Article 14

Ohtoshi, A., T. Miyake, K. Arai, & H. Masai (1997) Analyses of Saccharomyces cerevisiae Cdc7 kinase point mutants: dominant-negative inhibition of DNA replication on overexpression of kinase-negative Cdc7 proteins. Mo I. Gen. Genet. 254: 562-570.

1. State the specific amino acid changes made in each of the mutations listed in Table 1. (You will need to refer to a table of the single letter designations for the amino acids.) In each case, what reasons do the authors give for choosing to make the particular alteration?

2. The CDC7 genes under analysis in Table 1 are expressed from the GAL1 GAL10 promoter. In glucose-grown cells there is a very low rate of expression from this promoter and this is increased about 1000-fold by growth on galactose.

(a) What evidence presented in Table 1 indicates that even the very low rate of the wild-type allele of CDC7 expression is sufficient to complement the temperature-sensitive phenotype of cdc7-31

(b) What evidence indicates that each of the in vitro generated cdc7 mutant alleles encodes a defective Cdc7 protein compared with wild-type?

(c) What evidence indicates that Cdc7p alterations T281A, D182N, and D163N lack kinase activity while T281A and T167E have reduced activity?

(d) Western analysis of the galactose-grown cells using anti-Cdc7p antibody would have been an important control and would have strengthened the authors' conclusions. Why?

(e) Correct the underlined word in the following statement from the text of the article. 'However, all the mutants, including T281A and T167E, had lost the ability to complement temperature-sensitive growth of dbf4(ts) even when fully induced (Table 1).' Explain.

3. In a number of experimental situations it has been shown that an acidic residue, such as glutamate, mimics a phosphorylated serine or threonine residue. Based on this and on the authors' suggestion that activation of Cdc7p protein kinase requires phosphorylation of T281, predict the phenotype of the T281A and T281E mutant alleles. Did these predictions hold up to experimental testing? Upon which specific results in Table 1 do you base your answer? Discuss.

4. These questions relate to the experiments and results described in Table 2.

(a) Which promoter is driving the expression of the CDC7 alleles, the native promoter (i.e. the CDC7 promoter) or the GAL1-GAL10 promoter?

(b) Are all, some, or none of the transformed strains 209 (cdc7-3), KKY401-10B (dbf4-l), and 15Dau (CDC7 DBF4) carrying plasmids containing the indicated cdc7 mutants able to grow on glucose at 25°C (data not shown but implied in the text)? If you answered some, which ones?

(c) What carbon source is used to culture the cells in Table 2 and what effect should this have on the expression of the plasmid-borne cdc7 mutant genes?

(d) Based on the discussion of dominant negative mutations in Chapter 11, which of the two explanations best describes the results in Table 2? Why?

(e) Which results in Table 2 point to the depletion of titratable levels of Dbf4 protein as the basis of the dominant negative effect of Cdc7p-mutant overexpression? Discuss.

5. Describe the experiments demonstrating that the phenotype of overexpression of cdc7-D182N is similar to that of loss of Cdc7 kinase, that is, arrest at the Gl/S boundary.

6. Figure 3 indicates that the dominant negative effect of each of the three cdc7 mutants (D163N, D182N and T281E) is suppressed by overexpression of DBF4. Draw a diagram showing what the results would have looked like had overexpression of DBF4 not suppressed the D182N mutation but did suppress the other two.

7. If the authors had had no clues as to the identity of the titratable Cdc7p activator, what experimental approach might they have used to identify the gene encoding this activator? Be specific as to the type of library, the genotype of the host strain, and the phenotype (including growth conditions) to be selected.

8. Which results in Table 3 indicate that mutation T281E does not affect the ability of the mutant protein to bind Dbf4 protein?

9. If the Dbf4p binding site of Cdc7p had not been known, how might the dominant negative mutations been used to identify it?

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