UBC Theses and Dissertations
A genetic and biochemical study of a temperature-sensitive vermilion mutation in Drosophila melanogaster Camfield, Robert Graeme
The sex-linked vermilion (v) locus is probably the structural gene for the enzyme tryptophan pyrrolase. Mutations at the locus invariably are recessive and result in a bright-red eye colour phenotype accompanied by a loss of tryptophan pyrrolase activity. Extensive genetic, biochemical and developmental studies of v mutations have shown that the gene is a relatively small cistron controlling the catalytic activity of tryptophan pyrrolase which gives rise to kynure-nine, a brown eye pigment precursor, in the larval fat body during a defined developmental period. Alleles of the locus can be broadly grouped into two classes: 1) spontaneous v mutations, the majority of which are suppressible by mutation at the non-allelic suppressor of sable [su(s)] locus, 2) induced v mutations which are all unsuppressible by su(s) alleles. Alleles of both classes behave nonautonomously during development and all map within the definable limits of the v cistron. This investigation was initiated to recover conditional (temperature-sensitive) v alleles which could be used to study further the regulation of the activity of the v gene during development, and to extend our knowledge of the genetic functioning of the locus. A temperature-sensitive (ts) allele of a known structural gene, affecting the catalytic activity of an assayable enzyme, could also enable a determination of the factors responsible for temperature-sensitivity in Drosophila in terms of changes in the gene product. The temperature-sensitive period (TSP) of a ts mutant in Drosophila is defined as that period during development when exposure to the restrictive temperature commits the organism to a mutant phenotype. With a ts v allele, a correlation can be made between the TSP determined phenotypically and the variation in tryptophan pyrrolase activity during development, and thus contribute to a molecular understanding of the TSP. This study has consisted mainly of the following approaches: 1) mutagenesis and genetic screening to recover ts v alleles, 2) an examination of the phenogenetics of one ts v allele, including fine structure mapping, complementation properties, nonautonomous expression in gynandromorphs, and suppressibility, and a comparison of these properties with those exhibited by some non-ts v mutations, 3) a biochemical analysis of the effect of a ts v mutation on the properties of tryptophan pyrrolase, a deter-mination of the TSP of a ts v allele based on the eye phenotype. Both ts v alleles, v[sup ts1] and v[sup ts2], recovered in this investigation cause a vermilion phenotype if v flies are raised at the restrictive temperature (29°C), whereas v[sup ts] flies raised at the permissive temperature (17° or 22°C) have almost normal eye colour. The activity of tryptophan pyrrolase, extracted from [sup ts1] flies raised at 29°G and 22°C respectively, parallels the temperature-dependent phenotypic properties; enzyme activity is markedly reduced in flies raised at 29°C but is almost normal in flies raised at the permissive temperature. The v[sup ts1] mutation behaves like non-ts,induced v alleles at 29°C in its complementation, suppressibility and nonautonomy. Thus, it fails to complement any other v point mutant, is unsuppressible by su(s)² and is developmentally nonautonomous when present with v* tissue in gynandromorphs raised at 29°C. Since the v[sup ts1] allele is viable when heterozygous with deletions removing the v locus and maps within the v cistron as a point, it is assumed to be a point mutation in the v structural gene. Furthermore, the tryptophan pyrrolase controlled by the v[sup ts1] mutant has different in vitro kinetic and temperature-dependent properties when v[sup ts1] flies are raised at 29 C compared to either wild type or tryptophan pyrrolase extracted from v[sup ts1] flies raised at 22°C. The v[sup ts1] mutant demonstrates different phenotypic and enzyme properties between males and females raised at 29°C; hemizygous males are more mutant in phenotype and have lower tryptophan pyrrolase activity than their homozygous sibs. This result apparently is the reverse of the dosage compensation nor-mally demonstrated by wild type tryptophan pyrrolase in which males with one dose of the v⁺ gene have at least the enzyme activity obtained from females with two doses of the v⁺ gene. How-ever, the TSP for the v[sup ts1] mutant is the same for males and females and falls between the early third instar larva and early pupa stages of development. This period corresponds to the maximum pre-adult activity of tryptophan pyrrolase and also correlates with the formation of kynurenine in the cells of the fat bffidy. These results are discussed in relation to a molecular model explaining the genetic and molecular functioning of the v locus during development. The results are consistent with the hypothesis that v[sup ts] and nonconditional v mutations affect different aspects of active tryptophan pyrrolase structure rather than regulation of the rate of synthesis of the enzyme. Thus, suppressible v mutations affect allosteric or regulatory sites of the enzyme which interact with metabolic and develop-mental cofactors, whereas the nonconditional, unsuppressible, induced v mutations probably affect the catalytic sites of t tryptophan pyrrolase. The ts v mutation, v[sup ts1] , has genetic and biochemical properties which are compatible with an effect on the aggregation of enzyme subunits due to conformational changes during enzyme synthesis at the restrictive temperature.
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