We would plate on a plate lacking Pol and replica plate to a plate lacking Kat and one lacking Jan. We would then plate on a plate lacking Kat and replica plate to a plate lacking Pol and one lacking Jan.

6