GENETIC-CHARACTERIZATION OF A PHOSPHOLIPASE-C GENE FROM CANDIDA-ALBICANS - PRESENCE OF HOMOLOGOUS SEQUENCES IN CANDIDA SPECIES OTHER THAN CANDIDA-ALBICANS

Phospholipase C (PLC) enzymes are essential in regulating several impo rtant cellular functions in eukaryotes, including yeasts. In this stud y, PCR was used to identify a gene encoding PLC activity in Candida al bicans, using oligonucleotide primers complementary to sequences encod ing highly conserved amino acid regions within the X domains of previo usly characterized eukaryotic phospholipase C genes. The nucleotide se quence of the C. albicans gene, CAPLC1 (2997 bp), was determined from a recombinant clone containing C. albicans 132A genomic DNA; it encode d a polypeptide of 1099 amino acids with a predicted molecular mass of 124.6 kDa. The deduced amino acid sequence of this polypeptide (CAPLC 1) exhibited many of the features common to previously characterized P LCs, including specific X and Y catalytic domains. The CAPLC1 protein also exhibited several unique features, including a novel stretch of 1 8-19 amino acid residues within the X domain and an unusually long N-t erminus which did not contain a recognizable EF-hand Ca2+-binding doma in. An overall amino acid homology of more than 27% with PLCs previous ly characterized from Saccharomyces cerevisiae and Schizosaccharomyces pombe suggested that the CAPLC1 protein is a delta-form of phosphoino sitide-specific PLC (PI-PLC). PLC activity was detected in cell-free e xtracts of both yeast and hyphal forms of C. albicans 132A following 7 h and 24 h growth using the PLC-specific substrate p-nitrophenylphosp horylcholine (p-NPPC). In addition, CAPLC1 mRNA was detected by revers e transcriptase PCR in both yeast and hyphal forms of C. albicans 132A at the same time intervals. Expression of CAPLC1 activity was also de tected in extracts of Escherichia coli DH5 alpha harbouring plasmids w hich contained portions of the CAPLC1 gene lacking sequences encoding part of the N-terminus. Southern hybridization and PCR analyses reveal ed that all C. albicans and Candida dubliniensis isolates examined pos sessed sequences homologous to CAPLC1. Sequences related to CAPLC1 wer e detected in some but not all isolates of Candida tropicalis, Candida glabrata and Candida parapsilosis tested, but not in the isolates of Candida krusei, Candida kefyr, Candida guillermondii and Candida lusit aniae examined. This paper reports the first description of the clonin g and sequencing of a PLC gene from a pathogenic yeast species.