Genetic Analysis of the Saccharomyces Cerevisiae Centromere-Binding Protein CP1: a Thesis

CPl is a sequence-specific DNA binding protein of yeast (Saccharomyces cerevisiae) which recognizes the highly fonserved A glement I (CDEI) of yeast centromeres. We cloned and sequenced the gene encoding CPl. The gene codes for a protein of molecular weight 39,400. When expressed in E. , i coli, the CPl gene directed the synthesis of a CDEI-binding protein having the same SDS gel mobilty as purified yeast CPl. We have given the CPl gene the genetic designation CEPl centromere !!rotein 1). CEPI was mapped and found to reside on chromosome X, 2. 0 centimorgans from SUP4. Strains were constructed in which most of CEPl was deleted. Such strains lacked detectable CPl activity and were viable; however CEPl gene disruption resulted in a 35% increase in cell doubling time and a 9-fold increase in the rate of mitotic chromosome loss. An unexpected consequence of CPl gene disruption was methionine auxotrophy genetically linked to cepl. This result and the recent finding that CDEI sites in the MET25 promoter are required to activate transcription (Thomas, D., Cherest, H., and Y. Surdin-Kerjan, J. Mol. BioI. 9: 3292 1989) suggest that CPl is both a kinetochore protein and a transcription factor. INTODUCI0N \ \ \\! ! :'" CPl is a sequence-specific DNA binding protein of yeast (Saccharomyces cerevisiae) which binds to both gene promoter regions and centromeres (6). The DNA recognition sequence for CPl is the octanucleotide RTCACRTG (R=purine) which is found in all yeast centromeres and is named CDEI (fentromere lement I) (23). The conservation of CDEI strongly implies