The long term objective of the proposed research is to understand the mechanisms involved in temporal and tissue-specific gene expression during Drosophila development. We are approaching this problem by studying alcohol dehydrogenase (Adh) gene expression, and the function and expression of nautilus (nau), a presumptive myogenic regulatory gene. The Adh studies will involve characterization of cis-acting regulatory sequences involved in tissue-specific Adh expression in larvae and adults, and the identification of proteins that regulate Adh gene expression. We propose to study the structure, function and expression of proteins that bind specifically to DNA sequences required for normal Adh gene expression in larvae (BBF-1, BBF-2, ABF-1) and adults (AEF-1). The amino acid sequences of these proteins will be deduced from the DNA sequences of the corresponding cDNA clones, and their pattern of expression studied by in situ hybridization and RNAse protection methods. The function of these proteins will be determined by genetic studies, cotransfection experiments in cultured Drosophila cells, and by in vitro transcription analyses. A binding site for the mammalian transcription factor C/EBP has been implicated in the positive control of Adh gene expression in adults. We therefore plan to clone and characterize Drosophila genes which encode proteins that bind to this sequence to determine whether Adh gene regulation involves a functional analog of the mammalian C/EBP gene. If successful, the Adh studies should provide a detailed understanding of the protein-DNA and protein-protein interactions involved in regulating the complex pattern of temporal and tissue-specific expression of Adh. The primary goal of the nau gene project is to understand the regulatory mechanisms involved In the earliest stages of myogenesis in Drosophila. The DNA sequences required for nau expression in muscle precursor cells will be identified in P element transformation experiments, and these sequences will be used to identify genes that control nau gene expression. The latter will be accomplished by screening a lambda gt 11 cDNA expression library for clones that express proteins that bind specifically lo the regulatory sequences. A variety of molecular approaches will be used to determine whether the nau gene is directly involved in the regulation of gene expression in muscle precursor cells. Finally, we plan to identify the targets of nau gene function using the polymerase chain reaction in conjunction with methods to isolate specific DNA-protein complexes. The ultimate objective of the Adh and nau gene studies is to establish a link between the regulatory events involved in the terminal differentiation of specific cell types, and the well-characterized regulatory hierarchy involved in the establishment of specific segments in the early embryo.
Showing the most recent 10 out of 12 publications
