Date of Award:

5-1-1993

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Department name when degree awarded

Biology (Molecular Biology)

Committee Chair(s)

Neal K. Van Alfen

Committee

Neal K. Van Alfen

Committee

Gregory Podgorski

Committee

Anne Anderson

Committee

Dennis Welker

Committee

Charles Carpenter

Abstract

An efficient DNA-mediated transformation system has been developed for the chestnut blight fungus Cryphonectria parasitica. Twelve vectors, each containing one of three dominant selectable antibiotic genes, were tested for their ability to confer resistance to either hygromycin B, benomyl, or G418 sulfate. Transformants were obtained with each vector screened, and in each case, transformation occurred by integration of the transforming DNA into the host genome. The cosmid vector pMOcosX conferred hygromycin B resistance to transformants at an efficiency of greater than 104 transformants/µg DNA and was utilized for the construction of a wild-type genomic DNA library. This library was screened for two genes, a methionine gene (MET1) and a pigmentation gene (PIG), by transformation into a met1 pig double mutant. The met1 pig mutant is a methionine auxotroph (met1) that exhibits reduced asexual sporulation and orange pigmentation (pig). Selection for methionine prototrophy and screening for restoration of orange pigmentation to the met1 pig mutant resulted in the isolation of transformants complemented for each phenotype. The complementing DNA was rescued by in vitro packaging of transformant genomic DNA with bacteriophage lambda packaging extract. The MET1 gene of C. parasitica, conferring methionine prototrophy, was isolated on a cosmid clone containing a 39 kb insert and subcloned to a 3.3 kb BamHI/EcoRI fragment. The subcloned MET1 gene transformed the met1 pig mutant to methionine prototrophy with an efficiency of approximately 102 transformants/µg DNA. Additionally, asexual sporulation and orange pigmentation were partially restored to wild-type levels in Met+ transformants. The subcloned MET1 gene serves as a fourth selectable marker, and the first nutritional marker, for transformation of C. parasitica. A second cosmid clone, also isolated by functional complementation of the met1 pig mutant, conferred orange pigmentation to transformants. Preliminary data suggest that at least a portion of the PIG gene has been cloned to a 32 kb cosmid insert. These are the first reports of gene isolation by functional complementation in C. parasitica.

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