A) (5 points) As the starting step isolate genomic DNA from

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1 GS Final Exam Spring 00 NAME. bub ts is a recessive temperature sensitive mutation in yeast. At º C bub ts cells grow normally, but at º C they die. Use the information below to clone the wild-type BUB gene by complementation in yeast. A) ( points) As the starting step isolate genomic DNA from a) Ampicillin sensitive E. coli b) Embryonic stem cells c) Heterozygous bub ts/+ yeast cells d) Homozygous bub ts/ts yeast cells e) Homozygous BUB +/+ wild type yeast cells B) ( points) Choose one of the vectors below to create a genomic library of plasmids that can be maintained as free plasmids in yeast and bacteria: Vector - LEU, Amp r, ori Vector - LEU, ARS, Amp r, ori Vector - URA, Amp r, ori Vector - LEU, ARS, CEN C) ( points) Do a partial digest on the genomic DNA and a complete EcoRI digest on your vector. Mix the DNAs together and add ligase. Transform your recombinant molecules into a) Ampicillin sensitive E. coli b) trp - E. coli c) leu BUB yeast d) ura BUB yeast e) Something else D) ( points) Plate the transformants on a) - leucine plates b) + Ampicillin plates c) -trp - ampicillin plates at º C d) + X-gal plates º C e) Something else

2 E) ( points) To identify the possible BUB clones you prepare plasmid DNA from all of the clones and transform a) leu - E. coli b) leu bub ts yeast c) ura leu yeast d) Ampicillin sensitive trp - E coli F) ( points) you plate the cells on a) -leucine plates at º C b) Ampicillin plates º C c) -ura plates at º C d) -tryptophan plates at º C G) ( points) and replica plate the cells onto a) - tryptophan plates at 0º C b) - leucine plates at º C c) Ampicillin plates at º C d) -ura plates at º C H) ( points) How would you prove that the new recombinant plasmid has the BUB gene in it? a) Use the insert as a probe on a Southern blot of digested wild type and mutant genomic DNA and look for an RFLP difference b) Sequence the insert and look for an open reading frame c) Use the insert as a probe on a northern blot of mrnas from wild type cells d) Compare the sequence of the insert with the sequence from the same fragment cloned from bubts mutant cells and look for differences I) ( points) A mouse gene with homology to yeast BUB was identified by a BLASTP search. The E value is. x 0 -. From just this information you conclude a) This mouse gene and the yeast BUB gene are related by evolution. b) This mouse gene is an essential gene. c) This mouse gene and the yeast BUB gene share significant amino acid sequence similarity. d) The probability that this is a significant match is. X0 -. e) Some combination of the above answers. (a) Is probably acceptable as well

3 . P elements are mobile DNA elements that can excise from one locus on a chromosome and move to and integrate into another locus on a different chromosome. P elements, the type of transposon that you learned about in class and in quiz section, move by this cut and paste mechanism. When the P element is excised from the starting location, the chromosome can be repaired using information on the homologous chromosome, or from the sister chromatid. A male fruit fly that is heterozygous for a wild type P element at a locus on chromosome is shown below. Tnp-ase Draw the two pairs of homologous chromosomes shown above after the transposon on has hopped to assuming : A) (. points) That the original locus was repaired from its homolog (this happens if the transposition occurs during the G phase of the cell cycle). Tnp-ase B) (. points) That the original locus was repaired from its sister chromatid (this happens if the transposition occurs during the G phase of the cell cycle). Tnp-ase Tnp-ase

4 . Ty elements are yeast transposons that move via an RNA intermediate. Unlike P elements, transposons in flies that move by a cut and paste mechanism, cells in which a Ty element has hopped will contain the original Ty element, and a transposed copy at a new location. Shown below is a yeast strain before and after a transposition event induced by growing the cells on galactose. P GAL Reverse transcriptase HindIII LTR LTR galactose (0 points) Describe how you would clone the flanking sequences at the new TY insertion site and not the original TY insertion site. Easiest way is to cut with HindIII, ligate, transform E. coli, and select for Amp r. Then, use complementation in yeast to determine if the flanking URA sequence is functional (transform yeast ura cells). You could also sequence across the URA gene in the plasmid and determine if it has an intron. Complementation of ura - bacteria is also acceptable.

5 . You have identified a new species of wood rat that eats only juniper. You know that a diet consisting of only juniper is toxic to other species of wood rats, so you are interested in the mechanisms in which toxic compounds are metabolized in the new species. You identify proteins that are responsible for metabolizing the toxic compounds in juniper and have named these proteins Jun, Jun, Jun, and Jun. You decide to use the yeast two-hybrid system to determine if these proteins interact. You make plasmids to express each protein as either a fusion with the DNA binding domain (DB) or a fusion with the activation domain (AD). You use HIS and lacz as reporter genes. You mate the cells and analyze the diploids to determine which pairs interact. a) ( points) What types of plates would you use for the assay and why? -His plates that contain X-Ga Having markers reduces the number of false positivesl b) ( points) Based on the results of your assay (see figure), which proteins interact with each other? Jun forms a homodimer Jun and Jun Jun and Jun Jun and Jun Jun and Jun c) ( points) Using circles or other shapes, diagram a potential complex showing these interactions (Do not use ball and stick diagrams, indicate interaction by making interacting proteins touch).

6 . You decide you want to generate a mouse model for a rare disease. You clone the gene (TDG) from a mouse chromosomal library, generate a restriction map, and construct the following targeting vector shown below. The sizes refer to the distance between adjacent restriction sites. You electroporate the targeting vector into embryonic stem (ES) cells, select for Neo resistance, pick clones, harvest the chromosomal DNA, and genotype the cells using Southern blotting. Each of the lanes on the gel contains DNA from a different ES cell clone. PstI Targeting vector Exon Neo kb kb Dt toxin / / PstI PstI PstI Exon Exon Exon // kb kb. kb kb Probe A Chromosomal locus Probe B a) ( points) Which clone(s) shown below on the left have a successfully targeted locus? Clone is correct Clones and are random integrations b) ( points) Predict the bands you might find if you use Probe B. size standards Clone PstI Clone Clone size standards Clone PstI Clone Clone well well Probe A Probe B

7 Final Exam GS Spring 00 NAME Cont. c) ( points) You have decided that one of the clones is the result of a correctly targeted recombination event. You inject the ES cells into recipient blastocysts derived from the mating of mice with white coat color and transfer the blastocysts to a foster mother. After three weeks mouse pups are born. Included in the litter is a 00% black male, a % black and % white male, a % white and % black male, a 00% white female, and a 00% white male. You decide to choose two of the mice for breeding studies. Which two mice do you choose and why? [The ES cells were derived from blastocysts with black coat color alleles.] 00% black and % white/% black males because they have the highest contribution of ES cells d) ( points) What colored mice do you choose to breed with the mice that you chose in part c)? Why? All of the sperm from the chimera that are derived from ES cells will have a black coat color allele. Therefore, breed the chimeras to white females (homozygous for the recessive white allele). All black offspring (heterozygotes) will have been been fertilized by ES cell-derived sperm. Half of the black offspring will be heterozygous for the mutation in TDG (TDG/tdg). e) ( points) The offspring from one of your chimeras have either black or white fur. Offspring from the other chimera are all black. Suggest a hypothesis to explain the difference between the two chimeras. The chimera that has offspring with black or white fur must have both ES and recipient blastocyst derived germ cells (XY/XY chimera) The chimera that has only black offspring must have all the germ line contributed by the ES cells (probably XY/XX chimera)

8 Cont. f) ( points) You set up matings between a male and female who are heterozygous for the targeted mutation. In the Southern blot shown below predict all possible combinations of bands that you expect to see amongst the offspring. Assume that the mutation does not cause embryonic lethality. Circle the genotype that represents the homozygous mutant. The blot has been hybridized with probe A. size standards pup PstI pup pup well 0 9 Probe A

9 Final Exam GS Spring 00 NAME Extra Credit - Why is genetics easier to understand than economics? Because there is no logic to beer being less expensive than bottled water. In fact, even a gallon of gas is less expensive than a gallon of bottled water. Go figure! Page Points Total points exam All other points Total points for course Final Grade 9