POINT MUTATIONS THAT SEPARATE THE ROLE OF SACCHAROMYCES-CEREVISIAE CENTROMERE-BINDING FACTOR-I IN CHROMOSOME SEGREGATION FROM ITS ROLE IN TRANSCRIPTIONAL ACTIVATION

Centromere binding factor 1 (Cbf1p or CP1) binds to the CDEI region of Saccharomyces cerevisiae centromeres and is a member of the basic hel ix-loop-helix (bHLH)class of proteins. Deletion of the gene encoding C bf1p results in an increased frequency of chromosome loss, hypersensit ivity to low levels of microtubule disrupting drugs (such as thiabenda zole and benomyl) and methionine auxotrophy. By polymerase chain react ion-based random mutagenesis of the CBF1 gene we have obtained a numbe r of mutant alleles that make full-length protein with impaired functi on. The mutations in these alleles are clustered in or just downstream from the bHLH domain. Among the alleles obtained was a class that was more compromised for transcriptional activation and a class that was more compromised for chromosome loss and thiabendazole hypersensitivit y. These results indicate that at least some aspects of the role of Cb f1p in chromosome segregation and transcriptional activation are disti nct. In contrast, increased chromosome loss and thiabendazole hypersen sitivity were not separated in any of the alleles, suggesting that the se phenotypes reflect the same mechanistic defect. These observations are consistent with a model that suggests that one role of Cbf1p in ch romosome segregation may be to improve the efficiency with which conta ct between the kinetochore and spindle microtubules is established or maintained.