Characterizing Cyclin J By Identifying Conserved Protein-Protein Interactions

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\u27s 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