Isolation of a CDC28 homologue from Cryptococcus neoformans that is able to complement cdc28 temperature-sensitive mutants of Saccharomyces cerevisiae.
A partial cDNA fragment of the Cryptococcus neoformans homologue of the main cell cycle control gene CDC28/cdc2 was isolated using degenerate primer RT-PCR. A subsequent search in the C. neoformans genome database identified several sequences similar to CDC28/cdc2. A part of the sequence which showed the highest similarity to CDC28/cdc2 turned out to be identical to the partial cyclin-dependent kinase (Cdk) cDNA fragment isolated by degenerate RT-PCR. The full-length coding region of this Cdk homologue was amplified by RT-PCR using primers designed to target regions around start and stop codons, and the gene was named CnCdk1. To determine its function, an analysis of deduced amino acid sequence of the CnCdk1 was performed and its ability to rescue Saccharomyces cerevisiae cdc28-temperature sensitive mutants was tested. S. cerevisiae cdc28-4 and cdc28-1N strains transformed with the pYES2- CnCdk1 construct exhibited growth at 36.5 degrees C in galactose-raffinose medium, but not in glucose medium. Results of the sequence analysis and the fact that CnCdk1 is able to complement the S. cerevisiae cdc28-ts mutation support its assumed role as the CDC28/cdc2 homologue in C. neoformans.
Takeo
Kanji
K
Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan. takeo@faculty.chiba-u.jp
Ogura
Yasunobu
Y
Virtudazo
Eric
E
Raclavsky
Vladislav
V
Kawamoto
Susumu
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000235:genetics / Q000378:metabolism; D042846:CDC2-CDC28 Kinases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D017489:CDC28 Protein Kinase, S cerevisiae / Q000235:genetics / Q000378:metabolism; D003455:Cryptococcus neoformans / Q000201:enzymology* / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D004271:DNA, Fungal; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; D017489:CDC28 Protein Kinase, S cerevisiae