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Florida Genetics 2008 Organizing Committee Chair: Indra Vasil Members: Henry Baker, John Davis, Connie Mu lligan, Diana Nolte, John Pastor Douglas Soltis, Michele Tennant, Thomas Yang FG2008 Sponsors Gold Level University of Florida Genetics Institute, Center fo r Epigenetics, College of Engineering, Department of Molecular Genetics and Microbiology, Graduate Program in Plant Molecular and Cellular Biology, Health Science Center Libraries, Interdisciplinary Center for Biotechnology Research Silver Level Center for Research in Pediatric Immune Deficiency, Clinical and Translational Science Institute, Evelyn F. & William L. McKnight Brain Inst itute, Florida Center for AIDS Research Special Thanks To Jennifer Bald win, David Br umbaugh, Mickey Cuthbertson, Ned Davis, Martine Horrell, David Moraga, Connie Nicklin, Alan OMalley, Hope Parmeter University of Florida Genetics Institute Director: Kenneth Berns Associate Directors: Henry Baker, Donald McCarty, Connie Mulligan, Indra Vasil Executive Board: William Allen, Henry Baker, Kenneth Berns, John Davis, Robert Ferl, William Hauswirth, Julie Johnson, Donald McCarty, Michael Miyamoto, Connie Mulligan, Nicholas Muzyczka, Winfred Phillips, Pam Soltis, Doug las Soltis, Michele Tennant, Indra Vasil, Marta Wayne, and Thomas Yang Scientific Advisory Board: Jeffrey Bennetzen, Ph.D., Norman and Doris Giles Professor and Georgia Research Alliance Eminent Scholar, University of Georgia, Athens, Ga.; Ronald W. Davis, Ph.D., Director, Stanford Genome Technology Center, Stanford, Calif.; Rebecca W. Doerge, Ph.D., Professor, Departments of Agronomy and Statistics, Purdue University, West Lafayette, Ind.; Yoram Groner, Ph.D., The Dr. Barnet Berris Professor of Cancer Research, Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel; Peter M. Howley, M.D., George Fabyan Professor of Comparative Pathology and Chair, Department of Pathology, Harvard Medical School, Boston, Mass.; Patricia G. Spear, Ph.D., Guy an d Anne Youmans Professor and Chair, MicrobiologyImmunology, Northwestern Un iversity, Chicago, Ill.
UFGI Strategic Plan The discovery of the three-dimensional double helix architecture of DNA in 1953 was not only a defining moment for biology, but arguably one of the most significant scientific discoveries of all time. It fundamentally and permanently changed the course of biology and genetics. The unraveling of DNAs structure, combined with its elegant mechanism for self-replication and the existence of a universal genetic code for all living beings, have toge ther provided the basis for the understanding of fundamental cellular processes, mutation and genetic repair, genetic variation, the origin of life and evolution of species, and the structure/function/regul ation of genes. The double helix is also proving to be of immense significance to advances in agriculture, medicine and such other diverse fields as anthropology, criminology, computer science, engineering, immunology, nano technology, etc. It was the study of DNA that led to the development of tools that brought about the biotechnology revolution, the cloning of genes, and the sequenci ng of entire genomes. Yet, most knowledgeable people agree that what has been achieved in DNA science thus far is only the beginning. Bigger and better applications, which will impact directly on the quality of human life and sustainability of life on earth, are yet to come. In order to attain thes e objectives, the digital nature of DNA and its complementarity are beginning to be exploited fo r the development of biology as an informationbased science. Indeed, a paradigm shift is already taking place in our view of biology, in which the natural, physical, engineering and environmental sc iences are becoming unified into a grand alliance for systems biology. Indeed, biology in the 21st century will be surely dominated by this expanded vision. The Genetics Institute is committed to fost ering excellence in teaching and research, and in promoting cross-campus interdisciplinary interactio ns and collaborations. In the pursuit of these objectives, it offers a graduate program in geneti cs, and has identified the following four key areas for teaching, research and development: Bioinf ormatics, Comparative Genomics, Population and Statistical Genetics, and Epigenetics.
Florida Genetics 2008 Schedule Wednesday, October 29, 2008 Pre-conference Discussion Sessions 10:00 a.m. 11:00 a.m.: Bioinformatics and the National Center for Biotechnology Information, David Lipman, M.D. 11:00 a.m. noon: Interdisciplinary Center for Biotechnology Research, William Farmerie, Ph.D. Noon 1:00 p.m.: Check-in and poster set-up Session I Human Genetics Chair: Kenneth I. Berns, M.D., Ph.D. 1:00 p.m. 1:15 p.m. Opening Remarks: Indra K. Vasil, Ph.D., and Kenneth I. Berns, M.D., Ph.D. 1:15 p.m. 2:00 p.m. John M. Coffin, Ph.D. American Cancer Society Professor and Distinguis hed Professor, Department of Molecular Biology and Microbiology, Tufts University Co-evolution of retroviruses and their hosts 2:00 p.m. 2:30 p.m. William W. Hauswirth, Ph.D. Rybaczki-Bullard Professor of Ophthalmology an d Molecular Genetics, University of Florida Gene therapy restores light sensitivity to leber congenital amaurosis patients 2:30 p.m. 4:30 p.m. Poster Session and Reception 5:00 p.m. 6:00 p.m. HPNP Auditorium Francis S. Collins, M.D., Ph.D. Director, National Human Genome Research In stitute, National Institutes of Health Genetics, medicine, and society
Thursday, October 30, 2008 8:00 a.m. 3:00 p.m.: Check-in at UF s Cancer/Genetics Research Complex 8:00 a.m. 8:30 a.m.: Coffee Session II Quantitative and Computational Genomics Chair: Marta L. Wayne, Ph.D. 8:30 a.m. 9:15 a.m. Trudy F.C. Mackay, Ph.D. William Neal Reynolds Professor of Ge netics, North Carolina State University The genetic architecture of quantitative traits: lessons from Drosophila 9:15 a.m. 9:45 a.m. Charles F. Baer, Ph.D. Assistant Professor, Department of Botany and Zoology, University of Florida The effects of spontaneous mutations on phenotypic canalization in Caenorhabditis 9:45 a.m. 10:15 a.m. Lauren M. McIntyre, Ph.D. Associate Professor, Department of Molecular Genetics and Microbiology, University of Florida Modeling allele specific expression 10:15 a.m. 11:00 a.m. David J. Lipman, M.D. Director, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health Unexpected universals of protein evolution 11:15 a.m. 1:30 p.m.: Poster Session 11:30 a.m.: Lunch (for registered attendees) Session III: Plant Functional Genomics Chair: Robert Ferl, Ph.D. 1:30 p.m. 2:15 p.m. Joseph R. Ecker, Ph.D. Professor, Plant Molecular and Cellular Biology, Salk Institute for Biological Studies Sequencing 1,001 Arabidopsis genomes and epigenomes for functional and evolutionary studies 2:15 p.m. 2:45 p.m. A. Mark Settles, Ph.D. Vasil-Monsanto Associate Professor, Horticultura l Sciences Department, University of Florida Phenomics meets genomics: exploring maize seed development with high throughput technologies 2:45 p.m. 3:15 p.m. Kevin M. Folta, Ph.D. Assistant Professor, Horticultural Sciences Department, University of Florida Structural and functional genomics of strawberry: a gateway to the Rosaceae
Presentation Abstracts Co-evolution of retroviruses and their hosts Coffin JM Department of Molecular Biology and Microbiology, Tufts University, Boston, MA Retroviruses display a remarkable wealth of evol utionary phenomena, of which I will discuss only a few. First, the genomes of most or all anim al species contain remnants of retroviral DNA derived infection and integration, constituting a la rge fraction of total DNA (up to 8% in humans). The present relationship of these proviruses an d their host differs considerably among different vertebrate species. In mice, for example, there is considerable variation among individuals and among subspecies in location and structures of some groups, such as gammaretroviruses related to murine leukemia virus. In humans, by contrast, there is very little variation in provirus content from one individual to another, except for polymor phism due to post integration events, such as point mutation and solo LTR formation. Integratio n site polymorphism has been observed only in a handful of relatively recent members of the HE RV-K (HML-2) group, an d all these proviruses are widely distributed in the population, implying integration in the distant past. In the mouse, viruses produced by some endogenous provirus es, or recombinants among them, are known to cause disease, particularly lymphoma (MLV) an d mammary carcinoma (MMTV). At, present, although a number of human tumors (breast, germ cell, melanoma) are known to (at least occasionally) express one or more HER-K provir uses as RNA, proteins, or even virions (of questionable infectivity), genetic evidence connectin g this expression with etiology of the tumor has not yet been obtained. Second, as part of the coevolutionary arms race, two retrovirus groups (those found in chickens and mice) display a remarkable evolutionary adaptability in their ability to evolve to use completely different cell surface proteins as recept ors. This receptor switch involves many amino acid differences, so there must be many steps along the way, yet each step must be an infectious virus. To investigate this pathway, we have performed lengthy passaging experiments in cell culture, asking whether we can find in termediates in the process. In two different experiments, we identified mutations at nearby si tes in the receptor binding region of the avian retrovirus env protein, that confer on the virus the ability to infect cells without benefit of receptor. The mechanism of this altered ability and its evolutionary consequences will be discussed. Biography of John M. Coffin, Ph.D. Dr. Coffin is the American Cancer Society Pr ofessor and Distinguished Professor in the Department of Molecular Biology and Microbiology at Tufts University. He is also past director of and advisor to the highly successful HIV Drug Resistance Program for the National Cancer Institute, which he established in 1997. As a postdoctoral fellow, Dr. Coffin showed that retrovirus RNA is synthesized by cellular DNAdependent RNA polymerase II, fulfilling a key prediction of the DNA provirus theory. He went on to establish the size of the retrovirus genome at 10kb, and to develop and apply the first useful technique for mapping genes on retrovirus genomes. As a faculty member at Tufts University, Dr. Coffin extended these concepts and approaches and combined them with genetic studies to discover a critical step in reverse transcription the transfer of the nascent minus strand from one end of the genome to the other, mediated by the short redundant (R) sequence, which he discovered. This finding led him to propose the now well-accepted copy-choice model for retrovirus recombination. He also initiated studies of endogenous (germ-line inherited)
retroviruses, which his laboratory continues to pu rsue today. Genetic analysis of slow-growing and non-pathogenic endogenous avian retroviruses revealed that a region near the 3 end of the genome, now known as the U3 portion of the long terminal repeat (LTR), plays a key role in regulating virus expression by interacting with the cellular transcription machinery a well accepted principle of retrovirology. A major theme of his work has been endogenous pr oviruses as both agents and reporters of host evolutionary events. Using hybridization tools de veloped in the lab, he and his colleagues have identified and mapped over 150 endogenous muri ne leukemia (MLV) and mammary tumor virus (MMTV) proviruses in the genome of inbred mice, and demonstrated that a number of wellknown mutations in mice were caused by provirus integration. They also discovered that the mysterious endogenous superantigens in mice were encoded by endoge nous MMTV proviruses, thus settling some long-standing immunological, genetic, and virological puzzles. Dr. Coffin eventually turned to the study of HIV and AIDS, where he has been studying HIV evolution and applying that understanding to th e study of drug resistance, one of the most important factors limiting effective long-term therapy for HIV infection. His 1995 paper in Science described essential characteristics of the HIV-host interaction, in particular the evolution of drug resistance, and included the correct prediction that many drug resistance mutations preexist prior to therapy. This analysis established a framewor k for understanding the basic and clinical science of HIV resistance.
Genetics, medicine, and society Collins FS National Human Genome Research Institute, Na tional Institutes of Health, Bethesda, MD The successful completion of all of the goals of the Human Genome Project in April 2003 has opened the door to a host of follow-up projects that move us ever closer to widespread applications to medicine. With the cost of DNA se quencing falling rapidly, direct applications to medical problems, especially cancer, are becoming more and more feasible. Furthermore, the detailed catalogue of human genetic variation developed by the HapMap project has enabled a comprehensive approach to identifying common ge netic variants that confer risk for common disease. In just the last two years, more th an 300 such variants have been identified and validated, affecting diseases such as cancer, heart disease, and diabetes. These discoveries now place us in a position of being able to offer ind ividualized information about future risk of illness. The same strategy also offers the opportunity to be able to predict drug response, and to adjust the choice of the drug or the dose accordingly. Perhaps of greatest long term impact is the discovery of new pathways involved in diseas e, pointing towards previously unknown drug targets that may result in a revolution in th erapeutics in the next one or two decades. All of these discoveries hold great promise for medicine, but further emphasize the need to pay attention to the ethical, legal and social imp lications. The Human Genome Project has taken the lead in supporting research to identify steps that should be taken to maximize the benefits of these advances, and minimize the risks. The rece nt passage of federal legislation to prevent genetic discrimination is encouraging evidence of the effectiveness of such a prospective planning process. But many additional challenges, includ ing oversight of genetic testing, intellectual property applications to genetics, and access to genomic health care, will continue to need involvement of scientists, ethicists, pub lic policy experts, and the general public. Biography of Francis S. Collins, M.D., Ph.D. Dr. Francis S. Collins, a physician-geneticist note d for his landmark discoveries of disease genes and his leadership of the Human Genome Project, is Director of the National Human Genome Research Institute (NHGRI) at the National Instit utes of Health. With Dr. Collins at the helm, the Human Genome Project consistently met projected milestones ahead of schedule and under budget. This remarkable international project culminated in April 2003 with the completion of a finished sequence of the human DNA instruction book. Building on the foundation laid by the Human Genome Project, Dr. Collins is now leading NHGRIs effort to ensure that this new trove of sequence data is translated into powerful tools and thoughtful strategies to advance biological knowledge and improve human health. Dr. Collins is also known for his consistent emphasis on the importance of ethical and legal issues in ge netics. Dr. Collins laboratory has discovered a number of important genes, including those respon sible for cystic fibrosis, neurofibromatosis, Huntington's disease and most recently, genes for adult onset diabetes and the gene that causes Hutchinson-Gilford progeria syndrome, a dramatic form of premature aging. Dr. Collins has an intense interest in the interface between science and faith, and has written about this in The Language of God: A Scientist Presents Evidence for Belief (Free Press, 2006), which spent many weeks on the New York Times bestseller list. Dr. Collins received a B.S. from the University of Vi rginia, a Ph.D. in physical chemistry from Yale University, and an M.D. from the University of North Carolina. He has been elected to the Institute of Medicine and the National Academy of Sciences, and was awarded the Presidential Medal of Freedom in November 2007.
The genetic architecture of quantitative traits: lessons from Drosophila Mackay TF Department of Genetics, North Caro lina State University, Raleigh, NC Quantitative traits are affected by multiple interacting loci with individually small and environmentally sensitive effects. Understanding the genetic architecture of quantitative traits begins with identifying the genes regulating these traits, mapping the subset of genetically varying quantitative trait loci (QTLs) in natu ral populations, and pinpointing the molecular polymorphisms defining QTL alleles. Drosophila brings an impressive toolkit to the challenge of genetically dissecting quantitative traits: P tran sposable element mutage nesis to identify genes regulating these traits; artificial selection from natural populations to create extreme trait phenotypes; high resolution mapping to identify positional candidate genes corresponding to QTLs; linkage disequilibrium mapping to ident ify molecular polymorphisms that functionally define QTL alleles; and whole-genome transcriptional profiling to postulate interacting networks of genes affecting quantitative traits. Studies of any single Drosophila quantitative trait typically indicate a complex genetic architecture, with larg e numbers of pleiotropic genes that have sex-, environmentand genotype-specific effects. A systems genetics approach, in which multiple complex organismal phenotypes and whole ge nome transcript abundance are assessed simultaneously on the same panel of inbred Drosophila strains, identifies modules of correlated transcripts associated with quantitative traits yielding rich insights into unexpected genetic mechanisms underpinning trait variation. Thes e observations offer valuable lessons for understanding the genetic basis of variation for qu antitative traits in other organisms, including humans. Biography for Trudy F. C. Mackay, Ph.D. Trudy Frances Charlene Mackay has been the Willi am Neal Reynolds Distinguished Professor of Genetics at North Carolina State University since 1996. Dr. Mackay earned her Ph.D. in genetics from the University of Edinburgh in 1979 and perf ormed as a post-doctoral associate at Dalhousie University from 1979 through 1980. She has been em ployed in the Department of Genetics at NCSU since 1987. Dr. Mackays general research goal is to und erstand the genetic and environmental factors affecting variation in quantitative (or complex) traits. This is necessary for risk modification of multifactorial human diseases, in theory for a mo re comprehensive view of the genetic processes underlying phenotypic evolution and in practice for improving production traits in domestic species. Her current research focuses on Drosophila melanogaster, which has a wealth of genetic and genomic resources, and morpho logical, behavioral, physiologi cal and life history characters spanning the gamut of fitness profiles. Dr. Mackay has received a number of awards and honors including: Fellow of the American Association for the Advancement of Science (2003), the American Academy of Arts and Sciences, (2005), and the Royal Society (2006), the Genetics Society of America Medal (2004), and the O. Max Gardner Award from the University of North Carolina (2007). She has served as Executive or Associate Editor of the journals Genetical Research, PLoS Genetics, Genetics, and Evolution, and on boards or governing bodies for the following associations: the Genetics Society of America, the American Genetic Association, and the National Center for Biotechnology Informations Board of Scientific Counselors. She is current President of the Drosophila Board (through 2011). Dr. Mackay has published over 100 peer-reviewed journal articles, and has contributed to a number of books, book chapters, and invited conference proceedings and reviews.
Unexpected universals of protein evolution Lipman DJ National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD Proteins evolve at widely different rates due to a number of factors including function, three dimensional structure, and expression level. Surp risingly, the proteomes of organisms as different as human and E. coli share very similar distributions of prot ein evolutionary rates, i.e., they have similar proportions of proteins with rapid, medium and slow evolutionary rates. This steady state also implies that throughout most of the history of life, the evolutionary rates of gained and lost protein-coding genes must roughly balance out. New (gained) genes in a group, e.g. mammals, are those that do not have homologs detectable through sequence comparison outside of the mammals, and lost genes are those that for e.g. mammals can be traced to an ancestral group such as metazoans through detectable homologs in other metazoan branches such as insects and nematodes. Although the new pr oteins evolve more rapidly than older proteins, and so do proteins that apparently have been lost, the dist ributions of evolutionary rates of proteins of different ages are wide and strongly overlapping, i. e., a number of new proteins or lost proteins seem to have relatively slow evolutionary rates. I will discuss these findings on the process of protein gain and loss and other aspects of the evolutionary age of a protein. Biography of David J. Lipman, M.D. Dr. David Lipman is currently the Director of th e National Center for Biotechnology Information (NCBI), which is a division of the National Library of Medicine within the National Institutes of Health. NCBI was created by Congress in 1988 to do basic research in comp utational biology, and to develop computational tools, databases and in formation systems for molecular biology. After medical training, Dr. Lipman joined the Mathematical Research Branch of the National Institute of Diabetes, Digestive, and Kidney Diseases (NIDDK) as a Research Fellow. In his research on computational tools, he developed the most widely used methods for searching biological sequence databases. There are thousands of cita tions to Dr. Lipmans methods in papers which have used them to discover biological functions for unknown sequences and which have thereby advanced the understanding of the molecular ba sis of human disease. Since 1989, Dr. Lipman has been the Director of the NCBI, a leading re search center in computational biology, the creators of PubMed, and one of the most heavily used sites in the world for the search and retrieval of biomedical information. Dr. Lipman was elected to the National Academy of Sciences in 2003.
Sequencing 1,001 Arabidopsis genomes and epigenomes for functional and evolutionary studies Ecker JR Plant Molecular and Cellular Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA Deciphering the multiple layers of epigenetic regu lation that control transcription is critical to understanding how plants develop and respon d to their environment. Using sequencing-bysynthesis technology we directly sequence d the cytosine methylome (methylC-seq), transcriptome (mRNA-seq), and small RNA tran scriptome (smRNA-seq) to generate highly integrated epigenome maps for several Arabidopsis thaliana wild strains and mutants defective in DNA methyltransferase or demethylase activity. At single-base resolution we discovered extensive, previously undetected, DNA methyla tion, identified the context and level of methylation at each site, and observed local sequence effects upon methylation state. Deep sequencing of smRNAs revealed a direct relationship between the location of smRNAs and DNA methylation, perturbation of smRNA biogenesis upon loss of CpG DNA methylation, and a tendency for smRNAs to direct strand-specific DNA methylation in regions of RNADNA homology. Finally, strand-specific mRNA-seq revealed altered transcript abundance of hundreds of genes, transposons and unannotated intergen ic transcripts upon modification of the DNA methylation state. Biography of Joseph R. Ecker, Ph.D. Joe Ecker is a Professor in the Plant Biology Laboratory and the Director of the Genomic Analysis Laboratory at The Salk Institute for Biological Stud ies in La Jolla, California. He earned his Ph.D. in microbiology at the Pennsylvania State University College of Medicine and carried out postdoctoral studies with Ronald Davis at the Depa rtment of Biochemistry at Stanford University. He served on the Faculty at the University of Pennsylvania (1987-2000) before joining The Salk Institute for Biological Studies (2000). His resear ch on the gaseous plant hormone ethylene has yielded basic insights into the mechanisms of plan t growth control and its application has resulted in technologies that delay fruit ripening an d disease processes. He served as Principal Investigator and Chairman of the Arabidopsis Genome Initiative, a world-wide consortium of laboratories that determined the DNA sequence of the first plant genome (Arabidopsis thaliana) in 2000. His laboratory continues to explore the encyclop edia of DNA elements in genomes through the development and application of next generation DNA sequencing technologies for capturing genome/epigenome/transcriptome-wide information and applying systems biology approaches to the study of gene function in plant and human cells. Professor Ecker currently serves as associate editor of PLoS Genetics and Proceedings of the National Academy of Sciences, U.S.A. He has been the recipient of multiple honors, including: the Kumho Science International Award in Plant Molecular Biology and Biotechnology (2001), the International Plant Growth Substances Associ ation Distinguished Research Award (2004), the American Society for Plant Molecular Biology Martin Gibbs Medal (2005), and was chosen as the Scientific American 50: Research Leader of the Year in Agriculture in 2004. He was elected to the US National Academy of Sciences in 2006. In 2007 he received the John J. Carty Award for the Advancement of Science from the US National Academy of Sciences. He currently serves as President of the International Society for Plant Molecular Biology.
* = U.F. Genetics Institute Faculty Gene therapy restores light sensitivity to leber congenital amaurosis patients Hauswirth WW1,2,*, Kaushal S1,*, Cideciyan AV3, Aleman TS3, Byrne BJ2,4,*, Schwartz SB3, Boye SL1, Roman AJ3, Pang J-J1, Windsor EAM3, Sumaroka A3, Aguirre GD5, Fishman GA6, Heon E7, Flotte TR8, Stone EM9, Jacobson SG1 1Department of Ophthalmology, University of Florida, Gainesville, FL 2Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 3Scheie Eye Institute, Department of Ophthalmolog y, University of Pennsylvania, Philadelphia, PA 4Powell Gene Therapy Center, Univer sity of Florida, Gainesville, FL 5Department of Clinical Studies, University of Pennsylvania, Philadelphia, PA 6Department of Ophthalmology and Visual Scie nces, University of Illinois, Chicago, IL 7Department of Ophthalmology and Vision Sciences, Hospital for Sick Children, University of Toronto, Toronto, Canada 8Department of Pediatrics, University of Massachusetts Medical School, Worcester, MA 9Department of Ophthalmology, University of Iowa, Iowa City, IA The preclinical efficacy and safety basis for a recently initiated AAV vector clinical trial for LCA2 (RPE65 mutations) will be summarized and will be followed by a summary of our 3-month safety and visual function studies of the first three clin ical trial patients. AAV2-CBA-hRPE65 vector was delivered subretinally to affected dogs and mice and to normal rats and non-human primates to establish efficacy and safety parame ters for a clinical trial for LCA2. Three patients then received 150ul of GMP grade vector (5.9 exp10 vector genomes) and were then followed for three months. Efficacy studies in mouse and dog models of LCA2 in terms of vision restoration, both functionally and structurally, restoration of cort ical function and improvement in visually guided behavior will be summarized. Based on this data and safety/biodistribution studies in dogs, rats and nonhuman primates, the design of the trial, including the vector dosing rationale, will be discussed. Early-stage clinical tr ial results will then be presented for the first three patients, each for at least 3 months following subretinal vector delivery. In brief, there were no adverse events, either related to or not related to vector, no ted for any patient. All patients tolerated the procedure without incident. All three patients exhibited substantial and significant (more than three standard deviations above multi-patient test variation) improvement in light sensitivity, but only in the area of retina that received subretinal vector. High density threshold perimetry revealed that this improvement ranged from 2.2 to more nearly 5 logs higher sensitivity than respective pretreatment baselines. Based on this data, a second cohort of three patients is being treated. The effects of spontaneous mutatio ns on phenotypic canalization in Caenorhabditis Baer C* Department of Botany and Zoology, University of Florida, Gainesville, FL The evolution of canalization the robustness of the phenotype to environmental or genetic perturbation has attracted considerable re cent interest among both developmental and evolutionary biologists. A key step toward under standing the evolution of any phenotype is to characterize the rate at which mutation introduces genetic variation for the trait (the mutational variance, VM) and the average directional effects of mutations on the trait mean ( M). In this study, we quantify the effects of spontaneous mu tation on the canalization of three sets of
phenotypic traits in two sets of mutation accumulation lines of nematodes in the genus Caenorhabditis: (1) productivity and body volume, (2) a suite of traits involved in the development of the vulva, a classical model syst em in developmental biology, and (3) gene expression. For productivity and body volume, fo ur results emerge: first, spontaneous mutations consistently de-canalize the phenotype; second, the mutational parameters for de-canalization VM (quantified as mutational heritability) and M are of the same order of magnitude as the same parameters for the traits themselves; third, the mutational parameters for canalization are roughly correlated with the parameters for the trai ts themselves across taxa, and fourth, there is no evidence that residual segregating overdominant loci contribute to the decay of canalization. The trend is similar for vulval development. Su rprisingly, environmental variance for gene expression decreased in MA lines relative to the ancestral progenitor. These results suggest that canalization is readily evolvable, and that any ev olutionary factor that causes mutations to accumulate will, on average, de-canalize the phenotype. Modeling allele specific expression McIntyre L* Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL To date the majority of expression analysis has focused on the total amount of transcript. However, emerging evidence underlies the impo rtance of understanding the allele specific transcript. Incorporating a basic understanding of the allele-specific nature of expression will allows us to address fundamental questions ab out quantitative inheritance in novel ways. Drosophila is a model organism perfectly suited to understanding these basic questions. Full genome sequences for five divergent lines of D. simulans are readily available and importantly chromosomal content can be manipulated in order to test experimentally the impact of specific alleles on transcription. We combine the flexibility of this model system with modern technology and the development of analytic tools in order to examine the impact of maternal effects, dose response, additivity, and dominance on allele spec ific expression. The immediate objective of this work is to understand how spec ific alleles are expressed, how th at expression is influenced by cis and trans effects. The long term objective is to und erstand how genetic variation contributes to complex, quantitative phenotypes. Phenomics meets genomics: exploring maize seed development with high throughput technologies Settles AM1,2,* 1Horticultural Sciences Department, Un iversity of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL Seeds comprise the major source of our food and feed. The diversity in seed developmental programs among crop species leads to differences in their composition and nutritional value for human and animal consumption. We have de veloped both phenotyping and genotyping technologies to allow high-throughput screening for maize mutants that impact seed composition or weight. Specifically, we are screening the Un iformMu transposon-tagging population using single kernel near-infrared spectroscopy (NIR) and seed weights. NIR spectroscopy determines chemical composition and seed weight phenotypes of individual maize kernels non-destructively and at high-throughput. We built a semi-automated single-kernel NIR grain analyzer and developed partial least square (PLS) calibration models to predict the individual seed starch,
protein, and weight. We have developed a seed spectra and weight database as well as novel statistical tools to identify maize ears segregatin g for differences. We are focusing on phenotypes that show dosage-dependent or parent-of-origin changes to the kernels based on the hypothesis that these genes are the best targets for modifying the seed with transgenes. Finally, we developed 454 sequencing protocols to identify the transposon insertion sites in UniformMu mutants. We have used these insertion sites to clone multiple seed developmental mutants. Structural and functional genomics of st rawberry: a gateway to the Rosaceae Folta KM1,2,*, Davis TM3, Clancy MA1,2, Chatterjee M1,2, Bermudez Lozano C1,2, Zhang Q3 1Horticultural Sciences Department, Un iversity of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL 3Department of Plant Biology, University of New Hampshire, Durham, NH The family Rosaceae contains a variety of valuable fruit, nut, ornamental and lumber crops. Most of these species are large tree crops that requir e substantial time and space for propagation. These barriers have hindered genomics progress in this family. Today new challenges such as increased labor costs, wholesale price depression of fruit products, high fuel costs, new pests and a slate of other problems threaten sustained production of rosaceous crops. However, advances in genomics may offer remedies to these problems. Strawberry is a member of the Rosaceae family well suited for genomics study. It has a small genome, grows in limited space, is propagated by runners or seeds, progresses from seed to seed in 12-16 weeks and is routinely transformable. Therefore, the strawberry is an excellent system for understanding structural genomics as well as a rapid system for gene discovery and functional characterization. Our laboratory has participated in the multi-institut ional effort to sequence the genome of diploid strawberry (Fragaria vesca L.). Results indicate a gene density and arrangement much like that of Arabidopsis, as well as substantial colinearity with other major crops. We have characterized the factors that contribute to photoperiodic flowering and other traits of agricultural importance. One key study in our lab functionally assesse s the roles of genes that comprise the Fragaria unknown-ome, novel genes that are exclusive to the Rosaceae and may represent factors that underlie important traits. All of these findings are immediately relatable to other rosaceous crops, increasing our basic knowledge of plant physiolo gy and development, hastening production of new cultivars, and creating better products for consumers.
Poster Session = U.F. Genetics Institute Faculty 1. Peptide YY transgene expressed in murine salivary glands decreases food intake and body weight Acosta A1, Aslanidi G1, Voutetakis A2, Baum BJ2, Zolotukhin S1,* 1Division of Cellular and Molecular Therapy, Depa rtment of Pediatrics, University of Florida, Gainesville, FL 2Gene Transfer and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD Administration of satiety gut hormones, like peptide YY (PYY), is a novel therapeutic approach to reduce food intake in obese human patients. Ho wever, the current delivery methods are invasive and are associated with adverse side effects and low pharmacological adherence. Accordingly, we adopted a gene therapy approach to provide an advantageous and long-term delivery method of PYY to induce satiation and reduce food intake and body weight. Our experimental approach is based on the intrinsic physiological function of PYY to increase satiation without altering appetite. To achieve this goal a transgene encoding prepro-Peptide YY has been designed to target the regulatory granule secretion pathway such that the peptide will be released in response to normal food consumption. To test the regulatory granule secretion mechanism in vitro, we transduced neuro-endocrine-like NCI-H716 cells with a recombinant adeno-associated vector (rAAV) encoding murine pre-pro-PYY (rAAV-PYY) and measured PYY secretion with a radioimmuno-assay. After transfection and differentiati on, cells exhibited a 34-fold increase in PYY secretion under basal conditions; and a 180-fold increase after granule stimulation by meat hydrolysate. We further hypothesized that ta rgeting the salivary glands over conventional endocrine cells in the gut would 1) result in an earlier secretion of PYY and induce earlier satiation, 2) require a lesser amount of vector to achieve therapeutic PYY levels, and 3) provide a favorable intraoral delivery route for vector in jection. Towards this aim, we injected 1010 particles of AAV-PYY, AAV-leptin (positive cont rol), AAV-GFP or saline solution (negatives controls) to submandibular salivary glands of 8 week-old male Balb/c mice. We found that PYY and leptin transgenes decreased food intake compared to either negative control. Furthermore, 24 hour food intake following an overnight fast was lower in mice treated with PYY compared to leptin, indicating increased satiation. PYY treate d mice also had significantly lesser weight gain compared with the saline group 12 weeks after injection. These results indicate that ectopic expression of PYY in salivary glands decreases f ood intake and body weight, and is a potential new treatment for obesity. 2. Generation of transgenic, insect resistant seashore paspalum Altpeter F1,2,*, Neibaur I1,2, Zhang H1,2, Meagher R3, Gallo M1,2* 1Agronomy Department, University of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL 3Center for Medical, Agricultural, and Veterinary Entomology, Agricultural Research Service, U.S. Department of Agriculture, Gainesville, FL Seashore paspalum (Paspalum vaginatum Swartz) is a salt tolerant, fine textured turfgrass used on golf courses in coastal, tropical and subtropi cal regions. Targets for genetic engineering of seashore paspalum include improved disease and insect resistance. However, a genetic
transformation protocol for seashore paspalum was lacking. Our objective was to optimize callus induction and plant regeneration and develop a genetic transformation protocol for this commercially important turfgrass species. A multifactorial experiment showed that callus induction medium containing 3 mg L-1 dicamba and 1.0 mg L-1 BAP had a plant regeneration frequency that was 12 times higher than medium with 3 mg L-1 2,4 dichlorophenoxyacetic acid (2,4 D) alone, and 10 times higher than the combination of 3 mg L-1 2,4 D and 1.0 mg L-1 BAP. However, transgenic plants were regenerated on both 2,4 D and dicamba containing media following biolistic transfer of the hph selectable marker gene and selection with hygromycin. Transgenic plants grew vigorously and did no t show phenotypic differences compared to nontransformed controls. Stable transgene inte gration was confirmed by Southern blotting. Transgenic plants and their vegetative progeny stably expressed the transgene as indicated by RT-PCR. Co-transformation of the selectable marker gene with the insecticidal Bacillus thuringiensis (BT) cry 1F protein will be described as strategy to suppress fall armyworm (Spodoptera frugiperda). 3. Risk assessment of transgenic, apomictic bahiagrass (Paspalum notatum Flugge) Sandhu S1, Blount A2, Altpeter F1,3,* 1Agronomy Department, University of Florida, Gainesville, FL 2Agronomy Department, North Florida Research and Education Center, University of Florida, Marianna, FL 3Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL Bahiagrass (Paspalum notatum Flugge) is a warm-season perennial species which is widely grown in Florida as pasture and utility turf. The commercially important bahiagrass cultivar Argentine is an obligate apomict. Its asexual reproduction resulting in uniform seed progeny could reduce the risk of unintended gene dispersal by pollen. The primary objective of the present study was to investigate pollen-mediated gene transfer from apomictic tetraploid bahiagrass to sexual diploid bahiagrass using glufosinate resistance as a marker. Glufosinate resistant, fertile transgenic bahiagrass was gene rated by biolistic gene transfer and used as pollen donor in a field trial (USDA-Aphis perm it # 05-365-01r) in Marianna, Florida with two replications. Primary transformants and the ap omictic seed progeny was characterized by Southern blot analysis, immuno-chromatograph ic assay and glufosinate application. Openpollinated seeds were harvested from wild type sexual diploid bahiagrass surrounding the transgenic glufosinate resistant bahiagrass at 0.5-2.5 m distance. The average gene transfer from transgenic to non-transgenic sexual diploid bahiagrass was 0.03% (5 hybrids out of 17000 seedlings) under field conditions and 0.16 % (13 hybrids out of 8300 seedlings) under greenhouse conditions. Bar gene integration and expression in the hybrids were confirmed by Southern blot and immuno-chromatographic analys is and herbicide application respectively. All six of the so far analyzed hybrids have been co nfirmed as triploids by both flow cytometry and root-tip chromosome counting. Embryo sac analys is detected both apomictic and sexual embryo sacs, suggesting facultative apomixis. Fertility of hybrids will be evaluated. All triploid hybrids showed reduced vigor compared to diploid or tetraploid bahiagrass. The results suggests that gene transfer between tetraploid apomictic tr ansgenic and diploid sexual non-transgenic bahiagrass can occur under field conditions, although the frequency of hybridization is low as compared to other transgenic cross-pollinating grasses and the hybrids display reduced fitness.
4. Towards positional cloning an avirulence gene from Cronartium quercuum f.sp. fusiforme Anderson C1, Smith KE1,2, Smith JA1, Davis JM1,*, Kubisiak TL2, Nelson CD2 1School of Forest Resources and Conservation, University of Florida, Gainesville, FL 2Southern Institute of Forest Genetics, U.S. Forest Service, Saucier, MS Cronartium quercuum f.sp. fusiforme (Cqf) is the causal agent of fusiform rust on pines and is the most devastating fungal pathogen on pine tr ees in southeastern USA. Genetic resistance is the only economically feasible control method for this pathogen and several pine resistance genes have been identified and utilized in breed ing programs. However, as resistance genes function by recognition of specifically corresponding avirulence genes in the pathogen, successful deployment of resistant varieties will only occur if the corresponding avirulence gene is present at high frequency in the pathogen population. The Cqf avirulence gene, AvrFr1, specifically interacts with the Fr1 resistance gene in loblolly pine and a project has been initiated to isolate AvrFr1 by positional cloning. Four random amplified polym orphic DNA (RAPD) markers have been identified that are linked to the avirulence locus and these define the location of AvrFr1 within a 10.4 cM interval. We are currently searching for additional markers within this interval using fluorescent amplified fragment length polymorphism (AFLP) te chnology to facilitate our positional cloning efforts. AFLP fragments have been amplified using DNA from the parents of a mapping population that segregates for AvrFr1 and from bulks of progeny that either contain, or lack, the avirulence locus. To date, 114 AFLP primer comb inations have been tested using this bulked segregant analysis approach and 3946 amplified fragments have been scored, 198 of which are polymorphic between the parental isolates. Two AFLP markers have been identified that are linked to AvrFr1 and these will now be placed on the genetic map. 5. Evaluation of novel Rosaceae gene function in transgenic strawberry and Arabidopsis Bermudez Lozano CL1, Chatterjee M1, Clancy MA1, Zhang Q2, Davis TM2, Folta KM1,* 1Horticultural Sciences Department, Univ ersity of Florida, Gainesville, FL 2Plant Biology Department, University of New Hampshire, Durham, NH Rosaceae is unique plant family comprised of valuab le species that show a range of differences in form and function. Surprisingly, 10-15% of th e Rosaceae unigene is composed of novel transcripts that have not been functionally descri bed, and are unique to the Rosaceae family. The goal of this work is to leverage the rapid transformation of Arabidopsis thaliana and diploid strawberry (Fragaria vesca) to test for phenotypes associated with overexpression of these novel transcripts. The same constructs were suppressed in strawberry using RNAi. The resulting plants were examined using a defined regimen of phenotypic tests. The results indicate that a subset of the previously unclassified transcripts have potent biological consequences upon overexpression producing an array of phenotypes. Severe defect s in leaf development, trichome emergence, rosette architecture, plant stature, phyllotaxy, and root elongation were observed when these proteins were overexpressed in Arabidopsis. Phenotypes observed are reproducible between independent transgenic lines, although with varying degrees of phenotypic effects. Here a genomics level approach leverages the strength s of high-throughput sequencing, rapid cloning strategies, the most efficient transformation systems and careful physiological analysis to understand the roles for novel expressed genes in this valuable plant family.
6. Detection of seed dormancy quantitative tr ait loci (QTL) in 'Flordaguard' and 'Late Arkansas' Blaker KB, Chaparro JX* Horticultural Sciences Department, Un iversity of Florida, Gainesville, FL Dormancy is a condition that delays or inhibits growth in seed, vegetative buds, and floral buds. Growth resumes in vegetative and floral buds after winter bud dormancy has been met by the accumulation of chilling hours and heat uni ts necessary to break endodormancy and ecodormancy respectively. Seed germination occurs after seed dormancy has been met by sufficient accumulation of chilling hours. The number of genes involved in regulating seed dormancy and their location on the peach genomic map remains unknown. A recent study in our lab suggests that the date of seed germination and the same seedlings vegetative and floral bud chilling requirement are not correlated. Correlati ons have been reported, however, between mean bud chilling requirements and mean seed stratification requirements among families. Segregating F2 seed was collected from a Late Arkansas x Flordaguard F1 hybrid in 2005 and 2006 to study seed dormancy (stratification requirement) in peach. In the first of two experiments performed, all seed were strat ified simultaneously, and seed showing radical protrusion were planted in weekly intervals. In the second experiment, groupings of 50 seed were stratified at four day intervals representi ng 0, 100, 200, 300, 400, 500, 600, and 700 hours of chilling, and were removed from stratificatio n on the same day to be planted. Data on individual seed germination dates and stratific ation treatments were collected. Genomic DNA from resulting plants was screened with SSR markers selected from the Prunus genomic map at an average resolution of 20cM. QTL were dete cted for seed stratification requirement and compared with QTL previously detected in our lab for vegetative and floral bud chilling requirements. 7. Genetic examination of manatee evolutionary dispersal Bonde RK1,2, Kellogg ME2, McCann S3, Pause KC4, Clark AM5, McGuire PM3 1Florida Integrated Science Center, U.S. Geological Survey, Gainesville, FL 2Department of Physiological Sciences, University of Florida, Gainesville, FL 3Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 4College of Marine Science, University of South Florida, St. Petersburg, FL 5Genetic Analysis Laboratory, Interdisciplinary Ce nter for Biotechnology Research, University of Florida, Gainesville, FL Manatees have the ability to establish new popu lations within their subtropical range, as evidenced by their evolutionary history and geneti c traits. Although vicariance separated the taxa over time, the vagility of this unique group of aquatic mammals enabled populations to disperse through either deliberate migration or stochastic events. As temperature plays an important role in manatee fitness, the population size has cy clically fluctuated during climatic events. The phenomenon of population expansion is realized wh en the existing population is large enough to act as a source to populate new habitats. Although adjacent habitats are not always biologically optimal, new populations have persisted, probably due to the ability of manatees to adjust their behavior to adapt to new surroundings. Florida is an example of a more recently established manatee population (within the last 20,000 years) compared to outlier populations such as Puerto Rico and Belize. Previous genetic studie s suggest that Florida manatees have low to average genetic variation and a relatively homogeneous population. This could be explained by their movement patterns and seasonal breeding that result in adequate gene flow between
contiguous areas. Recent analys es suggest that low genetic diversity among all Florida manatees may be intrinsic to the population or is possibly due to inbreeding, a bottleneck event, or founder effect. Though a rapid increase in population size is thought to have occurred in recent decades, it is not known what possible genetic impact this accelerated growth may have on the well-being of the Florida population. Knowledge of the genetic composition of this group will determine whether breeding with parapatric populations is occurring, and may play a role in understanding the population structure by complementing efforts to model various life history strategies. Suitable habitat and the expression of health-rel ated genes will play significant roles in the ability of the Florida manatee population to persist, while facing future challenges of climate change and human population growth. 8. Plasmodium falciparum signal peptide peptidase (PfSPP) as a novel target for malarial chemotherapy Bonilla JA, Bonilla TD, Yowell CA, Dame JB* Department of Infectious Diseases and Pathol ogy, University of Florida, Gainesville, FL The P. falciparum signal peptide peptidase (PfSPP) is an intramembrane-cleaving aspartate protease and a viable therapeutic drug target. The intramembrane-cleaving proteases are unique in that they catalyze peptide bond cleavage in the hydrophobic lipid bilayer of a membrane. In Plasmodium spp. all three major classes of intramem brane-cleaving protease s, metallo-, serineand aspartate proteases, have b een identified. Modeling of PfSPP indicates it is an integral membrane protein that spans the membrane nine times by -helical transmembrane segments and retains the C-terminus in the cytosol and the N-terminus in the lumen. Quantitative RT-PCR and western blot analysis demonstrates that PfSPP is highly transcribed and translated throughout the asexual cycle and most abundan t in the trophozoite and schizont stages. Immuno-fluorescence microscopy localizes PfSPP primarily to the endoplasmic reticulum of the parasite in the trophozoite stage and at the plas ma membrane of the daughter merozoites within a late-stage schizont. Inhibitor st udies demonstrate that inhibition of PfSPP during all stages of development impairs parasite viab ility, and inhibitors specific to secretase activity do not inhibit the growth of the parasite. This important distin ction demonstrates PfSPP is a bona-fide signal peptide peptidase and suggests that the design of specific inhibitors to the parasite SPP versus human SPP and other presenilin-like homologs is feasible. Moreover, SPP is refractory to disruption in P. berghei, highly suggestive of a critical role in the parasite. 9. In situ visualization of the apicoplast and apicoplast nucleoid in asexual stages of Plasmodium falciparum Bonilla TD, Bonilla JA, Dame JB* Department of Infectious Diseases and Pathol ogy, University of Florida, Gainesville, FL Among the most striking sub-cellular morphological features of the Plasmodium falciparum parasite are the elaborate configurations taken on by the developing apicoplast. However, little is known regarding the mechanisms in which this organelle is replicated and divided in Plasmodium. At the culmination of schizogeny, a segment of the apicoplast is partitioned into each newly formed daughter cell. This process is expected to be directed in part by components of the eukaryotic division apparatus, and may also be linked to the structural characteristics and positioning of the nucleiod, to ensure proper segr egation of the apicoplast genome. In this study, combined immunofluoresence and fluorescent in situ hybridization were used to follow both
organellar morphology and nucleoid di stribution in asexual stages of P. falciparum. The DNA gyrase inhibitor ciprofloxacin was used to probe the effects of apicoplast DNA loss on organellar morphology. In untreated parasites, the apicopla st DNA was predominately organized as single discrete nucleoid structures in non-replicating rings and trophozoites. In maturing trophozoite stages when the apicoplast began to elongate, the nucleoid was often visualized as semi-discrete foci at both ends of the elongating apicoplast. As the apicoplast elaborated and began to branch, multiple nucleoids were observed within the apicoplast that varied in shape, size and hybridization intensity, and often appeared to encompass the entire stromal compartment. In ciprofloxacin treated parasites, the nucleoid signal was significantly reduced in size, intensity and number. Furthermore, in the ciprofloxacin trea ted parasites the apicoplast signal was highly fragmented. Also, in many instances the apic oplast DNA did not fully co-localize with the apicoplast. These data indicate that the apicopla st nucleiod is required for proper development and segregation of the apicoplast. 10. Kaposi's sarcoma-associated herpesvirus encodes an ortholog of miR-155 Boss IW1,2, Skalsky RL1,2, Plaisance KB1,2, Riva A1,2,*, Renne R1,2,* 1Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2University of Florida Shands Cancer Center, Gainesville, FL MicroRNAs are small, non-coding RNAs that post -transcriptionally regulate gene expression by binding to 3UTRs of target mRNAs. Kaposis sarcoma-associated herpesvirus (KSHV), a virus linked to malignancies including KS and primary effusion lymphoma (PEL), encodes 12 miRNA genes but only a few regulatory targets are currently known. We found that KSHV-miR-K12-11 shares 100% seed-sequence homology with hsa-miR-155, a miRNA frequently found upregulated in lymphomas and critically important for B-cell development. Based on this seedsequence homology, we hypothesized that both miRNAs regulate genes that are essential for terminal B-cell differentiation and as a re sult, KSHV-miR-K12-11 may mimic hsa-miR-155. Previously, our lab and others have published that ectopic expression of either miRNA inhibited expression of a BACH-1 3UTR luciferase report er, indicating that both miRNAs can indeed regulate identical gene targets. To further examine the role of viral miRNA mimicking miR-155 activities in B-cell differentiation, we identified target genes that are essential in this differentiation pathway. Using bioinformatic approaches in combination with 3UTR reporter assays, we found that CEBP/ ; and PU.1 are both targets for miR-K12-11 and miR-155. Because CEBP/ and Pu.1 have been shown to play essential roles in terminal B-cell differentiation, we suggest that viral miRNA mimics miR-155 in order to regulate post-germinal center differentia tion of B-cells. Ongoing experiments to directly evaluate the effects of KSHV-miR-K12-11 ex pression on B-cell differentiation, using in vivo and in vitro differentiation models, will also be discussed. Together, our findings indicate that KSHVmiR-K12-11 regulates gene targets that are important for terminal B-cell differentiation and in addition may contribute to lymphomagenesis as was shown for its human counterpart miR-155. 11. Using Drosophila melanogaster to investigate the effects of multiple introductions on adaptation to new environments Bouchard FA1, Marcus CB2, Wayne ML2,* 1Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2Department of Botany and Zoology, University of Florida, Gainesville, FL
During introductions to new environments, mala dapted populations must undergo rapid genetic adaptation to avoid extinction. It has been suggested that having multiple, independent introductions to the same region can increase the probability of a population establishing in new environments by increasing the rate of adaptation. Population introductions are difficult to study in the field due to lack of controls, lack of replication, and problems in sampling. In order to better explain the effects of multiple introductions on the rate of genetic adaptation, a series of artificial evolution experiments are conducted in the lab using Drosophila melanogaster. These experiments test the effects of multiple introducti ons from a single and from multiple sources by introducing different patterns of genetically dive rgent lines to a novel environment with high concentrations of ethanol and a control environment similar to the ancestral environment. The effect on mean egg to adult survival is then obse rved for several generations. Preliminary results show evidence that rate of adaptation to the novel environment is enhanced by having multiple sources. This experiment can act as a model for natural population introductions such as those of invasive species, biological control organisms, or endangered species reintroductions, and may help in guiding conservation decisions. 12. Analysis of the transcriptomic response to West Nile virus infection in the equine host Bourgeois MA1, Long MT1,*, Denslow ND2,*, Seino, KK3 1Department of Infectious Diseases and Path ology, University of Florida, Gainesville, FL 2Department of Physiological Sciences, University of Florida, Gainesville, FL 3Department of Veterinary Clinical Sciences Washington State University, Pullman, WA West Nile virus is one of the leading causes of ar boviral encephalitis in the United States. Despite the impact of WNV, little is known about diseas e pathogenesis and host response to central nervous system infections. The overall goal of this project is to gather and use host expression data from tissues of horses experimentally infected with WNV as a model to develop interventional strategies for viral encephalitis. It is hypothesized that there are gene pathways whose expression changes in a consistent manner during WNV infection, disease, and recovery. This hypothesis will be investigated through th e following aims: 1.) Create a tissue specific expression library from CNS tissues and spleen fr om normal horses and those infected with WNV (nave and non-nave), 2.) Create and validat e a custom high density equine brain WNV microarray, 3.) Utilize the WNV microarray to an alyze mRNA expression in the central nervous system and correlate this response to peripheral blood mononuclear cell gene expression during WNV infection, 4.) Develop a model of functional gene expression that predicts survival to WNV infection. The cDNA library from aim 1 has be en sequenced and assembled, revealing 41,040 sequences. BLAST analysis of these sequences identified 31,357 good sequence hits (e<10-4). 73.7% of these sequences had been previously iden tified by equine genome databases. 21.4% of the genes were either missed by EqCab2 predicted genes database or horse genome sequencing project and classified as novel. An equine specific microarray will be created from this library to analyze changes in gene expression which will be subjected to pathway analysis. Relationships between gene expression and WNV survival will be identified, and biomarkers that can lead to rapid identification of neurological disease establ ished. This information will be used to develop new interventional strategies, predict survival, and create better diagnostic tests for WNV and other viral encephalitides.
13. Analysis of locus-specific DNA methyl ation in response to chronic folic acid supplementation and withdrawal in Chinese women Brant JB1, Zhu J1,3, Nguyen D1, Crider K2, Rasmussen S2, Berry RJ2, Hao L3, Li Z3, Maneval D1, Quinlivan E1, Bailey LB1,*, Yang TP1,* 1Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA 3Peking University Health Science Center, Beijing, China The objective of this study was to evaluate the effect of chronic folic acid supplementation and withdrawal on DNA methylation at specific loci in a northern Chinese population. In a doubleblind randomized control trial Chinese women of reproductive age took folic acid (100, 400, 4000 mg/d) for 6 months followed by a 3 month withdrawal. Blood DNA from 5 subjects with the CC and 5 subjects with the TT MTHFR 677CT genotype in the 400 mg/d group were analyzed by methylation-specific PCR (MSP) for changes in DNA methylation status at the promoters of 5 tumor suppressor genes, DAPK, ESR1, HIC1, p 16 and TIMP3, genes known to be aberrantly silenced by DNA hypermethylation of their promoter regions in a variety of human cancers. Overall, no widespread changes in normal DNA methylation patterns (i.e., DNA hypermethylation or hypomethylation) were detected for any of the genes tested in all 10 subjects in response to 6 months of folic acid supplementation and 3 months of folic acid withdrawal. To further assess potential changes in DNA methylation in response to folic acid fortification, high resolution DNA methylation analyses by sodium bisulfite genomic sequencing at additional loci are currently in progress. 14. A novel macrophage-specific restriction of an HIV-1 clone is attributed to multiple domains outside of Env gp120 Bunger JC, Oshier JT, Goodenow MM* Department of Pathology, Immunology and La boratory Medicine, University of Florida, Gainesville, FL HIV-1 infection of macrophages is distinctly differe nt from infection of CD4+ T cells. CD4+ T cells die rapidly upon infection (within days) wher eas macrophages survive for weeks to months. Macrophage survival post-infection leads to long-term production of viral particles and acts as a reservoir for replication-competent virus, even wi th successful antiretroviral therapy. However, the ability of HIV-1 to infect macrophages is not shared by all viruses. While infection of macrophages is traditionally linked with viral entry, evidence from our lab shows that a particular molecular clone of HIV-1 with a macrophage-tro pic Env gp120 enters macrophages, but fails to establish a productive infection [M-restricted]. These observations support a model of a novel, macrophage-specific, post-entry restriction of HI V-1 and leads to the hypothesis that the HIV-1 genome has determinants outside of Env gp120 that are responsible for productive macrophage infection [M-permissive]. Two molecular clones displaying an M-permissive and M-restricted infection, MP and MR respectively, were used to generate chimeric replication-competent clones by restriction digestion. Infection of peripheral-blood mononuclear cells [PBMC] and elutriated monocyte-derived macrophages [MDM] were observed by supernatant p24 ELISA and real-time PCR. Results show restriction of MR is attributed to multiple domains outside of Env gp120 and is specific to macrophages, but not PBMC. Early-events in the viral life cycle, adsorption though reverse transcription, are similar between constructs at early time points. Interestingly, macrophage-produced virions from MR are able to infect PBMC alone, but are unable to infect
PBMC in the presences of infected macrophages. Understanding these unique post-entry interactions between HIV-1 and macrophages are cr ucial to identifying factors responsible for Mpermissive infection. The results from these stud ies may elucidate targets or pathways for novel drug therapies and interactions as well as fu rther our understanding of HIV pathogenesis. 15. Molecular characterization of blue light sensors in strawberry Chatterjee M, Folta KM* Horticultural Sciences Department, Un iversity of Florida, Gainesville, FL Cryptochromes are blue light and UV-A sensing phot oreceptors found both in plants and animals. Cryptochromes regulate diverse developmental pr ocesses in plants, such as inhibition of hypocotyl elongation, anthocyanin accumulation, cotyledon and leaf expansion, and flowering time. In spite of their major role in regulating agronomically important traits like plant stature and flowering time, not much work has been ca rried out on cryptochromes in agriculturally important crops. Genetic manipulation of CRY2 in commercially important fruit crops like strawberry will be of immense significance in regula ting traits like flowering. Thus, we have made an attempt to characterize and functionally validate cryptochrome2 from Fragaria vesca, a system highly related to cultivated strawberry and possibly relevant to the Rosaceae family. Full length FvCRY2 cDNA was obtained by screening a Fragaria vesca seedling library with a partial FvCRY2 as probe. FvCRY2 codes for protein of 645 amino acid residues and bears typical type I photolyase signature in the N-terminal region and a conserved DQXVP-acidic-STAES (DAS) domain towards the C-terminal. Phylogenetic ana lysis grouped FvCRY2 under eudicot CRY2 class and showed maximum resemblance with grape CRY2. FvCRY2 mRNA is ubiquitously expressed in all the tissues tested. Studies are in progress to dissect the exact role of Frageria cryptochrome2 by analyzing the over-expression and antisense transgenics in Arabidopsis and strawberry. 16. Analysis of simple sequence repeat variability to fingerprint major Florida strawberry cultivars Childers KS1, Seigo T2, Peres N2, Chandler CK2, Folta KM3,* 1Environmental Horticulture Department, University of Florida, Gainesville, FL 2Gulf Coast Research and Education Center University of Florida, Wimauma, FL 3Horticultural Sciences Department, Univ ersity of Florida, Gainesville, FL Cultivated strawberry (Fragaria ananassa) is a highly-valued commercial crop, bringing over $200 million annually to Florida alone. Plants are pr opagated in the summer in northern nurseries and then shipped to growers in the southern US for fall and winter cultivation. Recently there have been errors in the distribution of specific cultivars. Because all strawberry cultivars are vegetatively propagated, one lapse in proper phen otyping or labeling can have profound impacts in what is delivered to the farmers fields. In 2007 significant losses were incurred when mislabeled plants were delivered to farms throughout the Southeast, leading to significant losses. The plants flowered at the wrong time and labor was not available to harvest fruit that was produced in the incorrect time window. To avoid th is problem in the future it would be useful to have a reliable means to genotype these plants, so that cultivar identity is not based on phenotype alone. A set of reliable fingerprint s has been developed for a set of the major commercial cultivars grown in Florida based on DN A polymorphisms detected in simple sequence repeats (SSRs). Fluorescently-labeled primers were designed that flank a set of highly-variable set of diagnostic SSRs, and PC R was carried out on genomic DNA from plant lines. Specific
diagnostic DNA fragments representing the fin gerprint were resolved with high-resolution capillary electrophoresis. The results indicate that all major commercial cultivars are clearly discernible using this technique. These data will be extremely valuable to nurseries in cataloging materials and genotyping varieties. Growers ma y use these data to confirm genotypes in the field. Breeders may find these data useful in confirming crosses, and these tools will also be helpful in enforcing intellectual property protection. 17. Subgenome signatures: identifying di ploid contributions to the octoploid strawberry (Fragaria ananassa) genome Clancy MA1, Tombolato DCM1, Davis TM2, Folta KM1,* 1Horticultural Sciences Department, Univ ersity of Florida, Gainesville, FL 2Department of Plant Science, University of New Hampshire, Durham, NH The cultivated strawberry (Fragaria ananassa) maintains an octoploid genome composed of four independently segregating subgenomes. Thro ughout the last century analysis of meiotic configurations indicated that at least one of the subgeomes likely descended from Fragaria vesca, a wild North American diploid strawberry species. The identity of the other subgenomes has not been elucidated. Here genomic signatures defined by a series of unique hypervariable intergenic characters delineate the specific subgenome constituents and make them relatable to the potential diploid ancestors. In this study the order and frequency of single nucleotide polymorphisms (SNPs) and insertion-deletion events (indels) was assessed across several intergenic sequences in a set of cultivars and naturally-occurring diploids. Three trends are evident. The first indicates that at least one of the subgenomes most resembles the diploid Fragaria iinumae, suggesting that this Asian diploid is an ancient contributor to the polyploid. Surprisingly, the SNP and indel patterns were least similar to those of Fragaria vesca, the presumed genome donor to the polyploid. Another subgenome does not match well with diploid accessions, suggesting that it possibly belongs to a now-extinct donor. The results provide an insight into the ancient contributors to modern cultivated strawberry and emphasize the utility of analyzing intergenic regions. The variability in these non-genic areas has utility in molecular mapping and also provides a means to ident ify complex variable regions for comparative genomics studies. 18. Microsatellite libraries: How are th ey made? What can they do for you? Clark AM, Gomez A, Clark HC Genetic Analysis Laboratory, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL The ICBR Genetic Analysis Laboratory (GAL) design s and processes microsatellite libraries for any species requested. Microsatellites are a class of marker that takes advantage of genetic contributions from each parent to design a system for examining gene flow, population structure, paternity, and individual identification. This tech nology can be applied to questions about various aspects of conservation geneti cs, wildlife management, and fore nsics. Building a microsatellite library can be costly and time consuming. GAL uses a method that can be used to enrich the library for repeat DNA fragments or direct sequencing on the Roche 454 platform. The technique currently being used reduces the cost of excess sequencing and cuts the time necessary for creating the original library.
19. Greater expectations of a chorismate mutase gene in the flower of Petunia x hybrida cv Mitchell Diploid Colquhoun TA1, Verdonk JC1, Underwood BA1, Kim JY1, Reinhardt D2, Clark DG1,* 1Environmental Horticulture Department, University of Florida, Gainesville, FL 2Plant Biology, Department of Biology, University of Fribourg, Fribourg, Switzerland In plants the enzyme chorismate mutase (CM) directs the flux of metabolites from the shikimate pathway into the phenylpropanoid pathway by ca talyzing a [3,3]-sigmatropic rearrangement of chorismic acid to prephenic acid. Here we report the identification and characterization of two chorismate mutase cDNAs (PhCM1 and PhCM2) in Petunia x hybrida cv Mitchell Diploid (MD). PhCM1 is transcribed at high levels in floral tiss ues. PhCM1 transcription is up-regulated in MD flowers starting at anthesis and continuing until senescence. Exogenous ethylene treatment induces a rapid decrease in PhCM1 mRNA levels in MD flowers, but not in flowers of the ethylene-insensitive transgenic petunia line 44568 (CaMV 35S-etr1-1). In contrast, PhCM2 mRNA is detected in all plant organs. It is constitutively expressed in a developing MD flower from the earliest bud stage through senescence, when PhCM2 transcription is up-regulated. PhCM2 mRNA levels are unaffected by ethylene treatment in both MD and 44568 flowers. Collectively, these observations categorized PhCM1 as a potential regulator of the substantial metabolite supply required for volatile benzenoid/phenylpropanoid biosynthesis in MD flowers after anthesis. We generated RNAi knockdown lines for PhCM1 (PhC M1R) without affecting the expression of PhCM2. Floral volatile emissions from flowers of T1 plants from independent PhCM1R lines were reduced with differing levels of impact upon benzenoid and phenylpropanoid compounds. In the most severe lines methyl benzoate was reduce d by approximately 60%, phenylacetaldehyde by around 90%, while isoeugenol was not significan tly affected. These results demonstrate that PhCM1 is the principle CM gene responsible fo r the coupling of metabolites from the shikimate pathway to the synthesis of volatile benzenoi ds in MD, and PhCM2 is likely involved in the maintenance of prephenate levels required for vegetative growth and development. 20. High dynamic range (HDR) image proce ssing for visualizing gel-separated PCR products Croxton MD1, Andreu MG1,2 1School of Forest Resources and Conservation, University of Florida, Gainesville, FL 2Gulf Coast Research and Education Center PCC, University of Florida, Plant City, FL PCR products run on a gel and visualized using DNA-binding stains may vary greatly in luminance. Ideally, the image capture process will reproduce all of the bands on the gel, as seen by the viewer under transillumination. In reality, image capture is a carefully balanced function of resolution, bit-depth, and dy namic range (Martinec, 2008, http://theory.uchicago.edu/~ejm/pix/20d/tests/noise/ ). We evaluate the ability of high dynamic range (HDR) imaging software to combine the da ta from multiple image exposures of the same gel and generate an enhanced image that makes weak PCR bands more conspicuous in relation to strong PCR products. A different implementation of gel image combining has been described before (Taylor, 2003, United States Patent 6535624), but no product currently marketed expressly for gel imaging has the features or low price point of gene ral HDR software. These programs have mature user interfaces that are fle xible and relatively easy to use, comparable in practice to the level and contrast adjustments routinely performed on gel images with image editors. We report the types of gel imaging a pplications to which this method may be best applied, and discuss the technical requirements fo r obtaining the best effect with the software.
21. Lupus susceptibility locus Sle1a.1 contains a gene that controls peripheral Treg levels Cuda CC1, Dozmorov I2, Morel LM1 1Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 2Oklahoma Medical Research Foundation, Oklahoma City, OK Patients suffering from systemic lupus erythematosus present with several manifestations, one of which being a decrease in the number of circulatin g regulatory T cells (Tregs). However, it has been shown that the suppressive capacity of the remaining population is maintained. Major lupus susceptibility locus Sle1 in the NZM2410 murine lupus model comprises three independent loci (Sle1a, Sle1b, and Sle1c) and is associated with a decreased level of Tregs as well as decreased expression of the transcription factor Foxp3 among this population preceding autoantibody production. This phenotype is accounted for by Sle1a in a T cell-intrinsic manner. Examination of a truncated region of Sle1a, called Sle1a.1, presented with an intermediate phenotype, indicating the presence of two genes within Sle1a contributing to the observed decrease in Tregs. Both in vivo and in vitro functional studies indicate that the remaining Tregs maintain their suppressive capacity on a per-cell basis. Micr oarray analysis has shown an altera tion of retinoic acid signaling in Sle1a.1 CD4+ T cells. Treatment of Sle1a.1 CD4+CD25cells with IL-2, TGF, and retinoic acid results in a decreased aTreg population when compared to normal B6 CD4+CD25cells. These results indicate that the Sle1a.1 region contains a gene that controls the level of Tregs in the periphery. (Supported by NIH R01 AI1045050 and T32 AR007603.) 22. Proteomic analysis of leaves from alkali grass under salt stress Dai S1 1, Shi Y1, Sun G2, Chen S3,* 1College of Life Science, Northeast Forestry University, Harbin, China 2College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, China 3Department of Botany and Zoology, University of Florida, Gainesville, FL Salinity stress is the most severe environmental st ress that impairs crop production on at least 20% of irrigated land worldwide. Alkali grass (Puccinellia tenuiflora), a salt tolerance monocotyledonous halophyte, is widely distribute d in the saline-alkali soil of the Song-nen plain of northeastern China. The mechanisms underlying the salt tolerance phenotype have been highlighted by previous physiological and transc riptional studies. In this study, we took a comparative proteomics approach. Comparison of 2-DE profiles of leaf proteins between control and 0.4% or 1.0% Na2CO3 treated P. tenuiflora seedlings revealed 62 differentially-expressed protein spots. Fifty of the proteins were identified by MALDI TOF-TOF MS and ESI QTRAP MS. The identified proteins were mainly involved in photosynthesis, signaling, cytoskeleton, stress response, transport, energy and protein metabolism In addition, we determined the physiological characteristics of the leave material, including ph otosynthesis rate, activities of POD, CAT, SOD, and the K+ and Na+ content in the leaves. Collective ly, the results demonstrate that P. tenuiflora can activate its special ion and energy exchange mechanisms to regulate its photosynthesis and other processes in the course of adaptation to alkali salt conditions.
23. Genetical genomics applied to a popl ar pseudo-backcross pedigree associates differential expression of an ADP-ribosyla tion factor gene with leaf morphological variation Drost DR1,2, Novaes E2, Boaventura-Novaes C2, Benedict CI2, Peter GF1,2,*, Kirst M1,2,* 1Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL 2School of Forest Resources and Conservation, University of Florida, Gainesville, FL A primary goal of evolutionary and developmental genetics is to understand the molecular basis for morphological and adaptive differences between species or genera. Phylogenetic and systematic approaches adequately establish evolutionary relationships, but do not identify specific genetic factors contributing to these relati onships. However, novel molecular tools and experimental designs based on interspecific po pulations can be applied to identify genes and sequence elements contributing to phenotypic variance between species. The genus Populus is composed of five diverse evolutionary sections, yet leaf morphological variation is frequently diagnostic of relationships between the species. In a pseudo-backcross pedigree of narrow-leaf Populus trichocarpa and broad-leaf P. deltoides, a significant quantitative trait locus (QTL; Likelihood Ratio [LR] = 28.8) was identified which explained 40% of heritable variation in leaf width and spanned a genomic interval of 650 genes. To reduce the pool of candidate genes, we increased mapping resolution across the QTL interval. Next, we applied a genetical genomics strategy by measuring genome-wide gene expression in expanding leaves from 148 segregants and mapping resulting transcript variation as expr ession QTL (eQTL). An ADP-ribosylation factor (ARF) gene displayed a significant cis-eQTL (LR = 30.3) and was physically localized within the leaf width QTL interval. Furthermore, the ARF mRNA abundance correlated strongly (r2 = -0.37) with phenotypic variation in leaf width. We characterized sequence-level variation within and around the ARF gene sequence among alleles in the cross and identified polymorphisms in the 5 regulatory region that may contribute to the quantitative phenotype. Our preliminary data suggest that diversity in a basic cellular process could underlie evolutionarily relevant phenotypic variation. Additional comparative genomic experiments are expected to identify the specific ciselements that influence this variation in Populus species. 24. Effect of periplasmic nitrate reductase (Nap) on diauxic lag of Paracoccus pantotrophus Durvasula K1, Jantama K1, Fischer K1, Vega A1, Koopman B2, Svoronos SA1 1Department of Chemical Engineering, University of Florida, Gainesville, FL 2Department of Environmental Engineering Sciences, University of Florida, Gainesville, FL Paracoccus pantotrophus expresses two nitrate reductases membrane bound nitrate reductase (Nar) and periplasmic nitrate reductase (Nap). In growth experiments with two denitrifying species (Paracoccus pantotrophus and Alcaligenes eutrophus) that have both Nap and Nar and two species (Pseudomonas denitrificans and Pseudomonas fluorescens) with Nar only, it was found that diauxic lag is shorter for bacteria that express Nap. In P. pantotrophus, napEDABC encodes the periplasmic nitrate reductase. To an alyze the effect of Nap on diauxic lag, the nap operon was deleted from P. pantotrophus. The growth experiments with napmutant resulted in increased diauxic lag when switched from aero bic to anoxic respirat ion, suggesting Nap is responsible for shorter lags and helps in adapta tion to anoxic metabolism after transition from aerobic conditions.
25. Downregulation of 4-coumarate-CoA lig ase toward improve forage quality of bahiagrass Fouad MW1,2, Martin L1,2, Altpeter F1,2,* 1Agronomy Department, University of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL Bahiagrass is one of the most important warm season forage grasses. In Florida alone it is grown on more then 5 million acres most of these acre s support the beef cattle industry. However, the high lignin content in the bahiagrass biomass significantly reduces its forage quality. A key enzyme in the lignin biosynthetic pathway is th e 4-coumarate-CoA ligase (4CL). Reduction of 4CL and other lignin biosynthetic enzymes in various plant species resulted in reduced lignin content that was accompanied with increased digestibility of forage biomass. We cloned four 4CL cDNA`s from tetraploid bahiagrass cv. A rgentine. An RNAi construct ta rgeting a highly conserved domain in two out of the four genes was constructed using 200 bp of the coding sequences. The 4CLRNAi construct under the constitutive e35S promoter or under xylem specific promoter was introduced to bahiagrass callus by biolistic gene transfer. Following regeneration of plants their transgenic nature was confirmed using PCR. Si gnificant reduction of 4CL gene expression was detected in several transgenic lines by Northern blot analysis. Quantitative RT-PCR is currently used to precisely quantify the level of 4CL suppression and will be presented. 26. ZmUrp encodes a predicted splicing fact or and is linked to seed and seedling phenotypes in maize Fouquet R1,2, Fajardo D1,2, Martin F1,2, Settles AM1,2,* 1Agronomy Department, University of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL Seeds develop with coordinated growth of the endosperm and embryo. Cereal kernels have a large, persistent endosperm, which is the stor age tissue of the seed. We identified the rough endosperm3 (rgh3) locus as a mutant disrup ting coordination of endosperm and embryo development. rgh3 is tightly-linked to a Mu1 insertion in a putative U2AF35 Related Protein (ZmUrp). ZmUrp produces multiple splice products with only one variant encoding a predticted protein. The insertion disrupts the ZmURP reading frame suggesting that the rgh3 phenotype may be caused by loss of ZmURP protein. Homo zygous rgh3/zmurp seedlings are stunted and lethal 2-4 weeks after planting. However, in vitro endosperm cultures of rgh3/zmurp mutants are far more proliferative than normal endosperms. Thus, the Rgh3/Zmurp locus is necessary for normal seed and plant development, but rgh3/zmu rp does not reduce cell viability. Our attention is now focused on finding a second mutant allele for zmurp to test the hypothesis that ZmUrp is the Rgh3 locus. We are also in the process of determining the expression pattern of ZmURP at a cellular level as well as testing th e protein for nuclear localization. 27. Genetic control of floral morph in tristylous pickerelweed (Pontederia cordata L.) Gettys LA1, Wofford DS2,* 1Center for Aquatic and Invasive Plants University of Florida, Gainesville, FL 2Agronomy Department, University of Florida, Gainesville, FL
Pickerelweed (Pontederia cordata L.) is a diploid (2n=2x=16) tristylous wetland perennial native to Florida. Populations usually comprise three floral morphs that differ reciprocally in style length and anther height. All flowers are perfect and bear a single style and two sets of three anthers in one of three positions (long, mid and short). Plants bearing flowers with a style in the long position are referred to as L-morphs, while plants that produce flowers with a style in the mid or short position are called Mand S-morphs, re spectively. This floral polymorphism promotes disassortative mating among the three floral morp hs. The goal of this study was to determine the number of loci, number of alleles and gene action controlling floral morph in pickerelweed. Three parental lines (one each of the L-, Mand S-morph) were used to generate S1 and F1 populations. F2 populations were produced through self pollination of F1 plants. Progeny ratios of S1, F1 and F2 generations revealed that tristyly is controlled by two diallelic loci (S and M) with dominant gene action and expression is influenced by epistasis. The S locus is epistatic to the M locus, with the S morph produced by plants with the dominant S allele (genotype S _ _). Plants with recessive alleles at the S locus were either L-morph (ssmm) or M-morph (ssM_). The results of this experiment demonstrate that the inheritance of tr istyly in pickerelweed is the same as previously reported for several tristylous specie s in the Lythraceae and Oxalidaceae. 28. During HSV-1 infection of rabbits, the ability to express the LAT increases latentphase transcription of lytic genes Giordani NV1, Neumann DM2, Bhattacharjee PS2, McAnany PK1, Hill JM2, Bloom DC1,* 1Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2Department of Ophthalmology, Louisiana State Un iversity Health Sciences Center, New Orleans, LA HSV-1 recombinants with deletions of the LAT promoter and/or the 5 LAT exon region are severely restricted in adrenergically-induced reactivation in the rabbit eye model. In the present study, we determined that deletion of the LAT pr omoter alters the transcriptional status of the latent HSV-1 genome in latently-infected rabbits. Trigeminal ganglia from rabbits infected with either wild-type HSV-1 or the LAT promoter deletion mutant, 17 Pst, were assessed for their viral chromatin profile and transcript abundance during latency (>28 days post-infection). The wildtype 17syn+ genomes were approximately 2-fold more enriched in the transcriptionally permissive mark, dimethyl H3 K4, than the 17 Pst genomes at the 5 exon and ICP0 and ICP27 promoters. RT-PCR analysis revealed averages of at least 3-, 35, and 154-fold more RNA for ICP4, tk, and gC, respectively, for 17syn+ when compared to 17Pst. Our findings regarding the transcriptional status of a LAT-negative mutant during latency in the rabbit are in sharp contrast to those observed for the mouse, where the LAT appears to play a role in repression of the latent HSV-1 genome. Our results seem to indicate a difference in latent HSV-1 viral regulation between the rabbit and the mouse, and they suggest that in the rabbit the LAT does not act as a repressor but instead acts to keep the genome in a more transcriptionally permissive state. 29. Identification of subcellular distribution of the Arabidopsis 14-3-3 proteins using isoform specific antibodies Gokirmak T, Paul AP*, Ferl RJ* Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL
The 14-3-3 proteins are ubiquitous eukaryotic prot eins that are present as multiple isoforms and localized throughout the entire cell. The 14-3-3s are involved in a diversity of protein-protein interactions, which allows them to mediate a range of biological functions. Subcellular localization of specific 14-3-3s can provide important clues to possible roles of individual 14-3-3 family members. Arabidopsis cell suspensions were used to create sub-cellular and sub-nuclear fractions. Protein was isolated from cellular fracti ons comprising cell wall, whole cell, cytoplasm, whole nuclei, nucleoplasm and nuclear halos, and then used in westerns with isoform-specific 143-3 antibodies. The results were used to drive immunofluorescence experiments in Arabidopsis (Col-0) root tissues and protopla sts prepared from Col-0 suspensi on cell cultures. Isoform-specific antibodies were labeled directly wi th Fluors Alexa 488 or Alexa 568 and used in conjunction with laser scanning confocal microscopy (LSCM). The immunolocalization results were consistent with the results of the western analyses. The 14-3-3isoform is predominantly localized in the nuclear halos. The 14-3-3, 14-3-3, 14-3-3, 14-3-3, 14-3-3and 14-3-3isoforms are present both in nucleus and cytoplasm. These isoforms are shown to be localized to both nucleoplasm and nuclear halos. In situ fluorescent localization with labeled antibodies showed that 14-3-3and 14-3-3isoforms are compacted in the nucleus. On the other hand 14-3-3, is localized throughout the whole cell. The 14-3-3isoform is mainly localized in the cytoplasm, but it is also present in the nucleus. 30. Dynamics of HIV-1 diversity determines disease progression throughout natural infection Gray RR1, Salemi M1,*, Sleasman JW2, Goodenow MM1,3,* 1Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 2Department of Pediatrics, University of South Florida, Tampa, FL 3Division of Immunology, Rheumatology, and Infe ctious Disease, Department of Pediatrics, University of Florida, Gainesville, FL The clinical course of HIV-1 infection is genera lly well defined. However, the time from infection to development of AIDS varies among individuals the reasons for which have not been clearly elucidated. Two prevalent markers of disease pr ogression, viral load and CD4 cell count, have been shown to be correlated, although the causal relationship between the two is unclear. In this study, we investigated the longitudinal relation ship between CD4%, viral load, and viral diversity in nine pediatric patients prior to initiation of combination therapy. We estimated viral diversity using effective population size (Ne), an evolut ionary statistic that measures the number of effectively infectious virions within an individual. Skyline plots, which estimate the change in Ne over time, were compared with CD4% and viral lo ad throughout the course of natural infection for each patient. We present a model which provides in vivo evidence of Ne as the causal link between the two most common clinical indicators of disease progression, viral load and CD4%. Furthermore, because the increase in Ne precedes both the decline in CD4% and the increase in viral load, this measure is an earlier indicator of progression towards AIDS and as such, could be used to assess the optimal start of drug ther apy. Our results suggest that monitoring viral diversity should be considered as an important fe ature of clinical monitoring. Finally, this study provides a framework under which additional qu estions concerning HIV-1 pathogenesis can be addressed.
31. Identifying the components of regulatory divergence in D. simulans and D. mauritiana Graze RM1, Nuzhdin SV2,3 1Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2Center for Population Biology, Section of Evolut ion and Ecology, University of California, Davis, CA 3Section of Molecular and Computational Biology, De partment of Biological Sciences, University of Southern California, Los Angeles, CA Genetic variation in gene expression patterns may be caused by sequence variation in cis regulatory regions of a particular focal gene or the causal changes may result from sequence changes elsewhere in the genome (trans). We analyzed whole genome expression in D. simulans and D. mauritiana and their F1 hybrids to partition divergence in gene expression to cis and trans components. Allele-specific expression was measur ed for a set of species specific probes on the Drosophila 1.0 Affymetrix GeneChip. We first used the distribution of negative controls on the array to choose probes which hybridize in one of th e species, but have very little or no residual hybridization in the other. Statistical tests for cis and trans effects were constructed on the basis of a simple conceptual model for partitioning gene expression to cis trans and cis by trans components. We then tested for significant deviations from predicted relationships between parental or hybrid total gene expression and hybrid allele-specific expression by contrast analysis. While previous studies have found many more cis based differences in more distantly related species pairs, our results support a greater number of trans rather than cis effects on gene expression in this closely related species pair. 32. Cellomics Green L, Benson N, Kang B-H Cellomics Division, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL The Cellomics division of ICBR is comprised of the following research laboratories: hybridoma, flow cytometry, electron microscopy and bioima ging. This division is devoted to assisting investigators in the study of cell structure, func tion and generation and application of cellular products. The main focus of the hybridoma resear ch laboratory is developing new monoclonal antibodies. These antibodies can be used in many applications such as ELISA, western, IP, Co-IP, ChIP, immunoaffinity chromatography, flow cytome try, IHC, EM. The flow cytometry research laboratory offers services that are used for detecting cell surface or intracellular proteins, measuring apoptosis, cell cycle, cellular physiology The cell sorters available in this lab allow for isolation of specific populations of cells which can be put back in culture or used for single cell PCR. The electron microscopy and bioimaging laboratory provides ba sic services in both transmission and scanning electron microscopy. In addition, confocal laser scanning microscopy and basic optical microscopy services are also available. Recent acquisition of a high-pressure freezer and freeze-substitution system allows pres ervation of cell/tissue samples close to their native state. It is possible to visualize fine structures in the samples with a nanometer-level resolution.
33. Plastid interactions with the nucleus in leaf blade development Gustin J, Black J, Tseung CW, Settles AM* Horticultural Sciences Department, Univ ersity of Florida, Gainesville, FL The flat leaf blade of higher plants is an impo rtant adaptation for efficient photosynthesis. The leaf blade provides a porous tissue with large surface area for light capture and gas exchange. Growth of the blade is not yet fully understood, but key developmental events include polar flux of the phytohormone auxin, definition of the ad axial-abaxial (top-bottom) domains of the leaf, and regulated cell proliferation in the mediolateral (midvein-margin) axis. There is strong evidence that plastids have an integral role in leaf blade development. Plastids are cellular organelles required for cell survival. Many essent ial biosynthetic pathways are localized to the plastid, and the chloroplast is the site of phot osynthesis. In addition, mutations in multiple plastid-targeted and plastid-en coded genes suggest a specific role for this organelle in multicellular development. The underlying connection between the plastid and leaf morphogenesis is not known. We have identified a maize mutation that impacts development in multiple tissues including distinct narrow leaf and rough endosperm (nlr1) phenotypes. The nlr1 mutant reduces blade expansion and the number of chloroplasts in leaf cells. Based on these phenotypes, our central hypothesis is that the Nlr1 gene mediates the communication between the plastid and nucleus required for mediolateral growth. We have isolate an nlr1-linked transposon insertion from a Robertsons Mutator (Mu) flanking sequence tag. This transposon disrupts a gene encoding a Type C, J-domain protein that is specific to photosynthetic eukaryotes. J-domains activate Hsp70 ATPase domains and function in numerous protein complexes in the cell. Transient expression of a J-domain protein:GFP fusion in Arabidopsis mesophyll protoplasts indicates that the J-protein is not a plastid-localized protein. Based on these data, we propose that the Nlr1 gene mediates the communication between the plastid and nucleus required for mediolateral growth. 34. Characterization of a novel protein family involved in metal ion metabolism Haas CE, de Crcy-Lagard V* Department of Microbiology and Cell Science, University of Florida, Gainesville, FL Zinc is an essential cofactor for numerous proteins where it serves both structural and catalytic roles. The proper functioning of living cells depends on the acquisition of this metal while, at the same time, an overabundance of zinc can be toxic. The uptake mechanisms and efflux pumps employed by cells and the regulation thereof to maintain the intracellular concentration of zinc have been previously characterized. Conversely, little if anything is known regarding zinc trafficking within the cell, especially when the ce ll is faced with a zinc-limited environment like that found by pathogens during the initial stages of infection. After an extensive comparative genomic analysis of the COG0523 protein family, we have hypothesized that the members of this family are metal chaperones required for insertin g metal into essential metal-dependent proteins when cellular metal-ion concentration is low. Investigation into the role of YeiR, a member of COG0523 found in Escherichia coli, has further led us to theorize that this family of proteins is involved in zinc metabolism. Preliminary evidence supporting these hypothes es will be presented.
35. Phototropin 1 contributes to UV-C induced stem growth responses Hamner M1,2, Humphrey E3, Shinkle J3, Folta KM1,2,* 1Horticultural Sciences Department, Un iversity of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL 3Department of Biology, Trinity University, San Antonio, TX Plants maintain a suite of sensitive photosensors that monitor the light environment and adjust plant form and function to best match ambient co nditions. Analysis of the phototropin1 (phot1) receptor shows that UV-C (250-300 nm) illumination leads to alternative phosphorylation patterns than those induced by UV-A or blue light, yet no phot-dependent UV-C effect on plant physiology has been described. The rapid inhibition by blue light is mediated by the phot1 blue light sensor, so genetic and physiological tools exist to assess the role of UV-C and phot1 in this response. Stem growth kinetics were measured in cucumber and Arabidopsis thaliana using highly-sensitive monitoring of seedling growth rate. Here dark-gro wn seedlings were treated with a short, single pulse of UV-C illumination. The results of kinetic analyses sh ow that UV-C induces a rapid decrease in seedling growth rate. The fluence-resp onse kinetics and time course were similar to that observed following a blue pulse, only wi th a more pronounced recovery phase before reaching a terminal growth rate. In Arabidopsis the results were similar in cryptochrome and phytochrome mutants, genotypes that have de fects in sensing blue and red/far-red light, respectively, suggesting these receptor classes do not contribute to this response. However, the phototropin mutants exhibited defects in stem gr owth rate inhibition, achieving only 50% of the wild type response. The remainder of the inhibition could not be removed by testing genetic mutants, suggesting that it is under the direct ion of a novel sensor or redundant function. The work demonstrates that UV-C has an effect on a rapid plant photomorphogenic response and that the response is partially mediated by the phototropin1 photorecepto r. These findings could become increasingly relevant with a changing atmosphere or during plant cultivation in space. 36. The basis of gene specific activation by EBV SM protein Han Z1,2, Coulter C3, Dittmer DP3, Swaminathan S1,2 1University of Florida Shands Cancer Center, Gainesville, FL 2Department of Medicine, University of Florida, Gainesville, FL 3Lineberger Comprehensive Cancer Center, Unive rsity of North Carolina, Chapel Hill, NC Epstein-Barr virus (EBV) is a herpes virus that infects and persists in over 90% of the adult human population and is associated with a number of human malignancies such as Burkitts lymphoma, Hodgkinss lymphoma and nasopharyngeal carcinoma. The EBV SM protein is a posttranscriptional regulatory protein expressed early during lytic replication an d is essential for virus production. SM is an RNA binding protein which enhances accumulation of its target mRNAs but its exact mechanism of action remains to be determined. We had previously shown that SM enhances accumulation of some EBV transcripts over others. However the basis of such specificity has not been investigated. Understandin g the basis of gene specific activation by SM should provide insights into the regulation of lyt ic EBV replication and opportunities for specific therapeutic interventions. This study is aimed at determining the basis of specific RNA recognition by SM. To ask whether SM associated more efficiently with specific EBV transcripts, we employed an RNA immunoprecipitation/RT-PCR assa y. We used cell lines derived from lymphomas infected with EBV that have been modified to permit high le vel lytic EBV replication in an inducible manner.
Cells were induced to permit lytic replic ation, lysed and SM/RNA complexes were immunoprecipitated with SM antibody. RNA was isolated from each immunoprecipitation and analyzed by RT-PCR microarray for all EBV ORFs. We found that there is a general enrichment of EBV RNA in SM-immunoprecipitates, suggesting that SM has some non-specific RNA binding capability. However, there were several RNAs wh ich were highly enriched by SM, suggesting that SM does bind preferentially to specific RNAs. On e of the most highly enriched RNAs was encoded by the BFRF3 gene. In order to map high affinity SM-binding sites on BFRF3 RNA, protein-RNA crosslinking assays were employed to compare th e affinity of SM for various portions of the BFRF3 RNA. This assay utilized constructs that sp an different regions of BFRF3 from the 5UTR to the cleavage and poly-adenylation signal to genera te radiolabeled transcripts. The results of these studies will be presented. 37. Variations in experimental pain sensitivity and the A118G single nucleotide polymorphism of the mu opioid receptor (OPRM1) gene in a multi-ethnic sample Hastie BA1, Herrera DG1, Gladin W1, Herbstman D2, Wallace M2,*, Fillingim RB1,3 1Department of Community Dentistry and Behavioral Science, Universi ty of Florida, Gainesville, FL 2Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 3Malcom Randall V.A. Medical Center, Gainesville, FL Vast inter-individual variation in pain sensitivit y has been established although the distinct mechanisms explicating these differences have yet to be understood. Pain genetics suggests that pain perception may be partially mediated by single nucleotide polymorphisms (SNPs) of identified pain genes. Our previous data demo nstrated significantly higher pressure pain thresholds among those with the rare allele of A118G SNP on the OPRM1 gene. Allele frequencies at this locus vary by ethnicity, ranging from 20-30% in the general population, and may have different functional consequences across races. We sought to identify ethnic differences in allelic associations with pain sensitivity and to examin e race x sex interactions relating to genetic influence on pain responses. 249 healthy young adults (47.4% female) from three ethnic groups (81 African Americans/AA; 79 Hispanics/H; and 89 non-Hispanic whites/W) underwent testing with multiple experimental pain modalities. Genotyping of the A118G SNP was conducted and associations with pain response s, race and sex were analyzed. Few AA had the rare allele (10%) versus comparable frequencies in W and H (28. 1% vs 30.4%, respectively). Across the entire sample, effects for genotype alone were not significant, but analysis of standardized pain scores in each ethnic group revealed significant genotype effects for most pain modalities among W but not H or AA (Ps<.05). Specifically, the 118G alle le was associated with lower pain sensitivity among W but the opposite trend emerged in H. These findings demonstrate an ethnic-dependent association of OPRM1 genotype with pain sensitivit y that is independent of sex. The reasons for this pattern of results are not clear; however, it seems plausible that the A118G could be a tag SNP, and the true functional SNP may show differing allele frequencies across ethnic groups. Additional research is warranted to underco ver the mechanisms influencing these ethnicdependent and genetic associat ions with pain sensitivity. 38. Functional characterization of Arabidopsis isopropylmalate isomerases implicated in the biosynthesis of methio nine-derived glucosinolates He Y1,2, Zhu M2, Alvarez S2, Abraham L2, Chen, S1,2,3,* 1Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL 2Department of Botany and Zoology, University of Florida, Gainesville, FL
3Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL Methionine-derived aliphatic glucosinolates are a major class of secondary metabolites produced by plants in the order of Capparales including Arabidopsis. Enzymes that catalyze the methionine chain-elongation cycle in the biosynthesis of alip hatic glucosinolates with different chain-lengths show a close evolutionary relationship with those involved in leucine biosynthesis. Here we have identified AtLeuC and three AtleuD genes, predicted to encode the large subunit and the small subunits of isopropylmalate isom erases (IPMIs, EC 22.214.171.124), re spectively. Gene co-expression analysis showed that AtLeuC and AtleuDs are co-regulated with nearly all the known genes in aliphatic glucosinolate biosynthesis In addition, ProAtLeuC:GUS expr ession analysis revealed that AtLeuC was predominantly expressed in vascular tissues, a typical pattern for genes known to function in the formation of aliphatic glucosinolates. Mutation of AtLeuC in Arabidopsis led to a dramatic reduction of glucosinolates with C4 and longer side-chains and an increase of C3-short chain glucosinolates. This phenotype can be restored by the reintroduction of a functional AtLeuC gene driven by its native promoter. Moreover, AtLeuC KO mutant displayed a significantly accumulation of S-methylmethionine (SMM), while leucine/isoleucine and other free amino acid levels were not significantly affected in the mutant. Biochemical analysis of AtLeuC and AtleuDs are currently under way and the results will be presented. 39. The Interdisciplinary Center fo r Ongoing Research/Education (ICORE) Partnerships and the STEP program Hightower L1, Baker L1, Irani T1, Gallo M2,*, Myers B1, Telg R1 1Department of Agricultural Education and Communi cation, University of Florida, Gainesville, FL 2Agronomy Department, University of Florida, Gainesville, FL The Interdisciplinary Center for Ongoing Research /Education (ICORE) Partnerships is a 5-year project at the University of Florida (UF) funded by the Howard Hughes Medical Institute that introduces high school teachers to cutting-edge research methods that focus on an emerging pathogens theme, as well as state-of-the-art teaching methods. The UF Scientific Thinking & Educational Partnership (STEP) program is wo rking with the UF Center for Precollegiate Education and Training (CPET) on this project to develop an evaluation protocol, as well as a social networking wiki, that allows participants to learn from each other, and the experts, during the project and after the project has been complete d. During the initial year, participants were asked to evaluate their ICORE Partnerships 2-week workshop experience. They were asked what skills and knowledge they gained, their perceptions about how the project was conducted, and what they planned to do with the skills and kn owledge after the experience was over. The top rated skills and knowledge gained among partic ipants were related to pathogens, health, economics, and real world problems. The second rated skill was new lab protocols, and the third was how to use an on-line discussion forum. When participants were asked about their overall perceptions of how the project was conducted, the top ranked response was that they experienced an energy or excitement for lear ning that could be used in the classroom, the second ranked perception was that there was an extensive amount of preparation that had been made to develop the workshop. Though the majori ty of the responses were positive in nature, the third ranked perception of the program was the lack of Internet connectivity during the workshop on the UF campus.
40. Increase in neuron production in the hippocampus and substantia nigra in the adult brain during the dark phase of the circadian cycle Hoang Minh LB1, Jones BE1, Palmer TD2, Ormerod BK1,* 1J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL 2Department of Neurosurgery, Stanford University, Stanford, CA The entire adult brain has regenerative potential in the form of neural progenitor cells. A large body of data shows that significant neurogenesis exists in the hippocampus and olfactory bulbs of all mammals, including humans. Reports of lo w-level neurogenesis in other brain regions are controversial, such as in the substantia nigra which is the primary site of degeneration in Parkinsons patients. Previous studies have demonstrated that mice generate significantly more neurons during the dark phase of the light/dark cycle than during the light phase during which they are generally less active. We tested wh ether new cells born in the hippocampus and substantia nigra were more likely to generate ne urons during the dark phase of the circadian cycle. Adult female mice exposed to running wheels were injected with the cell synthesis marker bromodeoxyuridine (BrdU; 50mg/kg) once per day fo r 6 days, either 2 hours after lights on or 2 hours after lights off. Control groups were not exposed to running wheels. Sections were processed immunohistochemically so that total new cell number could be estimated stereologically and new cell phenotype assessed using confocal microscopy. Our results replicate previous work showing that more hippocampal ne urons are generated during the dark phase of the cycle, whether the mice were exposed to a running wheel or not (p<0.05). There was also a significant increase in the number of new cells in the substantia nigra during the dark phase, whether the mice were exposed to a running wh eel or not. Very few new cells exhibited a dopamine neuron phenotype. 41. LSD1-mediated epigenetic modification is required for TAL1 function and hematopoiesis Hu X1,2, Li X1, Ybarra R1, Valverde K1, Fu X2, Noguchi C3, Qiu Y4, Huang S1,5,* 1Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2Edmond H. Fischer Signal Transduction Laboratory, College of Life Sciences, Jilin University, Changchun, China 3Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 4Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 5University of Florida Shands Cancer Center, Gainesville, FL TAL1/SCL is critical for normal and abnormal hematopoiesis by regulating hematopoietic stem/progenitor cell growth and differentiation. Howe ver, it is still unclear how its transcriptional activities are controlled during hematopoiesis. He re, we undertook the biochemical isolation of TAL1-associated protein complexes in erythroleuke mia cells and showed that TAL1 interacts with histone demethylase LSD1 complexes cont aining LSD1, CoREST, HDAC1 and HDAC2. Interestingly, although TAL1 specifically colocalizes with LSD1 at the target gene promoter p4.2 in undifferentiated MEL cells, the recruitment of LSD1 is decreased at the p4.2 promoter upon induced MEL differentiation indicating that LSD1 may differentially regulate TAL1 target genes during differentiation. The siRNA-mediated knockd own of LSD1 in MEL and ES cells resulted in the derepression of p4.2 by increasing dimeH3K4 at its promoter region, respectively. Finally, we demonstrated that TAL1-associated LSD1 complexes, H3K4 demethylase, and histone
deacetylase activities are coordinately regulated during erythroid cell differentiation. Thus, the data suggest that LSD1 mediated epigenetic modification may affect hematopoiesis and leukemogenesis through its association with th e lineage-specific transcription factor TAL1. 42. Post-translational modification of subunits from 20S proteasomes of the haloarchaeon Haloferax volcanii Humbard M, Zhou G, Maupin-Fulow J* Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 20S proteasomes are a central protein degradation enzyme found in archaea, eukarya, and some bacteria. 20S proteasomes are made up of su bunits from two different superfamilies, and Haloferax volcanii encodes one and two subunits, 1 and 2. Both 1 and 2 are Nterminally acetylated. A small subset of 1 subunits is not modified by acetylation; rather the initiating methionine has been removed creating a new N-terminus that begins with a glutamine, Q2. Site-directed mutagenesis of the codon for the second amino acid created 1 Q2A, Q2D, Q2P, Q2S, Q2T, Q2V, and deletion of the N-terminal helix created 1 2 12. Whole cell activities of N-terminal acetyltra nsferases and methionine aminopep tidases were probed with the 1 variants. The 1 variants had different acetylation and cleavage patterns than unmodified 1. Q2A was completely acetylated after the initiator methionine was removed, 1 2 12 was not modified by either activity, while Q2D, Q2P, and Q2T were a mixture of acetylated, methionine removed, and unmodified proteins. All three subunits of the 20S core particle and an associated ATPase are also phosphorylated. In addition to phosphorylation and acetylation, the 1 subunit is also methylated. The roles of the post-translational modifications are currently unknown. Understanding the roles of these post-translational modifications will further our understanding of proteasome regulation in an ubiquitin-free system. 43. Gravitropic responses of peanut pegs James-Hurr VA1, Tillman BL2, Gallo M1,3,* 1Agronomy Department, University of Florida, Gainesville, FL 2Agronomy Department, North Florida Research and Education Center, University of Florida, Marianna, FL 3Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL Cultivated peanut (Arachis hypogaea L.) is a valuable food and oilseed crop that relies on correct perception and response to gravity for completion of its reproductive cycle. Fertilized ovules in the tip of the elongating gynophore, or peg, must be carried downwards into the soil, where fruit formation is completed. We have taken advantage of the peanut pegs ability to elongate and respond to gravity when cultured in vitro to study the gravitropic response of this unusual reproductive organ. Our results implicate auxin efflux in the correct gravitropic response of peanut pegs and show that at least one member of the pin-formed (PIN) gene family of auxin transport proteins is expressed in aerial pegs. Further characterization of auxin redistribution during gravistimulation and the role of PIN pr oteins in the gravitropic response will provide valuable insights into the mechanisms of altered gravitropic responses and potentially lead to manipulations to improve seed-set and yield.
44. Genome size of Metaseiulus occidentalis (Acari: Phytoseiidae) among smallest known in arthropods Jeyaprakash A, Hoy MA* Department of Entomology and Nematology University of Florida, Gainesville, FL The genome size of Metaseiulus occidentalis needs to be estimated before whole genome sequencing is done. Segments of actin and EF1-alpha sequences were amplified from M. occidentalis using degenerate primers and sequenced. This allowed designing species-specific primers. Both actin and EF1-alpha sequences were amplified by high-fidelity quantitative realtime PCR from M. occidentalis and a plasmid carrying these sequences. Comparing the amplifications allowed us to estimate the genome size of M. occidentalis as 88 Mb. When compared to other arthropod genomes, this appears to be very small. 45. The Escherichia coli yjfP gene encodes a carboxylesterase involved in sugar utilization during diauxie Johns N, Valladares R, Wrench AP, Gonzales CF* Department of Microbiology and Cell Science, University of Florida, Gainesville, FL The Escherichia coli gene, yjfP, encodes a 249 amino acid protein that belongs to the / fold hydrolase superfamily with potential esterase/prote ase activity. The amino acid sequence of YjfP contains the classical serine hydrolase signat ure motif. The highly conserved amino acids Ser115, Asp197, and His231 are fully conserved and in the right cont ext to form the catalytic triad at the active site of the protein. The potential stru cture of the catalytic pocket was confirmed by analysis of the structure of the closely rela ted homologous protein TT1662 (1UFO PDB code). YjfP gene was cloned and a ~28 kD YjfP-His6-tagged protein was overexpressed and purified by Ni-affinity chromatography. The purified homodime ric protein displayed carboxylesterase activity on several model substrates with classical hyperbolic saturation kinetics and high affinity for short acyl-chain esters. An analysis of gene expression by qRT-PCR and Western blot analysis showed peak expression of yjfP during diauxic lag when the cells were grown on a mixture of glucoselactose. No drastic effects on bact erial growth were detected in the yjfP strain. However, the diauxic lag of the mutant was to some extent retarded and the -galactosidase activity was detected later and at half the magnitude to that observed in the WT strain. The growth curve of the wild type and over producing strain were co mpared in several carbohydrate substrates. The YjfP overproducing strain showed a long lag phase (6 hrs) when the cells were cultured with lactose or galactose as sole carbon sources. The results obtained suggest that YjfP is a homodimeric short acyl-chain carboxylesterase with direct effects on sugar utilization. 46. Enabling high performance computing for the UF genetics research community through a web-based BLAST portal Kadirvel S1, Matsunaga AM1, Tong Y2, Chen K1, Yu F3, Liu L3, Farmerie WG3,*, Fortes JAB1 1Department of Electrical and Computer Engineering, University of Florida, Gainesville, FL 2Department of Computer and Information Science and Engineering, University of Florida, Gainesville, FL 3Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL
The explosive growth in genomic data has made the use of High Performance Computing (HPC) essential in the field of bioinformatics. The Ad vanced Computing and Information Systems (ACIS) Laboratory and Interdisciplinary Center for Biot echnology Research (ICBR) have devised a BLAST portal making the powerful computational capacity of the University of Florida (UF) HPC Center available to the UF genetics research community Designed with an emphasis on usability, our BLAST portal uses a familiar web-based interfac e allowing users to submit batch-scale BLAST jobs and retrieve BLAST results to their desktop. Our approach is a user-transpare nt middleware solution that allows the use of customized user databases in addition to the st andard NCBI databases in executing large and highly configurable BLAST jobs. The involved sequence of actions required to utilize an HPC resource such as job creation, resource estimation, job submission, progress monitoring, and results compression and retrieval are performed behind the scenes allowing the user to easily track job status and completion through log messages in the web fr ont-end and through email notifications. The success rate of job completion is improved by tr ansparently splitting large jobs into small-chunk jobs each of which acts on a subset of the inpu t queries. This approach has the added benefit of faster job completion times through decrease d queue wait-times and the ease of handling smaller result files. Furthermore, job failures are handled by an automated anomaly detection, correction and recovery scheme that requires no user intervention. This approach and middleware could be easily extended to additional applications and adapted to alternate HPC systems that rely on PBS-like submission and scheduling of jobs. 47. Electron microscopy analysis of maize basal endosperm transfer cells Kang B-H1,2, Xiong Y1, Williams DS1, Chourey P3,* 1Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 2Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 3Plant Pathology Department, Univers ity of Florida, Gainesville, FL As heterotrophs, developing maize seeds are dependent on mother plant for their carbon and nitrogen needs. Upon symplastic transport to phlo em termini in pedicel and a short distance postphloem transit, photoassimilates are unloaded in to growing maize seed by the basal endosperm transfer cells (BETCs). A ubiquitous feature of all transfer cells is labyrinth wall, the wall-ingrowth (WIG), which increases the plasma memb rane area and is believed to confer greater transport capacity. The symplastic discontinui ty from the mother plant necessitates much membrane transport capability and maternal-filial signaling at the BETCs. The functional importance of the BETCs is best evidenced by the Mn1-encoded cell wall invertase that localizes entirely and exclusively to these cells and abort ive seed development by loss of functional Mn1 gene. To better understand the formation of this crucial cell layer, we carried out electron microscopy imaging analyses of wild-type and mutant maize seeds preserved by high-pressure freezing and freeze-substitution. The WIG growth begins at 8 days after pollination (DAP) and remains active until 12 DAP. During this period, numbers of Golgi stacks and multivesicular bodies increase and multiple types of vesicles concentrate to the sites of WIG growth suggesting that polarized secretion contributes to WIG formation. Mitochondria accumulate at the basal side prior to WIG growth and enter interstices between the WIGs as the WIGs become elaborated. Endoplasmic reticulum (ER) is excluded from th e WIG but ER cisternae permeate rest of the cytoplasm thoroughly, anchored to that side an d apical walls by numerous plasmodesmata. When Mn1-encoded cell wall invertase was localized by immuno-electron microscopy, immunogold particles were seen in the WIGs but not in the primary cell wall. Disrupted WIG formation and organelle architecture in mn1 and sh1sus1-1 mutants indicate that monoand disaccharide conversion is essential for the BETC development. The mn1-1 mutant BETCs display abnormal
elaboration of the plasmamembrane without wa ll ingrowth and ER swelling. We are currently isolating BETC-transcripts to identify genes involved in the secretory/endocytic activities and photosynthetate metabolism in the BETC. 48. Muscular degeneration does not augmen t the ALS phenotype in SOD1 transgenic mice Karch CM, Borchelt DR Department of Neuroscience, University of Florida, Gainesville, FL Amyotrophic lateral sclerosis (ALS) is a late onset, neurodegenerative disease that results in progressive paralysis and death. Transgenic mice that overexpress human, mutant Cu,Znsuperoxide dismutase (SOD1) develop hindlimb paralysis similar to AL S patients. In SOD1 transgenic mice, SOD1 aggregates are detected in motor neurons and absent in oligodendrocytes and astrocytes. In symptomatic SOD1 transgenic mice, B crystallin is upregulated in oligodendrocytes and astrocytes. Thus, we sought to study the effect of B crystallin on SOD1 aggregation and disease progression in cell culture and the mouse model for ALS. In cell culture, we found that B crystallin reduces aggregation of mutant SOD1. Because B crystallin is expressed in cells that do no t form aggregates, and because B crystallin selectively reduces mutant SOD1 aggregation in cell culture, we asked if reducing or eliminating B crystallin from mutant SOD1 transgenic mice would alter the disease course and change the location of SOD1 aggregates. AlphaB crystallin knockout mice we re crossed to mutant SOD1 transgenic mice (Gn.SOD-G37R, Gn.SOD-L126Z, PrP.SOD-G37R). We found that muscular degeneration, a phenotype developed by B crystallin knockout mice, did not alter the disease progression in mutant SOD1 transgenic mice. Next, we found that the levels of detergent insoluble SOD1 species, which represent aggregated prot ein, did not change in mice lacking B crystallin. Finally, cellular localization of SOD1 was similar in mutant SOD1 transgenic mice when B crystallin was intact or eliminated. Thus, our study demonstrates that the expression of B crystallin is not sufficient to explain the cells specific localization of SOD1 aggregates. 49. Molecular isolation and characterizatio n of barley protein disulfide isomerase Kim JY1,2, Jeon WB1, Seo YW1 1Division of Life Sciences and Biotechnolo gy, Korea University, Seoul, South Korea 2Agronomy Department, University of Florida, Gainesville, FL In order to provide the molecular mechanisms inherent to barley kernel development, we conducted differential hybridization (DH) using two different tissues (grains as a tester and pericarps as a driver) 14 days after fertilization (DAF). The transcripts of Hordeum vulgare protein disulfide isomerase (HvPDI), a cDNA enco ding protein disulfide isomerase, were detected in abundance in the grains but were expressed only weakly in pericarps, stems, and leaves. The HvPDI gene expression was abundant in 5 DAF grains, gradually decreasing to 20 DAF. As evidenced by in situ hybridization, HvPDI transcripts we re detected primarily in the starch endosperm, located near the aleurone layer, duri ng the later stages of kernel development (i.e. 14 and 20 DAF). The southern blot analysis showed that the HvPDI genes contained at least 2 copies in barley. A whole coding region of HvPDI was cloned in the bacterial expression vector pET 32 and transformed into the host cells, BL21. Translational products of HvPDI were identified through 1D SDS-PAGE after induction with IPTG. Hybridization with an anti-HIS antibody at the expected size was obtained (65 kDa). In order to examine the localization of HvPDI protein,
green fluorescent protein (GFP) was fused in-frame to the C-terminus of HvPDI, and the fusion protein was allowed to express in the epidermal ce ll of onion. Expression of HvPDI recognized by GFP expression of 35S::HvPDI::GFP was detected in the ER, but the 35S::GFP (control vector) was detected in cytosol, nucleus, and cell wall. (Supported by a grant from the BioGreen 21 Program (20070301034016), Rural Development Administra tion, Republic of Korea, and partially supported by the Korea Research Foundation Grant (MOEHRD: KRF-2007-357-F00001) funded by the Korean Government.) 50. Myxoma virus oncolytic purging of cancer cells ex vivo for autologous hematopoietic cell transplantation in humans Kim M1, Cogle CR2, Madlambayan GJ2, Rahman MM1, Swaminathan S3, Joseph S2, Smallwood S1, Mohamed MR1, Bartee E1, Scott EW2,*, McFadden G1,* 1Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2Program in Stem Cell Biology and Regenerative Medicine, University of Florida, Gainesville, FL 3University of Florida Shands Cancer Center, Gainesville, FL Autologous blood and marrow cell transplantation (ABMT) following high-dose chemotherapy has gained extensive application as a therapeutic modality in the treatment of advanced stage cancer. However, contamination with malignant cells in autologous grafts is a major limitation because of malignant cell re-introduction and subs equent disease relapse. Myxoma virus (MV) is a rabbit-specific poxvirus that is considered a promising oncolytic virus platform for treating human cancer. The natural host tropism of MV is highly restricted to European rabbits, and the virus is nonpathogenic for all ot her vertebrate species tested, including humans. Despite this narrow host specificity, MV is capable of infecting a wide variety of malignant human cells, including lymphomas and leukemias. We have show n that the species barrier to MV infection in primary murine and human cells is mediated by the induced cellular antiviral responses (such as type I interferon or others), suggesting that no rmal hematopoietic stem cells might be spared from MV infection. This led us to test if MV can infect human hematologic malignancies while sparing normal blood stem and progenitor cells. We found that normal human cord blood stem and progenitor cells are highly resistant to MV ch allenge and their differentiation potential is not affected, as determined by human colony-forming cell (CFC) assays whereas MV infects a wide variety of human hematologic malignancies. Theref ore, MV holds great potential to specifically target and eliminate contaminating cancer cells ex vivo for ABMT. 51. Machine learning techniques for filtering low quality pyrosequencing reads Kumar D, Sun Y, Liu L, Farmerie W* Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL High throughput pyrosequencers such as th e 454 Life Sciences GS FLX have certainly revolutionized our approach to DNA sequencing. 454 technology is used extensively not only for genome sequencing but also transcriptome anal ysis, ultra-deep sequencing for detecting rare sequence variants, 16S ribosomal RNA profiling, and metagenomics. Pyrosequencing has a different sequence error profile when compared with conventional Sanger sequencing. Generally, in genome sequencing projects, building consen sus through multifold coverage masks individual base call errors. In metagenomics or transcript omics projects, where high coverage depth and consensus assembly is difficult or practically impo ssible, low read quality may have a significant impact on downstream analysis. Therefore, evalua tion of individual read quality is an important
step to prevent biased or incorrect interpretation due to sequencing errors. While 454 Life Sciences is continuously trying to make its base by base quality scoring method consistent with PHRED scores, we decided to approach the question of overall sequence accuracy by looking at sequence signatures. We built a mathematical model based on supervised machine learning techniques to predict read quality. This model includes and weights various sequence features, such as read length, GC ratio, homopolymer repeats, and presence of ambiguous bases. We use this evaluation method as a filter to remove lo w quality sequences prior to downstream analysis. Numerical experiments are presented that demonstrate the effectiveness of the newly proposed mode. 52. HSV-1 lytic genes are selectively repre ssed by facultative heterochromatin during latency in the mouse Kwiatkowski DL, Bloom DC* Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL The nature of repression surrounding Herpes Simplex Virus type 1 (HSV -1) latency remains unknown; however, previous studies implicate establishment of heterochromatin. It has been shown that dimethylated H3 lysine 9 (K9) accumula tes on the genome as it transitions from the lytic to latent phase. While this mark sugges ts the presence of transcriptionally repressed histones, it does not conclusively discriminate between euchromatic and heterochromatic regions. Therefore, other histone modifications must be examined to conclude 1) whether or not the latent genome is heterochromatic, and 2) if so whether the heterochromatin is facultative or constitutive. Both facultative and constitutive heterochromatin are transcriptionally silent but only facultative retains the ability to revert to eu chromatin. Since the HSV-1 genome appears to undergo chromatin remodeling in response to reactivation stimuli, suggesting facultative repression during latency, we investigated the presence of two known facultative marks, trimethylated (trime) histone H3 lysine 27 (K27) an d macroH2A, using murine dorsal root ganglia latently-infected with HSV-1 strain 17syn+. Chromatin immunoprecipitation analyses with antibodies to those marks revealed that compared to the LAT promoter, the lytic genes ICP27, ICP0 and gC are significantly more enriched in bo th macroH2A (p=0.01, all targets) and trime H3K27 (p=0.02, 0.006, 0.01, respectively). Howeve r, ICP4 is less enriched in both repressive marks, with levels similar to those of the LAT promoter. Additionally, ICP4 is significantly under enriched in macroH2A compared to ICP27, ICP0 and gC (p= 0.04, 0.04 and 0.03, respectively). This suggests that ICP4 may remain less repressed to readily permit reactivation in response to stress. In summary, it appears that 1) the late nt HSV-1 genome is associated with both trime H3K27 and macroH2A marks, indicating that the suppression of lytic genes is consistent with a facultative heterochromatic state, and 2) there is selective localization of these marks over the latent genome. 53. Characterization of a ferulic acid esterase isolated from Lactobacillus johnsonni, a strain with potential use in probiotics formulation against diabetes Lai K-K, Burrion A, Lorca G*, Gonzalez C* Department of Microbiology and Cell Science, University of Florida, Gainesville, FL According to the Centers for Disease Control and Prevention (CDC), about 23.6 millions subjects suffer diabetes in the United States. Previous studie s have reported that a low dose of ferulic acid can stimulate the secretion of insulin and help to lower the blood glucose level. Ferulic acid
together with cinnamic and p-coumaric acids are a group of bioactive phenolic compounds ester bonded to the carbohydrates of plant cell wall. These acids should be released from those macromolecular structures by the activity of specific enzymes prior to their absorption at intestinal level. The hydrolytic enzymes involve d are called feruloyl esterases (FAEs). The FAEs present in the human intestinal tract are produc ed only by symbiotic microorganism. However, the enzymes have not yet been isolated and characterized. We isolated several intestinal lactic acid bacteria that display excellent FAE esterase activity in solid agar plates amended with ethyl ferulate. A carboxylesterase of Lactobacillus johnsonni was cloned, purified and characterized. This enzyme showed high affinity (Km = 20 M) to wards ethyl ferulate compared to other model substrates used (aliphatic esters). In addition to these qualities, the enzymatic activity increased 5 times in presence of conjugated bile salts sugge sting that it is highly active in the conditions encountered in the intestinal tract. The enzyme and/or L. johnsonni described in this work are excellent candidates to be used as food additives to increase the levels of the antioxidant and bioactive ferulic acid for the treatment of diabetes. 54. Models of position-specific constrain ed neutral evolution of protein families Lee HW, Brocchieri L* Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Protein amino acid composition dependence on genome biases and over all measures of codonposition-specific mutation rates suggest that a significant fraction of compositional variability within a protein family is due to neutral substitu tions constrained by negative selection. This suggested the development of models of protein evolution that distinguish genetic drift from functional differentiation. We present a model of constrained neutral evolution of proteins that does not explicitly model evolution in terms of si te-specific and amino-acid-specific evolutionary rates. In this model generalized substitution matrices and evolutionary rates are replaced by proteinand position-specific profiles that define for each protein position subsets of substitutable amino acids. It is assumed that at each sequence position of a protein family a position-specific subset of amino acids ident ified by the profile are neutrally substitutable whereas there is purifying selection against th e complementary set of amino acids. These assumptions realistically model the evolution of a functionally conserved protein family where different sites (e.g., active site, structural core, hydrophilic loops) are subject to different levels and qualities of purifying selection. The profile and associated substitution model implicitly generate siteand amino-acid-specific distribution s of evolutionary rates for each protein family. To estimate the amino acid usage profile of a specific protein family, the frequency and distribution of position-specific amino acid su bsets of different size and composition were evaluated by analysis of the complete Pfam_A seed dataset of manually curated alignments. Given a profile and the evolutionary model, a tr ansformation associating pair-wise evolutionary distance with expected sequence similarity ca n be constructed, which may significantly diverge from other transformations classically used for distance-based tree reconstructions. The model provides insights in the interpretation of the role of long-branch attraction in evolutionary tree determination, and the effects of neutral evolution, functional differentiation and nucleotide compositional biases on protein differentiation.
55. Pleiotropic effects of a single gene mutation in sucrose utilization pathway extend beyond its tissueand metabolic-specificity in developing seeds of maize Li Q-B1, LeClere S1, Chourey P1,2,3* 1Chemistry Unit, Center for Medical, Agricultural, and Veterinary Entomology, Agricultural Research Service, U.S. Department of Agriculture, Gainesville, FL 2Agronomy Department, University of Florida, Gainesville, FL 3Plant Pathology Department, Univers ity of Florida, Gainesville, FL Loss-of-function mutations at the Miniature-1 (Mn1) locus that encodes a major cell wall invertase lead to a non-lethal mn1 seed phenotype marked by a loss of ~70% seed weight at maturity. Although the Mn1 expression is highly endosperm-specific, the mn1 seed phenotype is associated with several pleiotropic changes, mo st importantly, the reduced levels of auxin, Indole-Acetic-Acid (IAA), both in endosperm and embryo of developing kernels. Attempts to better understand the molecular details of IAA-deficiency have led us to describe here expression profiles of several genes, including the two major genes each of which represents the two major branches of tryptophan-dependent IAA pathway in plants. The ZmTARelated-1 gene is a maize ortholog of the recently discovered Trp-aminotransferase in Arabidopsis (TAA) a key enzyme in the indole-3-pyruvic acid (IPA) branch, and the ZmYUC1 that codes for a flavin mono-oxygenaselike enzyme a rate limiting step in the tryptami ne (TAM) branch of auxin biosynthesis. Reversetranscription quantitative PCR (q-PCR) analyses showed that although both ZmTAR1 and ZmYUC1 showed maximal levels of expression at 8 12 days after pollination (DAP), coincident with both an early peak of IAA levels as we ll as cell division and elongation phase, only the ZmYUC1 transcript levels were reduced in the mutant. Subsequent stages, at 20 28 DAP, showed a steep decline in the levels of ZmYUC1; however, the ZmTAR1 showed only moderate down-regulation. Additionally, changes were also seen in the two downstream biosynthetic genes and the two conjugation genes that yield IAA-co njugates for storage function. The invertasedeficiency also led to the changes in sugar composition in both endosperm and embryos of the mn1 mutant. Additionally, reduced endosperm mass was seen as early as 10 12 DAP in the mutant; more importantly, significant reductions were also seen in the mass of mn1 embryos. Overall, in addition to these pleiotropic chan ges, the results provide much insight on sugarhormone crosstalk and embryo-endosperm interdependence in seed development in plants. 56. USF1 recruits hSET1/NURF complex and is important for maintaining active chromatin domains in the -globin locus Li X1, Ybarra R1, Felsenfeld G2, Qiu Y3, Huang S1,* 1Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 3Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL The chicken -globin insulator element acts as a barrier to the encroachment of chromosomal silencing, preventing the spread of a 16 Kb condensed chromatin domain immediately upstream of the -globin genes in the chicken genome. We have previously shown that the transcription factors USF1/2 mediate the barrier activity by binding to the chicken 5'HS4 insulator element and maintaining a local active chro matin structure. In the mouse -globin locus, USFs play a critical role in regulating the developmental stage-specific transcription of the adult -globin genes. To further understand the diverse roles of USF protei ns, we undertook the biochemical isolation of USF1-containing multiprotein complexes. We found that USF1 associates with the hSET1/NURF
complex, which exhibits histone H3 methyltra nsferase enzymatic activity and a potential chromatin remodeling function. Consistent with the role of USF1 in recruiting histone modifying enzyme hSET1 to the 5'HS4 insulator, knockdown of USF1 expression in erythroid leukemia cell line 6C2 leads to the loss of histone H3K4 dimethylation at 5'HS4 of the chicken -globin locus. In addition, USF1, hSET1, as well as H3K4 methylation are colocalized on HS2 of the locus control region (LCR) and the promoter of the -major globin gene. Furthermore, the double ChIP analysis demonstrates that the histone methyltra nsferase hSET1 is recruited by transcription factor USF1 onto the mouse -globin locus. The results suggest that hSET1 may play an important role in establishing H3K4 methylations on the active -globin locus. Further characterization of USF1/hSET1/NURF complex may reveal the mechanisms of USF1 and its associated proteins in maintaining the open chro matin domain and in regulation of globin gene transcription. 57. Defective erythropoiesis in transgen ic mice expressing dominant negative upstream stimulatory factor Liang S1,2, Moghimi B1, Crusselle-Davis VJ1, Lin I-J1,2, Bungert J1,* 1Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2Advanced Concentration in Genetics, Interdiscip linary Program in Biomedical Sciences, University of Florida, Gainesville, FL The transcription factor USF (upstream stimulator y factor) is a ubiquitously expressed member of the helix-loop-helix family of proteins, and is known to be involved in the transcriptional regulation of many genes. USF binds with high affinity to E-box motifs and, through interaction with co-activators, aids in the formation of tran scription complexes. Previous work demonstrated that USF regulates genes during erythroid differentiation, including HoxB4 and -globin. It is shown here that erythroid-specific expression of a dominant negative mutant of USF, A-USF, in transgenic mice leads to diminished association of RNA polymerase II with the -globin gene promoter. Furthermore, expression of A-USF reduces expression of all -like globin genes as well as expression of several erythroid-specific tr anscription factors during yolk sac and fetal liver hematopoiesis. Preliminary data show that USF in teracts with control regions regulating EKLF and GATA-1 expression in erythroid cells. In summary, the data demonstrates that USF is required for erythropoiesis. 58. Calpeptin increases the stability of transc ription factor USF and induces high-level embryonic and adult beta-globin gene expression in erythroid cells Lin I-J, Zhou Z, Crusselle-Davis V, Moghimi B, Gandhi K, Pantick D, Huang S*, Bungert J* Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL Many proteins regulate expression of the beta-t ype globin genes during development of erythroid cells. Tissue-specific and ubiquitously expressed transcription factors together regulate the modification of chromatin domains and the recruitm ent of transcription complexes to the genes. The ubiquitously expressed transcription factor USF belongs to the family of helix-loop-helix proteins and interacts with E-Box elements located in the locus control region and the promoter of the adult beta-globin gene. Prev ious studies have shown that USF is required for the efficient recruitment of RNA polymerase II to the beta-globi n gene locus. Here we demonstrate that USF is subject to calcium-dependent proteolytic cleava ge by m-calpain. Treatment of erythroid cells with calcium ionophores leads to the enhanced degradation of USF and reduces expression of the
beta-globin gene. In contrast, the calpain inhi bitor calpeptin strongly induces globin gene expression and stabilizes USF. The results indicate that reducing calpain activity is required for erythroid differentiation and up-regulation of globin gene transcription. 59. Lysine specific demethylase 1 (LSD1) in snail-mediated gene repression Lin T, Lu J* Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL Epithelial to mesenchymal transition (EMT) is be lieved to be a key process in tumor progression to metastasis. Loss of E-cadherin expression is an essential event for EMT. Recently, the zincfinger transcriptional factor Snail emerged as a ma ster regulator of EMT. Snail directly binds to the E-box motifs at the E-cadherin gene promoter region and represses its expression. However, the molecular mechanisms by which Snail mediat es transcriptional repression are still largely unknown. Here we identified the protein lysine sp ecific demethylase 1 (LSD1) as a co-repressor of Snail. LSD1 is capable of removing the me thyl groups from the di-methylated lysine 4 of histone H3, a well-established marker for active transcription. LSD1 interacts with the SNAG domain of Snail. SNAG is essent ial for Snail's inhibitory function and a mutation in this domain disrupts the interaction. Depletion of LSD1 in the MDA-MB-231 metastatic breast cancer cells partially restores the expression of E-cadherin. Based on these observations we propose that Snail recruits the LSD1 complex to its target ge ne promoters and inhibits transcription through modulating chromatin structures. 60. Turf quality of transgenic bahiagrass under different field environments Lomba PN1,2, Agharkar M1,2, Zhang H1,2, Kenworthy K1,2,*, Sinclair T1,2, Altpeter F1,2,* 1Agronomy Department, University of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL Bahiagrass (Paspalum notatum Flugge) is a popular turf and fora ge grass in the southeastern US and other sub-tropical regions around the world. The popularity of this species is attributed to its strong persistence under low input conditions, su pported by its drought and heat tolerance and resistance against most insects and diseases. Howe ver, quality of bahiagrass turf is compromised by low turf density and its prolific production of long seedheads, during the summer. Two transgenic strategies were compared under field conditions relative to improvement of turf quality in bahiagrass: introduction of a gibbere llin catalyzing enzyme (AT-GA-ox1) or a repressor of cell expansion (ATHB16) into bahiagrass. Expr ession of ATHB16 or AT-GA-ox1 in bahiagrass resulted in phenotypes with significantly more t illers than non-transgenic bahiagrass. Transgenic plants also displayed decreased stem length and delayed flowering. We currently evaluate the persistence of the transgenic and non-transgenic bahiagrass plants under different irrigation regimes at the PSREC in Citra Fl under USDA-APHIS permit 06-219-01r. Data on establishment, irrigation and mowing requirements, turf density, biomass, chlorophyll content, and seed head production of th ese transgenic lines will be presented.
61. A computational model for functional mapp ing of genes that regulate HIV drug therapy and virus load Luo JT1, Hager WW1, Wu RL2,* 1Department of Mathematics, University of Florida, Gainesville, FL 2Department of Statistics, University of Florida, Gainesville, FL Genes have been recognized to control the development of HIV, but have been difficult to detect because the growth dynamic of it is sensitive to environmental changes. We present a statistical model for mapping and characterizing specific genes or quantitative trait loci (QTL) that affect treatment of HIV. This model integrates a system of differential equations into the framework for functional mapping, allowing for the hypothesis tests of the interplay between genetic actions and HIV drug therapy. We study the properties of the statistical model and a simulation approach based on treatment and virus load has been designed to test statistical properties of the model. The model will have great implications for probing the molecular genetic mechanism of HIV drug therapy through the detection of the corresponding QTL throughout the genome. 62. Development of diploid strawberry genotype 5AF7 as a functional genomics resource Mad Atari MF1, Schmitt K1, Slovin JP2, Folta KM1,* 1Horticultural Sciences Department, Univ ersity of Florida, Gainesville, FL 2Genetic Improvement of Fruits and Vegetables Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Henry A. Wallace Beltsville Agricultural Re search Center, Beltsville, MD Functional genomics efforts in strawberry have centered on the diploid genotype Hawaii-4. Hawaii-4 is currently being sequenced and has been used in many laboratory applications by multiple laboratories. However, we have observ ed significant variability in individuals within Hawaii-4 populations. Differences in flower number, thermo-tolerance, crown splitting, and other morphological attributes have been observed. The variable background observed strands to cloud interpretation of effects of a transgene or mutati on. The genotype 5AF7 is a 7-generation inbred that is substantially more homozygous than the Hawaii-4 line. In order to test its potential as a functional genomics system large quantities and sterile tissue are needed. In this study we define the culture conditions most amenable to in vitro micropropagation. Furthermore, regeneration protocols had been reviewed and modified to increase the transformation efficiency. The results of an extensive evaluation of culture conditions are presented. Mutagenesis of the diploid line may be facilitated using the TNT retrotransposon. This mobile DNA element transposes throughout the genome upon tissue damage, and has been a useful means to induce genetic lesions in other eudicot systems. The TNT retrotransposon has been introduced to strawberry tissue and regenerating plants will be evaluated for transposition. This wide series of preliminary tests will validate the use of the inbred 5AF7 line as a system to study functional genomics in strawberry.
63. KFR1, a novel Kelch-domain, F-box protein regulates blue-light-dependent transcript stability Madzima TF1,2, Kaufman LS3, Folta KM1,2,* 1Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL 2Horticultural Sciences Department, Univ ersity of Florida, Gainesville, FL 3Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL During early plant development, environmental sign als direct rapid changes in gene expression to accommodate growth in light. Changes in gene ex pression affect morphology and biochemistry to best optimize the plants ability to grow as an au totroph. One way to regulate gene expression is at the transcript level. RNA degradation regulates accumulation of Light-harvesting, chlorophyllbinding (Lhcb; formerly cab) transcripts. In the dark grown seedling, Lhcb transcript levels increase in response to a short, single low fluence pulse of blue light (104 mol m-2), yet they are destabilized by a single pulse of blue-high-fluence light (BHF; 105 mol m-2). The 65 b 5-UTR is necessary and sufficient to confer BHF-mediated destabilization. To ident ify potential regulatory proteins, a yeast-three-hybrid screen was performed using the Lhcb 5-UTR as an interaction target. Several bona fide interactors were obtained. One protein shown to associate with the transcript in yeast is a novel protein designated as KFR1 for Kelch domain, F-box RNA-associated protein. Genetic tests in the model plant Arabidopsis thaliana T-DNA insertion mutants indicate that Kfr1 is required for BHF-induced Lhcb transcript destabilization. Sinc e KFR1 does not possess described RNA-binding domains, we hypothesized that the F-box entity may be binding to a separate protein that stabilizes the transcript, and subsequently targets it for degradation in response to environmental cues. This hypothes is was tested by application of MG-132 (a proteosome inhibitor) prior to light treatment. The BLF response was not affected, but the inhibitor blocked the BHF response leading to abnormal accumulation of Lhcb transcripts. No other photomorphogenic defects were observed in kfr1 mutants. Taken together, the results indicate that KFR1 is active in ubiquitination -dependent, light-mediated RNA metabolism and not in light signaling directly. We are now explorin g the precise mechanism of KFR1s role in the regulation of transcript stability, as well as th e scope of its influence on other transcripts. 64. Comparing methods of estimating phylogenetic divergence times: the ostariophysan radiation as a test case Makinen TE1, Lpez JA2 1Ichthyology, Florida Museum of Natural Hist ory, University of Florida, Gainesville, FL 2University of Alaska Museum of the North, Fairbanks, AK The continuing development of new methods for estimating divergence times based on measured genetic differences and age constraints from fossil occurrences has resulted in competing hypotheses on the timing of the origin and divers ification of many groups of organisms. One such group is the Ostariophysi, a superorder of fish that includes such species-rich lineages as catfishes, minnows and characids. Initial mo lecular-based estimates of the age of the Ostariophysi and major ostariophysan clades ha ve indicated that these groups originated significantly earlier than the first fossil occurrences of members assigned to the relevant lineages would suggest. Published age estimates derived fr om mitochondrial genome sequences place the first major divergence between ostariophysan subgr oups at 251 million years ago, while fossils of these subgroups first appear in the Early Cretaceous about 105 mya. The same molecular estimates point to ages of 183 and 173 million year s for Cypriniformes (carp, minnows etc.) and
Siluriformes (catfishes), the two ostariophysan clad es with greatest extant diversity, although siluriform fossils are only recognized in the Late Cretaceous about 70 mya. In this study, we compare existing methods of estimating phyloge netic divergence dates, including the most popular software packages current ly in use. We also discuss limitations of age estimates derived from current molecular clock models. 65. A yeast two-hybrid screen to identify pr otein-protein interactions associated with the Lhcb 5-UTR Malone HC1, Madzima TF1,2, Folta KM1,2,* 1Horticultural Sciences Department, Univ ersity of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL RNA degradation regulates accumulation of Ligh t-harvesting, chlorophyll-binding (Lhcb; formerly cab) transcripts. In the etiolated seedling, Lhcb transcript levels increase in response to a short, single low fluence pulse of blue light (104 mol m-2), yet they are destabilized by a single pulse of blue-high-fluence light (BHF; 105 mol m-2). The 65 b 5-UTR is necessary and sufficient to confer BHF-mediated destabilization. To ident ify potential regulatory proteins, a yeast-threehybrid screen was performed using the Lhcb 5-U TR RNA as an interaction target. Several bona fide interactors were obtained. To test if the in dividual RNA-associated proteins formed higherorder intermolecular interactions around the 5-UTR, a yeast two hybrid screen was performed. The coding regions of the RNA-interacting proteins were PCR amplified, then transformed into E. coli utilizing the Gateway Cloning system. The activation domains and the DNA binding domains were then tranformed into yeast to screen for interactions using growth on dropout media as a principle screen. Further tests confirmed strength of interaction. The results indicate that a subset of the RNA-binding proteins show evidence of protein-protein interaction, indicating that Lhcb 5-UTR-based destabilizatio n may be mediated by a previously uncharacterized protein complex. 66. A knockout mutation of a Zea mays U2AF35 related protein alters splicing in a subset of maize genes Martin F1,2, Fajardo D1,2, Fouquet R1,2, Settles AM1,2,* 1Horticultural Sciences Department, Univ ersity of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL Alternative RNA splicing produces multiple mRNA species and allows genes to increase their range of functions. EST sequencing projects have shown that plants produce a significant fraction of alternatively spliced messages, but little is kn own about the control of alternative splicing. We have identified a Mutator insertion in a U2AF35 related gene from the UniformMu transposon tagging population. We are calling this gene ZmUrp for Zea mays U2AF35 related protein. U2AF35 proteins identify splice acceptor sites during RNA processing. The zmurp mutation is tightly-linked to the rough endosperm3 (rgh3) mu tation which causes both defective seed and seedling development. RT-PCR ana lysis from rgh3 mutant RNA indicates that the zmurp insertion allele accumulates a defective transcript with mu ltiple stop codons. RT-PCR analysis of normal RNA identified a series of alternatively spliced ZmURP mRNAs with only one variant predicted to encode a protein. We tested a series of alte rnatively spliced candidate genes to determine if mRNA splicing is disrupted in zmurp/rgh3 muta nts. Candidates were selected from maize orthologs of rice genes that were characterized in the ASIP database at PlantGDB. We selected
alternative splice variants that either had altern ative donor or alternative acceptor sites. No differences in alternative donor splicing patterns were detected in zmurp/rgh3 RNA. Consistent with U2AF35s role in defining splice acceptor site s, a subset of alternative acceptor candidates showed splicing differences in zmurp/rgh3 mutants. These data suggest that the ZmUrp/Rgh3 locus influences splice acceptor site select ion for a specific subset of maize genes. 67. Polymorphism of melanocortin 1 receptor gene in the common frog (Rana temporaria) Matsuba C1,2, Meril J1 1Ecological Genetics Research Unit, Department of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland 2Department of Botany and Zoology, University of Florida, Gainesville, FL The melanocortin 1 receptor (MC1R, or MSH receptor) on melanophore receives a pigmentation signal from the pituitary, and must play on a crucial role in background colour adaptation and pigmentation of dorsal colour-patterning in amphibians. To investigate possible existence of geographic cline in the MC1R gene variability in the common frog (Rana temporaria), the coding region sequence of the fully sequenced gene was analyzed across European populations spanning a latitudinal distance of ca 1800 km. Both Bayesian phylogenetic and parsimony network trees supported existence of a basal lineage consisting of German and Danish populations, and subsequent colonization of Fennoscandia. However, the resolution of the phylogeny was low within Fennoscandian populations. Higher variations in both nucleotide and predicted amino acid sequences were found on the eastern as compared to western side of Fennoscandia. A neutrality test detected a signat ure of ancestral population bottleneck in the eastern side where a post glacial colonization of northern Fennoscandia started. Although an admixture at secondary contact zone in wester n Fennoscandia was expected, the variation in western Fennoscandia was very low and nearly monoallelic. The results are thus consistent with a recent and rapid recolonization of northern Fennoscandia from South (Den mark), and/or strong selection on one favorable allele in the western side of Fennoscandia. 68. Sialidase activity is associated with the virulence of Mycoplasma gallisepticum May M1, Sczcepanek S2, Gates AE2, Frasca Jr. S2, Brown DR1,*, Geary SJ2 1Department of Infectious Diseases and Path ology, University of Florida, Gainesville, FL 2Department of Pathobiology and Veterinary Science, University of Connecticut, Storrs, CT Sialidase activity level correlates significantly with the virulence of Mycoplasma synoviae isolates, indicating a potential role for this enzyme in the pathogenicity of certain other mycoplasmas. Recent genome annotation of the poultry pathogen Mycoplasma gallisepticum strain R led to the identification of its putative sialidase gene MGA _0329, which has high sequence similarity to the annotated sialidase gene MS53_0199 of M. synoviae. We thus hypothesized that sialidase activity also contributes to the virulence of M. gallisepticum. Sialidase activity was quantitated in strains R, A5969, F, and S6 using the fluorogenic substrate 2-(4-methylumbelliferyl)-D-Nacetylneuraminic acid (MUAN). MGA_0329 was disrupted in strain R by insertional mutagenesis using the transposon Tn4001mod to create strain P1C5, which lost all sialidase activity. White Leghorn chickens (n=6 per group) infected intratracheally with P1C5 had tracheal lesion scores and tracheal thickenings significantly lower than th ose of chickens infected with equal doses of parent strain R, and 5log10 fewer CFU of strain P1C5 were cultured from the trachea, air sacs,
lungs, or ovaries at necropsy. Mini-Tn4001tet plasmid pTF20 carrying a functional copy of MGA_0329 with its promoter was then electroporat ed into P1C5 to complement the knockout. Three filtered clones, each having one copy of MGA_0329 transposed into a different mapped site in its genome, expressed sialidase activity (1.44 1.85x10-8 g/CFU) restored to wildtype levels (1.58x10-8 g/CFU). Experimental inoculation studies to examine the potential pathogenicity of these sialidase-complemented cl ones in chickens are currently ongoing. This represents the first use of a mini-Tn4001tet plasmid in M. gallisepticum, and also its first use for delivery of a stable gene expression construct in any Mycoplasma species. This work may lead to a basis for novel treatment and vaccination strategies focused on the role of sialidase in diseases associated with M. gallisepticum. 69. Proteomics and mass spectrometry applications in biomedical research McClung S, Chow M, Zheng R, Chung A, Bryant C, Zhu N, Chen S* Proteomics Division, Interdisciplinary Center fo r Biotechnology Research, University of Florida, Gainesville, FL Proteomics and mass spectrometry have provided unprecedented tools for fast, accurate, high throughput biomolecular separation and char acterization, which are indispensable towards understanding the biological and medical systems. Studying at the protein level allows researchers to investigate how proteins, thei r dynamics and modifications affect cellular processes and how cellular processes and the environment affect proteins. The mission of our facility is to provide excellent service and traini ng in proteomics and mass spectrometry to UF scientists and students. Here we present our ca pabilities in proteomics and other analytical services. The tools include a gel-based 2D-DIGE (Two Dimensional Difference Gel Electrophoresis) and gel-free iTRAQ (Isobaric Tags for Relative and Absolute Quantitation). Along with our capacity of separating thousands of proteins and characterizing differential protein expression, we have a suite of state-of-the-ar t mass spectrometers available for biomedical sciences and advanced technology research, including a tandem time-of-flight (4700 Proteomics Analyzer, AB), quadrupole/time-of-flight (QSTAR XL, AB), and hybrid quadrupole-linear ion-trap (4000 QTRAP, AB). These instruments are mainly used for protein identification, posttranslational modification characterization and protein expression analysis (e.g., Mass We stern). Our facility is also set up to provide Edman de novo N-terminal protein sequence analysis and Biacore biomolecule interaction analysis. We are fully set up to synthesize and purify peptides and have a good track record with this service as well. Pr oteomics and mass spectrometry are useful in large-scale suvey of proteome for hypothesis genera tion as well as in detailed analysis of target proteins for hypothesis testing. Our services al so include accurate molecular weight analysis, MRM-based protein screening and targeted metabo lite profiling. To ensure success and maximize productivity, the facility offers education, cons ultation, data processing and reporting, and support of grant application. 70. Genetic control of hydraulic proper ties and correlations with growth in Populus Miles B1, Martin T1, Peter G1,2,*, Huber D1, Dervinis C1 1School of Forest Resources and Conservation, University of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL There are many genetic studies on wood properties, growth and disease in Populus, but few genetic investigations of hydraulic properties. Genetic and phenotypic variation in stem hydraulic
properties, growth, foliar stable carbon isotope discrimination ( 13C), and interactions among these traits were quantified for twenty-two clona lly propagated genotypes (and the parents) of a pseudo backcross population of Populus deltoides and P. trichocarpa x P. deltoides. Mean hydraulic vessel diameter (Dh), vessels per sapwood area (VSA) density, and hydraulic conductivity were calculated from image analysis of stem cross-sections. Plant biomass, growth increment and 13C were also quantified. Stem diameter increment was positively genetically and phenotypically correlated with leaf specific hydraulic conductivity. Growth traits, leaf specific hydraulic conductivity and xylem anatomy traits were moderately heritable, while 13C was marginally heritable. VSA and Dh were negatively correlated, while Dh was positively genetically correlated with stem diameter increment. Specific leaf area and Huber value were negatively correlated with growth traits. This research is the first to quantify genetic control of xylem hydraulic traits in an angiosperm forest tree. Strong genetic correlations between hydraulic and growth traits suggest they share genes or biological pathways. 71. Coalescent simulations of human louse (Pediculus humanus) evolution reveal contact between archaic Homo species and modern humans Miro AT1, Kitchen A2, Toups M3, Reed D4,* 1Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2Department of Anthropology, University of Florida, Gainesville, FL 3Department of Botany and Zoology, University of Florida, Gainesville, FL 4Mammalogy, Florida Museum of Natural History, University of Florida, Gainesville, FL A central debate in human evolution is whether archaic Homo species and modern humans had contact throughout their colonization of the Old World. Attempts to address this debate by studying humans directly have achieved limited succe ss. A look at ectoparasitic lice, which have coevolved with hominids for millennia, reveals a story much older than human remains can. While human mitochondrial DNA (mtDNA) coalesces to a single lineage very rapidly (within ca. 100,000 years), human louse (Pediculus humanus) mtDNA lineages date back to the origin of the genus Homo (ca. 2 million years ago). Further, the same genetic signature of population expansion seen in human mtDNA is evident in louse mtDNA and dates to the same time. Therefore, inferences about human evolution can be made from the study of human louse evolution. Two hypotheses have been put forth to explain human louse evolution; 1) archaic Homo species (e.g. Neanderthals) carried louse lineages that st ill exist today due to host switching events or 2) founding populations of modern humans out of Africa carried at least three louse lineages into modern populations. We used coalescent simulations to model these and other hypotheses of human/louse coevolution to determine which models are more likely to produce gene trees like the observed mtDNA gene trees for lice. The results strongly support the host switching event hypothesis, thus suggesting contact between archaic Homo species and modern human. 72. Enzyme characterization of the soybean mitochondrial O-acetylserine (thiol)lyase Morriss C1, Kirst M2, Harmon A2,* 1Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL
2Department of Botany and Zoology, University of Florida, Gainesville, FL Sulfur metabolism is essential for plant growth and production of high nutritional quality food for both human and animal consumption. The cystei ne biosynthesis pathway lies directly at the junction between sulfur and nitrogen metabolism and is the location where inorganic sulfur is metabolized into an organic form. In a two step process, the two enzymes serine acetyltransferase (SAT, E.C. 126.96.36.199) and O-acetylserine (thiol)lyase (OASTL, E.C. 188.8.131.52) synthesize cysteine from serine. SAT transfers an acetyl group from acetyl-CoA to the hydroxyl group of serine to form O-acetylserine (OAS). The acetyl group of OAS is then replaced with sulfide by OASTL to form cysteine. Localization studies show unique isoforms of both SAT and OASTL in three subcellular compartments includin g the cytoplasm, chloroplast and mitochondria. In Arabidopsis cysteine synthesis has been proposed to be regulated by the reversible formation of a complex between SAT and OASTL. Characterization of a putative mitochondrial isoform of soybean (OASTL-M) suggests that the regulation of cysteine synthesis in soybean differs from that in Arabidopis. Unlike Arabidopsis OASTL, activity of free OASTL-M is unstable. Activity is stabilized by incubation with either OAS or S2-. Like Arabidopsis, OASTL-M activity is inhibited by addition of SAT, but a 500-fold molar excess of SA T is required for significant inhibition to occur. These data suggest that complex formation may not be as important as a regulatory factor in soybean as it is in Arabidopsis. (Supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2006-35318-17392.) 73. Bardet-Biedl syndrome proteins cons titute an anciently evolved machinery for ciliary transport Mukherjee K, Brocchieri L* Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL The Bardet-Biedl syndrome (BBS) is a human path ological condition affecting development and a wide range of functionalities associated with ciliary activities. BBS ha s been associated to mutations in twelve different genes, name d BBS1 to BBS12. Among these, BBS6, BBS10 and BBS12 have been recently characterized as a group of fast evolving genes related to eukaryotic Group-II chaperonin genes, widely known for fo rming in eukaryotes characteristic 16-mer complexes (chaperonins) involved in folding acti n, tubulin and other proteins. Experimental evidence indicates that many BBS proteins asso ciate with cilia, where some of them form a complex (the BBSome) involved in ciliary transpor t. Others associate wi th centrioles, possibly participating in transport from the centriole to the cilium through tubulin structures. The localization of BBS proteins explains their relevance in ciliary and developmental functionality. Little is known about presence of BBS proteins in species other than vertebrates. By thorough analysis of several complete ge nome representatives from all major eukaryotic branches, we determined that representatives of most BBS ge nes are present in several anciently diverged protist lineages. In particular, the seven members of the BBSome complex and BBS3 are found in all groups morphologically characterized by the pr esence of cilia. Chaperonin-like BBS proteins BBS6, BBS10 and BBS12 are also found in ancientl y-diverged ciliated groups, whereas BBS11 is only found in vertebrates. These findings suggest that the basic BBS-protein-machinery arose very early in eukaryote evolution, in primordial relation with the development of the eukaryotic cilium. The early appearance of the diverged chaperonin-like BBS proteins BBS6, BBS10 and BBS12 provides insights into the evolution and functional differentiation of the eukaryotic chaperonin protein family. In particular, some experimental evidence and our bioinformatics analyses indicate that these proteins do not function in a canonical chaperonin complex, suggesting an early development of new functionalities for eukaryotic chaperonin monomers.
74. Prevention of inhibitor formatio n in gene therapy for hemophilia B Nayak S1, Hoffman B2, Cooper M2, Atkinson M1, Cao O2, Herzog RW2,* 1Advanced Concentration in Immunology and Microbiology, Interdisciplinary Program in Biomedical Sciences, University of Florida, Gainesville, FL 2Division of Cellular and Molecular Therapy, Depa rtment of Pediatrics, University of Florida, Gainesville, FL Hemophilia B is an X linked bleeding disorder caused by the deficiency of coagulation Factor IX. Treatment is hindered by the onset of inhibi tor responses to the therapeutic protein/gene product. Regulatory T cells (Treg) are importan t for tolerance induction. A novel prophylactic antigen specific protocol was tested for AAV2 and AAV1 mediated muscle directed gene transfer using immune suppressive drug rapamycin (rapa) cytokine IL-10 and an antigen specific CD4+ T cell epitope. The mechanism of tolerance was determined using Treg deficient (DO.11.10tg Rag2-/-) BALB/c mice transgenic for ovalbumin-specific T cell epitope. 4-week injections of Rap/IL-10/specific peptide resulted in the deletion of ova specific CD4+ effector T cells with a concomitant increase of CD4+CD25+FoxP3+ Treg cells. Apopto tic cells expressed FasL and increased activation markers like CD69 indicating AICD. The protocol was tested in C57BL6 mice given IM injections of AAV2-CMV-hF.IX (1x1011vg) and formation of anti-hF.IX antibody was successfully blocked. F.IX knockout mice were injected IM with AAV1-CMV-F.IX (1x1011vg). Reduction of inhibitor titers was observed in the Rap/IL-10/specific antigen treated animals after 2 months (1-2 BU, which is the background of th e assay) with clotting time of 45-53 seconds. Rap/IL-10/irrelevant peptide controls showed 4-5 BU with clotting times of 61-86 seconds. Nontolerized gene transfer controls showed 4-18 BU with clotting times of 64-89 seconds. These results indicate that the prophylactic protocol tested may successfully reduce or eliminate deleterious immune responses in hemophilia B and may be extended to other protein deficiency disorders like hemophilia A and Pompe disease. 75. A microarray-based analysis of tran scriptional compartmentalization in the alimentary canal of Anopheles gambiae larvae Neira Oviedo M1, VanEkeris L1, Corena-McLeod MDP2, Linser PJ1,* 1Whitney Laboratory for Marine Bioscience, University of Florida, St. Augustine, FL 2Neuropsychopharmacology Laboratory Mayo Clinic, Jacksonville, FL The alimentary canal of the larval mosquito di splays a considerable degree of physiological compartmentalization among its different anatomic al sub-divisions. We performed a comparative microarray analysis in order to identify transcripts which are particularly enriched in each gut section of the 4th instar larva of Anopheles gambiae. Based on the available annotation of the selected transcripts, we suggest that the metabolism and absorption of proteins and carbohydrates takes place mainly in the gastric ca eca and posterior midgut, whereas the anterior midgut specializes in the metabolism and absorption of lipids. Transcripts encoding anti-microbial peptides were found to be enriched in the gast ric caeca, and a high enrichment of transcripts associated with enzymes involved in xenobiotic detoxification was found in the anterior midgut. Furthermore, our data supports the notion that the region encompassing the hindgut and Malpighian tubes plays important roles in avoidi ng the excretion of nutrients, as well as in xenobiotic detoxification.
76. The development of new molecular markers for population genetics: a pilot study using the central Florida endemic Polygala lewtonii Nelson CN1, Germain-Aubrey C1,2, Gitzendanner M2,3,*, Soltis D2,*, Soltis P3,* 1UF-HHMI G.A.T.O.R Program for Biomedical Science, University of Florida, Gainesville, FL 2Department of Botany and Zoology, University of Florida, Gainesville, FL 3Laboratory of Molecular Systematics and Evolutionary Genetics, Florida Museum of Natural History, University of Florida, Gainesville, FL The Florida scrub is a xeric ecosystem occurring on inland and coastal ridges throughout Florida. This ecosystem holds an exceptionally high level of endemism, with forty plant species, forty six arthropod species, and four vertebrate species not occurring anywhere else. Polygala lewtonii a Polygalacae (a family of over 900 species), is one of the federally listed endemics to the central Florida scrub. For that reason, we will investig ate it using the latest molecular techniques. We are proposing to do a population genetics study of P. lewtonii and of its nearest congener in order to develop a long-term conservation plan for the species. For interand intra-specific comparison in specie s, microsatellites (variable numbers of tandem repeats in DNA) are used because of they ar e evolutionarily neutral, transferable between generations, and have a high mutation rate. However, microsatellites have been shown to hold a high level of allele site homoplasy which can lead to an underestimation of genetic diversity. We propose to conduct a pilot study comparing compound microsatellites, which contain both flanking regions sequences and microsatellite repeat number to the classical microsatellites using P. lewtonii. Compound microsatellites have been successfully used in studies involving humans and fish. To accomplish this we will first develop primers encompassing both the microsatellite regions and their flanking regions. We will clone and sequence a subset of the population genetics study samples and evaluate the effect of adding the flanking region to the microsatellite on the accuracy in assessin g genetic diversity. Our study on compound markers on P. lewtonii and P. polygama will qualify the effectiveness and limitations for future use of this new molecular marker on plant species and overall further the field of conservation genetics. Without effective knowledge of scrubland species, we cannot preserve the remaining genetic divers ity of this highly valuable area. 77. Sigma virus in natural populations of Drosophila melanogaster Nguyen CV, Wayne ML* Department of Botany and Zoology, University of Florida, Gainesville, FL A common rhabdovirus sigma occurs in Drosophila melanogaster. Sigma, being vertically transmitted, is not contagious to humans and Drosophila that are infected by sigma tend to be sensitive to carbon dioxide, dying from exposure Because the sigma virus is able to be easily detected through carbon dioxide sensitivity assays and because the virus is known to be vertically transmitted, the Drosophila-sigma model system is useful in examining the relationship between viruses and their hosts.
Previous studies have shown that sigma virus af fects about 5% of flies in natural populations. Although female flies may transmit the virus to all their offspring, males affect only a small percentage of offspring. Through a viraemia stud y using wild-type males, we have seen this percentage in nature and in transmission to offs pring is higher than that detailed by previous literature. 78. Molecular detection of rAAV in blood from non-human primates Ni W1, Le Guiner C4, Baus J1, Bello-Roufai M3, Moullier P1,4, Snyder RO1,2,3,* 1Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2Department of Pediatrics, University of Florida, Gainesville, FL 3Center of Excellence for Regenerative Health Biot echnology, University of Florida, Gainesville, FL 4INSERM UMR649, Nantes Cedex, France Gene transfer of therapeutic genes has shown the potential to treat many serious human diseases; however, an emerging issue of this promising technology is misuse by athletes looking for an advantage over the competition. Gene doping is the transfer of genes to enhance athletic performance. Following intramuscular (IM) inject ion of a rAAV vector into non-human primates, our group has reported that vector DNA can be detected in serum, urine, feces, saliva, and nasal fluid for several days, even weeks post-injection In developing a test to screen athletes, detecting vector sequences from muscle would requ ire biopsy and would not be practical in terms of obtaining permission; therefore, the use of less invasive sampling and development of a sensitive detection technique is imperative to detect gene doping. We are currently determining the smallest dose of a rAAV vector injected IM that is able to be detected in blood from nonhuman primates. We also aim to discern the relationship between vector dose and longevity in blood. The test we are developing involves the collection of blood and the analysis of DNA by PCR and qPCR assays. Using oligos specific for erythropoietin (Epo) cDNA, we have optimized PCR conditions to detect at least 100 copies of the Epo transgene in the background of endogenous sequences. Data generated in the non-human primate is the basis for development of a legally defensible commercial qPCR assay that will eventually be used to test athletes. Given that gene transfer technology encompasses a variety of vectors, transgenes, expression cassettes, routes of admi nistration, as well as injection formul ations, the vector biodistributions can vary widely, thus an outcome of our work w ill be to better define assays that can be utilized to elucidate vector distribution for legitimate gene therapy applications. 79. Synthesis of genetic and GIS distance data Non AL1, Sanchez LF1, Raaum RL2, Al-Meeri A3, Mulligan CJ1,* 1Department of Anthropology, University of Florida, Gainesville, FL 2Department of Anthropology, Lehman College/CUNY, Bronx, NY 3Department of Biochemistry and Biology, Sanaa University, Sanaa, Yemen Genetic data are naturally embedded in a geogra phic context, but most analyses of human population history do not make direct use of geog raphic data. In our ongoing analysis of Yemeni population history, results from a pilot data set suggested a high proportion of African mitochondrial DNA (mtDNA) haplogroups (Hgs) (33%), and a visually distinct spatial pattern in which African mtDNA Hgs tend to cluster in the East. Further sequencing has revealed a more complex geographic pattern, making simple visual assessment of geographic structure difficult. To statistically analyze the geographic and gene tic structure, we applied three geographically
explicit methods to a dataset of 93 Yemeni mtDN A sequences with associated GIS data: 1) test for correlation of individual geographic and gene tic distances via Mantel test, Auto-Correlation Analysis, and Landscape Shape Plot (Alleles in Space), 2) hierarchical AMOVA to distinguish distinct Hg clusters, and 3) geostatistical anal ysis of interpolated contour maps with kriging (SURFERv8.0). Furthermore, we analyzed both individual and average population genetic admixture (STRUCTURE) using an Alu insertion polymorphism dataset to determine the relationship of our Yemeni samples to samples from worldwide populations. Our results suggest that the best method of incorporating GIS data may be use of a detailed map of genetic and GIS data to develop hypothes es to test in AMOVA. Binning of geographic distances (AIS) may be effective in very long-range analyses, but was less useful in this regionally focused study. Although it does not directly incorporate GIS da ta, STRUCTURE analyses provide data to develop testable hypotheses. Through this study, we hope to enable more refined analyses that explicitly incorporate geographic data in our reconstruction of human population history. 80. Gene compositional characterization of Pseudomonas aeruginosa pathovars Oden SM1,2, Brocchieri L1,* 1Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2Advanced Concentration in Genetics, Interdiscip linary Program in Biomedical Sciences, University of Florida, Gainesville, FL The genome of P. aeruginosa, an opportunistic pathogen of great nosocomial importance, exhibits great structural plasticity and variability in gene content, resulting in high adaptability to a wide range of environmental conditio ns and host tissues. The study of P. aeruginosa is of great clinical importance and theoretical interest from an evolutionary perspective. In an effort to correlate gene content with pathogenicity, the genomes of several different pathovars of P. aeruginosa have been sequenced since publication of the first P. aeruginosa genome (strain PAO1) in 2006. This has provided an opportuni ty to compare the gene content, genome structure, and patterns of evolvability among strains of differing virulence. Currently, among all predicted protein-coding genes of P. aeruginosa PAO1, only 30% are functionally characterized and only 10% have been experimentally verified. For meaningful genomics and comparativegenomics analyses it is highly relevant to obtain improved characterizations and measures of reliability of gene predictions. We have charac terized all genes annotated in the genomes of Pseudomonas aeruginosa strains PAO1 (5,568 genes), PA14 (5,892) and PA7 (6,286) in terms of non-randomness in the distribution among codon positions of compositional properties such as GC content and purine content, and of hexanucleotide representation. Comparing functionally characterized genes to uncha racterized (hypothetical) predictions, we identified considerable consistency in the dist ribution of compositional properties among functionally characterized genes of all strains. The hypothetical genes predicted in the genomes of P. aeruginosa PAO1 and PA14 are also compositionally similar to characterized genes. However, among the hypothet ical genes annotated in P. aeruginosa PA7, several genes show anomalous compositional properties compared to those exhibited by the corresponding genes of other strains, or compared to compositional ex pectations based on hundreds of other bacterial genomes. Tests of non-randomness of the compositional properties of these genes provide a measure of their reliability.
81. Is adult neurogenesis a product of the vascular environment? Ormerod BK*, Munikoti V J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL A growing body of evidence points to the possible involvement of vascular niches within discrete regions of the adult brain, in promoting neurogen esis. Several groups have observed an increase in endothelial cell proliferation, vascular growth fa ctor levels and angiogenesis in the brains of young adult animals, in response to exercise. Concurrently, other groups have noted enhanced hippocampal neurogenesis and neurotransmission, improved spatial learning, increased synaptic plasticity, and better overall cognitive function in physically active animals. Coupled with empirical observations such as age-related cognitive declin e, reduced synaptic plasticity, and diminished neurogenesis, as well as a decline in microvascular plasticity, these data imply that vascularity in the hippocampus and neurogenesis may not be mutually exclusive, and have provided strong motivation to explore a plausible relationship between the two phenomena. In this study, we hypothesize that there are nich es in the brain that support neurogenesis, i.e., that the vasculature within the hippocampus provides for an angiogenic niche wherein unique physical and chemical cues are expressed, that dr ive the differentiation of neural stem cells into neurons. We speculate that such a niche is situat ed in the subgranular zone (SGZ) of the dentate gyrus in the hippocampus, so that neurogenesis is localized to this region of the hippocampus. We will adopt a two-pronged approach to this project: (1) we will co-culture hippocampal and cortical microvessels with hippocampal progen itor cells to assess the role of hippocampal microvasculature in the process of neurogenesis. We expect progenitors co-cultured with hippocampal microvessels to differentiate into ne urons. (2) Next, we will evaluate the responses of hippocampal and cortical progenitor cells to hippocampal microvessels in co-culture. We hope to find out whether neurogenesis is indeed a product of the vascular niche, or if it is caused by fundamental differences between hippocampal progenitor cells and progenitors found elsewhere in the brain. 82. The Genomics Division within UFs ICBR offers the means for specialized genetic data acquisition at a reasonable cost Ostrow DG, Almira E, Clark AM, Moraga DA, Norton S, Shaw R, Shanker S Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL The ICBRs Genomics Division allows researchers to generate, store, and analyze genomic data without creating costly technical infrastructure within their own labs. The Genomics Division is composed of five groups: 1) DNA Sequencing usin g Sanger and next-generation technologies, 2) Fragment Analysis, 3) Gene Expression, 4) Real -Time PCR, and 5) SNP Genotyping. Together, we provide access to researchers ac ross campus to technology including genome sequencing on the 454 and SOLiD instruments, gene expression measurement on the Agilent and Affymetrix platforms, and high throughput SNP genotyping on the Illumina Bead Station. We are also able to provide expertise to assist with experimental de sign, data collection, and analysis. Having these resources on campus saves valuable time and money and allows researchers to focus on generating scholarly publications and external funding opportunities.
83. Refinement of the lupus susceptibility locus, Sle1c Perry DJ, Dozmorov I, Morel LM Department of Pathology, Immunology and La boratory Medicine, University of Florida, Gainesville, FL The homozygous NZM mouse strain develops a spon taneous and severe lupus-like disease with a higher mortality and penetrance than its heterozygous parent strain, NZB/WF1. We have utilized a congenic dissection strategy to discern the genetic components responsible for induction of this disease. This has led to the identification of se veral loci that confer various lupus phenotypes when congenically bred onto a non-autoimmune B6 background. Sle1c is one such locus. It is a NZW derived, 7.5 Mb interval located at the telomeric end of chromosome 1 and has been attributed to several autoreactive phenotypes incl uding elevated levels of activation markers of CD4+ T cells, elevated proliferation of CD4+ T ce lls, decreased percentages of regulatory T cells (Tregs), amplified chronic graft versus host di sease (cGVHD), decreased T-dependant immune response, and defective germinal center (GC) f unction and formation. New subcongenic stains have identified a 0.7 Mb interval at the centromeric end of Sle1c that retains the CD4+ T cell hyperactivation and proliferation, cGVHD amplific ation, and Treg deficiency phenotypes, and these phenotypes were found to be CD4+ T cell intrinsic. This subinterval, termed Sle1c-2, contains 2 candidate genes with no described f unction in the immune system. We are currently working to identify expression level, isoform, and coding sequence differences in the NZW and B6 alleles of these genes. Microarray analysis of CD4+ T cells has uncovered a plethora of genes that are differentially expressed between B6 and B6. Sle1c-2, many of which are described in Treg, Th17 and Tfh pathways of T cell differentiation. The dysregulation of these pathways have been implicated in several autoimmune diseases, including lupus. Our goal is to find the allelic difference(s) of the Sle1c-2 candidate gene and to characterize how it results in these autoimmune phenotypes. (Supported by NIH grant R01 AI1045050.) 84. Biosynthesis and physiological role of archaeosine (G*) in the halophilic archaeon Haloferax volcanii Phillips G1, Vimbai X2, Maxwell A1, Lyons B1, El-Yacoubi B1, Swairjo M3, Iwata-Reuyl D2, de CrcyLagard V1,* 1Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 2Department of Chemistry, Portland State University, Portland, OR 3Department of Basic Medical Sciences, Western University of Health Sciences, Pomona, CA Guanosines at position 15 of tRNA are modified to the 7-deazaguanosine derivative archaeosine in all archaeal tRNAs analyzed to date. The key en zyme in the insertion of this modified base is tRNA-guanine transglycosylase (Tgt). Tgt re places the guanine base with 7-cyano-7deazaguanine (preQ0 base) without breakage of the phosphodiester backbone yielding preQ0tRNA (Watanabe et al., 1997, J Biol Chem 272(32):20146-51). The preQ0-tRNA is then further modified by an unidentified pathway that adds an imidino group to pre-Q0 to generate archaeosine. The archaeosine mod ification is unique and is found in all branches of the archaeal phylogenetic tree; however, its biological role is poorly understood. It has been proposed that archaeosine affects the stability of th e tRNA molecule (Iwata-Reuyl, 2002, Bioorg Chem 31(1): 24-43). In order to determine the function of archaeosine in vivo, we embarked to construct Haloferax volcanii derivatives deficient in archaeosine. Compar ative genomic analysis revealed that most
archaeal genomes sequenced to date co ntain two gene families annotated as tgt encoding TgtA1 and TgtA2. TgtA1 corresponds to the experimentally characterized Tgt enzyme. Both TgtA1 and TgtA2 contain PUA tRNA binding domain, but TgtA 2 does not contain a transglycosylase domain. We predicted that TgtA2 must be the enzyme that converts preQ0-tRNA to archaeosine. Both tgtA1 and tgtA2 strains of H. volcanii were constructed. LC/MS analysis of digested bulk tRNA from H. volcanii mutants lacking the tgtA2 gene revealed a deficiency in converting the preQ0tRNA into a mature archaeosine. This is the first evidence that TgtA2 is the missing enzyme that catalyzes the last step of archaeosine synthesis. Complementation and biochemical studies confirmed that TgtA2 is the enzyme that converts preQ0-tRNA into archaeosine. Also, LC/MS analysis of digested bulk tRNA from H. volcanii mutants lacking the tgtA1 gene showed the absence of both archaeosine and preQ0-tRNA. Complementation and phenotypical studies are underway to reveal the biological role of archaeosine in halophilic archaeon H.volcanii. 85. Direct estimate of the rate and spectrum of microsatellite mutation in rhabditid nematodes Phillips N1, Salomon MP2, Custer A2, Ostrow G3, Baer CF2,* 1Department of Biology, Arcadia University, Glenside, PA 2Department of Botany and Zoology, University of Florida, Gainesville, FL 3Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL Simple sequence repeats (SSR, or microsatellites) are one of the most widely used classes of genetic markers, from genetic mapping and association studies to investigations of population structure and conservation. Despite their utilizat ion, many unresolved questions concerning the mutational processes shaping SSR evolution remain Here we describe a study aimed to provide unbiased estimates of the rate and spectrum of new SSR mutations in a novel mutation accumulation (MA) model system. The mutational properties of dinucleotide SSRs were characterized in two strains (= genotypes) of two species in the genus Caenorhabditis, C. briggsae and C. elegans after 250 generations of MA. Mutations have been allowed to accumulate in the (relative) absence of natural selection, thus allowing us to genotype paired control and generation 250 MA lines at approxima tely 40 dinucleotide loci of perfect, imperfect, and compound SSRs. Furthermore, we investigat e how the mutational pr operties of SSRs are correlated with the genomic mutation rate for fitness in these two species. 86. Variation in aggregation of disease variants of SOD1: correlation to human disease Prudencio M, Borchelt DR Department of Neuroscience, University of Florida, Gainesville, FL Familial amyotrophic lateral sclerosis (fALS) is a severe neurodegenerative disease that leads to the selective death of upper and lower motor neur ons. The most studied familial form of ALS is linked to mutations on a gene in chromosome 21 that encodes for the superoxide dismutase 1 (SOD1) protein. Mutations in SOD1 constitute nearly 20% of fALS cases. There are about 142 fALS-associated mutations in SOD1, from which only 9 have been expressed in transgenic animal models. All transgenic animals present a fALS ph enotype characterized by hindlimb paralysis that manifests at a time when the accumulation of SOD1 aggregates is prominent. The presence of aggregated forms of mutant SOD1, which can be isolated by high speed centrifugation in the presence of non-ionic detergents, correlate wi th the appearance of disease symptoms in
transgenic mice and human affected tissues. Nevertheless, fALS-linked SOD1 mutations are spread all over the 153 amino acid protein sequence, not being concentrated in any particular structure. Over 70 amino acids are known to be ta rgets of mutation that give rise to the fALS phenotype. This indicates the same amino acid can be mutated in different ways to lead to disease development. However, it is unknown, wh ether different types of mutations that affect the same codon have a different effect on aggr egation and/or disease. These aggregated SOD1 proteins (also called detergent-insoluble aggregat es) can be isolated from cultured cells that express mutated SOD1. Thus, we have used a cell culture system to study the aggregation propensity of 32 SOD1-linked fALS mutations; in cluding mutations that affect the same codon differently. We found that all mutant SOD1 proteins analyzed are able to aggregate. Mutations in the same amino acid have different impact on aggregation levels depending on the codon generated. Additionally a set of fALS mutations share more features with the wild-type SOD1 protein while remain soluble in detergent. The effe ct that different levels of aggregated mutant protein may have on disease has been evaluated. 87. Association genetics of pitch canker resistance in loblolly pine Quesada T1,2, Huber D2, Davis J1,2,* 1Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL 2School of Forest Resources and Conservation, University of Florida, Gainesville, FL Pitch canker is an economically important disease that affects most pine species. It is incited by the necrotrophic fungus Fusarium circinatum, causing resinous lesions, seedling mortality, and crown dieback. Plant responses to this disease are heritable, and suggest that resistance to pitch canker could be a multigenic complex trait. The ge ographical distribution and diversity of loblolly pine in southeastern United States makes it a good candidate for association studies, which rely on historical recombination events to identify relationships between phenotypes and genetic markers based on linkage disequilibrium decay. As a key step toward association mapping for pitch canker resistance in loblolly pine, disease resistance phenotyping was performed on 498 largely unrelated genotypes (association populati on). A randomized incomplete block design was implemented, with four replicates (rooted cuttings) of each clone. Lesion length measurements were taken 4, 8, and 12 weeks after inoculation with F. circinatum microconidia. Ranking of the genotypes based on their resistance to pitch ca nker was based on clonal estimates using best linear unbiased predictions (BLUP). The 50 most resistant and susceptible clones were reinoculated and data collected at four weeks post-inoculation showed significant differences between the resistant and susceptible tails (p < 0.0001). This suggests that the initial classification from the first experiment is reproducible after a second inoculation. Clonal repeatabilities ranged between 0.20 and 0.30; supporti ng evidence that pitch canker resistance is a heritable trait. The continuous distribution of pi tch canker resistance is also evidence that this trait is quantitative, possibly controlled by seve ral loci with relatively small effects. Future association tests using over 10,000 SNP genotypic ma rkers should provide further support on this hypothesis. 88. Changes in global DNA methylation in response to chronic consumption and withdrawal of folic acid is dependent on the MTHFR 677C T polymorphism Quinlivan EP1, Crider K2, Berry RJ2, Hao L4, Li Z4, Maneval D3, Rasmussen S2, Yang Q2, Zhu J3, Bailey LB3,* 1General Clinical Research Center, Un iversity of Florida, Gainesville, FL
2National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA 3Department of Food Science and Human Nutrit ion, University of Florida, Gainesville, FL 4Peking University Health Science Center, Beijing, China We evaluated the effect of the MTHFR C T polymorphism on global DNA methylation from a double-blind randomized controlled trial in wh ich Chinese women of reproductive age took supplements of folic acid (100, 400, 4000 g/d). DNA methylation was expressed as a % of methylated cytosines (measured by a novel LC-MS/MS assay) at enrollment, 1, 3, 6 months of supplementation, and after a 3 month washout period, stratified by MTHFR genotype (CC, CT, TT) (n=135; 15 subjects/genotype x 3 treatment gr oups). Baseline methylation levels (4.4 +/0.12) were similar (p>0.25) across the MTFHR geno types. Folic acid supplementation resulted in a 13.5% (p< 0.0001) decrease in global methylation after 1 month of exposure, independently of genotype and dose. After 6 months of supplementation, DNA methylation had returned to baseline in the TT-subjects taking 100 g, but not in CC-subjects, or in any subject receiving 4 mg/d. During the 3 month washout period DNA methylation decreased further in CC (40%) and CT (20%), but not significantly in the TT-subjects. This post-intervention decrease in methylation was both genotype and dose dependent. In conclu sion, global DNA methylation response to folic acid supplementation and withdrawal varied by MTHFR genotype with a complex genotype-dose interaction. 89. Predicting strawberry yield from early photomorphogenic phenotypes Salama DY1,2, Folta KM1,2,* 1Horticultural Sciences Department, Univ ersity of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL Light quantity (intensity), quality (color) and dura tion (photoperiod) all contribute to plant growth and development. Light has well-described roles in the regulation of plant stature and flowering, and studies in the model system Arabidopsis thaliana show that altered flux through various light signal transduction pathways results in clear seedling phenotypes and parallel effects on flowering. Here we initiate a test of the hypoth esis that analysis of early photomorphogenic seedling growth in strawberry (Fragaria) may allow researchers and breeders to predict genotypes likely to have higher yield. A fluence rate response curve was developed for F. vesca Hawaii-4 seedling stem growth inhibition usin g a variety of light wavebands and intensities. Light generally causes inhibition of seed ling stem growth, so variation in light sensing/transduction will result in aberrant stem phenotypes. Seedlings grown in 50 mol/m of red light exhibited a 50% decrease in hypocot yl elongation to that of seedlings grown in darkness. This is comparable to the response in Arabidopsis seedlings. However, strawberry seedlings are much more sensitive to far-red light (~730 nm) than Arabidopsis seedlings. The dual roles of the red light photoreceptor, phytochrome B, in the inhibition of hypocotyl elongation and the degradation of the CONSTANS protein, a critical positive up-regulator of flowering may provide a new baseline for cultivar selection. The he ightened sensitivity to far-red light in Hawaii4 strawberry may explain its robust, photop eriod-independent flowering, as the CONSTANS protein is stabilized by far-red light acting th rough phytochrome A. Individuals that are less responsive to red light or more responsive to far-red light may be sele cted as seedlings more likely to be highly productive as mature plants. In this scenario, 100,000 seedlings could easily be screened in the laboratory in one week, generating a focused population of several thousand candidate genotypes that are more likely to exhibit more intense flowering and higher yield. Such an approach would save breeders the time, money and effort necessary to select for this trait using conventional methods.
90. Investigating evolutionary and ecolog ical factors underlying HIV-1 epidemic history in east Africa by landscape phylodynamics Salemi M1,*, Gray RR1,2, Russell C3, Laeyendecker O4,5, Goodenow MM1,*, Quinn TC4,5 1Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 2Department of Anthropology, University of Florida, Gainesville, FL 3Land Use and Environmental Change Institut e, University of Florida, Gainesville, FL 4Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 5School of Medicine, Johns Hopkins University, Baltimore, MD The factors responsible for modern disease epidem ics are multi-factorial and involve ecological, political, social, as well as evolutionary influen ces. Here we introduce a new framework, called landscape phylodynamics, that combines genetic sequence data analysis of pathogens based on coalescent theory (phylodynamics) with geog raphic information system (GIS) data about the territory where the pathogen is found. Landscape refers to the myriad ways in which humans affect and are affected by environment, and incl udes geography, land use, infrastructure and migration on a temporal scale. We apply the fr amework to provide a comprehensive analysis of the social, ecological, and evolutionary causes behind the early emergence of HIV-1 subtypes A and D epidemic in east Africa. By using Bayesian skyline plots inferred from HIV-1 genealogies of the pol and env gene, we show that subtype A was introduced into Ug anda toward the end of the 1950s, 10-15 years earlier than the introduction of subtype D. This date precedes the earliest known cases of HIV-1 infection in the region by at least two decades, and occurs at the same time of massive migration out of Rwanda during the ethnic civil wars derived by a collection of spatial data layers. We also find that both subtypes grew exponentially duri ng the 1970s, coincident with high migratory outflux from, and political upheaval in Uganda. Furthermore, the current distribution of the two subtypes in east Africa and differences in effective population size suggest that the early epidemics of HIV-1A and D were driven by divers e socio-political and evolutionary factors, and spread differentially in urban and rural populations. In conclusion, our analysis illustrates how a landscape phylodynamic approa ch, which couples phylodynamic inference with GIS data, can offer a powerful framework for understanding and interpreting past epidemics as well as predicting newly emerging outbreaks of infectious diseases. 91. New genetic tools for improving SIT in Ceratitis capitata Schetelig MF1,2, Scolari F3, Handler AM2,*, Gasperi G3, Wimmer EA1 1Department of Developmental Biology, Georg-August-University, Goettingen, Germany 2Center for Medical, Agricultural, and Veterinary Entomology, Agricultural Research Service, U.S. Department of Agriculture, Gainesville, FL 3Dipartimento di Biologica Animale, University of Pavia, Pavia, Italy Environment friendly sterile insect technique (SIT) has been applied effectively as a component of area-wide integrated pest management (AW-IPM) for Ceratitis capitata since the 1970s. Nevertheless improved biological strategies are needed to increase the efficacy of AW-IPM. Transgenic approaches should increase and widen the applicability of such programmes to different pest species. In this respect we are following up several strategies:
1) Development of transgenic sperm marking for Ceratitis capitata. We have engineered two lines each with a green and a red fluorescent marker. One marker is under the control of the sperm specific beta2-tubulin promoter from Ceratitis capitata (turboGFP or DsRedExpress); the second is under the control of the polyubiquitin promoter of Drosophila melanogaster (DsRed or EGFP). Beta2-tubulin is transcribed selectively during spermatogenesis. Released males from these strains could be distinguished from wildtype ma les in the monitoring process during SIT. In addition monitoring of the mating success of sterile released males by trapping females and examining their spermathecae is possible with these strains. With this knowledge releasing strategies in SIT-based AW-IPM co uld be optimized. In principle these strains can also be used as sexing lines that could be sorted automatically for males and females in larval development. However, since Ceratitis already has an effective sexing strain, this might be more important for the development of SIT in other Tephritids. 2) Development of a transgenic embryonic lethal system for Ceratitis capitata. The system employs the ectopic expression of a hyperactive proapoptotic gene that causes embryo-specific lethality when driven by the tetracycline-controlled transactivator tTA under the regulation of a cellularization gene enhancer/promoter. The system has been tested successfully in Drosophila melanogaster (Horn and Wimmer, 2003, Nat Biotechnol 21(1):64-70). We transfered the system to Ceratitis to evaluate it in comparison with the effective conventional SIT using radiationinduced sterility. 92. New genomics systems in rosaceous crops Schmitt KS1,2, Folta KM1,2,*, Slovin JP3 1Horticultural Sciences Department, Univ ersity of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL 3Genetic Improvement of Fruits and Vegetables Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Henry A. Wallace Beltsville Agricultural Re search Center, Beltsville, MD The Rosaceae family contains a series of high-v alue plant species, ranging from herbaceous perennials (like strawberry) to large-architec ture tree crops (apple, cherry, peach), and ornamentals (rose, hawthorn). Despite their econ omic value, relatively few functional genomics resources exist to support studies in this family. The goal of this work has been to develop efficient transgenic systems to facilitate gene function studies in the family. Fragaria vesca, the diploid woodland strawberry, is currently used as a system for the study of functional genomics in the Rosaceae family. The strawberrys small size, ease of transformation, tiny genome, and fast regeneration time make it a highly desirable organism for testing gene function. One of the most frequently used Fragaria vesca accessions for genomic studies is Hawaii-4, but phenotypic variation among Hawaii-4 individuals has been ob served. These observations indicate that the Hawaii-4 line may maintain a degree of hete rozygousity that may obscure downstream phenotypic analyses. To help eliminate this problem of genetic variation, the Fragaria vesca AF7 line was developed by Dr. Janet Slovin of the USDA. 5AF7 has been inbred for seven generations, making it substantially more isogen ic, and hence phenotypically stable, than other accessions. Herein we provide a general descri ption of the 5AF7 genotype and accompanying transformation protocols that demonstrate its utilit y as a system to study gene function in the Rosaceae.
93. Glycerol metabolism and related metabo lic pathways in the halophilic archaeon Haloferax volcanii Sherwood KE, Maupin-Furlow JA* Department of Microbiology and Cell Science, University of Florida, Gainesville, FL Glycerol-rich waste streams are generated in subs tantial amounts by the biofuels industry. The competitive price of glycerol coupled with its highly reduced nature makes glycerol an attractive carbon source for bioconversion. While bacteria have been exploited by the biofuels industry, extremophiles capable of withstanding the harsh conditions associated with biomass saccharification remain an untapped bio-industrial resource. Haloferax volcanii is a GRAS, extremely halophilic archaeon that is easily cultured and genetically manipulated and is capable of growth on glycerol generated from biodiesel waste. Although Hfx. volcanii is an ideal industrial candidate, its central me tabolism has not been well-characterize d. The aim of this project was to elucidate glycerol metabolism an d related metabolic pathways in Hfx. volcanii. Upon uptake, microorganisms assimilate glycerol to dihydroxy acetone phosphate (DHAP) either via glycerol dehydrogenase/DHA kinase (DHAK) or via glycerol kinase/glycerol-3-phosp hate dehydrogenase. To better understand central metabolism, the fo llowing genes were targeted for knockout: glpK (encoding glycerol kinase), dhaL (encoding a regulator of DHAK), ptsI (encoding the PTS E1 component), and fruA (encoding the PTS IIBfructose component). Knockouts were analyzed for growth phenotype on HV-Minimal medium supplemen ted with various sole carbon sources. In contrast to parent H26, mutant H26 glpK did not exhibit growth on glycerol minimal medium, indicating glycerol metabolism proceeds via glycerol kinase/glycerol-3-pho sphate dehydrogenase. While Hfx. volcanii H26dhaL exhibited growth on DHA minimal medium, parent H26 did not, suggesting that DhaL may function as a negative regulator of DHA metabolism. Mutant H26 ptsIfruA displayed a growth reduction on glucose and fructose minimal medium, indicating that both PTS components contribute to sugar metabolism. Our results not only shed light on glycerol and DHA metabolism for Hfx. volcanii, but also suggest a novel archaeal DHAK-PTS. By first elucidating central metabolic path ways such as glycerol metabolism, Hfx. volcanii may prove a valuable microbial workhorse for the biofuels industry. 94. Phylogenetic analyses of Anopheles gambiae carbonic anhydrase and anion exchanger proteins Smith K1,2, Linser PJ1,2,* 1Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 2Whitney Laboratory for Marine Bioscience, University of Florida, St. Augustine, FL Mosquito larvae display a unique characteristic th at sets them apart from most other organisms. The anterior portion of the midgut generates a lu minal pH as high as 10.5, one of the highest known in any biological system. If well understood, this characteristic could be targeted in the development of highly specific larvacides. The mechanisms for midgut alkalinization are largely unknown; however strong evidence suggests roles for the enzyme carbonic anhydrase (CA) and membrane bound chloride/bicarbonate anion ex changers (AE). Here we report phylogenetic analyses of members of the -CA family and the AE SLC4 family from the Homo sapiens, Drosophila melanogaster, Aedes aegypti, and Anopheles gambiae genomes. Additionally, we report molecular characterization of three An. gambiae CA genes (AgCA6, AgCA9, and AgCAb) and two An. gambiae AE genes (AgAE2 and AgAE3).
95. Sequence-indexed Mutator transpos on insertion sites using 454 sequencing Spielbauer G1,2, Tseung C-W1,2, Shaw R3, Farmerie W3,*, Settles AM1,2,* 1Horticultural Sciences Department, Un iversity of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL 3Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL Gene knockouts are an essential resource for the functional analysis of the maize genome. A major challenge for the maize genetics community is to identify mutations in every gene in the genome. Multiple maize transposable element syst ems have been used as endogenous mutagens to generate knockouts on a genome-wide scale. These mutations are most accessible when the insertion sites are indexed with flanking sequence tags (FSTs) from individual seed stocks. Endogenous transposons exist as multicopy el ements within the genome. Identifying novel insertions relies on either highly redundant FST sequencing to identify a mix of parental and unique mutations or labor-intensive experimental manipulations to purify novel flanking DNA prior to sequencing. Here we show that the 454 Life Sciences sequencing technology is effective for achieving the level of redundancy needed to associate novel Mutator (Mu) insertions to specific plants. We sequenced FSTs from 144 mutagenized plants from the UniformMu population. The insertion sites were amplified from 24 pooled DN A samples arrayed in a 12 x 12 grid. Each pool was sequenced with a molecular barcode to assi gn individual reads to pools. We recovered 437,199 reads containing signature sequences indica ting a bona fide Mu FST. These FSTs had an average read length of 155 bp after removal of the pool barcode and Mu signature sequences. Less than 1% of the FSTs could not be assigned to specific pools due to sequencing or primer synthesis errors. Recovery of the same insertion site from row and column pools allows specific insertions to be addressed to individual plants. We have used site-specific PCR based on the FSTs to validate this addressing system. More detailed bioinformatic analysis of these FSTs will be presented. 96. Mature primary neural networks influence differentiation of adult neural progenitor cells and in turn progenitor-derived cells affect network wide activity Stephens CL, DeMarse TD, Ormerod BK* J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL Damaged or diseased neural tissue could potent ially be treated with autologous transplants of adult neural progenitor cells. Differentiation of these cells into neurons and glia has been shown to be influenced by neural activity. The microelectrode array (MEA) measures neural activity in vitro from a grid of 60 30 m electrodes arranged 200 m apart. Pairing the MEA system with fluorescent immunohistochemistry and confocal microscopy, we investigate the effects of adding GFP-expressing adult neural progenitor cells to an already established neural network of dissociated cortical tissue and the effects that primary cortical cells may have on progenitor differentiation. Fourteen days after the addition of progenitor cells, GFP-expressing neurons and glia were present. Network-wide activity significantly declined after progenitors were added but new activity was observed on previously inactive electrodes. Average spike rates were 1.37Hz (.34Hz) compared to the control of purely primar y cells (6.35 Hz), suggesting that cell addition compromises signaling for prolonged periods of ti me. These data suggest that transplanted cells will influence their immediate environment, potentia lly by buffering activity at least in the early stages.
97. Arsenate-induced high temperature stress tolerance in Arabidopsis thaliana Stokes D, Muthumani P, Rathinasabapathi B* Horticultural Sciences Department, Univ ersity of Florida, Gainesville, FL Contamination of arsenate, the most common form of toxic heavy metal arsenic is ubiquitous in aerobic soils because of both natural and anthropogenic activities. Howeve r, plant responses to arsenate stress have been poorly characterized. Because plants and yeast upregulate heat shock proteins upon treatment by arsenic, we hypothesized that plant tolerance to high temperature stress could be increased by exposing plants to non-lethal doses of arsenic. We tested this using a seedling bioassay in which 5-day old A. thaliana seedlings were exposed to mild and severe heat stress treatments (37 C and 42 C for 2 h, respectively) with or without a 24-h pre-treatment on medium containing 0.2 mM sodium arsenate Both root growth measurements and seedling survival data showed that arsenate pre-treatmen t significantly increased plant tolerance to high temperature stress. Like wild -type, mutants impaired in signaling -(salicylic acid, sid-2 and pad-4; jasmonic acid, jar-1 and ethylene, etr-1) -were also protected by arsenate pre-treatment suggesting that arsenate-induced high temperature stress tolerance is independent of these signaling pathways. 98. Glutaredoxin-mediated modulation of plant tolerance to high temperature stress: transgenic expression of fern glutaredoxin PvGrx5 in Arabidopsis thaliana increases stress tolerance and reduces oxidative damage to proteins Sundaram S1,2, Wu S1,2, Rathinasabapathi B1,2,* 1Horticultural Sciences Department, Univ ersity of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL Glutaredoxins are glutathione-dependent oxidoredu ctases protecting proteins from damage by oxidative stress. Functional roles for the fam ily of plant glutaredoxins are enigmatic. We implicated a glutaredoxin of the arsenic hyperaccumulating fern Pteris vittata PvGRX5 in arsenic tolerance (Sundaram et al., 2008, J Biol Chem 283(10):6095-101). Because of possible involvements of glutaredoxins in metabolic adapta tions to oxidative stress, and because many of the glutaredoxin target proteins are stress-related proteins, we hypothesized that glutaredoxins could be manipulated to improve plant stress tolerance. In a test of this, transgenic Arabidopsis lines constitutively expressing PvGRX5 were ev aluated for thermotolerance. Homozygous lines expressing PvGRX5 exhibited significantly greater tolerance to high temperature stress than the vector control and wild-type based upon growth during stress, which was positively correlated (R2=0.89) to leaf glutaredoxin specific activit ies. Measurements of tissue ion leakage, thiobarbituric acid reactive substances and protein carbonyl content showed that PvGRX5expressors were significantly (P<0.05) less affected by the high temperature stress treatment compared to wild-type and vector control lin es for damage to membranes and proteins. Immunoblots indicated that specific protein bands, carbonylated during the stress treatment in the control lines, were protected in PvGRX5-exp ressors, thus implicating PvGRX5 in heat tolerance, likely mediated through cellular protection against oxidative stress. Together our results suggest that engineering glutaredoxins could be a novel way to manipulate plant stress tolerance via possibly modulating pr otein-protein interactions.
99. The genetic potential of wild and cultivat ed strawberries for nitrogen uptake and reduction Taghavi TS1,2, Folta KM1,* 1Horticultural Sciences Department, Univ ersity of Florida, Gainesville, FL 2Department of Horticultural Science, University of Tehran, Karaj, Iran Strawberry genotypes have been selected for a lo ng time based on their fruit characteristics and their yield. Cultivated strawberries need a lot of nitrogen to grow and give a reasonable yield, because they have shallow roots which could not up take nitrogen from deeper areas of the soil. Little attention has been paid on the efficiency of strawberry for taking up nitrogen and its reduction as nitrogen has been inexpensive, easily applied and typically is not a limiting factor in cultivation. However, as fertilizer prices rise an d environmental impacts of nitrogen use become apparent, it is of interest to identify germplas m that may offer the ability to thrive with less nitrogen input. Many wild strawberries can thrive in rocky soils having little or no nitrogen. This germplasm may serve as a useful source of genes or alleles related to efficient nitrogen uptake or assimilation. In this study experiments have been conducted to study the physiological and genetic potential of wild strawberries and their efficiency in nitrogen uptake and reduction in comparison with cultivated strawberries. The gene s identified can be transferred by conventional or modern biotechnology methods to strawberry for improving the cultivated strawberries for reducing nitrogen usage in the field and reducing nitrogen pollution of the environment. 100. Sex-derived splicing in Drosophila Telonis-Scott M1, Kopp A2, Wayne ML3,*, Nuzhdin SV4, McIntyre LM1,5* 1Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2Section of Evolution and Ecology, Un iversity of California, Davis, CA 3Department of Botany and Zoology, University of Florida, Gainesville, FL 4Section of Molecular and Computational Biology, De partment of Biological Sciences, University of Southern California, Los Angeles, CA 5Department of Statistics, University of Florida, Gainesville, FL Many genes in eukaryotic genomes produce multiple transcripts through a variety of molecular mechanisms including alternative splicing. Alternatively spliced transcripts often encode functionally distinct proteins, indicating that ge ne regulation at this level makes an important contribution to organismal complexity. The mu lti-level splicing cascade that regulates sex determination and sex-specific development in Drosophila is a classical example of the role of alternative splicing in cell differentiation. Recent evidence suggests that a large proportion of genes in the Drosophila genome may be spliced in a sex-bi ased fashion, raising the possibility that alternative splicing may play a more general role in sexually dimorphic development and physiology. However, the prevalence of sex-specific splicing and the extent to which it is shared among genotypes are not fully understood. Genetic variation in the splicing of key components of the sex determination pathway is known to influence the expression of downstream target genes, suggesting that alternative splicing at other loci may also vary in functionally important ways. In this report, we used exon-specific microarrays to examine 376 multi-transcript genes for evidence of sex-specific and genotype-specific splicing in 80 different genotypes of D. melanogaster. Most of these loci showed sex-biased splicing, wher eas genotype-specific splicing was rare. 78 genes showed different ratios of alternative transcripts in males versus females in all genotypes. Realtime PCR of tissues, for sex-specific splicing of six genes chosen to represent a broad range of biological functions showed that most sex-biased splicing occurs in the gonads. However, somatic
tissues, particularly adult heads, also show evid ence of sex-specific splicing. Comparison of splicing patterns at orthologous loci in seven Drosophila species shows that sexual biases in alternative exon representation are highly conser ved, indicating that sex-specific splicing is an ancient feature of Drosophila biology. To investigate potential mechanisms of sex-biased splicing, we used real-time PCR to examine the expression of six known regulators of alternative splicing in males versus females. We found that all si x loci are themselves sp liced sex-specifically in gonads and heads, suggesting that regulatory hier archies based on alternative splicing may be an important feature of sexual differentiation. 101. A comparative phylogeographic analysis of five plant species endemic to the endangered Florida scrub ecosystem Thurston T1, Germain-Aubrey C2 1Horticultural Sciences Department, Univ ersity of Florida, Gainesville, FL 2Department of Botany and Zoology, University of Florida, Gainesville, FL Every year, thousands of species go extinct throughout the world, lowering the overall genetic diversity. The central Florida scrub is an endang ered ecosystem that provides a habitat for many rare and threatened plant species whose molecular phylogenetic analyses hold valuable information. This research project will perform a phylogenetic analysis using the internal transcribed spacer region of the DNA in five chosen plant species of high endemism to the scrub, and it will enable the completion of previously pu blished but incomplete phylogenetic trees. This project will lead to a better understanding of the evolutionary history of these endemics, and the system as a whole, along with determining the relationship of these species to the proposed nearest sister species. A comparative phylogeogra phic study will be then performed to hopefully determine the temporal and spatial origin(s) of th e endemic plant species. This project will help to better understand and therefore conserve this threatened ecosystem. 102. Role of RNA helicase A and SM protein in the post transcriptional regulation of gene expression by EBV Tirumuru N1,2, Gaillard M1, Boris-Lawrie K3, Swaminathan S1,2 1University of Florida Shands Cancer Center, Gainesville, FL 2Department of Medicine, University of Florida, Gainesville, FL 3Department of Veterinary Biosciences, Ohio State University, Columbus, OH Introduction: Epstein Barr virus (EBV) is a B-lymphocyte-tropic gammaherpes virus with a genome of 170 Kb that encodes over 90 viral proteins. SM is a post-transcriptional regulatory protein essential for lytic EBV replication. We iden tified SM-associated proteins by tandem affinity purification of SM, followed by mass spectrometri c analysis. SM was found in association with RNA helicase A, which is a multifunctional cellula r RNA processing protein. We investigated the functional significance of this association. Hypothesis: RNA helicase A is a multifunctional nuc lear cytoplasmic shuttling protein involved in several functions including transcription, RNA pr ocessing, translation an d ribosome biogenesis. We hypothesized that SM may co-opt and utiliz e RHA to enhance RNA processing (stabilization and nuclear export) or translation of EBV or specific cellular transcripts. Methods: Plasmids were utilized that encoded the luciferase gene and an RNA structure referred
to as a posttranscriptional control element (PCE) that is required for translational enhancement by RHA. Plasmids containing or lacking a PCE were transfected into Hela cells with or without SM. 48 hours after transfection, cells were separated into nuclear and cytoplasmic fractions and RNA was isolated. Protein lysates were also prepar ed from each sample and used to measure luciferase activity. Nuclear and cytoplasmic RNA levels were measured by Northern blotting analysis and quantitative RT-PCR. Results: Luciferase measurements were compared with cytoplasmic RNA levels to determine translational efficiency of the various RNAs in th e presence and absence of SM. As expected, the PCE-containing RNAs were translated with higher efficiency although the cytoplasmic RNA amounts were similar, due to the effect of RHA. Also as expected SM led to increased accumulation of RNAs with and without the PCE. In the presence of SM however, translational efficiency of the PCE-containing cons tructs was reproducibly increased. Conclusion: These results indicate that SM may synergize with RHA to enhance the effect of RHA on translation of RNAs contai ning structured RNA elements. 103. Epstein-Barr virus SM protein inhibits BHRF1 (viral Bcl-2) expression by downregulating the EBV transcriptional activator R protein Verma D1, Liu C1, Tirumuru N1,2, Swaminathan S1,2 1University of Florida Shands Cancer Center, Gainesville, FL 2Department of Medicine, University of Florida, Gainesville, FL EpsteinBarr virus (EBV) is an ubiquitous human herpesvirus associated with several malignancies that infects epithelial and lymphoid cells. During primary infection, EBV undergoes lytic replication in epithelial cells. The switch from latency to lytic replication is regulated by two EBV transcriptional activators; BZLF1 (Z) and BRLF1(R). SM is an RNA-binding EBV protein expressed after Z and R and is also essentia l for virus production. SM enhances EBV RNA accumulation by enhancing target mRNA stability and export. We used an epithelial cell line carrying an EBV recombinant deleted for SM to compare gene expression in the presence and absence of SM. We found that most EBV genes ar e upregulated by SM, but one gene BHRF1, is highly downregulated. BHRF1 is an anti-apototic pr otein that is homologous to cellular Bcl-2 and is expressed shortly after lytic EBV replication begins. To ask whether the inhibitory effect of SM on BHRF1 expression was post-transcriptional, we measured expression of BHRF1 from a heterologous promoter. SM was unable to decrease BHRF1 gene expression from a CMV promoter, suggesting that the effect was promoterdependent. We therefore analysed BHRF1 promoter activity with luciferase reporter assays and found that SM downregulated BHRF1 promoter act ivity, but only in EBV-positive cells, suggesting involvement of other EBV proteins in BHRF1 prom oter inhibition by SM. We further demonstrate that SM downregulates R protein and RNA levels in EBV positive cells and that the majority of BHRF1 promoter activity in 293 cells is R-dependent. Our results demonstrate that SM may specifically inhibit BHRF1 promoter activity by downregulating R expression during the lytic phase of EBV replication. Temporal modulation of BHRF 1 levels during lytic replication may thus be important for maintaining cell viability for a lim ited period during the early stage of EBV replication.
104. Genetic dissection of sorghum traits to enhance biofuel production Vermerris W1,*, Saballos A1,2, Lamb K1, Ejeta G2 1Agronomy Department, University of Florida, Gainesville, FL 2Department of Agronomy, Purdue University, West Lafayette, IN Sorghum (Sorghum bicolor (L.) Moench) has many characteristics that make it an attractive bioenergy crop. It is endowed with abundant geneti c diversity, has high tolerance to drought, is adapted to a wide range of environmental conditio ns, is responsive to inputs but requires less fertilizer and irrigation than corn and sugarcan e, and its potential for high biomass yield is comparable or superior to other alternative crops currently being considered for the production of lignocellulosic biomass. In addition, sorghum has a deep root system that may mitigate loss of soil organic matter due to biomass removal. We are developing dual-source bioenergy sorghums that can be used directly as a source of fermen table sugars, and that ge nerate bagasse suitable for the production of cellulosic ethanol. The gene tic basis of many of the abovementioned traits, including sugar accumulation in the stalks, is poorly understood, but with the availability of the sorghum genome sequence, it has no w become feasible to identify ke y genes. This is expected to further boost the potential of sorghum as a bioenergy crop, especially in Florida. 105. Post-transcriptional regulation of peripheral myelin protein 22 by miR-29a Verrier JD1,2, Lau P3, Hudson L3, Notterpek L1,2,* 1Department of Neuroscience, Unive rsity of Florida, Gainesville, FL 2Evelyn F. and William L. McKnight Brain Instit ute, University of Florida, Gainesville, FL 3Developmental Genetics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD Peripheral myelin protein 22 (PMP22) is a dose -sensitive, disease-associated protein primarily expressed in myelinating Schwann cells. Deletion or duplication of the PMP22 locus can result in hereditary peripheral neuropathy, suggesting a requirement for precise regulation. Studies demonstrate that PMP22 is post-transcriptionally regulated and the 3UTR of the gene exerts a negative effect on translation. The restricted expression of the PMP22 protein in comparison to its ubiquitously detected mRNA supports a role for post-transcriptional regulation. MicroRNAs (miRNA) are small regulatory molecules that functi on at a post-transcriptional level by targeting the 3UTR. Here we used cultured primary rat Schwann cells to demonstrate that not only do alterations in the miRNA biogenesis pathway a ffect PMP22 levels, but endogenous PMP22 is subjected to miRNA regulation. GW body formation and Dicer expression are co-regulated with the differentiation state of Schwann cells, both demonstrating increased levels in growing cells, as compared to differentiated cells. Furthermore, the levels of Dicer inversely correlate with PMP22, while Dicer suppression via siRNA leads to elevated PMP22. These results support a role for miRNA pathway in regulating PMP22 protein ex pression. We used microarrays to characterize the miRNA profile of actively growing and differentiated Schwann cells, and to identify differentially regulated miRNAs. These results, in conjunction with miRNA bioinformatics programs, identified candidate PMP22-targeting miRNAs. Using in vitro assays, we demonstrate that a differentially regulated miRNA, miR-29a, can bind to and repress PMP22 reporter expression through a specific miRNA seed bindin g region. Immunoprecipitation of Ago2 reveals that endogenous PMP22 RNA binds Ago2, and transfection of miR-29a enhances this association. Finally, transfection of miR-29a reduces stea dy-state PMP22 levels, while inhibition of endogenous miR-29a relieves the miRNA-mediated repression of the gene. These data reveal
another level of regulation of myelin gene expression and identify PMP22 as a target of miRNA repression. 106. Responses of wildtype Arabidopsis thaliana and transporter gene mutants to excess levels of magnesium sulfate ions; consequences for extra-terrestrial plant growth Visscher AM1, Paul AL1,*, Kirst M2,*, Ferl RJ1,* 1Horticultural Sciences Department, Un iversity of Florida, Gainesville, FL 2School of Forest Resources and Conservation, University of Florida, Gainesville, FL The ability to utilize in situ resources is crucial for the success of extended manned space exploration on other planetary surfaces such as the moon or Mars. Surface regolith represents a resource for plant growth in advanced life su pport systems that could not be replaced by materials from earth as the mass requirements wo uld be insurmountable. In this context, Martian regolith containing potentially phytot oxic levels of elements is being investigated as a medium for plant growth. Recent studies of surface materials on Mars have detected a variety of sulfate mineral salts, which are of interest because of th eir indication of aqueous weathering processes. Magnesium sulfate has been chosen as the focus of the current study because of the potential toxicity of magnesium to plant growth. Levels of magnesium ions toxic to crop plants have been described for soil types on Earth, such as serpentine soils. On Mars, localized weight percentages of magnesium sulfate can reach 10% in the regolith. Our overall research interest relates to what kinds of genetic variations and modifications ca n reduce plant growth constraints imposed by high levels of magnesium found in surface materials on Mars and Earth. These genetic adaptations would complement other options for stress reduction such as soil, water and nutrient management. Our starting hypothesis is that knockout variations of ion transporter genes expressed in the outer cell layers of Arabidopsis r oots result in plants that can thrive in soils containing hyper elevated levels of magnesium sulfate. An Arabidopsis SALK line was selected with a knockout T-DNA insertion in the MRS2-10 gene, which has been shown to encode a protein that can transport magnesium and which is located in the plasmamembrane of root cells. Ranges of increasing magnesium sulfate concentrations were established over which wildtype plant growth is progressively reduced. Resu lts show that the selected MRS2-10 T-DNA background does not mitigate the constraining impacts of high magnesium sulfate concentrations on wildtype plant growth over this range. Based on these findings, a microarray experiment has been done to determine what genes are expressed differently in wildtype lines under elevated concentrations of magnesium sulfate compared to normal concentrations. The CAX1-1 mutant line, which has been shown to exhibit increased tolerance for magnesium, was also included in the experiment to find genes that are differentially expressed from wildtype at elevated levels of magnesium sulfate. The results of this microarra y experiment will help form hypotheses about what genes are primarily responsible for magn esium ion uptake at excess levels, and, consequently, what genes may be likely candidates for follow-up experiments regarding magnesium tolerance improvement in crop plants. 107. Biofortification of strawberri es with anti-nematode proteins Wang HHY, Folta KM* Horticultural Sciences Department, Un iversity of Florida, Gainesville, FL Strawberry production in Florida faces a serious problem of sting nematode (Belonolaimus
longicaudatus Rau). Root tissues are the primary feeding sites for these pests. When the numbers of sting nematode in the soil reach a critical threshold duri ng the growing season, plants lose vigor leading to a significant decrease in yield. The goal of this work is to test the hypothesis that proven nematode antifeedants engineered into the roots of transgenic strawberry may reduce the damage caused by sting nematode Cysteine protease has been identified as a major digestive enzyme in nematode intestine. We have isolated several cystatins from strawberry genome and proven that one of the prot ease inhibitors FaCys8 has inhibitory activities against commercial cysteine proteases papain and legumain in vitro. The recombinant FaCys8 protein has also shown inhibitory activity agai nst protein extract from sting nematode. The FaCys8 gene has been overexpressed in strawberry, and the plants are being tested for resistance to nematode colonization. The goal of this work is to offer growers a safe, inexpensive and environmentally compatible alternative to nematocidic chemicals. 108. Mutations in the voltage-gated pota ssium channel KCNC3 cause degenerative and developmental CNS phenotypes Waters MF1,2,3, Minassian NA4, Durr A5, Brice A5, Papazian DM4, Pulst SM6 1Department of Neurology, University of Florida, Gainesville, FL 2Department of Neuroscience, Unive rsity of Florida, Gainesville, FL 3Evelyn F. and William L. McKnight Brain Instit ute, University of Florida, Gainesville, FL 4Department of Physiology, University of California, Los Angeles, Los Angeles, CA 5INSERM, Paris, France 6Department of Neurology, University of Utah, Salt Lake City, UT Objective: To determine the etiology of an inherited spinocerebellar ataxia. Background: Potassium channels influence many aspects of electrical excitability in nerve and muscle, and mutations in their genes have been described in episodic di seases. We have found that K+ channel mutations also cause disease phenotypes with neurodevelopmental and neurodegenerative features. These phenotypes are characterized in: a Filipino pedigree with progressive, adult-onset ataxia, and a Fren ch pedigree with childhood-onset ataxia. Design/Methods: A genome-wide linkage scan, followed by high-resolution mapping, was performed on the Filipino ataxia pedigree. Using a candidate gene approach, sequencing was performed to identify the causative mutation. Func tional data was generated with voltage-clamp experiments in a heterologous expression system. Results: The causative gene mapped to an 800Kb region of 19q13, overlapping the SCA13 locus previously described in a French pedigree with slowly progressive childhood-onset ataxia, mild mental retardation, and seizures. This region contains KCNC3, encoding a voltage-gated Shaw potassium channel with enriched cerebellar expr ession. Sequencing KCNC3 revealed mutations 1554G A (R420H) in the Filipino and 1639C A (F448L) in the French pedigree. Both mutations alter KCNC3 function in the Xenopus oocyte expression system. KCNC3R420H, located in the voltage sensor of the channel, has no detectab le channel activity when expressed alone and a strong dominant negative effect when co-expre ssed with wildtype KCNC3. KCNC3F448L shifts the activation curve in the negative direction and slows channel closing approximately 7-fold. Thus, R420H and F448L mutations are expected to change the output characteristics of fast-spiking cerebellar neurons, where KCNC channels co nfer capacity for high frequency firing. Conclusions/Relevance: Our results establish a role for KCNC3 in disease phenotypes ranging from developmental disorders to adult-onset neur odegeneration. Moreover, mutations in the Kv3-
channel family, which are important for the properti es of bursting neurons, are sufficient to cause neurodegeneration. These findings suggest voltagegated potassium channels as valid candidates for additional neurodegenerative disorders, particularly those involving fast-spiking neurons. 109. Evidence of extensive in vivo recombination in human immunodeficiency virustype 1 (HIV-1) subtypes B and C Williams WB1, Gray R1, Salemi M1,*, Yin L1, Aldrovandi G2, Goodenow MM1,* 1Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 2Childrens Hospital Los Angeles, University of Southern California, Los Angeles, CA Human immunodeficiency virus type 1 (HIV-1) is comprised of closely related but unidentical viruses, or quasispecies. The genetic diversity of HIV-1 is a result of both the high nucleotide misincorporation rate by the error-prone revers e transcriptase, and heterologous recombination between distinct HIV-1 genomes, during replicat ion. Recombination occurs in multiply infected HIV-1 targeted CD4+ cells and likely plays a major role in the diversification of HIV-1 quasispecies. Recombinant virions ha ve been associated with rapid spread of drug resistance and an accelerated progression to AI DS. Likewise, recombination provides an efficient mechanism for lateral transfer of genes that increase viral fitne ss and pathogenicity. Thus, an efficient and cost effective method of detecting recombinants in HIV1 quasispecies is critical for studies analyzing HIV-1 genetic diversity. Through limiting diluti ons, uncloned single genome copies of HIV-1 env V1-V5 were sequenced from diverse HIV-1 cDNA or proviral DNA preparations. This strategy avoids the formation of PCR generated recombinants and thus the sequences generated in this study are representative of in vivo generated viruses. This strategy was applied in validation of a previous report that detected high frequency of HIV-1 recombinants in the breast milk of HIV-1 subtype C infected women. The prior study utiliz ed cloning of bulk PCR-amplified genomes, which introduced the possibility of artifacts as observed recombinants. Application of our single genome sequencing approach confirmed the earlier findings by identifying similar frequency of HIV-1 env V1-V5 recombinants. A high frequency of recombinan ts was also detected in a HIV-1 subtype B sample with previously reported env diversity. Single genome recombinant screening will provide a reliable means of intra-study validation of high er throughput PCR-based sequencing methods, and provides evidence that recombinants play a major role in viral diversification and viral evolution. 110. A dominant temperature-sensitive proteasome subunit mutation for conditional lethality in tephritid fruit fly pests Nirmala X, Zimowska G, Handler AM* Center for Medical, Agricultural, and Veterinary Entomology, Agricultural Research Service, U.S. Department of Agriculture, Gainesville, FL Tephritid fruit flies are highly destructive insect pests that damage more than 200 fruit and vegetable plant hosts world-wide, and in recent years SIT has been the primary means of their biological control. Conditional lethal release (CLR ) is a variation of the sterile insect technique (SIT) for the biological control of pest insects resulting from the release of transgenic insects carrying dominant conditional lethal genes. Here we describe a heat-sensitive conditional lethal system that uses a mutant proteasome subunit, Pros 21, first described in Drosophila melanogaster as the DTS-7 mutation (Smyth and Belote, 1999, Genetics 151(1):211-20).
Proteasomes play a critical role in eukaryote development by regulating protein degradation. Ubiquinated proteins undergo proteolysis in a multi-subunit complex known as the 26S proteasome, which is comprised of a 20S core and 19S regulatory complexes (Yao and Cohen, 2002, Nature 419(6905):403-7). Missense mutations in the 20S subunits lead to the production of dominant temperature-sensitive (DTS) poi son subunits or antimorphs that disrupt proteasome function. Pros261 and Pros 21 are two such mutations identified originally in D. melanogaster that result in late larval or pupal lethality at 29 C (Saville and Belote, 1993, Proc Natl Acad Sci USA 90(19):8842-6; Smyth and Belote, 1999, Genetics 151(1):211-20). In order to study the potential of these genes to control tephritid population D. melanogaster Pros261 was used to genetically transform the Caribbean fruit fly Anastrepha suspensa. Heterologous expression of Pros261 resulted in approximately 90% late larval or pupal mortality in some transgenic strains (Handler, Oishi, and Krasteva, unpublished). In order to improve the temperature-dependent lethality we isolated and mutated the native Pros 2 gene cognate from the Caribb fly. The AsPros 2 transcript isolated from pupal cDNA is 1,022 nucleotides encoding 280 amino acids with more than 70% homology to the D. melanogaster protein and the predicted proteins from Aedes aegypti, Anopheles gambiae and Culex pipens. To study the use of aberrant AsPros 21 for CLR, the AsPros 2 transcript was mutated in vitro at position 723 resulting in a codon change from GGG to AGG creating the amino acid substitution Gly170Arg. Transgenic mutant strains were created by introducing the mutated gene with its own regulatory sequence into a piggyBac transformation vector to create pB[PUbDsRed.T3-AsPros 21] which was transformed into wild type A. suspensa. Homozygous lines with the mutant gene developed into normal appearing pupae at similar frequenc ies at both 25C and 29C, but viability was somewhat lower compared to non-transgenic wild type pupae. In tests for adult eclosion, at 25C 83% of wild type pupae reached adulthood comp ared to 74% at 29C. Survival was generally lower in the transgenic pupae, ranging from 54 to 61% at 25C, but 3% or lower survival at 29C. We show that a point mutation introduced into the AsPros 2 gene from A. suspensa, analogous to the Pros 21 mutation in D. melanogaster, results in a similar dominant temperaturesensitive lethal effect during pupal development. This temperature-dependent lethal system can potentially act as an environmentally benign insect management program, particularly in tropical and sub-tropical regions. 111. Development of a Drosophila simulans SNP chip for measuring allele specific expression Yang Y1, Walts B1, Graze R1, Nuzhdin S2, Wayne ML3,*, McIntyre L1,* 1Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2Department of Biological Sciences, University of Southern California, Los Angeles, CA 3Department of Botany and Zoology, University of Florida, Gainesville, FL For diploid organisms the two alleles of a particul ar gene can be expressed at different levels. Conventional arrays do not distinguish between the two alleles and instead measure total transcript abundance. However, recent reports indicate that array technology can be used for SNP detection and resequencing. Using information from a Drosophila simulans sequencing project, we are developing a custom array which measures the effects of two alleles separately. This will allow us to test for allele specific expression in Drosophila simulans transcripts. In this present project, the known SNPs have been cate gorized and evaluated and a rigorous selection process for SNPs has been developed. The se lected SNPs are then evaluated for their performance as probes in a resequencing design. Th e final selection includes more than half of all known transcripts in Drosophila. Chips will be available for purchase by the community shortly.
112. Inhibition of hepatitis C viral (HCV) replication by in vitro dimerization of IRF3 Yao L1, Zhu H2, Xu Y3, Lambiase L1, Nelson DR3, Liu C2, Li X1 1Department of Medicine, University of Florida, Jacksonville, FL 2Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 3Department of Medicine, University of Florida, Gainesville, FL Introduction: Type I interferon (IFN) has ability to induce an intracellular anti-viral state. Viral infection stimulates infected cells to produce the IFN via the RIG-I signaling pathway. Dimerization of IFN regulatory factors (IRF) plays a key step to produce IFN. The precise role of IRF in antiviral defense is not completely defined. In previous study, we have demonstrated that the fusion protein of STAT1 or STAT3 with the mouse estrogen receptor (ER) could form dimers by induction with the estrogen analog (4-HT). In th is study, we determined the role of IRF3 as a fusion protein with mouse ER to form the homodi mers in the establishment of intracellular antiHCV activity. Methods: The IRF3 open reading frame was fused with C-terminal domain of mouse ER, resulting in the expression of IRF3ER fusion protein. The cDNA plasmid was then transfected into the Huh7.5 cell line for subsequent infection with JFH1 HCV. The dimerization of IRF3 fusion protein was induced by 4-HT. IRF3ER dimerization was monitored by Western blot analysis. The antiHCV effects and IFN-beta expression of IRF3ER homodimers were determined by real-time RTPCR assay. Results: The IRF3ER fusion protein formed homodi mers upon 4-HT stimulation. The formation of IRF3ER homodimers substantially inhibited HCV RNA replication in correlation with the formation of homodimers, indicating the establishment of an tiviral activity in the hepatoma cell line Huh7.5. In the same study, we could not detect IFN-beta RNA in Huh7.5 cells transfected with IRF3ER plasmid and treated with 4-HT. Conclusion: We have established an inducible an d cytokine-independent IRF3 activation system. These studies can provide a valuable tool to understand the details of molecular events triggering the intracellular anti-HCV activity in liver cells. We will pursue the anti-HCV effects and IFN-beta expression induced by the IRF3ER fusion protein by establishing the stable cell line of Huh 7.5. 113. Inhibition of hepatitis C viral (HCV) replication by in vivo dimerization of STAT1 Li X1, Zhu H2, Butera M2, Nelson DR1, Liu C2 1Department of Medicine, University of Florida, Gainesville, FL 2Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL Introduction: Type I interferons (IFN) induce an intracellular antiviral state via the JAK-STAT signaling pathway. Dimerization of STAT1, STAT2, and STAT3 are key steps in this pathway. The precise role for each individual STAT in ant iviral defense is not completely defined. In our previous study, we have demonstrated that the fusion protein of STAT1 with the mouse estrogen receptor (ER) could form dimers by induction with the estrogen analog (4-HT). The IFN-induced genes play an important in anti-hepatitis C viral (HCV) activity. In this study, we determined the role of STAT1 as a fusion protein with ER to form the homodimers in the establish-ment of intracellular anti-HCV activity.
Methods: To create an inducible STAT1 dimeriza tion system, the STAT 1 open reading frame was fused with C-terminal domain of mouse ER, resulting in the STAT1ER fusion expression construct. The construct was then transfected into the Huh7.5 cell line for subsequent infection with JFH1 HCV. The fusion protein dimerization was induce d by 4-HT. 1 and 1.5 M of 4-HT was used to induce the dimerization of STAT1ER in Huh7.5. STAT1ER dimerization was monitored by Western blot analysis. The anti-viral effects were determin ed by real-time RT-PCR assay. The target genes of the STAT1ER dimers will be examined by cDNA microarray analysis. Results: The STAT1ER fusion protein formed homodi mers upon 4-HT stimulation. By transfection assay, the formation of STAT1ER homodimers substantially inhibited HCV full-length RNA replication in correlation with the formation of homodimers, indicating the establishment of antiviral activity in the hepatoma cell line Huh7.5. Conclusion: We have established an inducible and cytokine-independent STAT1 activation system. This system can provide a valuable tool to understand the molecular events triggering the intracellular anti-HCV activity in liver cells. The system will also allow us to identify STAT1specific target genes. 114. Functional significance of the epigene tic regulation of stress-induced cell death in Drosophila Zhang C, Lin N, Zhang Y, Zhou L* Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Epigenetic regulation plays an important role in stem cell maintenance, cellular differentiation, and aging process. Our lab recently demonstrated that during Drosophila embryogenesis, the cellular response to irradiation-induced apoptosis is also under the control of epigenetic blocking of an irradiation responsive enhancer region (IRER). However, the functional significance of this epigenetic regulation is still unknown. In order to test the significance of this regulation in response to environmental stress, we will generate mosaic clones deficient for IRER and study their fates in the lack of certain growth fact ors such as spitz (EGF). To fully understand the significance of this regulation in animal developm ent, we will also study the relationship between the IRER blocking and the cellular proliferation/differentiation. A newly generated transgenic strain containing an ubi-DsRed reporter within IRER allows us to monitor the accessibility of IRER in individual cells especially those undifferentiated quiescent stem cells. 115. Heat inducible expression of the gibberellin catabolizing enzyme AtGA2ox1 to improve turf quality of bahiagrass Zhang H1,2, Agharkar M1,2, Altpeter F1,2,* 1Agronomy Department, University of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL Bahiagrass is a popular low-input warm season turf species. However, its turf quality is limited due to its open growth habit and production of tall seedheads. Constitutive expression of the giberellin catabolizing enzyme AtGA2ox1 in bahiagrass reduced the endogenous bioactive gibberelin (GA) level and resulted in semi-dwarf plants with increased tillering, delayed flowering and shorter inflorescences (Agharkar et al., 2007, Plant Biotechnol J 5(6):791-801). However,
constitutive expression of AtGA2ox1 may compromise seed production. Therefore we cloned AtGA2ox1 under transcriptional control of a heat inducible promoter. The expression cassette was stably introduced into bahiagrass by biolistic gene transfer. The transgenic plants generated showed integration of AtGA2ox1and its differential expression under heat stress as compared to non stressed control plants. Transgenic lines are currently evaluated under controlled high and moderate temperature environments to study the effect of temperature on their phenotype. Field evaluation will follow to evaluate the seed production from transgenic lines. 116. Plant physiological and developm ental changes induced by green light Zhang TT, Maruhnich S, Folta KM* Horticultural Sciences Department, Univ ersity of Florida, Gainesville, FL Light not only provides plants with energy for me tabolism, it also regulates plants acclimatization to new environments and processes of plant grow th and development. Different light qualities have specific effects on plant growth and developm ent, and the effects of red, far-red, blue and UV light have been well described. Our laborato ry has shown clear, yet unexpected effects of green light that affect seedling and mature plant physiology. In this report the effect of green light on rosette architecture is tested, using ou r unique LED-based lighting system. Upon addition of green light to a background of red and blue, pl ants exhibit elongation of petioles, reduced leaf area, upward leaf reorientation, and decrease d leaf chlorophyll, symptoms associated with growth in shade. These phenotypes have only previously been associated with far-red light, mostly in an increase in the far-red to red light ratio. The phenotypes persist in known photoreceptor mutant backgrounds, suggesting that they may be mediated by redundant photosensor function or a novel photosensory system. Most importantly, this study shows that all parts of the spectrum contribute to plant growth and development. 117. Transcription factor NF-E2 and USF regulate recruitment, transfer, and phosphorylation of RNA polymerase II in the -globin gene locus Zhou Z, Viera KF, Levings PP, Li X, Huang S*, Bungert J* Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL -globin is an important subunit of the functional hemoglobin. Mutations in the -globin gene can cause sickle cell anemia and beta-t halassemia in mammals. The human -globin gene locus consists of five genes and a powerful long distance upstream enhancer, the locus control region (LCR). The LCR regulates downstream -like globin gene expression in a developmental stage specific manner. Many transcription factors play important roles in the process of LCR regulated -globin gene expression, such as GATA-1, EK LF, NF-E2, USF and TFII-I. Our laboratory proposed a model according to which transcription complexes are first recruited to a highly accessible LCR and subseque ntly transferred to the -globin gene promoter during differentiation of erythroid cells. Our recent da ta suggest that USF is required for the recruitment of RNA Polymerase II (RNA Pol II) to the LCR, and togeth er with NF-E2, facilitates the transfer of RNA Pol II to the -globin gene. More interestingly, even though NF-E2 is not required for the recruitment of RNA Pol II to the LCR, it plays an important role in the phosphorylation of RNA Pol II c-terminal domain in the -globin locus.
118. Low vitamin B12 status is associated with reduced global DNA methylation in young Chinese women Zhu J1,2,3, Hao L3, Li Z3, Quinlivan E2, Crider K4, Maneval D2, Yang TP1,* Rasmussen S4, Berry RJ4, Bailey LB2,* 1Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2Department of Food Science and Human Nutrit ion, University of Florida, Gainesville, FL 3Peking University Health Science Center, Beijing, China 4National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA Vitamin B12 is a required coenzyme for homocystei ne remethylation essential for generation of Sadenosylmethionine, the source of methyl groups for DNA methylation. The objective of this study was to assess the association of low vitamin B12 status with global DNA methylation in young Chinese women. Global DNA methylation wa s determined in two groups of women whose plasma vitamin B12 concentration was either deficient (<148pmol/L) (n=24) or normal ( 148pmol/L) (n=135). To adjust for the effect s of red blood cell (RBC) folate on DNA methylation, a subset (n= 24) of the vitamin B12normal subjects were matched for RBC folate concentration with the vitamin B12-deficient subjects. DNA methylation was expressed as a percent of methylated cytosines (as measured by a novel LC-MS/MS assay). The mean SD % methylcytosine to cytosine ratio for the B12-de ficient group (3.510.59) was lower (p<0.001) than the total B12-normal group (4.420.18), and the B12-normal RBC folate-matched group (4.40.17). These data suggest that impaired vitamin B12 status may reduce global DNA methylation and provide a rationale for future investigations. 119. Functional differentiation of Brassica napus guard cells and mesophyll cells revealed by comparative proteomics Zhu M1, Dai S1,2, McClung S3, Zhu N1,3, Chen S1,3,* 1Department of Botany and Zoology, University of Florida, Gainesville, FL 2College of Life Science, Northeast Forestry University, Harbin, China 3Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL Guard cells are highly specialized cells, forming ti ny pores called stomata on leaf surface. The opening and closing of stomata control leaf ga s exchange and water transpiration. Mesophyll cells are specialized for photosynthesis. Despite of the phenotypic and functional differences between the two types of cells, the full protein components and their functions have not been explored, but are addressed here through a comp arative proteomic analysis of purified guard cells and mesophyll cells. With the use of iTRAQ tagging and two-dimensional liquid chromatography mass spectrometry, we have identified 1458 non-redundant proteins in the guard cells of Brassica napus leaves. Numerous proteins were found to be differentially expressed between guard cells and mesophyll cells. Proteins involved in energy (respiration), transport, transcription (nucleosome), cell structure, and signaling are preferentially expressed in guard cells. By contrast, proteins involved in photosynth esis, starch synthesis, disease/defense/stress, and other metabolism (primary and secondary) are preferentially represented in mesophyll cells. This work represents the most extensive proteomic description of B. napus guard cells and has improved our knowledge of the functional specification of guard cells and mesophyll cells.