Access Type

Open Access Thesis

Date of Award

January 2013

Degree Type

Thesis

Degree Name

M.S.

Department

Biochemistry and Molecular Biology

First Advisor

Russell L. Finley

Abstract

Cyclins are proteins that bind to Cyclin-dependent kinases, or Cdks, through a conserved domain called the Cyclin Box. Many Cyclins regulate the cell cycle. A few Cyclins impact cellular processes outside of the cell cycle. Also, a few Cyclins have poorly understood functions.

Cyclin J is a member of the Cyclin superfamily of proteins. Cyclin J is conserved among all metazoans, but is presently not well understood. All the research done on Cyclin J has been done in Drosophila.

Its mRNA is present in the early embryo, then disappears, only to reappear in adult females. When probing protein extracts with antibodies, Cyclin J can be seen in unfertilized oocytes and embryos for the first few hours following fertilization. Immunoprecipitating Cyclin J from unfertilized oocytes and early embryos, Cdk2 co-immunoprecipitates. The same assay co-immunoprecipitates Cdk1 in unfertilized eggs only. Another group has observed very different results in regard to Cdk interaction. They observe Cyclin J to co-immunoprecipitate with Cdk1 and not to interact with Cdk2 in whole ovaries.

This project has one specific aim. It is to identify and test for biologically relevant Cyclin J protein-protein interactions (PPIs). I am using approaches that involve two assays to test PPIs. The assays I am employing are the yeast two-hybrid assay (Y2H) and co-affinity purification (CoAP). When a PPI is detected using two different assays, for example Y2H and CoAP, it is more likely to be a true positive. Orthologs of Drosophila Cyclin J's PPIs will also be tested. I am comparing Drosophila, mosquito, and human orthologs of PPIs. A PPI is also more likely to be a true positive when the PPI is conserved between more than one species.

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