East Carolina University : Research and Creative Achievement Week 2011
Expression of TLRR (lrrc67) and Protein Phosphatase-1 Isoforms as Fluorescent Fusion Proteins in Mammalian Cells, Caitlin Trumbo1, Rong Wang2, Ann O. Sperry2 Departments of Chemistry and 2 Anatomy and Cell Biology – Brody School of Medicine, East Carolina University, Greenville, NC 1
TLRR or testis leucine rich repeat (also known as Irrc67) has been recently identified in our laboratory and demonstrated to be primarily expressed in the testis associated with the spermatid cytoskeleton, in particular the microtubule manchette and the centrosome. TLRR contains 4 leucine rich repeats and a protein phosphatase-1 (PP1) docking site. These leucine rich repeats likely mediate protein-protein interaction. Our previous studies have shown that TLRR and PP1 reside in the same protein complex in the testis and that TLRR interacts with the testis specific isoform of PP1, PP1³2, which is required for successful spermatogenesis. TLRR may participate in regulating the phosphorylation state of proteins near the spermatid nucleus by localizing PP1 isoforms (PP1±, PP1³1, and PP1³2) to the manchette that encircles the developing spermatid nucleus. In addition, TLRR is expressed in cultured cells and localized to the centrosome in several cell types . This is consistent with our localization of this protein at the centrosome in spermatids and suggests a role for TLRR in biogenesis of cilia and flagella. We will test our hypothesis that TLRR and PP1 interact, with co localization studies of TLRR and PP1 (PP1±, PP1³1, and PP1³2) using fluorescent fusion proteins in cultured mammalian cells. We propose that TLRR is a regulatory subunit of PP1 in developing male germ cells and localizes this enzyme to the manchette and centrosome and therefore these two proteins will associate with one another in cultured cells.
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Using p-element excisions to determine the role of Ctf4 in Drosophila Melanogaster, Tabitha M. Reel, Tim W. Christensen, Department of Biology, East Carolina University, Greenville, NC 27858 DNA replication is essential process for all living organisms. DNA replication begins at origins with the unwinding of the antiparallel DNA strands. The complement of each strand is synthesized via polymerases. Various complexes are associated with DNA replication to ensure high fidelity such as Replisome Progression Complex and Fork Protection Complex. A central component of those two complexes is Ctf4. Prior research has shown that Ctf4 is necessary for proper chromatid segregation and cohesion, starting from their initial establishment during S-phase to later separation in anaphase. Ctf4 is essential for viability, and depletion results in cell cycle progression defects. Drosophila melanogaster is used as a genetic model organism not only because of their high homology to humans but also the short generation times, high fecundity, and chromosome manipulation make it ideal for genetic crosses. In order to study the role of Ctf4 in regards to its involvement in DNA replication and chromatin formation, a mutant allele needed to be generated. A strain of flies with a p-element, a transposable element, inserted just upstream of the gene is crossed with a strain containing a transposase. The transposase mobilizes the p-element. About 90% of the time the p199
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