Chromosome polymorphisms close to the cm-ADE1 locus of candida maltosa.

The imperfect yeast Candida maltosa has an ill-defined genetic constitution; it is nominally diploid, but probably highly aneuploid, in nature. We report on polymorphisms specifically affecting those chromosomes which bear the cm-ADE1 gene. This gene encodes phosphoribosylaminoimidazole-succino-carboxamide synthetase, an enzyme in the adenine biosynthetic pathway. By electrophoretic karyotype analysis, three differently sized chromosomes were demonstrated to carry cm-ADE1; the size (but not the number) of these chromosomes was also found to vary, both between strains and during the mitotic growth of a single strain. Four different alleles of cm-ADE1 have been cloned and sequenced from one prototrophic strain. DNA sequence divergence between these different alleles is as high as 8%, with the greatest divergence being found in the upstream region. Mitotic recombination events that led to changes in the karyotype were followed by using cm-ADE1 DNA as an hybridization probe. A recombination hot-spot in the neighbourhood of the gene appears to be responsible for the instability of the chromosomes on which it residues.

Molecular analysis of a leu2-mutant of Candida maltosa demonstrates the presence of multiple alleles.

Three different alleles of the beta-isopropylmalate dehydrogenase gene were cloned and sequenced from a leucine auxotrophic mutant, G587, of Candida maltosa. The cloning of functionally-intact wild-type genes from this mutant strain suggests the presence of silent gene copies. An interallelic-divergence comparison has provided evidence for new regulatory mechanisms. Sequence data and karyotype analysis argue for a highly-aneuploid genome of C. maltosa. An interpretation for the spontaneous auxotrophy-prototrophy-auxotrophy sequence of mutations in C. maltosa is suggested.

Efficient electropulse transformation of intact Candida maltosa cells by different homologous vector plasmids.

Conditions for efficient and quick transformation by electroporation were developed in Candida maltosa. To investigate the efficiency of transformation with integrative as well as with autonomously replicating plasmids, a series of vectors was constructed for homologous transformation of this species. Transformants were obtained with different plasmids as covalently closed circular molecules and as linearized DNA. The influence of recipient strain and plasmid type as well as of cell number and parameters of the supplied electrical pulse on the transformation efficiency have been investigated. A maximum of 7000 transformants per 100 ng of plasmid DNA was reached. The efficiency of transformation was compared with that of the LiCl method.

Correlation of biochemical blocks and genetic lesions in leucine auxotrophic strains of the imperfect yeast Candida maltosa.

The four enzymatic steps in the conversion of alpha-ketoisovaleriate to leucine were examined in the wild type and in 13 leucine auxotrophic strains of Candida maltosa. The genetic lesions in the auxotrophs, involve at least five different loci and are correlated with three enzymatic steps. This was confirmed by gene cloning, protoplast fusion, and enzyme assays. The pathway for leucine biosynthesis in C. maltosa shows general similarity to that of other lower eukaryotes but there are individual differences in the numbers of genes responsible for single enzymatic steps. A disomic state of the chromosomes carrying genes coding for alpha-isopropylmalate synthase and beta-isopropyl-malate dehydrogenase was elucidated.