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1 Florida Genetics 2007 Organizing Committee Chair: Indra Vasil Members: John Davis, Connie Mulligan, Diana Nolte, Jo hn Pastor, Michele Tennant, Thomas Yang FG2007 Sponsors University of Florida Genetics Institute, Center for Mammalian Genetics, College of Engineering, Department of Molecular Genetics and Microbiology, Evelyn F. & William L. McKnight Brain Institute, Graduate Program in Plant Molecular and Cellular Biology, Interdiscip linary Center for Biotechnology Research, UF Health Science Center Libraries Special Thanks To David Brumbaugh, Dwight Bennett, Mickey Cuthbertson, Ned Davis, Martine Horrell University of Florida Genetics Institute Director: Kenneth Berns Associate Directors: Henry Baker, Donald McCarty, Connie Mulligan, Indra Vasil Executive Board: William Allen, Henry Baker, Ke nneth Berns, Su-Shing Chen, John Davis, Robert Ferl, William Hauswirth, Julie Johnson, Donald McCarty, Mi chael Miyamoto, Connie Mulligan, Nicholas Muzyczka, Winfred Phillips, Pam Soltis, Doug las Soltis, Michele Tennant, Indra Vasil, Marta Wayne, and Thomas Yang Scientific Advisory Board: Jeffrey Bennetzen, Ph.D., Norman and Doris Giles Professor and Georgia Research Alliance Eminent Scholar, University of Ge orgia, Athens, Ga.; Ronald W. Davis, Ph.D., Director, Stanford Genome Technology Center, Stanford, Calif.; Rebecca W. Doerge, Ph.D., Professor, Departments of Agronomy and Statistics, Purdue University, West Lafayette, Ind.; Yoram Groner, Ph.D., The Dr. Barnet Berris Professor of Cancer Research, Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel; Peter M. Howley, M.D., George Fabyan Professor of Comparative Pathology and Chair, Department of Pathology, Harvard Medical School, Boston, Mass.; Eric N. Olson, Ph.D., Professor and Chairman, Department of Molecular Biology, The University of Texas Southwestern Medical Center at Dallas, Texas; Patricia G. Spear, Ph.D., Guy and Anne Youmans Professor and Chair, MicrobiologyImmunology, Northwestern Un iversity, Chicago, Ill.
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 biology and genetics. The unraveling of DNAs structure, combined with its elegant mechanism for se lf-replication and the existence of a universal genetic code for all living beings, have toge ther provided the basis for the understanding of fundamental cellular processes, mutation and genetic repair, genetic variation, the origin of life and evolution of species, and the structure/function/regulation of genes. The double helix is also proving to be of immense significance to advances in agriculture, medicine and such other di verse 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, and the sequencing of entire genomes. Yet, most knowledge able people agree that what has been achieved in DNA science thus far is only the beginning. Bigger and better applications, which w ill impact directly on the quality of human life and sustainability of life on earth, are yet to come. In orde r to attain these objectives, the digital nature of DNA and its complementarity are beginning to be exploite d for the development of biology as an informationbased science. Indeed, a paradigm shift is already taking place in our view of biology, in which the natural, physical, engineering and environmental sciences ar e becoming unified into a grand alliance for systems biology. Indeed, biology in the 21st century will be surely dominated by this expanded vision. The Genetics Institute is committed to fostering excellence in te aching and research, and in promoting cross-campus interdisciplinary interactions and collaborations. In th e pursuit of these objectives, it offers a graduate program in genetics, and has identified the following fo ur key areas for teaching, research and development: Bioinformatics, Comparative Genomics, Populati on and Statistical Genetics, and Epigenetics.
3 Florida Genetics 2007 Schedule Wednesday, November 7, 2007 Noon 6:15 p.m.: Check-in at UFs Cancer & Genetics Research Complex 1:30-1:45 p.m., Opening Remarks: Indra K. Vasil, Ph.D., and Kenneth I. Berns, M.D., Ph.D. Session I Developmental Genetics Chair: Brian Harfe, Ph.D. 1:45-2:30 p.m. Michael Snyder, Ph.D. Director, Yale Center for Genomics and Proteomics Analyze this and that: Genomes and proteomes 2:30-3 p.m. Martin Cohn, Ph.D. Associate Professor, Department of Zoology, University of Florida Molecular development of the external genitalia 3-3:45 p.m. Vicki Rosen, Ph.D. Professor and Chair, Department of Developmental Biology, Harvard School of Dental Medicine BMPs link skeletal developm ent and bone regeneration 3:45-4:15 p.m. Brian Harfe, Ph.D. Assistant Professor, Department of Molecular Ge netics and Microbiology, University of Florida Patterning a vertebrate embryo 4:15-6:15 p.m.: Poster Session and Reception
4 Thursday, November 8, 2007 8 a.m.-3:15 p.m.: Check-in at UFs Cancer & Genetics Research Complex 8-8:30 a.m.: Coffee Session II Plant Genetics and Evolution Chair: Matias Kirst, Ph.D. 8:30-9:15 a.m. Elizabeth Kellogg, Ph.D. E. Desmond Lee and Family Professor of Botanical Studies, Department of Biology, University of Missouri St. Louis Genes and morphology in diversificatio n of the cereals and their relatives 9:15-9:45 a.m. Matias Kirst, Ph.D. Assistant Professor, School of Forest Reso urces and Conservation, University of Florida Evolution of gene expressi on in flowering plants 9:45 a.m.-10:15 a.m. David Oppenheimer, Ph.D. Associate Professor, Department of Botany, University of Florida RAPs: Novel regulators of actin organization in land plants 10:15-11 a.m. Thomas G. Whitham, Ph.D. Regents' Professor of Biology, Department of Ecology and Evolution Director, Merriam-Powell Center for Environmental Research Northern Arizona University The genetic components of community structure and ecosystem processes, and their conservation implications 11 a.m.-1:30 p.m.: Poster Session and Lunch Session III: Evolutionary Genetics Chairs: Connie Mulligan, Ph.D., and Marta Wayne, Ph.D. 1:30-2:15 p.m. Henry Harpending, M.D., Ph.D. Distinguished Professor, Department of Anthropology, University of Utah Humans are evolving rapidly and the rate is accelerating 2:15-2:45 p.m.: Connie Mulligan, Ph.D. Associate Professor, Department of Anthropology, University of Florida Reconstructing human migrations: Projects from the Americas and from Africa 2:45-3:15 p.m. Marta Wayne, Ph.D. Associate Professor, Department of Zoology Director of the Graduate Program in Genetics and Genomics, University of Florida Genetical genomics and the X chromosome
5 Presentation Abstracts Analyze this and that: Genomes and proteomes Snyder M Department of Molecular and Cellular Biol ogy, Yale University, New Haven, CT A variety of approaches have been used to integrat e the functional elements of genomes of yeast and humans. Tiling arrays have been us ed to map new transcribed regions and transcriptional regulatory elements. This information has been used to construc t regulatory networks and identify key components in networks. We have also examined network evolution in related yeasts. In addition we have constructed and used proteome chips in yeast and humans to probe for novel biochemical activities, protein modification, and human disease. Integration of diverse data types re veals common regulatory modu les used by eukaryotes. Biography of Michael Snyder, Ph.D. Dr. Michael Snyder is the Lewis B. Cullman Professo r of Molecular and Cellular Biology and Professor of Molecular Biophysics and Biochemistry at Yale Universit y. He is also the Director of the Yale Center of Genomics and Proteomics. Dr. Snyder received his Ph.D. tr aining in the laboratory of Dr. Norman Davidson at the California Institute of Technology and carried out post doctoral training in Dr. Ronald Daviss laboratory at Stanford University. He is a leader in the field of f unctional genomics and proteomics. His laboratory study was the first to perform a large-scale functional genomi cs project in any organism, and currently carries out a variety of projects in the areas of genomics and pr oteomics both in yeast and humans. These include the large-scale analysis of proteins using protein microa rrays and the global mapping of the binding sites of chromosomal proteins. His laboratory built the first proteome chip for any organism and the first high resolution tiling array for the entire human genome. Dr. Snyder has published over 200 manuscripts and is editor of a number of journals including Functional and Integrative Genomics, Molecular and Cellular Proteomics, Proteomics, Drug Discovery Today, PloS Genetics and Genes and Development. He sits on many international advisory boards and was a co-founder of Protometrix Inc., a protein microarray company that was purchased by Invitrogen in 2004, and a new company, Affomix Inc.
6 BMPs link skeletal developm ent and bone regeneration Rosen V Department of Developmental Biology, Harvard School of Dental Medicine, Boston, MA Bone morphogenetic proteins (BMPs), memb ers of the transforming growth factor(TGF) super family, were originally identified based on their unique ability to induce bone formation at extraskeletal sites. Their presence in bone matrix and their osteoinductive na ture suggested that BMPs would be likely to influence bone repair, and true to that prediction, several reco mbinant human BMP proteins are currently approved for human use as bone regeneration agents. While we know quite a lot about the pharmacological actions of BMPs, our understanding of how BMPs act as endogenous mediators of skeletogenesis has been hampered by the fact that BMP signaling mediates many facets of embryonic development, so that global knockouts of individual BMPs may lead to early em bryonic lethality (BMPs 2, and 4), perinatal lethality (BMP7), or no phenotype (BMP6), in an unpredictable manner. To get ar ound this problem, we constructed a series of mice deficient singly or in combination in the ability to prod uce BMP2, BMP4 and BMP7 in the early limb. Results of these studies allowed us to conclude that there is a threshold level of BMP signaling required for proper skeletal development, with loss of individual BMP ge nes compensated for by othe r BMPs, and that postnatal limb bone growth, maintenance of bone mass, and ability to initiate fracture repair are dependent only on the presence of BMP2. Why BMP2 is central to bone homeostasis is a current focus in the lab. Biography of Vicki Rosen, Ph.D. After receiving a Ph.D. in cell biology/physiology and sp ending several years as a postdoc, Dr. Vicki Rosen started her career as an independent investigator as a scientist at a fledgling biotech company, Genetics Institute, in the fall of 1984. Her project was to identi fy the factors present in bone that were responsible for bone formation. This idea, named bone morphogenetic protein (BMP) by Dr. Marshall Urist in 1965, had remained an ill-defined concept for many years. As part of a research team that combined protein biochemistry, molecular cloning and cell biology, Dr. Ro sen and her colleagues were able to isolate the first BMPs genes and report on their act ivities in 1988. It was at this time that she became interested in the physiological roles that BMPs have in the skeleton, and the signaling pathways used by BMP proteins to exert these effects. Dr. Rosen continued to work on thes e questions at Genetics Institute until 2001 when she moved to Harvard School of Dental Medicine, shifting her lab from an industrial to an academic setting. She is currently professor and chair, Department of Developmen tal Biology. Her research remains focused on BMPs and the roles they play in musculoskeletal tissues.
7 Genes and morphology in diversification of the cereals and their relatives Kellogg EA Department of Biology, University of Missouri-St. Louis, St. Louis, MO Plant systematists have made great progress in recent years in describing phylogenetic relationships among plants, and these phylogenies are commonly used to describe patterns of morphological change through time. The morphological changes must be caused by changes in genes; identifying those genes is the challenge of evolutionary developmental genetics (ev o-devo). By focusing on a single plant family, the grasses, which contains excellent genetic and genomic resources in addition to considerable systematic knowledge, we have been able to identify genes underlyin g multiple morphological changes in the family. For example, the shift from tropical to temperate habitats that preceded the diversification of the cool-season grasses (subfamily Pooideae) correlates with developmen t of a vernalization requirement and recruitment of a floral meristem identity gene (FUL1) as a componen t of the vernalization signaling pathway. In another example, we have found that production of floral cl usters (spikelets) ending in a flower correlates with restricted expression of a different meristem identity gene (LHS1). In a third example, production of sterile branches, an evolutionary innovation of a group of se rious weeds, involves loss of meristem identity genes and truncation of an otherwise-conserved developmen tal program. Somewhat unexpectedly, comparative studies have allowed us to test hy potheses of gene function and deve lopmental role. Inference of gene function from model systems alone may be limited by available mutant alleles or reverse genetic tools, whereas evolution has produced a rich array of gene tic variation that can add to the toolkit of the developmental geneticist. Biography of Elizabeth A. Kellogg, Ph.D. Dr. Elizabeth Kellogg is the E. Desmond Lee and Family Professor of Botanical Studies at the University of Missouri-St. Louis, and an Associate of the Missour i Botanical Garden. She has spent her career studying cereal crops and their wild relatives in the grass fami ly, plants on which all of civilization depends. She has published well over 100 papers, and is co-author of a major textbook. In addition to her Ph.D. degree from Harvard University, she has been awarded an honora ry doctorate from the University of Cordoba in Argentina. She has recently received a Guggenheim Fellowship, as well as th e Chancellors Award for Research Excellence at the University of Missouri-St. Louis. She has been elected a Fellow of the American Academy for the Advancement of Science (AAAS) and has served as President of the American Society of Plant Taxonomists, and of the Society for Systematic Biology.
8 The genetic components of community structure and ecosystem processes, and their conservation implications Whitham TG Department of Biological Sciences and the Merriam-Powe ll Center for Environmental Research, Northern Arizona University, Flagstaff, AZ Because different genotypes of cott onwoods support different communities of arthropods and microbes, and affect ecosystem processes such as decomposition and nutrient cycling, these predictable effects are termed community and ecosystem phenotypes. These phenotypes are especially important to evaluate when they are expressed in foundation tree species as they are the drivers of community structure and ecosystem processes. Experimental forest studie s show that these phenotypes exhibit broad-sense heritability. The existence of these heritable phenotypes has broad implications. Fi rst, genetic diversity in co ttonwoods positively affects biodiversity. For example, the genetic diversity in stands of cottonwoods explains about 60% of the variation in the diversity of an arthropod community composed of 207 ar thropod species. Thus, the loss of genetic diversity in a common species could result in the extinction of species dependent upon those genotypes for their survival. Second, there are genetic components to ecosyste m services that explain about 50% of the variation in carbon storage, water cycles, and nutrient fluxes. Because the field of ecosystem science is largely genetics-free, it is important to understand how tree genetics affects carbon storage and other ecosystem services, which are important to the climate change debate. Third, the ef fects of climate change on the genetic structure of foundation species, is likely to alter their community and ecosystem phenotypes to affect a much larger community of organisms. Our studies with climate sensit ive insect resistant and susceptible pines suggest that ~1000 species from microbes to vertebrates have been a ffected by the recent record drought in the southwest United States. Fourth, because the phenotypes of ge netically modified organisms are likely to have community and ecosystem phenotypes, it is important to evaluate these higher order phenotypes before their release is approved. These and other findings are based upon the co mbined efforts of many colleagues and students who have been supported by an NSF FIBR grant. A key paper that illustrates the collaborative nature of our approach is: Whitham TG, Bailey JK, Schweitzer JA, Shuster SM, Bangert RK, LeRoy CJ, Lonsdorf E, Allan GJ, DiFazio SP, Potts BM, Fischer DG, Gehring CA, Lindroth RL, Marks J, Hart SC, Wimp GM, and Wooley SC: A framework for community and ecosystem genetics: From genes to ecosystems. (Nature Reviews Genetics 7:510-523, 2006). Biography of Thomas Whitham, Ph.D. Dr. Thomas Whitham grew up in a wholesale nursery in Iowa that developed new varieties of trees and shrubs that were derived from clones of somatic mutants found in the field. Observations of clonal variation in common trees and an interest in the insects found on these trees greatly influenced his education and research interests. After discovering that the distribution of insects was hi ghly sensitive to plant genotype, he began to explore how other factors such as plant development, interactions with other species, and climate change could interact with tree genotype to affect community structure and ecosyste m processes. To explore these interactions, large scale experiments were required. This led to collaborations with conservation agencies such as the Bureau of Reclamation, the Ogden Nature Center, and the Utah De partment of Natural Resources in which experimental forests were planted as part of riparian habitat rest oration. Because the underlying genetic makeup and/or pedigree of all trees used in these restoration efforts had been quantified, these landscape-level experiments allowed the cottonwood research group to map ecologically important traits and quantify the heritability of higher order traits such as biodiversity, community structure, stability and ecosystem processes. This leveraging of restoration and basic research resulted in the planting of costly landscape-level experiments that otherwise would have been difficult to establish. Th ese forests emphasize both restoratio n and a genes-to-ecosystem approach that has contributed to the emerging field of community and ecosystem genetics. This research is largely funded by an NSF Frontiers in Integrative Biological Resear ch (FIBR) grant to study the community and ecosystem genetics of poplars in the United States and parallel studies in Australia with eucalypts. Dr. Whitham has an undergraduate degree in Horticulture and Plant Pathology from Iowa State University, an M.S. in Zoology from Ohio State University, and a Ph.D. in Biology from the University of Utah. He is the recipient of the George Mercer Award from the Ecological Society of America and is a Re gents Professor at Northern Arizona University where he is the Executive Director of the Merriam-Powell Center for Environmental Research.
9 Humans are evolving rapidl y and the rate is accelerating Harpending HC Department of Anthropology, University of Utah, Salt Lake City, UT Many of us have harbored an implicit assumption th at modern humans appeared about 50,000 years ago and have not changed much since. Recent evidence from large genome surveys shows that a substantial portion of our genome is undergoing strong selection and that th e rate of change is increasing. We are not the same as our ancestors even one or several thousand years ag o. Since the spread of agriculture at the end of the Pleistocene human population sizes have increased ma nyfold and the chance of any favorable mutation occurring is proportional to population size; hence the recent speedup is probably a consequence of the size of our species. Since the great diaspora of modern humans from Africa, large scale gene flow has been restricted among major human groups so new advantag eous variants are geographically restricted. This means that continental populations are beco ming more different from each other. Biography of Henry C. Harpending, Ph.D. Dr. Henry Harpending is a distinguished professor an d the George Thomas presidential endowed chair of anthropology at the University of Utah, with current in terests in pre-industrial populations, the history of modern humans, and the evolution of human social life His most recent work includes genetic diversity within and between human populati ons where some genetic markers, such as mitochondrial DNA, microsatellites, and quantitative traits, evolve rapidly en ough to have information about size and isolation of subpopulations in the recent past. This work has foun d evidence that our species had only a few thousand members during the last interglacial and that there were several subsequent demographic expansions, the earliest among the ancestors of contemporary sub-Saha ran Africans. In addition, he has concentrated on family demographic histories with part icular interests on the effects of infectious infertility on population structure, consequences of preferential treatment of children by sex for mortality and for growth and development, and the relationships among wealth, fam ily organization, and individual reproductive outcomes. His current interests are the search for archaic human (e.g. Neanderthal) DNA in our species and the social consequences of human genetic diversity. Dr. Harpending is on the editorial boards of Human Biology, The American Journal of Human Biology, and HOMO: Journal of Comparative Human Biology. He is a faculty scholar medal awardee from Penn State University, a Fellow at the American Association for the Advancement of Science, and a member of the National Academy of Sciences. He has also been on the faculty of the University of New Mexico and Pennsylvania State University.
10 Molecular development of the external genitalia Cohn MJ* Department of Zoology and Department of Anatomy an d Cell Biology, University of Florida, Gainesville, FL Genitourinary malformations are among the most common birth defects in humans. Development of external genitalia requires tight coordination of proximodistal outgrowth, threedimensional patterning and tubular morphogenesis. Congenital malformatio ns of the penis arise when these processes are disrupted. The most common penile anomaly is hypospadias, which is characte rized by failure of urethral tube closure, and is often accompanied by abnormal dorsal-ventral patterning. Affected children can have mislocalized, multiple or oversized urethral openings, and males with severe hy pospadias are born with ambiguous genitalia. In the United States, the frequency of hypospadias double d, without explanation, from 1968 to 1993, and now affects 1:250 live births. Despite this, there is a relative ly poor understanding of the cell types that give rise to, and the molecular mechanisms that control morphoge nesis of, external genitalia. A major focus of our lab is the identification of the signals that control both outgrowth of the external genitalia and urethral tube formation. We have identified a new signaling region, the urethral epithelium, and found that its signaling activity is mediated by the Sonic hedgehog (Shh) prot ein. Shh has multiple roles in external genital development, and disruption of Shh function at different stages results in different classes of genital defect. In addition, our experiments are uncovering a complex ge ne network that functions to coordinate outgrowth and patterning of the external genitalia. The resu lts have implications for our understanding of the developmental basis of hypospadias and will be pertinent to tissue engineering and regeneration of urologic organs. Patterning a vertebrate embryo Harfe BD* Department of Molecular Genetics and Microb iology, University of Florida, Gainesville, FL How an individual cell decides what type of cell it will become has been a fundamental problem in biology for decades. The improper specification of just a single cell can have catastrophic consequences for the developing embryo. One of the goals of my laboratorys research is to elucidate how digits form in the developing limb using both the mouse and chick mode l systems. Two classes of molecules, the bone morphogenetic proteins (Bmps) and Sonic Hedgehog (Shh), are thought to play a role in this process. Using limb-specific mouse knockouts of these molecules we ha ve uncovered novel roles for these proteins in limb development. My laboratory is also interested in the role microRNAs play in development. Using a conditional allele of Dicer we constructed, we removed Dicer in a tissue-specific manner during mouse development. In these animals, numerous defects were observed demonstrating the important role microRNAs play in vertebrate development. The laborato ry has also begun to examine the molecular pathways responsible for intervertebral disk development using the mouse model system. = UF Genetics Institute faculty
11 Evolution of gene expressi on in flowering plants Kirst M* School of Forest Resources and Conservati on, University of Florida, Gainesville, FL The sequencing of the Populus trichocarpa genome creates the first opportuni ty to describe the complete transcriptome of a woody perennial species and compare it to the herbaceous model plant Arabidopsis thaliana. We characterized gene expression in a broad range of organs from the P. trichocarpa sequenced genotype Nisqually-1 and identified extensive transc ription diversification in paralogues, suggesting neofunctionalization, particularly in woody organs. A comparison to A. thaliana revealed very limited conservation in the expression pattern of orthologs, in dicating rapid evolution of gene function measured by gene expression variation. However, the expression of a small set of orthologs has remained surprisingly conserved in the two species, despit e separation of lineages for over 100 million years. These include genes implicated in essential plant mechanisms such as ph otosynthesis, as well as ge nes expressed in a broad range of plant organs in both species. For these genes there appears to be strong selection against diversification in transcription regulation. RAPs: Novel regulators of actin organization in land plants Oppenheimer DG* Department of Botany and the Graduate Program in Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL Control of actin dynamics underlies many processes ba sic to life including cellular movement and membrane trafficking. In plants, the actin cytoskeleton plays an im portant role in controlling the direction of cell growth in expanding cells. To further understand how regulation of actin dynamics controls plant cell expansion, we screened for mutants that affected the ex pansion of epidermal hairs (trichomes) on Arabidopsis plants, and examined these mutants for changes in actin organiza tion by immunofluorescence and confocal microscopy. One of these mutants, irregular trichome branch 3 (itb3), showed dramatic changes in actin organization during trichome development including an overabundance of actin cables and the formation of actin rings. Cloning of the ITB3 gene revealed that it encoded a novel plant-specific protein of unknown function that is conserved in land plants but not found in algae, fungi or animals. To identify proteins that interact with ITB3, we conducted a yeast two-hybrid screen using ITB3 as bait. We identified a plant homolog of actin depolymerizing factor/cofilin (ADF) as a potential bindin g partner of ITB3. The interaction of ITB3 with ADF was confirmed in vitro. We named this family of proteins RAPs for Regulators of ADF in Plants. RAPs comprise a large gene family in plants; Arabidopsis has 22 genes encoding RAPs, and rice has 33 RAP family members. ADF genes are also found in multiple copies in plant genomes with 12 genes present in Arabidopsis. We examined insertional mutation resources of Arabidopsis for mutants representing other members of the RAP and ADF families. Trichome phenotypes of these mutant s suggest that specific RAPs interact with specific ADF family members to regulate particular aspects of tr ichome cell morphogenesis. This work was supported by a grant from NSF (IOB 0352916)
12 Reconstructing human migrations: Projects from the Americas and from Africa Mulligan CA* Department of Anthropology, University of Florida, Gainesville, FL Anatomically modern humans have colonized all corners of the globe. These colonization events represent a wide range of processes from relatively recent and simp le, such as the single expansion into the Americas ~15,000 years ago, to ancient and highly complex, su ch as the initial migration out of Africa ~50,000100,000 years ago followed by multiple back-migrations an d expansions throughout the Horn of Africa and Arabia. Our research on the Americas suggests there was a long period (~20,000 years) of population equilibrium and genetic diversification in greater Beringia prior to a rapid expansion into the Americas. On the other side of the planet, our resear ch on Middle Eastern populations reve als that Bedouin, Palestinian, and Yemeni groups are genetically intermediate between African and other Old World populations. However, preliminary results suggest that th ese populations may not be equiva lently intermediate, thus we are performing simulation analyses to distinguish among anci ent isolation by distance, pre-historic admixture, and recent admixture. Investigation of two divergent co lonization scenarios informs our understanding of migrations in the study areas as well as migration processes in general. Genetical genomics and the X chromosome Wayne M* Department of Zoology, University of Florida, Gainesville, FL The maintenance of genetic variation remains a challenge throughout evolutionary genetics, but especially for the genetic variation of gene expression. Ironically, the complication likely stems from the fact that we can actually describe the mechanism of phenotypic variat ion at the molecular level for gene expression, in contrast to higher order phenotypic traits. Va riation in gene expression must result from cis, trans, and cis x trans effects; and each of these parameters may have its own genetic architecture, for each gene. The mechanisms and inheritance of sex differences in gene expression are of particular interest, because sexual dimorphism arises in large part from sex-specific tran scription, given that females and males share all genes except those on the Y chromosome. However, in flies and hum ans, males are hemizygous for the X, while females are not. This constrains the mode of inheritance for gene expression both for X-linked genes, and for autosomal genes with major X-linked transcription factors. The evolutionary implications of sex-specific mode of inheritance for expression include a molecular mechan ism for frequent observations of faster evolution in males.
13 Posters = UF Genetics Institute faculty 1. Phylogeny and diversification of rosids inferred from the plastid inverted repeat Alexandre R2, Brockington SF1, Moore MJ1, Soltis PS2,*, Soltis DS1,* 1Department of Botany, Universit y of Florida, Gainesville, FL 2Florida Museum of Natural History, University of Florida, Gainesville, FL Previous molecular phylogenetic analyses of the angi osperm clade, the rosids, employing approximately ~ 5000 base pairs, recovered a phylogeny in which many of the deeper level relationships were poorly supported. To clarify and provide support for interfamilial relationships with in the rosids, we amplified and sequenced the entire chloroplast inverted repeat (~26,000 bp) for 32 taxa. Th e inverted repeat was sequenced using the PCR-based method ASAP. This was combined with a dataset from pub lished chloroplast sequences to create a total dataset of 60 taxa. Various phylogenetic methods were used to ana lyze the data matrix including Parsimony and Maximum Likelihood. These analyses revealed that Vitaceae are si ster to all other rosids; the remainder form two large clades. Many poorly supported lineages received increased support as indicated by higher bootstrap values. The slower evolving plastid genes associated with the invert ed repeat of the chloroplast region proved useful in providing support and resolution at deeper levels within this clade. 2. Emerging trends in the evolutio n of primary endosymbiotic bacteria Allen JA1,2, Light JE2, Perotti MA3, Braig HR3, Reed DL2 1Department of Zoology, University of Florida, Gainesville, FL 2Florida Museum of Natural History, University of Florida, Gainesville, FL 3School of Biological Sciences, University of Wales, Bangor Primary endosymbiotic bacteria are thought to have increa sed the diversity of insects by supplementing their diet and enabling them to radiate into nutrient poor niches. Primary endosymbionts (p-endosymbionts) live their entire life cycle within the insect and are transmitted to the ne xt generation of hosts thro ugh the maternal lineage. Pendosymbionts also evolve faster (i.e., accumulate nucleoti de substitutions faster) than their insect hosts and other closely related but free-living bacteria. Th is faster rate has traditionally been at tributed to small population sizes, lack of recombination, genetic drift, and, more recently, re duced efficacy of selection. Based on original studies of the aphid p-endosymbiont (Buchnera), it has traditionally been thought that 16S rRNA of p-endosymbionts evolves at a rate of roughly 1-2% per 50 million years. More rece ntly, other host/endosymbiont assemblages have exhibited rates of evolution that greatly exceed this range, such as Blochmannia (ant endosymbiont), which is evolving at a rate 14 times faster than previously examined p-endosy mbionts. We have sequenced the 16S rRNA gene from Riesia, the p-endosymbiont in primate sucking lice, and found that it is evolving over 30 times faster than the traditional rate. When comparing rates of evolution amon g all characterized insect p-endosymbionts (including endosymbionts from aphids, co ckroaches, tsetse flies, whit eflies, psyllids, ants and lice), we find that the younger assemblages are evolving at a much faster rate than the host/endosymbiont assemblages that have older, longer term, associations. These results suggest an initial rate increase at the beginning of p-endosymbiont symbiosis with its insect host and a stabilization of the rate as the association ages.
14 3. Proteomics of redox regulated proteins in methyl jasmonate treated plants Alvarez S, Wilson G, Zhu M, Chen S* Department of Botany, University of Florida, Gainesville, FL Methyl jasmonate (MeJA) is a signaling molecule that play s a key role in the regulation of metabolic processes, reproduction and defense against insects and pathogens. It has been shown that MeJA induces accumulation of reactive oxygen species (ROS) by regulating the activities of antioxidant enzymes. ROS such as hydrogen peroxide may act through redox regulated protei ns to activate or inactivate diffe rent signaling pathways as well as transcription factors, which in turn regulate gene expres sion and cell-cycle processes. In this study, we aim to investigate the redox regulated proteins in methyl jasm onate treated plants using proteomic approaches. The proteins identified act downstream of MeJA and are possibly involved in plant defense processes. Here we report a proteomic approach used to identify redox regulated proteins in Arabidopsis leaves in response to a 24h treatment with MeJA. Several proteins involved in ROS detoxification we re identified. The implications of the changes of these proteins were discussed. In addition, we were able to map the disulfide bonds in several proteins using the monobromobimane labeling technology. 4. Is collagen present in Limulus polyphemus? Arce TR1, Zhang GJ1 and Cohn MJ1,2,* 1Department of Zoology, University of Florida, Gainesville, FL 2Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL In 2006, Zhang and Cohn found type II colla gen in lamprey. Their follow-up findi ng of type II collagen in hagfish, and amphiColA in lancelets suggested that the duplication and divergence of clade A collagen led to the origin of vertebrate chondrocytes, and thus a means for cartilage de velopment in these stem vertebrates. The main focus, thus far, has been on collagenous cartilage in vertebrate lineages. Cartilage, or cartilage -like tissue, has been found in many invertebrate phyla as well, including Cnidaria Annelida, Inarticulata, Mollusca, and Arthropoda. An arthropod, Limulus polyphemus, has been reported to have non-collage nous based cartilage. In this study we attempt to determine the presence of clade A fibrilla r collagen and SoxE genes in the horseshoe crab (Limulus polyphemus). We performed Mason Trichrome staining, proteonomic analysis of protein extract to screen for collagen, and initial immunohistochemical experiments on lim ulus. Our initial results have shown what appear to be chondrocytes in limulus gill tissue. Proteonomic analysis, as well as preliminary immunohistochemical study, has not confirmed the presence of collagen in li mulus tissue extract. In an attempt to identify additional non-vertebrate organisms that possess fibrilar collagen we utilized Mason Trichrome tissue analysis on the channeled apple snail (Pomacea paludosa). We will present our results and future studie s that will be needed to further explore the presence of collagen in invertebrates.
15 5. Conservation genetics of the endangered Okaloosa darter Austin JD1,2,*, Johnson A1, Jelks H3, Tate B4, Jordan F5 1Department of Wildlife Ecology and Conservation, University of Florida, Gainesville, FL 2Department of Fisheries and Aquatic Sciences, University of Florida, Gainesville, FL 3U.S. Geological Survey, Gainesville, FL 4U.S. Fish and Wildlife Service, Niceville, FL 5Department of Biological Sciences, Lo yola University, New Orleans, LA Molecular genetics play an important role in the management of threatened and endangered species. The endangered Okaloosa darter (Etheostoma okaloosae) occurs in only six streams that drain into two Choctawhatchee Bay bayous in northwest Florida. The Okaloosa darter was listed in 1973 due to its limited distribution, habitat degradation and loss, and concerns over competitio n with the common and widespread brown darter (E. edwini). Our research incorporates mitochondrial and nuclear ma rkers to examine demographic parameters and historic versus contemporary connectivity among creek and drai nage populations. We present initial mtDNA results (complete cytochrome b gene sequences) focusing on 1) the population history of brown darters within the Okaloosa darter range and 2) the population structure and level of isolation among the six Okaloosa darter creeks. Preliminary results based on mtDNA sequence data in corporating coalescent an d phylogenetic approaches, suggests that significant population structure exists amon g streams. Further, brown darters found in the lower reaches of the Okaloosa native streams are likely not introd uced as was assumed when the darter was first listed. 6. An epistatic model for mapping phenotypic plasticity of a count trait Berg A1, Drost D2, Novaes E2, Kirst M2, *, Wu R1,* 1Department of Statistics, University of Florida, Gainesville, FL 2School of Forest Resources and Conservation, University of Florida, Gainesville, FL Different expression of a given genotype in morphology physiology, and anatomy over changing environments is called phenotypic plasticity. The understanding of the gene tic basis of phenotypic plasticity has been one of the most important challenges facing modern biology. The fo rmation of phenotypic plasticity may be due to higher homozygosity at certain gene s (homeostasis hypothesis), environment-dep endent expression of the loci (allelic sensitivity hypothesis), or epistatic interactions between plasticity genes and trait genes (gene regulation hypothesis). Testing these three hypothes es requires different genetic designs an d statistical models. In this study, we develop a statistical model for detecting specific quanti tative trait loci (QTLs) that stimulate the phenotypic plasticity of a count trait through genetic regulations. Th e model was derived with a bivariate Poisson distribution with one variable for phenotypic means over different environments and the other for phenotypic differences between the same pair of environments. A multi-QTL model was implemented into a general mixture model framework, allowing the tests of ho w the same QTL affects the means and differences and whether the epistasis between different QTLs contribute to phenotypic plasticity A real example for the number of sylleptic branches on the main stems of poplar hybrids was used to elucidate the interpretation and utility of our model. The new model will provide a useful tool for dictating the picture of geno me by environment interactions that determine the final phenotypes of complex count traits.
16 7. Evolution and diversity of invertase genes in Populus trichocarpa Bocock PN1, Dervinis C2, Davis JM1,2,* 1Graduate Program in Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL 2School of Forest Resources and Conservation, University of Florida, Gainesville, FL Invertase plays a key role in carbon utilization as it catalyz es the irreversible hydrolysis of sucrose into glucose and fructose. The invertase family in plants is composed of two sub-families thought to have distinct evolutionary origins. These sub-families are distinguished by their pH opti ma for activity: acid invertases apparently originated in eubacteria and are targeted to the cell wall and vacuole, while neutral/alkaline invertas es apparently originated in cyanobacteria and function in the cytosol. The recently sequenced genome of Populus trichocharpa allows us to identify the genes encoding invertase in an economically im portant plant system, in order to understand both the evolutionary development of this family as well as its role in carbon allocation and partitioning in forest trees. Here we describe the identification of eight acid invertase genes; three of which belong to the vacuolar targeted group (PtVIN1-3), and five of which belong to the cell wall ta rgeted group (PtCIN1-5). Similarly, we report the identification of 16 neutral/alkaline invertase genes (PtN IN1-16). An examination of the micro-syntenic regions surrounding the poplar invertase genes reveals extensive colinearity with Arabidopsis invertases. Expression analysis using both whole genome microarrays as well as quantitative RT-PCR reveals a general conservation of expression profiles of genes encoding acid and neutral invertase in response to auxin, nitrogen, and wounding treatments, and during development. We also find evidence for expression of a novel intronless vacuolar invertase (PtVIN1), which apparently arose through reverse transcript ion of a processed PtVIN2 transcript that subsequently inserted in the genome. To our knowledge this is the first intronless invertase found in plants. 8. Sonic Hedgehog signaling in the apical ectodermal ridge is essential for proper formation of the vertebrate limb Bouldin CM1, Gritli-Linde A2, Scott WJ Jr.3, Harfe BD1,* 1Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2Department of Oral Biochemistry, Sahlgrenska Academy at Gteborg University, Gteborg, Sweden 3Division of Developmental Biology, Cinci nnati Childrens Hospit al, Cincinnati, OH The vertebrate limb develops along th ree axes: proximo-distal, dorso-ventral and antero-posterior. Proximo-distal patterning is regulated by the apical ectodermal ridge (AER ); antero-posterior patterning is controlled by the zone of polarizing activity (ZPA); and the dorso-ventral axis is determined by signals from the non-AER ectoderm. Sonic Hedgehog (SHH), a protein secreted from the ZPA, has be en shown to be both necessary and sufficient for ZPA function. Through the analysis of Shh target genes, for example Ptc1 and Gli1, it has become clear that the Shh signaling cascade is active in the limb mesoderm. Recent ly, array-based experiments have found that a number of target genes of the Shh signaling pathway were also present in the AER. For Shh signaling to occur in the limb AER, SHH protein must also be detectable in this region. Imm unohistochemistry using an anti-SHH antibody revealed the presence of SHH protein in the limb bud ectoderm. In addi tion, we have found that exceptionally low levels of at least one Shh target gene can be detected in the AER by methods not based on array technology. The potential role Shh signaling may play in the limb AER is unknown. To determine if Shh signaling within the AER plays a role in limb patterning, we used a conditional knockout approach to remove Shh signaling specifically from this region of the limb. Removal of Shh signaling from the AER resulted in the production of an extra digit, indicating that Shh signaling within the AER is required for proper format ion of the vertebrate limb. We are currently performing additional transgenic manipulations and using whole mount in situ hybridizations to elucidate the function of Shh signaling in the AER.
17 9. Comparison of the chondrogenic potential of BM P-4, BMP-7, IHH, Sox-9, and CTGF when delivered to mesenchymal stem cells as transgenes Bush ML, Anantharaman A, Levings PP, Kay JD, Watson RS, Gouze JN, Gouze E*, Dacanay EA, Currie TP1, Ghivizzani SC* Department of Orthopaedics and Rehabilita tion, University of Florida, Gainesville, FL Articular cartilage is a highly specialized tissue that protects the bones of diarthrodial joints from forces associated with load bearing, friction, and impact; however, there is lim ited capacity for regeneration in response to injury or disease. Adult mesenchymal progenitor cells (mesenchym al stem cells; MSCs) are readily available from many tissue sources and are capable of differentiating along mult iple lineages; thus, they are well-suited for cell-based therapies for cartilage repair. Gene transfer is a viable me thod of achieving sustained, local expression of specific protein factors, and it may be useful for in ducing chondrogenic differentiation of MSCs in vivo. Potent chondroinduction of MSCs in high density aggregate cult ure was previously observed following delivery of cDNAs for TGF1 and BMP-2. In this study, we have expanded these analyses to include cDNAs whose protein products are associated with chondrogenic differentiation during deve lopment, which include BMP-4, BMP-7, Indian hedgehog (Ihh), Sox-9, and connective tissue gr owth factor (CTGF). Using a high-density aggregate culture system, we performed a series of assays to examine the capacity of these candidate transgenes to induce chondrogenesis of bovine MSCs. We infected individual flasks of early pass age bovine MSCs with adenoviral vectors carrying the cDNAs for each transgene at doses ranging from 100 vp/ce ll to 10,000 vp/cell. At 24 hours post-infection, 2.0 x 105 cells were pelleted by centrifugation, and the resulting cell aggregates were cultured for 21 days in serum-free chondrogenic medium. Chondrogenesis wa s evaluated by examining aggregate mo rphology, histological staining for proteoglycans and by immunohistochemical analysis for co llagen types I and II. The grea test biological responses for each transgene were observed in the dose range of 100-1000 vp/cell; higher viral doses appeared to inhibit chondrogenesis. Robust chondrogenesis was observed fo llowing delivery of BMP-4, BMP-7, Ihh, and Sox-9; however, qualitative differences were evident among the treatment groups. 10. Spatial and temporal patterns in the flor al transcriptome of the basal angiosperm, Persea americana (avocado): an exploration of ancient whorls Chanderbali AS1, Altman N2, Soltis DE1,*, Soltis PS3,* 1Department of Botany, Universit y of Florida, Gainesville, FL 2Department of Statistics, Pennsylvania State University, University Park, PA 3Florida Museum of Natural History, University of Florida, Gainesville, FL The first transcriptional profiling of flowers in a basal angiosperm, Persea americana (avocado), has revealed a number of potentially fundamental elements as well as the novelties we expect of separate evolutionary experiments in flower development. Using custom micr oarrays we have identified 1081 genes with at least twofold up-regulation in particular floral organs and/or develo pmental stages relative to leaves. Hierarchical clustering suggests that these assemble into four regulatory units that accommodate homologs of AG, AP3 and PI, AGL6, and AP1, respectively. Members of the AG cluster are expresse d predominantly in stamens and carpels, often also in fruit, conceivably contributing to C-function in an evol utionarily conserved reproduct ive developmental program. Bfunction may be provided by members of the AP3/PI clus ter, most of which are expressed primarily in stamens, many also in the perianth, or in all the floral organs Also present are homologs of components of the SCFUFO complex, suggesting conservation in the promotion of AP3 and PI expression across angiosperms. The genetic regulators of perianth development may be found among the members of the AGL6 cluster. The transcriptional profiles of the two, morphologically alike tepal whorls are nearly identical, an d correlate strongly with the stamen transcriptome, conceivably, perhaps, harboring regula tory networks inherited from a stamen developmental program. If so, Persea tepals provide an unprecedented opportunity to examine the evolutionary genetics of andropetaloidy; perianth evolution through stamen sterilization. All Persea genes expressed primarily in inflorescence bud were assigned to the AP1 cluster, indicati ng potential evolutionary conservation in role of AP1 in floral meristem specification, but not A-function Comparisons with floral microarray data for Arabidopsis found few instances of potential evolutionary conservation in the downstream regulation of flo ral organ development, mostly relating to stamen and carpel developmental genetics, but in Persea, expression often extends to the tepals.
18 11. Genetic analysis of BMP2 and BMP4 in the limb apical ectoderm ridge Choi KS, Maatouk DM, Harfe BD* Department of Molecular Genetics and Microb iology, University of Florida, Gainesville, FL During early limb development the apical ectodermal ri dge (AER) expresses a number of signaling proteins including bone morphogenetic proteins (BMPs) 2, 4 and 7. BMPs in the limb are expressed in both the mesoderm and the ectoderm and have been proposed to play a role in anteroposterior digit patterning. To date, it has not been possible to directly examine the role Bmp ligands pl ay in limb development since mice deficient in BMP2 or BMP4 die before digit condensation occurs. Previous st udies have circumvented this problem by conditionally removing a BMP receptor (BMPR-IA) in the limb mesoderm or ectoderm or expressing a BMP antagonist (noggin) in the AER. The functions of each individual BMP remains unkno wn because each of these previous studies blocked all BMP signaling. Our studies take advantage of an Msx2-cre ectoderm-specific transgene to delete Bmp2 and 4 in the AER. Double knockout of Bmp2 and Bmp4 in the ectoderm results in limb polydactyly, syndactyly and interdigital webbing due to decreased programmed cell death (PCD ) and to increased cell proliferation. Using RNA in situ hybridization we showed that the expression domains of Fgf4 and Fgf8 in the AER were expanded spatially and temporally in double mutants. Further RNA in situ analysis showed the expression domains of 5 Hox genes were expanded anteriorly in the limb mesenchyme. Wnt7a and Lmx1b expression were normal in dorsal ectoderm and mesoderm, respectively. However, Engrailed 1 (En1) expressi on was absent in the ventral half of the AER although there was En1 expression in ventral ectoderm. Thus, En1 ex pression in the AER is not responsible for dorsoventral patterning. These results demonstrate that ectodermal BM P2 and 4 are partially functionally redundant in the limb ectoderm, function in the cell autonomously. and play an important role in interdigital PCD and anteroposterior patterning of the developing limb. 12. Are you taking full advantage of services offered by the GAL? Clark AM, Gomez A, Clark HC Interdisciplinary Center for Biotechnology Re search, University of Florida, Gainesville, FL The ICBR Genetic Analysis Laboratory (GAL) is known for its ability to do high quality DNA extractions and PCR reactions. DNA is extracted from many different sources (blood, tissue, bone s, teeth, etc.) and amplified using PCR for sequence analyses of mitochondrial and nuclear DN A and for fragment analyses. The lab designs new and optimizes or adapts published protocols to meet our clients needs. A very powerful tool that we offer to our clients is a microsatellite library enriched for di-, tri-, and tetranucleotide repeats for th eir organism of interest. Microsatellites are a class of DNA that consists of repeat ed DNA, i.e., CACAn or AGTAGTn. Since these repeats are biparentally inherited, they can be used to determine gene flow, fine scale po pulation structure, individual identity, and have been associated with some diseases such as H untingtons disease. A microsat ellite library incorporates a series of molecular techniques that must all be succes sfully completed to find loci that can be shown to be polymorphic. The individual services offered at the GAL, along with enriched microsatellite library production, include DNA isolations, PCR reactions (small and large sc ale), PCR cleanups for cycle sequencing, and fragment analysis of microsatellites, T-cell respon se, AFLPs, etc. In this poster, we will describe some of the more common services offered to clients and illustrate their va lue by highlighting their use in some projects.
19 13. Expression of Sle1a is required for observed T cell-intrinsic phenotypes Cuda CM1, Sobel ES2, Morel LM1 1Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, FL 2Department of Rheumatology, University of Florida, Gainesville, FL Patients suffering from Systemic Lupus Erythematosus pr esent with several manifestations, one of which being a decrease in the number of circulating regulatory T cells (T regs). However, it has been shown that the suppressive capacity of the remaining population is maintained. Loss of tolerance to nuclear antigens is associated with major lupus susceptibility locus Sle1 in the NZM2410 lupus model, comprising three independent loci (Sle1a, Sle1b, and Sle1c). Sle1-expressing T cells exhibit increased levels of ac tivation associated with increased levels of proliferation and cytokine production. The Sle1 locus is associated with a decreased level of Tregs as well as decreased expression of the transcription factor Foxp3 among this population preceding autoantibody production. This phenotype is accounted for by Sle1a. However, it was nece ssary to assess whether expression of Sle1a in T cells is required to produce the observed phenotype. While expression of the Sle1a interval results in decreased Treg levels as well as Foxp3 expression among this population, with a truncated region of Sle1a (Sle1a(15)) presenting intermediate levels, these cells maintain their suppressive capacity and show no difference regarding expression of other markers associated with the regulatory phenotype as compared to B6. Flow cytometric data from bonemarrow mixed chimeras only show enhanced proliferation and activation of the Sle1a-expressing T cells, as well as a decrease in Treg levels of B6.Sle1a origin. These data in dicate that expression of Sle1a results in T cell intrinsic activation. In addition, the observed decrease only in Tr egs expressing Sle1a suggests that Sle1a regulates Treg levels in a T cell specific manner. By examining Sle1a( 15), the observed intermediate phenotype suggests the presence of two genes within the Sle1a region contributing to the break in self-tolerance, and that this may be associated with decreased levels of Tregs. 14. A genetic model for predicting the incidence of cancer with age Kiranmoy D Department of Statistics, University of Florida, Gainesville, FL Cancer incidence increases with age. This epidemiologi cal pattern of cancer incide nce can be attributed to molecular and cellular processes for individual subjects. As an inherited disease, genes are thought to play a central role in shaping the incidence of cancer with age. In this article, we derived a dynamic statistical model for explaining the epidemiological pattern of cancer incidence based on individual genes that regulate cancer formation and progression. We incorporate the mathematical equation s of age-specific cancer incidence into a framework for functional mapping aimed to identify QTLs for dynamic ch anges of a complex trait. Th e mathematical parameters that specify differences in the curve of cancer incidence among QTL genotypes are estima ted within the context of maximum likelihood. We provide a series of testable quantita tive hypotheses about the initiation and duration of genetic expression for QTLs involved in cancer progre ssion. Computer simulation was used to examine the statistical behavior of the model. The new model proposed will provide a powerful tool to detect genes for cancer progression and a general framework for explaining the epidemiological pattern of cancer incidence.
20 15. Suppressor mutants of NPR1 restore salicylic acid tolerance and pathogen resistance in Arabidopsis thaliana DeFraia C, Zhang X, Mou Z* Department of Microbiology and Cell Scienc e, University of Florida, Gainesville, FL In plants, pathogen recognition triggers activation of defe nses, resulting in resistance. Signal transduction of the pathogen signal is essential for transcriptional reprogra mming and defense activation. NPR1 is a key component in several of these signaling pathways, and is essential for pa thogen resistance. The small molecule salicylic acid (SA) is another essential component of plant defense signalin g, and activates NPR1 followi ng pathogen recognition. NPR1 in turn controls SA synthesis and is essential for alleviating SA-related cytotoxic effects, though how NPR1 accomplishes this is unknown. In this study we took advantage of the toxic effects of SA on npr1 seedlings and isolated several suppressors of this mutation. Suppr essor mutants displayed several phenotypes including restoration of SA tolerance and pathogen resistance. Effo rts to identify these mutations by map-based cloning are underway. Identification of new signaling components thro ugh this approach will enhance our understanding of plant defense activation, and contribute to reducing harmful effects of pathogens on important crops. 16. Whole genome sequence analysis of the bacterial endophyte, Enterobacter cloacae P101 Drew JC, Triplett EW* Department of Microbiology and Cell Scienc e, University of Florida, Gainesville, FL Enterobacter cloacae P101 is a bacterial endophyte. Endophytes, which are microorganisms that colonize plants, can provide beneficial effects to their hosts such as incr eased plant growth and mineral absorption. The P101 strain was first isolated from a biomass crop known as Panicum virgatum L, and plants inoculated with P101 demonstrate increased growth versus wild type plants. To date, no Enterobacter or endophyte genome has been published and unlocking the genetic information will lead to an increase d understanding of how endophytes can provide benefits to their hosts. The Enterobacter cloacae P101 genomic DNA was isolated and sequenced with the Genome Sequencer 20 from 454 Life Sciences at the University of Florida Interdisciplinary Center for Biotechnology Research. Several sequencing runs generated 2 million in dividual sequence reads that total to 141 Mb of raw sequence data. Using the GS Paired End adaptors te chnology, 371,000 paired end reads were added to the sequence data to streamline genome assembly. The as sembly software, Newbler, a ssembled the reads into 520 contigs. The paired end read data wa s used by Newbler to assemble the contigs into 37 scaffolds. The average scaffold size is 143 kb and the scaffolds total 5.3 Mbp in length. The P101 genome size is estimated to be 5.25.4 Mbp in length. The assembled contigs and scaffolds will be verified with alternative genome assemblers: PCAP and SeqMan from DNASTAR. Genome closure is in progress and a variety of methods including PCR and Sanger sequencing are being used to close the gaps between sc affolds. Initial annotation of scaffolds by FGENESB identified 5,856 open reading frames. The sequence of the P101 genome will provide a valuable resource for comparative genomics and gene organization as a model for other Enterobacter species.
21 17. Resolving gene families, nearly-identical pa ralogs, and novel transcripts using 454-based 3'UTR profiling Eveland AL1,2, Kirst M1,3,*, McCarty DR1,2,*, Koch KE1,2,* 1Graduate Program in Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL 2Department of Horticultural Sciences, University of Florida, Gainesville, FL 3School of Forest Resources and Conservation, University of Florida, Gainesville, FL Differences in gene expression underlie central questions in eukaryotic biology extending from gene function to evolutionary mechanisms and quantitative traits. Howeve r, resolving expression of closely-related genes (e.g. alleles and gene family members) is challenging on a geno me-wide scale due to extensive sequence similarity and frequently incomplete genomic sequence data. We presen t a new expression-profiling strategy that utilizes longread, high-throughput sequencing to capture the informatio n-rich 3'-untranslated region (UTR) of mRNAs. Resulting sequences resolve gene-specific transcripts independent of a sequenced genome. Analysis of ~229,000 3'-anchored sequences from maize (Zea mays L) ovaries identified 14,822 unique transcripts represented by 2 sequence reads (consensus sequences). Total RNA from ovaries of drought-st ressed wild-type and viviparous-1 mutant plants was used to construct a multiplex cDNA library. Each sa mple was labeled by incorporating one of 16 unique threebase key codes into the 3'-cDNA fragments, and combin ed samples were sequenced using a GS 20 454 instrument. Transcript abundance was quantified by frequency of se quences identifying each unique mRNA. The 3'UTR profile resolved 12 unique transcripts representing nine Cellulose Synthase (CesA) gene family members in maize ovaries. In addition, this method distinguished near ly-identical paralogs, as illustrated by two Auxin Repressed Dormancy Associated transcripts, which showed reciprocal mRNA abundan ce in wild-type and mutant samples. We further evaluated 454-based 3'UTR profiling for global transcript ome analysis in parallel with a microarray approach. A multiplexed library of 12 samples representing four se quential stages of maize ovary development generated ~578,000 good-quality 3'-anchored sequences (including ~23,000 unique consensus sequences) using the FLX 454 technology. Resolution of nearly-identical paralogs, ident ification of 3'-RNA processing variants, and quantitative profiles of transcriptional changes over a dynamic range of expression provided an invaluable complement to microarray analyses. In addition, recovery of newly-en countered, non-arrayed genes enabled flexibility for gene discovery in a non-sequenced genome. 18. A putative role for RNA splicing in maize endosperm-embryo developmental interactions Fajardo D1, Gomez E2,, Royo J2, Tseung CW1, Martin F1, Hueros G2, Settles AM1,* 1Graduate Program in Plant Molecular and Cellular Biolog y Program and Department of Horticultural Sciences, University of Florida, Gainesville, FL 2Universidad Alcala de Henares, Spain Endosperm-embryo interactions are an important but p oorly understood aspect of seed development. The Rough endosperm3 (Rgh3) locus is involved in these interactions at a developmental level. Rgh3 seed mosaics marked with the pr1 anthocyanin gene indicate that Rgh3 is requ ired in the endosperm for the normal development of the embryo. Rgh3 also has an autonomous function in the embr yo and is required for seedling viability. We identified a tightly-linked transposon-tag from the rgh3-70 allele. This transposon insertion disrupts a predicted splicing factor with a U2 snRNP auxiliary factor homology motif (UHM). UHMs are RNA recognition mo tif (RRM)-like domains that function in protein-protein interactions. Analysis of Rg h3 cDNAs and genomic sequences suggest alternative spliced transcripts of Rgh3. Maize ESTs indicated that Rgh3 is expressed in endosperm tissue. Consistent with this expression pattern, the rgh3 phenotype is characterized by an overproliferation of the aleurone cells and aberrant development of the basal endosperm transfer cell layer. An analysis with endosperm cell type specific markers in mutant rgh3 seeds suggests that the basal endosperm transfer cell defect occurs after cell type specification. Rgh3 endosperm cultures grew forming homozygous mutant callu s. This data further supports Rgh3 possible role in cellular differentiation. Rgh3 maps to the long arm of chromosome 5. Complementation tests with other seed mutants mapped to the same region indicated that Rgh3 is a novel locus. Our data suggest that RNA splicing may have an important role in endosper m-embryo developmental interactions.
22 19. Discovery, the structure of haplotype blocks, and cladistic analyses of polymorphisms of natriuretic peptide receptor A (NPR1) gene Hua Feng H, Taimour L, Burkley B, Johnson JA* Department of Pharmacy Practice and UF Center for Pharmacogenomics, University of Florida, Gainesville, FL Genetic variation is increasingly recogniz ed as an important contributor to co mplex diseases. The variation of NPR1 gene has been suggested to be a potential risk factor with hypertension and cardiac hypertrophy. Systematically characterizing the variation of NPR1 leads to localization of the susceptibility disease sites with two approaches. An approach focuses on one marker at a time to test pote ntially causal variants directly; another one types several markers to locate causal loci via a ssociation between loci. The second approach utilizes the property of linkage disequilibrium and the structure of haplotype blocks. Haplot ypic methods play a powerful role at detecting casual variants, but the larger number of halotypes, the less po wer of locating the haplotypes having disease susceptibility sites. However, a cladistic approach can group haplotypes in nested clades, therefore to increase the power identifying clades containing potential candidate disease susc eptibility sites, consequentially, to pinpoint the causal site in those clades. In our study, we identified and charac terized 26 SNPs in the 5 UTR, exons, intron, and 3 UTR of NPR1 using DHPLC in combination with big-dye term inator chemistry-based direct sequencing from four populations including Caucasian, African American, Chinese and Native American. The results of functional analysis show that four non-synonymous SNPs and one synonymous SNP have medium to high risk levels of developing disease. Based on imperfect phylogeny, four blocks were identified in the combined population. The cladistic approach has shown the advantage to group the haplotypes into subgroups. The statisti cal significant analysis was also applied to test the significant difference of LD betw een populations. A lack of linkage disequilibrium might discourage us to apply haplotype ana lysis in association stud y. However, our study may present a novel way to detect the causal variants. 20. Ovariole number and biochemistry in Drosophila melanogaster Galantis S1, Bouchard F1, Moody K1, Miles C2, Wayne M1,* 1Department of Zoology, University of Florida, Gainesville, FL 2Department of Ecology and Evolution, University of Chicago, Chicago, IL An important goal in evolutionary genetics is to de termine how genetic variation is maintained in natural populations given that selection works to counter act it. It is known that ovariole number in Drosophila melanogaster is positively correlated with fecund ity and is under strong selection. Therefore, we would expect little variation for ovariole number; however, this is not the ca se. In addition, the progeny of starved females have more ovarioles than the progeny of unstarved females but decr eased early fecundity, possib ly promoting more efficient dispersal for longer periods. Moreover, differences between sexes within genotypes for flight ability exist, such that genotypes with the most efficient male flight have the le ast efficient female flight. There may be a tradeoff between flight ability and investment in fecundity within females because more fecund females are less streamlined. Thus, energy-rich genotypes might produce fast flying males, but slow flying and fecund females. One objective of this experiment is to quantify the amounts of glucose/glycogen, triacylglycerides, and protein in D. melanogaster with the purpose of examining the tradeoffs in energy between female flies of differing genotypes for ovariole number and differences in energy content betw een sexes for each genotype. Another ob jective is to quantify differences in energy content for each biological macromolecule between starved and unstarved D. melanogaster females and males with the purpose of investigating trade-offs between investments in progeny and dispersal in the environment.
23 21. UF Genetics.com: Enhancing the public understanding of genetics Gallo M1,*, Telg R2, Irani T2, Hightower L2 1Department of Agronomy, Univers ity of Florida, Gainesville, FL 2Department of Agricultural Education and Comm unication, University of Florida, Gainesville, FL Currently, much of the public receives their information about genetics from the news media. Unfortunately, newspapers and television reports tend to simplify scie ntific research and may omit important information. Researchers have cautioned journalists and other science educ ators to be mindful of the interpretation of genetics they offer both students and the general public. Accurate science education focusing on genetics can help the next generation become more informed citizens. To addres s this concern, the Scientific Thinking and Educational Partnership (STEP) program at the University of Florid a developed the ufGENETICS.com Web site to introduce educators, students and media professionals to gene tics research conducted at UF. The goal of the ufGENETICS.com project is to offer info rmation to the public about genetics research that is accurate and relevant to everyday life. The Web site contains monthly genetic fe atures on a variety of topics including biotechnology, gene therapy, conservation ecology, and genetic marker s. The Web site is geared towards educators and media professionals, groups that currently inform students and the public about science. The materials were developed with an entertainment-education approach that involves a strong interest approach with an educational message. Each monthly feature contains the following components: Series of three to five videos that run approximately three minutes each. News feature story and sidebar story highlighting a partic ular aspect related to gene tics, with accompanying highresolution photos. Lesson plans that integrate the videos, news feature stories, and other resources into high school science curricula. Downloadable print materials for use in classrooms. To date faculty featured in the videos and accompanying materials represent the following departments: pharmacy, veterinary medicine, microbiology, agronomy, and entomology. 22. Comparative phylogeography and population genetics of Lake Wales Ridge, FL, endemics Germain-Aubrey CC, Gitzendanner MA*, Soltis PS* Department of Botany and Florida Museum of Natura l History, University of Florida, Gainesville, FL The Lake Wales Ridge of Florida is one of the oldest ecosystems in North America and only 14% of it remains today. The age and uniqueness of the ecosystem has resulted in many endemic species of both plants and animals, most of which are threatened or endangered. I propose a comparative phylogeographic analysis of both plant and animal taxa endemic to the ridge comparing origin (bas ed on the range of the closest relative) and age of divergence. Common patterns can shed light on the orig ins of the high endemism on the ridge as well as mechanisms of speciation. Additionally, focusing on threaten ed plants, I will use nuclear loci to look at their genetic diversity in terms of global diversity and geographical ho tspots in order to better understand and conserve this system. This population genetic study will also enable me to try and relate genetic diversity to other factors such as nearest congener, time since speciation or extent of past bottlenecks. This will advance our understanding of genetic diversity, its origin and true meaning for conserva tion purposes beyond the species of focus or even this system.
24 23. Hydrilla genetic diversity survey in Florida Gioeli KT1, Overholt WA2, Williams D3 1St. Lucie County Cooperative Extension, Un iversity of Florida, Fort Pierce, FL 2Indian River Research and Education Center University of Florida, Fort Pierce, FL 3Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, Miami, FL Hydrilla verticillata is a serious aquatic invasive plant in Florida. This weed is found throughout Florida in freshwaters mainly in the southern and central regions. It is also found in all of the gulf states including Georgia and from Maryland to California. Hydr illa is capable of out-competing native aquatic vegetation. It grows very rapidly asexually from rootstocks, subter ranean turions, vegetative buds (tur ions), and vegetative nodes. Dense surface mats associated with hydrilla infestations cause pr oblems because they hinder navigation and flood control, interfere with recreational activities, and reduce the bi odiversity in aquatic ecosystems. According to the Florida Department of Environmental Protection, between 1980 and 2004, approximately $158 million in government funds was spent managing hydrilla in Florida public waters with non-biological control methods. With the recent discovery of herbicide resistance in hydrilla, there is renewed interest in biological control. Scientists at the University of Florida are undertaking an international effort to search for hydrilla biological control agents. The native range of hydrilla includes large parts of Asia, northern Australia an d a few lakes in east/central Africa. Historical evidence suggests that hydrilla arrived in Florida through the aqua rium plant trade in the 1950s, and probably came from Sri Lanka. The University of Florida Research & Extension, in co llaboration with the University of Miami, is conducting a study of the population genetics of hydrilla, which is belie ved will help to better determine the origin of Florida hydrilla. Identification of the native origin of Florida hydrilla will be useful for targeting surveys for biological control agents. The purpose of this presentation is to discuss the Extension Agents role in this genetic survey. The presenter will also describe sampling methodology and the use of genetic sampling in the global search for viable hydrilla biological controls. Preliminary results will be shared. 24. Deletions within the HSV-1 LAT promoter or enha ncer that impair reactivation result in increased histone H3 K4 dimethylation Giordani NV1, Amelio AL1, Neumann DM2, Bhattacharjee PS2, Hill JM2 and Bloom DC1,* 1Department of Molecular Genetics and Microbiology, Gainesville, FL 2Department of Ophthalmology, Louisiana State Unive rsity Health Sciences Center, New Orleans, LA During latency the HSV-1 lytic genes are shut down and only the latency-associated transcript (LAT) region is abundantly transcribed. Both the LAT promoter and the LAT 5 exon/enhancer are hyperace tylated relative to lytic genes at histone H3 lysines 9 and 14 (K9, K14) when exam ined in latently-infected murine dorsal root ganglia (DRG). This type of histone modification is generally asso ciated with transcriptionally permissive chromatin, while hypoacetylation is observed in less transcriptionally perm issive regions. Furthermore, the LAT region displays deacetylation of the LAT enhancer and a decrease in LA T abundance preceding an increase in ICP0 acetylation during early explant, a model of molecular reactivation. It has previously been shown that the latent genomes of LAT promoter mutants are altered in histone mo difications during latency in the mouse (Wang et al., PNAS 102:16055, 2005). To examine whether differences in histone modifications in LAT mutants are responsible for their decreased ability to reactivate, we assayed the chroma tin profiles of two reactiva tion-negative mutants, 17 Pst and 17 Sty, during latency and reactivation. 17 Pst contains a 202 bp deletion of the core LAT promoter and does not reactivate. 17 Sty contains a 371 bp deletion in the LAT 5 exon and while able to transcribe LAT, does not reactivate in the rabbit. Trigeminal ganglia (TG) from rabbits latently-infected with HSV-1 strain 17syn+, 17 Pst, or 17 Sty were assessed for dimethylated H3 K4, a marker of transcriptional permissiveness. Interestingly, 17 Pst demonstrated a dramatic increase in enrichment of dime thyl H3 K4 over wild-type upstream of the deletion. In 17 Sty the LAT promoter region and the 5 exon were also more enriched in H3 K4 dimethylation than wild-type. These increases in H3 K4 methylation suggest that deletion of either the LAT promoter or the 5 exon causes a loss of a repressor or other chromatin regula tory element. Contrary to observatio ns made following explant of murine
25 DRG, following epinephrine induction of rabbit eyes, ther e was no remodeling of histone modifications associated with the LAT or ICP0 promoters on the latent HSV-1 genome s within the rabbit TG within four hours post-induction. Unlike the changes in the chromatin induced by explant of murine DRG, epinephrine induction of latently-infected rabbits did not display chromatin-level changes of the LAT or ICP0 promoters. 25. Longitudinal evolution of HIV-1 in breast milk and plasma Gray RR1,2, Salemi M2,*, Aldrovandi GM3, Mulligan CJ1,*, Goodenow MM2,* 1Department of Anthropology, University of Florida, Gainesville, FL 2Department of Pathology, University of Florida, Gainesville, FL 3Childrens Hospital Los Angeles, University of Southern California, Los Angeles, CA International guidelines currently advise HIV-1 positive women in resource-poor settings to exclusively breastfeed their infants for several months followed by an abrupt wean ing to minimize overall infant mortality, despite the risk of transmitting the virus. These recommendations are based on the results of observational studies, although the benefits of abrupt weaning remain inconclusive. However, no studies have been publis hed to date describing the molecular evolution of the virus in breast milk. We an alyzed HIV-1 gp120 sequences from plasma and breast milk sampled over a two-year period from a Zambian individua l infected with a subtype C virus. Numerous recombinant sequences were identified with breakpoints primarily localized within the C2 region of the env gene. The majority of the recombinant sequences were found in the breast milk. An alignment of non-reco mbinant sequences spanning the 3' end of the env region (C2-V5 domains) was ana lyzed using Bayesian and maximum likelihood phylogenetic methods. At the initial time point corresponding wi th delivery, three well-supported viral sub-populations (virodemes) were present in the breast milk distinct from a fourth virodeme in the plasma. By the fourth month after delivery, breast milk and plasma vir uses appeared as a single panmictic population. Finally, by the twelfth to the twenty-fourth month after delivery, viral sequences from plasma and from breast milk separated again into two well-supported monophyletic clades. Furthe rmore, the later virus in the breast milk apparently shared a most recent common ancestor with the viruses circulating in both tissues four months after delivery. The loss of HIV-1 compartmentalization between breast milk and plasma four mo nths after delivery may be the result of intense viral gene flow between the two tissues. In contrast the re-emerg ence of distinct virodemes by month twelve suggests a reduced flow with consequent genetic drift during the ti me interval separating the samples. Understanding the complex population dynamic underlying HIV evolution within these tissues may be essential to uncover the molecular mechanisms responsible for the differential risk of transmission associated with duration and exclusivity of breastfeeding. 26. FoxA1 and FoxA2 are required during septa tion and development of the anogenital system Gray S1, Seifert AW1, Cohn MJ1,2,* 1Department of Zoology, University of Florida, Gainesville, FL 2Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL Congenital malformations of the anogenital system o ccur at a high frequency among newborn humans. Despite clinical descriptions of these birth defects, the basic developmental genetics underlyin g such malformations remains poorly understood. FoxA genes are transcription factors that pl ay a critical role in the establishment of the definitive endoderm, and are required during development of seve ral gut-derived structures such as the liver and the prostate. Embryos null for FoxA1 die two weeks after birt h and embryos lacking FoxA2 die between embryonic days 10-11, with severe defects in all three germ layers, includ ing the absence of a notochord and failure to form a gut tube. Given their importance during gut development, we examined the role of FoxA genes during development of the anogenital system. In order to circumvent the early lethality of FoxA2 mice, we utilized a tamoxifen-inducible cre driven by Shh (ShhCreERT2) to delete a conditional Fo xA2 allele on various FoxA1 null backgrounds. Here we show that loss of both FoxA1 and FoxA2 results in persiste nt cloaca. Additionally, we find that both alleles of FoxA1 are necessary to compensate for a loss of FoxA2. FoxA1 and FoxA2 are co-expressed with Shh throughout the gut endoderm. As FoxA1 and FoxA2 have been shown to be bo th regulators of and regulated by Shh, we further investigated whether loss of FoxA1 and F oxA2 results in a loss of Shh signaling.
26 27. Differential expression of A, B, and C chains of C1q in external genital and limb development of the mouse Gredler M1, Zheng Z1, Seifert A1, Cohn MJ1,2,* 1Department of Zoology, University of Florida, Gainesville, FL 2Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL Apoptosis plays a key role in external genitalia and limb development. C1q is a target recognition protein of a classical complement pathway crucial for the clearance of pathogens and apoptotic cells. It is involved in a number of immunological processes such as phagocytosis of ba cteria, neutralization of retroviruses, cell adhesion, maintenance of immune tolerance via clearance of apopto tic cells and modulation of dendritic cells, B cells and fibroblasts. C1q has a hexameric structure containing an Nterminal collagen-like region and a C-terminal globular (gC1q) domain. The gC1q domain, which is the main ligan d recognition domain of C1q, has a heterotrimeric structure composed of the C-terminal halves of the A, B and C chains. Little is known about the expression or function of C1q genes during embryonic development. We ha ve examined the expression patterns of the A, B and C chains of C1q at different stages of mouse development using in situ hybridization. Tissue specific patterns of expression were found in both the external genitalia and the developing limbs. These patterns are similar to those of macrophage expressed gene 1 (Mpeg1 ). Results suggest that C1q genes function to sculpt the genitalia and the digits during embryonic development. 28. Functional characterization of 3-isopropylmalate dehydrogenase in Arabidopsis He Y, Walker R, Alvarez S, Chen S* Graduate Program in Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL Glucosinolates are a class of plant secondary metabolites that function in the defense against herbivores and microorganisms. Upon tissue damage, glucosinolates ar e hydrolyzed by endogenous thioglucosidases called myrosinases to produce several biologically active compound s, typically isothiocyanates, thiocyanates and nitriles. Depending on their precursor amino acids, glucosinolate can be grouped into aliphatic, aromatic and indole glucosinolate, which derived from me thionine, tryptophan and phenylalanine respectively. Methionine-derived aliphatic glucosinolates are produced through a threestep chain-elongation cycle, which can add a single methylene group each time, leading to different side-chain structures. The enzyme involved in the formation of 3malate derivatives has not been characterized. Here we use bioinformatics, biochemist ry and reverse genetics to characterize the enzyme in Arabidopsis. We identified a candidate 3-isopropylmalate dehydrogenase, which probably functions as a heterodimer with another protei n. They catalyze the isomerization and decarboxylation reactions to produce 3-malate derivatives. This was support ed by analysis of glucosinolate profiles in a knockout mutant of the enzyme. Compared to wild type controls, all the long-chain aliphatic glucos inolates were decreased in the mutant. Future work includes studying the tissue-s pecific expression, subcellular localization and enzyme complex formation involving the enzyme.
27 29. Histone demethylase LSD1 regulates hematopoie tic differentiation by association with lineagespecific transcription factor SCL Hu X1, Valverde K1, Qiu Y2, Huang S1,* 1Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL The lineage specific basic helix-loop-helix transcription fact or SCL (Stem Cell Leukemia) is critical for the formation of hematopoietic lineages during normal hematopoiesis and is aberrantly activated in up to 60% of patients with Tcell acute lymphoblastic leukemia (T-ALL). To understand the mechanisms underlying the control of SCL function, we have purified SCL-containing multiprotein complexes an d identified the interacting polypeptides. We found that SCL forms a complex with histone demethylase LSD1, core pressor CoREST, and HDAC1/2, which exhibits histone demethylase and deacetylase activities. Furthermore, LSD1 enhances SCL-dependent transcriptional repression, and the demethylase activity of LSD1 is essential for the repr essive activity. More importantly, SCL association with LSD1 and CoREST complex declines upon DMSO-induced d ifferentiation of erythroid cells. Consistent with the decrease of the SCL and LSD1 interaction during erythrop oiesis, enforced expression of LSD1 not only stimulates MEL cell growth, but also perturbs DMSO-induced differe ntiation of MEL cells. The results demonstrate that chromatin demethylase LSD1 and cofactor CoREST regulate SCL transcriptional function during erythropoiesis, and suggest that chromatin modifying enzyme may affect hema topoiesis and leukemogenesis through association with the lineage-specific transcription factor SCL. 30. Using mechanistic growth models to map quan titative traits loci for developmental timing and duration Huang YJ1,2, Li Q2, Berg A2, Huang ZW3, Wang CG2, Gai JY3, Wu RL2,* 1School of Forestry and Biotechnology, Zhejiang Forestry University, Lin'an, Zhejiang, People's Republic of China 2Department of Statistics, University of Florida, Gainesville, FL 3National Center for Soybean Improvement, Nanjing Agricultur al University, Nanjing, Jiangsu, People's Republic of China A number of statistical methods have been proposed to map quantitative trait loci (QTLs) underlying complex phenotypes primarily based on the goodness of fit to observ ational data rather than on any biological mechanism. We have framed a new statistical strategy for the genetic mapping of QTLs that regula te developmental timing and duration through the incorporation of specific biological la ws behind the phenotypic ex pression of complex traits. The new strategy displays great potential in improving the precision, power and resolution of QTL mapping and generates a number of quantitative testable hypotheses a pplicable in any kind of organism. The model was used to analyze a real data set from a soybean genome project, lead ing to the identification of several QTLs that control the timing and duration of maximal growth rate for plant he ight. The new strategy can ask, answer and disseminate many more biologically interesting questions, and thus a llow QTL mapping to interface more directly with genetics, development and evolution.
28 31. N-terminal acetylation, methylation, and phosphorylation of Haloferax volcanii 20S proteasomes Humbard MA, Zhou G, Maupin-Furlow JA Department of Microbiology and Cell Scienc e, University of Florida, Gainesville, FL 20S proteasomes are large, energy-dependent protea ses that associate as four heptameric rings of and subunits ( 7777). The haloarcheon Haloferax volcanii synthesizes two -type subunits ( 1 and 2) and a single Two 20S proteasomes, the 1and 12, have been identified during stationary phase. An acidic isoform of the 1 subunit is also present in exponential growth, but abse nt in stationary phase. This study aims at identifying post-translational modifications of the and subunits of these 20S proteasomes. Multiple isoforms of the three subunits ( 1, 2 and ) have been identified using two-dimensional ge l electrophoresis. Tandem mass spectrometry experiments have revealed that both the 1 and 2 subunits are N-terminally acetylated and the 2 and subunits are phosphorylated (at T13 and S129, respectively). In a ddition to acetylation and phosphorylation, several methylesterification sites have been identified on the 1 subunit. Functions associated with the phosphorylation and methylesterification events have yet to be dete rmined. There are two detectable forms of the 1 N-terminus by mass spectrometry, the N-terminally acetylated form and an unacetylated form where the methionine has been removed. The two forms of the protein exist in a wild-typ e cell at a 100 to 1 ratio favoring the acetylated form. Amino acid substitutions in the penultimate position of the 1 influence this ratio. For example, Q2A is acetylated after the methionine is removed, several other substituti ons such as Q2T, Q2D, and Q2P exist as a mixture of unmodified, acetylated on M1, and methionine cleaved fo rms. These substitutions affect protein levels and peptidase activity of 20S proteasomes. A list of putative N-terminal acetyltransferases has been identified by BLAST and phylogenic analysis. Targeted chromosomal knock-outs have been generated in several of these candidate genes using the pyrE2 system. Many of these knock-outs affect the ratio of acetylated to unacetyled forms of the protein as well as protein levels. 32. The MAML1 co-activator: A novel regulator for NFB signaling and its potential role in hepatocyte survival Jin B, Shen H, Wu L Department of Molecular Genetics and Microbiology and UF Shands Cancer Center, University of Florida, Gainesville, FL NFB signaling regulates diverse biological processes incl uding immune and inflammatory responses, apoptosis, proliferation and differentiation. Deregulation of NFB signaling is associated with a variety of disorders such as autoimmune diseases and cancer. In this study, we identified a novel regulator, the MAML1 co-activator in mediating constitutive NFB activation. We showed that MAML1 influences the basal activity of NFB via two novel mechanisms. First, MAML1 interacts with I B (the inhibitor of NFB), and leads to its degradation. Second, MAML1 facilitates the nuclear import of NFB subunit p65 and enhances the transcriptional activation of NFB responsive promoters. In cultured cancer cells, we fo und that MAML1 expression level directly influences the strength and duration of TNF -induced NFB signaling. Importantly, mice that are deficient of the Maml1 gene display hepatocyte necrosis, indicating that Maml1 might be required for hepatocyte survival. Therefore, our overall data suggest that MAML1 might regulate he patocyte survival through its role in NFB signaling.
29 33. Producing transgenic Tifton-85 and TifEagle grasses using Agrobacterium vacuum infiltration of stolon nodes Joshi S1, Gallo M1,2,* 1Department of Agronomy, Univers ity of Florida, Gainesville, FL 2Graduate Program in Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL In the genetic transformation of monocotyledonous spec ies, callus culture is usually an unavoidable step. The initiation and maintenance of embryogenic callus is ge notype-dependent, time cons uming and laborious. We describe a protocol that expedites a straightforward and callus-free transformation procedure for two Bermuda grass cultivars, Tifton-85 and TifEagle. An efficient regene ration system was established for both cultivars using different concentrations of kinetin and auxins. St olon nodes were infected and co-cultivated with Agrobacterium tumefaciens strains GV3101 or AGLO containing the Cry 1Fa gene for insect resistance. Green shoots were directly produced from infected stolon nodes four to six weeks after hygromycin selection. Rooted putative transgenic plantlets were then obtained seven to eight weeks later. To date, 72 plants have been obtained using this procedure. The established plants are currently being screen ed for the transgene by PCR and the transgenic nature of five plants already has been demonstrated by Southern blot hybridization. Further research is focused on increasing the efficiency of transformation an d evaluating expression of the transgene. 34. Molecular genetics of mitochondrial function in maize Kamps TL1, Zhao L1, Chamusco K1, Read V1, Andersen A1, Hannah LC1,*, McCarty DR1,*, Gabay-Laughnan S2, Chase CD1,* 1Department of Horticultural Sciences, University of Florida, Gainesville, FL 2Plant Biology Department, University of Illinois, Urbana, IL The mitochondrial genome encodes protei ns essential for mitochondrial respirat ion and ATP synthesis. Nuclear gene products are, however, required for the expression of mitochondrial genes and the elaboration of functional mitochondrial protein complexes. To be tter understand the roles of nuclear ge nes in plant mitochondrial function, we developed a unique collection of nuclear mutations that disrupt mitochondrial functions in maize. These mutations restore male fertility to plants with the mitoch ondrial-encoded cytoplasmic male sterility type S (CMS-S) trait. Although restorer mutations rescue CMS-S pollen, many are homozygous lethal for maize kernel development and may, therefore, disrupt essential mitochondrial function s. These functions were inve stigated through studies of mitochondrial gene expression in CMS-S pollen restored to male fertility and in normal N pollen controls. Two mechanisms of fertility restoration are suggested by the re sults of these assays: Five unlinked restorer mutations conditioned global loss of mitochondrial gene products through post-transcriptional mechanisms. These restorer mutations are hypothesized to effect fertility by also conditioning the loss of the mitochondrial gene product responsible for CMS-S. These phenotypes indicate that maize pollen development can succeed in the absence of mitochondrial respiration and validate CMS-S maize as a sy stem for functional genomic analysis of mitochondrial biogenesis in plants. A second group of restorer mutations had no obvious effects on mitochondrial gene expression. These are hypothesized to disrupt mitochondria l-signaled cell death events that are associated with pollen collapse but necessary for maize seed development.
30 35. A broader role for E2F6 in the repression of meiosis-specific genes Kehoe SM1, Oka M1, Reichert N2, Gaubatz S2, Terada N1,* 1Department of Pathology, Immunology, an d Laboratory Medicine, Gainesville, FL 2Department of Physiological Chemistry I, Biocenter, University of Wrzburg, Am Hubland, Wrzburg, Germany E2F6, a member of the E2F family of transcription factors, was recently shown to play an essential role in the repression of six germ-cell-specific genes in somatic cells. Here we report that E2 F6 is required for the repression of another germ-cell-specific gene, Ant4. This discovery prom pted us to investigate whether additional germ-cellspecific genes require E2F6 for their repression. In partic ular, we focused on a subpopulation of germ-cell-specific genes whose expression is restricted to meiotic cells. We compiled a list of 24 meiosis-specific genes and observed that 19 of them (79.2%) have the core E2F6-binding element (TCCCGC) within 200bp upstream of their transcription initiation sites. The high frequency of bi nding element appearance relative to non-meiotic gene populations suggests that this site is of biological releva nce. Using embryonic stem cells, we demonstrate that E2F6 binds to the promoters and represses the transcription of these meiosis-sp ecific genes. These data suggest a broader role for E2F6 in the repression of meiosis-spec ific genes. However, the observation that E2F6 is not required for somatic cell repression of most of these ge nes supports the possibility that a functional redundancy exists whereby other safeguards are in place to ensure the repression of most meio sis-specific genes in somatic cells. 36. ICBR bacterial genome finishing programme Kumar D, Shankar S, Shaw R, Almira E, Farmerie W* Interdisciplinary Center for Biotechnology Re search, University of Florida, Gainesville, FL The ICBR is a well-known hub of research support for the Un iversity of Florida and other research partners. As the past 10 years have witnessed a dramatic increase in the number of sequencing projects, the ICBR sequencing group has gradually evolved as a high throughput-sequencin g centre with state of the art technology including pyrosequencing using 454 Life Sciences Corporation techno logy. In ICBR, 454 sequencers have been in operation for nearly two years. With the introduction of pair ed end reads and improved assembly software, 454 makes bacterial genome sequencing possible. Not only does the 454 sequencing method provide better coverage but also helps eliminate cloning bias. The ICBR is also acquiring the SOLiD System from Applied Biosystems, which will exponentially extend sequencing capability. This machine ca n produce up to one GB of sequence data from single run and will be installed shortly. Utilizing advancemen ts in technology and software, the ICBR communitysequencing program now also includes the publication qu ality finishing and detailed analysis of the genome of different representatives of the microbia l world. Complete genome information is a vital starting point for genome annotation, comparative analysis, deletion analysis, microa rray, etc. The finished genome is essential for analyzing previously unknown and difficult to cultivate microbes, for rearrangement analysis or for the creation of useful microbial strains. Thus, in order to take full advantage of an organism it is necessary to fully sequence the genome. Our pipeline encompasses all phases of a complete sequen cing effort, from library construction through genome assembly and annotation. The finishing process includes act ivities of informatics as well as wet lab experiments. It consists of manual editing through the use of both commercial and proprietary software for semi-automated assembly, including Phred, PHRAP and Consed. Paired end DN A sequence information is also used to resolve repeat sequences and maximize the level of contig ordering. Gaps are then filled by different methods such as, primer walking, alternative reaction chemistries, and/or direct PCR sequencing. Sequence data is finally inspected for quality, discrepancies, covera ge and contiguity error rate less than one in 10,000 bases.
31 37. The enhancer-blocking and silencing activities of a chromatin insulator element in the HSV-1 LAT region are physically separable Kwiatkowski DL, Amelio AL, Bloom DC* Department of Molecular Genetics and Microbio logy, University of Florida, Gainesville, FL A chromatin insulator-like element was previously identified in the repeat lo ng region of the HSV-1 genome and shown to bind the cellular insulator protein CTCF during latency in murine sensory ganglia (Amelio et al., J. Virol. 80:2358, 2006). This insulator element (CTRL2) is located between the LAT enhancer and ICP0 and demonstrates both enhancer-blocking and silencing activities. In th e present study, we sought to determine whether the enhancer-blocking and silencing activities map to overlappi ng or distinct portions of the 1.3-kb insulator and whether these activities were dependent on CTCF binding. Transient transfection assays performed with luciferase constructs containing subfragments of the insulator re gion have demonstrated that: 1) the enhancer-blocking activity of the insulator is located excl usively within the 5 half of the 1.3-kb fragment, 2) the silencing activity is located exclusively within the 3 half of the insulator elem ent, and 3) deletion of the CTCF-binding motifs from either the larger 1.3-kb fragment or a 3 750 bp subfragm ent abrogates the silencing ac tivity but not the enhancerblocking property of this insulator. Since the enhancer-bloc king and silencing activities of this element appear to be cell-type specific, work is currently under way using a yeastone hybrid screen to identify important cellular factors that may play key roles in regulating transcription of ICP0 and LAT during latency and reactivation. 38. Genome-wide in silico identification, comparative analysis and functional validation of human putative CSL-regulated genes Li JL1, Liu W2, Wu L2 1Interdisciplinary Center for Biotechnology Rese arch, University of Florida, Gainesville, FL 2Department of Molecular Genetics and Microbiology and Shands Cancer Center, University of Florida, Gainesville, FL The discovery of direct downstream targets of transcri ption factors (TFs) is essential for understanding the molecular mechanisms underlying complex and highly regu lated biological processes. Notch receptor-mediated signaling is an evolutionarily conserve d pathway regulating dive rse developmental processe s and its mis-regulation is implicated in a number of developmental disorders and canc ers. Currently, with the limited identified targets, it is difficult to understand the diverse re sponses of Notch receptor activation In order to understand the genetic mechanisms underlying Notch signaling-regulated complex processes, we conducted a genome-wide search for novel putative target genes of CSL, a key transcription fa ctor controlling the Notch signaling pathway. First, the CSL binding elements from those known Notch target genes were used to construct a position weight matrix (PWM) for the CSL protein. This PWM was then used for genome-wide screening to search for the potential binding elements in the entire collections of non-redundant human, mouse and rat promoter sequences. The conservation of the potential binding sites was assessed by comparing the promoter sequences from the orthologs of these three species. This analysis identified a set of genes containing several previously id entified Notch target genes as well as many potential novel targets, indicating that such ana lysis is valid for identifying Notch targets. Several novel putative genes were selected for functional validations. Ou r initial analysis reveals several genes as novel direct targets of CSL, thus increasing the sp ectrum of Notch targets that will allo w us understand the varied functional consequences of Notch signaling. Moreover, our results demo nstrate that the integrated strategy of bioinformatics and functional genomics is a powerful approach to iden tify direct downstream targets for transcription factors genome-wide.
32 39. The maize 6-phosphogluconate dehydrogenase 3 gene plays an important role during seed development Li L1, Tseung C2, Fajardo D2, Settles M1,2,* 1Department of Horticultural Sciences, University of Florida, Gainesville, FL 2Graduate Program in Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL The oxidative pentose phosphate pathway (OPPP) is a major source of reducing power and metabolic intermediates in central carbon metabolism. In this pathway, 6-ph osphogluconate dehydrogenas e (6PGDH) decarboxylates 6phosphogluconate to provide NADPH an d pentose sugars for metabolism. Re cent metabolic labeling studies of starch biosynthesis in the maize endosperm suggest that the OPPP plays a central role in starch accumulation. We have identified a transposon insertion in the maize Pgd3 locu s. Pgd3 is predicted to enco de the chloroplast-localized isoform of 6PGDH. Co-segregation an alysis showed that the pgd3-umu1 mu tant is tightly linked to a rough endosperm (rgh), reduced grain-fill phenotype. In additi on, a reverse genetics screen of other rgh mutants identified a second mutant allele, pgd3-umu2. Reciprocal crosses between these two alle les fail to complement the rgh mutant phenotype indicating that mutations in Pgd3 cause defective seed phenotype. Native-PAGE enzyme assays show that both mutant alleles cause an enzymati c knockout of the chloroplast-localized 6PGDH isozyme in maize. Combined, these data suggest that the chloroplastlocalized OPPP is likely to have a central role in seed development and may be a target for yield improvement in maize. 40. Functional mapping: How to study genotype by environment interactions for growth curves Li Q1, Huang Z2, Wang C1, Berg A1, Gai J2, Huang Y1, Wu R1,* 1Department of Statistics, University of Florida, Gainesville, FL 2National Center for Soybean Improvement, Nanjin g Agricultural University, Nanjing, Jiangsu Functional mapping has proven to be powerful in mapping quantitative trait loci (QTLs) that control biological processes. Functional mapping incorporates mathematical aspects of biological processes into a general QTL mapping framework, and a number of testable hypotheses about the genetic control of development can be made. In this study, we extend the idea of functional mapping to study genotype by environment interactions for growth curves. With this new model, we can characterize the dy namic patterns of the genetic effects of QTLs governing growth curves and estimate the global effects of underlying QTLs during the course of growth and development. In a real example with soybean data, our model has succe ssfully detected several QTLs that cause significant genotype by environment interactions for the plant he ight growth process. The model provides a basis for deciphering the genetic architecture of trait expression ad justed to different biotic and abiotic environments for any organism. 41. Histone methyltransferase PRMT1 is important for USF1-mediated transcriptional activation of beta-globin gene Li X, Zhou Z, Bungert J*, Huang S* Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL USF1 is a ubiquitously expressed helix-loop-helix transcri ption factor that binds to E-box elements. We showed earlier that USF1 mediates the barrier act ivity at the endogenous 5HS4 of the chicken -globin locus by recruiting histone modifying enzymes, including the histone H4R3-spe cific methyltransferase PRMT1, that maintain a local active chromatin structure. However, in the mouse -globin locus, USF1 interacts with conserved E-box elements within HS2 of the locus control region (LCR) and the -major promoter. It has been shown that USF1 plays a critical role in regulating the developmental stage-specific transcription of the -major globin gene. To further understand the diverse roles of USF1 protein, we investigated wh ether PRMT1 plays an important role in USF1-mediated
33 transcriptional activation of the -globin gene. We report that USF1 and PRMT1 are colocalized to the endogenous HS2 and the -major promoter of the mouse -globin locus in erythroid cells. Consistent with recruitment of PRMT1 by USF1, H4R3 methylation at these two sites is increased during differentiation of erythroid cells. Furthermore, knockdown of PRMT1 expression by shRNA leads to a loss of H4R3 methylation, as well as histone acetylation at these two sites. Accompanied with the loss of histone modifications, the expression of the -major globin is significantly inhibited in the PRMT1 kd ES cells. Taken to gether, these results show that H4R3 methylation by PRMT1 plays an important role in transcriptional activation of the -globin gene and suggest that H4R3 methylation by PRMT1 appears to be essentia l for the transcription of the -globin gene by establishing and maintaining an active open chromatin structure. 42. A statistical model for characterizing cisand trans-acting regulation by eQTL Li Y, Li J, Casella G*, Wu R* Department of Statistics, University of Florida, Gainesville, FL Two different types of eQTL regulation have been thought to contribute to genetic variation in gene expression. For the first type, cis-regulating, the DNA variants of a gene di rectly influence the transcript levels of that gene and, therefore, the underlying eQTL can be expected to map to the structural gene producing the transcript. For the second type, trans-regulating, the DNA variants of a gene exert an indirect effect on its expression, in which the underlying eQTL should co-localize to the structuring gene. In this study, we propose a general statistical model based on linkage analysis to discriminate between cis-an d trans-regulating eQTL and estimating their additive, dominant and epistatic effects, for the purpose of the identification of position al candidates in QTL studies. This model provides a useful tool for studying th e genomic architecture of complex traits. 43. Regulation of locus control region mediated tr anscription complex recruitment to the beta-globin gene locus by transcription factor USF Liang S, Zhou Z, Moghimi B, Crusselle -Davis VJ, Lin IJ, Anantharaman A, Dodev TS, Andersen F, Gandhi K, Huang S*, Bungert J* Department of Biochemistry and Molecular Biology, Powell-Gene Therapy Center, and Center for Mammalian Genetics, University of Florida, Gainesville, FL The human beta-globin genes are expressed exclusively in erythroid cells and regulated by a locus control region (LCR). The LCR is composed of several DNaseI hypersensit ive (HS) sites and located upstream of the globin genes. Our previous work demonstrated that transcription comp lexes are recruited to individual LCR HS sites prior to appearance at the globin gene promoters during erythr oid differentiation of embryonic stem cells. We also demonstrated that RNA polymerase II (RNA Pol II) can be transferred from immobilized LCR templates to globin gene promoters in an in vitro system. We found that the ubiquitously expr essed transcription factor USF is required for efficient recruitment of RNA polymerase II to the locu s control region and to the beta-globin gene. We now generated transgenic mice expressing a dominant negative mutant of USF exclusively or predominantly in erythroid cells. The transgenic mice exhibit a reduction in RNA polym erase II recruitment to the LCR and to the adult betaglobin gene. Expression of the adult be ta-globin gene is decreased in transgen ic mice; however, expression of the embryonic/fetal betaH1 gene is elevated, supporting the hypothesis that the genes are competitively regulated by the LCR. Furthermore, we present evidence demonstratin g that USF is subject to proteolytic degradation in undifferentiated erythroid cells. The data suggest that transc ription in the globin locus is regulated by tissue and stage-specific transcription factors that cooperate with ub iquitously expressed proteins. We propose that tissueor cell-type specific activities increase chromatin accessib ility in the globin locus and that proteins like USF subsequently mediate the recruitment of transcription complexes.
34 44. TaxaSorter: A solution to metagenomic projects Liu L, Sun YJ, Yu FH, Farmerie WG* Interdisciplinary Center for Biotechnology Rese arch, University of Florida, Gainesville, FL A metagenomic project generates a large collection of he terogeneous sequences. Assigning the correct taxonomic origin for each and every sequence is the key to answer all questions metagenomic pr ojects try to address. We have developed a program, TaxaSorter that classifies sequences into a hierarchical taxonomy tree based on sequence similarities. The algorithm is based on BLAST search. However, instead of simply taking the taxonomic origin of the first hit, probability valu es of all taxonomic origins from the top 100 hits are calculated for every query sequence. If only one taxonomic origin is assumed for each sequence, the one with the highest probability is selected. Otherwise, all possible origins are retained with their corresponding probability values. Then, all sequences are sorted through a hierar chical taxonomy tree where each taxon node is associated with the total count of linked and sub-linked sequences. TaxaSorter also includes functions to compare two taxonomy trees on all taxon nodes to identify nodes that are significantly over -represented or under-repre sented. Simulations were conducted to estimate the sensitivity and specificity. Taxa Sorter was applied on several metagenomic data sets, including both traditional Sanger sequences and 454 sequen ces. The sample result from a soil metagenomic project was presented. TaxaSorter is implemented as a module in BlastQuest available at UF/ICBR genomics server. 45. The role of histone deacetylase activity on GA TA-1 mediated hematopoiesis and leukemogenesis Luo Y1, Huang S2,3,*, Qiu Y1,3 1Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 2Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 3UF Shands Cancer Center; University of Florida, Gainesville, Florida Hematopoietic specific transcription factor GATA-1 is es sential for hematopoietic development. Misregulation of GATA-1 is linked to hematologic diseases including le ukemia. GATA-1 activity is required for erythroid and megakaryocytic cell differentiation. However, recent discov eries indicate that GATA-1 is associated with HDAC1 containing corepressor complexes throughout differentia tion of erythroid cells. We hypothesize that GATA-1 associated deacetylase activity is attenuated during eryt horid differentiation. We previously found that HDAC1 can be acetylated in vivo and acetylated HDAC1 completely lost deacetylase activity (Qiu et al., Molecular Cell 22:669679, 2006). In this study, we investigate the role of HDAC 1 in erythoid differentiation. Our results indicate that during erythroid differentiation, GATA-1 associated de acetylase activity is significantly decreased and further diminished at day 5 of DMSO induction in MEL cells. More interestingly, acetylated form of HDAC1 within the GATA1 complex increases during erythroid differentiation. Ov erexpression of HDAC1 mutant mimicking the acetylated HDAC1 promotes erythriod differentiation. These observat ions suggest a novel but rather general regulation mechanism of histone deacetylase contai ning complexes. Further studies will allow us to understand the molecular basis of the regulation of deacetylation of HDACs and their roles in hematopoiesis and general transcription regulation.
35 46. Preliminary genetic analysis of the Florida and Belize manatee populations McCann S1, Kellogg ME2, Pause KC1, Clark A3, Bonde RK2,4, Powell J5, Auil N5, McGuire PM1 1Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2Department of Physiological Sciences University of Florida, Gainesville, FL 3Genetic Analysis Laboratory, Interdisciplinary Center for Biotechnology Research, Univers ity of Florida, Gainesville, FL 4U.S. Geological Survey, Florida Integrated Science Center, Sirenia Project, Gainesville, FL 5Wildlife Trust, St. Petersburg, FL, and Belize City, Belize The endangered West Indian manatee contains two subspecies: the Florida manatee (Trichechus manatus latirostris) and the Antillean manatee (T.m. manatus), represented in this study by the Belize population. Morphological, ecological and biological data support the subspecies distinction and endangered status, but do not address current relatedness among po pulations. Integrative approaches are needed to identify and sustain ecological processes and evolutionary lineages of threat ened species. Therefore, in addition to traditional conservation studies, molecular analys es are used to aid in the assessment and management of the West Indian manatee. Previous mitochon drial DNA studies investigated historical genetic relationships within manatee populations. Low genetic diversity was observed with thr ee haplotypes in Belize and only one haplotype in Florida. Correspondingly, nuclear microsatellite DNA markers revealed little contemporary structure and high migration rates within Florida. In this study, microsatellite markers are us ed to examine the Belize population structure and assess relatedness and migration between the Florida and Belize populations. The genotypes of 91 Belize and 96 Florida manatees at 15 polymorphic microsatelli te loci were investigated. A Bayesian phylogenetic inference analysis was performed using the program STRUCTURE 2.2 to identify putative ancestral source populations for extant manatees. Additionally, neighbor-joining trees were constr ucted to illustrate genetic distances between individuals and populations. The genetic analyses detected structurin g within the Belize population and considerable diversity between the two subspecies. Results from this study sh ould benefit management and conservation decisions. 47. Proteomics: Advance technology for the analysis of cellular process McClung S, Chow M, Zheng R, Chung A, Alvarez S Proteomics Lab, Interdisciplinary Center for Biotechn ology Research, University of Florida, Gainesville, FL. In recent years, genomics and proteomi cs have emerged as key te chnologies in biological and biomedical research. Proteomics can be defined as the qualitative and quantita tive comparison of proteomes under different conditions to further unravel biological processes. By studying at the protein level, proteomi cs allows researchers to investigate how proteins affect cell processes and how cell processes and the environment may affect proteins. Proteomics includes the determination of localization, modifications, interactions, and activities of proteins and ultimately how these are related to their function. Proteomi cs developed initially from the study of comprehensive protein visualization on two-dimensional electrophoresis ge ls and has been expanded by mass spectrometry and the growth in searchable sequence databases providing sensit ive methods of characterizing and analyzing both small molecules and proteins. Currently, by use of more so phisticated technology such as a combination of multidimensional chromatography and mass spectrometry, thousands of proteins can automatically be identified. Using mass spectrometric based techniques, quantitative information on differential-protein expression can be obtained using isotope labeling methods such as iTRAQ, ICA T or SILAC. Along with our capacity of separating thousands of proteins and characterizing differential prot ein expression using 2D-DIGE technology, we have a suite of state-of-the-art mass spectrometers available for life sc iences and advanced technology research, including a tandem time-of-flight (4700 Proteomics Analyzer, AB), a hybrid quadrupole /time-of-flight (QSTAR XL, AB), and hybrid quadrupole-linear ion-trap (4000 QTRAP, AB). Thes e instruments are mainly used for protein identification, post-translational modifications characterization and differential protein expression using iTRAQ reagents. Proteomics also provides the tools to expand into more sophisticated biochemi cal approaches, such as the study of protein interactions that can be determined directly by perf orming a pull-down assay with a bait protein followed by mass spectrometric identification of the bound proteins. Prot eomics is useful for both large-scale surveys of proteins and detailed studies of the functional relationships among the proteins of interest.
36 48. Effects of metabolic rate on protein evolution McCoy MW, Gillooly JF* Department of Zoology, University of Florida, Gainesville, FL Since the Modern Evolutionary Synthesis was first propos ed early in the 20th century, attention has focused on assessing the relative importance of mutation versus natu ral selection to protein evolution. Here we test a model that yields general, quantitative predictions on rates of protein evolution by combining principles of individual energetics with Kimuras Neutral Theory. The model succe ssfully predicts much of the heterogeneity in rates of protein evolution for diverse eukaryotes (i.e. fish, amph ibians, reptiles, birds, mammals) from different thermal environments. Data also show that the ratio of non-synony mous to synonymous substitution is independent of body size, and thus presumably of effective population size. Together, these findings indicate that rates of protein evolution are largely controlled by mutation rates, which in turn are strongly influenced by individual metabolic rate. 49. Statistical properties of open reading frame distributions: A broad scale comparison of genome architecture McCoy MW, Gillooly JF* Department of Zoology, University of Florida, Gainesville, FL A better understanding of the size and abundance of open reading frames (ORFs) in whole genomes may shed light on the factors that control genome complexity. Here we ex amine the statistical distributi ons of open reading frames (i.e. distribution of start and stop codons) in the fully sequenced genomes of DNA viruses, prokaryotes, and unicellular and multi-cellular eukaryotes. Results show that the size-frequency distributions for ORFs are strikingly similar across all genomes, and highly correlated with GC co ntent. In addition, these results indicate that a large fraction of these distributions can be described by random processes. 50. Dominance and additive variation contributions to the maintenance of new mutations of ovariole number in Drosophila melanogaster Moody K, Galantis S, Edison M, Sylvestre L, Wayne ML* Department of Zoology, University of Florida, Gainesville, FL Mutation is the ultimate source of genetic variation. Genetic variation has multiple components, including dominance variation and additive variation. Both types of variance are subjected to selection, which works to rid a population of mutation, but have very different evolutio nary dynamics under the same strength of selection. Further, short-term response to selection is contribute d almost completely by additive variation. How then is genetic variation maintained in natural populations? The ma intenance of genetic variation is partially attributed to long-term selection response of dominance meaning dominanc e is affecting the fixation time of new mutations. For ovariole number of Drosophila melanogaster in natural population, additive variation makes up a much larger proportion of the genetic variation than dominance variation. Is there a mutational bias for additive variation, or is dominance variation being eliminated by selection? Here we estimate the relative contributions of additive and dominance variation to new mutations for ovariole number by crossing mutation accumulation lines from two genetic backgrounds to a reference line. The resulting F1 progeny were dissected and an epistatic interaction was found within one of the backgrounds, making it impossibl e to estimate additivity and dominance. Therefore, the experiment was repeated with a different reference line wh ere there is no epistatic interaction. The dominance to additive ratio will be calculated to determine if new mu tations for ovariole number are overdominant, completely dominant, mostly dominant, or mostly addi tive. The results could provide insight to the methods in which selection is acting and genetic variation is maintained.
37 51. Effects of genetic variation of ovariole number in Drosophila melanogaster on flight dispersal in the field Moody K, Wayne ML* Department of Zoology, University of Florida, Gainesville, FL Trade-offs between life-history traits he lp maintain genetic variation, and pl ay a central role in evolution. The examination of such trade-offs may contribute to the und erstanding of the continued coexistence of the opposing forces, natural selection and genetic variation. Trade-o ffs between reproduction and fitness are important for survival of many energy-limited species, such as Drosophila melanogaster, in which resource allocation influences reproduction, body size, and flight ability. In this presen t study, the trade-off between ovariole number, which is positively correlated to female fecundity (Bouletreau-Merle et al., Oecologia 53:323-329, 1982) and flight dispersal in D. melanogaster was examined. The first release and recapture experiment demonstrated that females with a high ovariole number were recaptured more frequently than females with a low/medium ovariole number. In contrast, males from the high ovariole number genotype were recaptured less frequently than the males from the low and medium ovariole number genotypes. The opposi ng trend between females and males within genotypes illustrates a genotype by sex interaction, which may contri bute to maintenance of genetic variation and hence the high heritability of ovariole number. Results from a second release and recapture experiment were consistent with the first. 52. High-throughput genotyping using Illumina bead arrays Moraga Amador D, Panayotova N, Zhang L Genomics Lab, Interdisciplinary Center for Biotechn ology Research, University of Florida, Gainesville, FL. An integrated, accurate and scalable high-throughput genoty ping system is now available to UF researchers at the ICBR Core facilities (First floor, South wing, CGRC Building) The system (Illumina) is based on a random array of 3micron glass beads lodged on 2-micron deep micro-wells that have been etched on either glass slides or the tips of fiber optic bundles. An array is composed of many bead -types, each represented at a 20-30 fold redundancy. Each bead-type contains 700-800 thousand copies of a two-part probe. One probe segment serves as a bead identifier (bead code) and another segment is designed to hybrid ize uniquely to a SNP locus. Detection is accomplished through allele-specific, fluorescent based assays and sc anning in a high-resolution (0.8 microns) scanner (BeadStation GX). Image data analysis is done using Il luminas BeadStudio software in conjunction with bead decoding files provided with each array. The system ha s proven to be highly robust and accurate. When DNA quality is not compromised, call rates of 99% and higher are easily accomplished. Depending on the nature of the project, researchers can choose from two different as say formats. First, the Go ldenGate assay allows the genotyping of a maximum of 1536 SNP sites on 96 different samples per array (SAM, sentrix array matrix). Second, the Infinium II assay is used for beadchips capable of genome-wide genotyping of up to one million SNP loci. Although Illumina currently offers only human, mouse and ra t genotyping arrays, the availability of a more diverse set of products is expected as the po pularity of this platform increases. 53. Ancient origin and functional differentiation of chaperonin-like genes involved in Bardet-Biedl syndrome Mukherjee K, Brocchieri L* Department of Molecular Genetics and Microb iology, University of Florida, Gainesville, FL Chaperonins are oligomeric complexe s that play important roles in de novo protein folding and renaturation and in protecting the cells from the deleterious effects of variou s forms of stress. They are divided into two structural classes, one typically found in Bacteria and eukaryotic or ganelles (Group I) and one found in Archaea and in the
38 eukaryotic cytosol (Group II). In eukaryotes, the Group II chaperonin complex is a double-ringed structure called CCT/TriC complex, which is composed of 16 monomers co ded by genes belonging to eight anciently-diverged paralogous families (cct1 to cct8). The Bardet-Biedl synd rome (BBS) is a human pathological condition that has been associated to mutations in twelve different ge nes (BBS1-BBS12). Among these, BBS6, BBS10 and BBS12 have been recently characterized as a group of fast evolving genes related to eukaryotic Group-II chaperonin genes that are only found in vertebrates. By thorough analysis of several complete genomes representative of all major eukaryotic lineages, we have been able to determine that BBS6, BBS10 and BBS12 are members of a family of genes found in phylogenetic groups as diverse as Op isthokonts (including animals and fungi), Amoebozoa (including Dictyostelium) and Heterokonts (including diatoms), placing their origin much earlier in eukaryotic history than previously suggested, as a distinct new gene family of chaperonin genes. This family of genes is characterized by rapid evolution and many of its members appear to be lost or are unrecognizable in various eukaryotic lineages. Synonymous vs. non-synonymous nucleotide substitution pa tterns suggest that the members of this gene family have been subject to intense processes of functional d ifferentiation. Homology modeling and sequence conservation analysis provide insights into the possible roles of thes e proteins in substrate-bind ing and participation to the chaperonin complex. 54. High-throughput annotation of genomic datasets Nuzzo A1, Riva A2,* 1Department of Computers and Systems Science, University of Pavia, Pavia, Italy 2Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL We describe the initial implementation of Genephony, an on line tool for the creation and manipulation of very large genomic datasets. Genephony allows the user to easily crea te sets containing biological entities (e.g. genes, SNPs, pathways, etc) and to combine them in a variety of wa ys in order to generate new sets. Sets are stored in a dynamic workspace through which the user can freely navig ate. Relying on an extensive underlying database of genomic information, the system makes it easy to integrat e, annotate and interpret the results of high-throughput experiments, providing automated op erations that would be otherwis e impractical if performed manually. Genephony will soon be made publicly ava ilable; our expectation is that it will become a useful tool for translational research, high-throughput biology, and for all knowledge -intensive data manipulation tasks in computational biology. 55. Custom gene expression microarray design pipeline Ostrow DG, Liu L, Farmerie WG* Interdisciplinary Center for Biotechnology Re search, University of Florida, Gainesville, FL Transcriptome libraries sequenced using either traditiona l Sanger technology or pyrosequencing technology (454 Life Science, Inc.) or both can be quickly transformed in to a custom gene expression microarray through our data processing pipeline. This pipeline is comprised of data cleaning, assembly (hybrid-assembly if both technologies were involved), annotation, coding region prediction, re dundancy reduction, orientation determination, probe design and array layout design. As a routine practice, a total of 250 probes designed from reverse complement strands of genes from five different organisms, namely, human, Arabidopsis, rice, zebrafish and yeast are included as negative controls. Another 1000 2000 probes were incl uded for QC/QA purposes. This pipeline employs several 3rd party programs such as Paracel Tr anscriptAssembler, Newbler, BLAST and Agilent eArray system, and in-house developed programs such as BlastQuest and Assembly Filter. Available formats include 244K, 2x105K, 4x44K and 8x15K. To date, eight custom Agilent microarrays were desi gned according to this prot ocol. And all of them have been used in gene expression research projects. Here, as an example, we presented a custom array design for a new non-model organism.
39 56. Clade A fibrillar collagen duplication in zebrafish Patel R1, Zhang GJ1, Cohn MJ1,2,* 1Department of Zoology, University of Florida, Gainesville, FL 2Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL In order for a diverse and complex number of organisms to arise over evolutionary time, genetic variation within populations was required for natural selection to act upon. A process by which loci can be replicated in an organism from ancestral generations to future generations is known as gene duplication. During gene duplication, loci are replicated and genes on those loci are either maintained deleted, or beneficial mutations can overwrite previous genes. This can result in subfunctionalization (b oth copies necessary to complement each other), neofunctionalization (locus with a new function), or nonfunc tionalization (non-functioning duplicate). Since it is well established that the teleost genome ha s undergone an extra round of genome duplication, we test the hypothesis that the clade A fibrillar collagen genes experienced gene duplication in zebrafish. We cloned and isolated six zebrafish clade A fibrillar collagen duplicates. Phylogeneti c analysis confirmed that gene duplication had taken place. We are currently investigating their expression patterns via in situ hybridization. The results will have implications for understanding the relationship between genome and skeletal evolution, and may account for the unusual complexity of teleost connective tissues. 57. Noise in the quorum-sensing mechanism of the marine bacterium V. fischeri Prez PD, Johnson E, Young J, Hagen SJ* Department of Physics, Universit y of Florida, Gainesville, FL Vibrio fischeri is a luminescent marine bacterium that naturally colo nizes several marine macroscopic life forms. It is mostly known for colonizing the light organ of the squid Euprymna scolopes in a symbiotic relationship. The bacteria start emitting light when they reach a critical cell density inside the light organ of the host. They sense cell density by means of the LuxIR quorum-s ensing mechanism that controls light production. Quorum sensing by this bacterium is of interest for its network has the same arch itecture as the ones possessed by a large number of other proteobacteria, including pathogens to humans. The objective of our research is to see how noise in the gene regulatory system affects the quorum sensing communication. Quorum sensing in V. fischeri has been extensively studied in bulk cultures, but little is known about how it is a ffected by gene noise, or the intercell variability in gene expression. Recent observations in other systems have shown that noise can drive switching between different modes of gene expression. It has been suggested, though not experimentally proved, that this may occur in the LuxIR system. We are measuring the light output of individual cells in time and under different conditions. We are using dark field microscopy imaging to find and focu s the cells, and an intensified CCD (iCCD) to collect the bioluminescence light of individual cells (or small clusters) in time. The analysis of the individual cell luminescence signal and its noise can give important information about time scales and the properties of the network; and eventually lead to a mathematical model for no ise properties of the quorum sensing system. 58. Genetic dissection of the lupus susceptibility locus, Sle1c Perry D, Morel LM Department of Pathology, Immunolo gy, and Laboratory Medicine, University of Florida, Gainesville, FL Systemic lupus erythematosus (SLE) is an autoimmune disease in which numerous genetic loci epistatically contribute, along with environmental and stochastic events to a break in self tolerance. SLE is characterized by high titers of autoantibodies, especially those that bind nuclear antigens such as dsDNA, histones/chromatin, and small nuclear ribonucleoproteins (snRNPs). The high levels of immune complexes that are formed by these antinuclear autoantibodies (ANA) bound to autoantigens are th e main culprits in the pathogenesis of the SLE. These
40 immune complexes can cause disease resulting in dermatit is, neuropathy, arthritis, cardiomyopathy, anemia, and nephritis. The genetic variables that contribute to SLE and other autoimmune diseases are numerous and complex, making it difficult to link disease related phenotypes to thei r corresponding alleles. In order to simplify this task, we have adopted a technique known as "Genetic Dissection" to study a murine model of SLE. Using this approach, we performed linkage analyses for various SLE phenotypes on F2 crosses between NZM2410 (NZM), a strain known to succumb spontaneously to a severe lupus-like disease, and C57BL/6 (B6), a healthy control strain. Three NZMderived lupus susceptibility loci were identified and named Sle1, Sle2, and Sle3 on chromosomes 1, 3 and 7, respectively. These individual loci were then congenically bred onto the B6 background to yield the new strains B6.Sle1, B6.Sle2, and B6.Sle3 and were each shown to ex hibit unique phenotypes found in the NZM mouse, but did not result in disease. Importantly, when the three loci we re combined on the B6 background to generate a triple congenic strain, B6.Sle1Sle2Sle3, the severe disease was re constituted. Unfortunately, it was found that these loci were, themselves, complex, and it was necessary to genera te subcongenic strains. Here we focus on Sle1c and its subcongenics. Using these subcongenic strains, we were able to narrow the range for candidate genes to ~1Mb. 59. Tmem16a is required for murine lung development Rock JR, Harfe BD* Department of Molecular Genetics and Microb iology, University of Florida, Gainesville, FL A recent screen in our lab compared gene expression be tween cells from the mouse zone of polarizing activity (ZPA) and cells from the rest of the limb using a combination of fluorescence activated cell sorting and the Affymetrix microarray platform. We have focused our efforts on characterizing one of the genes that this screen identified as upregulated in the ZPA. Tmem16a is a memb er of the TMEM16 family of proteins. Members of this family have eight predicted transmembrane domains and a highly conserved 3 domain of unknown function. RNA in situ hybridization has demonstrated expression of Tmem16 a in a variety of tissues during mouse development and human TMEM16A is overexpressed in many cancers. We have generated a null allele of Tmem16a by homologous recombination in mouse embr yonic stem cells. Mice homozygous fo r this deletion have no apparent limb defects, but die shortly after birth. We are currently in vestigating a severe lung defect in mutants that results from a failure of alveolar septation. 60. Conservation genetics of the endangered Miami blue butterfly Saarinen EV Department of Entomology and Nematology University of Florida, Gainesville, FL McGuire Center for Lepidoptera and Biodiversity, Florid a Museum of Natural History, University of Florida, Gainesville, FL The Miami blue butterfly, Cyclargus thomasi bethunebakeri (Comstock and Huntington) (Lepidoptera: Lycaenidae) is state-endangered in Florida and a candidate for federal lis ting. Extant colonies exist in a metapopulation structure and are limited to a single location, Bahia Honda State Pa rk in the lower Florida Keys. A captive breeding colony was initiated from Bahia Honda stock to safeguard the ta xon and to provide organisms for reintroduction. Wing fragment samples from both wild and captive colony spec imens were collected for DNA extraction and microsatellite analyses. These genetic data were used to infer populati on structure, overall genetic variability, and gene flow within the extant metapopulation for 2005 and 2006. Geneti c variability was also assess ed for several generations of the captive colony. Comparisons of microsatellite data between captive colony and wild-caught individuals reveal differences in allelic diversity and overall genetic variab ility. The conservation implications of using captive-bred individuals for reintroduction are discussed.
41 61. A new fast and dependable algorithm demons trates the high rate of HIV-1 intra-patient recombination Salemi M1,*, Gray RR1,2, Goodenow MM1,* 1Department of Pathology, Immunology, and Laborato ry Medicine, University of Florida, Gainesville, FL 2Department of Anthropology, University of Florida, Gainesville, FL Despite several studies demonstrating the occurrence of hum an immunodeficiency virus type 1 (HIV-1) intra-patient recombination, no general method exis ts to identify the most likely recombin ants in a data set of HIV-1 aligned sequences. Traditional phylogenetic based techniques such as bootscanning lack power and require the a priori identification of parental strains. We describe a simp le algorithmic procedure ba sed on split-decomposition networks and the PHI test, a robust statistical test for re combination. This new algorith m allows detection of HIV-1 intra-patient recombinant sequences in a fast and relia ble way. We re-analyzed sequence data longitudinally sampled from PBMC in four patients, as well as sequence data sampled from multiple tissues in two patients. Our results demonstrate that intra-patient recombination was si gnificantly underestimated in these studies, and that up to one third of the V1-V3 sequences longitudinally sample d from a given subject can be of recombinant origin. Since intra-patient recombination leads to the creation of mosaic genomes violating th e tree-like assumption of evolution, results and conclusions based on phylogenetic analysis of HIV-1 data sets including recombinants can be severely misleading. The results of our analysis not only in dicate that the algorithm could be a valuable tool for quick detection of recombinant sequences before proceeding to phylogenetic analysis, but also suggest that HIV-1 recombination in vivo is far more frequent and signifi cant than previously thought. 62. Standing genetic variance reflects mutational vari ance for fitness and body size in two species of Caenorhabditis Salomon M, Blanton D, Brown J, Bour W, Custer A, Keller T, Levy L, Ostrow DG, Phillips N, Rosenbloom J, Baer C* Department of Zoology, University of Florida, Gainesville, FL Over the past several years our lab has been investigatin g the properties of new mutations in a model nematode system. Mutations have been allowed to accumulate in the (relative) absence of natural selection, thus allowing us to estimate the genetic variance introduced by new mu tation (VM) for two species of Rhabditid nematodes, Caenorhabditis elegans and C. briggsae. However this begs the question, what is the relationship of VM to the standing genetic variance (VG) and what can this relation ship tell us about the nature and magnitude of selection acting on these species? To complement our previous studies and generate estimate s of VG, we assayed wild strains of both, C. elegans and C. briggsae, for fecundity at 20C and adult body size. Comparisons of VM to VG between our mutation accumulation lines and the natural is olates allow us to infer the magnitude and pattern of constraint on phenotypic evolution, as well as give insigh ts into the forces responsible for genetic diversity within and between these species. Our results suggest that the standing genetic variance in C. briggsae is much greater than in C. elegans, with C. briggsae displaying approximately twice the variance of C. elegans for both fitness and body size. The data also provides evidence for Mutation Se lection-Balance acting on the life history trait of fitness, with a VG to VM ratio much smaller than 4Ne. Also, the pe rsistence time of a new mutation was found to be longer in C. briggsae than in C. elegans.
42 63. Molecular characterization and risk asse ssment of apomictic, transgenic bahiagrass (Paspalum notatum Flugge) Sandhu S1, Altpeter F1,*, Blount A2 1Department of Agronomy and Graduate Program in Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL 2Department of Agronomy, North Florida Research and Ed ucation Center, University of Florida, Marianna, FL Bahiagrass is a predominant forage and low-input turf gr ass in the southeastern United States. Bahiagrass has apomictic tetraploid (2n=40) and sexual diploid (2n=20) genotypes, both of which have commercially important cultivars. Apomictic tetraploid cultivar Argentine was ch osen for genetic transformation. Its apomictic mode of reproduction should allow production of uniform seed progeny and might reduce the risk of unintended gene dispersal by pollen. To further investigate this, we co -transformed minimal unlinked nptII and bar expression constructs (MCs) into bahiagrass callus by biolistic ge ne transfer. The vector backbone was removed prior gene transfer to enhance co-expression and expression stabilit y and eliminate the prokaryotic antibiotic resistance expression cassette. Twenty-one nptII expressing plants were confirmed by ELISA following selection of 300 bombarded calli on paramomycin containing media. Relat ive simple integration patterns for the nptII gene, and higher copy numbers and more complex integration patte rns for the non-selected bar gene were detected by Southern blot analysis. Consistent with earlier report s the co-integration and co-expression frequency of the unlinked MCs was higher than 90%. Integration of quanti tative ELISA-, Southern blotand herbicide resistance data under greenhouse and field conditions indicated that MCs support high level expression of the bar gene and resistance to high glufosinate application rates. Molecular analysis of the seed progeny of the transgenic lines, will also be presented. Glufosinate resistance in apomictic A rgentine bahiagrass was used as a marker to study pollenmediated intraspecific gene flow from transgenic apomicti c Argentine bahiagrass to wild-type diploids and wildtype tetraploids under field and greenhouse conditions. Data on herbicide resistance of seed progeny, intraspecific gene transfer frequencies, ploidy, fertility and transgen e integration patterns of gene transfer events will be presented. 64. Multiphasic role of Shh during external genital development and cloacal septation Seifert AW1, Harfe B2,*, Cohn MJ1,* 1Department of Zoology, University of Florida, Gainesville, FL 2Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Loss of Sonic hedgehog (Shh) function during embryonic de velopment leads to a suite of congenital malformations, including holoprosencephaly, limb reduct ion, persistent cloaca, and agenesis of the external genitalia. During normal development of the urogenital system, Shh is ex pressed throughout the hindgut endoderm and has been proposed to act as an organizing signal during deve lopment of the external genitalia. The early and severe anogenital phenotype in Shh null mice has precluded an examination of Shh function during later stages of anogenital development. In order to ex amine the temporal role of Shh signaling during anogenital development, we employed a conditional knockout system that allows fo r the spatiallyand temporally-controlled removal of Shh signaling. We utilized mice with a tamoxifen-inducibl e form of cre-recombinase knocked into the Shh locus (ShhcreErT2) and crossed these to mice homozygous for a co nditional allele of Shh (ShhC/C). Injection of pregnant dams before, during, and after initiation of genital tuberc le outgrowth allowed for an examination of Shh function over the course of external genital and anorectal formation. Our results indicate that Shh is required during the first two phases of genital tubercle development (early outgrowt h and dorsoventral patterning of the phallus), but is not required during sexual differentiation of the penis and clitor is. Early removal of Shh function also results in improper partitioning of the cloaca. Failure of these embryos to form a bilaminar urethral plate is a secondary consequence of a disruption to cloacal morphogenesis. Removal of Shh at later time points leads to severe ventral hypospadias, uncovering a later role for Shh during urethral tube format ion and in dorsoventral patterning of the genital tubercle. These results implicate Shh signaling in a wide range of urogenital defects and reveal a role for Shh as an integrative mechanism involved in associative comple xes of the urogenital and anorectal systems.
43 65. Genetic and biochemical characterizatio n of a dihydroxyacetone kinase-linked phosphoenolpyruvate:protein transferase system in Haloferax volcanii Sherwood, K.E., Maupin-Furlow, JA* Department of Microbiology and Cell Scienc e, University of Florida, Gainesville, FL Dihydroxyacetone (DHA) kinases are a fa mily of widespread, sequence-conserved enzymes that phosphorylate DHA and other short-chain ketoses and aldoses. Two groups of DHA kinases have been well characterized based on the source of the high-energy phosphate utilized: ATP-de pendent (homodimeric, two-domain proteins found in eukaryotes and eubacteria) and PEP-dependent (tripartite proteins found in eubacteria). While the biochemical properties of DHA kinases have been elucidated for euba cteria, little is known of their function and regulatory mechanisms in archaea. The long-term goals of this stud y are to determine the metabolic function of a putative dihydroxyacetone kinase phosphotransferase system (DHAK-PTS) in the haloarchaeon Haloferax volcanii and establish whether this system is regula ted at the transcriptional and/or post -transcriptional level. Using the popin/pop-out, markerless gene deletion method, the gene en coding a DhaL homolog was deleted from the genome of H. volcanii H26. In E. coli, DhaL serves as the nucleotide-binding subunit of DHA kinase and the co-activator of DhaR, an enhancer binding protein. The resulting H. volcanii H26 dhaL mutant exhibited growth under microaerobic conditions on minimal medium supplemente d with 20 mM DHA, reaching a maximum O.D. (600 nm) of 1.7 and a displaying a doubling time of 6.9 h. The sole catabolic product was 2,3-bu tanediol, detected via HPLC using a BioRad HPX-87H column. Parent strain H26 did no t exhibit growth on minimal medium supplemented with DHA at any tested concentration. Both H26 and H26 dhaL exhibited similar growth phenotypes on minimal medium supplemented with 20 mM glycerol or 20 mM pyruvate, re aching a maximum O.D. (600 nm) of 1.6-1.7, suggesting that the H26 chromosomal dhaL knockout specifically effects DHA metabolism. Cells grown on pyruvate produced 2,3-butanediol as the sole catabolic pr oduct, while cells grown on glycerol ge nerated oxidative intermediates as well as 2,3-butanediol. Future endeavors include: perf orming growth complementation assays using the abovementioned growth media for an H26 dhaL strain complement (pJAM811) encoding DhaL; generating chromosomal gene knockouts in other components of this system and performing subsequent growth curve assays; performing enzyme activity assays; and determining whether this system is controlled at the transcriptional level and/or post-transcriptional level, partic ularly focusing on proteasomal control. 66. The roles of E2F6 and DNA methylation in the regulation of gene expression during germ cell development Smith EY, Resnick JR* Department of Molecular Genetics and Microb iology, University of Florida, Gainesville, FL Mammalian primordial germ cells (PGCs) are specified in the extraembryonic mesoderm, migrate to the developing genital ridge and subsequently differentiate into gonocytes. Colonization of the gonad coincides with a widespread DNA demethylation event in the germ cell genome, leading to the upregulation of a subset of gonocyte-specific genes. DNA methylation contributes to silencing these go nocyte genes in migratory PGCs and in somatic tissues. Additionally, recent studies suggest a role for the transcri ption factor E2f6 in silencing germ cell-specific genes in somatic tissue. A subset of germ cell-specific genes, including Tuba3, Stag3 and Smc1, is ectopically expressed in somatic tissues lacking E2f6 and DNA hypomethylation is found at the promoter region of at least one of these genes. These observations have led us to investigate a potential relationship between E2f6 and DNA methylation in the temporal regulation of gonocyte-specific gene expr ession in the developing germ line. Here we provide evidence that: 1) Members of the E2f family, including E2f6, are expressed in PGCs 2) E2f6-regulated genes are not activated as a result of the genome-wide DNA demethylation event coinciding with gonocyte differentiation 3) The Tuba3 promoter is highly methylated in PGCs Remarkably, unlike other known genes, Tuba3 DNA methylation appears to be stable through major stages of early embryogenesis and germ cell development. Current efforts are focused on creating an E2f6 gene trap mouse line to precisely determine the expression pattern of E2f6 in early development and its contribution to stability of DNA methylation in the germ line.
44 67. Conservation genetics of Crotalaria avonensis (Fabaceae), an endangered Lake Wales Ridge, FL plant Soria PS1, Germain-Aubrey C1, Weekely C2, Menges ES2, Soltis PS3,*, Soltis DE1,*, Gitzendanner MA1,* 1Department of Botany, Universit y of Florida, Gainesville, FL 2Archbold Biological Station, Lake Placid, FL 3Florida Museum of Natural History, University of Florida, Gainesville, FL The Lake Wales Ridge consists of sandy xeric uplands domi nated by pines, shrubby oaks, and hickory at the center of the Florida peninsula. Periodically isolated from North Am erica by rising sea levels du ring recent ice ages, many of these dune plants and animals remained restricted to the Ridge. Thus, the Lake Wales Ridge has one of the highest concentrations in North America of endemic species. One such endemic, Crotalaria avonensis (Fabaceae), is a federally listed endangered species. We have used microsatellite loci to investigate the genetics of C. avonensis populations. Genetic data can provide insight into distribution diversity, mating patterns and population substructuring. Additionally our data indicate that C. avonensis is tetraploid. Implications of these data for conservation planning will be discussed. 68. Estimating microbial population densities based on genomic signatures Sun Y, Yu FH, Liu L, Farmerie WG* Interdisciplinary Center for Biotechnology Rese arch, University of Florida, Gainesville, FL We consider the problem of estimating microbial composit ions of environmental samples for applications such as environmental monitoring and microbial disease diagno sis. Identifying the members of a community and their respective proportion is a challenging problem due to th e fact that the members are often dependent upon each other and thus cannot be isolated an d cultivated as pure cultures in a la boratory. The available computational methods to estimate species richness and abundance are limited. We propose a simple but effective method to overcome the limitations of the existing methods based on the concept of genomic signature. A simulation study is performed and the results de monstrate the effectiveness of the new algorithm. 69. Are head lice and clothing lice genetically distinct? A coalescent approach Toups MA1,2, Light JE2, Kitchen A3, Reed DL2,* 1Department of Zoology, University of Florida, Gainesville, FL 2Florida Museum of Natural History, University of Florida, Gainesville, FL 3Department of Anthropology, University of Florida, Gainesville, FL Humans are parasitized by two types of lice in the genus Pediculus: head lice, which live on the scalp, and clothing lice, which live in the clothing. Pesticide-resistant head lice are common among schoolchildren in developed countries as well as among the poor in both developed and undeveloped countrie s. Clothing lice are less prevalent, and occur mainly on those living in poor conditions. Precisely how and when clothing lice emerged from a head louse ancestor is not well known, but is clearly related to the habitual use of clothing among humans. Furthermore, it is unclear whether head and clothing lice have diverg ed genetically to the point that taxonomists would classify them as two distinct species. Phylogenetic analyses of mt DNA have consistently shown that clothing lice are nested within a single clade of head lice, however, Leo et al., (Heredity 95:34-40, 2005) recently us ed microsatellite data from 11 double infestations to demonstrate a lack of ge ne flow between head and clothing lice. To determine if head and body lice are genetically distinct, we analyzed pu blicly available data from three nuclear genes (18S, EF1alpha, and RPII) and one mitochondrial gene (COI) in a coalescent framework to measure migration using the software packages MDIV and IM. Preliminary results indica te that head and clothing lice have diverged, and only a small proportion of head lice initially colonized clothing. Ou r preliminary analyses also indicate that migration is unidirectional from head to clothing.
45 70. A statistical strategy for mapping imprinted quantitative trait loci: Implications for genetic mapping in mice Wang C, Berg A, Li Q, Wu R* Department of Statistics, University of Florida, Gainesville, FL The role of genetic imprinting in shaping development ha s been comprehensively investigated in plants, animals and humans. However, a statistical method that can detect and estimate the effects of imprinted quantitative trait loci (iQTLs) throughout the entire genome has not been ex tensively developed. In this study, we propose a general strategy for mapping and estimating the distribution and effects of iQTLs that are expressed from only one of the parental chromosomes. The experimental design used is based on four F2 po pulations, initiated with a pair of reciprocal crosses between two inbred lines, so that the inheritance of parent-specific alleles could be traced. The computing model and algorithm were implemented with the maximum likelihood approach. The new strategy presented was applied to study the mode of inheritanc e for iQTLs that control survival in mice. Monte Carlo simulation studies were performed to investigate the statis tical properties of the new model with the data simulated under different imprinting degrees. The F2-based design an d a series of analytical testing strategies provide a standard procedure for testing iQTL involvemen t in the genetic control of complex traits. 71. Development of a system to assess in situ switching in the Babesia bovis locus of active ves transcription (LAT) Wang X, Allred DR* Department of Infectious Diseases and Pathol ogy, University of Florida, Gainesville, FL Babesia bovis is an intraerythrocytic protozoal parasite that mainta ins persistent infection of its vertebrate host by at least two mechanisms: cytoadhesion and antigenic variation. Identification of the ves multigene family encoding the variant erythrocyte surface antigen-1 (VESA1) and the genomic locus from which ves genes are actively transcribed (LAT) enabled the demons tration of segmental gene conversion as one mechanism of antigenic variation. Previous observations reveal ed a similar organization and the presence of a highly conserved intergenic region structure among all the ves genes identified, including both the actively transcribed ves genes at LAT site and transcriptionally silent sequence dono r genes. In this study, we are attempting to determine whether antigenic variation may also occur by in situ switching of transcriptional activity from the current LAT to another previously silent gene pair. We propose to induce transcriptional switching of the LAT by selecting for a switch from the current active ves1a gene to another LAT-like locus. The activation of another ves locus will be characterized to clarify if mutually exclusive ves gene expression is occurring and controlled by in situ switching of the LAT. Toward the goals we have initiated the develo pment of a transfection system for B. bovis with the employment of a transfection vector carrying selectable markers. We have determined that B. bovis parasites are adequately susceptible to puromycin, blasticidin-s and pyrimethamin e, providing the possibility of positive selection and maintenance of transfected parasites. Transfection ve ctors have been constructed to allow identification in vitro and in vivo of suitable promoter and terminator sequences for tr ansfection. The characteristics of DNA uptake and its stability within bovine erythrocytes have been determined. Preliminary results revealed strong promoter activity of ves intergenic region. These data will provide crucial groundwork for the development of transfection in B. bovis. Supported by NIAID grant #R01 AI055864.
46 72. A model of arthrofibrosis using intra-articular gene delivery of TGF1 Watson RS1, Gouze E1,*, Levings PP1, Jorgensen M2, Gouze JN1, Bush ML1, Kay JD1, Dacanay EA1, Schultz G3,*, Ghivizzani SC1,* 1Department of Orthopaedics and Rehabilitatio n, University of Florida, Gainesville, FL 2Department of Pathology, Immunology, and Laborato ry Medicine, University of Florida, Gainesville, FL 3Department of Obstetrics and Gynecology University of Florida, Gainesville, FL Idiopathic adhesive capsulitis (IAC) of the shoulder is a disease of unknown etiology characterized by painful, chronic fibrotic expansion of the synovium and joint capsule, which gradually leads to loss of joint motion. Although IAC affects approximately 3% of the population, little is known about its pathogenesis. Similar to other fibrotic conditions, it is likely that the harmful effects of IAC are mediated by transforming growth factor1 (TGF1). In an effort to establish an animal model of this disease and develop an understanding of the cellular and molecular events contributing to arthrofibrosis, we used an adenovirus to over-express TGF1 cDNA in the knees of athymic nude rats. Various doses of Ad.TGF1were delivered into groups of rats wh ich were then sacrificed periodically. The joint tissues were examined macroscopically, histolog ically and for patterns of expression of extracellular matrix-associated genes. At days 5 and 10, TGF1 stimulated a dramatic fibrotic event, causing a rapid increase in knee diameter and the complete encasement of the joints in dense scar-like tissue, locking the joints at 90o of flexion. Histologically, massive proliferati on of resident synovial fibroblasts wa s seen followed by their differentiation into myofibroblasts. The fibrotic expansion was observed to overrun and displace the normal architecture of the joint capsule and fuse with the articular cartilage. The expr ession profiles showed exceptionally high levels of MMP production, as well as tenascin C, osteopontin and thromobospondins 1 and 2. By day 30 the predominant phenotype of the expanded tissue had changed to articular cartilage, indicated by cellular morphology, the matrix composition of the tissue, and 100 and 300 fold increase s in link protein and collagen type II expression, respectively. These results suggest that cellular and mole cular events leading to IAC share many aspects with tumorigenesis and metastasis. They also demonstrate the proliferative potentia l and plasticity of fully differentiated cells in adult tissues. 73. Mapping quantitative trait loci of complex tr aits based on zygotic linkage disequilibrium Wu S, Liu T, Yap JS, Hou W, Wu RL* Department of Statistics, University of Florida, Gainesville, FL. Linkage disequilibrium-based mapping that capitalizes on hi storical recombinant events has proven to be powerful for detecting quantitative trait loci (QTLs) that contro l a complex trait in a natural population. This approach, founded on the non-random association between markers an d QTL at the gametic level, requires the population mapped to be in Hardy-Weinberg equ ilibrium (HWE), which may not be a ca se for many genetically informative isolated populations. Here, we present a new QTL mappi ng approach based on linkage disequilibria at the genotypic or zygotic level by accommodating the deviatio n from HWE. This approach allows joint or separate estimation of Hardy-Weinberg disequilibrium at individ ual loci, gametic and non-gametic linkage disequilibria, trigenic, and quadrigenic linkage disequilibria between th e markers and QTLs. By testing these different types of disequilibria, we generalized framework for inferring the existence of the underlying QTL for a complex trait. We performed simulation studies and real data analyses to in vestigate the statistical properties of this approach and validate its utilization. This approach will open a genera l gateway for studying the detailed picture of the genetic architecture of quantitative vari ation in natural populations.
47 74. Bahiagrass with expression of HvWRKY38 tran scription activator displays improved drought tolerance Xiong X, James V, Altpeter F* Department of Agronomy and Graduate Program in Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL Bahiagrass (Paspalum notatum Flugge) is an important turf and forage grass in the south-eastern US and in subtropical regions around the world. The productivity and persistence of bahiagrass is limited by environmental stresses like drought, freezing and in salt affected areas. We isolated several transcription activators of genes involved in abiotic stress response and will present da ta on over-expression of the HvWRKY38 transcription activator in bahiagrass. Transcription factors, like HvWRKY 38 are capable of activating the expression of multiple genes involved in protection against environmental stre sses. A constitutive HvWRKY38 expression cassette was successfully introduced into bahiagrass cv. Argentine via biolis tic gene transfer as indicated by Southern blot or PCR analysis. Over-expression of this tr anscription factor was confirmed by real-time RT-PCR. We will present physiological data on dehydration tole rance including biomass production an d stress symptoms following severe dehydration, and photosynthetic efficiency during and after stress. 75. Deficiency in a cytosolic ribose-5-phosphate is omerase causes chloroplast dysfunction, cell death, and disease susceptibility in Arabidopsis Xiong Y, Zhang X, DeFrai a C, Williams D, Mou Z Department of Microbiology and Cell Scienc e, University of Florida, Gainesville, FL The oxidative pentose phosphate pathwa y (oxPPP) is part of the central metabolism, which not only produces the reducing equivalent NADPH for biosynth esis and maintaining the redox potential in the cell, but also generates erythrose-4-phosphate, a precursor of the shikimate pathwa y, leading to the biosynthesis of the antimicrobial compounds called phytoalexins and the defense signal mole cule salicylic acid (SA). It has been shown that the oxPPP is induced upon pathogen attack and elicitation, wh ich leads to the hypothesis that the oxPPP may play a role in plant defense responses. Howe ver, thus far no evidence is availa ble to demonstrate that a direct link between the oxPPP and plant defense responses exists. Here we report identification and characterization of Arabidopsis T-DNA knockout mutants of the RPI2 gene (At2g 01290), which encodes a cytosolic RPI that catalyzes the reversible interconversion of ribu lose-5-phosphate and ribose-5-phosphate in the nonoxidative stage of the oxPPP. Knockout of RPI2 interferes with the chloroplast function and causes premature cell death. The rpi2 mutants accumulate less starch in the leaves and flower significa ntly later than wild type when grown under short-day conditions. More interestingly, knockout of RPI2 also partially compromises SA accumulation following pathogen infection and leads to enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. The rpi2 mutant is the first plant oxPPP mutant that exhibits enhanced disease susceptibility, which supports the existence of a link between the oxPPP and plan t defense responses. Thus, further investigation of mutants of the genes that encode the oxPPP enzymes will allow us to identify the missing link between the oxPPP and plant defense responses, and gain insight into the role of the oxPPP in plants response to other environmental stresses.
48 76. Nonparametric covariance estimation in fu nctional mapping of quantitative trait loci Yap JS, Wu RL* Department of Statistics, University of Florida, Gainesville, FL Estimation of the covariance structure of longitudinal processes is a fundamental prerequisite for the practical deployment of functional mapping designed to study the ge netic regulation and network of quantitative variation in dynamic complex traits. We present a nonparametric approach to global estimation of the longitudinal covariance structure of a quantitative trait measured repeatedly at time s. This approach is formulated by regressing the trait variable on its predecessors, allowing for the estimation of a sequence of varying-coefficient and varying-order regression models. This approach is embedded within th e framework for functional mapping to genomewide scan for the existence of quantitative trait loci underlying a dyna mic trait, leading to enhance the breadth of use of this mapping method while preserving its biological relevance. A live example from a mouse genome project is analyzed to illustrate the methodology. Extensive simulation studie s are performed to reveal the statistical properties and advantages of the nonparametric covariance modeling for functional mapping. 77. Building a skeleton: The origin of vertebrate cartilage development Zhang GJ, Miyamoto MM*, Cohn MJ1,* Department of Zoology, University of Florida, Gainesville, FL The phylogenetic relationships of the vertebrates were established largely based on anatomical characters, particularly those of the skeleton. Skeletal development has been well studied in higher vertebrates (e.g., mouse and chicken), however, little is known about the developmen tal mechanisms responsible for evolutionary origin of the vertebrate skeleton. Here we investigate cartilage development in two jawless vertebrates, hagfishes and lampreys, and in a sister group to the vertebrates, la ncelets (amphioxus). We report that both lamprey and hagfishes have Col2a1-based cartilage, su ggesting that this type of cartilage was present in common ancestor of all crown vertebrates. Our analysis of lanc elets revealed the presence of an ancestral clade A fibrillar collagen (ColA) gene that is expressed in the notoch ord. Thus, during the chordate-vertebrate transition, an ancestral clade A fibrillar collagen gene underwent duplication and diversific ation, and this process may underlie the evolutionary origin of vertebrate skeletal tissues. 78. Quantitative PCR: A very useful tool for genomics research Zhang L, Holland K Interdisciplinary Center for Biotechnology Rese arch, University of Florida, Gainesville, FL Real-time quantitative PCR remains one of the most sensit ive and quantitative tools for genomics research used today. ICBR offers both custom and pre-optimized QPCR assays for an assortment of specific applications. We manage all aspects of the project from assay design to da ta analysis. This service is advantageous to researchers conducting the following studies: microRNA quantificat ion assays, SiRNA knockdown assay, gene expressionprimary validation/quantification, gene expression-confo rmation of microarray data, pathogen (viral/bacterial) detection/quantification, allelic discrimination/SNP geno typing analysis, transgene detection/quantification and biological diversity/contamination.
49 79. Genomic analysis of tissue compartmentaliza tion during development of the mouse external genitalia Zheng Z1, Seifert A1, Cohn MJ1,2,* 1Department of Zoology, University of Florida, Gainesville, FL 2Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL Development of external genitalia in mammalian embryos is a wellorganized morphogenetic process involving outgrowth, proximodistal and dorsoventral patterning, and epithelial tubulogenesis. Exte rnal genitalia development in males and females begin with formation of a genital tu bercle, an outgrowth of lateral plate mesoderm, surface ectoderm, and endodermal epithelium derived from the urog enital sinus. The molecular mechanisms that regulate morphogenesis of the mammalian external genitalia are poorly understood. Previous studies in our laboratory revealed that the urethral epithelium acts as a signaling center of the genital tubercle and Sonic hedgehog from the urethral epithelium is required for outgrowth, patterning, and cell survival in the developing external genitalia. In order to determine the transcriptional profile of this new ly-defined signaling region, we separated by flow cytometry the urethral epithelium and mesenchymal cells of E12. 5. shhgfpcre mouse embryos and performed microarray analysis. We identified 88 genes that are differentia lly expressed in the urethral epithelium and genital mesenchyme. Of these, 14.7% are cytoskeleton/structure proteins, 13.6% are membrane associate proteins with multiple activities, 11.4% are signal transduction protei ns, 8% are transcription factors, 9% are cell growth and proliferation regulators, 11.4% are metabolic enzymes, 15% belong to other identified protein classes and 25% are unknown proteins. Eighty-two genes were found more highly expressed in urethra and six genes more highly expressed in mesenchyme. More than 60 urethra expres sed genes were confirmed using both whole mount and section in situ hybridization. Interestingly, most of urethral expressed genes were also found to be strongly expressed in gut, suggesting a clos e relationship between the molecular mechanisms of gut and urethral patterning.