Evolution of Dosage Compensation for Amylase in Drosophila

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Sex-linked dosage compensation is a mechanism of gene regulation that results in the level of X-linked gene product being equal in both sexes, although males have a single active gene copy for the product while females have two. In Drosophila miranda, the structural gene for alpha-amylase, Amy, is sex- linked and located in a relatively "new" X chromosome, X2, which evolved in response to an ancestral translocation of one arm of an autosome to the Y chromosome. The unexpressed homolog of Amy is in the "neo-Y" chromosome of males. Male and female larvae express equivalent levels of amylase activity, suggesting full dosage compensation at this developmental stage. By contrast, the compensatory mechanism appears to have been, at best, only partially evolved in adults. Since Amy is not sex-linked in very closely related Drosophila species - pseudoobscura and persimilis, this group of three sibling species provides an opportunity to study evolution of a eukaryotic gene regulatory mechanism. The ultimate goal is to understand the molecular basis of dosage compensation for amylase in D. miranda and how it may have evolved through DNA sequence changes. Doane and Norman have cloned and partially characterized the Amy region of miranda and isolated a clone that contains the functional Amy gene from its X2 chromosome. They also have begun to characterize dosage compensation of Amy expression at the level of amylase messenger RNA. The aims of this proposal are to: (1) complete the amylase RNA studies and assay dosage compensation at the RNA level in male midguts during development; (2) clone and characterize the autosomal Amy region of a selected strain of D. persimilis; (3) sequence and compare the functional Amy gene and a putative Amy pseudogene from miranda and compare the former sequence with the functional Amy sequence of persimilis; (4) attempt to clone and characterize Amy-homologous sequences from the neo-Y chromosome of miranda; (5) establish a radiation-induced Amy null (no amylase) strain of miranda to provide hosts for Amy gene transformation experiments; and (6) delineate the regulatory sequences that are important in dosage compensation of Amy expression in males of miranda through transformation experiments coupled with the deletion or alteration of specific DNA sequences adjacent to the amylase coding region. Transformation experiments, intra- and interspecific, will include both transient expression assays ( somatic transformations) and P. element-induced germline transformations, both of which entail the injection of DNA sequences to be tested in Amy null host embryos..Z. The significance of this basic research lies in the limited understanding of gene regulatory mechanisms in higher animals. This is especially true of mechanisms like Drosophila dosage compensation which augments activity of an already transcriptionally active gene. The opportunity provided by the amylase system is unique in that the compensatory mechanism is not yet fully evolved, so an intermediate stage in DNA sequence reorganization leading to it may be analyzed. Information about this should add significantly to our understanding of how gene regulatory mechanisms may evolve and how important they may be to the process of evolution. Such knowledge would be of potential value in the application of genetic engineering techniques to species in which it would be useful to alter the expression of a given gene for beneficial or economic reasons, or for experimental purposes in population studies.