The Candida albicans group I intron ribozyme: Therapeutic target and molecular marker

Candida albicans a diploid asexual fungus, is normally a commensal organism in humans. However, in the immunodeficient human population, it can cause life-threatening diseases. Genotypic and phenotypic variation contribute, in part, to the pathogenic nature of C. albicans. The presence or absence of a group I intron ribozyme (Ca.LSU) in the 25S ribosomal RNA (rRNA) gene is one of the variations at the genomic level. It is used to group C. albicans strains in three genotypes: A, in which Ca.LSU is absent; B, in which Ca.LSU is present, and C, that contains both ribosomal DNA (rDNA) types, with and without Ca.LSU in the 25S rRNA. This genomic polymorphism has been correlated with fluorocytosine susceptibility, as intron-containing strains are more susceptible. This thesis shows that C. albicans strains harboring Ca.LSU are more susceptible to pentamidine than strains either without Ca.LSU or with both types of rDNA. The data strongly suggests that group I intron ribozymes, not yet found in the human genome but present in various human pathogens, are potential targets of antimicrobial agents.; C. albicans strain 221, which shows a level of susceptibility to pentamidine intermediate between that for strains 4-1 and 62-1, contains two types of rDNA units. One unit carries Ca.LSU on its 25S rRNA gene and the other does not. Southern blot analysis of pulsed-field gel electrophoresis (PFGE) karyotyping gels show that in strain 221, and presumably other genotype C strains, both types of rDNA units are present in both R (rDNA-containing) homologous chromosomes. Long-PCR amplification shows that the rDNA units are arranged in two tandem arrays of identical repeats with one located immediately adjacent to the other, within a single rDNA cluster on each chromosome R homolog. For a multicopy gene family, such as the rDNA genes, this arrangement is unusual since the units are expected to evolve in concert and show sequence homogeneity. This heteroallelic strain for the rDNA units also displays a high frequency of appearance of colonial morphology mutants, in comparison to the homoallelic strains tested. These data suggest a possible correlation between heteroallelic rDNA and phenotypic plasticity.; The asexual nature of C. albicans impedes a genetic analysis of the in vivo inhibition of the group I intron ribozyme by pentamidine. To carry out this investigation in Saccharomyces cerevisiae, a large plasmid (approximately 15 kb) carrying a single S. cerevisiae rDNA unit was engineered to contain Ca.LSU in the 25S rRNA gene in a homologous position by a novel methodology. (Abstract shortened by UMI.)...