The long term object of this proposal is to develop strains and techniques which will allow easy and rapid genetic manipulation of Candida albicans. This organism is the single most important fungal pathogen. Since it is diploid and no sexual cycle for it has been discovered, the only genetic approaches that are available are parasexual and molecular. Since the parasexual cycle involves spheroplast fusion, mitotic crossing over or chromosome loss, and segregation analysis, we will try to develop rapid ways of identifying segregants after crossing over or chromosome loss of constructing lac fusions which will be blue on x-gal medium. Segregation will be signalled by the appearance of white colonies. To improve the molecular genetics, we will use pulse-field electrophoresis and cloning to align the genetic linkage groups with the electrophoretic karyotype. We will also use the pulse-field system to create maps of the chromosomes using restriction enzymes with eight-base specificity and also some six-base-pair specific ones which cut Candida albicans DNA infrequently. Chromosome translocations and deletions, obtained by selection for recombination between gene fragments introduced by transformation will provide the opportunity to examine heterozygosities. The smallest chromosome of Candida stellatoidea will be used as the basis for a Yeast Artificial Chromosome (YAC) vector for cloning large pieces of DNA from Candida. At the completion of these experiments, the genetic map and the techniques for analyzing such aspects of C.albicans as its dimorphic life cycle, its phenotypic transition, and its virulence factors should be greatly advanced.
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