In this application Dr Herman proposes to study genetic mechanisms important in development, broadly defined. The experiments fall into three only loosely related groups. The first and largest group of experiments concern mec-8. mec-8 single mutants have three phenotypes: impenetrant cold-sensitive embryonic lethality, failure to respond to light touch (this is how the gene was initially identified), and failure of certain certain sensory neurons to take up dye, a phenotype usually associated with structural defects in the sensory endings. Lundquist and Herman showed in addition that mec-8 has a synthetic lethal interaction with certain weak mutations in the perlecan gene, unc-52. They cloned mec-8 and found that it encodes an RNA- recognition motif (RRM)-containing protein. Rogalski and Moerman have shown that unc-52 is alternatively spliced, and have evidence that the distribution of spliced products is changed in a mec-8 mutant. There is less complete evidence from the Moerman and Chalfie laboratories that MEC-8 is also necessary for alternative splicing of the mec-8 and mec-2 gene products. Dr Herman proposes that MEC-8 is necessary for the production of certain specific spliced products of certain target genes. In the absence of MEC-8, particular transcripts are not made, leading to the mutant phenotypes. Different phenes (Let, Dyf, and Mec) result from the absence of products of different target genes. For instance, MEC-8 may be necessary for the production of unc-52 mRNAs that lack exon 18. The cold-sensitive lethality might result from failure to make certain exon 18-lacking products that are important in embryogenesis. The lethality of mec-8; unc-52(weak) double mutants is explained by the observation that these weak mutations cause chain termination in exon 18: without the ability to skip exon 18, all unc-52 mRNAs are non-functional. To test this hypothesis, Dr Herman proposes to use PCR and anti-UNC-52 antibodies to find out what products are produced from unc-52 in wild- type and mec-8 mutant backgrounds. In addition, they will engineer an unc-52 gene that should bypass the synthetic lethality if the hypothesis is correct, and they will build reporter constructs that should express functional reporter protein only if MEC-8-dependent splicing occurs. Mutagenesis of the reporter constructs will define cis sites of MEC-8 action. A similar analysis will be done for mec-8, and existing mec-8 mutations will be defined by sequencing. Finally, immunocytochemistry and mosaic analysis will be used to define the focus of mec-8 action-- the hypothesis predicts that it will have distinct autonomous foci for each phene. Further experiments will attempt to identify mec-8 targets for specific phenes by isolating mutations in the target genes that cause MEC-8- independent expression of the normally MEC-8-dependent product--these will apear as dominant suppressors of specific MEC-8 phenes. In addition, they will screen for other genes involved in control of alternative splicing by looking for suppressors or enhancers of mec-8. Two genes, smu-1 and smu-2, have already been defined by suppressor mutations. These will be cloned. lin-44 was identified by Michael Herman and H Robert Horvitz on the basis of mutations that reversed the polarity of certain cell divisions in the tail. As a postdoc in Robert Herman's lab, Mike Herman cloned lin-44 and showed that it encodes a Wnt homolog. lin-44 will be studied with two goals, first to find out how it determines the polarity of asymetric divisions, and second to identify new genes important in Wnt signaling.
The final aim of this proposal is more exploratory. Dr Herman proposes to study for a collection of essential genes the effects of loss of gene function in a subset of the animal, i.e., in a genetic mosaic. He contends that lethal arrest phenotypes of essential genes are often uninformative, and that mosaic analysis will provide a clearer picture of essential gene function.
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