Molecular cloning of Saccharomyces cerevisiae CDC6 gene. Isolation, identification, and sequence analysis.
The CDC6 gene product is required for entering the S phase of the cell cycle in Saccharomyces cerevisiae. It has been isolated on recombinant plasmids by selection for complementation of temperature-sensitive alleles with a yeast genomic library. The entire complementing activity is carried on a 1.8-kilobase chromosomal DNA fragment, as revealed by deletion mapping. Northern blotting shows that the size of the CDC6 mRNA is about 1.7 kilobases. A Southern blot of yeast chromosomes which were separated by the field inversion gel electrophoresis method indicates that the isolated DNA fragment is derived from chromosome X. The locus from which the clone was derived was marked by integration with a nutritional marker and found by meiotic mapping to cosegregate with CDC6. Thus, we conclude that we have isolated the authentic CDC6 gene. Nucleotide sequence analysis of the CDC6 gene has revealed an open reading frame that encodes a protein with Mr = 57,969. There are five potential Asn-X-(Ser/Thr) glycosylation sites and a highly conserved nucleotide-binding site in the CDC6 sequence. Although computer surveys indicate overall sequence homology between S. cerevisiae CDC6 protein and Saccharomyces pombe CDC10 START protein, they may not be functionally equivalent as evaluated by the complementation assay.
Zhou
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Department of Pediatrics and Microbiology, University of Southern California School of Medicine, Los Angeles 90027.
Huang
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SH
Jong
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D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification*; D005656:Fungal Proteins / Q000235:genetics*; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014158:Transcription, Genetic
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D005656:Fungal Proteins; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins