Isolation of a chitin synthase gene (CHS1) from Candida albicans by expression in Saccharomyces cerevisiae

Chitin synthase activity was studied in yeast and hyphal forms of Candida albicans. pH-activity profiles showed that yeast and hyphae contain a protease-dependent activity that has an optimum at pH 6.8. In addition, there is an activity that is not activated by proteolysis in vitro and which shows a peak at pH 8.0. This suggests there are two distinct chitin synthases in C. albicans. A gene for chitin synthase from C. albicans (CHS1) was cloned by heterologous expression in a Saccharomyces cerevisiae chs1 mutant. Proof that the cloned chitin synthase is a C. albicans membrane-bound zymogen capable of chitin biosynthesis in vitro was based on several criteria. (i) the CHS1 gene complemented the S. cerevisiae chs1 mutation and encoded enzymatic activity which was stimulated by partial proteolysis; (ii) the enzyme catalyses incorporation of [14C]-GlcNAc from the substrate, UDP[U-14C]-GlcNAc, into alkali-insoluble chitin; (iii) Southern analysis showed hybridization of a C. albicans CHS1 probe only with C. albicans DNA and not with S. cerevisiae DNA; (iv) pH profiles of the cloned enzyme showed an optimum at pH 6.8. This overlaps with the pH-activity profiles for chitin synthase measured in yeast and hyphal forms of C. albicans. Thus, CHS1 encodes only part of the chitin synthase activity in C. albicans. A gene for a second chitin synthase in C. albicans with a pH optimum at 8.0 is proposed. DNA sequencing revealed an open reading frame of 2328 nucleotides which predicts a polypeptide of Mr 88,281 with 776 amino acids. The alignment of derived amino acid sequences revealed that the CHS1 gene from C. albicans (canCHS1) is homologous (37% amino acid identity) to the CHS1 gene from S. cerevisiae (sacCHS1).     

Isolation of the CAR1 gene from Saccharomyces cerevisiae and analysis of its expression

We isolated the CAR1 gene from Saccharomyces cerevisiae on a recombinant plasmid and localized it to a 1.58-kilobase DNA fragment. The cloned gene was used as a probe to analyze polyadenylated RNA derived from wild-type and mutant cells grown in the presence and absence of an inducer. Wild-type cells grown without the inducer contained very little polyadenylated RNA capable of hybridizing to the isolated CAR1 gene. A 1.25-kilobase CAR1-specific RNA species was markedly increased, however, in wild-type cells grown in the presence of inducer and in constitutive, regulatory mutants grown without it. No CAR1-specific RNA was observed when one class of constitutive mutant was grown in medium containing a good nitrogen source, such as asparagine. Two other mutants previously shown to be resistant to nitrogen repression contained large quantities of CAR1 RNA regardless of the nitrogen source in the medium. These data point to a qualitative correlation between the steady-state levels of CAR1-specific, polyadenylated RNA and the degree of arginase induction and repression observed in the wild type and in strains believed to carry regulatory mutations. Therefore, they remain consistent with our earlier suggestion that arginase production is probably controlled at the level of gene expression.     

The ICL1 gene from Saccharomyces cerevisiae.

The glyoxylate cycle is essential for the utilization of C2 compounds by the yeast Saccharomyces cerevisiae. Within this cycle, isocitrate lyase catalyzes one of the key reactions. We obtained mutants lacking detectable isocitrate lyase activity, screening for their inability to grow on ethanol. Genetic and biochemical analysis suggested that they carried a defect in the structural gene, ICL1. The mutants were used for the isolation of this gene and it was located on a 3.1-kb BglII-SphI DNA fragment. We then constructed a deletion-substitution mutant in the haploid yeast genome. It did not have any isocitrate lyase activity and lacked the ability to grow on ethanol as the sole carbon source. Both strands of a DNA fragment carrying the gene and its flanking regions were sequenced. An open reading frame of 1671 bp was detected, encoding a protein of 557 amino acids with a calculated molecular mass of 62515 Da. The deduced amino acid sequence shows extensive similarities to genes encoding isocitrate lyases from various organisms. Two putative cAMP-dependent protein-kinase phosphorylation sites may explain the susceptibility of the enzyme to carbon catabolite inactivation.     

Candida albicans HEX1 gene, a reporter of gene expression in Saccharomyces cerevisiae

A nonapeptide from IL-1β has been reported to be an immunostimulant and adjuvant. To investigate the possibility of enhancing the immunogenicity of recombinant antigens delivered by live-attenuated Salmonella strains, we inserted an oligonucleotide coding for the nonapeptide from murine IL-1β into the genes of three model proteins: LamB, MalE, and flagellin. The hybrid proteins were expressed and delivered in vivo by Salmonella aroA strains, and serum antibody responses were analyzed. The results showed that the nonapeptide induced an increase in the immune response against Salmonella-delivered flagellin, measured on day 28 post-immunization. However, the adjuvant effect was lost by day 42. In no case was an adjuvant effect detected for Salmonella-delivered LamB or MalE. Thus, by comparing the immune responses raised by purified MalE with and without the peptide, we investigated whether the insertion of the peptide affected the immunogenicity of the protein itself. Also in this case, a modest adjuvant effect was shown only after primary immunization and when very low doses of antigen were used. In conclusion, the immunomodulatory properties of the IL-1β peptide can also be detected when it is delivered in vivo by Salmonella; however, the effect is modest and antigen-dependent. Received: 30 May 1997 / Accepted: 15 September 1997     

CHS5, a gene involved in chitin synthesis and mating in Saccharomyces cerevisiae.

The CHS5 locus of Saccharomyces cerevisiae is important for wild-type levels of chitin synthase III activity. chs5 cells have reduced levels of this activity. To further understand the role of CHS5 in yeast, the CHS5 gene was cloned by complementation of the Calcofluor resistance phenotype of a chs5 mutant. Transformation of the mutant with a plasmid carrying CHS5 restored Calcofluor sensitivity, wild-type cell wall chitin levels, and chitin synthase III activity levels. DNA sequence analysis reveals that CHS5 encodes a unique polypeptide of 671 amino acids with a molecular mass of 73,642 Da. The predicted sequence shows a heptapeptide repeated 10 times, a carboxy-terminal lysine-rich tail, and some similarity to neurofilament proteins. The effects of deletion of CHS5 indicate that it is not essential for yeast cell growth; however, it is important for mating. Deletion of CHS3, the presumptive structural gene for chitin synthase III activity, results in a modest decrease in mating efficiency, whereas chs5delta cells exhibit a much stronger mating defect. However, chs5 cells produce more chitin than chs3 mutants, indicating that CHS5 plays a role in other processes besides chitin synthesis. Analysis of mating mixtures of chs5 cells reveals that cells agglutinate and make contact but fail to undergo cell fusion. The chs5 mating defect can be partially rescued by FUS1 and/or FUS2, two genes which have been implicated previously in cell fusion, but not by FUS3. In addition, mating efficiency is much lower in fus1 fus2 x chs5 than in fus1 fus2 x wild type crosses. Our results indicate that Chs5p plays an important role in the cell fusion step of mating.     

Lethal effect of the expression of a killer gene SMK1 in Saccharomyces cerevisiae

Expression of the SMK1 gene which encodes the yeast killer toxin SMKT is lethal in Saccharomyces cerevisiae. Effects of deletion and site-directed mutagenesis of SMK1 on the lethality and the secretion of the gene products were examined. Deletion of the interstitial gamma peptide or the C-terminal loop from Ala208 to the C-terminal Asp222 had no effect on the lethality. Those SMK1 products that lacked either the gamma peptide or the C-terminal loop were expressed in the cells but were not secreted into the culture medium, suggesting that these peptides may have a role in secretion or in protein stability. On the other hand, deletion of the signal sequence resulted in complete loss of the lethal activity. Entering the secretory pathway may be critical for the lethality. Further, deletion of the region from the C-terminus to Leu207 resulted in loss of the lethal activity. Leu207 is located at the C-terminus of the central strand of the beta-sheet structure of SMKT and its side chain is thrust into a hydrophobic environment between the beta-sheet and the alpha-helices. The result obtained upon substitutions of Ala, Ser or Glu for Leu207 suggested that the side chain of Leu207 stabilizes the hydrophobic environment that contributes to the overall structure of the SMK1 product.     

Cloning of the URA1 gene of Saccharomyces cerevisiae

A 5.7-kilobase segment of Saccharomyces cerevisiae deoxyribonucleic acid which complements both the yeast ura1 and Escherichia coli pyrD mutations in dihydroorotate dehydrogenase has been cloned in plasmid YRp7.