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1 Florida Genetics 2012 Organizing Committee Co Chair s : Connie Mulligan and Michele Tennant Members: Charlie Baer, Henry Baker, John Davis, Rob Ferl, D ouglas Soltis, Thomas Yang FG2012 Sponsors Platinum Level Department of Molecular Genetics and Microbiology, Interdisciplinary Center for Biotechnology Research Gold Level Center for Epigenetics, College of Liberal Arts and Sciences, Emerging Pathogens Insti tute, Graduate Program in Plant Molecular and Cellular Biology, Health Science Center Libraries University of Florida Genetics Institute Silver Level Animal Molecular and Cellular Biology Graduate Program Eve lyn F. & William L. McKnight Brain Institute Special Thanks To Matthew Daley Taum Hanlon, Rolando Milian, Kristyn Minkoff, Tessa Mitchell, Hope Parmeter, Connie Philebaum, Dustin Blanton, Bret Boyd, Tamar Carter, Mathew Citarella, Ana Carolina Costa Sa, Lauren Douma, Justin Fear Terry Felderhoff, Ruli Gao, Lara Ianov, Heather Rose Kates, Hye Won Lee Guang Y ao Li, Lana McMillan, Aida Miro, Katie O'Shaughnessy Marcio Resende, Justyna Resztak, Tatiana Salazar Andrew Smith, Jared Stees Ming Tang, Shaojun Tang, Carl Xiao, Yuanqing Yan University of Florida Genetics Institute Interim Director : Henry Baker Associate Directors: Donald McCarty and Connie Mulligan Executive Board: William Allen, Henry Baker, Brad Barbazuk, Jorg Bungert, Sixue Chen, John Davis, Robert Ferl, Curt Hannah, William Hauswirth, Julie Johnson, Donald McCarty, Michael Miyamoto, Connie Mulligan, Nic k Muzyczka, Douglas Soltis, Pamela Soltis, Michele Tennant, Mart a Wayne, and Thomas Yang Scientific Advisory Board: Rebecca W. Doerge, PhD Professor, Departments of Agronomy and Statistics, Purdue University, West Lafayette, IN Yoram Groner, PhD Barnet Berris Professor of Cancer Research, Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Peter M. Howley, MD George Fabyan Professor of Comparative Pathology and Chair, Department of Pathology, Harvard Medical School, Boston, MA Thomas J. Kelly, MD PhD Director, Sloan Kettering Institute, New York, NY Ronald L. P hillips, PhD Regents Professor, Agronomy and Plant Genetics, University of Minnesota St. Paul, MN Ron Sederoff, PhD Distinguished Univ ersity Professor of Forestry, North Carolina State University Forest Biotechnology Group, Raleigh, NC Thomas E. Shenk, PhD James A. Elkins, Jr. Professor in the Life Sciences, Molecular Biology, Princeton University, Princeton, NJ
2 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 bio logy and genetics. The replication and the existence of a universal genetic code for all living beings, have together provided the basis for the understanding of fundamental cellul ar processes, mutation and genetic repair, genetic variation, the origin of life and evolution of species, and the structure/function/regulation of genes. The double helix is also proving to be of immense significance to advances in agriculture, medicine a nd such other diverse fields as anthropology, criminology, computer science, engineering, immunology, nanotechnology, etc. It was the study of DNA that led to the development of tools that brought about the biotechnology revolution, the cloning of genes, a nd the sequencing 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 these objectives, the digital nature of DNA and its complementarity are beginning to be exploited for the development of biology as an information based science. Indeed, a paradigm shift is already taki ng place in our view of biology, in which the natural, physical, engineering and environmental sciences are becoming unified into a grand alliance for systems biology. Indeed, biology in the 21 st century will be surely dominated by this expanded vision. Th e Genetics Institute is committed to fostering excellence in teaching and research, and in promoting cross campus interdisciplinary interactions and collaborations. In the pursuit of these objectives, it offers a graduate program in genetics, and has ident ified the following four key areas for teaching, research and development: Bioinformatics, Comparative Genomics, Population and Statistical Genetics, and Epigenetics.
3 2012 Florida Genetics Symposium Schedule Florida Genetics 2012 is in honor of Kenneth I. Berns, MD, PhD, pioneer in the use of adeno associated virus (AAV) in gene therapy. Wednesday, November 28, 2012 11:00 a.m. 1 2 :00 p.m.: Check in and poster set up at the CGRC posters no 1 65 1 2 :00 p.m. 1 2 :15 p.m.: Opening Remarks: Connie Mulligan and Henry Baker Session I Gene Therapy Chair: Henry Baker 1 2 :15 p.m. 12:45 p.m., Nick Muzyczka* : Gene therapy, 1974 2012: a re we there yet? 12:45 p.m. 1:15 p.m., Mavis Agbandje McKenna : Tackling AAV gene delivery challenges with structural virology 1:15 p.m. 1:30 p.m. : Coffee break 1:30 p.m. 2:15 p.m.: Terence Flotte : enetic emphysema: from idea to p 2:15 p.m. 3:00 p.m.: Jude Samulski : Hairpins to helper virus and the impact on AAV vectors 3:00 p.m. 3:30 p.m.: Michael Linden : Site specific integration by AAV: back to the future 3:30 p.m. 5:00 p.m.: Poster Session I : Posters no. 1 65 award presentation for the Codified Art + Genetics competition and r eception (for registered attendees) ** 5:30 p.m. 6:3 0 p .m., David Baltimore : AAV to the rescue (Introduction by Bert Flanegan ) *= UF Genetics Institute Faculty **All activities will be at the Cancer/Genetics Research will be at 5:30 p.m. Wednesday in the auditorium of the HPNP building on the Health Science Center campus. Thursday, November 29, 2012 8 :3 0 a.m. 9:0 0 a.m.: Check in coffee set up posters no. 66 130 Session II Epigenetic Modification of Phenotype Chair: Thomas Yang 9:00 a.m. 9:4 5 a.m., Michael Skinner : Environmentally induced epigenetic transgenerational inheritance of reproductive phenotypes and disease: ancestral ghosts in your genome 9:45 a.m. 10:1 5 a.m., James Resnick : Imprinti ng mechanisms underlying Prader Willi and Angelman syndromes 10:1 5 a.m 10:3 0 a.m.: Coffee break 10:30 a.m. 11:15 a.m., Karen McGinnis : Epigenetic gene regulation and phenotype in maize 11:15 a.m. 11:4 5 a.m., Connie Mulligan : Epigenetic alterations and stress among new mothers and infants in war torn Democratic Republic of Congo 11:45 a.m. 1:30 p.m.: Poster Session II : Posters no. 66 130 11:45 a.m.: Lunch (for registered attendees) Session III Genetic Modification of P lants Chair: John Davis 1:30 p.m. 2:15 p.m., Steve Strauss : Transgenic forests: A funny thing happened on the way to the revolution 2: 15 p.m. 2:45 p.m., Curt Hannah : ADP glucose pyrophosphorylase: from bacterial selection to yield in the field 2:45 p.m. 3:15 p.m., Harry Klee : The chemist r
4 Presentation Abstracts (Listed in the order of presentation, external speakers followed by internal speakers) Developing gene therapy for g enetic emphysema: from idea to p hase 2 clinical trials Flotte T R Department of Pediatrics University of Massachusetts Medical School, Worcester, MA Department of Microbiology and Physiologic al Systems, University of Massachusetts Medical School Worcester, MA Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA Alpha 1 antitrypsin (AAT) deficiency is a common single gene disorder caused by mutations in the SERPINA1 gene which encodes an abu ndant serum antiprotease, AAT. AAT is normally synthesized predominantly in hepatocytes, and is secreted for activi ty in the plasma and interstitial fluid, where it protects the interstitial elastin of the lung from degra dation by neutrophil elastase. AAT deficiency is remarkably homogeneous genetically, with over 95% of patients possessing at least one copy of the spe cific mutant allele E3 42K (known as the PiZ allele). Z AAT forms stable polymers which are trapped within hepatocytes, resulting in a deficiency state in plasma, which in turn results in emphysema in the vast majority of symptomatic individuals. Studies in compound heterozygotes indicate that a plasma level of 11 microm olar or 571 mcg/ml is protective against the development of emphysema, and protein replacement products requiring weekly infusion, have been FDA approved based on achieving that level. 12 to 15% of Z homozygotes also develop liver disease, which appears to be linked to the retention of the mutant protein, although the phenotypic variation in the liver phenotype among Z homozygotes remains unexplained. Our laboratory began studies of gene the rapy for AAT deficiency, initially developing a series of rAAV2 AAT vectors utilizing different promoters and routes of administration. Targeting the lung disease initially, it was demonstrated that supra therapeutic levels of serum AAT could be achieved in mice using rAAV2 AAT in muscle, with the AAT driven from a CMV enhancer/beta actin promoter/hybrid intron (CBA or CAG) expression cassette. The intramuscular (IM) route of administration was chosen because of its safety and relatively high efficiency. Formal preclinical and a phase 1 clinical trial of rAAV2 AAT were performed. Safety was excellent, but levels were low and transient. A switch to rAAV1 AAT was made because of data from mice indicating a 1000 fold potency advantage over rAAV2 with IM deliv ery. Again, formal preclinical toxicology studies were completed, and subsequent phase 1 and phase 2 clinical trials have been undertaken. These clinical studies have demonstrated robust expression from IM administration, persistent at levels at 3 to 5% o f the target level for 12 months after a single IM injection. The immune response profile is complex, with both CD4 and CD8 responses to rAAV1 capsid epitopes, apparently not interfering with long term transgene expression. In parallel, a strategy for lon g term simultaneous allele specific knockdown of Z AAT and augmentation of wild type M AAT has been targeted to the liver using a bifunctional rAAV9 vector expressing both a synthetic miRNA targeting endogenous human AAT and an augmentation allele that has been altered in its codon usage to make it resistant to that miRNA while still expressing the wild type amino acid sequence. This construct resulted in 80% knockdown and therapeutic augmentation levels for three months after a single systemic intravenous (IV) injection. Further studies of gene therapy for both the lung and liver disease of AAT deficiency are planned. Supported by grants from the NHLBI, NIDDK, Alpha One Foundation, and AGTC. Biography of Terenc e R. Flotte, MD As Dean of the School of Medicine and Executive Deputy Chancellor of the University of Massachusetts Medical School, Terry Flotte, MD, serves UMMS as chief academic and administrative officer of the School of Medicine, overseeing all acade mic activities of the basic and clinical science departments, including education and research for the School of Medicine and the Graduate School of Biomedical Sciences. He was also recently appointed as the Celia and Isaac Haidak Distinguished Professor of Medical Education. Dr. Flotte joined UMMS from the University of Florida, where he was the Nemours Eminent Scholar and Chair of the Department of Pediatrics for the College of Medicine. He is a graduate of the University of New Orleans and the Louisiana State University School of Medicine. He served his residency in pediatrics at Johns Hopkins University, where he completed a pediatric pulmonary fellowship and postdoctoral t raining in molecular virology. nal Institutes of Health, the Cystic Fibrosis Foundation, the Juvenile Diabetes Research Foundation and the Alpha One Foundation. He has received numerous honors and awards and is a member of many professional organizations. In 2005, Dr. Flotte received th Mead Johnson Award for Outstanding Scientific Contributions. In 2010, he was elected to the Association of American Physicians and became a member of the Society for Clinical Investigation.
5 Hairpins to helper virus a nd the impact on AAV vectors Samulski RJ Department of Pharmacology, University of North Carolina, Chapel Hill, NC Gene Therapy Center, University of North Carolina, Chapel Hill, NC Adeno a ssociated virus (AAV) vector is demonstrating therapeut ic effect in a number of phase 1 clinical trials. Although AAV has very broad tropism, most humans have neut r alizing antibodies that restrict vector re administration. To further enhance AAV vector tropism and identify capsid variants that escape neutralizing antibody, we have generated synthetic AAV2 capsid by replacing a hexapeptide sequence in a previously identified heparan sulfate receptor footprint with corresponding residues from AAV8. The AAV2/AAV8 chimera designated AAV2i8 has displayed an altered ant igenic profile, readily traversed the blood vasculature, and selectively transduced muscle tissues including the heart with high efficiency in animal models. More remarkable, this capsid variant reduced hepatic tropism. In a parallel study, we demonstrated that a single amino acid (aa) insertion of Threonine into AAV2 capsid would convey high muscle transduction and changed the capsid immune profile as well. To explore whether the single aa insertion tropism in AAV2 was distinct from AAV2i8 capsid we insert ed aa at 265 (AAV2i8D), and injected AAV2i8D vector encoding the firefly luciferase transgene into mice via the tail vein. Compared to parental AAV2i8, liver tropism was rescued and muscle transduction was preserved. To address whether unique pharmacokinet ic, pharmacodynamic, and antigenic features of these synthetic AAV variants studied in mice translated across species, we screened thirty three gravid nonhuman primates for AAV antibodies and identified six for study. Pregnancies were monitored sonographic ally during gestation and newborns were delivered at term by cesarean section. Three AAV/luc vectors (AAV9, AAV2i8, and AAV2i8D) were administered intravenously at birth (N=2 per construct). Infants were raised in a nursery and transgene expression was mon itored monthly by bioluminescence imaging (BLI) immediately after the intravenous injection of D luciferin. Blood samples were also collected from a peripheral vessel monthly to monitor complete blood counts (CBCs) and clinical chemistry profiles. Animals remained healthy during the study period, to date, and two years post vector administration health, growth, CBCs, and clinical chemistry panels were within normal limits for the age group. Vector transgene expression determined by BLI has persisted without significant decline over time. Our results suggest that rational designed AAV variants can induce identical long term transgene expression without safety concerns across various species (mice to nonhuman primates). Further studies comparing species specif ic vector transduction tropism and immune profiles after systemic injection will be discussed. Bio graphy for R. Jude Samulski, PhD therapy. The major research effort utilizes adeno associated virus (AAV) which is a non pathogenic human parvovirus. In the absence of hel per adenovirus, AAV establishes latency by integration into the host genome. The research from this laboratory has identified critical steps involved in adeno associated virus (AAV) assembly and identified receptors for AAV type 1 6. These new findings, co upled with understanding vector persistence in vivo have played a role in the development of the fir st clinical trial for Duchenne muscular d ystrophy (DMD) in 2007 with Dr. Xiao Xiao. Efforts continue with important collaborations in the areas of: therapy for seizure (R01 NS035633), cystic fibrosis (P01 051818), pulmonary and hematologic disorders (P01 HL066973), and pharmacodynamics (P01 GM059299). Dr. Samulski graduated from the U niversity of F lorida from Dr. Nicholas Muzyczka laboratory followed by a post doc with Tom Shenk at SUNY Stony Brook and Princeton. He established his research lab at the U niversity of Pitt Department of Biology in 1986 as assistant p rofessor Dr. Samulski came to UNC in 1993 as an associate professor where he became t he first Director of the UNC Gene Therapy Center as member of the Department of Pharmacology. During this time he has served on many NIH committees (e.g. RAC, and recently served as 2012 President of the American Society of Cell and Gene Therapy ). Dr. Samu ransfer have been acknowledged by various societies including the National Hemophilia Foundation ociety's first awardee of Outs tanding Achievement Award. In the last 1 4 years he ha s trained more than 20 graduate students and over 33 postdoctoral fellows The lab oratory continues to focus on how to engineer AAV for gene therapy applications, generate novel AAV mutant vectors and map the structural determinants and mechanisms of AAV capsid cell entry.
6 Site specific integration by AAV: back to the future Linden R M Department of Infectious Diseases King's College London School of Medicine at Guy's, King's and St Thomas' Hospitals, London, UK Gene Therapy Consortium, University College London London, UK AAV is a virus that does not cause any known disease and has t he unique ability to insert its genome into one specific site in the human genome. These features render this virus an ideal vehicle to shuttle corrective genes into the genome of people with genetic diseases. However, genetic engineering of cells for ther apeutic purposes requires a thorough understanding of the viral mechanism of integration. We are testing the hypothesis that the mechanism underlying site specific integration is based on the evolutionarily ancient bacterial parasite DNA replication mode a nd, as a consequence, whether AAV genome integration is a byproduct from biochemical activities of the AAV Rep proteins that are necessary for viral genome replication. The proteins responsible for this mode of replication, the rolling circle replication ( RCR) initiators, are amongst the evolutionarily oldest proteins known. Their signature protein domain, the RCR domain, is present in the AAV Rep protein, which is the viral factor necessary and sufficient to mediate integration. In addition, we explore whe ther evolutionary adaptations have occurred to this ancient mechanism in order to secure stable integration within the complex chromosomal environment of the target site in the host. Biography for R. Michael Linden, PhD R. Michael Linden, Ph.D. is a Therapy Consortium at University College London. After graduating in Biochemistry and Molecular Biology at the University of Zrich, Switzerland, Dr. Linden trained in mo lecular virology under the guidance of Dr. Kenneth I. Berns at Cornell University Medical College, N.Y. Since then Dr. Linden has used a multidisciplinary approach to understanding the molecular mechanisms and the potential applicability of site specific g enome integration by adeno associated virus (AAV). His contributions to this field include the proposal for a mechanism of this potentially unique aspect of a viral strategy an d the structural determination of the AAV Rep proteins, which orchestrate all as pect s of this human parvovirus. During into complex viral mechanisms. More recently, Dr. Linden has engaged in the field of AAV mediated gene therapy and, in his role as the Director of the UCL gene therapy consortium is responsible for clinical grade vector production and the support of further development of UK gene therapy efforts. In this context Dr. Linden is actively engaged in trans lational projects aimed at the developmen trophy, lysosomal storage diseases and a variety of eye diseases using AAV based vectors. During his career Dr. Linden has served on many scientific advisory committees including the Virology and Experimental Virology study sections and review panels evaluating human embryonic stem cell programs. Dr. Linden is currently an associate editor of P LoS Pathogens AAV to the rescue Baltimore D Division of Biology, California Institute of Technology, Pasadena, CA Almost a decade ago, being convinced that standard routes to an HIV vaccine were unlikely to be effective, we committed ourselves to developing a new route to HIV prophylaxis. We believed, and have been vindicated in this belief, that broadly neutralizing anti HIV antibodies (bnAbs) would be found. Then the best was b12, an antibody that was very effective against clade B HIV. We thus u ndertook to find a way to use b12 to protect against HIV infection. Our approach was to take advantage of the power of viral vectors to express Ab genes in human cells. We had the generous support of the Gates Foundation. Although we started with lentivi ral vectors, when Alex Balazs joined my laboratory, he thought to use adeno associated v irus (AAV) as a vector. All I knew about AAV was that my old friend, Ken Berns, had worked on it for many years. I was not aware of how versatile and how powerful it wa s. We realized that it had enough genetic space to encode one heavy and one light chain antibody gene with a little left over for regulatory elements. When an AAV vector encoding b12 was injected into the muscle of mice, up to 1 mg/ml of serum could be ach ieved and maintained for a year. Using immunocompromised mice with an adoptive human immune system, we could completely protect against HIV infection with the high levels of b12 we could achieve. More broadly reactive and more potent anti HIV Abs then bec ame
7 available. We found that our platform is versatile; we can express a variety of Abs and can protect against a variety of HIV strains delivered by both the intravenous and vaginal routes. We are now developing a clinical trail of this form of anti viral prophylaxis, which we call VIP: Vectored ImmunoProphylaxis. We are also ext ending VIP to other pathogens. Biography for David Baltimore PhD David Baltimore, former president of the California Institu te of Technology (1997 2006), President Emeritus and the Robert Andrews Millikan Professor of Biology is an accomplished researcher, educator, administrator and public advocate Chemistry from Swarthmore College in 1960 and a PhD in 1964 from Rockefeller University. Awarded the Nobel Prize in 1975 in Physiology or Medicine for his research into viral replication that provided the key to understanding the life cycle of retroviruses, Dr. Baltimor e has profoundly influenced national science policy on such issues as recombinant DNA research and the AIDS epidemic. Dr. 1999 National Medal of Science and 2000 Warren Alpert Foundation Prize. He is Director of the Joint Center for Translational Medicine, a collaborative effort joining Caltech and UCLA in a program translating basic science discoveries into clinical realities. He is past President and Chai r of the American Association for the Advancement of Science a nd has published more than 650 peer reviewed articles. Environmentally induced epigenetic transgenerational inheritance of reprod uctive phenotypes and disease: a ncestral ghosts in your genome Skinner MK Center for Reproductive Biology, Washington State University, Pullman, WA School of Biological Sciences, Washington State University, Pullman, WA Transgenerational effects of environmental toxicants significantly amplify the impact and health hazards of t hese compounds. One of the most sensitive periods to exposure is during embryonic gonadal sex determination when the germ line is undergoing epigenetic programming and DNA re methylation. Previous studies have shown that endocrine disruptors can cause an i ncrease in a number of adult onset diseases in fertility, prostate, ovary kidney and cancers. Interestingly, this effect is transgenerational (F1, F2, F3 and F4 generations) and hypothesized to be due to a permanent (imprinted) altered DNA methylation of the germ line. The transgenerational epigenetic mechanism appears to involve the actions of an environmental compound at the time of sex determination to alter the epigenetic (i.e. DNA methylation) programming of the germ line that then alters the transcri ptomes of developing organs to induce disease development transgenerationally. Recently a variety of different environmental compounds have been shown to induce this epigenetic transgenerational inheritance of disease including: the fungicide vinclozolin, plastics BPA and phthalates, pesticides, dioxin and hydrocarbons. The suggestion that environmental factors can reprogram the germ line to induce epigenetic transgenerational adult onset disease is a new paradigm in disease etiology that is also relevant to other areas of biology such as evolution. Biography for Michael K. Skinner PhD Dr. Michael Skinner is a professor in the School of Biological Sciences at Washington State University. He did his BS in chemistry at Reed Col lege in Portland Oregon, his PhD in biochemistry at Washi ngton State University and his p o stdoctoral f ellowship at the C.H. Best Institute at the University of Toronto. Recently he did a sabbatical in bioinformatics at Rosetta/Merck. He has been on the faculty of the Pharmacolog y Department at Vanderbilt University focused on the area of reproductive biology and environmental epigenetics. Recent studies have e lucidated several critical events in the initiation of male sex differentiation, testis development and ovarian primordial follicle development. His current research has demonstrated the ability of endocrine disruptors and environmental toxicants to promot e epigenetic transgenerational inheritance of adult onset disease phenotypes, due to abnormal germ line epigenetic programming during gonadal development. Dr. Skinner has over 225 peer reviewed publications and has given over 220 invited symposia, plenar y lectures and university seminars. Dr. Skinner established and was the founding Director of the Washington State University and University of Idaho Center for Reproductive Biology (CRB) since its inception in 1996. The CRB has over 90 faculty and is one of the largest reproductive sciences research c enters in the world. Dr. Skinner also established and was the founding Director of the Center for Integrated Biotechnology (CIB). The CIB was established in 2002 and has over 170 active research faculty mem bers. In 2008 he s tepped down as Director of the
8 c enters to focus his efforts on his research. His research has been highlighted in BBC and PBS documentaries and selected as top 100 discoveries in 2005 and 2007 by Discover In addition, Dr Skinner has been actively involved with the start up of several biotechnology companies. Epigenetic gene regulation and phenotype in maize McGinnis K M Dep artment of Biological Science, Florida State University, Tallahassee, FL Epigenetic gene regulation is crucial for normal growth and development in a broad range of organisms. It can be both responsive to environmental conditions, and heritable from one generation to the next. Thus, epigenetic gene regulation represents importa nt mechanisms that bridge genotype to phenotype. In plants, one epigenetic regulatory pathway involves siRNA directed DNA methylation and chromatin modifications. In recent years, several components of this pathway have been genetically identified and exte nsively studied in the agriculturally and economically important crop, Zea mays These discoveries have facilitated the understanding of the mechanisms and impacts of RNA directed epigenetic gene regulation in plants. Results of recent genetic, genomic and bioinformatic approaches to assess the impact of this pathway on gene expression and genome regulation will be described. Biography for Karen M. McGinnis, PhD Dr. Karen McGinnis is an assistant professor in the Department of Biological Science at Florida State University. Prior to joining the faculty in 2008, she held postdoctoral positions at the University of Arizona and with the USDA ARS at Washington State Universit y. Dr. McGinnis received her PhD in 2000 in Plant Biology from Arizona State Uni versity. Her research focuses on epigenetic regulation of gene expression in maize. Transgenic forests: a funny thing happened on the way to the revolution Strauss SH Department of Forest Ecosystems and Society, Oregon State University Corvallis, OR Molecular and Cellular Biology Graduate Program Oregon State University, Corvallis, OR Because of their difficult genetic systems, transgenic methods had been touted as a revolutionary new means for conducting research and breeding of forest trees. The high heterozygosity, intolerance of inbreeding, and delayed onset of reproduction in forest trees makes conventional genetic approaches to genetic analysis nearly impossible. As a result, quantitative and whole genome approaches, rather than Mendelian app roaches, have dominated research and breeding. Transgenic methods could provide powerful new tools in bypassing the sexual cycle and enabling single gene gain of function or loss of function phenotypes to be readily produced. RNA interference has been fo und to be an effective means for reduced function studies for numerous genes, which I will demonstrate from our studies of genetic control of flowering time in poplar by its TERMINAL FLOWER/CENTRORADIALIS homologs. Gain of function transgenic mutagenesis and gene traps have enabled the discovery of novel genes that control complex traits, which I will illustrate with our results from activation tagging of tree size and dormancy traits in poplar. Of course, transgenic approaches also enable novel traits, s uch as for pest or abiotic stress tolerance, to be inserted based on genes from other species that would be impossible via breeding. However, transgenic approaches have not been widely adopted in research or breeding of forest trees for a number of reason s that were not widely appreciated when they were first introduced in the 1980s. First, extending transformation/regeneration methods outside of model species has proven much more difficult and costly than first expected. The inflexible developmental sys tem of trees poses obstacles that traditional in vitro horticulture and developmental biology have so far been unable to overcome, or are very costly to work around, for most species. Even species of immense economic importance, such as eucalypts and pine s, remain highly recalcitrant, limiting transgenic studies to very few laboratories and companies worldwide. Second, anti GMO campaigns have been far more widespread and influential than expected, leading to extremely onerous regulatory regimes and market place obstacles that treats all products of transgenic methods as putative environmental and food/feed safety threats, making essential field studies very difficult or impossible to carry out, including in the USA. Finally, the combination of extremely ra pid DNA sequencing and computation methods, and lack of social resistance to its applications in molecular breeding, has moved the field of
9 biotechnology toward genomic analysis and selection applications. Thus, transgenics have been pushed to the sidelin es for the foreseeable future, and there is a near absence of public funding and a declining capability for transgenic studies in academia. This is occurring despite the established power of transgenic methods to help modify traits such as pest and stress resistance, of direct and obvious value for helping forests cope with growing climate associated stresses. A scientific, regulatory, and marketplace revolution is needed if transgenic tools are to make a significant contribution to forest health and produ ctivity. Biography for Steven H. Strauss, PhD Dr. Steven H. Strauss is a Distinguished Professor of Forest Biotechnology in the Department of Forest Science at Oregon State University, and has a joint appointment in the Molecular and Cellular Biology Graduate Program. He is Director of the Tree Biosafety and Genomics Research Cooperative at OSU, a university public agency industry consortium formed in 1994. It conducts research on the biosa fety and physiology of genetically engineered trees used in plantation forestry and horticulture. Strauss directs the OSU Program for Outreach in Resource Biotechnology, aimed at promoting public understanding, and facilitating science based public debate s, in food and natural resources biotechnology ( http://agsci.oregonstate.edu/orb/ ). Strauss received the Barrington Moor e Memorial Award from the Society of American Foresters in 2001, which recognizes outstanding achievement in biological research leading to the advancement of forestry. He was recognized as a Fellow of the American Association for the Advancement of Science in 2009, for advancing of the science and policy regarding forest biotechnologies. He was the Forest Biotechnology Partners Forest Biotechnologist of the Year in 2009 recognizing his outstanding contributions to science, dialogue, and stewardship. In 2005 he was recognized as a Leopold Leadership Fellow taking part in a program aimed at training eminent environmental scientists to be more effective at in fluencing public policy and presenting science to news media. Dr. Strauss has earned degrees in biological sciences from Cornell, Yale, and the University of California at Berkeley. He has published 200 scientific papers, given more than 200 invited lectu res on biotechnology and genetics of trees, and obtained more than 17 million dollars of competitive grant support. His laboratory has trained 22 graduate students, 26 postdoctoral scientists, and more than 40 professional and technical staff. He has serv ed on panels at the United States National Research Council, National Science Foundation, and Department of Agriculture. Dr. Strauss' current research focuses on genetic engineering of flowering, stature, and transformation based "functional genomics" usi ng poplar trees as model organisms. He has also advised governments and written in many scientific journals about national and international regulations on field research and commercial development of genetically engineered crops and trees. He was an edi tor for the highly cited plant science journal New Phytologist for nine years.
10 *= UF Genetics Institute Faculty Gene therapy, 1974 2012: a re we there yet ? Muzyczka N Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Almost 40 years ago gene therapy was widely believed to be the next new therapeutic modality in medicine, but we have yet to have a single drug certified by the FDA. We now appear to be on the verge of success. We will discuss the issues that had to be resolved in the last 40 years, the major lessons learned, and the medical applications that are likely to emerge in the next decade. Tackling AAV gene delivery challenges with structural virology Agbandje McKenna M Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL The recombinant AAV gene delivery system is entering a crucial and exciting phase with the promise of more than 20 years of intense research now realized in a n umber of successful human clinical trials that report therapeutic efficacy. However, significant challenges have been highlighted in studies using animal models and in the human clinical trials, including difficulties in delivering genes efficiently to cer tain target cells/tissues and avoiding the detrimental effects of the pre existing host immunity to AAV capsids. Adopting a multidisciplinary approach, utilizing biochemistry, molecular genetics, structural biology, and in vivo e antigenic structures of the AAVs as they relate to capsid determinants of tissue tropism and transduction (gene expression). Such studies are aimed at obtaining information for rational capsid engineering of AAV vectors to achieve escape from host antibo dy neutralization while retaining their natural or targeted tropisms Our latest results will be presented. Imprinting mecha nisms underlying Prader Willi and Angelman syndromes Resnick J L Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Approximately 150 human genes are subject to genetic imprinting, the selective transcriptional silencing of one parental allele. Only a few diseases are known to result from deficits in imprinted genes. A 2 Mb region at 15q11 q13 harbors both paternally and maternally expressed genes that are responsible for both Prader Willi and Angelman syndromes (PWS / AS), two clinically distinct neurobehavioral disorders. PWS results from the lack of genes normally expressed from the paternal allele while AS results from the absence of maternal specific gene expression. Imprinted genes are mostly located in clusters and allele specific expression at each cluster is controlled by an imprint ing center. The PWS AS imprinting center contains two parts separated by 35 kb, the PWS IC and the AS IC. The PWS IC activates expression of genes transcribed from the paternal allele. The AS IC epigenetically modifies the PWS IC in oocytes, so that the PW S IC does not activate genes on the future maternal allele. Thus the AS IC functions in female germ cells to establish the imprint and the PWS IC functions in somatic cells to effect allele specific gene expression. We have developed mouse models to explor e imprinting defects arising from the lack of PWS IC or AS IC function. Surprisingly, we have found that presence of the PWS IC is required only transiently during embryonic development, but has durable effects even in its continued absence. We also found that AS IC activity resides in several oocyte specific promoters located upstream of the PWS IC, and that the establishment of imprinting at the locus requires transcription transiting the PWS IC in oocytes.
11 Epigenetic alterations and stress among new mothers and infants in war torn Democratic Republic of Congo Mulligan CJ Department of Anthropology University of Florida, Gainesville, FL Early life experiences, including those in utero have been linked to increased risk for adult onset chronic disease. The current study is the first to test the idea that extreme maternal psychosocial stressors, as observed in the war ravaged Democratic Republic of Congo, may modify epigenetic marks in newborns resulting in altered health outcomes. Maternal and u mbilical cord blood samples were collected from 25 mother infant dyads. Detailed ethnographic interviews and peri natal trauma surveys were administered to all mothers in order to develop three emic (culturally relevant) measures of prenatal stress. Both l ocus specific and genome wide methylation data were collected. The glucocorticoid receptor, NR3C1 has been previously implicated in newborn birthweight. In a locus specific study, we observed that increased methylation at the promoter for NR3C1 in newborn s was significantly correlated with maternal stress and newborn birthweight. This increased methylation may constrain plasticity in subsequent gene expression and restrict the range of stress adaptation responses possible in affected individuals, thus incr easing their risk for adult onset diseases. Our genome wide methylation analysis began with a pilot study using reduced representation bisulfite sequencing (RRBS) and Illumina GAIIx technology. One lane of data, for four indexed technical replicates, yield ed coverage for ~1.5 million CpGs per library. However, after estimating a minimum necessary coverage for accurate epi genotyping and identifying library overlaps, the number of informative sites was reduced to ~0.2 million. Observing that each additional library would Preliminary results indicate that data for almost 0.5 million CpG sites were acquired for 72 samples, exceeding what could be acquired from RRBS at a similar price. Further analyses are ongoing. ADP glucose pyrophosphorylase: from bacterial selection to yield in the field Hannah LC Horticultural Sciences Department, University of Florida, Gainesville, FL Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Historically, the cereal seed has played a critical role in feeding humans and animals. The need for even greater cereal yields is necessitated by population growth, shrinking agricultu ral acreage, global climate change and the use of cereals for biofuel production. Accordingly conventional breeding efforts have been intense and yield increases of ~ 1% per year have been realized from maize (corn) breeding programs. We have taken a bioch emical approach to this problem and focused on a rate limiting step in starch synthesis. Starch constitutes 70% of the seed weight and hence is the major contributor to yield. The rate limiting target is ADP glucose pyrophosphorylase, an allosterically co ntrolled, heat labile, heterotetrameric enzyme. Using initially in planta transposon mutagenesis and subsequently an E. coli expression system in which enzyme levels in bacterial colonies can be monitored by a glance of the eye, we selected variants that enhanced heat stability and reduced inhibition by allosteric effectors. One such variant produces a 38% yield increase in wheat, a 23% increase in rice and up to a 64% yield increase in maize, depending on growth conditions. Surprisingly this variant incr eases yield by enhancing the probability that an ovary will produce a seed rather than by increasing individual seed size. Other unexpected insights into the function of this gene as well as targets for breeding under elevated temperatures will be discuss ed.
12 The chemistry and genetics of tomato flavor Klee H* Horticultural Sciences Department, University of Florida, Gainesville, FL Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Flavor is a conscious appreciation of the total sensation derived from a food involving integration of multiple sensory inputs. We use tomato as a model to understand the chemical interactions that drive human preferences, exploiting the large range of flavor chemicals produced in heirloom cultivars. Many cultivars spanning a broad range of biochemical diversity have been evaluated by consumers, generating a subjective sensory profile of perceptions. Our models of human preferences provide hypotheses about the chemical contribut ions to flavor. While sugar is a major driver of flavor intensity, sweetness and liking, additional volatile chemicals contribute to these perceptions. Several volatiles widely accepted as being important contributors to flavor do not contribute to flavor intensity, liking or sweetness. Knowing how the components of flavor contribute to human preference constitutes a new direction in the chemistry of flavor preferences for tomato and, more broadly, food products. In parallel, we are developing a comprehens ive understanding of the pathways and regulation of synthesis of the most important volatiles. We have identified a large number of s in each pathway. The ultimate goal is to build a toolbox for the molecular breeder to use in restoring great flavor into elit e commercial varieties of tomato.
13 Poster sessions *= UF Genetics Institute Faculty. Presenters are underlined. Poster Session I, Posters no. 1 65 Wednesday, November 28, 3:30 p.m. 5:00 p.m. 1. Genome wide genetic diversity and demographic events shaping the evolutionary history of two Pinus species Acosta JJ 1 Neves LG 2 Fahrenkrog A 2 Davis JM 1,2, *, Holliday J 3 Kirst M 1,2, 1 School of Forest Resources and Conservation, University of Florida, Gainesville, FL 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 3 Department of Forest Resources and Environmental Conservation, Virginia Polytechnic Institute and S tate University, Blacksburg, VA Recent advances in genome complexity reduction and sequencing make possible the genome wide analysis and identification of g enes under natural selection. We applied DNA sequence capture to the analysis of two conifer species, Pinus taeda and Pinus e l liottii that are widely distributed in the south southeastern US. Haploid DNA from megagametophytes was extracted from seeds of 24 trees of each species, collected from native populations in 11 states representing their natural range. For genotyping, libraries w ere prepared by sequence capture of a small fraction (0.03%) of the Pinus genome that represents 14,729 genes. Six multiplexed pools, of eight barcoded DNA samples each, were hybridized to sequence capture oligonucleotides and sequenced in a high throughpu t identified from the sequence data, and fo r each individual on a gene level, DNA sequence alignment references were reconstructed. For each pine species, population statistics were calculated, and c oalescent simulations were made assum ing three demographic models (b ottleneck, growth a nd neutral equilibrium). Using approximate Bayesian c omputation simulation techniques, posterior probabilities for each demographic model given the observed population d ata were calculated. For Pinus elliottii results indicate population size expansion due to a bottleneck event (posterior probability 0.998966). Pinus taeda analyses are underway. The availability of data from both species will also allow the analysis of t he relationship between within species diversity and between species divergence, which has not yet been possible at this scale. 2. Nuclear genetic diversity in human lice ( Pediculus humanus ) reveals continental differences and high inbreeding among worldwide populations Ascunce MS 1 Toups M 1 Kassu G 1 Fane J 1 Scholl K 1 Toloza AC 2 Picollo MI 2 Gonzlez Oliver A 3 Reed DL 1 1 Mammalogy, Florida Museum of Natural History, University of Florida, Gainesville, FL 2 Centro de Investigaciones de Plagas e Insecticidas (CONICET CITEDEF), Buenos Aires, Argentina 3 Facultad de Ciencias, Universidad Nacional Autnoma de Mxico, Mxico Distrito Federal, Mxico The ability to control human parasites has been hampered by the scarce knowledge of parasite populati on dynamics and structure. This is the case for human lice, where despite growing knowledge about their ancient coevolution with humans, little is known about their current genetic structure. Pediculosis is a prevalent infectious disease caused by the two ecotypes: the head louse ( Pediculus humanus capitis ), and the clothing (body) louse ( Pediculus humanus humanus ). Hundreds of millions of head louse infestations affect children every year, and this number is on the rise in part because of increased resista nce to insecticides. Here, we present the first assessment of the genetic structure of human lice by analyzing the nuclear genetic variation at 15 microsatellite loci in 93 human lice from 11 sites worldwide. Bayesian clustering analyses assigned lice to f o ur genetic clusters that were geographically structured. Clothing lice clustered within or close to the Asian head louse cluster. Although overall gene flow values were low, some pairwise comparisons between clothing and head lice showed more similar Nem values than between two populations of head lice, reflecting a consistent pattern with cluster assignments. The geographical and ecological structure among human louse populations could affect the evolution of insecticide resistance as well as disease tran smission, thus our results are relevant to strategies for the control of human lice. Furthermore, our panel of microsatellite markers provides powerful data to elucidate not only ecological and evolutionary processes in lice, but also may elucidate those p rocesses in humans because of their long term coevolution. 3. Correlation between development time and virus titer in Drosophila melanogaster Atkinson E 1 Brusini J 2 Wayne ML 1, 1 Department of Biology, University of Florida, Gainesville, FL
14 2 Department of Ecology and Evolutionary Biology, University of California, Santa Cruz, Santa Cruz, CA which states that pathogens will tend to evolve toward an intermediate level of virulence, relies heavily on the assumption that there exists a causal relationship between the number of pathogens in a host (titer) and the Drosophila melanogaster system, we performed an artificial selection experiment to test the rel iability of this assumption. When we selected on lines for high and low titer, we saw a correlated response for host development time, such that flies with high titer had longer development time than flies with low titer. We now test the generality of this result by performing the inverse experiment: selecting on high and low development time, and testing for a correlated response in terms of titer. Lines responded quickly to selection on development time. Analysis of titer by qPCR is in progress. 4. Map based cloning and anatomical analysis of a maternal effect seed m utant mre* 594 Bai F 1 Bagadion A 1 Evans M 2 Settles AM 1 1 Horticultural Sciences Department, University of Florida, Gainesville, FL 2 Department of Plant Biology, Carnegie Institution for Science, Stanford, CA Genomic imprinting in plants is an epigenetic phenomenon by which a subset of genes is expressed in a parent of origin dependent manner. Imprinted gene expression primarily occurs in the endosperm and is thought to influence seed size and embryo development. We identified a maternal effect seed mutant, named as maternal rough endosperm 594 (mre* 594). When inherited from the female parent, mre* 594 seeds show a rough, etched, or potted endosperm surface as well as a reduced seed s ize and weight. Transmission of the mre* 594 allele through the male does not affect seed development. The severity of mre* 594 embryo defects is correlated to seed size with the least severe mutants developing near normal embryos and seedlings. Microscopi c examinations of developing seeds from 4 days after pollination (DAP) to 10 DAP revealed d evelopmental delay of basal en dosperm transfer cell layer (BETL) as well as starchy endosperm defects. The BETL in normal 4 DAP kernels is a single cell layer of elo ngated cells with extensive secondary cell wall ingrowths. The mutants did not develop BETL cells until 8 DAP. Moreover, mre* 594 starchy endosperm cells failed to accumulate starch granules. mre* 594 maps to chromosome 4S, and we are in the process of pos itional cloning this gene Based on mre* 594 inheritance patterns, we expect the mutant to encode an imprinted gene required for normal cell differentiation and grain fill. 5. Evolution of alternative splicing across land p lants Mei W # Chamala S # Walts B, Barbazuk WB Department of Biology, University of Florida, Gainesville, FL # These authors contributed equally to this work Alternative splicing (AS) is the process by which a pre mRNA is processed into several alternate isoforms. AS contributes to tra nscriptome and proteome diversity, and also involves in gene regulation. Alternative splicing has been extensively investigated in human and mouse, but its role in plants has not been extensively studied. We are interested in examining the evolution of alt ernative splicing in the land plants by taking a comparative genomics approach to identify conserved alternative splicing between Amborella Arabidopsis thaliana Oryza sativa and Physcomitrella patens By identifying AS isoforms in all four genomes we are able to compare the distribution of alternative splicing, detect AS th at is specific to angiosperms, eudicots, m onocots, and enable examination of how AS is changed by whole genome duplication. Our analysis uses RNA Seq to identi fy splice isoforms within four species, and then examines the alternative splicing potential of each member of a set of orthologous gene s In terms of Amborella there are 26 846 genes in the current annotation, 17 089 of these contain multi exon s Considering only five major types of alternative splicing events (alternative donor, alternative acceptor, exon skipping, intron retention and alternative exon), 6 407 (37.5%) of intron containing genes produce AS transcript. The total number of predicted AS transcripts is 17,095. Intron retention is the most prevalent and occurs within 7 860 (46.0%) of the isoforms, while alternative donor, alternative acceptor, exon skipping, and alternative exon events contribute to 4 673, 6 206, 2 540 and 4 096 isoforms, respectively. The AS analysis i n three other species is currently underway. 6. How does obligate symbiosis shape bacterial genomes? Comparative genomics of closely related bacterial symbionts of insects provides clues Boyd BM 1,2 Allen JM 3 de Crecy Lagard V 4, *, Reed, DL 1, 1 Mammalogy, Florida Museum of Natural History, University of Florida, Gainesville, FL 2 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 3 Illinois Natural History Survey, University of Illinois at Urbana Champaign, Champaign, IL
15 4 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL Candidatus Riesia is one of the fastest evolving and youngest known heritable bacterial symbionts of insects. Found in symbiosis with human, chimpanzee, and gorilla lice, these recently acquired symbionts represent either a symbiont replacement or novel symbiosis wit hin lice. Why louse symbionts have multiple origins remains unknown, but presents a unique opportunity to study how bacterial genomes evolve in symbiosis. We assembled the genome of the symbiont of the chimpanzee louse, Candidatus Riesia pediculischaeffi, from genomic DNA extracted from whole lice. Comparisons between the genomes of C Riesia pediculischaeffi and the closely related C Riesia pediculicola, symbiont of the human body louse, revealed gene loss, duplicated regions, and numerous unique short co ding sequences of unknown function. In contrast genome synteny and the majority of identified genes are maintained across the two taxa. Tests for selection showed that all but two shared genes are under strong purifying selection. We conclude that despite being young and rapidly evolving, gene content and order is largely static and maintained by purifying selection. 7. Fruit specific genes in strawberry: physiology and protein protein i nteractions Brunings A Chambers A, Wang Y, Folta KM* Horticultural Sciences Department, University of Florida, Gainesville, FL Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Strawberry ( Fragaria x ananassa ) is an excellent model plant to study the function of novel, fruit specific genes. Strawberry transcriptome data yielded 50 genes whose expression is greatly enriched in fruit. RNAi and over expressor lines were developed in our transgenic system and the ir phenotypes analyzed. These genes include a mini zinc finger protein, a number of hypothetical proteins, and others providing only inferred function. One gene encoding a putative F box protein was used as bait in a yeast 2 hybrid screen to identify pot ential interaction partners, and interacting candidates were cloned, and sequenced. Most importantly, the new genes characterized in strawberry may be applied to other members of the Rosaceae family (like apple, rose and peach) that are not as easily gene tically manipulated and studied because of their longer generation times and large size. 8. Detection of sickle hemoglobin in febrile patients in Logne Haiti Carter TE 1 3 Von Fricken M 4,5 Mulligan CJ 1, *, Memnon G 6 Romain JR 6 Okech BA 4,5 1 Department of Anthropology, University of Florida, Gainesville, FL 2 Department of Epidemiology, University of Florida, Gainesville, FL 3 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 4 Emerging Pathogens Institute, Univers ity of Florida, Gainesville, FL 5 Department of Environmental and Global Health, University of Florida, Gainesville, FL 6 Hopital Sainte Croix, Logne Haiti Sickle cell disease and trait are common erythrocyte disorders that are caused by a mutation in the hemoglobin beta gene. Sickle cell trait (i.e. heterozygous for the sickle cell allele) is selectively advantageous against severe malaria. Haiti is a malari a endemic country, yet little is known about the prevalence of sickle cell disease and trait in this country. The purpose of our study was to detect the presence of sickle cell disease and trait in Haiti, as part of a larger epidemiological study on malari a in Haiti. Sixty two individuals at Hopital Sainte Croix were first screened for sickle hemoglobin using a solubility based rapid diagnostic test (RDT), SickleHeme (Michlone Associates, INC). RDT solution turbidity, as an indicator of sickle hemoglobin, w as assessed visually and with spectrophotometry. Samples were also genotyped for hemoglobin beta gene mutations. Of the 62 individuals screened for sickle hemoglobin using the RDT kit, 11 (17.6%) were positive. Of the 11 RDT positive samples, only six actu ally carried the sickle cell mutation, and as a heterozygote only. All other samples carried the wild type in some RDT samples after sitting for eight hours, but only in sickle hemoglobin samples that were confirmed by genotyping. We conclude that genotyping is the most accurate method to estimate the frequency of sickle hemoglobin. However, genotyping is expensive and observation in the RDT assay ma y be the best option for detecting sickle hemoglobin in Haiti and similar resource limited regions. 9. Assembly and validation of an evolutionary reference genome for flowering plants ( Amborella trichopoda ) Chamala S 1 Walts B 1 Chanderbali A 1 Albert V 2 Ayyampalayam S 3 Burnette J 4,5 dePamphilis C 6 8 Der J 6 8 Estill J 3 Lan T 2 Leebens Mack J 3 Lee S 9 Ma H 6 8 Moore R 10 Palmer J 11 Ralph P 6 8 Rice D 11 Rounsley S 12,13
16 Schuster S 14 Tomsho L 14 Wessler S 4,5 Wing R 9,12,13 Yu Y 9 Jiao Y 6 8 Xiao N 10 Soltis DE 1,15, *, Soltis PS 15, *, Barbazuk WB 1, 1 Department of Biology, University of Florida, Gainesville, FL 2 Department of Biological Sciences, University at Buffalo, State University of New York, Buffalo, NY 3 Department of Plant Biology, Univers ity of Georgia, Athens, GA 4 Department of Botany and Plant Sciences, University of California, Riverside, Riverside, CA 5 Center for Plant Cell Biology, University of California, Riverside, Riverside, CA 6 Intercollege Graduate Degree Program in Plant Biolo gy, Pennsylvania State University, University Park, PA 7 Institute of Molecular Evolutionary Genetics, Pennsylvania State University, University Park, PA 8 Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA 9 Arizona Ge nomics Institute, University of Arizona, Tucson, AZ 10 OpGen, Inc., Gaithersburg, MD 11 Department of Biology, Indiana University, Bloomington, IN 12 School of Plant Sciences, University of Arizona, Tucson, AZ 13 BIO5 Institute, University of Arizona, Tucson, AZ 14 Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 15 Laboratory of Molecular Systematics and Evolutionary Genetics, Florida Museum of Natural History, University of Florida, Gainesville, FL Am borella trichopoda as the sister to all other extant angiosperms, occupies a crucial phylogenetic position, and its genome sequence is therefore an important reference for comparative genomic studies across the angiosperms. A complete genome sequence of A mborella will help in understanding the evolution of key angiosperm traits and provide a baseline to examine genome organization throughout the angiosperms. We are using a whole genome shotgun strategy to sequence the ~731 Mb Amborella genome. We assembled over 48 Gb of DNA sequence, comprising single end (SE) 454 FLX, SE 454 FLX+, 11 kb paired end (PE) 454 FLX, and 3 kb PE Illumina HiSeq, and 69,466 BAC end reads. These sequences were extensively screened and filtered for organellar contaminants, short rea d lengths, poor quality, artificial duplicates, and chimeras. De novo sequence assembly was performed on these filtered sequence sets (~23 Gb) using Newbler. This resulted in 5,745 scaffolds totaling 706 Mb with a mean scaffold size of 123 kb and N50 size of 4.9 Mb covering over 96% of the Amborella genome. Significantly, about 90% of our assembled sequence resides within 155 scaffolds, each greater than 1.1 Mb in length. In the absence of genetic and physical maps for Amborella we employed a combination o f chromosomal FISH analysis and optical mapping (OpGen) to evaluate the fidelity and the chromosomal positioning of our current scaffolds. Using this approach, our assembly continuity increased by 2X (N50 from 4.9 Mb to 9.3 Mb and N90 from 1.2 Mb to 2.9 Mb ). We are further evaluating the potential of this approach to serve as a surrogate to using long range PE libraries to increase the contiguity of an assembly. 10. The r ole of USF2, BRG1 and GATA 2 in the early globin locus control r egion Chamales P 1 Morton S 1 Liang S 1 Stees J 1,2 Fan X 1 Varn F 1 Huang S 1, *, Deng C 1 Bungert J 1, 1 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL globin genes is regulated by a locus control region (LCR). The LCR is composed of several DNase I hypersensitive sites that function together to mediate high globin genes during development and differentiation. Previous work demonstrated that the LCR HS form before globin gene expression during differentiation. We found that LCR HS2 and HS3 recruit RNA polymerase II (Pol II) transcription complexes early on during differentiation of erythroid cells before the genes are expressed. Furthermore, efficient recruitment of Pol II required the function of transcription factor USF. Subsequent studies demonstrated that USF interacts with the nucleosome remodeling factor BRG1. We thus hypot hesized that USF recruits BRG1 to the LCR and that the re organized nucleosome structure lead s to HS formation and Pol II r ecruitment. However, we found that in transgenic embryos expressing dominant negative USF (AUSF) the association of BRG1 with LCR HS2 is increased. Furthermore, preliminary data suggest that expression of GATA 2 is enhanced in A USF expressing d10.5 yolk sac cells. Previous studies have shown that GATA 1 interacts with BRG1. We thus propose that GATA 2 recruits BRG1 to the LCR in undiff erentiated erythroid cells. This allows USF mediated recruitment of Pol II. Transcription of HS2 and HS3 keep these regions in an open configuration and during subsequent differentiation steps erythroid specific protein complexes associate with the LCR est ablishing a domain that mediates efficient transcription of the globin genes. 11. Identifying genes for strawberry flavor with a breeding genetics genomics a pproach Chambers A 1 Pollard H 1 Bai J 2 Plotto A 2 Whitaker VM 1,3,4 Folta KM 1,4,5,
17 1 Horticultural Sciences Department, University of Florida, Gainesville, FL 2 Gulf Coast Research and Education Center, University of Florida, Balm, FL 3 Agricultural Research Service, U.S. Department of Agriculture, Fort Pierce, FL 4 Plant Innovation Program, University of Florida, Gainesville, FL 5 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL C ultivated strawberry ( Fragaria x ananassa ) is prized for complementation of desserts and add ing attractive color to foods. However, commercial strawberries have been bred for shipping, disease resist ance and product size, leaving flavor and a roma as areas for improvement. Several flavor volatiles have been recognized as desirable for consumers, namely methyl anthranilate (grap e like) and gamma decalactone (peachy) as well as other fruity, tropical terpenes and esters. We have taken an approach leveraging breeding, genetics and genomics to identify the genes that control the production of these volatiles. First, a candidate gen e approach was used. An uncharacterized alcohol acyl transferase was overexp ressed and suppressed by RNAi. Analysis of fruits show s a decrease in several esters and methyl anthranilate, suggesting a role for this enzyme in the process, and ability to use several substrates. In a second approach a non commercial variety containing several important volatiles was crossed with a F lorida variety possessing a different set. Progeny segre gated for increased volatiles. Bulk RNAseq on progeny fruit expressing an d not expressing different volatiles has identified several candidates with intuitive connections to flavor volatile synthesis that are now being validated in transgenic plants. 12. Characterization of phosphoproteins in signaling pathways using phosphop eptide enrichment mass spectrometry Silva Sanchez C 1 Zhu M 2 Diaz C 1 Chen S 1,2 1 Proteomics Division, Interdiscipli nary Center for Biotechnology Research, University of Florida, Gainesville, FL 2 Department of Biology University of Florida, Gainesville, FL Protein phosphorylation and dephosphorylation constitutes one of the reversible post translational mechanisms that control gene expression, signal transduction, cell cycle, apoptosis and cytoskeletal regulation. Due to its dynamic nature, stoichiometry of phosphorylation is usually low. This presents a challenge for mass spectrometry analysis of phosphoproteins. To overcome the challenge, a phosphopeptide enrichment step becomes necessary prior to mass spectrometry analysis. The goal of thi s work was to develop an effective methodology for phosphopeptide enrichment and phosphorylation site mapping through comparing the capabilities of different TiO2, Zro2 and Phos tag resins. The protocol for each media was tested with casein to determine the efficiency. Then, we used a phosphorylated SnRK to optimize the protocol for de tecting low phosphorylation sto ichiometry in biological samples. The protein samples (SnRK and SnRK pho) were separated in a gel. After Coomassie staining and in gel digesti on, the phosphopeptides were enriched using different media. All phosphopeptide samples were run on a nanoflow UPLC coupled to a LTQ Orbitrap mass spectrometer. The results led to an optimized procedure for analyzing phosphoproteins in signaling pathways. 13. Aberrantly activated AREG EGFR signaling is required for the growth and survival of CRTC1 MAML2 fusion positive mucoepidermoid carcinoma Chen Z Wu L* Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Salivary gland tumors (SGT) are a group of highly heterogeneous head and neck malignancies with widely varied clinical outcomes and no standard effective treatments. The CRTC1 MAML2 gene fusion, encoded by a specific chromosomal translocation t(11;19)(q14 21;p12 13), is a highly specific genetic alteration in more than 50% of mucoepidermoid carcinoma s the most common malignant SGT. To date, it remains poorly elucidated regarding the importance of the CRTC1 MAML2 fusion gene in supporting the growth and sur vival of MEC and its critical downstream mediators. In this study, we showed that depletion of the CRTC1 MAML2 fusion oncoprotein reduced the growth and survival of fusion positive human MEC cancer cells in vitro and the in vivo growth of human MEC xenogra ft tumors. Since CRTC1 MAML2 is a nuclear protein with no known enzymatic activity, we then investigated critical target genes and pathways downstream of the fusion oncoprotein for targeting opportunities. We performed gene expression profiling analyses an d functional studies and iden tified that an autocrine AREG (a mphiregulin) EGFR signaling is a downstream mediator of the CRTC1 MAML2 fusion oncogene and is essential for fusion positive MEC cells. Excitingly, inhibition of EGFR signaling with an anti EGFR antibody, Cetuximab, significantly blocked human fusion positive MEC cell growth in vitro and in vivo Therefore, our study revealed a critical role of aberrantly activated AREG EGFR signaling axis in mediating oncogenic functions of the CRTC1 MAML2 fusion Our study provides a molecular justification for the clinical evaluation of anti EGFR therapeutic agents in treating CRTC1 MAML2 po sitive mucoepidermoid carcinoma
18 14. MALDI mass spectrometry of nematode s urfaces Clendinen CS 1 Menge r R 2 Yost R 2 Edison AE 1 1 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2 Department of Chemistry, University of Florida, Gainesville, FL Like many other organisms, nematodes synthesize and use small molecules to communicate wi thin and between species. In Caenorhabditis elegans ascarosides have been found to c ontrol dauer formation (Butcher et al., 2007 Nat Chem Biol 3(7):420 2), mating (Srinivasan et al., 2008 Nature 454(7208):1115 8), aggregation (Macosko et al., 2009 Nature 458(7242):1171 5), and olfaction (Yamada et al., 2010 Science 329(5999):1647 50). Several behaviors, like aggregation, may be mediated or enhanced by chemical cues on the cuticle surface of the nematodes. Using a combination of MALDI MS and princi pal component analysis (PCA), we present an alternative approach for C. elegans chemical biology studies that uses small numbers of individual animals. To demonstrate the potential for this approach, we have used multivariate analysis to compare MALDI MS d ata from several mutants that were predicted to produce different compositions of small molecule metabolites. 15. Phenotyping a segregating pseudo F2 population of elephantgrass for b io mass related t raits Corsato CE 1,2 Sinche M 1 Kannan B 1 Vilarinho AA 1,3 Altpeter F 1 1 Agronomy Department, University of Florida, Gainesville, FL 2 Departamento de Cincias Agrrias, Universidade Estadu al de Montes Claros, Jana ba, Brazil 3 Embrapa Roraima, Empresa Brasileira d e Pesquisa Agropecuria, Boa Vista Brazil Elephantgrass ( Pennisetum purpureum Schum.) is one of the best adapted and highest biomass yielding perennial C4 grasses in the costal SE. Due to the very high biomass yields the emerging biofuels industry is curre ntly preparing to grow elephant grass as fe edstock for their new biofuel conversion facility. To enhance biosafety of elephantgrass we are developing elephantgrass accessions that combine late flowering and high biomass yields. Genetic distance of elephantgrass accessions can be estimated with the help of molecular markers and allows the identification of two contrasting parents for flowering and biomass yield. Biomass related traits (e.g. stem diameter, number of tillers, stem length) are quantitative traits, expressed as a continuous distribution between maximum and minimum levels, and are normally under the control of a larger number of minor genes, each with small additive effects. Positions on the genome that can be attributed to detectable differences in phenotype due to gene effects are known as quantitative trait loci (QTL). To identify and locate possible QTL associated with these traits there is a need to identify co segregation of phenotypic traits in the mapping population and markers on the genetic map. Hence accurate phenotyping of the m apping population is a crucial prerequisite for marker developmen t and for locating putative QTL s. Therefore, we generated an F1 population (segregating pseudo F2 population) from contrasting parents. This F1 population was vegetatively propagated and phen otyped under replicated field conditions at the UF PSREC in Citra, FL. The statistical evaluation and distribution of the data describing the flowering time, flo wering frequency and biomass related traits (e.g. stem diameter, length of tillers and number of tillers, biomass yield) within the mapping population will be presented. 16. Decreased survival following combined exposure to heat and oxidative stress is correlated with attenuated expression of glutathione S transferase Crombie TA Julian D Depa rtment of Biology, University of Florida, Gainesville, FL We investigated the combined effects of heat stress and oxidative stress on survival and induction of the oxidative stress response in the nematode model Caenorhabditis elegans To generate oxidativ e stress, nematodes were exposed to the redox cycling compound juglone at six concentrations (0 100 M). Heat stress was administered by increasing the incubation temperature from 20 to 25, 30 or 35 C. We found that heat stress and oxidative stress, when administered simultaneously for 4 h, interacted synergistically. This synergy caused a marked reduction in survival a bove 30 C at all juglone concen trations. Glutathione S transferase 4 (gst 4) helps detoxify juglone by conjugating it with glutathione, in creasing its solubility and aiding in its excretion. Induction of gst 4 is via the SKN 1 transcription factor. We examined w hether heat stress impairs the response to juglone induced oxidative stress in a reporter strain containing GFP fused to a SKN 1 pr omoter fragment upstream of gst 4. We found that induction of gst 4 by juglone was attenuated at temperatures greater than 30 C. We validated this finding using qPCR, which showed a 3 fold loss in gst 4 induction capacity at 33 C. These findings suggest that temperature dependent inhibition of gst 4 induction is at least one mechanism by which heat
19 and oxidative stress synergize to reduce nematode survival. 17. Membrane locali zation of a novel regulator of actin depolymerizing f actor Cuddy KK , Grey PH, Zhang X, Oppenheimer DG Department of Biology, University of Florida, Gainesville, FL Section of Cell and Developmental Biology, Division of Biological Sciences, University of California San Diego, San Diego, CA Plant Molecular and Cellula r Biology Program, University of Florida, Gainesville, FL Actin filament turnover is required for many actin dependent cellular processes including cell motility and membrane trafficking. Members of the actin depolymerizing factor/cofilin (ADF) family of actin binding proteins are essential for severing/depolymer izing actin filaments, and thus regulating actin dynamics. We recently identified a new regulator of ADF in Arabidopsis named IRREGULAR TRICHOME BRANCH 3 (ITB3). Our in vitro analysis of ITB3 function showed that it interacts with ADF and inhibits actin bi nding to ADF. ITB3 is one of 22 family members in Arabidopsis Interestingly, about half of the ITB members possess a putative signal sequence (SS). Given that signal sequences direct proteins for secretion, we have to reconcile the fact that ADF, which in teracts with ITB3, is not secreted. We thus hypothesize that the putativ e signal sequences are novel N terminal transmembrane anchors. To test this hypothesis, we constructed YFP fusions to the ITB3L 10 protein, which contains a putative SS, and used parti cle bombardment to transiently transform Arabidopsis epidermal cells. Results sho w putative localization to the endoplasmic r eticulum. We are also con ducting in vitro transcription/ translation of ITB3L 10 in the presence of eukaryotic microsomes to test fo r co translational membrane insertion. 18. Cross immunity in Drosophila melanogaster for bacteria and viruses Culbreth E 1 Harshman L 2 Wayne ML 1, 1 Department of Biology, University of Florida, Gainesville, FL 2 School of Biological Sciences, University of Nebraska Lincoln, Lincoln, NE Is the response of the host to a parasite specific to the given parasite, or a more general function of the host? Host responses include tolerance or resistance. There are a number of genetic models suggesting t hat tolerance and/or resistance are parasite specific. One also finds terms like response that might translate to a spectrum of parasites. We attempt to distinguish between these two ideas by evaluati ng infection rates by sigma virus of lines of Drosophila melanogaster artificially selected for increased resistance to Bacillus cereus Also, two sets of controls exist, exactly as the selected lines ; and an unselected control, such that animals were not treated at all. If response to expect the selected lines to be more difficult to infect with sigma virus than either of the control lines. If r esponse to selection has been in the form of a parasite specific genetic change, we do not expect to see any difference in our ability to infect the selected lines rather than the control lines. Preliminary data suggest that there is no difference between treatments, and thus that response to selection for resistance to B. cereus is specific. Further experiments, both challenging thes e selection lines against parasites other than sigma and evaluating other selected lines, are needed to draw conclusions. 19 Investigating the role of the WICH chromatin remodeling complex in maintaining facultative heterochromatin at the human inactive X chromosome Culver Cochran AE Chadwick BP Department of Biological Science, Florida State University, Tallahassee, FL X chromosome inactiv ation (XCI) is the mammalian for m of dosage compensation that balances the levels of X linked gene expression between the sexes. Early in female development, transcription from one X is shut down as the chromosome is repackaged into facul tative heterochromatin. Once established, the same X is maintained as the inactive X chromosome (Xi) throughout subsequent somatic cell divisions. While much is known about the initiation of XCI, our understanding of how the Xi chromatin is faithfully main tained through each cell cycle is limited. We have identified the multifunctional Williams syndrome transcription factor (WSTF) as a protein that transiently associates with the Xi as it is replicated in late S phase, and, therefore, is a prime candidate f or maintaining XCI. We have previously shown that WSTF acts at th e Xi as a component of the WST F ISWI chromatin remodeling complex (WICH). Interestingly, we have found that WICH and two other H2A.X and BRCA1, sequentially associate with the Xi, suggesting that each plays a unique role in maintaining the role of WICH at the Xi, we have compared the localization of WICH with the spatially distinct t ypes of heterochromatin at the Xi. We have characterized the localization of WICH in relation to XIST RNA as well as to
20 X linked BAC clones. Additionally, we have determined the H2A.X, and BRCA1 associate with the Xi. We are actively investigating the precise function of WICH at the Xi. 20. DNA content increases with cell size in h umans Gillooly JF*, Hein A Damiani R Verster K Department of Biology, University of Florida, Gainesville, FL Endopolyploidy has been observed in many human cell types, but still is often viewed as exceptional, tissue specific, or indicative of pathology. Here we show that nucl ear DNA content varies by over two orders of magnitude across 19 different human cell types, and that it is positively c orrelated with cell volume. We also show that this increase in DNA content with cell size is similar to intraspecific relationships between DNA content and cell size in other species, and interspecific relationships between diploid genome size and cell siz e. Our results therefore suggest that endopolyploidy is a general, size dependent feature of cells, which may have important consequences for cell function. 21. Characterizing a lncRNA associated with the X linked macrosatellite DXZ4 Darrow EM Tremblay DC, Moseley SC, Chadwick BP Department of Biological Science, Florida State University, Tallahassee, FL DXZ4, a macrosatellite located at Xq23, is one of at least three extensive tandem repeats located on the human X chromosome that adopt a euchromatic ch romatin state on the otherwise heterochromatic inactive X chromosome. We have identified several DNA sequences located both proximal and distal to DXZ4 that are candidate regulatory elements for the epigenomic organization of the macrosatellite. A lucifera se reporter assay revealed strong promoter activity for one of these sequences, located approximately 80 kb distal to DXZ4. The promoter drives transcription of a long non coding RNA (lncRNA) toward DXZ4. Numerous alternatively spliced transcripts are gene rated, which remarkably include variants that transcribe across the entire macrosatellite, terminating on the proximal edge. RT PCR of pooled tissue samples and human embryonic stem ce lls suggests that expre ssion of the DXZ4 traversing transcript is tissue specific and independent of monomer copy number. Active transcription of the lncRNA across the array may contribute to chromatin organization and suggests that DXZ4 adopts an alternate chromatin configuration in different tissues and cell types. In order to assess the role of the distal promoter in establishing and maintaining DXZ4 chromatin, we are generating TAL effector nucleases to induce removal of the promoter sequence via homology mediated genome editing, thereby disrupting lncRNA transcription. 22 Novel regulators of cellular antioxidation and detoxification genes identified by RNAi screening in C aenorhabditis elegans Deonarine A Choe KP* Department of Biology, University of Florida, Gainesville, FL Animal cells share a common inducible transcriptional collar (CNC) transcription factors. CNCs play a central role in stress defense, drug metabolism, redox homeostasis, tumorigenesis, neurodegenerat ion, and longevity. Regulation of CNC factors is highly complex and coordinated with numerous physiological and developmental processes. Understanding CNC factor regulation will yield fundamental insights into understanding how animal cells detect changes to environmental conditions and lay the foundation for therapeutic modulation. We take advantage of the genetic and molecular tractability of C. elegans to identify and characterize novel regulators of CNCs. C. elegans has one functional CNC named SKN 1 th at plays a central role in longevity and resistance to oxidant s. A genome wide RNAi screen y ielded eleven novel regulators of gst 4, a SKN 1 target, in response to an oxidant. Four are thought to be required for general transcription or translation and are thus unlikely to play a specific role in CNC regulation. The remaining seven gst 4 regulators are predicted to function in transcription, ubiquitin labeling of proteins, and inositol 1,4,5 trisphosphate signaling. We are now testing these genes for SKN 1 regulation, stress resistance, and longevity. Characterization of these will define novel mechanisms for regulation of a conserved stress defense and longevity pathway. 2 3 Role of multiple quorum sensing signals for regulation and synchronization of group behaviors in Sinorhizobium meliloti populations Dilanji GE 1 Madhavan R 1 McLeod LC 1 Teplitski M 2, *, Hagen SJ 1, 1 Department of Physics, University of Florida, Gainesvil le, FL 2 Soil and Water Science Department, University of Florida, Gainesville, FL Quorum sensing (QS) bacteria use small molecule signals to regulate and synchronize collective behaviors. Sinorhizobium meliloti synthesizes several N acyl L homoserine lactones (AHLs) as QS signals that differ in their carbon side chain length (C8 C18) and molecular
21 structure (i.e. saturated vs. un saturated or keto substituted). These signals differ in diffusivity and solubility. S. meliloti uses these signa ls to coordinate complex behaviors necessary for invasion of plant roots. We have studied the regulation of exopolysaccharide (EPS) production utilized in swarming colonies of S. meliloti We hypothesized that S. meliloti exploits the physical and chemical properties of AHLs to segregate behaviors among cells within a population and swarm more efficiently. To test this hypothesis we studied the activation kinetics of the EPS genes in the presence of AHLs, investigated the single cell EPS gene activation in well mixed AHL environments, and measured the spatial and temporal range of the diffusing signal in a spatially extended system. We found only three AHLs regulated EPS production, each inducing different kinetic responses of the EPS genes. The different AH Ls elicit different distributions of EPS expression levels (e.g. broad vs. narrow) from populations of cells. Moreover, in a spatially extended colony, diffusing AHLs readily activate EPS genes over time scales of ~20 h and over distances of ~0.5 cm (oxoC1 4 and C16:1 HSL) or ~1 cm (oxoC16:1 HSL). Our results suggest that S. meliloti uses multiple AHLs to generate differences in population wide expression profiles. Furthermore, variation in AHL diffusivity may allow for control of long range synchronization. 24. Effect of colony stimulating factor 2 (CSF2) during the morula blastocyst stages of development on gene specific methylation in inner cell mass (ICM) and trophectoderm (TE) cells of the bovine blastocyst as determined by high resolution melting analy sis Dobbs K 1 ,2 Sudano M 1 3 Hansen PJ 1 ,2, 1 Department of Animal Sciences, University of Florida, Gainesville, FL 2 DH Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, FL 3 Universidade Estadual Paulista, Campus de Botucatu, Sao Paulo, Brazil CSF2 can enhance the competence of the bovine embryo for establishment and maintenance of pregnancy. In particular, treatment of in vitro p roduced embryos with CSF2 from d ay 5 7 post insemination (pi) increased ICM ce ll number, conceptus survival and th e proportion of pregnancies at d ay 35 that developed to term. One possible mechanism by which CSF2 improves post transfer survival is alterations in DNA methylation of the embryo. Here we used ICM and TE cells isola ted f rom bovine blastocysts at d ay 7 after in vitro fertilization to test the hypothesis that exposure to CSF2 alters the methylation status of specific genes in a cell type specific manner. Pools of blastocysts from embryos treate d with 0 or 10 ng/ml CSF2 from day 5 7 pi were harvested on d ay 7 pi, and subject to magnetic activated cell sorting to separate ICM and TE Genomic DNA was extracted, b isulfite converted, and subjected to methylation sensitive high resolution melting analysis (MS HRM) to examine CpG i slands within IGF2, DNMT3B and SNRPN. There was no effect of cell type or interactions of CSF2 and cell type on degree of methylation for any gene. CSF2 reduced methylation for IGF2 (2.8 + 0.3% vs. 1.6 0.3%). There was no effect of CSF2 on methylation of DNMT3B or SNRPN. In conclusion, exposure to CSF2 during the morula blastocyst stages of development can alter methylation of specific genes in the bovine embryo. The functional significance of the change in IGF2 methylation needs to be determined by exami ning whether transcription is affected. It was also demonstrated that MS HRM could be used with low amounts of ICM and TE cells to examine DNA methylation. 25. B and T cell immunomodulation attenuates anaphylaxis in a n ovel mouse model of Pompe d isease Doerfler P 1 ,2 Nayak S 1 3 Herzog RW 4, Byrne BJ 1,3, 1 Department of Pediatrics, University of Florida, Gainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 3 Powell Gene Therapy Center, University of Florida, Gainesville, FL 4 Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida, Gainesville, FL glucosidase (GAA) causes cardiac, respiratory, and skeletal muscle dysfunction in Pompe patients. Enzyme replacement therapy (ERT) using recombinant human GAA (rhGAA) can lead to life threatening immune reactions. We utilized a novel huma n CD20 (hCD20) Gaa / mouse to evaluate immunomodulatory therapies using rituximab (Ritux) concomitant with rapamycin (rap) or methotrexate (MTX). hCD20Gaa / mice received Ritux one week prior to ERT to deplete B cells; MTX (10 mg/kg) or Rap (4 mg/kg) wer e gavage fed daily for a month with once/week IV rhGAA (20 mg/kg). Controls received rhGAA only. After six weeks, mice were re challenged with ERT. Controls developed high Ab titers (IgG1 1.2x105 ng/mL by four weeks P<0.01). The Rap and MTX cohorts had ~1 8 fold reduction in anti rhGAA IgG1 that increased on secondary challenge. Pulse oxymetry revealed that without immune suppression, arterial oxygen saturation, pulse distention, breath distention, body temperature and heart rate decreased significantly (P< 0.0001) within 5 minutes of IV rhGAA. MTX group had decreased survival despite low anti rhGAA titers. Ritux cohorts had a reduction in B cell numbers and activation compared to controls. MTX cohort showed increased CD4+ and CD4+CD69+ cells (P<0.05),
22 Rap c ohort showed an expansion of CD4+CD25+ T cells compared to MTX (P<0.05). Overall, Ritux and Rap administration reduced anaphylactic response, T cell activation and maintained prolonged low anti rhGAA titer while expanding CD4+CD25+ T cells more effectively than Ritux MTX cohort. Although preliminary, these results suggest Pompe immunomodulatory therapy should include Ritux and Rap to preclude the adverse effects of ERT. 26. Examining the roles of bacterial clamp loaders in DNA replication and repair Douma LG 1,2 Bloom L B 1, 1 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL To increase DNA polymerase processivity during replication, sliding clamps anchor DNA polymerase to the DNA template. These sliding clamps are ring shaped proteins that are loaded onto the DNA by clamp loaders which harness the power of ATP hydrolysis. In E. coli the two clamp loaders are complex and complex, named for the or subunits in the complex that perform the required ATP hydrolysis. Both subunits are products of the same gene, dnaX The smaller subunit is produced through a programmed rib osomal frameshift that results in a premature stop codon, while subunit is the full gene product. The subunit is essential for viability, coordinating interactions at the replication fork by its C terminal end (absent in ). It has been debated if co mplex has any real role. Temperature sensitive cells that contained mutations in the dnaX gene were found to have defects in template switching repair. Plasmids expressing not full length dnaX restored the template switching repair phenotype. Recombinant proteins harboring the mutations were expressed, and gel filtration analysis of these proteins show that the subunits aggregate to form a large, stable oligomer (equivalent to 16 and 13 su bunits), whereas the wild type DnaX subunits forms a tetramer in solution. Oligomerization may reduce the efficiency of DnaX2016 subunits assembling into clamp loader complexes in vivo. These mutants will serve as useful tools to define cellular functions of and complex. 27. Cancer progression is linked to altered chromatin structure at multiple scales Druliner BR 1 Fincher JA 1 Sexton BS 1 Roche M 2 ,3 Lyle S 2 ,3 Dennis JH 1 1 Department of Biological Science, Florida State University, Tallahassee, FL 2 Department of Cancer Biology University of Massachusetts Medical School, Worcester, MA 3 Department of Pathology, University of Massachusetts Medical School, W orcester, MA Chromatin structure plays a fundamental role in regulating nuclear processes, and is a critical point of reference in the development and progression of cancer. We used nucleosome distribution and chromatin sensitivity assays to analyze the genome wide chromatin structure of patients with different grades and stages of lung adenocar cinoma as compared to matched normal tissue. Here, we report that low grade adenocarcinoma displays widespread nucleosome alterations compared to matched normal tissue at 50% of 886 genes studied. These altered nucleosome arch itectures are consistent among patients indicating that they may serve as important early cancer markers. We demonstrate that the nucleosomal alterations are driven by the underlying DNA sequence. Conversely, substantial disruptions in chromosomal sensitivity were seen in a p atient wit h an aggressive tumo r, while low grade and stage tumors are nearly identical to normal. Importantly, these chromatin sensitivity results are concordant with folding principles of the human genome determined by long range interactions (Hi C). Our data show that chromatin structure changes during the progression of lung adenocarcinoma. We have developed a model in which low grade lung adenocarcinoma is linked to changes in nucleosome distributions, whereas aggressive tumors are linked to high order chromosoma l changes. Moreover, we identified the underlying signals that explain altered nucleosome distribution profiles as features intrinsic to the DNA sequence itself. These results provide a foundation to identify a new class of chromatin based diagnostic, prog nostic, and therapeutic markers in cancer progression. 28 Studying the interaction between actin depolymerizing factor (ADF) and a novel ADF regulator Emmanuel M 1 Grey PH 1 Oppenheimer DG 1,2, 1 Department of Biology, University of Florida, Gainesville, FL 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL The actin cytoskeleton is required by all eukaryotic cells to carry out key functions such as cellular motility and intracellular trafficking of cellular components. The actin binding proteins are important in controlling actin dynamics: the ability of the actin cytoskeleton to remodel by polymerization and depolymerization in response to cellular signals. One of the most important actin binding proteins r esponsible for actin remodeling is actin depolymerizing factor (ADF). Earlier work in our lab led to the identification of a novel regulator of ADF in plants, called IRREGULAR TRICHOME BRANCH 3 (ITB3). In
23 addition to ITB3, Arabidopsis has 21 additional ITB3 l ike genes (ITB3L1 ITB3L21). In this study we will conduct tests on one of the ITB3 like family members, ITB3L04, to quantify binding to ADF. To do this, we will first construct multiple gene fusions, express and isolate the protein s of interest and then have the option of performing various tests of interactio n such as pull down assays and bio layer i nterferometry. Studying the interaction between ITB3L04 and ADF will allow us to confirm its effect as a regulator of ADF and quantify its binding properties. This is the first step in characterizing this new pathway that contributes to actin dynamics. 29. Exome resequencing in a Populus deltoides association population Fahrenkrog AM 1 Neves LG 1 Barbazuk W B 1,2, *, Kirst M 1,3, 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 Department of Biology, University of Florida, Gainesville, FL 3 School of Forest Resources and Conservation, University of Florida, Gainesville, FL Populus deltoides (eastern c ottonwood) is a model species to study the biology and genetics of wood formation and biomass growth in woody, perennial plants. The availability of a reference genome and ease of genetic transformation and vegetative propagation make poplars especially su ited to conduct association genetics studies to identify the genes determining bioenergy traits. In this study we are resequencing the coding fraction of the genome in 579 individuals of a P. deltoides association approach is based on the generation of genomic DNA libraries where each individual is identified by a barcode sequence. The exome is captured by hybridization of 12 multiplexed genomic DNA libraries with 204,108 oligonucleotide probes designed to capture 18,153 genes. This highly multiplexed, genome complexity reduction by sequence capture allows high throughput characterization of a large number of individuals, and redu ces the sequencing c ost significantly. The information generated will be used to identi fy polymorphisms such as single nucleotide polymorphisms (SNPs) and copy number variants (CNVs), which will be used for population genetics and association studies. 30. In utero caffeine exposure alters DNA methylation patterns in adult hearts Fang X 1 Xue H 2 Rivkees SA 1 Wendler CC 1 1 Child Health Research Institute, Department of P ediatrics, Universit y of Florida, Gainesville, FL 2 Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA Our previo us research demonstrated that caffeine exposure on embryonic day (E) 8.5 increased body weight, altered cardiac morphology and function in adult male mice. However, these adverse effects were not observed in adenosine A1 receptor knockout (A1AR / ) mice. O ur hypothesis is that A1AR action mediates changes in DNA methylation patterns in mice exposed in utero to caffeine and that these changes in methylation lead to long term effects in adult mice. To test this hypothesis, DNA Methylation 2.1M Deluxe Promoter Arrays (NimbleGen) were used to examine the methylation patterns of DNA isolated from left ventricles of the A1AR knockout line exposed to 20 mg/kg caffeine at E8.5 in utero In A1AR+/+ mice, 4,896 hypermethylated and 2,823 hypomethylated regions were dis covered in the left ventricles of the caffeine group compared to the normal saline group. In A1AR / mice, 1,024 hypermethylated and 1,757 hypomethylated regions were found in the caffeine group. The differentially methylated regions (DMRs) in A1AR+/+ were mapped to 6,148 genes, 4,853 promoters, 4,111 primary transcripts, 816 CpG islands, and 98 miRNAs. Functional annotation clustering of genes revealed that many genes were involved in the development of hypertrophic cardiomyopathy and other heart diseases, which may explain our earlier findings of thickening of the left ventricle after in utero caffeine treatment. The methylation changes in several DMRs, mef2c ins2 tnnt2 and myh6 were validated by bisulfite sequencing. In summary, in utero caffeine expo sure caused DNA methylation changes in adult left ventricles and that these changes were possibly mediated by A1ARs. 31. Transcriptome wide profiling of gene expression using single molecule real time (SMRT) sequencing on the Pacific Biosciences RS Zhang Y 1 Panayotova N 1 Shanker S 1 Moraga D 1 Underwood J 2 Farmerie W 1 1 Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 2 Pacific Biosciences Inc., Menlo Park, CA Alternative RNA splice forms create dynamic blends of gene transcripts derived from a single gene locus. Microarrays, and more recently, highly parallel NGS DNA sequencing methods (RNA Seq) are the most common methods for discovering alternative splicing e vents. Transcriptome characterization by DNA sequencing requires no prior knowledge of gene structure, and as such can be used to discover unknown gene expression motifs. Massively parallel, short read (ca ~ 100 150 bp) DNA sequencers (Illumina, Applied Bi osystems, and Ion Torrent) use statistical analysis of reads mapped to a
24 reference genome to infer alternative exon usage and minor transcript frequency. While extremely powerful, short DNA sequence reads cannot infer the complete end to end structure of s ingle, full length mRNA transcripts. Pacific Biosciences (Pacbio) Singl e Molecule Real Time (SMRT) DNA sequencing technology generates DNA sequencing reads having a median length of 3,000 4,000 bp, which is sufficient to fully characterize the bulk of matu re gene transcripts. Hence Pacbio SMRT cDNA sequencing can be used to examine the complexity of gene expression in ways heretofore not possible. Here we describe methods for cDNA pre paration and sequence analysis u sing the exceptionally long reads of the P acbio SMRT system. The UF ICBR study is part of a larger comprehensive multi center project being conducted by the Association of Biomolecular Resource Facilities (ABRF) with support from the ABRF and Pacific Biosciences. 32 Identifying the activity of m ale specific isoforms of Drosophila fruitless Fear JM 1 ,2 Dalton JE 3 4 McIntyre LM 2 *, Arbeitman MN 3 1 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 3 Program in Neuroscience, Biomedical Science Department, Florida State University, Tallahassee, FL 4 Department of Biological Sciences, University of Southern California, Los Angeles, CA In Drosophila melanogaster the transcriptio n factor fruitless ( fru ) produces male specific isoforms (Fru M ) required for courtship behaviors. fru is part of the somatic sex determination hierarchy. Fru M isoforms are produced alternative different DNA binding domains. To understand differences in activity, each Fru M isoform was overexpressed or perturbed in fru expressing neurons. We identified differences in expression downstream of each Fru M isoform and distinct differences in Fru M activity between males and females. Genes that are significantly induced (repressed) by Fru M are highly enriched (depleted) on the X chromosome in males, but not in females. Different isoform activities in fru may be explained by differences in DNA binding. Each Fru M isoform has different DNA binding specificities. We examined enrichment of these motifs in genes induced (repressed) by different Fru M isoforms. In males there was an enrichment of Fru M binding motifs in genes that were significantly induced by overexpression of Fru M while genes repressed by Fru M overexpression showed no such enrichment. This pattern indicates that Fru M isoforms likely induce gene expression by acting as a transcription factor, while repressing gene expression indirectly through other factor(s). This pattern did not exist in females, indicating a distinct difference in Fru M activity between males and females. 33. Monitoring chromatin organization and regulation of the X linked macrosatellite DXZ4 in human embryonic stem cells Figueroa DM Chadwick BP Department of Biological Science, Florida State University, Tallahassee, FL DXZ4 is an epigenetically regulated X linked macrosatellite that ad opts alternate chromatin states in primates, in response to X chromosome inactivation (XCI), the mammalian dosage compensation process. On the female inactive X chromosome (Xi) DXZ4 is arranged into euchromatin, whereas on the active X chromosome (Xa) the macrosatellite is package d into heterochromatin. Most female human embryonic stem cells (hESCs) have alre ady undergone XCI and possess a Xa and Xi. Thus, in female hESC, the DXZ4 chromatin is expected to resemble that of terminally differentiated female somatic cells. However, ChI P in female hESCs failed to show heterochromatin marks characteristic of DXZ4 on the Xa. Nevertheless, bisulfite sequencing revealed both DXZ4 alleles are extensively hypermethylated at CpG dinucleotides in independent female hE SC. It is possible that 5 hy dr oxymethylcytosine (5 hmC), an epigenetic modification often associated with regulatory elements in hESCs, may be partly responsible for the CpG methylation detected at DXZ4. Preliminary data suggests this is indeed the case. Immediately proximal to DXZ4 is a putative promoter, IMPX, which drives expression of a long noncoding RNA (lncRNA) towards the array in hESCs, but is shut down by Polycomb on differentiation and remains silent in somatic cells. Polycomb signals and slight reactivation of IMPX activit y is observed in male cancers that shift DXZ4 packaging from a heter o chromatic to euchromatic state. IMPX silencing coincident with dynamic differentiation associated chromatin change at DXZ4 combined with transformation associated deregulation make this l ncRNA a good candidate for regulating DXZ4 chromatin states. 34. Escape from siRNA induced silencing in an orthobunyavirus, Tensaw virus Fitzpatrick DM Maruniak JE* Department of Entomology and Nematology, University of Florida, Gainesville, FL The inf ectivity of populations of short interfering RNA (siRNA) induced virus escape mutants from Vero cells was assessed in both vertebrate and mosquito cell cultures,
25 using a model orthobunyavirus, Tensaw virus (TSV). RNA interference is a major component of an tiviral immunity in dipteran insects, including mosquitoes. Virus specific siRNAs are being considered as a potential therapy to provide resistance to viral infection in vivo However, due to the high mutation rate of RNA viruses, antiviral siRNA treatments often result in the emergence of escape mutants and a delayed reestablishment of infection. Yet, few studies have examined the infectivity profiles of viral escape populations, especially in arboviruses, where host alternation during arboviral t ransmission cycles constrains major genomic shifts. SiRNAs targeting the o verlapping nucleocapsid/nonst r uctural protein coding regions of TSV significantly reduce viral titer in Vero cells for 48 h. However, viral escape is seen at 72 h, and this viral pop ulation establishes infection. According to tissue culture infective dose assays, viral supernatants collected at 120 hpi from siRNA treated, TSV infected Vero cells were significantly less infective in human HeLa cells than parent virus but did not show s ignificantly different growth curves in Vero and mosquito (C6/36) cells compared to parent virus. Further experiments will utilize short hairpin RNA expressing plasmid constructs comparable to siRNA treatments to prolong antiviral RNA expression, and exam ine escape mechanisms by quantitative PCR of immune genes, changes to particle to infectivity ratio, and genomic mutations associated with small RNA induced selection. 35. EMO s environmentally modified o rganis ms: controlling consumer desired traits with l ight Childers KS 1 Knitter E 1 Evans S 1 Pate D 1 Carone C 1 Folta KM 1,2, 1 Horticultural Sciences Department, University of Florida, Gainesville, FL 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Plants are anchored to the earth and have limited capacity to move. To meet this challenge plants have evolved intricate sensory networks that carefully monitor the ambient environment and guide adjustments in physiology as necessary. The light sensing net works that regulate growth and development have been well defined in the model plant Arabidopsis thaliana The effects of light on pigmentation, stature and composition are well understood. In this work we test the hypothesis that non natural light conditi ons may be applied to expose latent genetic programs in plants that increase traits with significant horticultural value. This study analyzes subjected to a range of narrow bandwidth light treatments. The results show that significant differences in pigmentation, leaf iness, size and nutraceutical production may be achieved with simple, progressive treatments that excite molecular and biochemical pathways leading to enhancement of consumer desired traits. T hese techniques increase their attractiveness and nutritional quality for the consumer, adding value for the grower. Here we bridge basic and applied biology, implementing the well established rules of photomorphogenesis to design the next generation of co nsumer produce. 36. Role of the proteasome in the phytopathogenic bacterium Streptomyces scabies Francis I Loria R L Plant Pathology Department, University of Florida, Gainesville, FL Unlike most prokaryotes, streptomycetes and other actinobacteria contain a proteasome similar to the 20S core particle found in eukaryotes. Proteasomes are essential to eukaryotic life but, since bacteria have specialized proteases and peptidases to degrad e unneeded or damaged proteins, their function in bacteria remains to be elucidated. Nevertheless, mutation of the mpa gene, that encodes the proteasome associated ATPase, renders Mycobacterium tuberculosis more susceptible to nitric oxide (NO) and reactiv e oxygen species (ROS) produced as a defense response by the macrophages, leading to an attenuated virulence phenotype in mice. The primary virulence determinant of the plant pathogen S. scabies is a family of nitrated dipeptide phytotoxins called thaxtomi ns; these novel molecules inhibit cellulose biosynthesis in expanding plant cell tissue in both monocots and dicots, at nanomolar concentrations. Thaxtomin A, the predominant form, is synthesized from L phenylalanine and 4 nitro L tryptophan by the non rib osomal peptide synthases TxtA and TxtB. A nitric oxide synthase (NOS) generates NO, some of which is incorporated into the tryptophan moiety specifically at position 4 of the indole ring by a unique cytochrome P450, TxtE. We hypothesized that the proteasom e would have a role in defending the bacterium against the deleterious effects of NO on proteins. Experimental results show that a deletion mutant in the mpa homolog showed reduced growth and thaxtomin production under thaxtomin, and therefore NOS, inducin g conditions, or when challenged with NO. 37. Deep sequencing of maize seedling transcriptomes to understand the impact of the ROUGH ENDOSPERM3 splicing factor Gault CG 1 Mei W 2 Martin F 1 Fouquet R 1 Barbazuk WB 1 2 *, Settles AM 1 ,3, 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 Department of Biology, University of Florida, Gainesville, FL
26 3 Horticultural Sciences Department, University of Florida, Gainesville, FL More acres of maize were harvested in 2011 than any other crop in the United States (USDA NASS, 2012). Because maize is such an economically important crop, a deeper understanding of the molecular mechanisms that govern maize development can lead to great benefits, such as improvements in maize yield an d grain quality. We study the maize rough endosperm3 ( rgh3 ) mutant, which exhibits defective kernel and seedling development, in order to gain insight into maize development. rgh3 is a hypomorphic allele of the U2AF 35 related protein (ZmURP). Prior characterization of the rgh3 mutant indicates that the gene is required to repress cell proliferation and promote cell differentiation. In humans, URP is associated with the minor and major spliceosomes, but its role in maiz e unclear. To test whether rgh3 affects mRNA splicing, we sequenced the rgh3 and normal seedling transcriptomes. Twelve cDNA libraries were constructed from the roots and shoots of rgh3 and normal seedlings, and the libraries were sequenced on the HiSeq 20 00 platform. The run produced 149 million paired end 100 bp reads that mapped to more than 35,000 genes. mRNA splicing in rgh3 and normal tissues was analyzed at the mRNA isoform level using the Mosaik2, GSNAP, and Cufflinks software programs. Reverse tran scription PCR validation of altered splicing in rgh3 seedlings reveals that the rgh3 mutant fails to efficiently splice introns from a select group of pre mRNA transcripts. 38 How does Pectobacterium carotovorum promote proliferation of Salmonella in tom atoes? George A Noel J Teplitski M Soil and Water Science Department, University of Florida, Gainesville, FL In tomatoes infected with a soft rot plant pathogen Pectobacterium Salmonella can grow to numbers 10 fold higher than in intact tomatoes without Pectobacterium There are three hypotheses under investigation which may adequately explain this phenomenon. The first hypothesis is that pectinolytic activities increase nutrient availabi lity allowing for increased growth of Salmonella The second hypothesis is that rotting of the tomato increases the pH of the fruit thereby reducing the acid stress on Salmonella which may allow it to grow to higher final densities. Lastly, we also tested whether cell to cell signaling between Salmonella and Pectobacterium promotes increased proliferation of Salmonella within soft rots. A series of one week long in vitro experiments have indicated that Salmonella grows to higher n umbers in the presence of b oth polygalacturonic aci d (PGA) and GA. Further experiments conducted with a deletion mutant of the metabolic repressor KdgR, indicated a possible increase in growth when the repressor is non functional. The role of Pectobacterium in this is not yet clear, leading us to the second hypothesis. These hypotheses are being tested using co inoculation and fold change experiments. The co inoculation experiment compares the fitness of type strain Salmonella enterica sv Typhimurium 14028 with the mutants that lack genes with functions in the acid tolerance response (ATR) or are involved in the use of PGA monomers. Rotting by Pectobacterium tends to increase fruit pH, which should allow the mutants to be more competitive due to a decrease of acid stress. Furthermore, this same process should make PGA oligomers and monomers more readily available. 39. Differences in AAV serotype dependent distribution and t ropism w ithin the CNS of wild type and mutant mice in the MPSIIIB mouse m odel Gilkes JA Bloom MD, Kolarich AR, Heldermon CD* Division of Hematology and Oncology, Department of Medicine, University of Florida, Gainesville, FL Mucopolysaccharidoses IIIB (MPSIIIB) is an autosomal recessive lysosomal storage disease caused by mutations in the gene N acetyl glucosaminidase (NAGLU). Defective NAGLU activity results in aberrant retention of heparan sulfate within lysosomes and progre ssive central nervous system (CNS) degeneration. Treatment options are limited by the need to overcome the blood brain barrier and gain successful entry to the CNS. Here, we assess the relative effectiveness of various intracranial delivery methods and sev eral adeno associated virus vectors for gene therapy based treatment of this disease. Using the MPSIIIB mouse model, we perform a comparative study between intracranial six site (IC6) injections and thalamic injections of AAV GFP serotypes 5, 8, 9 and 10, to determine the best treatment modality as determined by tropism and distribution. In both wild ty pe and MPSIIIB animals, neurons were primarily transduced by all serotypes, while microglia were the least transduced. Interestingly, the broadest distri bution patterns and tropism were exhibited by AAV 8 in both IC6 and thalamic treatment groups. However, a greater number of cells and cell types were transduced in the IC6 treatment group. Considerably fewer cells were transduced in the hippocam pus of MPSI IIB compared to wild type mice. Transduction and distribution patterns of AAV 8 and AAV 9 were comparable in the thalamus of wild type and mutant animals. Importantly, this data suggests that AAV 8 exhibits the better distribution and tropism with the IC6 method for use in future gene therapy studies. Fur ther, the data strongly suggest that distribution and tropism are altered in MPSIIIB compared to wild type brains.
27 40 Daxx and USP7: novel regulators of mitosis, taxane sensitivity and genomic stability Giovinazzi S 1 ,2 Morozov VM 1 ,2 Reinhold WC 3 Ishov AM 1 ,2, 1 Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 2 University of Florida Shands Cancer Center, Gainesville, FL 3 Genomics and Bioinformatics Group, Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD Daxx is a multifunctional protein that plays a pivotal role in both physiological and pathological cellular processes. We previously demonstrated that cells wit h low levels of Daxx have reduced sensitivity to taxanes, powerful chemotherapeutic agents, by persisting in a pro metaphase block that allows cells to escape taxane induced cell death. In this study we dissected the mech anisms of Daxx dependent taxane res istance that also suggests function of this protein in mitotic progression. We show that Daxx interacts and cooperates with ubiquitin specific processing p rotease 7 (USP7) to regulate mitosis. We demonstrate that depletion of USP7 promotes stabilization of cyclin B, aneuploidy and mitotic anomalies, as it was previously observed for Daxx. We further demonstrate that USP7 depletion results in reduced stability of the mit otic E3 ubiquitin ligase Check point with Forkhead and RING finger (CHFR). Consequently ce lls depleted by USP7 accumulate CHFR substrate, Aurora A kinase that has a crucial role in mitotic progression. We conclude that Daxx and USP7 are necessary to regulate proper execution of mitosis and their effects are at least partially mediated by CH FR a nd Aurora A kinase. Results from colony formation assay and in silico analysis show that USP7 expression negatively correlates with response to taxanes in cancer cell lines indicating that this protein can be used as a predictive factor for taxane response in cancer patients. 41 Fasting glucose loci associated with glucose response to antihypertensives results from the Pharmacogenomic E valuation of A ntihypertensive R esponses (PEAR) study Gong Y 1 ,* Moore MJ 1 Karnes JH 1 McDonough CW 1 Wang Z 2 Langaee TY 1 Beitelshees AL 3 Turner ST 4 Chapman AB 5 Gums JG 1, 6 Bailey KR 4 Boerwinkle E 2 Johnson JA 1, 6 Cooper DeHoff RM 1,6 for the PEAR investigators 1 Department of Pharmacotherapy and Translational Research, Unive rsity of Florida, Gainesville FL 2 IMM Center for Human Genetics, University of Texas at Houston, Houston, TX 3 Division of Endocrinology, Diabetes and Nutrition, University of Maryland, Baltimore, MD 4 Division of Nephrology and Hypertension, Mayo Clinic, Rochester, MN 5 Renal Medicine Division, Department of Medicine, Emory University, Atlanta, GA 6 Division of Cardiovascular Medicine, Department of Medicine, University of Florida, Gainesville FL BACKGROUND: Genome wide association studies have identified 36 loci to be associated with fasting glucose in nondiabetic white individuals. We assessed the association of these loci with plasma glucose response to atenolol (ATEN) and hydrochlorothiazide (HCTZ), antihypertensives that have been associated with adverse metabolic effects. METHODS : PEAR evaluated blood pressure and glucose response in hypertensive patients randomized to either ATEN or HCTZ monotherapy then the combination. Genotypes of 33 loci were obtained from Illumina 50K cardiovascular or Omni1M GWAS chips. Associations with AT EN or HCTZ induced glucose change was evaluated in 450 white patients using linear regression. Genetic risk scores based on number of glucose increasing allele at each SNP were created to assess the collective genetic effect of nominally associated SNPs. P ermutation tests were performed to validate the p values of the genetic risk scores. RESU LTS:Three SNPs (PROX1rs340874, ARA P1rs11603334 and PCSK1rs4869272 ) were nominally associated with ATEN induced glucose increase. Two SNPs (MTNR1B rs10830963 and SLC2A2 rs11920090) were nominally associated with HCTZ induced glucose increase. The corresponding genetic risk scores were strongly associated with glucose increase after ATEN monotherapy (p=6.4E 6, beta=2.1) or HCTZ monothera py (p=8.3E 5, beta=2.8) The permut ation p values for both scores were < 0.0001. CONCLUSION: Five fasting glucose loci were also associated with ATEN or HCTZ induced increase in plasma glucose. These data highlight that atenolol and HCTZ may constitute an environmental risk factor similar to those known to be associated with dysglycemia and diabetes. 42. Diverse roles of strigolactone signaling in maize architecture and the uncoupling of a branching specific sub network Guan JC Suzuki M Klee HJ*, Koch KE*, McCarty DR* Horticultural Sciences Department, University of Florida, Gainesville, FL Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Strigolactones (SLs) control lateral branching in diverse species by regulating transcription factors ortholo gous to Teosinte branched1 (Tb1). In maize, however, selection for a strong central stalk during domestication is attributed primarily to the Tb1 locus, leaving the architectural roles
28 of SLs unclear. To determine how this signaling network is altered in m aize, we first examined effects of a knock out mutation in an essential SL biosynthetic gene that encodes Carotenoid Cleavage Dioxygenase 8 (CCD8), then tested interactions between SL signaling and Tb1. Comparative genome analysis revealed that maize depen ds on a single CCD8 gene ( ZmCCD8 ), unlike other panicoid grasses that have multiple CCD8 paralogs. Function of ZmCCD8 was confirmed by transgenic complementation of Arabidopsis max4 ( CCD8 ), and by phenotypic rescue of the maize mutant ( zmccd8::Ds ) using a synthetic SL (GR24). Analysis of the zmccd8 mutant revealed a modest increase in branching that contrasted with prominent pleiotropic changes that include (1) marked reduction in stem diameter, (2) reduced elongation of internodes (independent of carbon supply), and (3) a pronounced delay in development of the centrally important, nodal system of adventitious roots. Analysis of the zmccd8 tb1 double mutant revealed that Tb1 functions in an SL independent sub network that is not required for the o ther diverse roles of SL in development. Our findings indicate that in maize, uncoupling of the Tb1 sub network from SL signaling has profoundly altered the balance between conserved roles of SLs in branching and diverse aspects of plant architecture. 43. The role of flagella in virulence of Vibrio vulnificus Gulig PA 1, *, Tucker M 1 Thiaville P 1,2 Rezaie N 1 Comiskey J 1 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL The bacterium Vibrio vulnificus is the leading cause of reported death from ingestion of seafood in the United States. It causes sepsis after ingestion of oysters and skin infection from contamination of wounds. Comp romised patients can die within days of initial symptoms. The mechanisms by which this opportunistic pathogen causes such rapid death with extensive tissue destruction are mostly unknown, although some virulence factors have been identified. We used signat ure tagged transposon mutagenesis to identify genes essential for virulence in a mouse model of infection. One insertion was in a gene involved with flagellar biosynthesis. V. vulnificus possesses six genes encoding the major protein subunit of flagella: f laFBA and flaCDE. Through deletional mutagenesis we determined that only flaC and flaE were necessary and sufficient to enable full vir ulence in our mouse model. Interestingly, when the bacteria encoded only flaC, their growth was inhibited; they were non motile, non flagellated, and exhibited a more severe attenuation than if they expressed no flagella at all. This result suggests that the FlaC and FlaE flagellins must interact to form intact and functional flagella. We also determined that flagellated bac teria that are non motile or are defective at chemotaxis are attenuated for infection of mice. Ongoing studies are aimed at determining the structure/function relationships of the flagella in motility and virulence. 44. Advancing complex phenotype analyses through machine vision and computation Gustin J 1 Settles AM 1, *, Miller N 2 Subramanian R 2 Yoshihara T 2 Baier J 1 Durham Brooks T 3 Ferrier N 2 Spalding E 2 1 Horticultural Sciences Department, Univers ity of Florida, Gainesville, FL 2 Departmen t of Botany, University of Wisconsin Madison, Madison, WI 3 Department of Biology, Doane College, Crete, NE Phenotyping methods frequently limit functional genomic studies. Phenotypes are currently not studied with the same degree of sophistication or throughput as genomes or processes more proximate to the genome such as gene expression. Because phenotypes are such an important source of information about gene function, we are integrating multiple machine vision platforms to study seed and seedling phe notypes. Machine vision utilizes information contained in an image or other optoelectronic signal, such a reflectance spectroscopy, to collect quantitative measures of phenotypes. We are focusing on the interrelationships of maize kernel traits with seedli ng growth traits. We have developed a semi automated pipeline to collect kernel weight, near infrared reflectance (NIR) kernel spectra, kernel color and 3D shape, and dynamic seedling root growth. Serial phenotyping on indexed kernels will provide greater statistical power to detect interrelationships that have a physiological basis. Computational workflows are being developed to automatically extract biologically relevant data from each phenotyping platform and to interrelate the machine collected data. Th e combination of high throughput serial phenotyping and diverse genetic resources available in maize allow access to a wide variety of relationships between seed and seedling characteristics. As an example, preliminary studies show that aspects of the NIR spectra of a kernel are correlated with seedling root gravitropism response. We are also using this pipeline to identify quantitative trait loci (QTL) underlying the phenotypes and physiological relationships within the maize Nested Association Mapping (NA M) population.
29 45. The effects of silver nanoparticle administration on intestinal microbiota composition in C57/B7bl6 mice Gutalj A 1 ,2 Ukhanova M 2 Hinkley G 3 Roberts S 3 Yao J 2 Wang X 2 Mai V 1,2 1 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 2 Emerging Pathogens Institute, University of Florida, Gainesville, FL 3 Clinical Toxicology Graduate Program, University of Florida, Gainesville, FL Silver nanoparticles are currently being explored for their known antimicrobial properties and their potential as a novel medical tool. Several studies have been conduc ted using silver nanoparticles to investigate their effect on different organs such as the liver, brain, and kidneys. However, to date, few studies have a nalyzed their effects on the composition and diversity of gut microbiota. Here we investigate, in a mouse model, the hypothesis that administering silver nanoparticles will alter the microbiota composition. 46. The phytochrome gene family of s trawberry ( Fragaria vesca ) Hart JE 1 Brunings A 1 Folta KM 1,2, 1 Horticultural Sciences Department, University of Florida, Gainesville, FL 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Phytochromes are red/ far red light sensing pigments that inform plants of ambient light conditions. Phytochromes exist as a small multigene family in each plant species. Family members tend to have specific roles with some functional overlap. The phytochromes control horticulturally importa nt processes such as control of flowering, plant stature and fruit/vegetable ripening. It is therefore of great interest to characterize the phytochromes of strawberry. Five phytochromes were identified in the diploid strawberry ( Fragaria vesca ) genome. On e phytochrome closely resembles phyA, the far red active photoreceptor in other species. Another resembles phyB, and another phyC. Two other similar sequences match best with phyE, and no phyD is present. Tissue specific expression has been examined and sh ows that some family members are greatly enriched in specific plant parts. To test the functions of these molecules in vivo transgenic RNAi lines were created. Loss of function phentoypes reveal the roles for individual light sensors in the control of various biological processes. Currently we have many independent lines of the phyA RNAi plants. These plants exhibit difference s in runnering, as phyA RNAi plants produce more stolons than wild type plants. After 16 h in intense far red light wild type plants show strong leaf inclination toward the light source. The phyA plants do not. These preliminary observations show some of t he likely responses to far red light. Future studies will examine seedling responses and the effects on flowering and fruit development, with the goal of understanding how strawberry light sensors contribute to these processes. 47. Optimization of a direc t organogenesis system from the cotyledonary node explants of peanut ( Arachis hypogaea L.) Hsieh Y F 1 Jain M 2 Wang J 1,2 Gallo M 1 3 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 Agronomy Department, University of Florida, Gainesville, FL 3 Current address: Molecular Biosciences and Cultivated peanut ( Arachis hypogaea L.) is the second most important food and oilseed legume in the world. Pub lished direct regeneration protocols for transgenic improvement in peanut are highly genotype dependent and have provided limited utility for peanut cultivar development. An alternative repetitive somatic embryogenesis system is suitable only for biolistic gene delivery and requires prolonged in vitro subculturing. We have optimized a facile and rapid method for obtaining direct shoot organogenesis from cotyledonary node (CN) explants excised from peanut seedlings germinated on cytokinin supplemented Murash ige and Skoog (MS) basal salt medium. A mass of multiple shoot initials formed at the axillary bud region of CN explants derived from seedlings germinated in the presence of 4 mg l 1 6 benzylaminopurine (BAP) within th ree weeks of culture on the sa me cytok inin containing medium. Adventitious shoots elongated rapidly over the next three weeks, and rooted efficiently on MS medium supplemented with 1 mg l 1 naphtha leneacetic acid (NAA). Although adequate initiation of adventitious shoot buds was also observe d from the CN explants in the presence of another commonly used cytokinin, thidiazuron (TDZ), the elongation of shoot buds was negatively impacted, thus compromising overall regeneration potential. Starting from mature seed, the described protocol yielded rooted plantlets within 12 15 weeks, in contrast to 15 18 months required for initiating and regenerating somatic embryogenic cultures. This procedure is highly reproducible and worked for several peanut genotypes tested.
30 48. Targeting nucleocytoplasmic shuttling mechanisms in esophageal cancers Hu C Wu L* Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL University of Florida Shands Cancer Center, Gainesville, FL Esophageal squamous cell carcinoma (ESCC) is usually drug resistant tumor and has a dismal 5 year survival rate of <10%. Therefore, there is an urgent need to develop effective therapeutic approaches. In this study, we investigated whether CRM1 (XPO1), a major mediator of nuclear export, represents a therapeutic target in ESCC. CRM1 is responsible for the nuclear export of a variety of proteins including tumor suppressors and cell growth and survival regulators. CRM1 overexpression was correlated with poor prognosis in many cancers. Specific blockade of CRM1 caused selective cell growth suppression and apoptosis in several hematological malignancies and solid cancers, in part due to nuclear accumulation and activation of tumor suppressors. In this study, we evaluated CRM1 expression in ESCC cancer cell s and determined whether CRM1 is required for maintaining cancer cell phenotypes of ESCC. We found that CRM1 is highly expressed in the majority of ESCC cancer cell lines. Blocking CRM1 expression and activity, through respective RNA interference and pharm acological inhibition with the CRM1 inhibitor LMB, reduced esophageal cancer cell growth and survival. The molecular action of CRM1 in ESCC cancer cells was further demonstrated by nuclear retention of the CREB coactivator CRTC2 and activation of CREB tran scription when there was loss of function of CRM1. Therefore, CRM1 has an essential role in the maintenance of esophageal cancer cells, suggesting that CRM1 is a therapeutic target in ESCC. Our data thus warrant the preclinical investigations into the effi cacies of newly developed small molecule CRM1 inhibitors with better specificity and less toxicity in blocking ESCC. 49. Development of an adeno associated virus vector targeting the Keap1 Nrf2 pathway for dry age related macular d egeneration Ildefonso CJ 1 Jaime H 2 Lewin AS 1 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Department of Biology, University of Florida, Gainesville, FL Age related macular degeneration (AMD) is a leading cause of blindness i n developed countries. This ocular disease has two forms known as wet AMD (characterized by the growth of blood vessels in the retina) and dry AMD (characterized by drusen accumulation). Currently there is no treatment available for dry AMD. This form of A MD has been associated with an increase in oxidative stress within the eye retinal pigmented epithelium. The Keap1 Nrf2 signaling pathway is involved in the modulation of antioxidant genes. In this pathway the repressor Keap1 binds and sequesters the trans cription factor Nrf2 within the cytoplasm and targets it for degradation. The present study hypothesizes that the expression of antioxidant genes can be increased by knocking down the levels of Keap1 and increasing the levels of Nrf2 therefore alleviating the oxidative stress. Several siRNA sequences that target the murine Keap1 were screened by co transfection into HEK293T cells. One siRNA sequence which decreased the levels of murine Keap1 by 90% was selected. This sequence was embedded within the sequenc e of the pre miRNA30 and cloned in an AAV plasmid between the Nrf2 cDNA and its poly A signal. The co transfection of this vector with the murine Keap1 in HEK293T cells resulted in a 50% knock down of Keap1. Future experiments will determine if this knock down of Keap1 and overexpression of Nrf2 results in a significant increase of antioxidant genes and its correlation with cell viability under acute oxidative stress conditions These in vitro studies will lead to future preclinical safety and efficacy testing in a model of dry AMD. 50. Exposure to the G protein coupled estrogen receptor 1 (GPER) selective agonist G 1 results in elevated levels of vitelloginin in adult fathead minnows ( Pimepha les promelas ) Jayasinghe BS 1 Denslow N D 1,2, *, Sabo Attwood T 1 1 Center for Environmental and Human Toxicology University of Florida, Gainesville, FL 2 Department of Physiological Sciences, University of Florida, Gain esville, FL Using a variety of human cell lines and animal models, several research groups have shown that G protein coupled estrogen receptor 1 (GPER) (formerly known as estradiol (E2) activation of signal transduction through non genomic membrane initiated pathways. As the GPER is not well characterized in fish compared to mammalian organisms, we began studies to; (1) assess the tissue specific expression of GPER in adult fathead minnows (FHM) ( Pimephales promelas ), (2) determine the effect of GPER activation on vitellog enin synthesis in adult FHM males and females using a selective GPER agonist (G 1) and (3) to study at molecular level whether the GPER antagonist, G 15, can block G 1 induced effects. Results from these studies show that GPER mRNA is detectable by qRT PCR in many vital organs, and is most highly exp ressed in the brain followed b y gall bladder, trunk kidney, intestine, liver, heart, ovary and muscle. Exposure to G 1 (5, 30 and 100 ng/L) for 48 hours
31 resulted in increased level of vitellogenin mRNA expressio n compared to vehicle control in both male and female FHM. These data suggest that GPER activation or inhibition result in activation of estrogen receptor (ER) down stream signaling or G 1 and G 15 directly activate ERs. To begin to understand alternate do wnstream targets of GPER, we are currently performing microarrays on various organs using G 1 exposed fish. Our findings to date suggest that control of vitellogenin synthesis likely involves both nuclear and membrane receptors that are sensitive to E2 act ivation. 51. Functional differentiation of AFL and VAL B3 transcription factors regulating the transition from embryo formation to germination Jia H 1 Suzuki M 1 ,2 McCarty D R 1 ,2, 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 Horticultural Sciences Department, University of Florida, Gainesville, FL The transition between seed and seedling is coordinately regulated by a family of plant specific B3 transcription factors (TFs). As master regulators of embryo deve lopment, AFL B3 TFs (ABI3, FUS3 and LEC2) and two HAP3 family TFs (LEC1 and L1L) positively regulate embryogenesis and embryo maturation, whereas VAL B3 factors (VAL1, VAL2 and VAL3) repress the AFL/LEC1 network during germination. AFL TFs recognize the Sp h motif through their B3 DNA binding domain. The non redundant roles of individual AFL and VAL B3 TFs in regulating the transition process have not been fully dissected. val1 val2 double mutant seedlings exhibit embryonic callus. Seed specific genes in the AFL/LEC1 network are highly up regulated in the val1 val2 mutant. Therefore, a series of triple mutants ( val1 val2 abi3 val1 val2 fus3 val1 val2 lec2 val1 val2 lec1 and val1 val2 l1l ) were created in order to study the genetic interactions of AFL/LEC1 and VAL TFs. We found that fus3 and lec2 mutants can completely suppress the val1 val2 embryonic seedling phenotype, while abi3 lec1 and l1l are partial suppressors. The seedling phenotype of the partially suppressed triple mutants is strongly dependent on the embryo developmental stage: seedlings rescued early in embryo development (7 9 DAF) uniformly show embryonic phenotypes; embryos rescue d at mid stage (10 12 DAF) have intermediate phenotypes; while embryos rescued late (13 14 DAF) produce normal seedlings. Consistent with the genetic results, expression of FUS3 and LEC2 is elevated in seedlings of partially suppressed triple mutants that are rescued early compared to seedlings from late rescued embryos (12 14 DAF). Our results indicate that FUS3 and LEC2 expression is required for the embryonic seedling phenotype. These results highlight the distinctive functional roles of AFL and VAL B3 T Fs in regulating the progression from seed formation to germination. 52 Structural insights into B ocavirus Kailasan S 1 Kantola K 2 McKenna R 1 Chipman P 1 Brown K 3 Kapoor A 4 Sderlund Vener mo M 2 Agbandje McKenna M 1 1 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2 Department of Virology, University of Helsinki, Helsinki, Finland 3 Virus Reference Department, Health Protection Agency, London, United Kingdom 4 Mailman School of Pu blic Health, Columbia University, New York, NY Human Bocavirus (HBoV1) was first isolated in 2005 from nasopharyngeal aspirates of children (< 2 years of age) with acute respiratory tract infections (ARTI). During 2008 10, several strains of HBoV, namely HBoV2 4, were identified in children suffering more frequently from gastrointestinal infections than ARTIs. HBoV1 4 occurrence has been reported in more than 17 countries worldwide with limited knowledge regarding its transmission and temporal dependence. HBoVs belong to the small, non enveloped, ssDNA virus family of Parvoviridae Other members of genus Bocavirus like Porcine Bocavirus (PBoV1), Bovine Parvovirus (BPV1), Gorilla Bocavirus (GBoV1), and Canine Minute Virus (CnMV or MVC) are known to cause sim ilar respiratory and gastrointestinal infections in their respective hosts. These viruses encode two structural proteins called VP1 and VP2; the latter being the predominant protein of an icosahedral capsid (T=1). Our efforts to purify baculovirus/ Spodopte ra frugiperda expressed VP2 virus like particles (VLPs) of HBoV1 4, BPV1, PBoV1, MVC and GBoV1, and determine high resolution three dimensional structures of these viruses using X ray crystallography and/or cryo electron microscopy (cryo EM) will be presen ted here. 53 Breeding genetically improved non invasive elephantgrass hybrids for lignocellulosic biofuel production Kannan B Sollenberger L, Altpeter F Agronomy Department, University of Florida, Gainesville, FL Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Elephantgrass ( Pennisetum purpureum ) is best adapted perennial grass and has the ability to produce large amounts of high quality forage biomass. The biofuels industry has identified elephantgrass as one of the
32 productive feedstocks for lignocellulosic biofuel production in the southern US. However, elephantgrass is listed as invasive in Florida by the Florida Exotic Pest Plant Council. Plant propagation and establishment of new elephantgrass plantings occurs through vegetative plant parts. Therefore, unlike most seeded crops, seed production is not necessary for elephantgrass biomass production and its suppression will significantly reduce its potential for invasiveness. To develop non invasive genetically im proved genotypes of elephantgrass we made interspecific hybridization between elephantgrass (2n=4x=28) and pearl millet (2n=2x=14) to result in drought tolerant, high biomass with male and/or female sterile triploid hybrids (2n=3x=21) which will eliminate production of wind dispersed seeds. Tall, stress tolerant elephantgrass parents were chosen to generate interspecific hybrids with good productivity and persistence. Pearl millet (AA genome) multiline population with A4 CMS was pollinated with allopolyploi d napiergrass than 3000 triploid, interspecific hybrids between elephantgrass and pearl millet. Phenotypic variability present in these hybrids allowed selecting lines which produced similar or higher b iomass amounts as the seed producing elephantgrass cultivar Merkeron. We will present data describing the biomass yield and related traits of interspecific hybrids evaluated in replicated field trials. 54 RcrRPQ affect s development of genetic c ompetence in Streptococcus mutans through multiple p athways Kaspar J Ahn S J, Burne RA Department of Oral Biology, University of Florida, Gainesville, FL Streptococcus mutans displays complex regulation of the development of natural genetic competence. The central regulator in the development of com petence is ComX, an alternative sigma factor that activates late competence genes. Expression of comX is activated by a small hydrophobic peptide (ComS) that is secre ted, re internalized and bound by ComR, the proximal regulator of comX. Previously, the rcrRPQ operon, which encodes a MarR transcriptional repressor and a pair of co transcribed ABC transporters has been shown to link stress tolerance, (p)ppGpp production and genetic compet ence in S. mutans Notably, an rcrR polar transformable, but the rcrR non transformed; consistent with the inability of the latter strain to activate late competence genes. To explore these phenotypes further and dissect the mechanisms by which RcrRPQ affect competence development, we show that overexpres sion of comS in the rcrR mutant backgrounds impacts growth inhibition by competence stimu lating peptide (CSP) in complex medium, but does not affect th e transformation phenotypes. In contrast, in a comX overexpressing background, the rcrR non polar mutant is able to be transformed and late competence genes are activated. Based on these results and o ur prior work, we conclude that RcrRPQ govern ComX depen dent activation of late competen ce genes, that hyper expression of comX can overcome the effects of the non polar rcrR mutatio n, and that levels of effectors that are inhibitory to the activity of ComX are modulated by the RcrPQ transporters. 55 Variabil ity in Wolbachia prevalence among human louse ( Pediculus humanus ) populations Kassu G Ascunce MS, Reed DL* Mammalogy, Florida Museum of Natural History, University of Florida, Gainesville, FL Wolbachia p roteobacteria infecting a wide range of arthropods and filarial nematodes. Wolbachia can manipulate host reproduction resulting in reproductive abnormalities such as cytoplasmic incompatibility (CI), parthenogenesis, feminization and male killing, thus aff ecting biology, ecology and evolution of its hosts. In addition, Wolbachia can affect host mitochondrial DNA evolution, because of the linkage between Wolbachia and associated mitochondrial haplotypes, and thus confound host phylogeny based on mtDNA. Previ ous research has shown that human lice have Wolbachia infections; therefore, we wanted to know whether Wolbachia could influence the divergence of mitochondrial haplotypes in human lice. We surveyed 59 human lice collected from 7 sites throughout the Ameri cas. Nine Wolbachia primers ta rgeting seven loci were evalua ted in this study including fbpA, 16 S2 and coxA. None of the PCR primers tested in these populations of human lice yielded PCR products related to Wolbachia Therefore, we conclude that Wolbachia are not present in the samples surveyed here, or that they are present at undetectable levels. This does not exclude the possibility that some other populations do harbor infections. It may be possible that Wolbachia or i nfection pattern within the species, such that populations have either a very high (>90%) or very low ( < 10%) prevalence of Wolbachia We suggest that metapopulation dynamics and environmental fluctuations may partially explain the abrupt difference in prevalence reports of Wolbachia in human lice. 56 Expression of alpha and beta subunits of colony stimulating factor 2 receptor during preimplantation embryonic development in the cow Khan FA Moss JI, Hansen PJ*
33 Department of Animal Sciences, University of Florida, Gainesville, FL DH Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, FL Colony stimulating factor 2 (CSF2) is an important cytokine regulating preimplantation embryonic development in several mammalian species. Studies on bovine in vitro produced embryos have shown that addition of CSF2 to culture medium improves development. In hematopoietic cells, CSF2 signaling subunit of CSF2 subunit (CSF2RB). However, studies in mice and humans subunit is absent in preimplantation embryos. There is paucity of information on expression of CSF2RA and CSF2RB in bovine embryos. The present study was, therefore, conducted to evaluate expression of CSF2RA and CSF2RB during preimplantation stages of development. Embryos cultured in vitro w ere harvested at 2 cell [32 40 hours post insemination (hpi)], 3 4 cell (48 hpi), 5 8 cell (48 hpi), 9 16 cell (72 hpi), morula (120 hpi), and blastocyst stages (168 hpi). Matured oocytes were also collected. RNA was extracted, reverse transcribed, quantif ied by real time PCR, and product was visualized using 1.5% (w/v) agarose gel electrophoresis. Five replicates, each involving 25 30 oocytes/embryos at each stage, were used. Leukocyte RNA was used as a positive control. CSF2RA was expressed at all stages of development examined while CSF2RB was not expressed at any stage. Expression of CSF2RA varied with development (P<0.0001), with maximum expression at the 9 16 cell stage (i.e., coincident with embryonic genome activation). These findings suggest that CSF2 signaling in bovine embryos may occur independently of subunit of CSF2R and through an alternate cytokine receptor coupled signaling pathway. 57 Role of glutaredoxins in high temperature stress tolerance in Oryza sativa Krishnamurthy A Nichols G, Rathinasabapathi B Horticultural Sciences Department, University of Florida, Gainesville, FL In the present global warming scenario, development of crop varieties with improved tolerance to high temperature stress is an important goal for mod ern agriculture. High temperature stress is a major limitation for productivity in cereals. Rice being a major food crop in the world, serves as an excellent model crop to test biotechnologies to improve heat tolerance in cereals. The objective of our proj ect is to evaluate the role of glutaredoxins in imparting oxidative and heat stress tolerance in rice. We found heat shock induced glutaredoxin specific activity in young rice seedlings. In a previous study, PvGrx5 cDNA encoding a glutaredoxin was isolate d from an arsenic hyper accumulator fern, Pteris vittata Transgenic expression of PvGrx5 in Arabidopsis resulted in improved whole plant tolerance to high temperature stress. To test whether a similar approach can be used to improve heat tolerance in rice we generated stable transformants of Oryza sativa cv. Nipponbare expressing PvGrx5 via agrobacterium mediated transformation. We also generated transgenic rice lines over expressing Arabidopsis glutaredoxins for comparison. The expression of glutaredoxin transgenes were confirmed by RT PCR and enzyme activity assays. PvGrx5 over expressing lines and control lines were evaluated for high temperature stress tolerance by using a seedling bioassay. PVG19 2 line over expressing PvGrx5 was significantly less in hibited to a heat stress treatment compared to the vector control line VC19 6. 58 Development of an open urethra: comparative external genitalia development in the mouse and turtle Larkins C 1 Cohn MJ 1 3, 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Department of Biology, University of Florida, Gainesville, FL 3 Howard Hughes Medical Institute, University of Florida, Gainesville, FL Hypospadias is a congenital defect characterized by an ectopic op ening of the urethra on the ventral side of the penis. It is one of the most common congenital defects and can be caused by both environmental and genetic factors, although, despite its prevalence, it is still unclear how hypospadias develops embryonically Interestingly, mammals are the only group of amniotes that normally develop a closed urethra, while all other groups have an open urethral groove or sulcus. Therefore, hypospadias in mammals may be interpreted as a reversion to a more primitive condition We are taking advantage of this evolutionary characteristic as a means to identify genes and processes that are important for the generation of a closed urethra by comparing external genitalia development in mice and turtles. We show that during normal e mbryonic development of the red eared slider turtle, Trachemys scripta the urethra is initially closed but later opens along the entire ventral side of the penis generating the urethral groove. Importantly, we show that the morphology, with the exception of the open urethra, and transcription of several gene s within the turtle penis are highly similar to that in mammals. Interestingly, our comparative analysis has led to the identification of the Bmp and Fgf signaling pathways as potential regulators of ur ethral opening in both mice and turtles.
34 59 Effects of mode of transmission on v irulence Larsen JS Bermudez AL, Culbreth EM, Abbas R, Wayne ML* Department of Biology, University of Florida, Gainesville, FL Is virulence of parasites evolvable? Multiple theories have been proposed to answer this qu estion, such as the trade off hy pothesis, which proposes on optimal balance its negative effects on the host organism. This experiment seeks to s tudy the effects of mode of vertical transmission on virulence, and hence its evo lution. We use the Sigma virus/ Drosophila melanogaster system. Three different vertical transmission modes were studied: uninfected male with infected female, infected male wi th uninfected female, and infected male with infected female. A control was also included (neither parent infected). Data were collected for egg to adult viability, development time, fecundity, and body size, all of which are components of fitness in Droso phila melanogaster as well as for viral titer (load) using quantitative PCR. This data could serve to increase our understanding of the evolut ion of virulence by quantifying the effects of mode of transmission. The results could also be applied to other v ertically transmitted viruses carried by dipteran vectors that infect human hosts. 60 Novel regulatory genes affect thaxtomin production and p athogenesis in Streptomyces scabies Laskaris P Francis IM, Loria RL Plant Pathology Department, University of Florida, Gainesville, FL Potato scab disease, caused by Streptomyces scabies and other streptomycetes, has a global distribution and causes significant economic losses. Production of thaxtomin A, a nitrated dipeptide phytotoxin that potentiates virulence, is regulated by the txtR gene. Additional regulatory genes likely play a rol e in the production of thaxtomin, or in the expression of other genes involved in pathogenicity; their discovery may suggest new methods of combating potato scab. A total of 21 previously unstudied regulatory genes, that are conserved among the plant patho gens S. scabies S. turgidiscabies and S. ipomoeae were deleted in S. scabies 87 22 and mutants were evaluated for production of thaxtomin in oat bran b roth (OBB). One knockout was reduced in thaxtomin production, while two overproduced thaxtomin. RT QPCR revealed that the thaxtomin biosynthetic cluster was over expressed in all three knockouts, though other pathogenicity genes were unaffected. Two of the regulators belong to the GntR family, a member of which is a global developmental regulator. Their eff ect may be due to the link between development and secondary metabolism. The third regulator belongs to the ribonuclease III family, a member which is a global regulator of secondary metabolism; the homolog in S. scabies may have a similar role. The overex pression of thaxtomin biosynthetic genes in the knockout with reduced production indicates that the regulation of thaxtomin production is a complex process. 61 Comparative analysis of the limb specific enhancer of the Sonic hedgehog (Shh) gene and its re lation to vertebrate limb evolution Leal F 1 Cohn MJ 1 3, 1 Department of Biology, University of Florida, Gainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 3 Howard Hughes Medical Institute, University of Florida, Gainesville, FL Digit and limb loss have occurred many times during vertebrate evolution, however the underlying molecular mechanisms are largely unknown. Pythons lack forelimbs and have only rudime ntary pelvic girdles and femora. We showed previously that the arrest of python hindlimb bud development is associated with absence of an AER and lack of Shh expression. However, Shh can be induced in python limb bud cells grafted under a chick AER. Shh ex pression in the ZPA is regulated by a long range, limb specific enhancer known as the ZRS. Previous studies have been unable to identify a ZRS in limbless reptiles, linking loss of this enhancer to the evolution of limblessness. We isolated the ZRS contain ing region from the basal snake Python reticulatus and identified a well conserved ZRS domain. We therefore revisited whether Shh is expres sed at very early stages of py thon hindlimb development, and found a transient, posterior domain of Shh in early limb buds. Interestingly, the Shh expression domain extends further anteriorly than the typical ZPA pattern; consistent with our finding that polarizing potential is widespread in python limb buds. Together, these results suggest that the genomic regulatory el ements required for digit development have been retained during the 100+ million years of snake evolution 62 BLIMP1 binds the ZRS enhancer and negatively regulates Shh expression in the limb bud Lee C Harfe BD* Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL
35 The hedgehog signaling protein SHH is responsible for patterning many different types of tissues during organogenesis. In the limb buds, Shh is expressed in the zone of polarizing a ctivity (ZPA) located in the posterior mesenchyme of the developing limb bud and is required for normal limb patterning. Expression of Shh in the limb bud is controlled by the limb specific ZPA regulatory sequence (ZRS). A few positively acting factors hav e been identified that can activate expression from this enhancer, however it has been unclear how ZPA specific expression is achieved since the positively acting factors are all ex pressed more broadly than Shh. We uncovered a negative regulator of Shh exp ression, BLIMP1. Blimp1 is expressed in cells proximal to the ZPA. BLIMP1 protein binds three sites within the ZRS enhancer and is required for repressing Shh expression in proximal posterior cells in the limb bud. Our data suggests that BLIMP1 is a negati ve regulator of Shh expression in cells that will later migrate into the ZPA and express Shh. 63 Measures of sequence diversity for sizing taxon sampling Lee HW 1,2 Brocchieri L 2, 1 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL With the increasing size of genome, gene and protein databases, evaluation of the size of the available samples for different taxa, of how densely e ach taxon is represented, and of how much diversity is represented in each sample, is becoming increasingly relevant for optimization of sampling strategies. Thorough taxon sampling is crucial in increasing accuracy of phylogenetic tree reconstructions and for estimating patterns of functional differentiation. Taking into consideration the evolutionary relations hips among sampled sequences, we developed several metrics based on alignments and phylogenetic trees and estimated size and diversity of available samples of bacterial phyla. We expect our metrics are useful for constructing balanced samples of phylogenetic g roups. 64 Combinatorial splicing regulation by muscleblind like proteins in development and disease Lee K Y 1,2 Li M 1 Manchanda M 1 Chamberlain C 3 Charizanis K 1 Mohan A 1 Hong H 1 Shiue L 4 Ares M 4 Swanson MS 1 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Department of Neurology, Chang Gung Memorial Hospital at Keelung, Keelung, Taiwan 3 Department of Genetics, Cell Biology and Development, Universi ty of Minnesota, Minneapolis, MN 4 RNA Center, Department of Molecular, Cell and Develo pmental Biology, University of California, Santa Cruz, Santa Cruz, CA The muscleblind like (MBNL) proteins regulate the alternative splicing of hundreds of genes during development and loss of MBNL splicing activit y is a key pathogenic feature underlying the neuromuscular disease myotonic dystrophy (DM). Since the MBNL gene family consists of three paralogs, which vary in temporal and spatial tissue expression patterns, we have proposed that the multi systemic disea se manifestations of DM result from combinatorial loss of MBNL proteins. To test this hypothesis, we have generated conditional Mbnl1, Mbnl2 and Mbnl3 knockout (KO) mice and studied the effects of single and multiple Mbnl knockouts on embryonic and postnat al development. Splicing microarrays, RNA seq and HITS CLIP analyses demonstrate that the Mbnl1 and Mbnl2 proteins regulate hundreds of splicing events in skeletal muscle and the brai n, respectively, via the recogn ition of a similar RNA core sequence motif YGCY. While Mbnl1 KOs develop DM associated muscle and eye pathologies, loss of Mbnl2 expression does not significantly affect muscle function but instead has a profound impact on the brain with spatial learning/memory deficits on a hippocampal dependent task, a decrease in NMDAR synaptic transmission, impaired hippocampal synaptic plasticity and REM sleep abnormalities. Mbnl1 / ; Mbnl2 / double KOs are embryonic lethal but Mbnl1 / ; Mbnl2+/ are viable and show enhanced pathological phenotypes in skelet al and cardiac muscle suggesting that Mbnl2 partially compensates for Mbnl1 loss in these tissues. The molecular basis of this compensatory function will be discussed. 65 Proteomic analysis of high NaCl induced changes in abundance of nuclear proteins Li J 1,2 Ferraris JD 2 Yu D 2 Singh T 2 Izumi Y 2 Wang G 2 Gucek M 2 Burg MB 2 1 Proteomics Division, Interdiscipli nary Center for Biotechnology Research, University of Florida, Gainesville, FL 2 Systems Biology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD Mammalian cells are normally stressed by high interstitial NaCl in the renal medulla and by lesser elevation of NaCl in several other tissues. High NaCl damages proteins and DNA and can kill cells. Kn own protective responses include nuclear translocation of the transcription factor NFAT5 and other proteins. In order to better understand the extent
36 and significance of changes in nuclear protein abundance, we extracted nuclear and cytoplasmic proteins se parately from HEK293 cells and measured by LC MS/MS (iTRAQ) changes of abundance of proteins in the extracts in response to high NaCl at three time points: 1 hour, 8 hours, and adapted for two passages. We confidently identified a total of 3190 proteins. 1 63 proteins changed significantly at least at one time point in the nucleus. We discerned the biological significance of the ch anges by Gene Ontology and pro tein network analysis. Proteins that change in the nucleus include ones involved in protein folding and localization, microtubule based process, regulation of cell death, cytoskeleton organization, DNA metabolic process, RNA processing, and cell cycle. Among striking changes in the nucleus, we found a decrease of all six 14 3 3 isoforms; dynamic changes proteins, suggestive of nucleoskeletal reorganization; rapid decrease of tubulins; and dynamic changes of heat shock proteins. Identification of these changes of nuclear protein abundance enhances our understanding of high NaCl induced c ellular stress, and provides leads to previously unknown damage and protective responses. Poster Session I I, Posters no. 66 130 Thursday, November 29 11:45 a.m. 1:3 0 p.m. 66 Unexp ec ted role of Rad51 in Babesia bovis antigenic variation Mack E Xiao Y Allred D R Department of Infectious Diseases and Pathology, University of Florida, Gainesville, FL Babesia bovis is an intraerythrocytic protozoan parasite of cattle that causes pathology sharing many parallels with falciparum malaria. B. bovis undergoes antigenic variation of at least one virulence factor, VESA1, using a segmental gene conversion type mechanism. Rad51 is a canonical DNA repair protein that plays a major role in gene conversion mediated repair in most organisms. To determine whe ther Rad51 similarly plays a key role in the segmental gene conversion process associated with antigenic variation in B. bovis we created a Rad51 knockout clonal line, rad51 ko1 using a double crossover gene replacement strategy. The success of gene repla cement was confirmed by PCR and Southern blotting, and the lack of rad51 transcripts by RT PCR. Preliminary experiments with B. bovis rad51 ko1 revealed several unexpected results. First, no apparent defects in parasite growth rate or morphology were observ ed following recovery from transformation with the plasmid used for targeted gene disruption, despite the significance of Rad51 to DNA replication. Perhaps more significantly, there was no difference in the survival rate of B. bovis rad51 ko1 compared with the CE11 parental line after exposure to DNA irradiation. Finally, the absence of Rad51 appears to have a stabilizing effect on the locus of active ves transcription (LAT), which encodes the VESA1 protein. This result raises the possibility that in wild type parasites Rad51 may serve to target actively transcribed ves genes for dsDNA breaks, focusing antigenic variation associated gene conversion mutational events on this family. 67 Genome wide effect of the mop1 1 mutation on chromatin structure in maize Madzima TF 1 Fincher JA 1 Vera DL 1 Dorweiler JE 2 Bass HW 1 Dennis JH 1 McGinnis KM 1 1 Department of Biological Science, Florida State University, Tallahassee, FL 2 Department of Biological Sciences, Marquette Uni versity, Milwaukee, WI The mop1 gene encodes a putative RNA dependent RNA polymerase required for several examples of epigenetic regulation of endogenous genes and transgenes in maize. Its orthology to the Arabidopsis RDR2 and pleiotropic mutant phenotypes suggest that Mop1 influences chromatin structure via an RNA dependent silencing pathway and plays an important role in maize development. Nucleosome occupancy and higher order chromatin structure have been characteri zed using micrococcal nuclease (MNase) protection and sensitivity assays. We developed two types of Nimblegen microarray based assays to characterize chromatin structure responses in maize. One assay (nucleosome occupancy) produces data on promoter/TSS arc hitecture. The other assay (nuclease sensitivity) produces genome wide data on global chromatin accessibility. Here we describe new fi ndings from our chromatin acce ssibility assay used to characterize the global changes in chromatin structure in response t o the mop1 1 mutation. Using the mop1 1 mutation segregating in a B73 genetic background we observed two large trends in genomic response, an increase in chromatin accessibility in gene rich areas, and an intriguing decrease in accessibility around centrom eres, which are enriched in LTR retroelements. These global regulation of chromatin structure. This work demonstrates the utility of genome wide, microarray based nuclease sensitivity assays to deter mine changes in chromatin structure. 68. Foxa1 and Foxa2 are required for intervertebral disc formation Maier JA Lo YT, Harfe BD* Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL
37 The intervertebral disk (IVD) is composed of the collagenous annulus fibrosus, which surrounds an inner, gel like nucleus pulposus (NP). The NP is derived from the notochord. Disc degeneration results in back pain, for which effective treatments are limited. Uncovering the mechanisms o f IVD development and degeneration could lead to improved treatments for back pain. The forkhead box ( Fox ) genes are expressed in the early embryo and function in development and post natal life. Both Foxa1 and Foxa2 are expressed in the notochord, but due to embryonic lethality of the Foxa2 null mouse, their role in IVD formation has not been characterized. We removed Foxa2 from the notochord using a conditional Foxa2 allele under control of the ShhcreER T2 allele, allowing us to inactivate Foxa2 where Soni c hedgehog was expressed. This mouse was combined with the Foxa1 null allele to make double knockouts. Histology and fate mapping with the Rosa26 reporter allele were also done. Mice null for Foxa1 and lacking Foxa2 in Shh expressing cells have a severely deformed NP and a shortened tail. Fate mapping in these mice suggests defects in the notochord to NP transition in Foxa1; Foxa2 knockout mice, cell death studies indicate cells of the posterior midline and somites are dying. Hedgehog signaling is decreased in the double mutants, and the neural tube is abnormally patterned. Study of the role of Foxa family action in IVD development may provide insight into new treatments for disk degeneration. 6 9 Structure function analysis of the RGH3 splicing factor in m aize Martin F Fouquet R, Fajardo D, Gault C, Settles AM Horticultural Sciences Department, University of Florida, Gainesville, FL Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Alternative RNA splicing produces multiple mRNA species from individual genes increasing protein diversity and regulating gene expression. About 42% of intron containing genes in plants are alternatively spliced, yet little is known about the biological fu nction of alternative splicing. The rough endosperm3 (rgh3) mutant causes lethal seed and see dling developmental defects and encodes a U2AF35 related protein (ZmURP). U2AF35 identifies splice acceptor sites during RNA processing by interacting with U2AF65 and other SR proteins. Rgh3 is alternatively spliced, producing at least 19 different isoforms. Only one variant is predicted to encode a full length URP ortholog containing an N terminal acidic domain followed by two zinc fingers flanking a UHM domain and a C terminal RS like domain. Several Rgh3 splice variants produce tru ncated proteins missing one to several domains. GFP fused to full length RGH3 localized to the nucleolus and nuclear speckles. Functional analysis with endogenous truncated protein variants and artificial domain deletions fused to GFP showed that the acidic domain contains a nuclear loc alization signal and the RS like domain is important for its localization to speckles. These results suggest Rgh3 is regulated by splicing to produce truncated variants that render the protein unstable and excluded from spliceosomal speckles. 70 Differe n tial regulation of Salmonella t yphimurium genes involved in O antigen capsule production and their role in persistence within tomatoes Marvas i M Noel JT, Teplitski M* Soil and Water Science Department, University of Florida, Gainesville, FL Enteric pathogens including non typhoidal Salmonella and enterovirulent E. coli are capable of persisting within plants and multiplying within fruit tissues consumed by their animal hosts. Yet, little is still known about the mechanisms that Salmonella use s to colonize and persist within plants including crops important to humans. This study identified the Salmonella yihT gene (involved in synthesis of the O antigen capsule) as involved in persistence in immature tomatoes. Deletion of yihT reduced competiti ve fitness of S. enterica sv Typhimurium by over 3 logs. The yihT RIVET reporter was strongly resolved in immature tomatoes. Expression of yihT in mature tomatoes (regardless of their color) was low, and yihT did not affect competitive fitness within matur e fruit. Fitness tests within tomato rin, Nr mutants (defective in synthesis and perception, respectively, of the plant hormone ethylene essential in ripening and defense responses) suggest a role for functional ethylene mediated signaling in the persisten ce of Salmonella within tomato fruit. 71 Is mutation rate fitness dependent? Fitness decay and mutation rate in muta tion accumulation lines of the n ematode worm Caenorhabditis elegans Matsuba C 1 Salomon MP 1,2 Ostrow D 1 ,3 Sylvestre L 1 Ungvari Martin J 1 Tabman B 1 Lewis S 1 Baer CF 1, 1 Department of Biology, University of Florida, Gainesville, FL 2 Section of Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, CA 3 Mol Hospital Los Angeles, Los Angeles, CA Disparate lines of evidence suggest that physiological condition may affect mutation rate, which in turn suggests that the mutation rate may depend on the underlying
38 fitness of the organism. To investigate that possibility we chose five high fitness and fi ve low fi rst order (1 MA) lines that had previously generations subsequent to diver gence from a common ancestor. Forty eight second order MA (2 MA) lines were initiated from each 1 MA progenitor and additional 150 generations. On average, the rate of decay of fitness of the 2 MA lines did not differ between high fitness and low fitness lines, but se veral non significant trends are consistent with low fitness lines having greater mutation rate. Re sequencing of the genomes of 2 4 2 MA lines and their 1 MA progenitors revealed that the base substitution mutation rate does not differ between high and low fitness groups, but mutations in the mtDNA genome may partially explain the observed fitness patterns. 72 Assessment of atenolol induced HDL change in the Pharmacogenomic E valuation of Ant ihypertensive Responses (PEAR) s tudy McDonough CW 1, 2 Gillis NK 1, 2 Alsultan A 1, 2 Bu ford TW 3 Chang SW 1,2 Lang JE 4 Shahin MHA 1, 2 Suzuki Kawaguchi M 1, 2 Gong Y 1, 2, *, Langaee TY 1, 2, *, Gums JG 5 Chapman AB 6 Turner ST 7 Coop er DeHoff RM 1,2,8 Johnson JA 1,2,8, 1 Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville, FL 2 Center for Pharmacogenomics, University of Florida, Gainesville, FL 3 Department of Aging and Geriatric Research, Universi ty of Florida, Gainesville, FL 4 Division of Pulmonary and Sleep Medicine, Nemours Ch 5 Department of Community Health and Family Medicine, Universi ty of Florida, Gainesville, FL 6 Renal Medicine Division, Department of Medicine, Emory University, Atlanta, GA 7 Division of Nephrology and Hyper tensi on, Mayo Clinic, Rochester, MN 8 Division of Cardiovascular Medicine, Department of Medicine, University of Florida, Gainesville, FL We sought to identify novel pharmacogenomic markers for HDL response to atenolol in patients with mild to moderate hypertension. We genotyped 768 hypertensive patients from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) study on the Il lumina Human CVD Beadchip. During PEAR, patients were randomized to receive atenolol or hydrochlorothiazide first as monotherapy and then as combination therapy. Blood pressure and additional laboratory measures, such as HDL, were evaluated at baseline, af ter monotherapy, and after combination therapy. This analysis focused on patients treated with atenolol during monotherapy. Association with atenolol induced HDL change was evaluated in 232 Caucasian patients and 152 African American patients using linear regression. Top regions were selected as those with a P value < 10 3 in one race, and a nominal signal (P value < 0.05) in the gene region in the other race. We identified 26 regions that replicated across races. Most interesting were seven gene regions wi th prior associations with HDL or other metabolic traits, or functional implications in the lipid pathway: GALNT2 STARD3NL ABCB1 LEPR FTO LRP5 LIPC and ESR1 In conclusion, we identified multiple gene regions associated with atenolol induced HDL cha nge that replicated across race groups, several with functional implications or prior associations with HDL. 73. A dual transgene system as an approach to study DNA methylati on and silencing mechanisms in m aize McGivern J 1 Irsigler A 2 Madzima T F 1 Stroud L 1 McGinnis K M 1 1 Department of Biological Science, Florida State University, Tallahassee, FL 2 Embrapa Recursos Genticos e Biotecnologia, Parque Estao Biolgica, Braslia, Brasil In maize and other organisms, transcriptional gene silencing acts to prevent gene expression. This epigenetic pathway can be mediated by siRNAs acting in association with directed DNA methylation. Transgenes have also been demonstrated to be regulated by these pathways. We are developing a two transgene system to study these pathways. The first transgene encodes a reporter gene whose expression results in a distinct purple phenotype. The second transgene contains a DNA methyltransferase fused to protein sequence which binds to the first transgene. When the two transgenes are present and expressed, the fusion protein binds to the reporter construct, allowing methylation and silencing of adjacent promoter regions. Independent transgenic lines were created for each of these constructs, and plants containing the transgenes we re crossed to create segregating populations of plants with either one or both transgenes. In a segregating population, plants with both transgenes exhibit hypermethylation in the promoter of the reporter gene and reduced gene expression. These results are consistent with what we would expect given successful tethering. Ho wever there is evidence of DNA methylation of the selectable marker cassette that each transgene encodes, silencing by small RNAs or chromosome pairing. Additional experiments are being carried out to demonstrate whether methyl ation is occurring by tethering dependent or independent mechanisms.
39 74 Y chromosome library construction for next generation s equencing McNulty S 1 Miro Herrans A 2, 3 Mulligan CJ 2 1 Biotechnology Track, Biology Major, College of Agriculture 2 Department of Anthropology, University of Florida, Gainesville, FL 3 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL The Y chromosome is particularly useful for p opulation history inferences through the investigation of haplogroup diversity in males. These haplogroups are defined by single nucleotide polymorphisms (SNPs). The cost of high throughput sequencing of the entire Y chromosome of multiple individuals for population analyses renders it prohibitive for SNP i nvestigation. Selectively enriching for areas of the Y chromosome that provide the most useful information, via library construction, can make next generation sequencing cost effective. We are creating a protocol in which we enrich for Y chromosome fragments containing lineage defining SNPs through hybridization with PCR products of selected haplogroups. Two thousand base pair fragments were generated from these PCRs to serve as probes to capture complemen tary sequences on the Y chromosome. The resulting PCR products were sheared to increase sequence diversity and coverage during hybridization. Individual samples were tagged with identifying sequences, allowing multiple samples to be pooled and sequenced on a single lane of the Illumina GAIIx after capture on the custom probes. Sixty three primer pairs were designed to produce probes containing over 150 SNPs. SNPs were chosen that define major haplogroups, as well as more divergent subgroups of E and J haplo groups, which are of particular interest in our samples. Ninety five samples were uniquely tagged and pooled for library construction. Approximately 1.824 billion bases will map to the Y chromosome, allowing for haplogroup determinations of all 95 samples. This method provides an unprecedented amount of sequence data in order to address questions that have been intractable. 75 Investigating the alternative splicing landscape in maize analysis from B73/Mo17 hybrids and B73 x Mo17 recombinant inbred line (IBM RIL) population Mei W 1 Liu S 2 ,3 Yeh C Y 2 ,3 Li X 2 ,3 Springer NM 4,5 Schnable PS 2 ,3 Barbazuk WB 1, 1 Department of Biology, University of Florida, Gainesville, FL 2 Center for Plant Genomics Iowa State University, Ames IA 3 Department of Agronomy Iowa State University, Ames IA 4 Microbial and Plant Genomics Institute, University of Minnes ota, Saint Paul, MN 5 Department of Plant Biology, University of Minnesota, Saint Paul, MN Alternative s plicing (AS) produces multiple isoforms from a single pre mRNA through selective use of splice sites. AS can influence protein diversity or affect protein levels by regulating message processing. AS is known to play roles during plant development, stress response, and flowering, but the extent of AS in plants is not well understood. Maize the major crop plant in the USA, lack s information about genome wise alternative splicing. We are examining RNA Seq data to investigate tissue specific AS within endosperm and embryo in B73 and Mo17 maize, identify parent of ori gin alternative splicing events or isoforms, cis and trans regulation in the hybrids and sQTL in the IBM RIL population. The results identified here will help us to further understand the cross talk between endosperm and embryo during seed develo pment an d embryogenesis, the r egulation of alternative splicing in the hybrids and IBM RIL population. By comparing AS between B73 and Mo17 we can test for the existence of genotype specific AS. Currently we are developing a new computational approach to detect pa rent of origin effects in B73/Mo17 hybrids, cis and trans regulation in the IBM RIL to identify genome wide patterns of regulation of isoform abundance and alternative splicing. Our analysis centers on huge amount of RNA Seq read alignment, allele specifi c splice site detection, alternate isoform detection and identification of allelic gene structures and isoforms. Our current results have identified differences in transcript isoform abundance between embryo and endosperm as well as some evidence for genot ype specific AS and parent of origin AS. 76 HD ZIP III lost the HD: e volution of a transcription factor regulator Miles NW 1 Soltis D E 1,2 Soltis P S 2 1 Department of Biology, University of Florida, Gainesville, FL 2 Laboratory of Molecular Systematics and Evolutionary Genetics, Florida Museum of Natural History, University of Florida, Gainesville, FL LITTLE ZIPPER (ZPR) is a leucine zipper gene that regulates the activity and expression of Class 3 Homeodomain Leucine Zipper (HD ZIP III) genes.This interaction is well studied in Arabidopsis and is known to affect the polarity of lateral organs. However, little is known about its evolution in plants. To get a phylogenetic history of these genes, we sampled sequences from ferns and gymnosperms from the 1KP Transcriptome Project. The genome of the basal angiosperm, Amborella was also sample d to understand the evolution in a ngiosperms and
40 intron/exon evolution. We have found that ZPR evolved fro m a paralog of HD ZIP III in g ymnosperms by truncation of the HD ZIP III gene down to just the leucine zipper region. This means that ferns do not have ZPR genes whic h might have implications for interpreting the evolution of lateral organ development in vascular plants. 77 High throughput sequencing of pre historic Tano s amples Miro Herrans A T 1,2 Wang A 3 Mulligan C J 2 1 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2 Department of Anthropology, University of Florida, Gainesville, FL 3 Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY DNA allows us to reconstruct the evolutionary history of pop ulations. A challenge in inferring evolutionary histories is identifying, from the current genetic variation, the different processes that have shaped the genetic variation of a population. Ancient DNA (aDNA) allows the unique opportunity to reconstruct an event from the genetic variation present at the moment the event occurs. Until recently, it was unfeasible to accurately infer ancient events because of the cost and time required to generate a large aDNA dataset. Next generation sequencing platforms now make it possible to generate large amounts of aDNA data. We test the use of an indexed whole mitochondrial DNA (mtDNA) enriched library to generate sequence data for a pre historic Native American population of Puerto Rico. Twenty seven Tano samples from the Punta Candelero archaeological site were simultaneously sequenced for whole mtDNA genomes, on one lane of the Illumina GAIIx, using an indexing and library enrichment approach. Sequence reads were sorted into samples according to the index identifier, trimmed to remove the index and adapters, and mapped to the NC012920.1 reference whole mitochondrial genome. Sequence coverage was highly variable among samples (725bp to 10,358bp) with mostly 1x coverage for each read. Sequences showed very low variabili ty with only 21 variant sites among all samples, of which at least 4 sites are artifacts of C to T deamination conversions. These results show that indexed library approaches are useful for aDNA data generation, but will require further improvement to addr ess the particular challenges of aDNA, such as DNA degradation and low quantities of DNA. 78 Dualistic function of Daxx at centromeric and pericentromeric heterochromatin in normal and stress conditions Morozov VM 1 Gavrilova EV 1 3 Ishov AM 1 1 Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 2 Faculty of Biology and Soil Science, St. Petersburg State University, St. Petersburg, Russia 3 Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia Heterochromatin architecture is essential for the proper orchestration of nuclear processes, while transcription from this part of the genome is required for its own maintenance. Here we present the first evidence that depletion of protein Daxx affects tra nscription of human heterochromatin, reducing accumulation of centromeric (CEN) RNA in normal conditions and pericentromeric (periCEN) RNA after heat shock (HS) application. Searching for the mechanism of Daxx dependent regulation of heterochromatin transc ription, we found that depletion of Daxx decreases incorporation of transcription associated histone H3 variant, H3.3, into both CEN and periCEN. In normal conditions, Daxx is mostly accumulated at ND10/PML nuclear bodies, with minor association with CEN/p eriCEN in subpopulation of cells. HS changes this balance forcing very robust accumulation of Daxx on CEN/periCEN. Surprisingly, this transient redistribution of Daxx does not further elevate the incorporation of H3.3 that remained steady during HS and rec overy. Instead, depletion of Daxx leads to HS induced changes in the balance of epigenetic modifications at heterochromatin, most dramatically elevating levels of H3K4Me2 at periCEN. We propose dualistic function of Daxx containing complexes at CEN/periCEN : 1) regulation of H3.3 loading in normal conditions, and 2) protection of epigenetic status upon stress application, thus collectively guarding epigenetic identity of heterochromatin and genome integrity. 79 Biocomputing resources at UF Research Computi ng Moskalenko O 1 Gitzendanner MA 2, 1 Research Computing University of Florida, Gainesville, FL 2 Department of Biology, University of Florida, Gainesville, FL The University of Florida High Performance Computing Center, part of UF Research Computing, provides comprehensive computing infrastructure and suppo rt including an instance of Galaxy to UF biological researchers. Galaxy is a web based platform for accessible and reproduci ble biologi cal computing. We will provide an overview of Galaxy; the modules system for easy command l ine software environment set up; s oftware for biological research; and support, consulting and training services.
41 80 Carbohydrate availability modifies gene expression and virulence t raits in Streptococcus mutans Moye Z Zeng L Burne RA Department of Oral Biology, University of Florida, Gainesville, FL Human oral biofilms are usually limited for carbohydrates during fasting periods of the host, but lar ge quantities of sugars can be intermittently introduced via the diet. Here, we investigated the phenotypic and transcriptional responses of Streptococcus mutans the primary pathogen in dental caries, to carbohydrate availability. Steady state planktonic populations of S. mutans UA159 were generated using continuous chemostat culture in conditions of excess (100 mM) or limiting (10 mM) glucose; with pH, temperature and growth rate held constant. Cells growing under glucose limitation were found to be able to acidify the environment more rapidly and to a greater extent than cells growing with excess glucose. Further, transport of glucose, fructose or mannose via t he phosphoenolpyruvate: sugar phosphotransferase system (PTS) was dramatically more active in glu cose limited cells. Microarrays revealed that 123 genes were differentially expressed in cells grown in the presence of limiting versus excess carbohydrate (p < 0.001). Glucose limited cells upregulated genes involved in energy metabolism and carbohydrate transport, as well as those encoding two component systems. Cells grown in excess glucose showed elevated levels of transcripts associated with carbohydrate storage and biosynthesis of certain amino acids. Thus, carbohydrate availability dramatically influ ences physiological and biochemical pathways that contribute directly to the virulence of S. mutans 81 The re discovery of non additive effects with g enomic relationship matrices and its implication in b reeding Munoz P 1 Resende M 2 Gezan S 3 Resende M 4 Kirst M 1, 3, *, Dudley H 3 Peter G F 1,3, 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 3 School of Forest Resources and Conservation, University of Florida, Gainesville, FL 4 Department of Forest Engineering, Federal University of Vicosa, Vicosa, Brazil Non additive effects have been highly overlooked in breeding, mainly because traditional methods for variance estimation y ield usually small if ever significant parameters compared to the additive. This has cause that many breeding programs make progress using only breeding values. We compare estimation, partition and prediction of different linear mixed models under addi tive and full (additive plus non additive) assumptions with the use of either the pedigree (BLUP) or marker derived relationship matrices (GBLUP) in a clonal population of the tree species Pinus taeda Our results show that additive REML/GBLUP increased the precision of the heritability estimate compared to additive REML/BLUP. We studied the partition of the components and found that the marker based models were able to partition the estimations more efficiently. Finally, we evaluated all models in a cros s validation and found a 36 and 30% higher predictive ability of the marker based models that include additive, dominance and two way epistasis, when compared to the traditional GBLUP and to the additive BLUP models. We conclude that the use of relationshi p matrices derived from markers in a model including additive and non additive effects had the best performance not only to partition the genetic variances but to improve considerably the breeding value prediction ability in trend, magnitude and top indivi dual selection. 82. Regulation by KaeR mediated through interaction with flavonoids Murdoch C Chen A Pagliai F, Lorca GL* Department of Microbiology and Cell Science, University of Florida, Gainesville, FL The focus of this study will be LVIS1989 (Ka eR), a LysR family transcriptional regulator identified in Lactobacillus brevis Through previous work, it was discovered that interaction with flavonoids (kaempferol and myricetin) stabilizes the regulator's binding to its cognate DNA inducing transcripti on. Two binding sites were identified through DNase I footprinting, one at the intergenic region between LVIS 1989 and LVIS 1988 and the other within LVIS1988. The aim of this work will be to determine critical residues responsible for KaeR's ability to re spond to the presence of flavonoids. Using structure guided, site directed mutagenesis the following residues will be altered: glutamic acid at position 127, proline at position 201, and serine at position 203. These residues will be changed to aspartic ac id, alanine, and alanine respectively. It is hypothesized that these mutations will result in a lack of response of KaeR to the ligand. In vivo and in vitro approaches will be utilized to compare the wild type protein and its mutant versions on ligand bind ing. It is also possible that mutations on KaeR may result in lower affinity of the protein to DNA or changes in the oligomeric state of the transcriptional regulator. To test these effects, electrophoretic mobility shift assay (EMSA) and size exclusion ch romatography experiments will be performed
42 with the purified mutant proteins. Ligand binding will be tested in EMSAs by varying concentrations of kaempherol. In vivo lacZ fusions to the KaeR regulated promoter will be used to test the effect of kaempferol and myricetin on activation of KaeR. 83 Genetic diversity of w o rld collection of sugarcane and its related g rasses Nayak SN 1 Villa A 1 Pathak B 1 Todd J 2 Ayala Silva T 3 Glaz B 2 Wang J 1, 1 Agronomy Department, University of Florida, Gainesville, FL 2 Sugarcane Field Station, Agricultural Research Service, U.S. Department of Agriculture, Canal Point, FL 3 Subtropical Horticulture Research Stat ion, Agricultural Research Service, U.S. Department of Agriculture, Miami, FL Sugarcane ( Saccharum spp.) cultivar improvement programs have not yet systematically utilized most of the genetic sources of yield potential and resistance to biotic and abiotic stresses that may exist in the Saccharum germplasm. Two collections of genetic material potentiall y useful to sugarcane breeding programs, which are maintained in India and USA, are known collectively as the objectives of this study are 1) to genotypically evaluate the World Collection of Sugarca ne and Related Grasses maintained at the National Plant Germplasm System in Miami, FL, and 2) to select representative accessions in the collection that sugarcane breeders can utilize as a core breeding collection. In total, 1 002 accessions in the world g ermplasm collection, comprising 16 species, w ere sampled for genotypic eval uation using microsatellite markers. Initial screening of 192 microsatellite markers on eight species of sugarcane accessions yielded 76 polymorphic markers with polymorphic informa tion content ranging from 0.22 to 0.89. Of these, 26 polymorphic markers were used to genotype the 1 002 accessions yielding around 200 alleles. Genotyping data was used for diversity analysis of the 1 002 accessions. Meanwhile, these accessions will be ph enotypically evaluated by taking the observations on several plant growth parameters. Based on diversity parameters of the cluster and phylogenetic analysis, the sugarcane core collection will be formed, which will be useful in sugarcane breeding programs after thorough evaluation and later for genetic studies such as association mapping and genomic selection. 84 KBAS: a s oft ware tool for knowledge based association s tudies Nazarian A 1 Riva A 2 1 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Genome wide association studies (GWAS) have revolutionized the genotype phenotype analysis of complex traits in which multiple genetic and non genetic factors act jointly to form a phenotype. However, despite the successes of GWAS in detecting a number of previously unknown susceptibility loci, a remarkable amount of missing heritability still exists for most of the studied traits, in par t because GWAS are unable to reliably detect small contributions of individual genetic risk factors, and to capture the interactions existing among genetic factors. We have developed a framework for analyzing the complexity underlying the genetic architect ure of complex diseases, based on a hypothesis based method which integrates hypotheses about the genetic basis of a complex phenotype, generated from pre existing biological knowledge, with a genetic algorithm based search engine (GA) which generates and optimizes multi SNP models associated with the trait under investigation. We implemented the method in the KBAS sof tware package, a command line 64 bit executable. The program provides users with an interface to generate sets of SNP s related to the hypothe ses of interest, and to test their association with the phenotype through the GA engine. Finally, the best multi SNP model generated by the GA engine is written to a fully annotated output file that also contains the GA parameters and the results of the mo del validation. KBAS is freely available at: http://genome.ufl.edu/rivalab/kbas 85. Characterization of an n type ATP pyrophosphatase required for thiolation of tRNA in the haloarchaeon, Haloferax volcan ii Nembhard NE 1 Englert M 2 Elbanna D 1 Holman M 1 Sll D 2 Maupin Furlow JA 1, 1 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 2 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT Two small archaeal modifier proteins (SAMP1and SAMP2) and their activating enzyme, UbaA, were recently described in Haloferax volcanii SAMP 1 participates in molybdenum cofactor biosynthesis and SAMP2 is important for thiolation of tRNAs. It is apparent f rom our previous work that SAMPs function in the mobilization of sulfur for the biosynthesis of sulfur containing molecules. However, the enzymes mediating the thiolation of tRNA remain unclear. In this study, we generate an in frame markerless deletion in H. volcanii of an Urm1 associated tRNA thiolation protein Ncs6p homolog belonging to the n type
43 ATP pyrophosphatase family. The n type ATP pyrophosphastase family is characterized in adenylation of tRNA leading to acceptance of activated sulfur in biomole cule synthesis. Mutant strain analysis revealed the requirement of the n type ATP pyrophosphatase (HVO_0580) in H. volcanii for 2 thiouridine synthesis and the deletion strain exhibited high temperature sensitivity similar to other n type ATP pyrophosphata ses which are required for thiolation of tRNA at high temperatures. 86 Whole genome genotyping of gene copy number variation in forest trees Neves LG 1 Davis JM 1,2 *, Barbazuk WB 1,3 *, Kirst M 1,2 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 School of Forest Resources and Conservation, University of Florida, Gainesville, FL 3 Department of Biology, University of Florida, Gainesville, FL Trees present a remarkable adaptation to a variety of environments. The diversity in their genome might explain their broad plasticity. For instance, most flowering plants have undergone one or more event s of whole genome duplication. As a consequence, in a population of plants from the same species, genes may be present in different copies in the genome, referred to as copy number variation (CNV). What is not known, however, is the extent to which CNV is responsible for changing the phenotype of the plant, particularly for complex traits. We are currently addressing this topic by developing a high throughput, genome wide, genotyping pipeline for CNV based on targeted resequencing and, subsequently, genotyping a large population of 579 unrelated trees of Populus deltoides For the genotyping, targeted genes are captured fro m the genome using complementary pro bes and sequenced using next generation sequencing. The resequencing data is being analyzed in a mixed model to identify genes for which some individuals are consistently sequenced with higher or lower depth, chara cteriz ed by both CNV and presence /absence variation. Data from several species are being analyzed, namely, Pinus taeda Pinus elliottii Populus t ric h ocarpa and Populus deltoides with 24, 24, 48 and 10 individuals, respectively. The optimized model will be used to genotype the P. deltoides population containing 579 individuals, with the objective of associating population level CNV with phenotypic variation. 87 Comparison of SIV in vitro recombination rates using traditional clonal sequen cing and single genome s equencing Nolan DJ 1 ,2 Watson AR 1 ,2 Norstrom MM 3,4 Strickland SL 1 ,2 Lamers SL 5 Salemi M M 1 2, 1 Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 2 Emerging Pathogens Institute, University of Florida, Gainesville, FL 3 Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden 4 Center for HIV Research, Department of Laboratory Medicine, Karolinska Institu t et, Stockholm, Sweden 5 Bio Info Experts, Thibodaux, LA Simian immunodeficiency virus (SIV), like human immunodeficiency v irus (HIV), can interchange portions of genetic sequence between viral genomes to generate recombinant progeny in vivo Recombination rates are commonly calculated t o assist in the interpretation of viral evolution, but sequences obtained by traditional cloning could be skewed by PCR mediated recombination, where Taq polymerase can switch templates during cycles creating in vitro recombinants. The potential for in vit ro recombination can be reduced with single genome sequencing (SGS) using limiting dilution nested PCR. By diluting cDNA until there is just a single copy to amplify during SGS, the in vitro recombination effect is minimized due to sequence similarity of every amplified product. Using cDNA generated from SIV isolated from 4 plasma samples, we performe d triplicate cloning and SGS e xperiments to produce six alignments of ~22 env gp120 sequences for each of the cDNA samples. The resulting 24 alignments were analyzed with SplitsTree, an application that uses a split decomposition network and a powerful statistical test to identify likely recombinants within the population. After doing chi square a nalysis, no statistically significant difference was found between the numbers of recombinants in the cloning and SGS experiments. Given our experimental results, we concluded that PCR meditated recombination occurs at a rate not high enough to be signific ant against the background rate of in vivo recombination. 88 Bioinformatics, data management, and general information needs of clinical and translational science researchers Norton HF Garcia Milian R Tennant MR*, Lyon JA, Botero C E Health Science Center Library University of Florida, Gainesville, FL The UF Health Science Center Library (HSCL) strives to researchers, including those affiliated with the Clinical and Translation al Science Insti tute (CTSI). In the fall of 2011 we began a comprehensive assessment of CTSI online assessments, focused discussions and one on one
44 interviews, and concentrated on bioinformatics, data management, a nd general information needs. Preliminary analysis of the bioinformatics results suggests that researchers are interested in training on both the free bioinformatics tools available through the National Center for Biotechnology Information and commercial t ools. Data suggest researchers tend to use the resources with which they are familiar, and tend not to explore alternatives to those resources. From the data management assessment, we learned that the resources most needed for effective management and anal ysis of research data include computing expertise, storage capacity, organizational systems, and training on best practices in data management. The general information needs assessment addresses areas of interest including assessing research impact, facili tating research collaboration, meeting the NIH Public Access requirements, and various activities related to community engagement. We will present results from these assessments and indicate future directions for library services to support CTSI researcher 89 Hormonal regulation of chondricthyan fin development: implications for the evolution of copulatory organs O'Shaughnessy KL 1,2 Dahn RD 3 Cohn MJ 2,4,5, 1 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 3 Countrywood Lane, Madison, W I 4 Howard Hughes Medical Institute, University of Florida, Gainesville, FL 5 Department of Biology, University of Florida, Gainesville, FL The most primitive vertebrate copulatory organs are penis like extensions of the posterior pelvic fins, known as claspers. Claspers are found in the fossil record extending back to the arthrodires, jawed fishes that predate the origin of sharks by 25 mil lion years. Today only male members of the class Chondricythes develop claspers, and this represents an interesting sexual dimorphism of the fins that suggests a potential role for sex steroids during fin development. We reported previously that Sonic Hedg ehog (Shh), Hoxd12, and Hoxd13 expression persists in the developing clasper of male fins after downregulation in the rest of the fin. This study seeks to determine the potential role of sex hormones in clasper development by examining the distribution of androgen receptor, as well as sexually dimorphic patterns of developmental genes in chondricthyan embryo s. Immunohistochemistry and in situ hybridization results reveal the presence of androgen receptors within the developing pelvic fin buds, suggesting th eir competence to respond to androgens. We find that the apical ectodermal ridge (AER) persists in the posterior region of male pelvic fins, suggesting that clasper formation is achieved by prolongation of Shh Fgf8 feedback loop. These results suggest that primitive copulatory organs evolved by hormonal regulation of the fin development program in early jawed vertebrates. 90 Gene identification in Pseudomonas aeruginosa : from bioinformatics to experimental analysis Oden S 1 Zhang Y 1 Jin S 1, *, Tornaletti S 2 Brocchieri L 1, 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL Gene coding regions generally exhibit contrasting gl obal compositional properties in the three codon positions, depending on the overall base composition of the sequence. General rules of the base content at the three codon positions as a function of the overall base content of the sequence can be identifie d and exploited to score sequence regions for their coding potential. We developed procedures to predict and quantify coding regions in prokaryotic sequences. These procedures augment the visual approach of frame analysis for detecting significant contrast s in nucleotide usage among codon positions. The procedures are implemented in the web based tool N PACT (N Profile Analysis Computational Tool) available at http://genome.ufl.edu/npact/ N PACT produces gene pr edictions and characterizations, and a graphical output of predictions and sequence compositional features. We applied N PACT to the genome of Pseudomonas aeruginosa PAO1 and other P. aeruginosa strains, identifying several potential new genes. Many of these new predictions consist of short sequence length and are conserved in different bacterial species. We compared our computational predictions with experimental evidence of transcription obtained by RNA seq analysis. RNA seq results revealed that many of the predicted genes were undergoing transcription, furthering the validity of their prediction as protein coding. 91 A dual role of TstR transcriptional regulator provides insights into cyanide detoxification in Lactobacillus brevis Pagliai FA M urdoch CC, Nath R, and Lorca GL Department of Microbiology and Cell Science, University of Florida, Gainesville, FL In this study, a dual activity for a member of the MarR family of transcriptional regulators was discovered in Lactobacillus brevis ATCC 367. TstR modulates the expression and activity of the downstream encoded thiosulfate:cyanide sulfurtransferase (TstT). DNase I footprint identified the TstR binding site in a region of 34
45 nucleotides ( 1 to +33, from tstR transcriptional start site) EMSA assays revealed that while sulfite, an end product of the reaction catalyzed by TstT, improved the interaction between TstR: PtstR iron impaired this interaction. A structural model of TstR was used to identify putative ligand binding sites. Site di rected mutagenesis identified M64 as a key residue in the sulfite recognition, while a pocket comprising the residues H136 H139 C167 M171 was the site coordinating iron. Besides its role as a transcriptional repressor, TstR played a role in the thiosulfate :cyanide sulfurtransferase activity of TstT. Using a two hybrid system, we demonstrated that TstT and TstR undergo a physical interaction promoted by iron. Enzymatic assays proved that this novel interaction increased the activity of TstT by 3 fold, compar able to domain sulfurtransferases. The protein protein interaction between TstR and TstT described herein represents a novel mechanism of regulation of enzymatic activity performed by a transcriptional regulator. 92 Bulk s egregant a nalysis generates markers linked to a virulen ce gene in fungal p athogen Pendleton AL 1 Smith KE 2 Nelson C D 2 Davis JM 1,3, 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 Southern Institute of Forest Genetics, Southern Research Station, U.S. Forest Service, Saucier, MS 3 School of Forest Resources and Conservation, University of Florida, Gainesville, FL Cronartium quercuum f.sp. fusiforme (Cqf), the fungus that incites fusiform rust disease of oak and southern pine, produces galls on stems and branches of southern pines. Gene for gene interactions between the Pinus taeda resistance gene Fr1, and the corresponding pathogen avirulence gene A vr1 have been documented. Obtaining markers for avirulence loci would allow pathogen populations to be surveyed where plantations were to be established, enabling growers to plant trees with corresponding resistance genes that ensure the stand is resistant to rust. We have coupled the power of bulk segregant analysis (BSA) with next generation sequencing to identify markers that segregate with avirulence to Fr1. Spores collected from resistant (Fr1/ ) and susceptible (fr1/fr1) hosts were bulked and sequence d using the Illumina GAIIx platform. Following alignment to the ref erence Cqf genome, single nucleotide polymorphisms (SNPs) were identified in the bulked sequencing data. Significant deviation f rom a 1:1 ratio of all SNPs was calculated to determine marke rs tightly linked to Avr1, which generated an approximately 70kb interval with candidate Avr1 genes. Syntenic analyses with a closely related rust pathogen ( Melampsora larici populina ) validated the interval generated through BSA. Subsequent bulked sequenc ing and comparative analyses will be completed to refine the interval. 93 Comparing PCR conditions that detect rAAV harboring human e rythropoie tin cDNA: applications for gene d oping Perez IC 1 Ni W 1 Le Guiner C 2 Moser D 3 Simon P 3 Moullier P 1,2 Snyder RO 1,2,4, 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 nce 3 Department of Sports Medicin e Johannes Gutenberg University, Mainz, Germany 4 Center of Excellence for Regenerative Health Biotechnology, University of Florida, Alachua, FL The development of molecular assays that help detect gene doping in sports can serve as a deterrent and can identify athletes who may be illicitly using gene t ransfer for performance enhancement. Promising technologies to detect foreign DNA with high reliability, sensitivity, and specificity include TaqMan real time, nested PCR, an d internal threshold c ontrol PCR. Previous studies conducted in cynomolgus macaque s (cm) have shown that optimized conditions for TaqMan real time PCR detected as low as three copies of foreign cm erythropoietin (EPO) in white blood cells with a false positive rate of 0% and false negative rate of 13%. In this present study, the reliabi lity, sensitivity and specificity of three different PCR assays are being investigated for the detection of a recombinant adeno associated virus (rAAV) vector harboring a pro moterless human EPO (hEPO ) complementary DNA (cDNA) administered to cynomolgus mac aques, and on samples of known copy numbers of rAAV promoterless hEPO plasmid spiked into nave human genomic DNA. The first two assays involve a one step real time PCR, each having different primer sets. The third assay involves a nested PCR approach. Pre liminary data shows that the nested PCR has the most sensitivity, detecting as low as two copies of the hEPO cDNA in human gDNA spiked with plasmid, and the presence of human na ve genomic DNA did not interfere. Future work involves injecting macaques intr amuscularly with a rAAV promoterless hEPO vector in order to investigate the longevity and biodistribution of hEPO, as well as the sensitivity and specificity of the three different assays. 94. A MarR transcriptional regulator from Candidatus shape and biofilm formation Potts AH 1 Melanson N 1,2 Lorca GL 1, 1 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL
46 2 Undergraduate Honors Program, College of Agricultural and Life Sciences, University of Florida, Gainesville, FL intracellular pathogen of citrus and the causative agent of citrus greening disease. Although this pathogen has e is known about this bacterium, and it has yet to be cultured. Our approach Ca. identify protein targets for the development of small molecule therapeutics. In order to demonstrate the biological role o f these small molecules, we will use the closely related Sinorhizobium meliloti 1021 as a surrogate. Ca. has undergone reductive evolution and thus has much less complex regulatory networks than related species, presenting a target for the development of treatments. One of these few regulators is CLIBASIA_01180, a member of the MarR family of transcriptional regulators. Ca. mobility shift assays sugges t that it may regulate the gene directly downstream, CLIBASIA_01175. This gene encodes a hypothetical protein that contains a YkuD domain that is associated with L,D transpeptidase activity as well as a PG_binding_1 peptidoglycan binding domain. This domai n structure suggests CLIBASIA_01175 is likely a transpeptidase involved in cross linking peptidoglycan. In a S. meliloti strain with a mutation in this regulator, more biofilm formation is seen, potentially because the cells are aggregating as a response t o stress. Thus, we hypothesize that disrupting the regulation of this gene may result in Ca. 95 Transcriptional r egulation of wdr 23 Prasse S Choe KP* Department of Biology, University of Florida, Gainesville, FL Tight regulation of inducible gene transcription responses is essential for basic animal function and for survival of environmental stress. In the nematode Caenorhabditis elegans the gene wdr 23 tightly represses a stress inducible transcr iption factor named SKN 1. Loss of wdr 23 activates SKN 1 and enhances stress resistance, but also impairs growth and reproduction. Interestingly, we have found that expression of the wdr 23 gene is regulated by SKN 1 in a feedback mechanism and by other u nknown factors. Chromatin immunoprecipitation assays indicate that PHA 4, a transcription factor previously known to mediate development and longevity, associates with the promoter of wdr 23. Interestingly, PHA 4 and SKN 1 both associate with the same regi on of the wdr 23 promoter suggesting that the two transcription factors might act as co regulators. This research is currently on going, and is focused on being able to define the regulatory pathway for wdr 23. 96 The molecular evolutionary rate of scombr oid fishes is predicted by the body size (but not metabolic rate) hypothesis Qiu F 1 Kitchen A 2 Burleigh JG 1, Miyamoto MM 1 1 Department of Biology, University of Florida, Gainesville, FL 2 Department of Anthropology, University of Iowa, Iowa City, IA The study of variable molecular evolutionary rates is confounded by the fact that the life history traits of groups typically co vary with each other. Interestingly, in contrast to other groups (i.e., mammals), the mass specific metabolic rate of scombroid fishes trends positively (not negatively) with body size. This study exploits this divergent pattern and its corresponding opposing predictions for scombroid rate variation to distinguish between the metabolic rate and body size hypotheses. This investigation uses a supermatrix of molecular and morphological characters to infer robust phylogenies, which are subsequently incorporated into two recent comparative methods that directly account for the phylogenetic non independence of species in their tests of trait and substitution rate. As predicted, a negative correlation is supported between body size a nd substitution rate. Conversely, a negative (not positive) association is also unexpectedly found between metabolic and substitution rates. These re lationships are consistent across all molecular data, genomes, and genes with large sample sizes. On the basis of these consistent relationships, we conclude: (i) that scombroid rate variation may be explained by the body size hypothesis; and (ii) that met abolic rate is tracking body size and not evolutionary rate. This study highlights how groups with divergent traits hold special promise for the isolation and testing of typically co varying factors. 97. Genomic selection and association mapping identify candidate genes linked to fusiform rust resistance (Fr) loci in loblolly pine Quesada T 1 Resende M 1,2 Muoz P 1,3 Gezan S 1 Kirst M 1, *, Peter GF 1, *, Davis JM 1, 1 School of Forest Resources and Conservation, University of Florida, Gainesville, FL 2 Gra duate Program in Genetics and Genomics, University of Florida, Gainesville, FL 3 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Fusiform rust is a pine disease caused by the fungus
47 Cronartium quercuum f.sp. fusiforme ( Cqf), producing galls in stems and branches. It affects survival in young trees and seedlings, generating significant economic losses to the timber industry. Studies in loblolly pine showed that resistance to fusiform rust involves major genes based on Men delian cosegregation of resistance with RAPD markers in seedlings inoculated with single spore lines of Cqf; however, no fine mapping has been achieved. We used a genomic selection approach, using Bayes C to pre select SNPs for association analyses based on their effects on breeding values for gall score in two loblolly pine tests. One test was inoculated with (Cqf) spores from a single gall and the other was inoculated with spores from a mixture of ten galls. Significant associations were detected for gal l score using BAMD software at 99% confidence. We observed nine and 13 significant SNPs for the single gall and 10 gall test, respectively, of which two appear to be linked to Fr1. They are significant at high confidence, showed high LOD scores from single marker regression analyses for parent 17, which is known to be heterozygous for the Fr1 gene, and both mapped to the same linkage group that harbors Fr1. Additionally, other significant SNPs mapped to different linkage groups, suggesting the presence of n ew fusiform rust resistance loci. 98 Functional characterization of a candidate gene for hydraulic conductivity in Populus Ribeiro CL 1 Dervinis C 2 Kirst M 1,2 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 School of Forest Resources and Conservation, University of Florida, Gainesville, FL In a previous study, moderate heritability and significant QTL for vessels per sapwood area, hydraulic vessel diameter, and specific hydraulic conductivity showed that the se physiological traits are under genetic control in a Populus hybrid population. Co localization between QTLs for hydraulic conductivity traits and growth increment further suggested a functional linkage between these traits, supporting the hypothesis that they are correlated in forest trees. To uncover potential re gulators for these traits we used a genetical genomics approach that integrated hydraulic conductivity and growth trait QTLs with gene expression measured in the poplar hybrid population. This analysis identified a previously uncharacterized gene, referred hereafter as HC1 (hydraulic conductivity 1) as the most probable gene regulator. Currently, e vidence of the functional role of this gene is being obtained by the analysis of poplar genetically modified plants, up regulated in HC1 expression levels with th e use of 35S promoter. Down regulation (RNAi) of this gene was lethal in transgenic lines. Up regulated plants grown under controlled environment have significant higher height and vessel diameter, based on analysis on a micro CT scan, in comparison to wil d type plants. Further characterization will be obtained from analysis of T DNA lines in Arabidopsis and vessel analysis under light microscope. 99 siRNA preservation in rapidly progressing a uto somal dominant r etinitis Rossmiller B Mao H, Lewin AS* Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Purpose : We are interested in characterizing and treating the rapidly degenerating autosomal dominant retinitis pigmentosa (ADRP) mouse model, T17M. Work by our lab has shown this strain to be highly susceptible to light damage (White et al., 2007, Invest Ophthalmol Vis Sci 48(5):1942 51). We hypothesize that the knockd own of transducin alpha in rods will reduce rod degenerat ion and preserve cone function. It is ther efore the purpose of this study to determine if siRNA mediated knockdown of transducin alpha can inhibit the phototransduction cascade in rod cells preventing apoptosis resulting from exposure to bright light. Methods : A cell culture assay was conducted to determine the siRNA with greatest knockdown of the transducin alpha gene (GNAT1). Two small hairpin RNAs (shRNAs) were designed to target homologous regions between mouse and dog GNAT1. Transfections were done in HEK293 cells, six replicates, using CMV GN AT1 and H1 shRNA GNAT1 given at 1:0, 1:2, 1:4, and 1:6 ratios. Using a CBA GFP control miRNA expression plasmid as carrier, the total amount of DNA transfected was constant. The amount of transducin produced was observed at 24 and 72 hours post transfectio n using western blot with a mouse transducin specific antibody. The percent knockdown was then calculated using tubulin as the loading control and normalized again st the control plasmid. Results: siRNA GNAT1(a) showed the greatest knockdown with 24 .0 %, 28. 6% and 30.9% at the 1:2, 1:4 and 1:6 ratios, respectively, after 72 hours. Conclusions : We demonstrated siRNA GNAT1(a) to be a viable siRNA for testing in T17M RHO transgenic mice. 100 Detection of primary cilia in human g lioblastoma Sarkisian MR 1 Siebzehnrubl D 1 Deleyrolle L 2 ,3 Silver DJ 2 ,3 Siebzehnrubl FA 2 ,3 Guadiana SM 1 Steindler DA 2 ,3 Reynolds BA 2 ,3 1 Department of Neuroscience, University of Florida, Gainesville, FL 2 Department of Neurosurgery, University of Florida, Gainesville, FL
48 3 Evelyn F. and William L. McKnight Brain Institute, University of Florida, Gainesville, FL Glioblastoma (GB) is the most common adult brain tumor and carries a poor prognosis due to primary and acquired resistance. While many cellular features of GB have been documented, it remains unknown if cells within these tumors possess primary cilia, a cellular organelle reported to promote cell proliferation and support survival of neural precursor cells. We used immunohistochemical and western blot analyses to scr een both hum an GB biopsies and primary cell lines for presence of cilia and cilia associated proteins. In two primary cell lines, we found that ~10 20% of cells contained acetylated alpha tubulin positive cilia. By combining immunostaining for cilia with a FUCCI cell cycle indicator, we found that cilia extended from cells in G0/1. Other key cilia genesis/function proteins, such as IFT88 and KIF3a, were detectable with in the primary cilia and withi n protein lysates generated from >20 GB biopsies. Notably, c ilia were also obser ved in several tumor biopsies (two primar y and one recurrent). The cilia were especially identifiable within and surrounding the vasculature but also in necrotic areas. Collectively, our data indicate that ciliated cells and proteins re quired for ciliogenesis are detectable in human GB. The contribution of these organelles to GB requires further investigation. 101 The influence of genetic background and natal environment on testes development Sasson D 1 Miller C W 2 1 Department of Biology, University of Florida, Gainesville, FL 2 Department of Entomology and Nematology, University of Florida, Gainesville, FL up and natal environment can play a large role in its morphological, physiological, and behavioral development. These genetic and environmental effects on development are often studied separately. However, we know that the expres sion of genes changes across environments and thus any developmental outcome genetic make up and its environment. In this study, we have explored the interaction of genes and the environment on testes development in the cactus bug, Narnia femorata We are interested in three questions: 1) nd influence testes development?; 2) do males from high and low quality environments differentially allocate resources to testes developme nt? ; 3) are there interactions between the genetic background and n atal environment such that some male lines do better in certain environments than other male lines? Preliminary results suggest that genetics affect testes development. Additionally, males from the low quality environment are smaller but allocate proportionally more resources to testes development than do males from high quality environments. This finding suggests that small males may shift resources to testes development in an effort to com pete for reproductive success with other males through sperm competition rather than through direct fights. We are currently analyzing the degree to which genes and the environment interact to influence testes development. 102 Tissue regeneration and bla stema formation in a mammal, the African spiny mouse ( Acomys ) Seifert AW 1 ,2 Kiama SG 2 Seifert MG 1 Goheen JR 3,4 Palmer TM 1 Maden M 1, 1 Department of Biology, University of Florida, Gainesville, FL 2 Department of Veterinary Anatomy and Physiology, University of Nairobi, Nairobi, Kenya 3 Department of Zoology and Physiology, University of Wyoming, Laramie, WY 4 Department of Botany, University of Wyoming, Laramie, WY Evolutionary modification has produced a spectrum of animal defense traits to escap e predation, including the ability to autotomize body parts to elude capture. Following autotomy, the missing part is either replaced through regeneration (e.g. urodeles, lizards, arthropods, crustaceans) or is permanently lost (mammals). While most auto tomy involves the loss of appendages (e.g. leg, cheliped, antennae, tail), skin autotomy can occur in certain taxa of scincid and gekkonid lizards. Here we report the first demonstration of skin autotomy in Mammalia (African spiny mice, Acomys ). Mechanical testing revealed a propensity for skin to tear under very low tension and the absence of a fracture plane. Following skin loss, rapid wound contraction was followed by hair follicle regeneration in dorsal skin wounds. Surprisingly, we found reg enerative capacity in Acomys extended to ear holes where they exhibited complete regeneration of hair follicles, sebaceous glands, dermis, and cartilage. Salamanders capable of limb regeneration form a blastema (a mass of lineage restricted progenitor cell s) following limb loss, and our findings suggest that ear tissue regeneration in Acomys may proceed through assembly of a similar structure. This study underscores the importance of investigating regenerative phenomena outside of traditional model organism s and suggests that mammals may retain a higher capacity for regeneration than previously believed. As re emergent interest in regenerative medicine seeks to isolate molecular pathways controlling tissue regeneration in mammals, Acomys may prove useful in identifying mechanisms to promote regeneration in lieu of fibrosis and scarring.
49 103 DNA s equence directs nucleosome r ed istribution in the immune r esponse to KSHV Sexton B S Avey D, Druliner B, Fincher J, Zhu F, Dennis JH Department of Biological Science, Florida State University, Tallahassee, FL In eukaryotic cells, DNA is organized into chromatin, and the fundamental subunit is the nucleosome: 150 base pairs of DNA wrapped 1.6 times around a histone octamer. The distribution of nucleosomes can re gulate access to DNA and influence transcription. Although various chromatin regulatory complexes are known to regulate nucleosome occupancy, the role of primary DNA sequence in this regulation remains unc lear, particularly in mammals. To address this pro blem, we measured nucleosome distribution at high temporal resolution in human cells at hundreds of genes during the reactivation associated herpesvirus (KSHV). We show that nucleosome redistributions are widespread and transient. To c larify the role of DNA sequence in this regulation, we compared the genes with altered nucleosome distribution to a sequence based computer model and in vitro assembled nucleosomes. We demonstrate that both the computational model and the in vitro assembl y accurately predict the nucleosome distributions at a majority of genes studied. The surprising findings that nucleosome redistributions are transient and DNA directed shifts the current perspective regarding regulation of nucleosome distribution in human s, and suggest that DNA sequence plays a more considerable role in the regulation of nucleosome positions than was previously appreciated. 104. VKORC1 Asp36Tyr geographic distribution and its impact on warfarin dose requirements in Egyptians Shahin MHA 1 ,2 Cavallari LH 3 Perera MA 4 Khalifa SI 5 Dvorak A 6 Langaee TY 1,2, *, Patel S 3 Perry K 4 McLeod HL 6 Johnson JA 1,2, 1 Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville, FL 2 Center for Pharmacogenomics, Univers ity of Florida, Gainesville, FL 3 Department of Pharmacy Practice, University of Illinois at Chicago, Chicago, IL 4 Department of Medicine, University of Chicago, Chicago, IL 5 Pharmaceutical Sciences Section, College of Pharmacy, Qatar University, Doha, Qa tar 6 Institute for Pharmacogenomics and Individualized Therapy, University of North Carolina, Chapel Hill, NC Identifying predictors of higher warf arin dose has been challenging. VKORC1 Asp36Tyr single nucleotide polymorphism (SNP) is one of the most promising predictors of high warfarin dose, but data on its population prevalence is incomplete. We determined the frequency of this SNP in participants from seven countries on four continen ts and investigated its effect on warfarin dose requirement. A total of 992 samples were analyzed to define the population prevalence of this SNP. Those samples included individuals from Egypt, Ghana, Sudan, Kenya, Saudi Arabia, Peru and African Americans from the United States. 206 Egyptian samples were then used to investigate the effect of this SNP on warfarin dose requirements. This SNP was highest among Kenyans and Sudanese, with a minor allele frequency (MAF) of 6% followed by Saudi Arabians and Egypt ians with a MAF of 3.0% and 2.5%, respectively. It was absent in West Africans, based on our data from Ghana, and a large cohort of African Americans. Egyptian carriers of the VKORC1 Tyr36 showed higher warfarin dose requirement (57.1 29.4 mg/week) than those with the Asp36Asp genotype (35.8 16.6 mg/week; P<0.0001). In linear regression analysis, this SNP had the greatest effect size among the genetic factors (16.6 mg/week increase in dose per allele), and improved the warfarin dose variability explaine d in Egyptians (model R2 from 31% to 36.5%). The warfarin resistant VKORC1 Asp36Tyr appears to be confined to north eastern Africa and nearby Middle Eastern populations, but in those populations where it is present, it has a significant influence on warfar in dose requirement and the percent of warfarin dose variability that can be explained. 105 Comparative proteomics of maize basal endosperm in miniature1 seed mutant and its wild type Mn1 Silva Sanchez C 1 Chen S 1,2, *, Zhu N 2 Li Q B 3 Chourey PS 3,4 1 Proteomics Division, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 2 Department of Biology, University of Florida, Gainesville, FL 3 Center for Medical, Agricultural, and Veterinary Entomology, Agricultural Re search Service, U.S. Department of Agriculture, Gainesville, FL 4 Plant Pathology Department, University of Florida, Gainesville, FL Developing endosperm in maize seed is a major site for biosynthesis and storage of starch and proteins, and of immense econ omic importance. The basal part of endosperm is however unique as it performs a major role in solute, water and nutrition acquisition from mother plant. The miniature1 (mn1) mutation is a loss of function
50 mutation of the Mn1 encoded cell wall invertase, wh ich is entirely expressed in the basal endosperm and is essential to many of the above functions. Here we report a comparative proteomic study between Mn1 and mn1 basal endosperm to better understand the possible basis of pleiotropic effects on many divers e traits in the mn1 seed. Specifically, we used iTRAQ based quantitative proteomics combined with Gene Ontology classification and the KEGG bioinformatics to conduct functional analysis of the proteomic information. A total of 2 ,518 protein s were identifie d from soluble and cell wall associated protein fractions, and 131 proteins were differentially expressed in the two genotypes. Based on the gene ontology classification, the main functional groups of proteins that were significantly enriched were those in volved in the carbohydrate metabolic and catabolic process, and cell homeostasis. 106 Genetic mapping of transgene reactivated mutant 1 ( tgr1 ), a novel allele of the largest subunit of RNA polymerase IV in maize Sloan A Madzima T F Mills ES, McGinnis K M Department of Biological Science, Florida State University, Tallahassee, FL Stably silent transgenic lines containing the b1 genomic transgene (referred to as BTG s) were used in a forward genetic screen to identify transgene reactivated (Tgr) mutants. In the M2 generation recessive homozygous mutants were identified by purple pigmentation, characteristic of BTG expression. In addition to the loss of transcriptional silencing of BTG s, tgr1 1 individuals exhibit hypomethylation of the promoter region of BTG s and a reduction in 24 nucleotide siRNAs. These molecular phenotypes are consistent with Tgr1 encoding a component of the RNA directed DNA methylation gene silencing pathway in maize. Genetic mapping of Tgr1 indicates that the mutation lies within a 12Mbp interval on chromosome 1, which also includes the Rmr6 locus. Complementation assays were used to demonstrate that tgr1 1 is an allele of Rmr6 We confirmed that tgr1 1 plants do not contain any of the cloned Rmr6 mutant alleles. Together, these results demonstrate that tgr1 1 is a novel allele of Rmr6 a gene that has previously been shown to encode the largest subunit of RNA polymerase IV. 107 Intra host p hylodynamics in S IVmac251 infected CD8 depleted rhesus m acaques with different n europathology Strickland SL 1,2 Veras NM 1,2 Prosperi MC 1,2 Suchard M A 3 5 Williams K 6 Salemi M M 1,2 1 Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 2 Emerging Pathogens Institute, University of Florida, Gainesville, FL 3 Department of Biomathematics University of California Los Angeles, Los Angeles, CA 4 Department of Biostatistics University of California Los Angeles, Los Angeles, CA 5 Department of Human Genetics, University of California Los Angeles, Los Angeles, CA 6 Biology Department, Boston College, Chestnut Hill, MA Determining the intra host evolutionary factors leading to the emergence of neurovirulent strains continues to be of importance for understanding development of neuroAIDS. We applied high resolution Bayesian phylodynamics analysis to longitudinal samples collected from SIV infected CD8 depleted rhesus macaques, a rapid disease model for AIDS related neuropathology. The mode of transmission, the tem po of the appearance of neurovirulent strains and the spatial distribution of the virus were determined. Molecular clock analysis showed rate of evolution along the branches differed slightly among primates. The average evolutionary rate of the virus was 3 .0e 4 substitutions per site per day, one log higher than what is usually observed in non CD8 depleted primates. Bayesian skyline plots were inferred to quantify the dynamics of the effective population size (Ne) over time. Two primates (D03 and D06) showed a peak in the Ne at about 50 dpi. Whereas one primate (D04) showed two peaks at 50 and 85dpi and the last primate (D05) had three peaks occurring around 20, 50, and 85 dpi. Phylogeography and gene flow analysis showed multiple brain seeding events o ccurred throughout the infection, with the initial brain infection occurring as early as the first week post infection, and the median of the of the transitions taking place between 52 68 dpi. The data indicate that the depletion of CD8 immune response in the infected macaques, all of which developed neural damage, results in an accelerated evolution rate of the virus leading to an increase of genetic heterogeneity and the emergence of effective viral variants where the majority migrate to the brain several weeks post infection. 108 The inducible antioxidant transcription response has context dependent effects on stress resistance Tan g L Choe KP* Department of Biology, University of Florida, Gainesville, FL Pre exposure to one type of stress often confers resistance to a distinct type of stress suggesting that inducible stress transcription factors are principle regulators of antioxidant
51 and detoxifica tion defense genes in animals ranging from nematodes to mammals. Caenorhabditis elegans has a single functional CNC named SKN 1 that is directly suppressed by the WD40 repeat protein WDR 23. The role of SKN 1 in mediating oxidative and xenobiotic stress re sistance has been widely reported, but it is unclear what role SKN 1 has during other types of stress. Juglone (175 uM), sodium chloride (425 mM), and heat exposure (34C, 12 hrs) were used as models for oxidative, osmotic, and heat stress, respectively. W orms with increased SKN 1 activity due to wdr 23 deletion were resistant to juglone and had slightly increased longevity. Alternatively, loss of wdr 23 sensitized worms to osmotic and heat stress. We also found that loss of wdr 23 dramatically reduced surv ival of diapause, a starvation induced developmental state. All of the stress resistance phenotypes of wdr 23 were suppressed by skn 1(RNAi) Taken together, our studies demonstrate that SKN 1 has complex effects on stress resistance that vary by the natur e of the stressor. Surprisingly, these results also suggest that individual stress responses may be beneficial only under a narrowly defined set of conditions. 109 SFMBT1 is required in Sn ail1 mediated transcriptional repression during epithelial mesenchymal t ransition Tang M 1 2 S hen H 3 Lin T 1 2 Jin Y 1 Cai QS 1 Quyen T 4 Lin S 5 W u L 5 *, Lu J 1 1 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 3 Zhongshan Ophthalmic Center, Sun Yat Sen University, Guangzhou, China 4 Department of Biology, Agnes Scott College, Atlanta, GA 5 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, F L Epithelial mesenchymal transition (EMT) is a reprogramming event reflecting the plasticity of epithelia l cells. EMT is a critical step during embryonic development and tumor progression. During thi s transition, epithelial markers such as E cadherin, a gatekeeper of the epithelial phenotype and a su ppressor of tumor invasion, are down regulated. We previously demonstra ted that the Snail1 zinc finger transcription factor represses epithelial gene expr ession by recruiting the LSD1 demethylase complex to re move dimethyl histone H3 lysine 4 (H3K4me2), a mark associated with act ive transcription. Here we show that the Snail1 protein complex also con tains the malignant brain t umor (MBT) domain protein SFMBT1. We have confirmed that SFMBT1 associates with Snail1 through the LSD1 compl ex. Depletion of SFMBT1 impairs Snail mediated E cadherin repression and induced EMT. Importantly, knockdown of SFMBT1 blocks demethylation of the H3K4Me2 mark at the E cadherin promoter by Snail1 LSD1. Be cause the MBT domains r ecognize dimethyl lysines, we propose that S FMBT1 functions as an essential histone reader of the LSD1 d emethylase complex required for Snail1 mediated transcriptional repression. 110 Computational analysis of alternative splicing regulation using RNA Seq data from a mouse model Tang S 1,2 Riva A 2, 1 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Characterization of the RNA population plays an important role in studying the spatial and temporal expression patterns of gene isoforms and elucidating protein functions. Differential inclusion of exons and alternative usage of splice sites causes genes in metazoan organisms to produce multiple isoforms with distinct biological functions and compositions. In this study, we report on the application of the software package PASTA (Patterned Alignments for Splicing and Transcriptome Analysis) to study alternative splicing and differ ential expression of isoforms using RNA Sequencing (RNA Seq) data from an Mbnl2 knockout mouse. Results from PCR validation show that PASTA is very sensitive in discovering alternative splicing events, and has the ability to detect non canonical splicing. The use of PASTA can lead to a more accurate cha racterization of alternative s plicing and its regulation, and can be used to analyze changes in RNA isoform expression levels in a variety of applications including cancer research. 111 The genetic program for cartilage development evolved in the common ancestor of Bilateria Tarazona OA 1,2 Slota L 1,2 Cohn MJ 1 3, 1 Department of Biology, University of Florida, Gainesville, FL 2 Howard Hughes Medical Institute, University of Florida, Gainesville, FL 3 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Cartilage has been proposed to be a defining character of vertebrates, however this tissue type has evolved independently in a number of invertebrate lineages. Studi es of vertebrate chondrogenesis have revealed critical proteins for the structure of the cartilage extracellular matrix (ECM) and the regulation of chondrogenesis. Collagen2A1 is the major structural protein in the cartilage
52 ECM, for instance, and the Coll agen2A1 gene is directly regulated by Sox9, Sox5 and Sox6 transcription factors. Signaling proteins such as Sonic hedgehog regulate chondrogenesis by acting upstream of Sox genes. To test the hypothesis that invertebrates and vertebrates use a common genet ic program to build cartilage, we studied chondrogenesis in the cephalopod Sepia pharaonis and the horseshoe crab Limulus polyphemus two distantly related Protostome invertebrates. We cloned the invertebrate orthologs of the ancestral (pre duplication) Co llagen2A1, Sox9, Sox5/6 and Hedgehog genes and examined their expression during embryonic development by in situ hybridization. We found that Clade A collagen and Sox genes are expressed in pre chondrogenic cells of both species, and the expression of Hedg ehog in adjacent tissues suggests a possible regulatory role. The expression data presented here demonstrate that invertebrate cartilage is collagen based and chondrogenesis is probably regulated by SoxE and SoxD genes. Therefore, invertebrate cartil ages s hare homologous molecular building blocks, which supports the hypothesis of suggest the independent evolution of cartilage by a deeply conserved genetic program for chondrogenesis for Bilateria. 112 Genome engineering using ZFN and TALEN t echnology Tremblay DC Culver Cochran A, Horakova A, Chadwick BP Department of Biological Science, Florida State University, Tallahassee, FL Significant progress in the field of genome engineering has recently be en fueled by advances in the design of novel zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs). Targeted gene disruption using ZFNs and TALENs can be useful for gene correction, mutation, insertion or deletion via homology mediated repair (H MR) and/or non homologous end joining (NHEJ). While these powerful technologies have been extensively used to induce NHEJ based mutations in various model organisms, there has been limited use of ZFNs and TALENs in HMR of human cells. As proof of principle that ZFNs and TALENs can be used to induce HMR in human cells, we have constructed a ZFN and a TALEN that target the human XIST and SETDBI genes, respectively. The ZFN targets the minimal promoter of the XIST gene, and was introduced into RPE1 cells in co m bination with a homologous tem plate that replaces the endogenous promoter with an SV 40 driven neomycin gene. We isolated one heterozygous clone in which the promoter has been successfully deleted from one allele. Alternatively, the TALEN targets the ATG containing exon 2 of SETDB1, and was introduced into RPE1 cells with a promoter trap repair template that fully replaces the exon. Two independent clones from 74 total (2.3%) were isolated that successfully deleted exon 2 on both alleles. Our findings supp ort the continued use and further development of both nuclease platforms for homology mediated genome editing in human cells. 113 Bacterial expression of a plant regulator of actin dynamics Turcotte M 1,2 Grey PH 1 Emmanuel M 1 Cuddy KK 1 Oppenheimer DG 1,3, 1 Department of Biology, University of Florida, Gainesville, FL 2 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 3 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL The ability of the actin cytoskeleton to rapidly polymerize and depolymerize in response to various cellular signals underlies many important cellular processes including membrane trafficking and intracellular transport. Members of the actin depolymerizing factor/cofili n (ADF) family of proteins play an important role in regulating the depolymerization of actin filaments. We recently discovered a family of ADF regulators in plants that we named RPA for Regulator of Plant ADF. We previously demonstrated that the founding member of the family, RPA1, could regulate ADF in vitro by using bacterially expressed RPA1. An analysis of the protein sequences of other RPA family members revealed that about half of the members possess a putative membrane anchor sequence at their N te rminus. To determine if these RPA family members also regulate ADF, we attempted to express RPA11, which possesses a putative membrane anchor, in bacteria and purify the protein to use in our in vitro ADF activity assays. Unfortunately, the N terminal memb rane anchor sequence appears to inhibit growth of the bacteria and expression of RPA11. To overcome this problem, we created a bacterial protein expression vector that contains a yellow fluorescent protein tag in addition to the affinity purification tags. Using this vector will allow the expression levels of the RPA11 protein to be monitored under different bacterial growth conditions prior to its purification. This will allow us to determine appropriate bacterial media and growth temperature to optimize R PA11 expression levels. Subsequent purification and in vitro analysis of RPA11 function will shed light on this new family of ADF regulators. 114 Bayesian divergence dating of Yemeni mitochondrial haplogroups Vyas DN 1 Kitchen A 2 3 Al Meeri A 4 Mulligan CJ 1
53 1 Department of Anthropology, University of Florida, Gainesville, FL 2 Department of Anthropology, University of Iowa, Iowa City, IA 3 Archaeogenetics Laboratory, Institute of Archaeology of the Academy of Sciences of the Czech Republ ic, Prague, Czech Republic 4 Department of Clinical Biochemistry, Faculty of Medicine The southern dispersal route (SDR) out of Africa posits that anatomically modern humans (AMHs) first left Africa by c rossing the southern extent of the Red Sea. Yemen would be the first stop outside Africa. If AMHs did follow the SDR and left modern descendants, one would expect to see deep divergences in the Yemeni mitochondrial gene tree. On the other hand, if AMHs fol lowed the SDR but no modern descendants remain or if AMHs did not pass through Yemen on their route, one would expect more re cent divergence dates instead. We used data from a wide array of haplogroups that were previously sequenced using traditional Sange r sequencing. We used multiple methodologies within the BEAST software package to generate divergence dates. One method, which we took from published literature, used a relaxed clock model and a prior on the tree root, but restricted analyses to the coding region. We also used a novel method that attempts to integrate both the coding and control regions by using multiple substitution models. Dates were assessed to see how they related to various theories, hypotheses, and models of human migration out of Afr ica. 11 5 Phototropin 1 and cryptochrome action in response to green light in combination with other wavelengths Wang Y 1 Maruhnich SA 1 Mageroy MH 1 Justice JR 2 Folta KM 1 2 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 Horticultural Sciences Department, University of Florida, Gainesville, FL Light signals from different portions of the spectrum converge to shape seedling development. Studies in Arabidopsis thaliana have delineated discrete roles for far red, red, blue and green light in the control of hypocotyl growth inhibition. In this study the effect of green light (525 550 nm) was examined in concert with other light qualities. During photomorphogenic growth, na rrow bandwidth far red, red or blue light inhibit hypocotyl (stem) elongation. Conversely, green light induces elongation. Blue green reversibility has been shown to occur via the cryptochrome receptors. While early hypocotyl growth inhibition during de et iolation is triggered by blue light acting through cryptochromes, it is not green light reversible. Instead, blue and green light act additively to enhance inhibition. On the other hand, green light reverses the effects of red and far red phytochrome actio n on hypocotyl growth inhibition in both early (min h) and late (h d) responses. The effects of green light requires the presence of phototropin 1, suggesting that this was not a green light response, but instead a response to extremely low fluence rate bl ue light. Tests with dim blue light (<0.1 mol/m 2 s) confirm a phot1 dependent promotion of stem growth, opposing the effects of red/far red light. The results show that green light works additively with blue light via cryptochromes during initial acclimati on to light, and those red and far red responses are actually opposed by dim blue light, in a manner that requires phot1. These findings demonstrate how enriched green environments may adjust red and blue light photomorphogenic responses through both the c rys and phot1 receptors, and define a new role for phot1 in stem growth promotion. 116 scAAV mediated gene transfer of interleukin 1 receptor antagonist to synovium and articular cartilage in large mammalian j oints Watson R S 1 Broome TA 2 Levings PP 1 Colahan PT 2 Ghivizzani SC 1 1 Department of Orthopaedics and Rehabilitation, University of Florida, Gainesville, FL 2 Department of Large Animal Clinical Sciences, University of Florida, Gainesville, FL With the long term goal of developing a gene based tr eatment for osteoarthritis (OA), we performed studies to evaluate the equine joint as a model for AAV mediated gene transfer to large, weight bearing human joints. A self complementary AAV2 vector containing the coding regions for human interleukin 1 recep tor antagonist (hIL 1Ra) or green fluorescent protein (GFP) was packaged in AAV capsid serotypes 1, 2, 5, 8 and 9. Following infection of human and equine synovial fibroblasts in culture, we found that both were only receptive to transduction with AAV1, 2 and 5. For these serotypes, however, transgene expression from the equine cells was consistently at least 10 fold higher. Analyses of AAV surface receptor molecules and intracellular trafficking of vector genomes implicate enhanced viral uptake by the equi ne cell s. Following delivery of 1 x 10 11 vector genomes of serotypes 2, 5 and 8 into the forelimb joints of the horse, all three enabled hIL 1Ra expression at biologically relevant levels and effectively transduced the same cell types, primarily synovial fibroblasts and, to a lesser degree, chondrocyte s in articular cartilage. These results provide optimism that AAV vectors can be effectively adapted for gene delivery to large human joints affected by OA.
54 117 Selection for cold tolerance alters the maintenance of metabolic homeostasis during cold expo sure in Drosophila melanogaster Williams CM 1 Watanabe M 2 Morgan T 3 Edison AS 4 Boroujerdi A 2 Hahn DA 1 1 Department of Entomology and Nematology, University of Florida, Gainesville, FL 2 Department of Chemistry, Claflin University, Orangeburg, SC 3 Division of Biology, Kansas State University, Manhattan, KS 4 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL Low temperatures induce in insects a state of paralysis (chill coma), which is reversib le following the return of favo rable conditions, although the time taken to recover varies widely both inter and intra specifically. This variation may result from differences in the degree to which insects can maintain metabolic homeostasis during cold exposure. We selec ted replicate lines of Drosophila melanogaster for either fast or slow recovery from chill coma (cold tolerant or susceptible lines), then profiled and compared the polar metabolome before, during and after cold exposure using nuclear magnetic resonance s pectroscopy. We found that the cold tolerant lines were smaller, and maintained a higher degree of metabolic homeostasis during cold exposure. Pathways that responded differently to the cold exposure between cold tolerant and susceptible lines included am ino acyl tRNA biosynthesis (indicating differential levels of translation during co ld stress), proline and alanine metabolism, starch and sucrose metabolism, and the TCA cycle. Our results suggest that adaptation to cold environments results in evolution t owards energetic pathways that function better in the cold. These lines are fully genotyped, allowing us to look for single nucleotide polymorphisms among the selection lines in genes belonging to these pathways. 118 Evaluation of a grobacterium mediated and biolistic gene transfer to s ugarcane Wu H 1 ,2 Awan FS 1 3 Zeng Q 1,2,4 Vilarinho A 1,2 Phipps T 1,2 McCuiston J 5 Wang W 5 Caffall K 5 Altpeter F 1 ,2, 1 Agronomy Department University of Florida, Gainesville, FL 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 3 Current address: Centre of Agricultural Bioch emistry and Biotechnology University of Agriculture, Faisalabad, Pakistan 4 Current address: Biochemistry and Biotechnology Department, Yunnan Agricultural University, Kunming, China 5 Syngenta Biotechnology Inc., Research Triangle Park, NC Sugarcane ( Saccharum spp. hybrids) is one of the most productive crops and is extensively utilized for table sugar or biofuel production. Both agrobacterium mediated tran sformation or biolistic gene delivery have been successfully used for efficient gene transfer to sugarcane. For biolistic gene transfer method we have previously employed various optimizations including reduced amounts of DNA and/or removal of the vector b ackbone prior to gene transfer. These modifications resulted in reduced copy number and backbone insertion of the transgenic loci as well as reduced soma clonal variation. Data fro m a biolistic and agrobacterium mediated gene transfer of minimal nptII expr ession cassettes to sugarcane will be presented. Callus derived from six weeks culture of immature leaf whorl cross sections of commercial cultivar CP88 1762 was used as target for either b iolistic gene transfer of the minimal nptII expres sion cassette or agrobacterium mediated gene transfer of the nptII expression cassette in five independent experiments. Selection of transgenic events followed the same concentrations and types of selective agents for both transformation procedures. PCR amplification by np tII specific primers confirmed a total of 383 transgenic plants. The number of transgenic plants generated per tissue unit, variability from experiment to experiment, the time required from explant to transgenic plants in soil, the number of non transgenic escapes, the frequency of complex or simple transgenic loci and the transgene expression level of simple and complex loci events for both agrobacterium mediated and biolistic gene transfer were compared. 119 Anti antitrypsin single chain variable fragment prevents Z AAT toxicity and a ggregation Xiao K 1 ,2 Levites Y 3 Wang RL 1 Oshins RA 1 Rouhani FN 1 Song S 4 *, Brantly ML 1 1 Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, University of Florida, Gainesville, FL 2 Graduate Program in Genetics and Genomics, University of Fl orida, Gainesville, FL 3 Center for Translational Research in Neurodegenerative Disease, University of Florida, Gainesville, FL 4 Department of Pharmaceutics, University of Florida, Gainesville, F L Alpha 1 antitrypsin (AAT) is a neutrophil elastase inhibitor. The Z mutation (E342K) of AAT causes retention of the mutant protein as an inclusion body within the ER of hepatocytes, which may cause severe liver disease such as fibrosis and cirrhosis. Curre nt methods of blocking the loop sheet polymerization of Z AAT with ligands or small peptides have been proven to be feasible in vitro but have
55 limitations in the delivery method to the cell. We designed a new strategy using a single chain variable fragm ent (scFv) derived from a monoclonal antibody to inhibit AAT polymerization; we can further improve the scFv ZAAT complex degradation by adding a tag recognized by proteasome machinery. Anti AAT scFv was constructed and validated based on a parent hybridom a. The mRFP Z AAT stable expression CHO cells were transfected with scF v expression constructs or a G FP expression vector as a control and Z AAT accumulation levels and cell stress were evaluated. The ability of the scFv to bind AAT was validated in vitro and ex vivo To facilitate degradation of the complex, Hsp70 binding motif, FKBP12, or KDEL sequence was attached to the C terminus of the scFv; of these, only scFv FKBP12 treated cells showed significant reduction of polymers within the ER. In addition, t he scFv FKBP12 Z AAT complexes were degraded by the proteasome pathway and reduction of Z AAT polymerization lead to decreased cell stress levels. Furthermore, although the scFv can recognize both M and Z AAT, the expression of scFv FKBP12 does not interfere with the secretion or activity of normal M AAT in the cells. 120. Analysis of the maternally expressed gene1 family provides novel insight into the evolution and expression of tandem gene duplicates in maize Xiong Y 1 Mei W 2 Kim D H 3 Mukherjee K 1 Hassanein H 1 Barbazuk WB 2 *, Kolaczkowski B 1 *, Sung S 3 Kang B H 1,4 1 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 2 Department of Biology, University of Florida, Gainesville, FL 3 Cell and Molecular Bio logy Graduate Program, Universi ty of Texas, Austin, TX 4 Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL Maternally expressed gene1 ( Meg1 ) encodes a small cysteine rich peptide that is required for the normal development of the basal endosperm transfer cells (BETCs) in maize. Twelve copies of Meg1 like genes were identified to be a gene cluster near the Meg1 gene. Here we analyzed the genom ic organization, gene structure, expression, and chromatin status of the family. Except Meg1 and Meg13 one of the Meg1 like gene, all the other members are flanked by homologous sequences varying in lengths from a few hundred bp to >5 kb, indicating that the gene family expanded through unequal crossing over. Eight genes with intact gene structures are transcriptionally active and exhibit the BETC specific expression pattern. Chromatin immunoprecipitation revealed activating and repressive marks of these g enes in BETC and non BETC samples, respectively, indicating that chromatin modifications contribute to the cell type specific expression of the Meg gene family. Phylogenetic analysis and synteny mapping suggest that the major duplication events took place after the maize allotetraploidy at 4.8 million years ago (mya) but prior to the emergence of teosinte. The oldest member displays a tissue expression pattern different from rest of the members suggesting that Meg genes acquire new functions as they expand and evolve. A strong signature of positive selection in the youngest members suggests further functional diversification in the family. 121 JAZ2 and JAZ7 function in jasmonate signaling through different mechanisms Yan H 1,2 Yoo M J 1 Chen Y 1 Koh J 3 Acikgoz D 1 Wang Q 2 Chen S 1,3,4, 1 Department of Biology, University of Florida, Gainesville, FL 2 Department of Horticulture, Zhejiang University, Hangzhou, China 3 Proteomics Division, Interdiscipli nary Center for Biotechnology Research, University of Fl orida, Gainesville, FL 4 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Jasmonates (JAs) are recognized as one of the important plant hormones, which play a vital role in plant growth, development and stress response. In this study, we focused on characterizing JAZ2 (JASMONATE ZIM DOMAIN 2) and JAZ7, which are two of the 12 JA Z family members presumably functioning as repressors in JA signaling in Arabidopsis thaliana We have isolated homozygous T DNA insertion mutants of JAZ2 and JAZ7 and observed different growth phenotypes between the mutants and wild type (WT) in the pres ence or absence of methyl jasmonate (MeJA). We have also tested the effects of other phytohormones and conditions. To understand the phenotypes and elucidate the molecular mechanisms underlying the functions of JAZ2 and JAZ7 we have employed functional genomics approaches to analyze different types of molecules and their dynamic changes in the above plant materials. Here we will report our recent findings from RNA Seq, proteomics and metabolomics experiments. These data can be used to construct a molecul ar network of JA signaling. 122. Genetic suppressors of plastid translation mutants in m aize Yang J 1, *, Suzuki M 1,2 McCarty D R 1,2, 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 Horticultural Sciences Department University of Florida, Gainesville, FL
56 Genetic studies indicate that plastids have an essential role in plant embryo development. Our recent findings indicate that in maize, the embryo lethal phenotypes typical of a large class of mutants that have defe cts in plastid ribosome assembly and protein translation can be suppressed in some inbred backgrounds. At least one genetic modifier of a non lethal class plastid mutant is already known. Inhibitor of Striate 2 ( Isr1 ) gene is a suppressor of the white leaf striping mutant, striate 2 (sr2). Isr1 encodes a putative plastid localized hydrolase, has been proposed to modify striping by suppressing proliferation of a subset of cells in leaves of the sr2 mutant that lack pr otein translation competent plastids. Hence, Isr1 may serve as a model for suppressors of the embryo lethal plastid mutants. To understand the molecular mechanism responsible for the formation and suppression of the white striping, we have characterized na tural allelic variation of Isr1 in diverse maize inbreds as well as molecular cloning of sr2 gene. A candidate gene for sr2 identified by bioinformatic analysis of linked genes in the maize genome is shown to encode a putative plastid localized nudix hydro lase homolog 26 (NUDX26). To test whether NUDX26 is sr2 we have sequenced alleles from wild type and sr2 mutant plant. In addition, genetic analyses are underway to map modifiers of the embryo lethal plastid translation mutants. Together these studies w ill illuminate the relationship if any between Isr1 and background suppressors of embryo lethal plastid mutants in maize. Our long term goal is to understand the role of plastids and plastid derived signals in regulation of cell proliferation and embryo fo rmation. 123 Objective measures of variability and reliability in ChIP seq Yang Y 1,2 Fear J 1,2 Hu J 2,3 Haecker I 2,3 Jacobs D 2 ,4 Bloom D C 2 *, Renne R 2,3 *, McIntyre L M 2,5, 1 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 3 University of Florida Shands Cancer Center, Gainesville, FL 4 Interdisciplinary Program in Biomedical Sciences, University of Florida, Gainesville, F L 5 Department of Statistics, University of Florida, Gainesville, FL ChIP seq is a new technology which enables rapid identification of protein DNA binding. The technique is noisy, requiring biological replicates to confirm peak identification and to quant ify binding. Biological replicates have been found to be critical and are required for deposit of data in the ENCODE and MODENCODE projects. Yet, to date no consensus has emerged about how to determine peak presence/absence among multiple biological replic ates. In addition, there is no consensus about how to quantify peaks among replicates. We use objective measures of variability and reliability to determine the best approach to consensus peak identification among biological replicates. We used several dif ferent ChIP seq datasets including PolII, transcription factors and histones from the ENCODE and MODENCODE projects to illustrate the utility of objective measures in the determination of consensus peaks. 124 De novo transcriptome assembly and proteome profiling of the recently formed allopolyploid Tragopogon mirus (Asteraceae) and its diploid parents Yoo M J 1 Koh J 1,2 Soltis DE 1,3, *, Soltis PS 3 *, Barbazuk WB 1, *, McIntyre LM 4,5, *, Chen S 1,2, 1 Department of Biology, University of Florida, Gainesville, FL 2 Proteomics Division, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 3 Laboratory of Molecular Systematics and Evolutionary Genetics, Florida Museum of Natural Hist ory, University of Florida, Gainesville, FL 4 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 5 Department of Statistics, University of Florida, Gainesville, FL Polyploidy (whole genome duplication) is recognized as an important evolutionary process in speciation and genome evolution of diverse organisms, particularly plants. However, much of our current understanding of polyploidy is based on analyses of crop species. Here, we examined the transcriptomes of naturall y occurring allopolyploid T. mirus and its diploid parents ( T. dubius and T. porrifolius ) using Illumina HiSeq 2000 technology. In parallel, we employed iTRAQ LC MS/MS to investigate the global proteomes of the three species. A total of 480 million 100 bp paired end reads was generated from leaf transcriptomes of the three species, which corresponds to 34 Gb of sequence. These reads were assembled de novo by the Trinity short read assembler, and the assembled contigs were annotated by Blast2GO software. Thi s assembly was uti lized as a reference for RNA S eq and proteomic data analyses. Differential gene expression between the allopolyploid and its diploid parents was analyzed and compared at transcriptomic and proteomic levels. Together with ongoing analysis of transcriptome profiles of another young allopolyploid, T. miscellus and its diploid parents ( T. dubius and T. pratensis ), this study will provide valuable insights into transcriptomic as well as proteomic changes in recently formed allopolyploids. In a ddition, the transcriptome data set generated here provides the most comprehensive sequence resource for
57 the Tragopogon polyploid system, which enables further study of gene and protein expression patterns in different tissues and under different conditions. 125 Systematic characterization of multiple sucrose metabolizing pathways in Streptococcus mutans Zeng L Burne RA Department of Oral Biology, University of Florida, Gainesville, FL Sucrose is perhaps the most efficient carbohydrate for the promotion of dental caries in humans, and the primary caries pathogen Streptococcus mutans encodes multiple enzymes involved in the metabolism of this disaccharide. Here, we engineered a series of mutants lacking individual or combinations of sucrolytic pathways to understand control of sucrose catabolism and to identify novel or alternative sucrases. Growth phenotypes indicated that gtfBCD (glucan exopolysaccharide synthases), ftf (fructan exopolysaccharide synthase) and scrA scrB (sucrose PTS permease and sucrose 6 P hydrolase, respectively) constitute the majority of the sucrose catabolizing activity of S. mutans ; however mut ations of any of these genes alone did not negatively impact planktonic growth on sucrose. Conversely, the multiple sugar metabolism pathway ( msm ) was shown to play only a minor role in growth on sucrose. Notably, loss of scrA led to growth stimulation in fructooligosaccharide based medium, due in large part to increased expression of the fruAB (fructanase) operon (shown as a P fruA cat fusion). Using the LevQRST four component signal transduction system as a model for carbohydrate dependent gene expression in strains lacking combinations of sucrases, a P levD cat reporter was shown to be activated by pulsing with sucrose. Interestingly, ScrA was required for activation of levD expression by sucrose through components of the LevQRST complex, but not for activ ation by the cognate LevQRST sugars, fructose or mannose. Collectively, these results revealed novel regulatory circuitry for control of sucrose catabolism, with a central role of the ScrA PTS permease as an effector. 126. Map based cloning of the maize d efective kernel5 (dek5) mutant Zhang J 1 Settles AM 1,2, 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 Horticultural Sciences Department, University of Florida, Gainesville, FL Defective kernel 5( dek5 ) is one of the classic dek mutants in maize, but little is known about the biochemical and molecular function of the dek5 gene. The dek5 mutant has a shrunken and collapsed seed phenotype with an opaque, chalky endosperm and a normal embryo. Mutant seeds germinate poorly, and seedling leaves are pale green with white stripes. The seed phenotype is similar to starch biosynthesis mutants such as brittle1 and brittle2 and we hypothesize that dek5 affects endosperm starch biosynthesis. Starch accumulates in t he amyloplast, which is a non green form of the chloroplast. The pale green seedling phenotype of dek5 suggests the gene is also required for chloroplast development. We are cloning this gene to understand the function of dek5 in seed development. We deve loped F2 mapping populations from crosses of the dek5 25 allele with the Mo17 inbred. Fine mapping identified a 460 kb interval on chromosome 3 as containing the dek5 locus. Thirteen gene models are within this map interval and are currently being evaluate d as candidates for the dek5 gene. 127 Biochemical characterization of Brassica napus MAP kinase 4 Zhang T 1 Zhu M 1 Song W Y 2,3, *, Harmon AC 1,3 Chen S 1,3,4 1 Department of Biology, University of Florida, Gainesville, FL 2 Plant Pathology Department, University of Florida, Gainesville, FL 3 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 4 Proteomics Division, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL Mitogen activate d protein (MAP) kinase pathway includes MAP triple kinases (MAPKKKs), MAP double kinases (MAPKKs) and MAP kinases. This kinase cascade plays an important role in plant growth, development and defense. We show here that a MAP kinase from Brassica napus BnM PK4, has autophosphorylation activity in the presence of manganese. The phosphorylation sites were mapped to the tyrosine residue in the activation loop using mass spectrometry. In addition, BnMPK4 can phosphorylate myelin basic protein (MBP) and the B. na pus MAP kinase substrate (MKS1) in vitro Compare to MBP, the in vivo substrate MKS1 was a better substrate for MPK4. While substrate phosphorylation activity of BnMPK4 is much stronger than the autophosphorylation activity, the presence of MKS1 can enhance the autophosphorylation activity of BnMPK4. BnMPK4 exi sts in both o xidized and reduc ed forms in vitro and both showed kinase activity. BnMPK4 aggregated under oxidized conditions, such as in the presence of hydrogen peroxide and cupric chloride, but it still retained the kinase activity. These results indicate that BnMPK 4 is an active kinase and its activity may not be affected by redox in vitro The functional
58 significance of substrate enhancement and aggregation of BnMPK4 will be discussed. 128. pHA4C plasmids: a new modular series of vectors for constitutive and GAL4 mediated expression in Drosophila Zhang Y 1 Arcia S 1 3 Perez B 1,3 Fernandez Funez P 1,4, *, Rincon Limas DE 1, 1 Department of Neurology, University of Florida, Gainesville, FL 2 Howard Hughes Medical Institute UF Science for Life Program, University of Florida, Gainesville, FL 3 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 4 Department of Neuroscience, University of Florida, Gainesville, FL Control of gene expression relies on a variety of strategies, including gene overexpression, gene rescue, and gene silencing. The UAS/GAL4 system has become a popular choice for genetic manipulation. However, there are cases in which ubiquitous expression is desirable independently of GAL4. Traditionally, constitutive expression ha s been achieved by cloning cDNAs under control of ubiquitous promoters such as the Actin5C or Hsp70 genes. Unfortunately, the Actin5C promoter displays heterogeneous expression and the Hsp70 promoter requires heat induction, which may affect certain experi ments. In a search for more homogeneous Tubulin, Armadillo or EF containing essential regulatory information were used together with the first exon containing the ATG and a fragment of the second exon for ligation of the cDNA, further complicating cloning. To overcome these for expression of cDNAs under control of a minimal Tubulin promoter carrying key regulatory regions and the Tubulin. We then incorporated an optimized polylinker to offer flexible cloning options. Finally, we added the option of double labeling the expressed proteins with two C terminal tags, the viral epitope hemagglutinin (HA) and a syn thetic tetracysteine (4C) tag that binds small fluorescent compounds. To expand the application of this optimized polylinker, we also subcloned it into the typical pUAST vector to allow GAL4 dependent expression as well as att mediated integration of the t ransgene. The properties and advantages of the new series of vectors will be presented. 129 High Performance Computing Center overview Taylor CA, Zhang Y Research Computing, University of Florida, Gainesville, FL The High Performance Computing Center, part of the University of Florida Research Computing, is a faculty directed facility with the focused mission of providing high performance computing and storage resources, including support, to faculty whose research depends on large scale computing. We w ill provide an overview of the facilities, services, and resources available within the High Performance Computing Center. 1 30 Thiol based redox regulation of serine/threonine protein kinase in Brassica napus Zhu M 1 Zhang T 1 Silva Sanchez C 2 Song WY 3 ,4, *, Harmon A C 1,4 Chen S 1, 2,4 1 Department of Biology, University of Florida, Gainesville, FL 2 Proteomics Division, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 3 Plant Pathology Department, University of Florida, Gainesville, FL 4 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Protein kinase mediated phosphorylation events have been recognized as pivotal regulatory processes in stomatal function under abiotic and biotic stress conditions. A serine/threonine protein kinase belonging to the SnRK2b subfamily was identified to be AB A and MeJA responsive in a proteomic analysis of B. napus guard cells. Heterologously expressed BnSnRK2b exhibited in vitro kinase activity in a manganese dependent manner and casein. Phosphorylatio n of multiple residues during the kinase autophosphorylation was detected by mass spectrometry analysis, including the serine and threonine in the activation loop. Interestingly, the in vitro autophosphorylation activity was inhibited by treatment of oxida nts, including hydrogen peroxide, oxidized glutathione (GSSG) and nitri c oxide donor S nitrosoglutath ione (GSNO). Isotope tagging analysis revealed that specific cysteines are responsive to certain treatment and contributes to the redox response. Additiona lly, the recovery of the inhibited kinase activity by reductant such as dithiothreitol (DTT) or thioredoxin isoforms indicated the in vitro autophosphylation activity is reversibly regulated by redox status of the microenvironment. Futhermore, the ability to phosphorylate a B. napus slow anion channel protein (BnSLAC1 NT) is also reversibly redox regulated. All these data suggested redox regulation of (auto)phosphorylation process in guard cell signal transduction.