Dominant negative selection of heterologous genes: isolation of Candida albicans genes that interfere with Saccharomyces cerevisiae mating factor-induced cell cycle arrest.
We have used a genomic library of Candida albicans to transform Saccharomyces cerevisiae and screened for genes that act similarly to dominant negative mutations by interfering with pheromone-mediated cell cycle arrest. Six different plasmids were identified from 2000 transformants; four have been sequenced. One gene (CZF1) encodes a protein with structural motifs characteristic of a transcription factor. A second gene (CCN1) encodes a cyclin homologue, a third (CRL1) encodes a protein with sequence similarity to GTP-binding proteins of the RHO family, and a fourth (CEK1) encodes a putative kinase of the ERK family. Since CEK1 confers a phenotype similar to that of the structurally related S. cerevisiae gene KSS1 but cannot complement a KSS1 defect, it is evident that dominant negative selection can identify proteins that complementation screens would miss. Because dominant negative mutations exert their influence even in wild-type strain backgrounds, this approach should be a general method for the analysis of complex cellular processes in organisms not amenable to direct genetic analysis.
Whiteway
M
M
Eukaryotic Genetics Group, National Research Council of Canada, Montréal, Québec.
Dignard
D
D
Thomas
D Y
DY
D000595:Amino Acid Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002176:Candida albicans / Q000235:genetics*; D002453:Cell Cycle; D016384:Consensus Sequence; D016213:Cyclins / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000235:genetics; D005799:Genes, Dominant; D005800:Genes, Fungal; D000072235:Mating Factor; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics*; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011506:Proteins / Q000235:genetics; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D016415:Sequence Alignment; D013045:Species Specificity; D014157:Transcription Factors / Q000235:genetics*; D016335:Zinc Fingers
D016213:Cyclins; D004271:DNA, Fungal; D005656:Fungal Proteins; D010455:Peptides; D011506:Proteins; D014157:Transcription Factors; C077339:CEK1 protein, Candida albicans; C077336:CZF1 protein, Candida albicans; C077337:CLN1 protein, Candida albicans; C077338:CRL1 protein, Candida albicans; D000072235:Mating Factor; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019204:GTP-Binding Proteins