Date

February 1990

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Biochemistry and Molecular Biology

Institution

Oregon Graduate Institute of Science & Technology

Abstract

Genetic recombination occurred between auxotrophic strains of the lignin-degrading basidiomycete Phanerochaete chrysosporium. Cytological studies demonstrated that the basidiospores are binucleate. Six leucine auxotrophic strains of P. chrysosporium were characterized genetically and biochemically. The mating system and formation of basidiospores in P. chrysosporium were studied using wild-type and auxotrophic strains. The prototrophic basidiospore progeny of crosses between auxotrophic strains were shown to be homokaryotic recombinants rather than complementary heterokaryons. Various wild-type strains were shown to have multinucleate cells lacking clamp connections and to possess a variable number of sterigmata per basidium. Single basidiospores from three wild-type strains all produced fruit bodies and basidiospores. Nonfruiting as well as fruiting isolates were obtained from single basidiospores of five other wild-type strains. Basidiospores from fruiting isolates always yielded colonies that fruited, again indicating that the spores are homokaryotic. Nonfruiting isolates from the same strain did not produce basidiospores when paired, indicating that these isolates do not represent mating types. Basidiospores from strain OGC1O1 also gave rise to colonies which did not grow on cellulose (Cel[superscript-]). This evidence suggests that at least some strains of P. chrysosporium are heterokaryons with primary homothallic mating systems. A DNA.transformation system was developed for P. chrysosporiwn. Swollen basidiospores of an adenine auxotroph were protoplasted and transformed to prototrophy using a plasmid containing the gene for an adenine biosynthetic enzyme from Schizophyllwn commune. Southern blot analysis demonstrated that plasmid DNA was integrated into the chromosomal DNA in multiple copies. Recombination experiments demonstrated that integration occurred at site(s) other than the resident adenine biosynthetic gene. A second P. chrysosporium adenine auxotroph was transformed to prototrophy with a second adenine biosynthetic gene from S. commune. Fragments containing both adenine biosynthetic genes were subcloned into the plasmid pUC18 and transformants obtained with these subclones were analyzed. The subclones were mapped for restriction sites and the approximate locations of the complementing genes were determined. One of these plasmids was used to transform the Neurospora crassa ade2 strain, thereby identifying the complementing adenine biosynthetic gene as encoding phosphoribosylamdnoimddazole synthetase.

Identifier

doi:10.6083/M4R20Z9Z

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