Candida albicans is a dimorphic fungus that is pathogenic for humans. No sexual cycle has been reported for this fungus, and earlier reports have differed on whether typical strains of C. albicans are haploid or diploid. Previous estimates of the DNA content of C. albicans varied by one order of magnitude. We used three independent methods to measure the kinetic complexity of the single-copy DNA from a typical strain of C. albicans (strain H317) to determine the DNA content per haploid genote; we obtained values of 15 and 20 fg per cell by using S1 nuclease and hydroxyapatite assays, respectively. Optical assays for DNA reassociation kinetics, although not definitive in themselves, yielded values in this range. Chemical measurements of the DNA content of several typical strains, including strain H317, yielded values clustered about a mean of 37 fg per cell. We concluded that these strains are diploid.Candida albicans is a dimorphic fungus, classified among the fungi imperfecti because no sexual cycle has been observed in strains of the species. Most members of the species exist in the yeast form, but the mycelial form may develop under certain conditions (23,24). Both forms of the species are pathogenic for human hosts, although the details of the pathogenicity of the two forms may be very different. Candida is the most common fungal pathogen of humans (1); clinical reports concerning the genus number in the hundreds each year (23).In contrast to the clinical reports, biochemical studies of Candida are relatively sparse. In particular, little has been established concerning the nature and organization of Candida genomes, including those of the most extensively studied species, C. albicans. Even the ploidy of C. albicans is in dispute (27,32,35,46). Several recent reports (27,28,45,46) conclude that at least some C. albicans strains are diploid, whereas results from other reports (32,35), are more simply interpreted in terms of haploidy.There have also been confficting reports of the DNA content of C. albicans strains; even values published during the last 2 years (35,39,46) disagree by nearly one order of magnitude. We present direct measurements of the total DNA content and sequence complexity of C. albicans.The determination reported here was designed specifically to include a large number of measurements to ensure a high degree of precision (2) or Stewart (41); the former procedure was modified by replacing the 0.2 M PCA wash with a 0.2 M PCA extraction (10 to 15 min on ice). We refer to these two extraction procedures as method A and method B, respectively. The supernatants of the hot PCA extractions from both methods were centrifuged at 20,000 x g for 30 min to remove substances that interfered with the diphenylamine assay. DNA was assayed colorimetrically with a diphenylamine reagent (5) and, for some experiments, also with diaminobenzoic acid reagent (37). Highly polymerized calf thymus DNA (type V, Sigma Chemical Co., St. Louis, Mo.) was used as a standard. The DNA was dried by heating at 70°C overnight...

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We report the mobilization by cointegration of the gonococcal 5.2 kb beta-lactamase plasmid pSJ5.2 in an Escherichia coli background. Transfer of pSJ5.2 was measured by filter mating assays with five different conjugative plasmids from Enterobacteriaceae and the gonococcal 41 kb tet(M). Plasmid pSJ5.2 was mobilized to E. coli at frequencies of 1.7x10(-6), 9.3x10(-8) and 2.7x10(-5) by the tet(M), R64 drd-33 and N3 conjugative plasmids, respectively. Mobilization of pSJ5.2 by the 41 kb tet(M) conjugative plasmid resulted in stable Amp(R) E. coli transconjugants consisting of pSJ5.2 plasmid with an insertion located in the 2.4 kb BamHI-BamHI fragment. Mobilization of pSJ5.2 by R64drd-33 and N3 conjugative plasmids involved stable cointegrates as detected by Southern Blot with a DIG-labelled PstI-digested pSJ5.2 probe. Restriction analysis of the R64::pSJ5.2 and N3::pSJ5.2 cointegrates and Southern Blot with the pSJ5.2 probe showed that cointegrates formed by deletion of DNA regions within the 1.8 kb BamHI-HindIII fragment of pSJ5.2. The plasmid thus appears to use multiple recombination mechanisms for cointegration with different conjugative plasmids. The complete nucleotide sequence of pSJ5.2 was determined, and will be a useful tool to further investigate the molecular mechanisms leading to its cointegrative transfer.

Protoplasts have been obtained in high yields from the yeast and mycelial forms of a variety of strains of Candida albicans by enzymic digestion of cells with commercially available lytic enzymes. The protoplast formation procedure was equally effective for exponential and stationary phase cells. Pretreatment with dithiothreitol and Pronase in the presence of EDTA and Tris was necessary. Other thiol reagents and conditions did not release protoplasts from all the strains of C. albicans tested. Treatment with digestive juice of the snail Helix pomatia required the addition of chitinase for the release of protoplasts from most strains tested. Conditions for maximizing the yield of protoplasts and the activities of P-glucuronidase and chitinase were determined. Electron microscopy of C. albicans showed that the pretreatment conditions removed the outer layers and the treatment itself completely removed the inner layers of the cell wall. More than 90 % of the protoplasts produced by this method were viable as assessed by vital staining with Janus Green B.