Date of Award:

5-1-1990

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Biology

Department name when degree awarded

Biology

Committee Chair(s)

Joseph K. Li

Committee

Joseph K. Li

Committee

Neal Van Alfen

Committee

Jeffery K. Kondo

Abstract

Cryphonectria parasitica is the fungal pathogen that causes chestnut blight. Hypovirulent variants of this fungus have been isolated, and this phenotype has been shown to be associated with a cytoplasmically transmissible doublestranded RNA (dsRNA). The dsRNA is not encapsidated but is contained within membranous vesicles of fungal origin. These vesicles contain an RNA polymerase activity which produces both single ( ssRNA) and dsRNA products. In the study reported here, the strandedness of the dsRNA in vitro products of the polymerase was determined by hybridization to plus and minus copies of a 194-base-pair fragment from the homopolymer end of the dsRNA that was cloned into the phage M13. The ssRNA and dsRNA products of the polymerase hybridize strongly to the M13 DNA containing the insert of the minus strand, indicating that the single-stranded products are plus-sense. There is slight but detectable hybridization to the clone containing the insert of the plus strand. Probes prepared from 5’ end-labeled fragmented dsRNA isolated from vesicles also hybridize strongly to the clone containing the insert in the minus orientation, suggesting preferential labeling of the plus strand of the dsRNA or that the plus strand is present at a higher molar ratio with respect to the minus strand. We have been unable to detect the presence of free ssRNA within or associated with the isolated vesicles.

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