GENES CONTROLLING CONJUGATION AND MITOTIC CELL DIVISION IN YEAST SACCHAROMYCES CEREVISIAE by

New mutations were isolated that affect nuclear fusion during conjugation in the yeast Saccharomyces cerevisiae. These mutations, called kem (Kar enhancing Mutation), enhance the nuclear fusion defect of the karl-l mutation. In crosses of karl-l strains, 10 15% of zygotes undergo nuclear fusion forming stable diploids whereas 85 90% of zygotes fail to have nuclei fused. The nuclear fusion defect of karl-1 leads to formation of a heterocaryon which buds off a cytoductant with a parental haploid nucleus and a mixed cytoplasm. Newly isolated kem mutations block the residual nuclear fusion of karl-l crosses. This block in nuclear fusion has been confirmed by the genetic assay measuring cytoductant formation and the cytological observation of nuclear DNA in zygotes stained with DAPI. Nuclear fusion is normal in crosses of kem x KAR. Eight independent kem mutations were isolated. These eight mutations define at least three genes KEM1, KEM2, and KEM3. The KEM1 and KEM3 genes are located on the left arm of chromosome VII; CenV metl3 (16 cM) kem3 (18 cM) lys5 (13 cM) keml. I have characterized mutations in KEM1 in a Kar+ background. Several phenotypes of the keml mutants suggest that keml mutations affect the microtubule or spindle pole body function. The keml-1 and keml-5 mutations cause a bilateral nuclear fusion defect. In crosses where both parents are keml, about 30% of the zygotes fail to fuse nuclei with the result that cytoductants are produced at high frequency. The growth of keml mutants was examined at various concentrations of the antimicrotubule drug, benomyl. As compared with KEM1, the keml mutants are hypersensitive to benomyl. keml/keml diplids lose chromosomes with a frequency of 10 20 fold higher than Kem diploids. These results are consistent with the idea that KEM1 has a role in microtubule function. The KEM1 gene was disrupted by replacing the internal fragment of the KEM1 gene with a fragment carrying the URA3 gene. The null allele keml::URA3 is viable but causes a slow-growth phenotype and an altered cell morphology during the mitotic cell cycle. Microscopic examination of cell culture has shown that about 80% of keml::URA3 cells have an elongated rod-shape and twice the size of a wild type cell. In these cells spindle pole body duplication/separation is defective as visualized by indirect immunofluorescence microscopy using anti-tubulin antibodies. A high proportion of large budded cells in keml: :URA3