The proposed research will continue our efforts to define the genetic elements which specify the normal assembly of the myofilament lattice during muscle development in nematode Caenorhabditis elegans. Our studies will use a combination of molecular and genetic methods to attack the problem at three different levels. Mutants of the myosin heavy chain gene, unc-54, and the actin genes, act-1, -2 and -3 will be investigated in detail. Specific mutant myosin genes affecting assembly or contraction will be analyzed to determine domains of myosin important in assembly and contraction. Deletions removing various members of the three clustered actin genes will be recovered and used to discover the roles of the three different actins in muscle development. At an intermediate level attempts will continue to associate additional genes with specific muscle components by two different methods: One will use the transposable element Tel as an insertional mutagen to tag genes already defined in previous genetic studies. These Tel induced mutants will subsequently be used to recover DNA for the gene and assign a protein product following methods developed in the study of unc-22. Recombinant phage containing the unc-22 gene have already been recovered this way, and future studies will define the unc-22 function in muscle and explore means for obtaining Tel mutants in other genes. The second means of associating genes with products will begin with known protein constituents of the muscle cell and identify in turn the DNA specifying the polypeptide, the location of that DNA on the genetic map and finally mutants affecting that polypeptide function. At the most general level new genes essential for muscle development will be discovered in screens for recessive lethal mutations. Those mutants with defective muscle will be recognized by the unique terminal phenotypes found associated with known recessive lethal muscle mutations. These genes will be studied further by the variety of techniques developed in the other parts of this proposal. By defining in detail the role of specific genes in muscle assembly, by identifying mutants in additional components and by extending our studies to as yet undefined functions, we will begin to learn the general principles involved in the genetical specification of the muscle structure, not only in Caenorhabditis elegans but in all higher organisms.
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