Point mutations that separate the role of Saccharomyces cerevisiae centromere binding factor 1 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 helix-loop-helix (bHLH) class of proteins. Deletion of the gene encoding Cbf1p results in an increased frequency of chromosome loss, hypersensitivity to low levels of microtubule disrupting drugs (such as thiabendazole and benomyl) and methionine auxotrophy. By polymerase chain reaction-based random mutagenesis of the CBF1 gene we have obtained a number of mutant alleles that make full-length protein with impaired function. 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 hypersensitivity. These results indicate that at least some aspects of the role of Cbf1p in chromosome segregation and transcriptional activation are distinct. In contrast, increased chromosome loss and thiabendazole hypersensitivity were not separated in any of the alleles, suggesting that these phenotypes reflect the same mechanistic defect. These observations are consistent with a model that suggests that one role of Cbf1p in chromosome segregation may be to improve the efficiency with which contact between the kinetochore and spindle microtubules is established or maintained.
Foreman
P K
PK
Department of Biochemistry, Stanford University School of Medicine, California 94305.
Davis
R W
RW
D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002503:Centromere / Q000378:metabolism*; D015825:Chromosomes, Fungal; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D005838:Genotype; D018257:Helix-Loop-Helix Motifs; D008969:Molecular Sequence Data; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014158:Transcription, Genetic
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C064042:CBF1 protein, S cerevisiae; D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins