Hartwell, L.H. (1980) Mutants of Saccharomyces cerevisiae unresponsive to cell division control by polypeptide mating hormone. J. Cell Biol. 85: 811-822.
In this article Hartwell uses the G1 arrest response to isolate mutants in the pheromone response pathway. Since a-factor is easily purified from the culture medium of mating type a cells, Hartwell was able to add the pheromone directly to the culture medium. Hartwell avoided the problems in genetic analysis encountered by MacKay & Manney as follows. First, he isolated temperature-sensitive mutants. In this way, crosses could be carried out at the permissive temperature at which even the mutants were able to mate but phenotype testing could be done at the non-permissive temperature. Second, he used a genetic manipulation method to convert the MATa/MATa diploids, in which the phenomone sensitivity of the ste mutations could not be determined, to MATa/MATa homozygous diploids. Despite the fact that these strains are diploid for all chromosomes and at all loci, they are mating type a because they are homozygous for the MA 7a allele and they exhibit the same phenotypes as haploid MATa strains. They arrest as unbudded cells in the presence of a-factor, they schmoo in the presence of a-factor, they mate with mating type a strains, etc. MATa/MATa diploids heterozygous for different ste mutations thus can be tested for all of these mating type a phenotypes and therefore can be used for complementation analysis.
The conversion of MATa/MATa to MATa/MATa diploids relies on a type of gene conversion event or 'loss of heterozygosity' that occurs at low frequency at any site in the genome but perhaps is more common at the MA T locus because of the mating-type switching process that occurs in this region. The conversion of the MATa locus to MA7a involves a rather large section of the chromosome and clearly extends beyond the MA T locus to the linked CR Y1 gene. Hartwell cleverly uses the recessive cryl cryptopleurine resistant mutation to select for this 'loss of heterozygosity' event. The MATa CRYl/MATa cryl diploid strain is sensitive to cryptopleurine but, if a conversion event occurs in this region that replaces the CRY1 sequence with the cryl sequence from the homologous chromosome, then one can select these cells based on their resistance to cryptopleurine. Because of the linkage of MATa to cry I, these cells are frequently simultaneously converted from MATa to MATa.
1. Describe the method used to isolate mutants resistant to a-factor arrest. Include in your answer:
(a) List the complete genotype and phenotype of the parent strain 381G. Discuss the SUP4-3 mutant allele.
(b) Are these spontaneous or induced mutants and why?
(c) Is this a selection or a screen and why?
(d) Describe the conditions of this selection/screen.
(e) Define the conditional nature of the phenotype exhibited by the mutants and describe the permissive and nonpermissive conditions.
(f) How does the author ensure that the mutants are independent?
2. After the primary selection/screening of mutant clones, secondary testing was used. Describe this test and the desired phenotype. Give one reason why this secondary testing was carried out.
3. Before complementation analysis could be carried out, the author needed to demonstrate that each mutant clone contained only a single mutation. Describe the cross you would do to demonstrate this. Give the genotype and phenotype of the parents and the results of tetrad analysis of this diploid if the mutant contains a single alteration in a ste gene that is not mating-type specific.
4. Before complementation analysis could be carried out, the author needed to demonstrate that the mutation in each mutant clone was recessive. Describe a method Hartwell might have used to determine this. Base you answer on the methods presented in Figure 1.
5. Describe in detail the method for complementation analysis summarized in Figure 1.
(a) List the complete genotype and phenotype of the 381G mutant parent, the 382-31 parent, and the diploid produced by mating these two strains.
(b) Describe the conditions for mating these strains and for the selection of diploids.
(c) Describe the conditions for selecting MATa cry 1/ MATa cry I homozygotes.
(d) Describe how you would determine whether the two ste mutations in your cross are or are not complementing.
6. Hartwell (1980) tested his ste mutants for the following phenotypes: mating ability, biochemical synthesis of macromolecules, schmoo formation, budding patterns, agglutination, and mating factor production and destruction. List the different phenotypes of mutations in ste2, 4, 5, 7, 8, 9, 11, and 12. Why are ste8 and ste9 mutants considered different from the rest? SIR3 and SIR4 encode proteins required for silencing the normally repressed copies of MATa and MA Ta, called HMRa and HMLa, located near the telomeres of chromosome
III. Explain why mutations in sir3 or sir4 would give the demonstrated phenotypes of ste8 and ste9 mutations.
7. In Article 3, Hartwell (1980) identified mutations in STE2, 4, and 5 that were also identified by MacKay & Manney (Article 2). None was isolated in STE3. Explain.
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