UBC Theses and Dissertations
Developmental studies in Drosopnila melanogaster Holden, Jeanette Jeltje
The study of development and differentiation has been greatly facilitated through the use of mutations which affect this process at various discrete stages. The gross morphological effects produced by the mutations are indicative of their role in development. The conditional expression of, for example, a lethal phenotype, permits an examination of the effects of the mutation at different times during development— even after an initial lethal phase has been passed. In this respect, a temperature-sensitive expression of developmental mutations is a useful property which is subject to relatively simple yet extremely variable experimental procedures. Thus, dominant temperature-sensitive (DTS) lethals were induced in Drosophila in order that the nature, i.e. the molecular basis, of dominant lethal mutations, as well as their genetic and developmental characteristics, could be investigated. It was hoped, also, that loci within heterochromatin might be detectable among such mutations. Ten DTS-lethal mutations on chromosome 3 which are lethal when heterozygous at 29°C but survive at 22°C were recovered. Eight of the mutations were mapped, 7 were tested for complementation; these mutations probably define eight loci. Only DTS-2 survived in homozygous condition at 22°C; homozygous DTS-2 females expressed a maternal effect on embryonic viability. Two of the mutant-bearing chromosomes, DTS-1 and DTS-6, exhibited dominant phenotypes similar to those associated with Minutes. Each of the seven mutants examined exhibited a characteristic pheno-type with respect to the time of death at 29°C and the temperature-sensitive period(s) during development. Only DTS-4 expressed a dominant lethal phenotype in triploid females. In addition to the DTS-lethals, a special class of recessive temperature- sensitive mutations, which are lethal when homozygous or in combination with the multiply-inverted TM2 chromosome, were recovered, Only one combination of the four mutations in this category failed to complement at 22°C whereas all trans-heterozygotes were lethal at 29°C. These were found to be closely linked to one another and to differ only slightly in their developmental characteristics. Together they provide an excellent model system for genetic fine structure analysis. Although such studies provide an indication of the morphological changes occurring during development, and the use of temperature-sensitive mutations gives some suggestion as to the time of action of the gene product, a determination of the precise biochemical defect is not possible short of exceedingly extensive biochemical analyses. Alternatively, then, development may be viewed as a series of precisely regulated changes in the species of macromolecules present within the organism. Thus, differences in the amounts of certain RNAs during the cell cycle or with specific developmental stages provide a measure of transcriptional changes. Since tRNAs represent primary gene products which can be readily analyzed qualitatively and quantitatively, their role(s) in development may be indicated by an absence or reduced amount of a specific tRNA species. The extensive array of mutations available in Drosophila provides the material from which tRNA mutations might be detected. However, it is first essential to establish a pattern for the synthesis of specific isoaccepting tRNAs during the development of wild-type Drosophila so that a comparison can be made with those from various mutants. The reversed-phase chromatographic system (RPC-5) was used to compare the 20 aminoacyl-tRNAs from first instar, third instar, and adult flies. While some of the aminoacyl-tRNAs remain essentially unchanged throughout these stages, others show marked quantitative changes. One group of tRNAs, including tRNA[sup Asp], tRNA[sup Asn], tRNA[sup His], and tRNA[sup Tyr], showed similar differences in the relative proportions of certain chromatographically distinct (i.e. isoaccepting) forms. In addition, these four tRNAs show a further quantitative change in flies of the su(s)² v; bw genotype. Two-dimensional thin-layer chromatograms of pancreatic RNase digests of the two major tRNA[sup Asp] chromatographic forms revealed almost identical patterns whereas such chromatograms of piperidine hydrolysates revealed a difference of one altered nucleotide. It is suggested that these chromatographic forms are homogeneic (i.e. they are transcribed from the same gene) but that they differ only in the extent of the modification of a single nucleotide. The su(s)²v; bw mutant would, then, appear to be defective in some aspect of this modification process. Possible models describing the activity of the modification enzyme are discussed.
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