Date of Award

1988

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

William R. Lee

Abstract

Wild type alcohol dehydrogenase polypeptides (ADH) from Drosophila melanogaster transformants were examined using western blots and polyclonal antiserum specific for Drosophila melanogaster ADH. These data indicate that ADH from Hawaiian drosophilids was similar in molecular weight to Drosophila melanogaster ADH, but that the isoelectric points of the Hawaiian electromorphs were distinct. Therefore it was evident that western blots could be used to characterize ADH from different species of Drosophila. Mutants induced in Drosophila spermatozoa at the alcohol dehydrogenase (Adh) locus using X-rays, 1-ethyl-1-nitrosourea (ENU) or ethyl methanesulfonate (EMS) were characterized using genetic complementation tests, western blots, Southern blots, northern blots and enzymatic amplification of the Adh locus. Genetic complementation tests showed that 22/30 X-ray induced mutants, and 3/13 ENU and EMS induced mutants were multi-locus deficiencies. Western blot analysis of the intragenic mutations showed that 4/7 X-ray induced mutants produced detectable polypeptides, one of which was normal in molecular weight and charge. In contrast 8/10 intragenic ENU and EMS induced mutants produced normal polypeptides. Southern blot analysis showed that 5/7 intragenic X-ray induced mutants and all 10 of the intragenic ENU and EMS induced mutants were normal with respect to the alleles they were derived from. These data indicated that X-rays primarily induced large deletions, while the alkylating agents ENU and EMS predominantly induced missense mutations. Therefore the Adh forward mutation assay recovered a broad spectrum of mutations ranging from missense to multi-locus deficiencies. Enzymatic amplification of the Adh locus using the polymerase chain reaction (PCR) procedure provided further evidence for classification of mutants, as missense, deletions or intragenic chromosomal rearrangements. Enzymatic amplification of Adh alleles represents a specific, rapid method to analyze newly induced mutations. Only 3 of 43 mutations produced polypeptides detectable as electrophoretic variants. Therefore it is evident that screening heterozygotes for electrophoretic variant polypeptides is inefficient. Most mutants that alter phenotype would either make a polypeptide that comigrates with the normal allele or not produce a detectable polypeptide.

Pages

110

DOI

10.31390/gradschool_disstheses.4613

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