The aim of this research is to understand how spatial regulation of gene expression is achieved and to hereby learn how networks of interacting genes form, communicate, and maintain themselves during development. A molecular biological and genetic approach is to be used for investigating the control mechanisms operating within the bithorax complex of Drosophila melanogaster. Specifically, we will use element mediated transformation, and a novel scheme for constructing and altering large portions of the complex, to identify and assess the importance of particular regions and products to the overall function of the complex. Regulatory sequences will be identified by fusions of putative control elements to a hybrid Ubx-lacZ reporter gene. The expression patterns of the fusion product will be analyzed by immunohistological staining of whole mount embryos and various tissues such as imaginal discs. Initially, we will study fusions of those regions which have been identified by mutation to affect the spatial regulation of the Ubx product. Subsequently we will examine other regions for which no mutations have yet been identified in order to test the notion that much of the large size of the complex is due to very large regulatory units. Once the general picture had emerged by transforming in large fragments of DNA, we will begin to focus on important elements by dissecting relatively small regions which faithfully reproduce some aspect of the wild type pattern. To elucidate the molecular architecture within the fragment we will use several approaches, including sequence comparison to an equivalent region from the species D. virilism, as well as DNA footprinting and in vitro mutagenesis of conserved and protected sites. To understand how and if these sites tie into the network of the early acting gap, segmentation, and segment polarity genes, and the later acting maintenance genes of the Pc and Pc-like classes, we will examine changes in the pattern of expression of various fusion protein constructs introduced into backgrounds mutant for these genes. Because all developmental processes ultimately lead to the specification of individual tissues, understanding the regulation of the bithorax complex should yield general insights into developmental mechanisms.
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