Research Project
3290047
The purpose of this proposal is to understand the genetic regulation of the expression of specific genes in individual cell lineages during development. In the first part, we describe experiments to improve the DNA transformation system of C. elegans. These experiments include selecting for drug resistant transformants, stable transformants, and transformants that have integrated the exogenous DNA. They also include developing new methods for assaying lineage specific expression. In the second part of the proposal, we describe the construction of several DNA vectors for assaying expression. These constructs involve fusions of developmentally regulated genes with the E. coli gene encoding beta-glucuronidase. The developmental genes are derived from the nematode Caenorhabditis elegans. The bacterial enzyme is used as the indicator of expression because extremely sensitive colorimetric and fluorimetric assays are available. We will transfer the gene fusions into the nematodes by DNA mediated transformation. Using sensitive histochemical methods, we will assay expression of the gene fusions in specific cell lineages at different developmental stages. In this manner, we will establish the coordination of the expression of the different genes with their lineage histories. By manipulating the DNA, we will identify the sequences essential for lineage specificity. We will also transform mutants with abnormal cleavage patterns in order to assess the degree of dependency on cell organization. We will search for mutant nematodes that fail to regulate the transformed DNA either temporally or spatially on the assumption that such mutations represent regulators of lineage specific expression. In the third part of the proposal, we use DNA transformation to facilitate isolation of genes with central roles in the early development of C. elegans. We will construct fusions of these genes with beta-glucuronidase in order to study the regulated expression of these genes during normal and mutant development. We also propose to study the distribution of the gene products during early development.
