Article 11

Hardy, C.R.J., O. Dryga, S. Seematter, P.M.B. Pähl, & R A. Sclafani (1997) mcm5!cdc46-bob-1 by-passes the requirement for the S phase activator Cdc7p. Proc. Natl Acad. Sei. USA 94: 3151-3155.

Jackson et al. (1993) isolated bobl-1 as a suppressor of the temperature-sensitive growth defect of cdc7-l. They found that bobl-1 can by-pass null mutations in CDC7 and DBF4 but not temperature-sensitive mutations in CDC28 or CDC4. They conclude that bobl-1 cannot suppress all events that occur in the G1 phase of the cell cycle and that Cdc7p and Dbf4p act at the same step, which the epistasis analysis of Hereford & Hartwell (1974) places downstream of CDC28. This article continues the characterization of bobl-1 and finds that it is an allele of a gene known as CDC46 (now officially called MCM5).

1. Describe how you would undertake the isolation of additional suppressors of cdc7-l such as bobl-1.

2. Diagram crosses to demonstrate the following. In each case give the genotype and phenotype of the parent strains, the diploid, and of each spore in a tetratype tetrad.

(a) bobl-1 is a suppressor of dbf4-l (demonstrated in Jackson et al., 1993).

(b) bobl-1 does not suppress the temperature-sensitive cdc28-l mutation (demonstrated in Jackson et al., 1993).

(c) bobl-1 does not suppress the temperature-sensitive orc2-l mutation (a conclusion stated here but the data are not shown).

3. bobl suppresses a cdc7A::HIS3 null mutation (Jackson et al., 1993). This was demonstrated by sporulating a diploid strain homozygous for bobl and heterozygous for edc7A::HIS3.

(a) Describe the construction of this diploid strain starting with strains of the following genotype of opposite mating types: bobl-1 CDC7 his3.

(b) Give the genotype and phenotype of the spores resulting from the sporulation of this diploid. Include bobl-1, cde7, and HIS3 in your cross.

(c) What would the tetrad analysis results be if bobl-1 does not suppress cdc7A::HIS31

4. From the results of the CDC46 gene disruption experiments carried out prior to the cloning and sequencing of the bobl-1 mutant allele, the authors had concluded that bobl-1 is not a null allele. What is the evidence in support of this?

5. Which result in Table 1 indicates that bobl-1 is recessive?

6. Describe the cloning of BOBl. How does genetic mapping provide evidence consistent with their finding that BOBl is CDC461

7. Using the diploid strain (dbf4-lldbf4-l bobl-HBOBl) the authors confirm their finding that bobl-1 is a mutant allele of CDC46. Describe.

8. Deletion/disruption of BOBl is lethal but the Pro83Leu alteration of Cdc46 protein found in the bobl-1 mutation produces only a modest phenotype.

(a) What is the phenotype of the bobl-1 mutation in the absence of cdc7-ll

(b) On what basis do the authors suggest that bobl-1 cells enter the S phase prematurely?

(c) Suggest a model to explain the different phenotypes of these alleles that takes into consideration the fact that bobl-1 is recessive.

9. This article demonstrates that BOB1 is CDC46. It also shows that cdc46-l, isolated as a temperature-sensitive suppressor of cdc45-l, does not suppress a cdc7A null allele while the bobl-1 allele does. Propose a model explaining these results that includes a discussion of the possible role of Cdc7-Dbf4 kinase in Cdc46p activation.

REFERENCES

Hereford, L.M. & L.H. Hartwell (1974) Sequential gene function in the initiation of

Saccarhomyces cerevisiae DNA synthesis. J. Mol. Biol. 84: 445-461. Jackson, A.L., P.M. Pähl, K. Harrison, J. Rosamond, & R.A. Sclafani (1993) Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein. Mol. Cell. Biol. 13: 2899-2908.

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