A colony color assay for Saccharomyces cerevisiae mutants defective in kinetochore structure and function.
We have designed a colony color assay for monitoring centromere DNA-protein interactions in yeast (Saccharomyces cerevisiae). The assay is based on the ability of centromere DNA sequences to block (in cis) transcription initiated from a hybrid CEN-GAL1 promoter. Using a IacZ reporter gene under control of the CEN-GAL1 promoter, we screened colonies derived from mutagenized cells for a blue color phenotype indicative of derepression of the hybrid construct. A limited screen in which a 61-bp CEN11 DNA fragment containing an intact CDEIII subregion plus flanking sequences was used as the "pseudo-operator" led to the identification of mutations (blu) in three complementation groups. The blu1 mutants exhibited a decrease in activity of the major CEN DNA-binding proteins in vitro. The BLU1 gene was shown to be identical to the previously isolated SPT3 gene, known to be involved in the transcriptional regulation of a subset of yeast genes. Our results indicate that the BLU1/SPT3 gene product may also be required to maintain optimal levels of functional centromere DNA-binding proteins.
Périer
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Department of Biological Sciences, University of California, Santa Barbara 93106.
Carbon
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D000167:Acriflavine; D001483:Base Sequence; D002503:Centromere; D003124:Colorimetry; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005799:Genes, Dominant; D005800:Genes, Fungal; D005808:Genes, Recessive; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D001616:beta-Galactosidase / Q000235:genetics
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C075582:SPT3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D000167:Acriflavine; D001616:beta-Galactosidase