The proposed project analyzes gene regulation during the early development of C. elegans. There are three main parts to the project. First, techniques, methodologies, and tools for the analysis of gene expression will be developed. Second, cell type-specific promoters are analyzed and the factors that regulate them are identified. Third, the asymmetries in gene expression in the early lineage will be identified. Dr. Fire proposes to continue his development of technology for analysis of gene expression. In the previous project period an in situ hybridization protocol was developed for embryos. This will be optimized for handling small numbers of animals. The widely used set of expression vectors will be expanded to include more targeting signals. The utility of lacZ reporter genes will be increased in two ways. First, the differences between good and bad expressing constructs with respect to apparent mosaic expression will be determined. Second, germline expression of lacZ reporter constructs will be obtained by trying different lacZ genes and variants of the E. coli gene with, for example, synthetic introns. Lastly, an attempt to obtain targeted gene disruption by homologous recombination will be made. Previous work on this project identified several candidates for myogenic regulators, hlh-1 (by molecular cloning in another laboratory), cey-1 (by cloning a protein that binds the unc-54 body wall myosin gene enhancer), mylVL (by a deficiency screen), and ceh-22 (by cloning a protein that binds the myo-2 pharyngeal myosin gene enhancer). These proteins will be analyzed in parallel to define their roles in regulating both body wall and pharyngeal muscle gene expression. For each, unless already done, both loss-of-function mutants will be analyzed and the effects of ectopic expression determined. Target genes will be identified. For each factor, its regulation will be studied. In the cases, such as cey-22, in which the factor itself is regulated, another round of cis- regulatory sequence analysis and factor identification will be carried out. Additional regulators involved in myogenesis will be identified by four types of screens. One method seeks nematode sequences that confer desired expression pattern on a lacZ reporter construct. A second method is screening of an expression library with DNA probes. This will be done with the pharynx-specificity element (C) of myo-2 enhancer, body wall and hypodermal specific element (site II) of unc-54 enhancer, and one of the posterior-muscle elements of hlh-1 control region. A third method is screening additional deficiency homozygotes for myogenic defects. Lastly, a screen for ectopic expression effects will be attempted. In this screen, random cDNAs will be expressed under a heat shock or hlh-1 promoter and animals stained for defects in myosin expression. Early asymmetries in gene expression will be analyzed. The pes-10 gene, identified as a gene expressed in 3 of 4 blastomeres at the four-cell stage, will include genetic analysis. A second asymmetry is the stability of the cey-2 mRNA, which is degraded in somatic cell lineages. A control will be the dpy-30 mRNA. These observations will be followed by genetic screens for mutations that fail to exhibit the asymmetry in pes-10 transcription or cey-2 mRNA degradation.
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