Recent studies are beginning to delineate those pathways by which the important pathogen Candida albicans switches from one growth form to another; at the same time, insights are being gained into the importance of growth form in pathogenesis.

The heat shock protocol described in this paper causes mitotic instability in log phase Candida albicans cells. Such instability is induced in diploid, aneuploid and tetraploid strains. The strains analysed are multiple heterozygotes which facilitates the detection of mitotic instability as manifested by the formation of homozygotes. Strains previously shown to be carrying cis linked mutant alleles show coincident segregation of the linked alleles. Conversely, strains which carry unlinked mutant alleles display no such coincident segregation. This segregation of complete linkage groups suggests that heat shock is inducing chromosome loss in C. albicans. The application of this protocol to the genetics of the imperfect fungus C. albicans has produced evidence of at least three chromosomes.

We report here the separation of chromosomal DNA molecules from the diploid pathogenic yeast C. albicans by field inversion gel electrophoresis (FIGE)(1). Our earlier work on C. albicans ATCC strain 10261 revealed a pulsed field gel electrophoretic pattern of six bands, two of which were possibly doublets (2). In the present study we found that FIGE gave superior resolution with uniform DNA mobility between lanes, and therefore could be used to examine the possibility of electrophoretic karyotype variation in this asexual yeast. The extension of our study to other strains of C. albicans showed that local clinical isolates, strain 22114 (fromM.D.Richardson, Birmingham, England) and our ATCC 10261 reference strain exhibit considerable differences in their FIGE banding patterns (Figure). However, certain similarities are apparent, especially in bands 5 and 6. Probing Southern blots with C. albicans DNA probes which complement the HIS3, ADE2 and URA3 genes in S. cerevisiae gave results supporting this conclusion. Close examination of the variant chromosomes, including further probing, suggested that they may have had common origins, but have undergone reanrangements which involve either one or both homologues of a diploid pair. However, we do not believe that C. albicans chromosomes are overly unstable, since ATCC 10261 strains freeze-dried in 1960 and 1965 gave the same banding pattern as an ATCC 10261 strain in laboratory culture for the last 15 years.

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The Saccharomyces cerevisiae CDC9 gene encodes a DNA ligase protein that is targeted to both the nucleus and the mitochondria. While nuclear Cdc9p is known to play an essential role in nuclear DNA replication and repair, its role in mitochondrial DNA dynamics has not been defined. It is also unclear whether additional DNA ligase proteins are present in yeast mitochondria. To address these issues, mitochondrial DNA ligase function in S.cerevisiae was analyzed. Biochemical analysis of mitochondrial protein extracts supported the conclusion that Cdc9p was the sole DNA ligase protein present in this organelle. Inactivation of mitochondrial Cdc9p function led to a rapid decline in cellular mitochondrial DNA content in both dividing and stationary yeast cultures. In contrast, there was no apparent defect in mitochondrial DNA dynamics in a yeast strain deficient in Dnl4p (Deltadnl4). The Escherichia coli ECO:RI endonuclease was targeted to yeast mitochondria. Transient expression of this recombinant ECO:RI endonuclease led to the formation of mitochondrial DNA double-strand breaks. While wild-type and Deltadnl4 yeast were able to rapidly recover from this mitochondrial DNA damage, clones deficient in mitochondrial Cdc9p were not. These results support the conclusion that yeast rely upon a single DNA ligase, Cdc9p, to carry out mitochondrial DNA replication and recovery from both spontaneous and induced mitochondrial DNA damage.