Isolation and characterization of Candida albicans homologue of RAP1, a repressor and activator protein gene in Saccharomyces cerevisiae

To study the function of RAP1, a Candida albicans gene (CaRAP1) that shows sequence similarity to RAP1 of Saccharomyces cerevisiae was isolated by colony hybridization. DNA sequencing predicted an open reading frame of 429 amino acids with an overall identity of 24% to the ScRap1p. The DNA binding domain (DBD) was highly conserved, and EMSA using a GST-CaRap1p fusion protein confirmed its binding ability to the RPG-box of S. cerevisiae ENO1. In contrast, the N-terminus was less conserved and a moderate homology was observed in the BRCT domain. Interestingly, CaRap1p did not contain the C-terminal activation/repression region of ScRap1p.     

Candida albicans homologue of GGP1/GAS1 gene is functional in Saccharomyces cerevisiae and contains the determinants for glycosylphosphatidylinositol attachment

The GGP1/GAS1/CWH52 gene of Saccharomyces cerevisiae encodes a major exocellular 115 kDa glycoprotein (gp115) anchored to the plasma membrane through a glycosylphosphatidylinositol (GPI). The function of gp115 is still unknown but the analysis of null mutants suggests a possible role in the control of morphogenesis. PHR1 gene isolated from Candida alibicans is homologous to the GGP1 gene. In this report we have analysed the ability of PHR1 to complement a ggp1Δ mutation in S. cerevisiae. The expression of PHR1 controlled by its natural promoter or by the GGP1 promoter has been studied. In both cases we have observed a complete complementation of the mutant phenotype. Moreover, immunological analysis has revealed that PHR1 in budding yeast gives rise to a 75–80 kDa protein anchored to the membrane through a GPI, indicating that the signal for GPI attachment present in the C. albicans gene product is functional in S. cerevisiae.     

Molecular cloning of the RPS0 gene from Candida tropicalis.

The Saccharomyces cerevisiae RPS0 A and B genes encode proteins essential for maturation of the 40S ribosomal subunit precursors. We have isolated a homologue of the RPS0 gene from Candida tropicalis, which we named CtRPS0. The C. tropicalis RPS0 encodes a protein of 261 amino acid residues with a predicted molecular weight of 28.65 kDa and an isoelectric point of 4.79. CtRps0p displays significant amino acid sequence homology with Rps0p from C. albicans, S. cerevisiae, Neurospora crassa, Schizosaccharomyces pombe, Pneumocystis carinii and higher organisms, such as human, mouse and rat. CtRPS0 on a high copy number vector can complement the lethal phenotype linked to the disruption of both RPS0 genes in S. cerevisiae. Southern blot analysis suggests that CtRPS0 is present at a single locus within the C. tropicalis genome.     

Molecular cloning of a third chitinase gene (CHT1) from Candida albicans

Here we report the complete nucleotide sequence of a third chitinase gene (CHT1) from the dimorphic human pathogen Candida albicans. The deduced amino acid (aa) sequence of Cht1 consists of 416 aa and displays 36% protein sequence similarity to chitinases Cht2 and Cht3, from C. albicans. Interestingly the domain structure of Cht1 is truncated when compared to the other chitinases of C. albicans and lacks a Ser/Thr-rich region. The sequence data will appear in the GenBank Nucleotide Sequence Data Library under the accession number U36490.     

Functional characterization of the Candida albicans homologue of secretion-associated and Ras-related (Sar1) protein

Secretion-associated and Ras-related protein (Sar1p) plays an essential role during the protein transport from the endoplasmic reticulum to the Golgi apparatus. The cDNA sequence of the Sar1 gene has been identified and characterized from the human yeast pathogen, Candida albicans. This cDNA encodes a protein of 190 amino acids, which shares a 78% sequence identity with Saccharomyces cerevisiae Sar1p and contains the conserved GTP-binding motifs of the small GTPase superfamily. Complementation studies confirmed that this cDNA encodes the functional homologue of ScSar1p. The recombinant C. albicans Sar1p exhibits GTP-binding activity in vitro that was abolished by deletion of one of the three GTP-binding motifs.     

Molecular cloning of the CRM1 gene from Candida albicans

In a screen for Candida albicans genes capable of supressing a ste20Delta mutation in Saccharomyces cerevisiae, a homologue of the exportin-encoding gene CRM1 was isolated. The CaCRM1 gene codes for a protein of 1079 amino acids with a predicted molecular weight of 124 029 and isoelectric point of 5.04. Crm1p from C. albicans displays significant amino acid sequence homology with Crm1p from Saccharomyces cerevisiae (65% identity, 74% similarity), Schizosaccharomyces pombe (55% identity, 66% similarity), Caenorhabditis elegans (45% identity, 57% similarity), and Homo sapiens (48% identity, 59% similarity). Interestingly, CaCRM1 encodes a threonine rather than a cysteine at position 533 in the conserved central region, suggesting that CaCrm1p is leptomycin B-insensitive, like S. cerevisiae Crm1p. CaCRM1 on a high copy vector can complement a thermosensitive allele of CRM1 (xpo1-1) in S. cerevisiae, showing that CaCrm1p and S. cerevisiae Crm1p are functionally conserved. Southern blot analysis suggests that CaCRM1 is present at a single locus within the C. albicans genome. The nucleotide sequence of the CaCRM1 gene has been deposited at GenBank under Accession No. AF178855.     

Isolation and Characterization of the Candida albicans PFY1 Gene for Profilin

We have isolated the Candida albicans gene for profilin, PFY1. Degenerate oligonucleotide primers based on regions of high homology were utilized to obtain a polymerase chain reaction-amplified copy of the gene. This was then used as a probe to isolate the gene from a C. albicans genomic library. Our studies indicate that the full-length gene is unstable in Escherichia coli. Several clones were sequenced, and the predicted amino acid sequence demonstrated homology with profilin proteins from other organisms, most notably Saccharomyces cerevisiae. Northern analysis revealed that the gene is expressed in C. albicans. Attempts to express the gene in S. cerevisiae cells were unsuccessful until the C. albicans promoter was replaced with an S. cerevisiae promoter. Functional complementation of the gene was demonstrated in S. cerevisiae profilin-requiring cells. Antibodies raised to isolated C. albicans profilin protein recognized a protein of the predicted molecular weight when the gene was expressed in S. cerevisiae cells. The sequence of the C. albicans PFY1 gene has been deposited in the Genome Sequence database under Accession Number L3783. © 1997 John Wiley & Sons, Ltd.     

Functional analysis of the Candida albicans ALS1 gene product

ALS1 encodes a cell surface protein that mediates adherence of Candida albicans to endothelial cells. The predicted Als1p has an N-terminal region, which contains a signal peptide; a middle region, which contains 20 36-amino acid tandem repeats; and a C-terminal region, which contains a glycosylphosphotidylinositol-anchorage sequence. We used site-directed mutagenesis to delineate the regions in Als1p required for endothelial cell adherence and cell surface expression of the protein. Mutant alleles of ALS1 containing either deletions or insertions were expressed in the normally non-adherent Saccharomyces cerevisiae. These transformants were analysed for endothelial cell adherence and cell surface expression of Als1p. We found that mutations centred around amino acid 285 in the N-terminus completely abolished adherence, but had no effect on cell surface expression of Als1p. Deletion of 15 of the tandem repeats reduced adherence by 50%, whereas deletion of all abolished adherence completely, even though cell surface expression of the N-terminus of Als1p was maintained. Insertions into the C-terminus at amino acids 413 and 254 upstream of the stop codon resulted in a modest loss of adherence, while cell surface expression of Als1p was maintained. An insertion at amino acid 249 in the C-terminus caused complete loss of both adherence and cell surface expression, even though the glycosylphosphotidylinositol-anchorage sequence remained intact. These data suggest a model of Als1p in which the endothelial cell binding region is localized within its N-terminus, the tandem repeats are essential for the proper presentation of the binding site, and the C-terminus is required for localizing Als1p to the cell surface.     

The major exoglucanase from Candida albicans: a non-glycosylated secretory monomer related to its counterpart from Saccharomyces cerevisiae.

Exoglucanases secreted by two different strains from Candida albicans have been purified to homogeneity. The purified enzyme from each strain behaved as a non-glycosylated monomer (molecular weight 38,000) that was identical in terms of sodium dodecyl sulphate/polyacrylamide gel electrophoresis comigration, amino acid analysis and amino terminal sequence. The amino acid composition was similar to that of the major exoglucanase from Saccharomyces cerevisiae. In addition, these two enzymes displayed a 50% homology in the first 35 amino acids of the amino terminus. Antibodies against the deglycosylated exoglucanase (treated with Endo H) from S. cerevisiae were reactive with the exoglucanase from C. albicans and vice versa. Immunoblotting proved to be a semiquantitative method to detect C. albicans antigen in culture fluids. The exoglucanase from C. albicans appears to enter the secretory pathway without undergoing N-glycosylation.     

The SPL1 tRNA splicing gene of Candida maltosa and Candida albicans

The tRNA splicing gene SPL1-1 has been cloned and sequenced in Saccharomyces cerevisiae (Kolman and Soll, 1993). Sequence adjacent to the LEU2 gene in Candida maltosa showed some homology to the SPL1-1 gene of S. cerevisiae. This work describes the sequencing of the SPL1 tRNA splicing genes from C. maltosa and C. albicans and the analysis of these genes. Comparison of these sequences and the relationship observed between the LEU2 and SPL1 genes in these yeasts suggests that there may be some synteny amongst various species of yeasts. The coding region of the C. maltosa SPL1 region described in this work differs from previously described partial sequences in that it is a complete uninterrupted open reading frame. Two strains of C. maltosa were each shown to contain different alleles, one uninterrupted open reading frame and one disrupted open reading frame. The sequences have been deposited in the GenBank/EMBL data libraries under Accession Numbers X72940, AF000115, AF000116, AF000117, AF000118, AF000119 and AF000120. © 1998 John Wiley & Sons, Ltd.     

Isolation and sequence of the MIG1 homologue from the yeast Candida utilis

The Mig1p repressor from the food yeast Candida utilis has been isolated using a homologous PCR hybridization probe. This probe was amplified with two sets of degenerate primers designed on the basis of highly conserved motifs in the DNA-binding region (zinc-finger domain) from yeast Mig1p and fungi CreA repressors. The cloned gene was sequenced and found to encode a polypeptide of 345 amino acids which shows significant identity with other yeast and fungus repressors in the DNA-binding domain and also with the yeast Mig1 proteins in the C-terminal region (effector domain). The MIG1 repressor gene from C. utilis was able to complement functionally the mig1 mutation of S. cerevisiae. The sequence presented here has been deposited in the EMBL data library under Accession No. AJ277830.     

Cloning of Candida albicans SEC14 gene homologue coding for a putative essential function

The yeast SEC14 gene product is required for the transport of proteins from the Golgi complex. We have cloned the homologous Candida albicans SEC14 gene (CaSEC14) by functional complementation of a Saccharomyces cerevisiae thermosensitive mutant, sec14. Some putative TATA boxes have been identified in CaSEC14 and, contrary to S. cerevisiae SEC14, no introns were found in the Candida homologue. Sequence analysis revealed that CaSec14p is a 301 amino acid protein, 67% identical to S. cerevisiae and Kluyveromyces lactis Sec14p, and 61% identical to the 300 amino-terminal residues of Yarrowia lipolytica Sec14p. Hydrophatic profile analysis of CaSec14p suggests a soluble protein without transmembrane domains, as has been described for the S. cerevisiae counterpart. While it was easy to disrupt one allele of SEC14 in C. albicans, repeated attempts to disrupt the second allele were unsuccessful, thus suggesting that the gene could be essential for vegetative growth in C. albicans. The sequence has been deposited in the EMBL data library under Accession Number X81937.     

Molecular cloning and analysis of autonomous replicating sequence of Candida maltosa.

A Candida maltosa chromosomal DNA fragment which confers high frequency transformation of C. maltosa and autonomous replication of recombinant plasmids was cloned and sequenced. Analysis of the nucleotide sequence of the cloned DNA revealed a sequence homologous for C. maltosa autonomously replicating sequence (ARS) elements. Vector pRJ1 for C. maltosa was constructed, which contained a 1.3 kb ARS sequence, pICEM-19H and the ADE1 gene of C. maltosa. Southern blot analysis suggested that the copy number of pRJ1 in C. maltosa was approximately 20 per genome. The sequence analysis also revealed an open reading frame, encoding a polypeptide with high homology (70%) to the RS15 protein of Brugia pagangi. This open reading frame has an intron with canonical sites for correct splicing in Saccharomyces cerevisiae.     

Isolation of the YAP1 homologue of Candida utilis and its use as an efficient selection marker

The industrially important yeast Candida utilis is widely used in the production of food and medical materials, but its practical host-vector system has not been well developed. In order to construct a food-grade host-vector system, we isolated the YAP1 homologue, CuYAP1, of C. utilis IAM4264 and evaluated its use as a selection marker in transformation. A DNA probe was obtained by PCR using degenerate primers and the CuYAP1-encoding 438 amino acid protein was isolated by hybridization. Although the amino acid identity of Yap1 and CuYap1 was 28.7% as a whole, the characteristic bZip region and two cysteine-rich domains (CRDs) showed a higher homology. CuYAP1 was inserted in a CuGAP1 expression cassette of the C. utilis ARS vector pRI177, and C. utilis AHU3053 was transformed with this plasmid. A number of transformant colonies grew in the presence of cycloheximide, which indicated that CuGAP1-CuYAP1 is an effective selection marker. The transformant also showed higher resistance to other agents, including cadmium and fluconazole. The overexpression of CuYAP1 in S. cerevisiae also resulted in increased resistance to various types of drugs.     

Isolation and sequence of the gene encoding ornithine decarboxylase,SPE1, from Candida albicans by complementation of a spe1Δ ctrain of Saccharomyces cerevisiae

The gene encoding ornithine decarboxylase, SPE1, from the pathogenic yeast Candida albicans has been isolated by complementation of an ornithine decarboxylase-negative (spe1Δ) strain of Saccharomyces cerevisiae. Four transformants, three of which contain plasmids with the SPE1 gene, were isolated by selection on polyamine-free medium. The C. albicans ornithine decarboxylase (ODC) showed high homology with other eukaryotic ODCs at both the amino acid and nucleic acid levels. The GenBank accession number for this gene is U85005. © 1997 John Wiley & Sons, Ltd.