The nuclear and mitochondrial genomes of Candida albicans : a study of the yeast phase cell

Author

John W. Wills

Date of Award

8-1982

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

W. S. Riggsby

Committee Members

Jeffrey Becker

Abstract

The purpose of this study was to characterize the nuclear and mitochondria] genomes of the pathogenic, dimorphic fungus Candida albicans. Buoyant density measurements of whole cell DNA revealed two components: a major band of 32.8% G + C and a satellite band of 40.3% G + C. Cation-exchange chromatography of 5'-deoxyribonucleotides from whole cell DNA showed an average G + C content of 34.4%. Electrophoresis of restriction endonuclease EcoRI digested whole cell DNA revealed three distinct repetitive DNA bands (containing fragments 6.6, 3.3, and 2.6 kbp in length) which proved to be of nuclear DNA origin. Reassociation experiments using whole cell DNA and the HAP method of assay showed the presence of four kinetic components in randomly sheared DNA (300 bp, average length). The component composition of the DNA was found to be: 3.4% foldback, 2.5% fast repetitive (K_pure = 742), 11.0% slow repetitive (K_pure = 13.1), and 83.6% single-copy sequences (K_pure = 0.063). The total kinetic complexity (haploid genome size) was found to be 1.85 X 10⁷ base pairs. Comparison with previously reported DNA content values indicates that Candida albicans is a diploid organism.

Methods were developed which enabled the successful purification of mitochondria] DNA (mtDNA) in large amounts from yeast phase cells of C. albicans strain H317. Buoyant density measurements revealed a G + C content of 38%. Digestion of mtDNA with restriction endonuclease EcoRI was found to produce six fragments, and digestion with PvuII was found to produce seven fragments. Based upon restriction endonuclease analysis, the mtDNA was found to have a length of 41 kbp. The EcoRI fragments of this mtDNA (except for the largest fragment) were cloned in plasmid pBR322 without any detectable alterations. Hybridization experiments showed that each of the six EcoRI fragments contain sequences homologous to one or more of the other five fragments. Restriction endonuclease mapping of the EcoRI and PvuII sites proved that the mtDNA of this organism is circular and contains a large, inverted duplication. Comparison with EcoRI digests of the mtDNA of three other strains of C. albicans (ATCC, H350, and WD-18-4) suggests that all have the same restriction map. Thus, it appears that the map reported here is that of wild type C. albicans mtDNA.

Recommended Citation

Wills, John W., "The nuclear and mitochondrial genomes of Candida albicans : a study of the yeast phase cell. " PhD diss., University of Tennessee, 1982.
https://trace.tennessee.edu/utk_graddiss/13351