The Alpha-amylase system of Drosophila melanogaster is being studied as a model for the analysis of genetic regulatory mechanisms in a higher eukaryote. Genetic elements that control the temporal and spatial expression of the structural gene for amylase, Amy, during development are being sought. The system is complex, including cis and trans acting regulatory loci. Emphasis will be placed on a trans control locus, map, which controls Amy expression in the posterior midgut at the level of translatable amylase mRNA. The interaction between this tissue-specific regulatory locus and the Amy gene will be analyzed at the molecular level to illucidate the exact control mechanisms by which it regulates Amy expression. Other control elements in the amylase system will also be isolated and analyzed. Understanding of gene regulation is basic to understanding the biology of cell differentiation, genetic disease, and neoplasia.
Specific aims of this program include: (1) continuation of efforts to precisely define the Amy and map loci genetically and cytogenetically; (2) use of a new and efficient selective screen for mutants with low or no amylase activity, or which are overproducers, to isolate Amy and map mutations for use in aim (1) and in structure-function analyses of both loci; (3) study of the relationship between base sequence structure and function at the Amy locus through (a) analysis of mutants isolated by experimental mutagenesis, (b) analysis of chromatin structure with reference to the location of nuclease sensitive and hypersensitive sites in the presence of different Amy and map alleles, and (c) analysis of transformed flies utilizing experimentally modified Amy sequences; (4) analysis of the tissue-specific transcription of Amy structural genes in different stages of development through qualitative and quantitative studies of amylase mRNA, relating the analysis to the presence of different map alleles and levels of amylase activity and translatable amylase message; (5) analysis of the posttranscriptional modification of amylase mRNA in the presence of different Amy and map alleles; (6) an attempt to isolate, clone and sequence the map locus and its flanking regions; (7) identification of other elements in the amylase control system which will be defined genetically, cytogenetically and molecularly; and (8) identification of the map control signal. The methodology used to achieve these aims will include the tools of genetic engineering at two levels, that of the whole fly (genetic, cytogenetic) and that of the base sequences, utilizing the tools of gene splicing, cloning and transformation.
