A study of genes involved in carbon metabolism in Candida albicans
In this study, several approaches were utilised in attempts to isolate the gene encoding glucose 6-phosphate dehydrogenase from C. albicans (CaZWF1). PCR amplification using degenerate primers yielded a complex mixture of products of the expected length, but amongst these was found a 291 bp fragment which, when cloned and sequenced, was found to have strong homology (77.9%) to the appropriate region of the S. cerevisiae ZWF1 gene. Therefore, the appropriate region of the CaZWF1 gene had been successfully amplified and cloned. This CaZWF1 PCR clone was used as a probe in Southern analysis to show that the CaZWF1 locus might exist at single copy per haploid genome in C. albicans. At this point, a second gene of interest was studied. Two C. albicans cDNA clones shown to have a strong homology to S. cerevisiae PYKI have been isolated. The clone with the largest insert, cDNA2, was characterised by DNA sequencing and this sequence analysed using various computer packages. The 1,834 bp sequence contained a partial open reading frame with the potential to encode 611 amino acids. The presumed amino acid sequence showed strong homology over its entire length to pyruvate kinase from various organisms including S. cerevisiae (67.2%), human liver (55.6%), and rat (60.8%). Southern analysis had shown that the cDNA was encoded by a single locus in the C. albicans genome. Therefore, cDNA2 was presumed to correspond to the functional CaPYKI sequence in this fungus. Pyruvate kinase specific activity and CaPYKI mRNA levels reached their maximum in mid-exponential growth phase, declining to negligible levels late in stationary phase. Therefore, the regulation of pyruvate kinase synthesis during growth appears to be similar in the yeasts S. cerevisiae and C. albicans.