Doctoral Dissertations

Date of Award

6-1985

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

W. Stuart Riggsby

Committee Members

Jeffrey M. Becker, Dorothy Skinner, Karl Sirotkin

Abstract

During the course of my studies on the regulation of morphogenic control of the dimorphic pathogen Candida albicans, I have cloned the actin gene to be used as a probe of actin gene transcription during the yeast-to-mycelial conversion. To clone the C. albicans actin gene, whole cell DNA was digested with Sal I, the fragments were fractionated by size on a sucrose gradient, fractions were electrophoresed, blotted to nitrocellulose and hybridized with labeled cerevisiae actin gene probe. DNA from fractions enriched for fragments containing the actin gene sequences were cloned into the Sal I site of plasraid pBR322 and transformed into E. coli DHI. Clones containing the actin gene were identified by hybridization to two known actin genes, one from S. cerevisiae and the other from Drosophila melanogaster. Genomic blots hybridized with the cloned C. albicans actin gene produced no new signals, suggesting no unidentified actin sequences and that the major portion of the C. albicans actin gene was cloned. The existence of a single actin gene in the C. albicans genome was confirmed by reconstruction experiments. The cloned C. albicans actin gene has been mapped with several restriction endonucleases. RNA blot analysis of yeast and mycelial phase cells was used to detect the length and abundance of actin gene transcripts. A 3.3-fold increase in actin transcriptionm was detected during the first half hour of the yeast-to-mycelial conversion; actin gene transcription reached a maximum at two hours then declined. Actin transcription from pH 4.5 yeast increased approximately 30% during the first half hour at 37 C, then gradually increased. Models for enhanced actin gene transcription iii were explored. Southern blotting demonstrated that rearrangement of the act in gene is not a mechanism for enhanced act in gene synthesis for yeast or mycelial cells. Southern blotting of yeast and raycelial DNA with methylation isoschizomers demonstrates no difference in methylation pattern of the actin gene during the yeast-to-mycelium conversion.

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