Date of Award:

5-1-1991

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Biology

Department name when degree awarded

Biology

Committee Chair(s)

Nabil N. Youssef

Committee

Nabil N. Youssef

Committee

Dennis L. Welker

Committee

Joseph K. K. Li

Abstract

An extrachromosomal DNA plasmid was isolated from both mating types of the entomopathogenic fungus Ascosphaera apis and named pAaL. The subculture first identified to carry pAaL originated from mummified honey bee larvae from an apiary in Wyoming, USA. Very similar, homologous plasmids were found in 13 out of 14 A. apis isolates collected from diverse geographic locations. Only one isolate obtained from New York was found to be plasmid-free. DNA hybridization indicated that pAaL is not homologous with another plasmid we isolated from Ascosphaera aggregata. This represents the first report of the presence of plasmids in entomopathogenic fungi. The pAaL element was found to have the same buoyant density as mitochondrial DNA in bisbenzamide-CsCI gradients. Extraction of DNA from intact mitochondria followed by gel electrophoretic analysis suggests pAaL is located inside the mitochondria. From hybridization tests it was concluded that pAaL did not contain sequences homologous to either mitochondrial DNA or genomic DNA. This implies that the plasmid was self-replicating. Electron microscopy and restriction digestion data indicated that the plasmid was linear, double-stranded and 12 kilobase pairs (kb) long. Based on the intensity of staining with ethidium bromide, pAaL had a higher copy number than the mitochondrial DNA. Endonuclease digestion suggested the presence of an inverted repeat at each terminus. The lack of digestion in an exonuclease assay indicated that pAaL had two blocked 5' ends, probably due to the presence of terminal binding proteins. Restriction site data showed pAaL to be AT-rich. No apparent difference in growth rate, culture appearance or reproductive cycle was found between plasmid-bearing or plasmid-free A. apis isolates. pAaL was stably inherited in the plasmid-bearing strains, but it was lost in the progeny of crosses and reciprocal crosses between the plasmid-free strain and plasmid-bearing strains. The biological function of this plasmid has not yet been determined.

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