Neigeborn, L. & M. Carlson (1987) Mutations causing constitutive invertase synthesis in yeast: genetic interactions with snf mutations. Genetics 115: 247-253.

1. The constitutive expression of a gene indicates that the expression is unregulated. The authors isolated mutant strains that express invertase constitutively.

(a) Describe the normal regulation pattern of invertase expression.

(b) Define repressed conditions.

(c) Define derepressed conditions.

(d) Define the term constitutive as it relates to invertase expression.

2. Describe the method used to obtain constitutive invertase mutants.

(a) Is this a selection or a screen and why?

(b) What is the role of the 2-deoxglucose in the medium, and what other carbon sources are in the medium?

(c) What is the mutation rate? On average, if a brute force screen had been used, how many individual clones would have to have been tested to find one mutant?

3. A total of 210 mutant strains were obtained which produce >10% of the wildtype derepressed level of invertase when grown in repressed conditions.

(a) Diagram the genetic cross carried out to determine whether each mutation is recessive or dominant to the wild-type allele. Include in your answer the genotype and phenotype of the parental and diploid strains. The phenotype should include resistance versus sensitivity to 2-deoxyglucose and whether invertase is expressed in repressed and derepressed conditions.

(b) All of the mutations were recessive to the wild-type allele. What is the evidence?

(c) Do these genes encode positive or negative regulatory factors and why?

4. Next, the authors placed the mutations into complementation groups.

(a) Diagram the genetic cross used to determine whether two constitutive mutations (call them mutant 23 and mutant 152) were in the same or different complementation groups. Include in your answer the genotype and phenotype of the parental and diploid strains.

(b) Four of the genes identified in this article had been identified in previous studies. List them.

(c) Of the 210 mutant strains, 140 were found to contain mutant alleles of REG1. Select one of these 140 mutants and diagram the genetic cross used to demonstrate that it is an allele of REG1. Include the specific allele name of the regl tester strain.

5. CID1 is a new gene defined by this mutant hunt.

(a) How many mutant alleles of CID1 were obtained?

(b) cidl mutations are pleiotropic. What results demonstrate this?

(c) The most downstream effect of the cidl mutations is on the transcription of SUC2. How do the authors demonstrate this? (Please read the section of Chapter 1 on Reporter Genes.)

6. The authors carried out an epistatic analysis of cidl, hxk2, regl, and ssn6 mutations.

(a) Are the phenotypes of strains carrying mutations in these genes distinguishable and if so how?

(b) What do the authors mean when they say, 'Segregants carrying combinations of cidl, regl, and hxk2 showed no unexpected phenotypes'?

(c) What results indicate that ssn6 is epistatic to cidl, regl, and hxk2'}

7. Since snf mutations also affect the regulation of SUC2 the authors were interested in carrying out an epistasis analysis with the snf mutations and constitutive mutations in cidl, hxk2, regl, and ssn6. Double mutant strains were constructed by mating single mutant strains, sporulating the diploid, dissecting the haploid segregants, and determining the phenotype of the double mutant strain with regard to invertase expression. The results are shown in Tables 5 and 6.

(a) Except for the snf3 mutant (which is too complex to consider here), 'all of the snf cidl and snf regl combinations exhibited the phenotype of the snf parent'. Give one example from Table 5.

(b) In this analysis, which genes are epistatic to which?

(c) Is SNE1 upstream or downstream of REG 11 Of CID11

(d) Is SSN6 epistatic to SNF1 or vice versa, and why?

(e) Is SSN6 upstream or downstream of SNF1, and why?

8. Diagram the regulatory pathway showing the epistatic relationships of the following genes and indicate whether their action is as a positive (arrowhead) or negative (vertical line) regulatory factor controlling SUC2 expression: CID1, HXK2, REG1, SNF1, SSN6, and TUP1. In your diagram, SUC2 expression is the downstream response and, if the relationship between two or more genes is unknown but at a similar step, list the genes separately.

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