Dr. Sherman and his group have recently demonstrated that standard laboratory strains of Candida albicans spontaneously gave rise to different types of colonial morphological mutants that are associated with single and multiple gross chromosomal rearrangements. Some of the mutants were unstable and gave rise to additional colonial forms after further subcloning. More recently, they have shown that the electrophoretic karyotypes of four independent clinical isolates all differed from each other, and some of the chromosomal differences superficially resembled the alterations uncovered in the morphological mutants. They suggest that the chromosomal aberrations arising from normal strains provide a means for genetic variation in this asexual microorganism. This proposal deals with a systematic investigation of the patterns of electrophoretic karyotypes and chromosomal rearrangements associated with morphological mutants and comparisons of these mutants to a variety of clinical isolates. High resolution procedures of pulse field electrophoresis to separate the closely running chromosomes and to detect slight variations of abnormal lengths will be carried out with orthogonal-field alteration-gel electrophoresis (OFAGE) and contour-clamped homogeneous-electric-field gel electrophoresis (CHEF). The chromosomal rearrangements will be characterized by hybridizing separated NotI and SfiI restriction fragments to probes corresponding to sites distributed along each chromosome. The number of rDNA repeats, that could be responsible for the variation of chromosome VIII length observed in the four clinical isolates and in many morphological mutants, will be estimated simply by digesting total DNA with BamHI (or another restriction endonuclease that does not cleave rDNA), separating the fragments with OFAGE or CHEF, and hybridizing blots with a rDNA probe. The results of the comparisons of the mutants and independent clinical isolates may provide credence to the hypothesis that the chromosomal aberrations are providing genetic variation that allows C. albicans to adapt to new conditions of their host. The patterns and types of chromosomal aberrations in the stable and unstable mutants my shed light on mechanisms by which they arise.
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