East Carolina University : Research and Creative Achievement Week 2012
pulldown assays that the levels of GTP-bound RhoA have been increased in the GPR4 cells when exposed to pH 6.4 indicating that the Rac GTPase levels could be repressed. Microarray analysis has revealed a 8-fold increase in the expression levels of the p21 activated kinase, Pak3, in B16/GPR4 cells. Recently Pak3 has been shown to inhibit Rac1/2 mediated lamellipodia formation as well as act downstream of Cdc42. These findings indicate that the Rho family GTPases such as Cdc42 and Rac, along with the p21-activated kinase Pak3, could be potentially altered when the GPR4 receptor is activated. Psf2: A Role In Chromosome Condensation, Jeffrey Chmielewski, Laura Henderson, Tim Christensen, Department of Biology, East Carolina University, Greenville, NC 27858 In D. melanogaster, the CMG complex is a group of proteins that function as the DNA helicase during replication. The CMG complex is composed of cdc45, MCM2-7, and the GINS complex. The GINS complex is a heterotetrameric complex composed of the protein subunits Psf1, Psf2, Psf3, and Sld5. Recent research in human dermal fibroblasts shows GINS is essential for the initiation and elongation stages of chromosomal replication. Working with a null mutation, I have designed a series of experiments aimed at elucidating the function of Psf2 in vivo. Using phosphoH3 immunostaining and M-phase indices, I have shown that heterozygous mutants exhibit a significant M-phase delay. EdU incorporation assays in third instar larval brains shows no significant difference in the number of cells in S-phase. However, when compared to wild type, the pattern of EdU incorporation indicates cells take longer to replicate euchromatin; possibly resulting in the improper packaging of euchromatin as heterochromatin. To corroborate the data seen in the pattern of EdU incorporation, we designed a novel technique to establish the packing ratio of salivary gland polytene chromosomes. Using this novel technique, we are able to show that heterozygous mutants exhibit a significant increase in packing ratio compared to WT. Additionally, in the later stages of egg chamber development, nurse cell nuclei display overly condense polytene chromosomes during the psuedo M-phase at the end of endocycle 5. We have evidence that indicates these instances of overly condensed chromosomes results in apoptotic egg chambers, seen later in development. We have also taken a genetic approach to further substantiate our cytological characterizations. Heterozygous Psf2 flies show a significant decrease in viability with complete homozygous lethality. Position effect variegation analysis show Psf2 enhances variegation an indication that chromosomes are overly condensed. This data, when combined, suggests Psf2 has a role during replication that ultimately determines how DNA is packaged.
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