The purpose of this research is to learn how committed states of gene expression are attained during metazoan development. A molecular genetic approach will be used to investigate the control mechanisms operating within the bithorax complex (BX-C) of Drosophila melanogaster, a locus involved in determining the identities of several segments of the fly. Previous studies have demonstrated the importance of two types of sequences in regulating BX-C gene expression. The parasegmental control elements (PCE) respond, in part, to cues from segmentation gene products to initiate transcription of BX-C gene expression within precise spatial domains at the blastoderm stage of embryogenesis. Due to the transitory nature of the cues from early segmentation gene products, a second class of sequences is needed to maintain proper expression boundaries at late developmental stages. A current model suggests that the Polycomb-group gene products act at these maintenance sites to nucleate localized changes in chromatin structure. These alterations in chromatin conformation are proposed to repress BX-C gene expression in those regions of the embryo where initiation of transcription did not take place. The role that various segmentation gene products play during the initiation process at PCE's will be addressed by DNA footprinting studies employing segmentation gene products produced in E coli. Protected sites will be mutagenized in vitro and tested for in vivo function by P element mediated transformation experiments. Additional factors which bind to PCE's will be identified by DNA footprinting experiments using embryonic nuclear extracts. P element mediated transformation and reporter gene fusion experiments will also be used to identify a minimal maintenance sequence. The assay will employ a PCE that lacks a maintenance site. Various BX-C DNA fragments will be inserted next to the PCE and tested for their ability to stabilize proper expression boundaries at late developmental stages. A similar strategy will L be used to see how abundant maintenance sites are within the Drosophila genome. A P element containing the yellow gene under control of a minimal PCE will be transposed to numerous chromosomal locations. Insertions which land near Polycomb-group response elements will be identified by virtue of a particular type of y+ mosaic pattern. The final set of experiments will be to provide a genetic and molecular description of a new gene, enlarged halteres, which appears to act as a positive regulator of the BX-C gene Ubx. These studies will contribute to our understanding of the molecular mechanisms that are involved in the determination of cell fate and may suggest models by which aberrant forms of cell determination lead to birth defects and cancer.
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