Mutagenesis in Drosophila Melanogaster. I. Scheduled Synthesis of Nuclear and Mitochondrial Dna and Unscheduled Dna Synthesis. II. Dosimetry Studies Using Phosphorus-33. IIi. Molecular Analysis of Induced Mutations at the Adh Locus (Alcohol Dehydrogenase, Restriction, Cloning, Southern, Blotting).

Abstract

Presented here are three distinct, but interwoven areas of mutation research and molecular genetics using Drosophila melanogaster as the test organism. The first section describes the characterization of the DNA replication process and DNA repair in the oocyte. This involved sex-linked recessive lethal tests on ethyl methanesulfonate (EMS) treated females and unscheduled DNA synthesis (UDS) studies involving EMS, methyl methanesulfonate (MMS) and N-ethyl-N-nitrosourea (ENU). Oocytes were positive for UDS with EMS and MMS in a DNA repair competent strain and negative in excision repair deficient strains. ENU was negative in a DNA repair competent strain. Since UDS is not quantitative the development of a molecular dosimetry assay for oocytes is a necessity. The second section describes equilibrium labeling of Drosophila with ('33)P. This technique relies on the theory that Drosophila, living their life on media with ('33)P, will reach an equilibrium between the ('33)P and unlabeled phosphorus in the media, leading to a uniform labeling of the DNA. This has been achieved and may eventually replace (('14)C-dT) deoxythymidine as a DNA label. Analysis of the types of mutations makes up the third section and involves the study of x-ray induced mutations at the Alcohol dehydrogenase (Adh) locus. Only two of the eight mutants (25%) analyzed here or two out of the original 31 (6%) induced mutants produced a detectable protein using the O'Farrell two-dimensional gel technique. Restriction endonuclease and Southern blot studies showed five out of the eight (63%) of the mutants previously determined not to be large deletions had an altered DNA restriction pattern, including one spontaneous mutant. These alterations were all outside of the Adh structural gene region. Only one of these produced a detectable ADH protein. Of the three normal appearing mutants after restriction endonuclease and Southern blot analysis only one made a detectable ADH protein. All of the mutants made a normal sized mature mRNA which hybridized to the genomic probe. These alterations in the five mutants not producing a detectable ADH protein may be due to very small deletions resulting in frameshifts.