H Luks, J Wright, C Cooper, J Foot, J Thames, B Cortes, Dr. S Baviskar

1. Characterization of TBC1D20 Homolog in Dictyosteliumdiscoideumvia Gene Knockout H Luks, J Wright, C Cooper, J Foot, J Thames, B Cortes, Dr. S Baviskar Department of Biological Sciences, University of Arkansas – Fort Smith BACKGROUND INFORMATION CURRENT PROGRESS INTRODUCTION TO D. discoideum The trafficking of lipids, membrane and soluble proteins between membrane bound compartments is regulated by proteins such as RabGTPases. RabGTPases, or Rabs, are membrane associated monomeric proteins found on the surfaces of distinct membrane bound compartments. Rabs coordinate transport vesicle formation from the donor compartment, motility, docking, and fusion with the target compartment. RabGTPases cycle between active and inactive states. They are activated by guanine nucleotide exchange factors (GEFs) and deactivated by GTPase activating proteins (GAPs) . Dictyosteliumdiscoideumis an amoeba found in forest soils. The National Institute of Health (NIH) has recognized D. discoideumas a model organism to study various cellular, developmental, and evolutionary processes because its life cycle is simple, genome size is small and haploid, and most of its genes are homologous to human genes. D. discoideumcan be infected by various human pathogens as D. discoideumare efficient phagocytes and all their mechanisms of phagocytosis and membrane trafficking are similar to mammals.4 We have successfully extracted D. discoideum genomic DNA and amplified the gene of interest by PCR. We designed the PCR primers with artificial restriction sites. The pBSR-19 plasmid that is to be engineered with the amplified gene is in our possession and is awaiting restriction digestion and subsequent ligation. Over the course of the semester, we have gained experience in maintaining axenicD. discoideumcultures in various media, splitting cultures, harvesting, and freezing cells. We have also increased our knowledge of PCR and gel electrophoresis.  Based on sequence similarity, a homolog of TBC1D20 is found in Dictyosteliumdiscoideum genome on chromosome 4. This unnamed gene in D. discoideum(Gene ID #DDB_G0283055) has not been characterized (molecular data, function and location not known). 1598 bp Amplicon of TBC1D20 Homolog Successful Extraction of Genomic DNA METHODS AND MATERIALS 100 bp DNA ladder GOAL The goal of this project is to characterize the gene in Dictyosteliumdiscoideumduring unicellular and multicellular stages of its life cycle. The characterization will be done by disrupting the gene via homologous recombination, also known as gene knockout. The knockout mutants will be compared with normal cells at various stages of their life cycle. TBC1D20 HOMOLOG KNOCKOUT CONSTRUCT 100 bp DNA ladder Genomic DNA 1598 bp TBC1D20 is the human gene coding for the GAP for Rab1 which is responsible for trafficking between Endoplasmic Reticulum and Golgi. TBC1D20 contains a TBC (Tre-2, Bub2, and Cdc16) domain present in most known RabGTPase-activating proteins (GAPs). Overexpression of catalytically active TBC1D20 in human cells disrupts the integrity of the Golgi complex and hence vesicular transport. It also plays a role in allowing viruses like Hepatitis C and Human Immunodeficiency virus (HIV) enter cells and mediate their replication.2,3 REFERENCES 1. J. P. DiNitto, T. C. Cronin, D. G. Lambright. “Membrane Recognition and Targeting by Lipid-Binding Domains.” Sci. STKE 2003, re16. 2003. 2. Nachmias et al. "Human immunodeficiency virus type 1 envelope proteins traffic toward virion assembly sites via a TBC1D20/Rab1-regulated pathway." BioMed Central 9 (7). 2012. http://www.ncbi.nlm.nih.gov/pubmed/22260459 (accessed January 28, 2012). 3. Sklan, EH et al. TBC1D20 is a Rab1-GTPase-activating protein that mediates Hepatitis C virus replication.  The Journal of Biological Chemistry.  282 (50),  36354-36361. 2007. 4. Steinert, M and Heuner, K. Dictyostelium as host model for pathogenesis. Cellular Microbiology.  7(3), 307-314. 2005. We would like to thank the Department of Biological Sciences at UAFS for the support and facilities provided. In addition, we would like to thank dictyBase.org for the information on D. discoideum as well as the Dicty Stock Center for providing the AX2 strain and the plasmid pBSR-19 for the experiments. ACKNOWLEDGEMENTS