An allele of SID2 was identified in a screen for mutations lethal in combination with deletion of the Clb-Cdc28 kinase inhibitor SIC1 (Jacobson et al., submitted). sid2-1 sic1D cells arrest as large budded cells with a single nucleus and short spindle, and an approximately 2C DNA content. There is a delay in the completion of S phase in synchronized sid2-1 strains, suggesting there is a defect in DNA replication or repair. This hypothesis is also supported by genetic interactions between sid2-1 and CLB5/CLB6, and sid2-1 and RAD9. Here, we develop two different approaches to elucidate the molecular function of SID2.

A two-hybrid screen was used to identify proteins that interact with SID2. An initial screen by Claudia Muñoz '99 using wild-type SID2 as "bait", isolated a number of interacting proteins. To determine which of these have biological relevance, we are analyzing their interaction with three different sid2 mutants. Proteins found to interact with wild-type SID2, but not with the mutants, may be responsible for the decrease in cell viability that is observed with these alleles. The results from this analysis will be presented.

Characterization of phenotypes associated with either the over- or under-expression of SID2 provides a second approach to learning more about its function. The wild-type chromosomal allele has been placed under the control of the inducible GAL1 promoter. A second construct which has a 3HA epitope tag in addition to the GAL promoter has also been constructed. This construct may be used to confirm the tentative localization of Sid2 in the cytoplasm. Growth on galactose of cells heterozygous for the GAL-SID2 construct appears normal. The viability and other characteristics of haploid cells containing the GAL-SID2 construct on various media and under different conditions will be presented.

The utilization of an actomyosin contractile ring for cytokinesis is conserved evolutionarily; however, many questions about the mechanism and regulation of contraction remain unanswered. The actomyosin system is essential for cytokinesis in animal cells and in Schizosaccharomyces pombe. In contrast, it appears that cytokinesis in Saccharomyces cerevisiae can occur either via contraction of the actomyosin ring or through formation of a septum. Because the actomyosin system is redundent in S. cerevisiae, null mutations that affect ring contraction can be isolated and monitored. This should allow a more complete understanding of the function of genes required for actomyosin ring contraction.

The HOF1 gene appears to be required for the septum formation pathway of cytokinesis and yeast with a hof1 deletion are dependent on the actomyosin system for cytokinesis (Vallen et al. 2000). We are exploiting yeast lacking hof1 to identify genes and proteins that are important for the function of the actomyosin pathway. Mutagenized hof1 ade2 ade3 ura3 cells that contain a plasmid with HOF1 ADE3 URA3 were screened for colonies that could not lose the plasmid using a sectoring assay. Nonsectoring colonies were confirmed to be dependent on the plasmid by analyzing sensitivity to 5-FOA. Amongst these mutants should be yeast that are defective in the actomyosin pathway. We have screened approximately 32,000 colonies and have identified about 60 mutants that are dependent on the HOF1 URA3 ADE2 plasmid for viability. Non-sectoring putative mutants will be analyzed by transformation with a HOF1 LEU2 plasmid to determine which specifically require HOF1 for viability. Mutants that require HOF1 will be characterized to determine whether they are known participants in the actomyosin sytem (i.e., MYO1, BNI1 and CYK3) or represent novel genes. Results of these experiments will be presented.

SIC1, a nonessential gene, encodes a cyclin dependent kinase inhibitor known to block the kinase activity of the B-type cyclin/Cdc28 complexes.By inhibiting the B-type cyclin/Cdc28 complexes, Sic1 functions both by releasing a block on the exit from mitosis as well as preventing the premature onset of DNA replication in the budding yeast Saccharomyces cerevisiae. A screen designed to isolate mutants which are dead in the absence of SIC1, but viable in its presence, was carried out. Fifteen putative mutants were isolated. Two have been eliminated for not truly being dependent on Sic1 levels. Twelve of the remaining thirteen mutants have been confirmed to be dependent on Sic1 levels and are called SID, for SIC1 Indispensable. Nine of the isolated mutants diploidized spontaneously, perhaps resulting from the unregulated entry into S phase or a failure to exit mitosis. One of the mutant strains has been determined to contain a dominant mutation. The remaining recessive mutants fall into at least three complementation groups. The sid1 complementation group contains four alleles. A phenotypic analysis of strains containing two of the sid1 alleles reveals that mutants arrest as large budded cells in the absence of Sic1, possibly resulting from a failure to exit mitosis.

DBF2 and DBF20, two previously described homologous protein kinases, have been shown to be involved in exit from mitosis in Saccharomyces cerevisiae. The lethality of the double deletion indicates that the genes perform an essential activity. DBF20 requires SPO12 to function, whereas SPO12 does not appear to be essential to DBF2 activity. To help clarify the role of DBF2 and SPO12, characterization of the Spo12 homologous protein, Ygr230w,was carried out.

mec1-1 Saccharomyces cerevisiae, which are mutant for the DNA damage checkpoint gene MEC1, are inviable in the presence of GAL-CLN1 or GAL-CLN2 in cln1cln2CLN3 strains. A previous screen identified plasmids that rescue this lethal phenotype; these experiments were undertaken to identify which gene on the overlapping plasmids 48A and LB80 is the suppressor. Four subclones were created by removing BglII, AgeI, SacI and EcoNI fragments from the suppressing plasmids. Subclones were transformed into mec1-1 strains; testing for viability on media that induced the overexpression of CLN2 indicated that YDR451 was the suppressor. As LB80 and 48A are weak suppressors, YDR451 was overexpressed via the inducible GAL1 promoter to determine if increased YDR451 would provide a higher level of suppression. mec1-1 transformants with GAL-YDR451 were unable to grow in the presence of an excess CLN2. Neither LB80 nor GAL-YDR451 were found to alter the rate of the cell cycle.