1887

Abstract

Summary: Phospholipase C (PLC) enzymes are essential in regulating several important cellular functions in eukaryotes, including yeasts. In this study, PCR was used to identify a gene encoding PLC activity in , using oligonucleotide primers complementary to sequences encoding highly conserved amino acid regions within the X domains of previously characterized eukaryotic phospholipase C genes. The nucleotide sequence of the gene, (2997 bp), was determined from a recombinant clone containing 132 A genomic DNA; it encoded a polypeptide of 1099 amino acids with a predicted molecular mass of 124.6 kDa. The deduced amino acid sequence of this polypeptide (CAPLC1) exhibited many of the features common to previously characterized PLCs, including specific X and Y catalytic domains. The CAPLC1 protein also exhibited several unique features, including a novel stretch of 18-19 amino acid residues within the X domain and an unusually long N-terminus which did not contain a recognizable EF-hand Ca-binding domain. An overall amino acid homology of more than 27% with PLCs previously characterized from and suggested that the CAPLC1 protein is a δ-form of phosphoinositide-specific PLC (PI-PLC). PLC activity was detected in cell-free extracts of both yeast and hyphal forms of 132A following 7 h and 24 h growth using the PLC-specific substrate -nitrophenylphosphorylcholine (-NPPC). In addition, mRNA was detected by reverse transcriptase PCR in both yeast and hyphal forms of 132A at the same time intervals. Expression of CAPLC1 activity was also detected in extracts of DH5x harbouring plasmids which contained portions of the gene lacking sequences encoding part of the N-terminus. Southern hybridization and PCR analyses revealed that all and isolates examined possessed sequences homologous to Sequences related to were detected in some but not all isolates of and tested, but not in the isolates of and examined. This paper reports the first description of the cloning and sequencing of a gene from a pathogenic yeast species.

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1998-01-01
2021-04-14
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