The long-term objectives of the research outlined in this proposal are to understand the mechanisms which lie behind the successful pathogenesis of the dimorphic asexual diploid yeast, Candida albicans. This yeast causes infections ranging from mucosal thrush to systemic fungemia. In immunocompromised patients, infections by Candida albicans are life- threatening. No virulence factors are known for this organism, leading to the conclusion that its pathogenicity is a very complex phenomenon. One aspect of biology which may play a role in virulence is the capacity of C. albicans to vary its karyotype. It has been speculated that this karyotypic instability may provide a means of genetic variation. The mechanism of the karyotypic variation is unknown. It is manifested in changes in chromosomal length and in chromosomal translocations.
The aim of this project is to investigate the mechanism of karyotypic variation in C. albicans. The hypothesis to be tested is that chromosomal rearrangements take place preferentially at a repeated sequence called RPS. To test this hypothesis, the junction points of known chromosomal translocations will be cloned and characterized to see whether the repeated sequence is found at the breakpoints. PIasmid recombination systems will be constructed to test for the frequencies of recombination between homologous sequences in the presence and absence of RPS. Recombination between heteroalleles inserted on heterologous chromosomes will be tested in order to see whether RPS increases the frequency of these events. Finally, the junctions of translocations which have occurred in the test system will be cloned and characterized to see whether the structure is consistent with the hypothesis. Because karyotypic rearrangement seems to be much more frequent in strains which undergo the phenotypic transition, the experiments described above will be carried out in """"""""switching"""""""" and """"""""non-switching"""""""" strains, to see whether translocation is indeed elevated in the former compared to the latter. These experiments will give information on recombination in C. albicans, information which will be useful in a variety of studies bearing on the pathogenicity of the organism, including the examination of phenotypic instability and adaptation to various niches in the host. They will also greatly aid in developing the genetics of this important pathogen.
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