Molecular cloning of a glutathione S-transferase overproduced in an insecticide-resistant strain of the housefly (Musca domestica).

We report the cloning and sequencing of a glutathione S-transferase (GST) gene from the housefly Musca domestica. A cDNA lambda gt11 library was prepared from the organophosphate insecticide-resistant housefly strain Cornell-R--a variant that has elevated GST activity. The lambda phage GST clone was identified on the basis of its ability to cross-hybridize to a GST DNA probe from Drosophila melanogaster. Based on amino acid homology to other GSTs and expression of GST activity in Escherichia coli, the Musca GST gene (MdGST-1) belongs to the GST gene family. Although organophosphate resistance in Cornell-R is largely due to one of the GSTs, MdGST-1 is probably not the enzyme responsible for resistance. The mutation that controls resistance to organophosphate insecticides in Cornell-R is highly unstable and we isolated spontaneous variants to both insecticide sensitivity and to even higher levels of resistance. This provided us with an isogenic set of three strains. We found that MdGST-1 transcript levels as measured by Northern assays are higher in all three Cornell-R strains relative to the sensitive wild type, but that the sensitive Cornell-R strain has more MdGST-1 transcript than does the highly resistant Cornell-R strain. These data as well as Southern analysis of genomic DNA allow us to conclude: (1) there are multiple GST genes in M. domestica; (2) the natural variant Cornell-R overproduces excess transcript from two and probably more of these genes; and (3) the unstable mutation in Cornell-R influences the levels of multiple GSTs.

Loss of electrophoretic variation in serially bottlenecked populations.

Experimental bottleneck lines were generated from a single outbred population of the housefly using one, four, or 16 pairs of flies. These lines were followed through one, three, or five successive founder-flush cycles. After each flush 30 individuals from each line were assayed for electrophoretic variation at four initially polymorphic loci. Electrophoretic variation largely followed neutral expectation, the major exception being that the differentiation among 16-pair lines was greater than expected. All bottleneck sizes showed significant losses in genetic variation compared to the control, particularly for losses in numbers of alleles. These findings contrast theoretical predictions in that few lines and loci would be required to detect historical bottlenecks.

Temporal control of transposition in Tn5.

IS50R is an insertion sequence associated with the transposon Tn5. IS50R carries the structural genes for two proteins; one (P1) is the Tn5 transposase, and the other (P2) is an inhibitor of transposition. These two proteins are translated from two different transcripts, m1 and m2. When bacteriophage lambda::IS50R DNA was introduced into a bacterial cell, m1 and m2 were initially at relative levels of about 1 to 2. As time progressed the amount of m1 fell, whereas the amount of m2 continued to increase, until after about 3 h the ratio of m1 to m2 was about 1 to 80. The temporal changes in the levels of these transcripts correlated with temporal changes in P1 and P2 levels and Tn5 transposition that have been documented in other studies. We measured the stability of the messages and showed that the differences in the levels of m1 and m2 must reflect real differences in the strengths of their promoters and that the changes in transcription kinetics are mediated by the dam methylation system of the cell and are not determined by IS50R products. Our results show that the 5' end of m2 is about twice as stable as that of m1, which raises the possibility that differential message stability does, in part, influence the ratio of inhibitor to transposase.

The Effect of an Experimental Bottleneck upon Quantitative Genetic Variation in the Housefly.

Effects of a population bottleneck (founder-flush cycle) upon quantitative genetic variation of morphometric traits were examined in replicated experimental lines of the housefly founded with one, four or 16 pairs of flies. Heritability and additive genetic variances for eight morphometric traits generally increased as a result of the bottleneck, but the pattern of increase among bottleneck sizes differed among traits. Principal axes of the additive genetic correlation matrix for the control line yielded two suites of traits, one associated with general body size and another set largely independent of body size. In the former set containing five of the traits, additive genetic variance was greatest in the bottleneck size of four pairs, whereas in the latter set of two traits the largest additive genetic variance occurred in the smallest bottleneck size of one pair. One trait exhibited changes in additive genetic variance intermediate between these two major responses. These results were inconsistent with models of additive effects of alleles within loci or of additive effects among loci. An observed decline in viability measures and body size in the bottleneck lines also indicated that there was nonadditivity of allelic effects for these traits. Several possible nonadditive models were explored that increased additive genetic variance as a result of a bottleneck. These included a model with complete dominance, a model with overdominance and a model incorporating multiplicative epistasis.

Morphometric Differentiation among Experimental Lines of the Housefly in Relation to a Bottleneck.

Differentiation in morphometric traits among experimental populations of the housefly subjected to an experimental bottleneck was examined for replicate lines founded with one, four or 16 pairs of flies. Differentiation among lines within a bottleneck size was significantly greater than predicted by drift in relation to the additive genetic variation for these traits within the founding population. Two models of nonadditive genetic variance were investigated to interpret these results, one involving dominance of allelic effects within loci and another incorporating multiplicative epistasis. Both models generated more variation among lines as a direct result of sampling during the bottleneck than predicted by a model with additive gene action. The pattern of differentiation among our experimental lines in relation to these models conformed more to the model incorporating epistasis. Nevertheless, it may be difficult to distinguish differentiation among lines occurring during a bottleneck as a result of nonadditive gene action from that caused by diversifying selection among lines after the bottleneck.

A taxonomic approach to evaluation of the charge state model using twelve species of sea anemone.

The charge-state model of electrophoretic variation was tested by comparing the distances between nearest electromorphs of five enzyme loci within polymorphic species and among pooled species of sea anemone. If the charge-state model is generally true, and in particular if it allows linear distance between electromorphs to be used as a measure of genetic distance, then electromorphs of different species should be on the same "mobility ladder". Therefore, distances between adjacent electromorphs should be approximately equal for the two sets of comparisons. It was found that distances between adjacent electromorphs for each locus were significantly smaller for the pooled comparisons than within polymorphic species. Thus, it was concluded that much of the variation detected among different species does not conform to the charge-state model, and therefore that distance between electromorphs per se would not be a good measure of genetic distance. However, the charge-state model does appear to adequately account for most of the variation existing as common polymorphisms within species, or between very closely related species. Possible reasons for this apparent difference in the nature of the variation seen within and among species are discussed.