Advanced Genetics Susan Dutcher

Yeast Problem Set March 2, 2016

1. Explain how each term in the mapping equation 6NPD + TT/2 Total is derived. Remember to include a counting of all single crossover tetrads and all double crossover tetrads.

2. You have decided to study octopus genetics and would like to map its centromeres using what you have learned about yeast genetics. Octopi do NOT make tetrads. Previous workers in octopi were interested in meiosis and identified four kinds of meiotic mutants. Which one would be most useful for you to map centromeres and why or why not for each one?

a. Recessive mutant that causes about 10% nondisjunction in meiosis II and has normal rates of recombination

b. Recessive mutant that causes about 10% nondisjunction in meiosis I and has normal rates of recombination

c. Recessive mutant that causes a reduction in recombination to about 10% of normal levels and 50% nondisjunction in meiosis I

d. Dominant mutant causes about 10% nondisjunction in meiosis II and has 10% of normal rates of recombination

3. Sapropterin is a drug being marketed for patients with phenylketonuria (PKU). These patients have mutations in the enzyme phenylalanine hydroxylase and so they build up high levels of phenylalanine in the blood that results in mental retardation as well as other symptoms. Treatment is generally a diet that has low levels of phenylalanine. Sapropterin acts as a cofactor to

increase activity of the mutant enzyme. You want to set up a screen to find mutants that confer super-sensitivity to the action of sapropterin as a possible means to find off-target effects of this drug.

a. You screen by replica plating for colonies that are killed by sapropterin and find 10 strains.

b. You mate each of them by a wild-type tester strain and select for diploid strains. Eight of the diploids are able to grow on sapropterin and two are unable to grow. What do you conclude? Which strains in the table below correspond to these categories?

c. You cross each of the strains by the other strains and get the complementation results in the table below. How many different genes have you identified that cause sapropterin sensitivity? Which mutants are likely to be in the same complementation groups? If you have uncertainty about the number, explain why and propose a method to test your hypothesis

Mutant Wild-type

1 2 3 4 5 6 7 8 9 10

1 + - + - + + + + - + -

2 + + - - + + + - - - +

3 - - - - + - - - -

4 + - - + + - + +

5 + - + + - + +

6 + - + - + +

7 + - - + +

8 - - - -

9 + - +

10 + -

+ indicates growth (resistance) on sapropterin

- indicates lack of growth (super-sensitivity) on sapropterin

d. Explain the complementation results for the diploid between mutant 3 and e. You cross mutant 7 by mutant 5 and get the tetrads below scored for sapropterin sensitivity. Fill in the table on the right with the genotypes for the progeny on the left.

Growth on sapropterin Genotypes

- + -

- + -

+ + -

+ + -

65 17 18 65 17 18

f. You want to identify the genes that are mutated by using transformation with a library. What kind of plasmid library would you use and why?

g. After sequencing a plasmid that you get by rescue of the sapropterin phenotype of mutant 2, you find you have the gene PDR5. How might a mutation in this gene cause sapropterin sensitivity?

4. You perform a cross of met1 x ura5. You get 6 PD: 7 NPD: 87 TT. What can you conclude about these two genes?

5. You perform a cross with several auxotrophic markers (leu, tyr) and dissect 100 tetrads. Below is the scoring of the tetrads.

Leu- Tyr- Leu-Tyr+ Leu-Tyr-

Leu- Tyr- Leu-Tyr+ Leu-Tyr+

Leu+ Tyr+ Leu+Tyr- Leu+Tyr-

Leu+ Tyr+ Leu+Tyr- Leu+Tyr+

1 82 17

a. What are the likely genotypes of the two parents?

b. Are these genes linked or unlinked?

c. If they are unlinked, what can you say about distance to the centromere(s)?

d. If they are linked, what is the map distance between them?

6. You now cross one of the Leu- Tyr- progeny by strain ww-45 from Beth Jones’s lab that has no auxotrophic phenotype. You obtain the following tetrads. Leu- Tyr- Leu+Tyr- Leu+Tyr- Leu-Tyr+

Leu- Tyr- Leu+Tyr- Leu-Tyr- Leu-Tyr-

Leu+ Tyr+ Leu+Tyr+ Leu+Tyr+ Leu+Tyr-

Leu+ Tyr+ Leu+Tyr+ Leu+Tyr+ Leu+Tyr+

14 17 62 7

a. What is the major change in the segregation patterns in this second cross?

b. What is your hypothesis?

c. If you cross a Leu+ Tyr- spore from the one of the tetrads in the second group (the class with 17 examples) to the other Leu+Tyr- spore in the same tetrad (assuming the mating-type alleles will allow this), what does your hypothesis

predict will appear in 100 tetrads?

d. What molecular experiments (not sequencing) would you propose to identify this change in segregation?

7. You are interested in splicing and want to use a genetic approach. a. Describe a screen and a selection for mutants that alter the fidelity of splicing

at the 5’ splice site. b. Describe how you will identify the genes mutated in these mutants. Assume

you identify 10 independent mutants.