Isogenic strain construction and gene mapping in Candida albicans.

Genetic manipulation of Candida albicans is constrained by its diploid genome and asexual life cycle. Recessive mutations are not expressed when heterozygous and undesired mutations introduced in the course of random mutagenesis cannot be removed by genetic back-crossing. To circumvent these problems, we developed a genotypic screen that permitted identification of a heterozygous recessive mutation at the URA3 locus. The mutation was introduced by targeted mutagenesis, homologous integration of transforming DNA, to avoid introduction of extraneous mutations. The ura3 mutation was rendered homozygous by a second round of transformation resulting in a Ura- strain otherwise isogenic with the parental clinical isolate. Subsequent mutation of the Ura- strain was achieved by targeted mutagenesis using the URA3 gene as a selectable marker. URA3 selection was used repeatedly for the sequential introduction of mutations by flanking the URA3 gene with direct repeats of the Salmonella typhimurium hisG gene. Spontaneous intrachromosomal recombination between the flanking repeats excised the URA3 gene restoring a Ura- phenotype. These Ura- segregants were selected on 5-fluoroorotic acid-containing medium and used in the next round of mutagenesis. To permit the physical mapping of disrupted genes, the 18-bp recognition sequence of the endonuclease I-SceI was incorporated into the hisG repeats. Site-specific cleavage of the chromosome with I-SceI revealed the position of the integrated sequences.

Cloning and characterization of ECE1, a gene expressed in association with cell elongation of the dimorphic pathogen Candida albicans.

The gene ECE1 (extent of cell elongation 1) was isolated by differential hybridization screening of a Candida albicans cDNA library by using probes derived from populations of yeast cells or hyphae. Expression of this gene was not detected when C. albicans grew as a budding yeast cell but was observed within 30 min after cells had been induced to form hyphae. In all strains tested, regardless of the induction signal, ECE1 expression correlated with the extent of cell elongation. The genomic version of ECE1 was cloned and sequenced. The deduced 271-amino-acid polypeptide consisted of eight tandem repeats of a degenerate 34-amino-acid sequence which contained no discernible homology with other known sequences. An ECE1 null mutant displayed no morphological alterations, which demonstrated that ECE1 is not essential for cell elongation or hypha formation despite the strict morphological association of its expression.

The yes proto-oncogene is present in amphibians and contributes to the maternal RNA pool in the oocyte.

Low stringency screening of a cDNA library from the frog Xenopus laevis with a viral yes oncogene probe resulted in the isolation of clones with restriction maps which were distinct from previously identified X. laevis src cDNA clones. The polypeptide produced by in vitro translation of RNA synthesized from one of the clones was slightly larger in size than X. laevis pp60src, but gave protease cleavage patterns which were nearly identical to those yielded by pp60src. Determination of the DNA sequence of this clone revealed that it is derived from a X. laevis yes gene homologue. Transcripts from the yes gene were found in the ovary and consisted of a major 3.4 kb species and additional minor species. These findings indicate that the duplication event separating the src and yes genes occurred prior to the appearance of amphibians and that the yes gene product is likely to play an important role in oogenesis or early development.