Identification of Candida albicans clinical isolates by PCR amplification of an EFB1 gene fragment containing an intron-interrupted open reading frame.

The use of a single pair of primers, deduced from the intron and exon nucleotide sequences of the Candida albicans EFB1 gene, in polymerase chain reaction (PCR) assays performed with whole cells of both laboratory strains and clinical isolates of Candida species, resulted in the species-specific amplification of a 785 bp DNA fragment in C. albicans strains. Clinical C. albicans isolates were tested, and 85 out of 86 generated the expected PCR-amplified product; other Candida species, both laboratory strains and clinical isolates, as well as laboratory strains belonging to other fungal genera, including medically relevant taxa, failed to amplify any DNA fragment. In addition, unusual C. albicans isolates (glucosamine- and N-acetylglucosamine-negative) from Africa also yielded the expected PCR-generated DNA fragment. These results indicate that genes containing intron sequences may be useful to design species-specific primers for the identification of fungal strains by PCR.

Complementation of Saccharomyces cerevisiae mutations in genes involved in translation and protein folding (EFB1 and SSB1) with Candida albicans cloned genes.

We have demonstrated that the expression of Candida albicans genes involved in translation and protein folding (EFB1 and SSB1) complements the phenotype of Saccharomyces cerevisiae mutants. The elongation factor 1beta (EF-1beta) is essential for growth and efb1 S. cerevisiae null mutant cells are not viable; however, viable haploid cells, carrying the disrupted chromosomal allele of the S. cerevisiae EFB1 gene and pEFB1, were isolated upon sporulation of a diploid strain which was heterozygous at the EFB1 locus and transformed with pEFB1 (a pEMBLYe23 derivative plasmid containing an 8-kb DNA fragment from the C. albicans genome which contains the EFB1 gene). This indicates that the C. albicans EFB1 gene encodes a functional EF-1beta. Expression of the SSB1 gene from C. albicans, which codes for a member of the 70-kDa heat shock protein family, in S. cerevisiae ssb1 ssb2 double mutant complements the mutant phenotype (poor growth particularly at low temperature, and sensitivity to certain protein synthesis inhibitors, such as paromomycin). This complementation indicates that C. albicans Ssbl may function as a molecular chaperone on the translating ribosomes, as described in S. cerevisiae. Northern blot analysis showed that SSB mRNA levels increased after mild cold shift (28 degrees C to 23 degrees C) and rapidly decreased after mild heat shift (from 28 degrees C to 37 degrees C, and particularly to 42 degrees C), indicating that SSB1 expression is regulated by temperature. Therefore, Ssb1 may be considered as a molecular chaperone whose pattern of expression is similar to that found in ribosomal proteins, according to its common role in translation.

Molecular cloning of a Candida albicans gene (SSB1) coding for a protein related to the Hsp70 family.

We have cloned and sequenced a Candida albicans gene (SSB1) encoding a potential member of the heat-shock protein seventy (hsp70) family. The protein encoded by this gene contains 613 amino acids and shows a high degree (85%) of sequence identity to the ssb subfamily (ssb1 and ssb2) of the Saccharomyces cerevisiae hsp70 family. The transcribed mRNA (2.1 kb) is present in similar amounts both in yeast and germ tube cells of C. albicans.

Molecular cloning and characterization of a Candida albicans gene (EFB1) coding for the elongation factor EF-1 beta.

A Candida albicans gene homologous to Saccharomyces cerevisiae elongation factor 1 beta was isolated by screening a genomic DNA library using a C. albicans cDNA as a probe. This cDNA was previously obtained by immunoscreening of an expression library with polyclonal antibodies raised against candidal cell wall components. Sequence analysis of the cDNA and the whole C. albicans gene (EMBL accession number X96517) revealed an intron-interrupted open reading frame of 639 base pairs that encodes a 213 amino acid protein. Exon sequences are highly homologous (74%) to S. cerevisiae EFB1, whereas intron sequence is less conserved (34% identity), and the predicted amino acid sequence shares about 73% identity.