Isolation and characterization of a Caenorhabditis elegans SRC loss-of-function allele using reverse genetics

The vertebrate proto-oncogene Src is a protein-tyrosine kinase that has been implicated as a component of receptor-mediated signal transduction pathways important for cell growth and differentiation. Consistent with this notion, overexpression or activation of Src by mutation induces neoplastic transformation in cell culture, leads to tumorigenesis in laboratory animals, and has been observed in a number of human tumors. Despite years of intensive investigation, neither its role in oncogenesis nor its normal, biological role is understood. To diminish the issue of redundancy that has complicated analysis of Src function in vertebrates and Drosophila, I have chosen to study Src function in the genetically less complex nematode Caenorhabditis elegans. The gene src-1 encodes a protein with significant structural and sequence identity to vertebrate c-Src. The first part of this dissertation describes a set of biochemical experiments supporting the hypothesis that SRC-1 is the ortholog of vertebrate pp60c-src. The second part of this dissertation describes the isolation and characterization of a src-1 loss-of-function allele using a reverse genetic approach. src-1(cj293) is a deletion allele generated by N-ethyl-N-nitrosourea mutagenesis that encodes a truncated, kinase-inactive protein. It is a recessive maternal effect lethal allele that interacts genetically with components of a conserved Wnt signaling pathway providing the first direct connection between two intensively studied vertebrate signaling pathways: Src and Wnt. The genetic interactions identified in combination with the embryonic defects observed in src-1( cj293) animals suggest that SRC-1 normally plays a role in regulating spindle orientation, morphogenesis, and cell fate decisions of developing C. elegans embryos. This study lays the ground work for additional genetic and biochemical analyses that will help define the molecular details of SRC-1 signaling. Such experiments will shed light not only on the biological role SRC-1 plays in C. elegans development, but also the biological role of Src-family kinases in general. Ultimately, these analyses may provide insight into the mode of action of oncogenic Src mutants