Specification of pharynx precursors in Caenorhabditis elegans depends on pha-4, a transcription factor characterized by a winged-helix DNA binding domain shared with the human HNF-3 and fly Fork head proteins. In pha-4 mutants, pharynx cells are transformed into ectoderm. While the DNA binding domain of HNF-3 proteins has been well characterized, relatively little is known about other functional domains. Two classes of pha-4 mutations will be characterized to identify important domains. Class I consists of nonsense mutations that truncate the pha-4 carboxyl terminus to varying extents. Analysis of these mutations indicates that the carboxyl terminus of pha-4 is not essential for viability, but may play a regulatory role. Class II is a semi-conservative missense mutation in the pha-4 amino terminus, suggesting a functional role for the amino terminus. To define the roles of the pha-4 amino and carboxyl termini, pha-4 mutants will be subjected to i) PHA-4 antibody staining to determine if mutant protein is made and properly localized, both in the pharynx and subcellularly; DNA binding assays will also be performed to determine the integrity of mutant proteins; ii) microscopy to determine if different pharynx cell types are made; iii) antibody staining to see if ectodermal genes are repressed in cells that would normally become pharynx; iv) genetic screens to identify new genes involved in pharynx development. First, the lethal Class II allele will be used to screen for second site suppressors that allow homozygous pha-4 mutants to live. A second screen will utilize a viable class I allele as a sensitized background to search for lethal, pharynx-less mutants. Our analyses will elucidate the function of pha-4 in C. elegans and may identify new pharynx regulators.
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