C. elegans is a small free nematode which is studied both as a model for development in higher organisms and in order to understand related parasitic nematodes. We have developed gene transfer technology for C. elegans and initiated a study of muscle cell development. The gene transfer techniques developed include selections for both integrative and extrachromosomal transformation as well as assays for transient expression. We also present preliminary analyses of two homologous integration events. The transformation assays have been used to define signals required for expression of the major myosin gene (expressed in muscle in the body wall of the animal). A set of beta-galactosidase fusion vectors with nematode expression signals have been developed for identifying signals that regulate gene expression, and these have been used to define tissue specific promoter and enhancer segments in the major myosin gene and two minor myosin genes. The conclusion from these studies is that the genes are controlled positively by elements which can activate expression in a single muscle type. For two of the genes we have found multiple positive elements independently capable of activating expression in the correct set of cells. The research in the laboratory will continue to focus on three general areas: 1) Continued development of DNA transformation technology: This part of the project will be directed toward a) the development of marker systems for detecting and localizing gene expression in live animals, b) techniques for finding and analyzing regulatory elements such as enhancers in the genome, and c) developing generally applicable techniques for homologous gene replacement. 2) Analysis of general aspects of gene expression in C. elegans: The structure and function of general G. elegans signals for transcription and RNA processing will be investigated using transformation and biochemical assays. Understanding of these elements should prove useful both in understanding the molecular genetics of C. elegans and in studies of other nematode species. 3) Events leading to the determination of muscle cell fate: our eventual goal is to understand how a reproducible subset of cells in the embryo is chosen to become muscles. In this period we plan to identify the trans- activating regulatory proteins controlling myosin synthesis, clone and characterize the corresponding genes and determine the basis for the tissue specificity of the trans-activation. In addition, we will use genetic screens in order to identify genes required at various stages in the developmental pathway producing the distinct pattern of muscle cells.
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