The role of Rtr1 in the regulation of genomic integrity Gabriela Cabello1 , Melanie Fox 2, Jerry Hunter2 , Jason True2 , and Amber L. Mosley, PhD2 1Ben Davis High School, Indianapolis, IN, and 2Department of Biochemistry and Molecular Biology, Indiana University, Indianapolis, IN ABSTRACT: Genomic instability is a hallmark of human cancers. Not only is genome stability required for DNA repair in mitosis, for the generation of genetic diversity, and for proper reducing division in meiosis, but it is responsible for chromosomal defects associated with some types of cancer and hereditary diseases, many of which happen because of recombination between DNA repeats. The identification of the genes and functions that control initiation of recombination between repeats is therefore necessary to understand the origin of chromosome aberrations and genome instability.   Rtr1 is a C-terminal domain phosphate which regulates the termination phase of transcription. Functional characterization of Rtr1 reveals its role as a CTD phosphatase essential for the S5-to-S2-P transition. Despite our poor understanding of the molecular basis of transcription-induced recombination, it seems clear that transcription is an important potential source of spontaneous recombination between repeats leading to genomic instability and needs to be investigated. OBJECTIVES: This ongoing research investigates the effects of Rtr1Δ in the regulation of genome integrity in yeast cells and the extent to which the Rtr1 phosphatase increases levels of recombination and translation. RESULTS: SUMMARY: PCR amplification produced ≈8mg of TAP tag plasmid and Rpb11-TAP PCR product The difference in amount of transformants between wild-type and rtr1Δ was significant. However, the transformation will have to be repeated in order to prove further statistical significance. METHODS: A maxiprep of the PGLG plasmid (Leu-) prepared and transformed into various strains. Colonies were restreaked . 25 mL cultures were grown in YNB Leu minus 2% glucose or YNB Leu minus 2% galactose Preparation of Homologous Recombination Casette PCR amplification performed using either the TAP tag plasmid or the Rpb11-TAP PCR product as DNA template on a 1.5% agarose gel CONCLUSION: Regarding the Rpb11-TAP casette transformations, less transformants were obtained from the cells in comparison to the wild-type cells. This could indicate that Rtr1 increases homologous recombination We were unable to obtain any transformants using the pGLG plasmid DNA (n=3). This data indicates that there is a undetermined problem with the plasmid that will need to be resolved before proceeding. 3: INTRODUCTION: Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. FUTURE WORK: The transformations performed with wild-type (A) or rtr1Δ (B) yeast will have to be repeated in order to prove further statistical significance. Also, due to undetermined difficulties with the PGLG plasmid, further studies will need to be done in order to solve problems before proceeding. Figure 2: Direct repeat recombination assay using GFP direct repeat plasmid (pGLG) from Andrés Aguilera lab. To test direct repeat recombination, we attempted to transform the pGLG plasmid into wild-type and rtr1Δ yeast. In the presence of galactose, transcription induced recombination could occur in a percentage of the yeast cells. Direct-repeat recombination would result in a full length GFP gene which could then be transcribed and then translated into protein. Only the full length GFP will produce a fluorescent signal. Figure 4: Rpb11-TAP PCR cassette transformations grown on Ura minus media for 3 days at 30°C. The transformations were performed with wild-type (A) or rtr1Δ (B) yeast. ACKNOWLEDGEMENTS: Megan Zimmerly, Michael Berna, Mary Cox, Elmer Sanders, Project SEED staff and mentors, Andrés Aguilera Barcelona lab for PGLG plasmid, Charlie Dong lab for flouremeter REFERENCES: Aguilera, A., 2001. “The connection between transcription and genomic instability.” The EMBO Journal 21: 195-20 Mosley, A.L., Pattenden, S.G., Carey, M., Venkatesh, S., Gilmore, J.M., Florens, L., Workman, J.L., Washburn, M.P. Rtr1 is a CTD phosphatase that regulates RNA polymerase II during the transition from serine 5 to serine 2 phosphorylation. Molecular Cell. 2009. 34(2):168-178. Kuehner, J., et al. 2011. “Unravelling the means to an end: RNA polymerase II transcription termination.” Nature Reviews: Molecular Cell Biology 12. Gómez-González, B. and Aguilera, A., 2007. “Activation-induced cytidine deaminase action is strongly stimulated by mutations of the THO complex.” PNAS 104(20): 8409-8414 Figure 1: Description of homologous recombination using a PCR product. For homologous recombination to occur, the PCR product must have two stretches of homologous sequence (at least 50 base pairs in length) to direct the PCR product to the correct area of the genome. Figure 5: Sent PGLG plasmid with respective primers (HIS3-F, T7, T3, MI3) out for sequencing.