Date of Award

Fall 2005

Project Type

Dissertation

Program or Major

Genetics

Degree Name

Doctor of Philosophy

First Advisor

John Collins

Abstract

The vertebrate proto-oncogene, Src, is the prototype of a family of membrane-associated protein tyrosine kinases involved in cell signaling pathways that control cell growth, development, and differentiation. Activation of Src by mutation or overexpression has been implicated in a range of cancers, particularly breast and colon cancer. Inappropriate activation of Src has pronounced oncogenic effects on cell morphology, adhesion, and motility. Despite its implications in cancer, the normal biological role of Src is not well understood.

I used the nematode C. elegans to investigate the expression, function, and evolution of two src genes (src-1 and src-2) utilizing a combination of genetic, reverse genetic, molecular, biochemical, and proteomic approaches. A previously characterized src-1 knockout confers a maternal effect lethal phenotype. Arrested embryos exhibit abnormalities suggesting that SRC-1 plays an essential role in cell fate decisions, spindle orientation, and morphogenesis in the early embryo. src-2 mutant animals appeared wild type under all conditions examined. The src-1 src-2 double knockout phenotype was indistinguishable from the src-1 single mutant, suggesting that src-1 has a unique, essential embryonic function.

Experiments using in-situ hybridization revealed that src-1 (but not src-2) is present in the meiotic region of the hermaphroditic gonad, but that mRNA transcripts from both genes are present in the embryo. Real-time RT-PCR and expression data from a SRC-1::GFP reporter construct showed that src-1 and src-2 expression was widespread in all larval stages, with the src-1 transcript being detected at considerably higher levels than src-2 .

In addition to the work described above, I also performed a pilot study examining the evolutionary history of this important gene family. Phylogenetic comparisons revealed that vertebrate Src and Fyn group together, suggesting that they share a common ancestor with one of the two invertebrate SFK. Finally, I developed a novel proteomic approach to identify targets of Src tyrosine kinase signaling as part of a long term goal to understand the pathways and biological role of SFKs in metazoans. Collectively, this research contributes to a better understanding of the normal expression and function of SFKs and provides the groundwork for future delineation of the pathways and biological outcomes of Src signaling.

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