Cell division proceeds by a tightly regulated cycle that includes a precise distribution of the chromosomes to each daughter cell. Errors in chromosome segregation are the basis of several human birth defects and are the cause of half of spontaneous abortions. Although many of the components involved in the complex process have been identified, chromosome segregation is not understood at the molecular level. Evidence from animals, plants and fungi suggest one component is the small Ca2+-binding protein calmodulin, but its role is unknown. Saccharomyces cerevisiae cells carrying heat-sensitive mutations in calmodulin survive at the nonpermissive temperature until mitosis and then rapidly lose viability as the chromosomes become aberrantly arranged. Our long term objective is to exploit biochemical, cytological, and genetic techniques to determine why defects in calmodulin lead to chromosome missegregation in S. cerevisiae. A recent breakthrough was our discovery that changes in a component of the nuclear matrix can suppress mutations in calmodulin. Dominant mutations in NUF1 permit the growth of all heat-sensitive calmodulin mutants tested. The protein encoded by NUF1, Nuf1p, has previously been identified as part of the nuclear matrix. A primary objective of the proposed research is to understand the relationship between Nuf1p and calmodulin. Genetic experiments already suggest a direct interaction between the two proteins. Biochemical techniques will further examine the interaction. The different mutant alleles of NUF1 that suppress the defective calmodulins will be sequenced to define the regions of Nuf1p that are responsible for suppression. Heat-sensitive mutations in NUF1 will be isolated to determine if the loss of NUF1 function has the same effect on mitosis as the loss of calmodulin function. Finally, the studies on the heat-sensitive calmodulin mutant will be continued, but with a new focus on the nuclear matrix. Two other extragenic suppressors of the calmodulin mutant have been identified, SCM2 and HCM1. The SCM2 gene will be cloned and sequenced as a first step in understanding its function. The protein encoded by HCM1 is a new member of the fork head family of DNA-binding proteins. The DNA recognition sequence for HCM1p will be identified by random- sequence selection. HCM1 is not an essential gene and thus a related gene may perform the same function. A fragment that hybridizes to HCM1 homolog. Finally, heat-sensitive calmodulin mutants of Schizosaccharomyces pombe will be isolated and characterized to discover if calmodulin performs a similar function in fission yeast.
Showing the most recent 10 out of 58 publications
