By genetic analysis of a thermosensitive autolytic mutant whose phenotype was complemented by osmotic stabilization with sorbitol, we identified gene LYT2 of Saccharomyces cerevisiae, which is probably involved in cell wall formation. A yeast gene complementing lyt2 strains was cloned and shown to carry an open reading frame coding for a 484-amino-acid protein exhibiting all the characteristic domains of serine/threonine protein kinases and highly homologous to other yeast protein kinases involved in control of the mitotic cycle. Mutants disrupted in the cloned gene also displayed an autolytic phenotype complemented by osmotic stabilization with sorbitol. However, genetic comparison of lyt2 mutants and disruptants of the protein kinase gene revealed that the cloned gene is not the structural gene LYT2 but a suppressor of the lytic phenotype, named gene SLT2, that was mapped to chromosome V. The product of gene SLT2 is the first protein kinase to be described in relation to the yeast cell-wall functions.

In order to develop plasmids adequate for non-integrative genetic transformation of Candida albicans, a DNA fragment of 15.3 kb was cloned from this organism on the basis of its capacity to convert the integrative Saccharomyces cerevisiae vector YIp5 into a non-integrative one. Southern hybridization analysis, carried out with a labelled DNA probe of 3.6 kb derived from the cloned fragment, showed that it consisted of C. albicans DNA, the hybridization pattern indicating that the corresponding sequences were homologous to several chromosomal regions. The size of the C. albicans DNA promoting autonomous replication in S. cerevisiae was substantially reduced by subcloning. A 5.1 kb subfragment, defined by BamHI and SalI restriction sites, retained autonomous replication sequences (ARS) functional in the heterologous S. cerevisiae system and in C. albicans, when inserted in plasmid constructions that carried a S. cerevisiae trichodermin-resistance gene (tcm1) as selection marker. C. albicans transformants were both of the integrative and the non-integrative type and the plasmids recovered from the latter very often carried a reorganized ARS, indicating that recombination of the inserted ARS DNA had occurred in the homologous host. Successive reorganizations of the ARS insert in C. albicans eventually led to a more stable and much smaller fragment of 687 bp that was subsequently recovered unchanged from transformants. Sequence analysis of the 687 bp fragment revealed four 11-base blocks, rich in A+T, that carried the essential consensus sequence considered relevant for yeast ARS elements in addition to other features also described as characteristic of yeast replication origins.

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Considerable progress has been made in the study of the factors that contribute to the pathogenicity of fungi. Phenotypic variability, adherence to host tissues, toxins and enzymes have been listed as candidates for virulence factors.Substantial evidence is available to show that pathogenic fungi secrete various hydrolytic enzymes such as proteinase, lipase and phospholipase into culture medium. Extracellular hydrolytic enzymes may play a part in the pathogenesis of infection by causing damage to host cells and by supplying nutrients in a nutrient-restricted environment. Acid proteinase from Candida albicans has been investigated extensively as a virulence factor in candidiasis. However, only limited information is available on other hydrolytic enzymes in Candida species and other pathogenic fungi.We report here a series of experiments that provide evidence for the role of hydrolytic enzymes in fungal pathogenesis and demonstrate the development of gene cloning techniques using C. albicans as the host system. Proteinase from Nattrassia mangiferae (Hendersonula toruloidea)N. mangiferae (H. toruloidea) is a dematiaceous mould commonly causing diseases of plants and fruits in the, tropics and subtropics. The infection in humans shows clinical features resembling chronic dermatophytosis due to Trichophyton rubrum but is restricted to nail and thickly keratinized glabrous skin such as palms and soles [5]. In dermatophyte and other fungal infections, there is clear evidence to suggest that proteinases produced by these organisms play an important role in the pathogenesis of tissue invasion [10,16]. Although N. mangiferae causes a similar infection this organism had not, until recently, been reported to produce a proteinase [12].In this study, an extracellular proteinase from a fast-growing strain of N. mangiferae was demonstrated when the fungus was grown in liquid medium, containing bovine serum albumin (BSA) as the sole nitrogen source, in a rotary shaker at 27°C for 2 weeks. Proteinase activity was assayed with Azocoll as a substrate at 37°C for 30 min. Liberated dye was measured at 520 nm. Purification of the proteinase to homogeneity was performed by CM cellulose, CM Sephadex G-50 and Sephacryl S-200 column chromatography in 5mM phosphate buffer (pH 7-0) [15]. Final purification was 3,230-fold and the yield was 37.5%. The results of SDS-polyacrylamide gel electrophoresis showed the purified enzyme to have the appearance of a single band. The molecular mass was estimated to be 34 kDa, the same value as estimated from 189 Med Mycol Downloaded from informahealthcare.com by McMaster University on 11/20/14For personal use only.

We report the sequence of a 9000 bp fragment from the right arm of Saccharomyces cerevisiae chromosome VII. Analysis of the sequence revealed four complete previously unknown open reading frames, which were named G7587, G7589, G7591 and G7594 following standard rules for provisional nomenclature. Outstanding features of some of these proteins were the homology of the putative protein coded by G7589 with proteins involved in transcription regulation and the transmembrane domains predicted in the putative protein coded by G7591.