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.     

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.     

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.     

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.     

Nucleotide sequence analysis of the 5·8S rDNA and adjacent ITS2 region of Candida albicans and related species

We have determined the nucleotide sequence for the DNA encoding the 5·8S RNAs and downstream internal transcribed spacer (ITS2) regions for Candida albicans and the taxonomically related species C. parapsilosis, C. tropicalis, C. glabrata and C. krusei. Phylogenetic analysis of all known fungal 5·8S RNA sequences revealed a close relationship between C. tropicalis and C. parapsilosis, and to a lesser extent C. albicans within the yeast-like fungi. This group can itself be delineated from predominantly filamentous species. The more distal relationships between Candida (torulopsis) glabrata and C. krusei support previous findings based on small (18S) ribosomal RNA sequence analysis, suggesting a greater degree of evolutionary divergence of these species from the C. albicans group. Among strains of C. albicans we observed conservation of the ITS2 region of the nucleotide level. Conservation was also observed for a more limited number of C. parapsilosis strains. Although the 3′ region of the ITS spacer was species specific, sequence homology was observed in the 5′ end within the albicans/parapsilosis/tropicalis group. Our findings suggest a rapid approach to species identification through the use of non-conserved regions flanked by highly conserved, functional domains.     

Characterization of Candida albicans orthologue of the Saccharomyces cerevisiae signal-peptidase-subunit encoding gene SPC3

The Candida albicans orthologue of the SPC3 gene, which encodes one of the subunits essential for the activity of the signal peptidase complex in Saccharomyces cerevisiae, was isolated by complementation of a thermosensitive mutation in the S. cerevisiae SEC61 gene. The cloned gene (CaSPC3) encodes a putative protein of 192 amino acids that contains one potential membrane-spanning region and shares significant homology with the corresponding products from mammalian (Spc22/23p) and yeast (Spc3p) cells. CaSPC3 is essential for cell viability, since a hemizygous strain containing a single copy of CaSPC3 under control of the methionine-repressible MET3 promoter did not grow in the presence of methionine and cysteine. The cloned gene could rescue the phenotype associated with a spc3 mutation in S. cerevisiae, indicating that it is the true C. albicans orthologue of SPC3. However, in contrast with results previously described for its S. cerevisiae orthologue, CaSPC3 was not able to complement the thermosensitive growth associated with a mutation in the SEC11 gene. The heterologous complementation of the sec61 mutant suggests that Spc3p could play a role in the interaction that it is known to occur between the translocon (Sec61 complex) and the signal peptidase complex, at the endoplasmic reticulum membrane.     

Isolation and sequencing of the Candida albicans MSI3, a putative novel member of the HSP70 family

We have reported previously that the expression of CGR1 increased at an early stage of the yeast-mycelial transition (morphogenesis) in Candida albicans. We now show that Cgr1p interacts in a yeast two-hybrid system with the C. albicans Msi3p (CaMsi3p), a putative novel member of the heat shock protein 70 (HSP70) family. The DNA sequence of CaMSI3 encodes a predicted protein of 702 amino acids with a molecular mass of 78.6 kDa. The amino acid sequence of CaMsi3p is 63% identical to Msi3p/Sse1p of the HSP70 family of Saccharomyces cerevisiae. Further, CaMSI3 complemented the temperature-sensitive phenotype of the msi3(-) mutant of S. cerevisiae. Other heat shock proteins of C. albicans are required for morphogenesis and are highly antigenic. These observations suggest that CaMSI3 may well provide functions for this organism unrelated to a heat shock function. The DDBJ Accession No. for the sequence reported in this paper is AB061274.     

Sequence,map position and genome organization of the RPL17B gene,encoding ribosomal protein L17b in Saccharomyces cerevisiae

Sequence analysis of the newly defined SSU81 gene revealed an adjacent open reading frame (ORF) encoding a protein whose deduced amino acid sequence is identical to that of ribosomal protein L17. The DNA sequence of this region is different from that of the RPL17A gene and therefore represents a duplicate gene encoding L17. We have designated this gene RPL17B. The RPL17B coding region is split by an intron that occurs in the same position (codons 14/15) as the intron in RPL17A. The RPL17B promoter region includes two TATA boxes, a canonical UAS_RPG motif, and several pyrimidine-rich tracts. RPL17B was mapped by CHEF and lambda clone grid hybridization blots to the right arm of chromosome V, linked to the TRP2 and RAD51 genes. A partial ORF was identified adjacent to RPL17B and SSU81 that is homologous to an ORF (designated A509) physically linked to RPL17A. This observation, and the identical position of the introns within the RPL17 genes, suggest that one RPL17 locus arose by duplication and translocation of the other. The complete 3·8 kbp DNA sequence encompassing RPL17B has been entered in the GenBank data library under Accession Number U15653.     

Identification of potential cell-surface proteins in Candida albicans and investigation of the role of a putative cell-surface glycosidase in adhesion and virulence

Cell-surface proteins are attractive targets for the development of novel antifungals as they are more accessible to drugs than are intracellular targets. By using a computational biology approach, we identified 180 potential cell-surface proteins in Candida albicans, including the known cell-surface adhesin Als1 and other cell-surface antigens, such as Pra1 and Csa1. Six proteins (named Csf1-6 for cell-surface factors) were selected for further biological characterization. First, we verified that the selected CSF genes are expressed in the yeast and/or hyphal form and then we investigated the effect of the loss of each CSF gene on cell-wall integrity, filamentation, adhesion to mammalian cells and virulence. As a result, we identified Csf4, a putative glycosidase with an apparent orthologue in Saccharomyces cerevisiae (Utr2), as an important factor for cell-wall integrity and maintenance. Interestingly, deletion of CSF4 also resulted in a defect in filamentation, a reduction in adherence to mammalian cells in an in vitro adhesion assay, and a prolongation of survival in an immunocompetent mouse model of disseminated candidiasis. A delay in colonization of key organs (e.g. kidney) was also observed, which is consistent with a reduction in virulence of the csf4-deletion strain. These data indicate a key role for extracellular glycosidases in fungal pathogenesis and represent a new site for therapeutic intervention to cure and prevent fungal disease.     

Sequence and Analysis of a 36·2 kb Fragment from the Right Arm of Yeast Chromosome XV Reveals 19 Open Reading Frames Including SNF2 (5′ end), CPA1, SLY41, a Putative Transport ATPase,a Putative Ribosomal Protein and an SNF2 Homologue

The complete sequence of a 36 196 bp DNA segment located on the right arm of chromosome XV of Saccharomyces cerevisiae has been determined and analysed. The sequence includes the 5′ coding region of the SNF2 gene, the CPA1 leader peptide sequence and 17 open reading frames (ORFs) of at least 100 amino acids. Two of these correspond to previously known genes (CPA1, SLY41), whereas 15 correspond to new genes. The putative translation products of three ORFs show significant similarity with known proteins: one is a putative transport ATPase, another appears to be a ribosomal protein, and the third is an Snf2p homologue. The sequence has been deposited in the EMBL databank under Accession Numbers: Z75198, Z75199, Z75200, Z75201, Z75202, Z75203, Z75204, Z75205, Z75206, Z75207, Z75209, Z75210, Z75211, Z75212, Z75213, Z75214, Z75215, Z75216. © 1997 John Wiley & Sons, Ltd.     

Cloning and sequence of a 3·835 kbp DNA fragment containing the HIS4 gene and a fragment of a PEX5-like gene from Candida albicans

We have isolated the Candida albicans HIS4 (CaHIS4) gene by complementation of a his4-34 Saccharomyces cerevisiae mutant. The sequenced DNA fragment contains a putative ORF of 2514 bp, whose translation product shares a global identity of 44% and 55% to the His4 protein homologs of S. cerevisiae and Kluyveromyces lactis, respectively. Analysis of CaHIS4 sequence suggests that, similarly to S. cerevisiae HIS4, it codes for a polypeptide having three separate enzymatic activities (phosphoribosyl-AMP cyclohydrolase, phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase) which reside in different domains of the protein. A C. albicans his4 strain is complemented with this gene when using a C. albicans-S. cerevisiae-Escherichia coli shuttle vector, thus enabling the construction of a host system for C. albicans genetic manipulation. In addition, upstream of the sequenced CaHIS4 sequence, we have found the 3′-terminal half of a gene encoding a PEX5-like protein. The EMBL/DDJB/GenBank Accession Number of this sequence is AJ003115. © 1998 John Wiley & Sons, Ltd.     

The Genes Encoding the Transcription Factor yTAFII60, the G4p1 Protein and a Putative Glucose Transporter are Contained in a 12·3 kb DNA Fragment on the Left Arm of Saccharomyces cerevisiae Chromosome VII

We report the nucleotide sequence of a DNA fragment of 12 325 base pairs from the left arm of the Saccharomyces cerevisiae chromosome VII. Inspection of the coding capacity revealed 11 open reading frames (ORFs) longer than 100 amino acids. Five ORFs are significantly homologous to known proteins. The region encoding ORF G2985 corresponds (100%) to the gene encoding the yeast TATA binding protein-associated factor TAF_II60. The G3075 ORF is 47·8% identical to the hypothetical yeast protein YB88. G3080 shows 36·7% identity to the eel calmodulin. G3085 shows 94·9% identity with the published sequence of the quadruplex DNA binding protein G4p1. G3090 reveals 46·7% identity with the probable glucose transport protein yBR1625. The DNA sequence has been submitted to the EMBL data library under Accession Number X97644. © 1997 by John Wiley & Sons, Ltd.     

Purification and characterisation of the 7S globulin storage protein from sesame (Sesamum indicum L.)

The 7S globulin from sesame seeds was purified by means of selective precipitation and anion-exchange chromatography on Q Sepharose Fast Flow. The 7S globulin migrated as a single band on native PAGE, which suggested homogeneity of the sample. The isolated protein was composed of at least eight polypeptide chains, ranging from 12.4 to 65.5 kDa, judged by SDS–PAGE analysis, and did not contain disulphide bonds. Furthermore, comparison of the polypeptide bands of the 7S and 11S globulins by SDS–PAGE indicated that the purified 7S globulin was free of legumin-like contaminant polypeptides and of 2S albumin. The identity of the purified polypeptides was verified by comparing the N-terminal amino acid sequences of the main polypeptide bands with the amino acid sequence deduced from a cDNA clone, which encoded the sesame 7S globulin precursor. Purification of the 7S globulin from sesame has not previously been reported.     

Sequence Analysis of a 33·2 kb Segment from the Left Arm of Yeast Chromosome XV Reveals Eight Known Genes and Ten New Open Reading Frames Including Homologues of ABC Transporters,Inositol Phosphatases and Human Expressed Sequence Tags

The complete nucleotide sequence of a 33 221 bp segment, contained in cosmid pEOA1044, derived from the left arm of chromosome XV of Saccharomyces cerevisiae, appears in public databases between coordinates 177013 and 210234 (http://speedy.mips.biochem.mpg.de/). Computer analysis of that sequence revealed the presence of the previously known genes IRA2, DEC1, NUF2, HST1, RTG1, RIB2 and HAL2, one previously partially sequenced open reading frame (ORF) of unknown function (SCORFAC) and ten newly identified ORFs. One of the new ORFs is similar to the Drosophila melanogaster white gene and other transmembrane ABC transporters, another one has similarities to inositol phosphatases and others are similar to ORFs of unknown function from various organisms, including human Expressed Sequence Tags (ESTs). Potential transmembrane regions, ATP/GTP-binding and WD motifs have also been identified. The existence of yeast ESTs for two of the newly identified ORFs indicates that they are transcribed. © 1997 John Wiley & Sons, Ltd.