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OCTOBER 27-28Cancer and Genetics Research Complex University of Florida Gainesville, FLTHE SIXTH ANNUAL SYMPOSIUM OF THE UF GENETICS INSTITUTE GOLD SPONSORSCenter for Epigenetics College of Liberal Arts and Sciences Department of Molecular Genetics and Microbiology Graduate Program in Plant Molecular and Cellular Biology Health Science Center Libraries Interdisciplinary Center for Biotechnology Research UF Genetics Institute SILVER SPONSORSEmerging Pathogens Institute Evelyn F. and William L. McKnight Brain Institute
Florida Genetics 2010 Organizing Committee Co Chair s : Connie Mulligan and Michele Tennant Members: Henry Baker, John Davis, Rob Ferl, John Pastor D ouglas Soltis, Indra Vasil, Thomas Yang Mimi Zarate FG2010 Sponsors Gold Level University of Florida G enetics Institute, Center for Epigenetics, College of Liberal Arts and Sciences Department of Molecular Genetics and Microbiology Graduate Program in Plant Molecular and Cellular Biology, Health Science Center Libraries, Interdisciplinary Center for Biot echnology Research Silver Level Emerging Pathogens Institute, Evelyn F. & William L. McKnight Brain Institute Special Thanks To David Brumbaugh Mickey Cuthbertson, Martine Horrell, Hope Parmeter, Dustin Blanton, Tamar Carter, Lauren Douma, Justin Fear Rolando Garcia Milian, Xiaoxia Han, Hye Won Lee Guangyao Li, Tong Lin, Katie O'Shaughnessy Beverly Osborn, Connie Philebaum, Yan Ren Tatiana Salazar Jared Stees Ming Tang, Patrick Thiaville, Yuanqing Yan University of Florida Genetics Institute Di rector: Kenneth Berns Associate Directors: Henry Baker, Donald McCarty, Connie Mulligan, Indra Vasil Executive Board: William Allen, Henry Baker, Kenneth Berns, John Davis, Robert Ferl, William Hauswirth, Julie Johnson, Donald McCarty, Michael Miyamoto, Co nnie Mulligan, Nicholas Muzyczka, Winfred Phillips, Pam ela Soltis, Douglas Soltis, Michele Tennant, Indra Vasil, Wilfred Vermerris, Marta Wayne, and Thomas Yang Scientific Advisory Board: Rebecca W. Doerge, Ph.D., Professor, Departments of Agronomy and S tatistics, Purdue University, West Lafayette, IN 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 Com parative Pathology and Chair, Department of Pathology, Harvard Medical School, Boston, MA Thomas J. Kelly, M.D., Ph.D., Director, Sloan Kettering Institute, New York, NY Ronald L. Phillips, Ph.D., Regents Professor, Agronomy and Plant Genetics, University of Minnesota St. Paul, MN Ron Sederoff, Ph.D., Distinguished University Professor of Forestry, North Carolina State University Forest Biotechnology Group, Raleigh, NC Thomas E. Shenk, Ph.D., James A. Elkins, Jr. Professor in the Life S ciences, Molecular Biology, Princeton University, Princeton, NJ
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 sc ientific discoveries of all time. It fundamentally and permanently changed the course of biology and genetics. The unraveling of DNA's structure, combined with its elegant mechanism for self replication and the existence of a universal genetic code for all living beings, have together 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 doubl e helix is also proving to be of immense significance to advances in agriculture, medicine and such other diverse fields as anthropology, criminology, computer science, engineering, immunology, nanotechnology, etc. It was the study of DNA that led to the d evelopment of tools that brought about the biotechnology revolution, the cloning of genes, and the sequencing of entire genomes. Yet, most knowledgeable people agree that what has been achieved in DNA science thus far is only the beginning. Bigger and bett er applications, which will impact directly on the quality of human life and sustainability of life on earth, are yet to come. In order to attain these objectives, the digital nature of DNA and its complementarity are beginning to be exploited for the deve lopment of biology as an information based science. Indeed, a paradigm shift is already taking place in our view of biology, in which the natural, physical, engineering and environmental sciences are becoming unified into a grand alliance for systems biolo gy. Indeed, biology in the 21 st century will be surely dominated by this expanded vision. The Genetics Institute is committed to fostering excellence in teaching and research, and in promoting cross campus interdisciplinary interactions and collaborations. In the pursuit of these objectives, it offers a graduate program in genetics, and has identified the following four key areas for teaching, research and development: Bioinformatics, Comparative Genomics, Population and Statistical Genetics, and Epigenetic s.
2010 Florida Genetics Symposium Schedule Wednesday, October 27, 2010 Noon 1:00 p.m.: Check in and poster set up at the Cancer/Genetics Research Complex 1:00 p.m. 1:15 p.m.: Opening Remarks: Indra Vasil and Kenneth Berns Session I Microbial Genomics Chair: Val rie de Cr cy Lagard 1:15 p.m. 2:00 p.m., Folker Meyer : Reversing the paradigm t he complete genome sequence for Candidatus Sulfuricurvum sp. derived from a complex short read metagenome enables characterization of this novel pr oteobacterium 2:00 p.m. 2:30 p.m., Eric Triplett : Defining the autoimmune microbiome for type 1 diabetes 2:30 p.m. 4:0 0 p.m.: Poster Session and Reception (for registered attendees) ** 5:00 p.m. 6:00 p.m., Tom Caskey : Schizophrenia gene dis covery by complete genome sequencing of families *= UF Genetics Institute Faculty ** All activities will be at the Cancer/Genetics Research Complex except for Dr. Caskey 's presentation, which will be at 5 :00 p.m. Wednesday in the auditorium of th e HPNP building on the Health Science Center campus. Thursday, October 28, 2010 8 :00 a.m. 8:30 a.m.: Check in coffee Session II Pathogen Genomics Chair: Glenn Morris 8:30 a.m. 9:15 a.m., Marshall Bloom : "Pathogenesis of tick born flavivirus i nfections: probing the pathogen vector host interface" 9:15 a.m. 9:45 a.m., Jorge Giron : Hide n sick strategies of a human pathogen to colonize animal and plant cells 9:45 a.m. 10:30 a.m., David Rasko : Genomic and transcriptomic characterizatio n of E. coli/Shigella : novel insights into established pathogens" 10:30 a.m. 11:00 a.m., Marco Salemi : On the temporal structure of longitudinal sample genealogies: from fast evolving viruses to ancient DNA 11:10 a.m. 1:30 p.m.: Poster Session 11 :30 a.m. Lunch (for registered attendees) Session III Genetics of Plant Reproduction Chair: Doug Soltis 1:30 p.m. 2:00 p.m., Chris tine D. Chase : Cytoplasmic male sterility: window to mitochondrial nuclear interactions in plants 2:00 p.m. 2:4 5 p.m., Richard Amasino : "Vernalization: an environmentally induced epigenetic switch for flowering" 2:45 p.m. 3:15 p.m., Bernie Hauser : Reactive oxygen scavengers regulate reproductive traits in Arabidopsis 3:15 p.m. 3:45 p.m., Don McCarty : Structure and evolution of the B3 transcription factor network controlling the transition between the seed and vegetative phases of the plant life cycle"
Presentation Abstracts Reversing the paradigm t he complete genome sequence for Candidatus Su lfuricurvum sp. derived from a complex short read metagenome enables characterization of this novel proteobacterium Meyer F Mathematics and Computer Science Division and Institute for Genomics and Systems Biology, Argonne National Laboratory, Argonne, IL ! Characterizing genomes of unculturable microbes requires new approaches for genome assembly from environmental samples. I will describe the successful assembly of a complete microbial genome from a complex sample (>200 OTUs). The assembly via short rea d metagenomics req uired novel approaches for assembly based on simple statistical principles. Traditional approaches fail because uneven numbers of sequence reads from common and rare spe cies, and pan genome variation, confuses traditional genome assembler s; species without close relatives sequenced cannot co assemble: without our approach, additional sequence data hurts rather than helps assembly. Metabolic reconstruct ion of this genome will permit cultiv ation of this dominant organism from an ecosystem re levant to bioremediation. This general approach will allow the assembly of genomes and cultivation of key species from diverse environments/enrichment cultures, thus providing new pangenomic insights into processes ranging from biofuel generation to identi fication of emerging pathogens. Biography of Folker Meyer Ph.D. Folker Meyer is a C omputational B iologist and A ssociate D ivision D irector of the Institute of Genomics and Systems Biology at Argonne National Laboratory He also is a senior fellow at the Computation Institute at the University of Chicago. He train ed as a computer scientist through the computer science and biology departments at Bielefeld University in Germany. His early work with biologists heightened his interest in building software sys tems to tackle biological problems, mostly in the field of gen omics or post genomics. H e has played an active role in the design and implementation of several high performance computing platforms including a 500 plus CPU high performance bioinformatics com puting facility at Bielefeld University. While there, he also initiated and oversaw the Applied Bioinformatics Group. He is known for his leadership role in the development of the GenDB genome annotation system for prokaryotic genomes. The system was devel oped as a use r friendly framework for both bioinformatics researchers and biologists to use in their genome projects. His current work focuses on the analysis of shotgun metagenomics data sets known as Meta Genome Rapid Annotation using Subsystem Technol ogy ." C urrent advances in sequencing technology have led to dramatic growth in the number of scientists using shotgun metagenomics as well as the number and size of the data sets being produced. MG RAST is a fully automated service for annotating metagenom e samples, providing annotation of sequence fragments, their phylogenetic classification, metabolic reconstructions and comparison tools. Complementing his work with shotgun metagenomics, he has an interest in microbial genomics and the analysis of complet e microbial genomes
Schizophrenia gene discovery by complete genome sequencing of families Caskey CT 1 "#$% !" & '! ()*+,-.+ / (,0,1 !2 & '! 2,0#,* !34 & '! !5,%%.6 !3 7 '! "8,** !3 7 '! 5#--#,$% !9 7 1 Brown Foundation Institute of Molecular Medicine University of Texas Hea lth Science Center, Houston, TX 2 University of Texas Harris County Psychiatric Center (UTHCPC), Houston, TX The genetic basis of schizophrenia is well established clinically and by molecular population studies using SNP and copy number variant (CNV) analy sis. Heterogeneity of the genetic basis of the schizophrenia phenotype is anticipated. We have studied families with multiple affected individuals from the Harris County Psychiatric Center, in Houston, by complete genome sequencing with two approaches 1) whole exome capture and deep sequencing (Illumina GAIIx), and 2) whole nuclear genomic DNA seque ncing (Complete G enomics, Inc. Drmanac et al., 2010, Science 327 (5961 ) :78 81) The observed sequence changes are being validated by comparison of results fr om both sequencing approaches. Where appropriate any candidate variants found are further confirmed by still a third method (genotyping or Sanger seque ncing). Our study utilizes human genetics and complete genome sequencing to identify disease gene candi da tes for individual families. Families have been selected and the sequencing has been completed. Bioinformatic analysis focuses on non synonymous coding and splice junction alterations, in addition to CNV analysis. We correlate any sequence variants discove red with known SNPs, CNVs, and regional brain gene ex pression of candidate alleles. Thus, we utilize a systems biology approach to candidate gene identification, taking into account family pedigrees, sequence variants, knowledge of pathways involved and ex pression information. The studies identify gene candidates for schizophrenia. New disease/gene candidates are the focus of our presentation. Biography of C. Thomas Caskey, M.D., F.A.C.P. C. Thomas Caskey attended the University of South Carolina as an un dergraduate and received his medical degree from Duke University Medical School. His exposure to world renowned scientists began early. At Duke, he was a Biochemical Fellow with James B. Wyngarden, a pioneer in the study of the biochemical basis of gout, a metabolic disease. Shortly afterward, while he was a Research Associate with the National Institutes of Health from 1965 to 1971, Dr. Caskey became a close colleague of Nobel Laureate Marshall Nirenberg, who had received his undergraduate and master's deg rees in zoology at the University of Florida. His research with Dr. Nirenberg proved the universality of the genetic code for all living organisms. Dr. Caskey joined Baylor College of Medicine in 1971 where he served as Chief of the Section of Medical Gene tics and Professor of Medicine and Biochemistry. He was a Howard Hughes Medical Institute Investigator from 1976 to 1994. During his career, Caskey discovered 11 genetic disease genes most notably, those for "triple repeat" diseases myotonic dystrophy an d fragile X syndrome, the most common form of retardation. From there, he quickly unraveled the phenomenon of "anticipation," a characteristic of fragile X in which the disease worsens each generation. Dr. Caskey left academia in 1994 to assume the positio n of Senior Vice President for Research at Merck Research Laboratories, and Trustee and President of the Merck Genome Research Institute. A research team under Dr. Caskey's direction developed the adenoviral vector HIV vaccine He returned to Texas in 2000 and became Founding Director and Chief Executive Officer of Cogene Biotech Ventures and Cogene Ventures, venture capital funds designed to support early stage biotechnology and life sciences companies using genome technology for drug discovery. He served on Texas Governor Rick Perry's Advisory Committee for two years, which provides expertise for the Texas Emerging Technology Fund. He also serves on the World Health Organization's Expert Advisory Panel on Human Genetics and as a special advisor to the Here ditary Diseases Program. Caskey was part of the original group that started the human genome initiative, now called the Human Genome Organization. The FBI adopted his forensic analysis system for identifying people based on their digitized genetic informat ion. The method, first used during the Persian Gulf War to identify soldiers killed in battle, is now employed worldwide to solve crime and missing persons cases. In 2006, Dr. Caskey was appointed Chief Operating Officer and Director of the Brown Foundatio n Institute of Molecular Medicine for the Prevention of Human Diseases, and Executive Vice President of Molecular Medicine and Genetics at the University of Texas Health Science Center Houston. In 2007 Dr. Caskey was named CEO of the Brown Foundation Insti tute and the George and Cynthia Mitchell Distinguished Chair in the Neurosciences. But of all the brilliant moments in his career, some of his most treasured memories are from his work with the late Marshall Nirenberg. "Marshall Nirenberg was very special to me," he recalls. "We cracked a major problem no one knew how to get information on the genetic code. We did that. And we did that in an environment with smart young people. He was a great young leader of that lab."
Pathogenesis of tick born flavivir us infections: probing the pathogen vector host interface Bloom M E Tick borne Flavivirus Pathogenesis Section Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases National Institutes of Health, Hamilton, MT Vector borne fl aviviruses are the source of considerable misery, morbidity and mortality worldwide. These positive sense single stranded 11 kb RNA viruses encode a large polyprotein that is post translationally cleaved into 3 structural and 7 non structural proteins. Cli nical syndromes form a spectrum from mild febrile illnesses to severe, sometimes fatal, meningoencephalitides and viral hemorrhagic fevers. Although the emergence of West Nile Virus and the resurgence of Y ellow Fever and Dengue Virus have sparked renewed i nterest in mosquito borne agents, the tick borne flaviviruses (TBFV) also pose serious problems in Europe, Eurasia and Asia. Indeed, the TBFV Powassan virus is emerging in the US. The natural history of TBFV infections is a complicated interplay involving the virus, the arthropod host (Ixodid ticks), and various mammalian species. The virus must cope with or subvert features of both the vertebrate and invertebrate hosts for successful maintenance and replication. This talk will describe two areas of TBFV pa thogenesis. One section will relate the limited number of TBFV genetic alterations specific to serial passage in either arthropod or mammalian cell lines, and how these mutations impact viral pathogenicity. Another section will detail studies intended to r elate tick salivary gland gene expression to pathogenesis. Tick saliva is known to augment transmission of various pathogens, including TBFV, to mammalian hosts. Using recently available information about the ixodid genome and the expression profile in tic k salivary glands, we are defining the specific components of tick saliva responsible for this phenomenon. A multi faceted approach will inform opportunities for understanding and controlling TBFV infections. This work is supported by the NIAID Division o f Intramural Research. Biography of Marshall E. Bloom, M .D. Marshall Bloom received his medical degree in 1971 from Washington University School of Medicine at St. Louis. He joined Rocky Mountain Laboratories (RML) of the National Institute of Allergy and Infect ious Diseases in Hamilton, Montana in 1972 as a R esearch A ssociate. From 1975 to 1977, he was a postdoctoral fellow in the NIAID Laboratory of the Biology of Viruses on the National Institutes of Health campus in Bethesda. He returned to R ocky M ountain Laboratories as Medical Director for research and tenured investigator in 1977 RML is perhaps best known for its research into vector borne diseases, such as Rocky Mountain spotted fever, Q fever and Lyme disease three illnesses caused by microb es whose names pay tribute to the former RML scientists who discovered them. In 2002, Dr. Bloom was appointed Associate Director for RML in NIAID's Division of Intramural Research, responsible for program supervision of the permitting, construction and sta ffing of the NIAID's first Biosafety Level 4 facility, among other duties. He has also served as Acting Chief of Laboratory of the NIAID's Laboratory of Virology and Acting Chief of the NIAID Laboratory of Human Bacterial Pathogenesis. Dr. Bloom was named Associate Director for Science Management for RML in NIAID's Division of Intramural R e search in 2008, and in the same year, Dr. Bloom was named Chief of the Tick borne Flavivirus Pathogenogenesis Section In 2009, Dr. Bloom was appointed Associate Director for Science Mana gement. He is a world expert in the molecular biology and pathogenesis of parvoviruses, and is considered an authority in biocontainment. His major areas of research include interactions between tick borne encephalitis (TBE) viruses and in nate immune responses, including interferon and apoptosis signaling pathways; viral determinants of neuro virulence and neuro invasiveness; and viral determinants of effective transmission from tick to mammalian host D r. Bloom's laboratory investigates th e pathogenesis of viruses belonging to the TBE virus complex of flaviviruses. Endemic throughout much of the Northern Hemisphere, these viruses can cause severe encephalitis, meningitis, or hemmorhagic fevers with relatively high mortality rates. The labor atory's research utilizes two animal models of infection, the mouse and the tick, to examine viral determinants of transmission and virulence. Understanding these aspects of pathogenesis will provide targets for the design of therapeutics and vaccines. !
Genomic and transcriptomic characterization of E. coli/Shigella : novel insights into established pathogens Rasko D A Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD Escherichia coli/Shigella exist as the major facultative anaerobe in the human gastrointestinal tract, but also can cause serious disease es pecially in young children and travelers. While E. coli/Shigella has been a model microbial system in use for decades, the advent of the genomic era has allowed unprecedented insight into the variation of this important species. There are currently five readily identifiable and generally agreed upon pathogenic variants of diarrheagenic E. coli/Shigella We have determined that there are ~2200 genes that all E. col i/Shigella share and sequencing each novel genome will provide approximately 300 new genes. These two factors together indicate that the pan genome of E. coli/Shigella is open and ever expanding. Genomic studies have focused on the enterotoxigenic E. coli (ETEC) pathovar and Shigella Using an unbiased whole genome comparison we have identified approximately 2.7 million bases of the E. coli/Shigella genome that is highly conserved in all isolates examined to date. These analyses have provided new insights i nto the evolution of ETEC as a pathogen. Additionally, we have examined a collection of Shigella isolates from lethal diarrheal outcomes in an effort to understand if there was a molecular basis for increased virulence. Using both comparative genomic hybri dization and whole genome sequencing we have identified genomic regions that may explain the unfortunate outcomes in some cases. While the genome can identify the potential of any isolate, examination of the transcriptome will provide insight into the gene s that are actually expressed. Again we have focused on ETEC and Shigella as model systems to examine genes expressed in vivo and in vitro in an attempt to examine the regulatory networks of these important pathogens. This expression data combined with com parative genomics has identified a number of targets that are both transcriptionally active and species restricted that we can utilize as therapeutic targets. Comparative genomics and transcriptomics has reached a new era where the number of isolates that can be rapidly characterized will begin to address epidemiological questions and become a more integrated component of the global health care system. Biography for David A. Rasko Ph.D. David Rasko is an Assistant Professor at the newly formed Institute for Genome Sciences at the University of Maryland School of Medicine. His laboratory focuses on the functional genomics and transcriptome analysis of Escherichia coli and Shigella Prior to joining the group at Maryland, Dr. Rasko had a Research Assistant Professor position at the University of Texas Southwestern Medical Center at Dallas, where he examined the functional genomic aspects of enterohemorrhagic E. coli and Francisella tularensis Dr. Rasko gained extensive comparative genomics experience at The Institute for Genome Research studying the genome structure and evolution of multiple human pathogens including Escherichia coli/Shigella and Campylobacter Listeria Yersinia species as well as Bacillus anthracis and B. cereus Dr. Rasko's post doctoral s tudies were completed at the University of Maryland School of Medicine in the Department of Microbiology and Immunology with Harry L.T. Mobley on genomics and functional characterization of virulence factors of urinary tract pathogens. In addition, his und ergraduate and graduate studies were completed at the University of Alberta on the regulation and expression of Lewis blood group antigens by the gastric pathogen Helicobacter pylori
Vernalization: an environmentally induced epigenetic switch f or flowering Amasino RM Department of Biochemistry University of Wisconsin, Madison, WI Howard Hughes Medical Institute, University of Wisconsin, Madison, WI Certain plants, such as biennials or winter annuals, require relatively long periods of cold ex posure during winter to initiate flowering the following spring. Cold exposure renders the meristem of such cold requiring species competent to flower (and in Arabidopsis it also permits young leaves to express the FT gene which encodes "florigen"). This a cquisition of competence to flower is known as vernalization. Before competence is achieved, plants must measure exposure to a sufficient number of days of cold to represent a complete winter; this ensures flowering only occurs when spring has arrived, rat her than during a temporary w arming in the middle of winter. Our studies have revealed that, in Arabidopsis vernalization mediated meristem competence is a function of the expression level of the MADS box gene FLOWERING LOCUS C (FLC). FLC is a repressor o f flowering. Exposure to prolonged cold causes epigenetic silencing of FLC, thus rendering the shoot apical meristem competent to flower. During cold exposure, chromatin remodeling complexes catalyze covalent modification of histones of FLC chromatin resul ting in silencing of expression. Studies in other groups of plants reveal that the vernalization requirement is based on components different than those in Arabidopsis but to date all vernalization pathways are "overlaid" on a conserved flowering pathway that is often involved in photoperiod sensing. Biography of Richard M. Amasino, Ph.D. Richard M. Amasino is a Distinguished Professor of Biochemistry at the University of Wisconsin Madison and a Howard Hughes Medical Institute Teaching Professor. H e be came interested in plants as a youngster Today, he addresses the mystery of how a plant "knows" that the dangers of winter have passed and the lengthening days of spring have made it safe for the plant to flower Through those studies his lab discovered the molecular epigenetic switch that prevents a plant from flowering until it has experienced a long period of cold weather. His lab also has projects on other aspects of flowering time regulation such as the mechanism of day length perception. Using Arabi dopsis thaliana he identified two dominant genes that work together in vernalization in flowering plants. His work has lead to a molecular understanding of epigenetic memory of winter in Arabidopsis Dr. Amasino received his bachelor's of science degree i n biology from The Pennsylvania State University and his master's and doctoral degrees from Indiana University, Bloomington. He was Senior Fellow in the laboratories of M.P. Gordon and E.W. Nester of the Departments of Biochemistry and Microbiology at the University of Washington, Seattle. In 1985, he was appointed Assistant Professor at the University of Wisconsin Madison and in 1991 he was named an Associate Professor. He was appointed Wis consin Distinguished Professor of Biochemistry in 1996 and HHMI Tea ching Professor since 2006 where he is working on a project to develop a practical classroom model to teach classical, molecular, developmenta l and biochemical genetics to undergraduates.
*= UF Genetics Institute Faculty. Presenters are underlined. D efining the autoimmune microbiome for type 1 diabetes Giongo A 1 Gano KA 1 Crabb DB 1 Mukherjee N 2 Novelo LL 2 Casella G 2 ,* Drew JC 1 Neu J 3 Wasserfall CH 4 Schatz D 3 ,* Atkinson MA 4 ,* Triplett EW 1,* 1 Department of Microbiology and Cell Science, Uni versity of Florida, Gainesville, FL 2 Department of Statistics, University of Florida, Gainesville, FL 3 Department of Pediatrics, University of Florida, Gainesville, FL 4 Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Ga inesville, FL Several studies in rodent models have shown that gut bacteria play a role in diabetes. However, it is unknown whether human intestinal microbes play a role in the development of autoimmu nity that often leads to type 1 diabetes (T1D), an auto immune disorder in which insulin secreting pancrea tic islet cells are destroyed. High throughput, culture independent approaches identified bacteria that correlate with the development of T1D associated autoimmunity in young children who are at high geneti c risk for this disorder. The level of bacterial diversity diminishes over time in these autoimmune subjects relative to that of age matched, genotype matched, non autoimmune individuals A single species, Bacteroides ovatus comprised nearly 24% of the t otal increase in the phylum Bacteroidetes in cases compared to controls. Conversely, another species in controls, represented by the human firmicute strain CO19, represented nearly 20% of the increase in the Firmicutes compared to cases over time. Four l ines of evidence are presented that support the notion that, as healthy infants approach the toddler stage, their microbiomes become healthier and more stable; whereas, children who are destined for autoimmunity develop a microbiome that is less diverse an d stable. Compared to the healthy microbiome, the autoimmune microbiome is less diverse, has fewer unclassified bacteria, appears to contain more pathogens, and is a less stable community. These data also suggest bacterial based markers for the early diagn osis of T1D. In addition, bacteria negatively correlated with the autoimmune state may prove useful in the prevention of autoimmunity development in high risk children. Hide n sick strategies of a human pathogen to colonize animal and plant cells Giro n J* Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a food borne pathogen that uses a variety of adhesive filamentous structures to adhere to the human large inte stine and injects many effector molecules that inflict damage to the gut mucosa. The Shiga toxins produced by this E. coli are responsible for the hemorrhagic colitis caused in the intestine and the glomeruli occlusion that leads to renal failure in th e kidney (hemolytic uremic syndrome). The bacteria live as commensal organisms in the gut flora of cattle and other farm animals. Thus, consumption of contaminated ground beef, milk, juices, water or leafy greens has been associated with outbreaks of diarr heal disease. Our investigations of the cellular, molecular, and ultrastructural aspects of the interaction of EHEC O157:H7 with host cells has led to the identification of 3 new adherence factors and understanding how these new pili are regulated and sync hronized during human, bovine, or plant colonization is central to our research. The new pili include the E. coli common pilus (ECP), a type IV pilus called hemorrhagic coli pilus (HCP) and the E. coli laminin binding fimbriae (ELF). Of interest as well, i s to study the molecular mechanisms of pili biogenesis and the identification of specific cellular receptors. Our investigations on the E. coli spinach interaction show that this human pathogen establishes a niche in leaves' organelles called stomata and i nternal tissues where they are protected from decontamination processes and environmental foes, being able to survive in the environment and gain access to the human host. An exciting aspect of our EHEC research was the discovery that a secreted protein, E spP, oligomerizes into novel macroscopic rope like structures that exhibited adhesive and cytopathic effects on cultured epithelial cells, served as a substratum for bacterial adherence and biofilm formation, and protected bacteria from antimicrobial compo unds. We hypothesize that these ropes play a biologically significant role in the survival and pathogenic scheme of these organisms.
On the temporal structure of longitudinal sample g ene alogies: from fast evolving viruses to a ncient DNA Salemi M Depa rtment of Pathology, Immunology and Laboratory Medicine University of Florida, Gainesville, FL Emerging Pathogens Institute, University of Florida, Gainesville, FL The genealogies of fast evolving RNA viruses and retroviruses are shaped by the inter action of selective pressure, spatial and population dynamics occurring on multiple scales over relatively short time intervals (months to years). Dated tip genealogies from longitudinally sampled sequences can be inferred by enforcing either a strict or a relaxed molecular clock. Trees from genes under significant selection typically display strong temporal structure, which reflects the continuous turnover of the viral population. Examples of this may be seen in genealogies inferred from intra host HIV 1 o r inter host influenza sequences sampled over time, where the shape of the genealogy often resembles a staircase characteristic of continuous selection leading to antigenic shift and periodic population bottlenecks. On the other hand, weak selective pressu re usually results in genealogies exhibiting star like topology and poor temporal structure with sequences from different sampling times highly intermixed. Two classes of statistics have been employed so far to compare genealogical shapes: those that use b ranch length information, and those that measure purely topological characteristics. However, none of these statistics is able to evaluate genealogies from longitudinal data. The temporal structure (TS) index, on the other hand, was developed to quantify t he degree of temporal structure of serial samples genealogies. The TS index is based on the Slatkin Maddison trait tip association test, which was modified to measure the distribution of the sequences sampling times, treated as characters rather than discr ete traits, in the genealogy. The behavior of the TS index was assessed by analyzing sequence data simulated with the serial samples coalescence. Several empirical genealogies inferred from HIV 1 intra host and HCV inter host data, as well as mammalian seq uences including ancient mtDNA were also investigated. The results demonstrate that the TS index is a robust statistic that can provide significant insights on the evolutionary and population dynamic processes of dated tips genealogies at different time sc ales. Cytoplasmic male sterility: window to mitochondrial nuclear interactions in plants Chase C D Horticultural Sciences Department, University of Florida, Gainesville, FL Plant Molecular and Cellular Biology Program, University of Florida, Gainesvill e, FL In hybrid seed production, pollen sterility is often achieved through the use of cytoplasmic male sterility (CMS), encoded by the mitochondrial genome. Expression of the CMS trait can be conveniently controlled by the presence or absence of nuclear restorer alleles, to condition large, uniform populations of pollen fertile or pollen sterile plants within one generation. The mitochondrial CMS genes of diverse plant species encode dissimilar proteins that effect pollen sterility by different mechanisms including cell death and the homeotic transformation of male floral organs. The S type of cytoplasmic male sterility in maize (CMS S) is characterized by a pollen collapse phenotype associated with a mitochondrial open reading frame predicting a peptide similar to ATP synthase subunit 9, and by a large collection of nuclear mutations that restore pollen function but condition homozygous lethal kernel phenotypes. Our work addresses the hypotheses that CMS S pollen collapse results from mitochondria signal ed, apoptotic like programmed cell death, and that nuclear restorer mutations rescue CMS S pollen by disrupting mitochondrial gene expression and signaling functions that are expendable in the pollen but essential to seed development. Beyond the practical importance of CMS, mitochondrial CMS and nuclear restorer genes therefore afford opportunities to investigate the nuclear regulation of mitochondrial gene expression and mitochondrial signaling pathways in plants.
Reactive oxygen scavengers regu late reproductive traits in Arabidopsis Hauser B 1 Wang Y Y 1 Mohammed Y 1 ,2 Head DJ 1 Sun K 1 ,3 1 Department of Biology, University of Florida, Gainesville, FL 2 Department of Biology, University of Miami, Coral Gables, FL 3 Department of Plant Biology, M ichigan State University, East Lansing, MI Reactive oxygen species (ROS) are naturally produced during photosynthesis, respiration, and other metabolic processes. In plants, moderate amounts of ROS act as redox signaling molecules; however, excessive accu mulation of ROS is toxic. High levels of ROS damage DNA and proteins, and induce programmed cell death (PCD). Plant stress causes ROS accumulation around the egg cell, which subsequently spreads through an ovule and ultimately causes PCD. Accumulation of R OS in ovules is a binary process ovules either have high levels of ROS or very little. Interestingly, the frequency of high ROS levels in ovules correlates with the ovule abortion rate. ROS scavengers neutralize those toxic molecules and protect plants fro m cellular damage and PCD. Mutation of ROS scavengers that are expressed in ovules affects ovule abortion, seed germination, and seedling growth. In per17, per28, and per29 mutants, as well as their double and triple mutants, the ovule abortion rate signif icantly increased. Furthermore, peroxide accumulation in the pistils of these peroxidase mutants was higher than wild type controls. In these three peroxidase mutants, germination and seedling growth are unchanged. In contrast, an ascorbate peroxidase apx4 mutant shows reduced seedling growth and seed germination, but this locus does not significantly change fertility. Tetrazolium tests show that wild type and apx4 seeds are metabolically active, however, the seed coat of the apx4 mutant is more permeable t han wild type. In the future, we will determine whether increased ROS scavenging activity leads to increased plant fertility or yield. Structure and evolution of the B3 transcription factor network controlling the transition between the seed and vegetati ve phases of the plant life cycle McCarty DR Jia H, Suzuki M Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL The B3 domain proteins are plant specific transcription factors that have centr al roles in plant developm ent. Sub families of B3 proteins implicated in major hormone signaling pathways appear early in the evolution of land plants. Genes in the AFL/VAL group regulate the fundamental transition between seed and veg etative phases of development. The AFL B3 facto rs are principally activators of the embryo maturation program leading to developmental arrest and acquisition of desiccation tolerance in the developing seed; whereas, the sister clade of VAL B3 genes function as repressors of the AFL network prior to ger mination. AFL class mutants have viviparous embryos that bypass seed maturation and associated dorman t phases of plant development. In contrast, VAL mutant embryos are trapped in the late embryogenesis developmental program and are unable to transition fr om s eed to vegetative development. Key downstream AFL targets include components of seed specific abscisic acid (ABA), gibberellin (GA), and auxin signaling pathways. ABA feeds back into network via ABI3 interaction with ABI5. GA promotes repression of th e AFL network by the VAL repressors with involvement of the PICKLE (PKL) chromatin remodel ing factor before germination. Genetic studies in maize show that VP8 (ortholog of Arabidopsis AMP1) signaling acts upstream of the AFL network.
Poster session s *= UF Genetics Institute Faculty. Presenters are underlined. 1. Characterization of TMEM16 Ca2+ activated Cl channels in C. elegans Alam T 1 Hill Harfe K 1 Harfe BD 2,* Choe KP 1,* 1 Department of Biology, University of Florida, Gainesville, FL 2 Departm ent of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Ca2+ activated Cl channels (CaCCs) have been found to function in transmitter release from photoreceptors, excitability in neurons and myocytes, sensory signal transductio n, and epithelial membrane transport. Despite their importance, the molecular identity of CaCCs remained unknown until recently. Three labs have now independently identified members of the TMEM16 transmembrane protein family as CaCCs in cell culture. Curre ntly, the main goals of the field are to characterize the physiological functions of TMEM16 proteins and define the molecular mechanisms that regulate channel activity. Forward and reverse genetics are extremely powerful approaches for characterizing gene function and defining cellular signaling pathways. Unfortunately, there are 10 TMEM16 family members in mice with overlapping expression and potentially redundant functions that can hinder genetic analysis. To overcome these obstacles, we are exploiting th e genetic and molecular tractability of Caenorhabditis elegans. C. elegans only has two TMEM16 family members. Transgenic analysis demonstrates that these genes are expressed in sensory neurons and reproductive structures of the nematode. Interestingly, kn ockdown of one TMEM16 family member disrupts the ability of worms to detect osmotic gradients suggesting a role in sensory perception or signal transduction. We are currently generating null alleles for both TMEM16 genes in C. elegans and designing genetic screens to identify pathway components. 2 Generation and evaluation of mutagenized, apomictic bahiagrass ( Paspalum notatum Flugge) Kannan B, Lomba P Altpeter F Agronomy Department, University of Florida, Gainesville, FL Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Improvement of the popular bahiagrass ( Paspalum notatum Flugge) cultivar Argentine' by conventional breeding is very difficult due to its apomictic reproduction mode. Our objective was to explore t he potential of chemical and tissue culture derived mutagenesis for genetic improvement of apomictic bahiagrass for generation of uniform mutagenized seed progeny with improved turf and forage quality. Scarified and surface sterilized bahiagrass seeds were treated with the mutagen sodium azide at various concentrations and exposure times. Callus was induced from these seeds and regenerated via somatic embryogenesis to obtain uniformly mutagenized plants. 2,000 of the 20,000 regenerated seedlings were select ed based on their vigor or morphological characteristics and transferred to soil. 46 independently mutagenized lines with reduced stem length, higher tiller density or reduced or delayed seedhead formation were established under field conditions in 1.2m x 1.2m plots in a randomized block design with 4 replications for further evaluation of density, leaf texture, tiller length, color, growth pattern, biomass, seedhead and seed production, as well as seedling vigor. Several mutagenized bahiagrass lines with improved characteristics and viable seed production have been identified and are currently evaluated in larger plots. Greenhouse and field data from selected mutagenized lines will be presented in comparison to non mutagenized bahiagrass. 3. The role of g ut mic r obiota in Clostridium difficile infection Antharam V 1 Li E 1 Rand K 2 Wang G 1,* 1 Division of Infectious Diseases, Department of Medicine University of Florida, Gainesville, FL 2 Department of Pathology, Immunology and Laboratory Medicine, Univers ity of Florida, Gainesville, FL Clostridium difficile causes 3 million cases of diarrhea and colitis in the US annually and accounts for most cases of colitis associated with antibiotic therapy. Human gut microbiota is widely believed to be important in t he protection against C. difficile infection (CDI), but the basic features of this association remain poorly understood. Using Roche/454 pyrosequencing, we examined 109,197 partial prokaryotic 16S rRNA gene sequences from feces of 14 individuals with C. di fficile infection and 32 individuals with diarrhea not related to CDI, compared with 36,804 sequences from feces of 6 healthy individuals without diarrhea. We obtained taxonomic assignments of bacterial rDNA using a refined algorithm for sequence processin g. Our analysis indicates that CDI is associated with a reduced microbial diversity and richness, accompanied by a significant alteration of organismal lineages in the gut microbiota. The gut microbial structure in subjects with CDI is distinct from that o f healthy individuals, but is similar to subjects with diarrhea unrelated to CDI. Members of the Faecalibacterium genus were significantly reduced in abundance compared to normal subjects, consistent with a hypothesis that these commensal bacteria are crit ical for protection against CDI. These results provide a framework for longitudinal analyses of these diverse gut microbial communities in well defined
subjects, a critical next step in elucidating the role of gut bacteria in relapsing CDI. 4. Manipulatio n of the non canonical N F B pathway in human monocyte derived dendritic cel ls transduced by AAV2 vectors: s trategies to achieve enhanced transgene expression and reduced cytotoxic T cell response to transgene products Aslanidi G 1,2 Jayandharan GR 3 Perr in GQ 4 Martino AT 1 ,2 Zhong L 5 Herzog RW 1,2,6,* Srivastava, A 1,2,6,7,* 1 Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida, Gainesville, FL 2 Powell Gene Therapy Center, University of Florida, Gainesville, FL 3 D epartment of Haematology and Centre for Stem Cell Research, Christian Medical College, Vellore, India 4 Department of Urology, University of Florida, Gainesville, FL 5 Gene Therapy Center and Division of Hematology/ Oncology, Department of Medicine, Univers ity of Massachusetts Medical School, Worcester, MA 6 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 7 University of Florida Shands Cancer Center, Gainesville, FL There are conflicting reports on the transduction ef ficiency of primary human dendritic cells (DCs) by single stranded AAV2 vectors. Since self complementary AAV2 vectors have been shown to transduce various cell types more efficiently, and since in our recent studies with HeLa cells, we achieved efficient transduction following treatment of cells with small molecule activators of the transcription factor, nuclear factor kappa B ( NF B ), we compared the transduction efficiency of scAAV2 vectors in primary human DCs in the absence or the pres ence of VP16, an activator of NF B VP16 led to ~19 fold increase in EGFP expression suggesting the involvement of the NF B pathway in AAV mediated transduction of DCs. Western blot analyses with cytosolic and nuclear fractions prepared from transduced primary human DCs further corroborated that the alternative pathway of NF B activation was operational. Additional studies revealed reduction of expression levels of DC maturation markers, CD83 and CD86, by an NF B inhibitor, Bay11, without reduction in EGFP expression. T his reduction in maturation marker expression diminished the main function of DCs to process antigenic material, and dramatically reduced T cell activation and proliferation as well as IFN production in antigen specific CTLs. We speculate that the use of NF B inhibitors during transduction with AAV vectors would lead to dampened host cell immune response in general, and cytotoxic T c ell in particular by arresting antigen presenting cells maturation process, which has significant implication in the optima l use of these vectors in human gene therapy. 5 Direct assessment of the mutational spectrum in Caenorhabditis Wilhelm LJ 1 Howe DK 1 Baer CF 2 Denver DR 1 1 Department of Zoology, Oregon State University, Corvallis, OR 2 Department of Biology, Univers ity of Florida, Gainesville, FL A robust understanding of the mutation process is key to understanding virtually all evolutionary phenomena and the underlying nature of genetic disorders and cancers. Differential mutation rates among species are a highly plausible and testable hypothesis for the observation that different species evolve at different rates. Here we expand on previous work that characterized the mutation rate and spectrum in a common laboratory strain of the nematode Caenorhabditis elegans In these studies we applied high throughput sequencing technologies to provide an unprecedented volume of data to bear on this central question. Here we present the results of expanding this an alysis to a natural strain of C. elegans and to two natural st rains of the close relative Caenorhabditis briggsae to characterize within and between species variation in the rate and molecular spectrum of spontaneous mutations. Mutation accumulation (MA) lines for these species were employed as they provide a system to study mutational phenomena in which the confounding effects of natural selection are reduced to the maximum extent possible. 6 Analyzing the r ole of #$ globin gene locus a ssociated cis regulatory DNA elements using artificial DNA binding d omains Barrow J Liang SY, Lin IJ, Bungert J Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL The drive to understand the regulation of the globin gene locus has been warranted by the need to improve the treatment of hemoglobinopathies. Our work focuses on exploring the role of various cis regulatory DNA elements within the #$ globin locus. Employing a novel technology, we are generating zinc finger DNA binding domains (ZF DBD) that can bind with high affinity and specificity to 18bp recognition elements a sequence adequate in length to recognize a unique signature within the genome. By blocking cis regulatory DNA elements, w e are able to delineate regions of DNA within the beta globin gene locus that are essential for regulation and activation of the globin genes. Our main research goal is to identify key cis DNA elements that can ultimately be altered in order to restore the synthesis of the fetal beta globin genes to improve the treatment of hemoglobinopathies. To date, we have successfully designed a ZF DBD that binds to the EKLF binding site in the murine beta maj globin gene
promoter. We have also designed ZF DBDs that wi ll neutralize the function of additional putative regulatory DNA elements in the beta globin gene locus, including the +60 Ebox in the beta maj globin gene promoter as well as E box and MARE sequences in the locus control region. We will present data on th e functional characterization of the ZF DBDs. 7 Transient expression of mitochondrial targeted peptides linked to cell death signaling and pollen collapse in S male sterile maize Bhan K 1 Smith P 2 Chamusco K 3 Seib J 1 Moreira C 2 Gallo M 1 ,* Chase CD 3 ,* 1 Agronomy Department University of Florida, Gainesville, FL 2 Department of Biology, Bennett College for Women Greensboro, NC 3 Horticultural Sciences Department, University of Florida, Gainesville, FL In maize, the S type of cytoplasmic male ste rility (CMS S) conditions pollen collapse at the early bi cellular stage. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay verified nuclear and chromatin fragmentation in pre collapse CMS S pollen consistent with mitochondrial s ignaled programmed cell death. The main aim of this research is to identify the mitochondrial open reading frame (orf) responsible for these events. Mitochondrial transcripts are associated with CMS S pollen collapse, but post transcriptional RNA editing c reates different versions of the orfs suspected to be responsible for pollen cell death. The up stream frame ( orf355 ) is not edited, but the down stream frame ( orf77 ) is edited to create two shorter orfs ( orf17 and orf26 ). Also, partial editing within orf1 7 creates S14 and L14 coding variants. Thus far, plant mitochondrial genomes cannot be genetically transformed, so a transient expression approach was taken. Candidate orfs were fused to a mitochondrial targeting sequence, the ATP9 double leader (ATP9DL) f rom Neurospora crassa and expressed behind the 35S or ap3 promoter. Fluorescence microscopy of stable Arabidopsis transformants demonstrated that ATP9DL targets GFP to mitochondria creating the potential to successfully target orf translation products. Tr ansient experiments conducted by Agrobacterium infiltration will test whether mitochondrial targeted peptides condition cell death lesions in tobacco leaves and petunia petals. 8 ZmNlr1: a function and structural analysis Black JB Gustin JL, Fajardo DS Settles AM Horticultural Sciences Department, University of Florida, Gainesville, FL The flat leaf blade of higher plants is an important adaptation for efficient photosynthesis. We recovered a maize mutant that impacts development in multiple tissues including distinct narrow leaf and rough endosperm (nlr1) phenotypes. We identified a Robertson's Mutator (Mu) transposon ins ertion tightly linked to nlr1 (s ee poster no. 43 ). The transposon disrupts the coding sequence of a J domain containing protein l eading to reduced accumulation of the full length transcript. J domains activate Hsp70 ATPase activity and proteins containing these domains have diverse functions in the cell. In addition to the J domain, the protein contains an Arginine/Serine (RS) rich domain at the N terminus and nuclear localization signals (NLS) at the C and N termini. RS domains are enriched in pre mRNA splicing factors and presence of this domain suggests the protein is involved in transcriptional regulation. Transient expression o f N and C terminal fusions with GFP shows subnuclear localization within nuclear speckles. Nuclear speckles are enriched with pre mRNA splicing factors. Deletion of the nested NLS and RS domains eliminates the speckling pattern and relocates the modified p rotein to the nucleoplasm. Deletion of either the C terminal NLS or J Domain has no affect on the localization pattern. Initial yeast two hybrid screens retrieve a putative snRNP, further suggesting Nlr1's involvement in transcriptional regulation. 9 Ef fects of endocrine disrupting compounds on development of external g enitalia Boger LJ 1 Cohn MJ 1 3, 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Department of Biology, University of Florida, Gainesville, FL 3 Howard Hughes Medical Institute, University of Florida, Gainesville, FL Hypospadias is a congenital malformation of the external genitalia in which the urethral tube fails to close and the prepuce (foreskin) and ventral penis do not completely form. D ue to the dramatic increase in the incidence of hypospadias over the past 40 years, it has been proposed that endocrine disrupting compounds (EDCs) may be the underlying cause. In this study, we administered the EDCs Bisphenol A, Methoxychlor and p,p' DDE as well as the type II 5 % reductase inhibitor Finasteride during the androgen dependent stage of development of external genitalia. We also examined the effect of the fungicide Vinclozolin administered during development and the possible ameliorative effects of % tocopherol (vita min E) when given with Vinclozoin. Here we report the range of effects and phenotypes and the implications for the environmental effects of EDCs on external genital development.
10 A supermatrix p ersp ective on the Amphiesmenoptera p hylogeny Boyd BM 1, 2 Burleigh G 3 ,* 1 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2 Mammalogy, Florida Museum of Natural History, University of Florida, Gainesville, FL 3 Department of Biology, University of Florida, Gainesville, FL The A mphiesmenoptera are a highly diverse clade of endopterygote insects that includes Lepidoptera (butterflies and moths) and Trichoptera (caddisflies). We attempted to build a comprehensive phylogenetic framework of Amphiesmenotpera that reflects both the rap idly growing sequence data and the computational advances that allow for large scale phylogenetic analyses. We first assembled all potentially phylogenetic informative sequence data from Amphiesmenotpera and selected outgroups in GenBank. After pruning que stionable or anomalous sequences and editing the alignments, homologous sequence clusters with more than 100 taxa were concatenated into a single phylogenetic "supermatrix" that contained 5609 taxa, representing by far the largest Amphiesmenoptera data set Maximum likelihood phylogenetic analyses on the supermatrix revealed a credible phylogenetic hypothesis that raises intriguing evolutionary questions and suggests directions for future data collection. The supermatrix phylogenetic hypothesis provides not only a starting point for large scale evolutionary and ecological analyses of the hyper diverse Amphiesmenoptera clade, but will hopefully be a resource to promote and guide future phylogenetic studies within Amphiesmenotpera. 11 Evaluation of accessibl e cellulose binding sites on maize cell wall mutants by using fluorescently labeled cellulose binding proteins Caicedo HM Vermerris W Agronomy Department, University of Florida, Gainesville, FL Lignin content and composition have a significant impact on efficiency of the enzymatic saccharification during the conversion of lignocellulosic biomass into fermentable sugars. In previous studies, we have demonstrated enhanced biomass conversions of genetically altered plant cell walls of maize brown midrib ( bm ) mutants compared to wild type controls. The modification of the lignin subunits composition can considerably increase the yield of fermentable sugars from maize stover. To investigate the basis of the enhanced biomass conversion obtained for all bm mut ants relative to the wild type control, we have developed two different methods based on the detection of fluorescently labeled proteins that bind specifically to cellulose. The accessible binding sites for cellulolytic enzymes on the cell walls were assa yed through adsorption equilibrium based methods by using either a recombinant fusion protein consisting of the green fluorescen t protein (GFP) fusion to a cellulose binding module (CBM) of endoglucanases obtained from the white rot fungus Trichoderma rees ei or an Atto 488 conjugated cellobiohydrolase I (CBH I) purified from Trichoderma reesei The amount of protein adsorbed on the plant cell walls from equilibrated solutions was determined by a subtraction assay of the detected free fluorescently labeled protein against a known fluorescence level of its respective standards. The results suggest that the increase of cellulose conversion during the enzymatic hydrolysis on the mutant genotypes is associated with the enhanced number of accessible binding sites on the cell walls. 12 Essential role for the MAML1 co activator in T ALL Cao C 1 Tian L 1 Li J L 2 Griffin JD 3 Huang S 4 ,5 ,* Wu L 1 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Sanford Burnham Medi cal Research Inst itute at Lake Nona, Orlando, FL 3 Dana Farber Cancer Institute, Boston, MA 4 University of Florida Shands Cancer Center, Gainesville, FL 5 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL T cell acu te lymphoblastic leukemia is the most common malignancy in children and accounts for nearly one third of all pediatric cancers. Two common genetic alterations frequently associated with this disease are mutations in the NOTCH1 cell surface receptor and abe rrant expression of the TAL1 transcription factor. In this study, we investigated the role of MAML1, in regulating NOTCH1 and TAL1 transforming activities in leukemic cells. We found that MAML1 is a novel interacting partner for TAL1. MAML1 also enhanced T AL1 transcriptional activities, suggesting a role for MAML1 in TAL1 regulated transcription and leukemogenesis. A subset of T ALL leukemic cells exhibit aberrant activation in both the NOTCH1 and TAL1 activities; thus, it suggests that these two genetic al terations cooperate in promoting leukemic cell growth. Indeed, we found that the combined inhibition of both the pathways results in synergistic responses in leukemic cells that carry genetic alterations in both the NOTCH1 and TAL1 genes. We next determine d whether MAML1 expression level affects leukemic cell growth and survival. Gene knockdown studies suggest that MAML1 is essential for leukemic cell growth and survival by possibly regulating NOTCH1 and TAL1 mediated transcription. Overall, our data reveal s a novel common regulatory mechanism for both NOTCH1 and TAL1 oncogenic pathways, and suggest that the manipulation of MAML1 expression or functional activities will affect leukemia initiation and progression.
13 Genetics of tree structure in interspec ific hybrids of peach Carrillo Mendoza O Chaparro JX Horticultural Sciences Department, University of Florida, Gainesville, FL Horticultural tree crops are typically pruned to manage tree size, distribute fruit load, enhance color development and to r ejuvenate bearing trees. The location, frequency and type of branching have a major impact on labor costs in fruit orchards. Phenotypic variation for tree architecture exists in peach, however the low polymorphism rate in peach hinders genetic studies. To overcome these difficulties, peach was crossed with Prunus kansuensis and P. dulcis high and low branching species, respectively. Several F1 hybrids were selected and backcrossed to peach to generate segregating populations. Data on blind node production and branching frequency was collected on backcross progeny from P. persica x ( P. persica x P. kansuensis ) and P. persica x ( P. persica x P. dulcis ) backcross hybrids. Differences in blind node and branching frequency within and between interspecific hybrid families were detected and progress in the analysis of these results will be presented. 14 Extending genetic predictors of warfarin dose: APOE and CALU Carris N 1 ,2 Gawronski BE 1,2 Shahin MH 3 Khalifa SI 4 Gong Y 1,2,* Langaee T 1,2,* Johnson JA 1,2,* 1 Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville, FL 2 Center for Pharmacogenomics, University of Florida, Gainesville, FL 3 Department of Pharmacy Practice and Clinical Pharmacy, Misr International University, Cairo, Egypt 4 College of Pharmacy, Qatar University, Doha, Qatar Genetic variation in the genes APOE and CALU have previously been studied as predictors for therapeutic warfarin dose. We hypothesized that the additional information of SNPs in APOE and CA LU could lead to better prediction of therapeutic warfarin dose. Methods: Two study cohorts, a 195 patient warfarin treated cohort from Cairo Egypt and 350 patient cohort of warfarin treated patients from Gainesville, Florida, were genotyped using pyrosequ encing for two SNPs in APOE (rs429358 and rs7412) and one SNP in CALU (rs339097). Results: In the Egyptian cohort variation in APOE was divided into haplotypes (e2 TT, e3 TC, e4 CC). Only the e2 haplotype showed a significant effect on dose requirement (pa rameter estimate 0.3364mg/week, p=0.0215, r !=0.0238). Using the same haplotype analysis in the Florida cohort, e4 showed a significant effect on dose requirement (parameter estimate .052185 mg/week p= 0.0179, partial r!=0.0092). Patients in the Florida c ohort showed no relation between rs339097 genotype and dose. Patients in the Egyptian cohort with variant allele at rs339097 showed an increased warfarin requirement (p=0.04), but for the stepwise linear regression did not meet the criteria to stay in the final model. Conclusions: The Egyptian data suggest these SNPs might explain an additional 4% of warfarin dose variability and the Florida cohort also shows associations making the case for genotyping of APOE and CALU in future pharmacogenomic warfarin stu dies. 15 Universal fingerprinting platform for strawberry ( Fragaria species) Chambers AH 1 Chamala S 2 Barbazuk WB 2,* Whitaker VM 3 Folta KM 1,* 1 Horticultural Sciences Department, University of Florida, Gainesville, FL 2 Department of Biology, Universi ty of Florida, Gainesville, FL 3 Gulf Coast Research and Education Center, University of Florida, Wimauma, FL Strawberries ( Fragaria species) are grown around the world for their delicious taste, aesthetic appeal, and nutritional benefits. Developing molec ular tools for strawberry will help improve these and other important characteristics in cultivated strawberry ( Fragaria x ananassa ). Simple Sequence Repeats (SSRs) are DNA markers defined by repeating, tandem nucleotide patterns found throughout a genome, and are useful for determining diversity between related genotypes. Our current work includes developing a SSR universal fingerprinting platform of high nucleotide ( > 4) repeats for strawberry. The results could provide greater insight into the evolutiona ry relationships between diverse genotypes specifically in the strawberry "supercore" used in our studies. Initially, we hope to broadly link desirable phenotypes to clusters of related species in the "supercore". We are especially interested in the long t erm goal of improving flavor in cultivated strawberry and investigating the volatile profiles of diverse strawberry genotypes. Our results will be useful to strawberry breeders and molecular researchers, as well as to those developing similar platforms in related systems. 16 Mitochondrial features of pollen development in male fertile and S male sterile maize Chamusco KC Siripant M, Tan YB, Chase CD Horticultural Sciences Department, University of Florida, Gainesville, FL In S male sterile maize, co transcribed mitochondrial open reading frames ( orf355 orf77 ) were associated with a programmed cell death (PCD) event characterized by
nuclear and chromatin fragmentation and culminating in the collapse of pollen development at the bi cellular stage. To be tter understand this mitochondria signaled PCD pathway, the accumulation of mitochondrial proteins was investigated through the course of normal and S male sterile maize pollen development. Mitochondria encoded respiratory subunits NAD9, SDHB, COB, COXII, ATP4, ATP6, ATP8 and ATP9 were of low abundance in both normal and male sterile microspores, and became more abundant during later (bi cellular and tri cellular) stages of normal pollen development. Male sterile pollen collapsed prior to any detected incre ase in the accumulation of these proteins. In contrast, two nuclear encoded, mitochondrial antioxidant proteins (alternative oxidase and MnSOD) were more abundant in microspore than bi cellular stage pollen. Maize pollen development was therefore character ized by a shift in mitochondrial metabolic capability. Sterile pollen collapsed at the initiation of this shift and was accompanied by loss of cytochrome c from the mitochondrial inter membrane space. An integrated model of mitochondrial function in steril e and normal pollen development will be presented based upon these findings. 17 A strawberry KNOX gene regulates leaf, flower and meristem architecture Chatterjee M Bermudez Lozano C, Clancy MA, Folta KM Horticultural Sciences Department, University of Florida, Gainesville, FL Plant Molecular and Cellular Biology Program, University of Florida Gainesville, FL The KNOTTED LIKE HOMEOBOX (KNOX) genes play a central role in plant development, as they shape the morphology of simple and compound leaves. S trawberry ( Fragaria spp.) leaves undergo a developmental transition from juvenile simple leaf to mature compound leaf, so this system provides a means to study the function of KNOX genes in leaf development. FaKNOX1 was isolated from a cultivated strawberr y ( Fragaria ananassa ) flower EST library and belongs to Class I KNOTTED LIKE HOMEODOMAIN proteins. The gene was mapped to linkage group VI of the diploid strawberry genome. Steady state transcript levels were highest in flowers and fruits. During strawbe rry leaf morphogenesis the FaKNOX1 transcript were detected as leaf progression changes from a simple to trifoliate leaf form. Transgenic strawberry plants suppressing or overexpressing FaKNOX1 exhibited conspicuous changes in the leaf morphology. The FaKN OX1 RNAi strawberry plants presented a dwarfed phenotype with deeply serrated leaflets and exaggerated petiolules. The severely malformed RNAi plants exhibited a high level of disorganization of shoot apical meristem and cellular organization in mature lea ves. Overexpression of FaKNOX1 caused a dwarfed stature with wrinkled leaves. These results in strawberry functionally illustrate the role of a KNOX domain protein in a rosaceous plant, as there are clear effects on leaf form and plant stature throughout d evelopment. 18 Phylogeny of the North American plums ( Prunus Rosaceae) Chavez DJ Chaparro JX Horticultural Sciences Department, University of Florida, Gainesville, FL The genus Prunus L. belongs to the subfamily Amygdaloideae (=Prunoideae) of the R osaceae. This genus includes plums, cherries, almonds, apricots and peaches. It is distributed around the world, with approximately 200 species. North America is an important center of diversity for plum species as having adapted (native) to widely diverge nt climates and soils. These species represent an important potential source of genes for disease and for pest resistance, and for adaptation. Early research in the 1900's increased the number of described plum specimens, taxonomic discrepancies started to grow, new species were added and old species eliminated, and relationships at species level became unclear. The Stone Fruit Breeding and Genetics Program at University of Florida recognize s this tremendous breeding p otential in the North American p lums. T he main objective of this research is to study the subgenus Prunus (in particular section Prunocerasus) and to clarify the phylogenetic relationships (relatedness, evolution, and character/trait diversification) of the native species. The study of the subg enus Prunus sect. Prunocerasus will allow us to identify, to support, and to preserve these species as important genetic resources (gene pool) of unique traits that could be used in the near future. Specific objectives and methodology proposed for this research will be presented. 19 Application of proteomics and mass spectrometry in biomedical research Diaz C 1 Chow M 1 Zheng R 1 Silva Sanchez C 1 Koh J 1 Chen S 1,2, 1 Proteomics Division, Interdisciplinary Center for Biotechnology Research, Universi ty of Florida, Gainesville, FL 2 Department of Biology, University of Florida, Gainesville, FL Proteomics and mass spectrometry have provided unprecedented tools for fast, accurate, high throughput biomolecular separation and characterization, which are i ndispensable towards understanding the biological and medical systems. Studying at the protein level allows researchers to investigate how proteins, their dynamics and modifications affect cellular processes and how cellular processes and the environment a ffect proteins. The mission of our facility is to provide excellent service and training in proteomics and mass spectrometry to UF scientists and students. Here we present our capabilities in
proteomics and other analytical services. The tools include a ge l based 2D DIGE (Two Dimensional Differential Gel Electrophoresis), gel free iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) and other quantification strategies. Along with our capacity of separating thousands of proteins and characterizing di fferential protein expression, we have a suite of state of the art mass spectrometers available for biomedical sciences and advanced technology research, including a tandem time of flight (4700 Proteomics Analyzer, AB), quadrupole/time of flight (QSTAR XL and Elite, AB), quadrupole linear ion trap (4000 QTRAP, AB) and a high resolution LTQ Orbitrap MS (Thermo Scientific Inc.). These instruments are mainly used for protein identification, posttranslational modification characterization and protein expression analysis (e.g., Mass Western). Our facility is also set up to provide Edman de novo N terminal protein sequence analysis and Biacore biomolecule interaction analysis. Proteomics and mass spectrometry are useful in large scale su r vey of proteome for hypoth esis generation as well as in detailed analysis of target proteins for hypothesis testing. Our services also include accurate molecular weight analysis, MRM based protein screening and absolute quantification. To ensure success and maximize productivity, t he facility offers education, consultation, data processing and reporting, and support of grant application. 20 Genetic variation in the L type calcium channel (LTCC) and association with blood pressure response to antihypertensive therapy in the pharmac ogenomic evaluation of antihypertensive responses (PEAR) study Chen Yin K 1 Davis H 1 Gong Y 1 ,* Langaee T 1 ,* Chapman AB 2 Turner ST 3 Gums J 1 Cooper DeHoff RM 1 Johnson JA 1 ,* 1 Department of Pharmacotherapy and Translational Research, University of Flo rida, Gainesville, FL 2 Department of Internal Medicine, Emory University, Atlanta, GA 3 Department of Internal Medicine, Mayo Clinic, Rochester, MN We investigated the association of genetic variation within CACNA1C and CACNB2 of the L type Ca 2+ channel ( LTCC) and blood pressure (BP) response to antihypertensive therapy in uncomplicated hypertensives. Two SNPs from each gene were selected based on prior association with cardiovascular phenotypes and genotyped in subjects enrolled in the Pharmacogenomic Eva luation of Antihypertensive Responses study on TaqMan ( CACNB2 n=542) or Illumina BeadChip ( CACNA1C n=519) platforms. We assessed home diastolic (DBP) and systolic BP (SBP) response to monotherapy, add on of alternate study drug and combination therapy in subjects randomized to atenolol (ATEN) or hydrochlorothiazide (HCTZ) treatment arms. We used ANCOVA to compare BP response between genotype groups, with p<0.05 considered significant. Analyses were stratified by race and initial treatment arm, and adjuste d for baseline BP, age and gender. In blacks, the CACNB2 rs2357928 promoter variant was associated with decreased DBP response to ATEN + HCTZ combination therapy (p=0.0369). There was a similar trend for decreased DBP response to HCTZ add on therapy (p=0.0 717). Additionally, black CACNA1C rs3794284 variant carriers had a decreased SBP response to HCTZ monotherapy (p=0.0097), an effect that was consistent when HCTZ was administered as mono or add on therapy (p=0.0121). These data suggest that variation with in LTCC genes may influence BP response to antihypertensive therapies, and that these effects may differ by race. 21 A high throughput screen for inhibitors of xenobiotic detoxification in nematodes Choe KP Deonarine A Department of Biology, Universi ty of Florida, Gainesville, FL Multidrug resistance is a growing problem in parasitic nematodes. In pathogenic organisms and tumor cells, multidrug resistance is mediated by enzymes that detoxify xenobiotics. The transcription factor SKN 1 activates the e xpression of multiple xenobiotic detoxification genes in Caenorhabditis elegans SKN 1 is also essential for development. Pharmacological compounds that target SKN 1 would provide new tools for studying multidrug resistance and have the potential to inhibi t development in embryos and drug resistance in adults. We recently used genome wide RNAi screening to identify the WD40 repeat protein WDR 23 as a principal regulator of SKN 1. Importantly, the homologous mammalian transcription factor Nrf2 is regulated b y a distinct molecular mechanism. We propose the WDR 23/SKN 1 pathway as a promising target for drugs that inhibit embryonic development, xenobiotic detoxification, and drug resistance in nematodes without affecting mammalian hosts. The small size, simple culturing characteristics, and genetic tractability of C. elegans make it an ideal system in which to screen for pharmacological inhibitors of SKN 1. These inhibitors would provide tools for studying the function of SKN 1 in parasitic species. We have deve loped a robust assay for SKN 1 activity using in vivo fluorescent reporters in C. elegans and plan to use it in high throughput screens of small molecules. 22 Deregulation of cellular miRNA by KSHV latent gene product Choi HS Renne R Department of Mo lecular Genetics and Microbiology, University of Florida, Gainesville, FL Kaposi's sarcoma associated herpes virus (KSHV), a DNA tumor virus, is the etiological virus of primary effusion
lymphoma (PEL), multicentric Castleman disease (MCD), and Kaposi's s arcoma (KS). Limited numbers of KSHV genes are expressed during latency, including KSHV miRNAs, which modulate host cellular gene expression. MiRNAs are small non coding RNAs, which regulate gene expression post transcriptionally. It has been revealed that aberrant expression of cellular miRNA contributes to tumorigenesis by regulating the expression of cancer related genes. We hypothesized that KSHV latent genes might affect upregulation of cellular miRNA expression. According to miRNA profiling data and l uciferase assay, expression of miR 17/92 cluster and miR 194 is upregulated in KSHV infected SLK cells. Therefore, we cloned KSHV latent genes into expression vectors and performed co transfection assays with miRNA promoter containing luciferase constructs We show that among the KSHV latent genes, vFLIP and vCyclin are involved in upregulation of both miRNAs. Further investigation is needed to elucidate the signal pathways leading to upregulated miRNA expression and the targets of these miRNA, which may co ntribute to pathogenesis and tumorigenesis by KSHV infection. 23 The role of hedgehog signaling in intervertebral disc development Choi KS Harfe BD Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL The verteb rate notochord is a transient embryonic structure that serves as a signaling center in the midline of the developing early embryo. During later mouse embryogenesis the notochord derives all cells found within the nucleus pulposus, which is located in the c enter of each intervertebral disc. The nucleus pulposus is essential for maintaining disc function as damage to this structure leads to disc degeneration. In this study we demonstrate that the hedgehog signaling pathway is essential for formation but not t he maintenance of the notochord sheath that surrounds the notochord during mid embryogenesis. Removal of hedgehog signaling from the notochord resulted in loss of the notochordal sheath and aberrant migration of notochord cells throughout the vertebral col umn. Mis migration of notochord cells resulted in the formation of small, abnormal nuclei pulposi. Our results demonstrate that hedgehog signaling is required for formation of the notochord sheath and patterning of nuclei pulposi along the vertebral column 24 Empirically designed tools for molecular biology: exploiting a deeply sequenced transcriptome of octoploid strawberry Clancy MA 1 ,2 Brunings AM 1,2 Chamala S 3 Barbazuk WB 3,* Davis TM 4 Folta KM 1,2,* 1 Horticultural Sciences Department, Universit y of Florida, Gainesville, FL 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 3 Department of Biology, University of Florida, Gainesville, FL 4 Department of Biological Sciences, University of New Hampshire, Durham, NH A transcriptome accounting was designed to capture the full transcriptional potential of cultivated strawberry, Fragaria ananassa Developmental stages and tissues were subjected to a wide range of physical and chemical stresses and treatments. RNA was p repared from nine plant tissue pools. Resulting cDNAs were MID tagged to specify tissue of origin and sequenced using Roche 454 GS FLX technology. A key feature of our transcriptome analysis was deconvolution of each contig into its component fragments sor ted by tissue of origin. By evaluating distribution and abundance of sequence contributions from the nine tissue pools, we identified contigs with apparent tissue specific or constitutive expression. This analysis formed an empirical foundation for develop ment of two sets of molecular tools designed for utility in strawberry gene expression studies: (1) constitutively expressed normalization controls for qRT PCR; (2) promoters with tissue specific activity. Genomic sequences found upstream of putative tissu e specific transcripts were cloned, linked to reporter coding sequences and transiently expressed in strawberry tissues to test for promoter activity. To discover ideal strawberry controls for qRT PCR, contigs assembled from fragments of all nine tissue ty pes in equal proportions were chosen as candidates. Several candidates identified by transcriptome analysis performed well as normalization controls, in some cases better than typical housekeeping gene choices. 25 Combined treatment of CD4+ T cells and S OCS1 KIR mimetic peptide reverses SOCS1 KO disease lethality by restoring peripheral Foxp3+ Treg homeostasis Collins EL Jager LD, Dabelic R, Benitez P, Holdstein K, Lau K, Haider MI, Johnson HM, Larkin J Department of Microbiology and Cell Science, Univ ersity of Florida, Gainesville, FL Suppressor of Cytokine Signaling 1 deficient mice die of a T cell mediated autoimmune disea se within 3 weeks after birth. This SOCS1 knockout disease is characterized by excessive interferon signaling, lymphopenia, and leukocyte infiltration which mediate destruction of many vital organs. Numerous models of autoimmunity, where lymphopenia is present, have been inhibited by Foxp3+ regula tory T cell adoptive transfer. Our observations show that SOCS1 / mice have a reduc tion in peripheral Tregs, despi te enhanced thymic development. The adoptive
transfer SOCS1 +/+ Tregs or CD4+ T lymphocytes mediated an increased, yet limited, survival in the SOCS1 / mice. However, the adoptive transfer of CD4+ T lymphocytes in conjunctio n with SOCS1 KIR, a mimetic peptide sufficient to partially restore SOCS1 function, resulted in a 3 fold increase in the lifespan of 30% of treated SOCS1 / mice. Additionally, the combined treatment mediated both a decrease in leukocyte infiltration into vital organs and an increase in peripheral lymphocyte populations, includ ing Foxp3+ regulatory T cells. Collectively these results suggest that the combined adoptive transfer CD4+ T cells/SOCS1 KIR treatment synergistically promote the long term survival of SOCS1 / mice by restoring Treg. Moreover, these data propose a relationship between the prevention of autoimmune disease, SOCS1, and Treg homeostasis. 26 Looking for regulators of regulators: screening for proteins that interact with a new regulator of actin depolymerizing factor Cuddy KK Grey PH, Zhang X, Roney JC, Dugas SM, Oppenheimer DG Department of Biology, University of Florida, Gainesville, FL Actin filament turnover is required for many actin dependent cellular processes including cell motility and membrane trafficking. Members of the actin depolymerizing factor/cofilin (ADF) family of actin binding proteins are essential for severing/depolymerizing actin filaments. Because ADF plays a central role in severing actin filaments, understand ing the regulation of ADF activity is essential for understanding actin dynamics. We recently identified a new regulator of ADF in plants named RPA for R EGULATOR OF P LANT A DF. The RPA1 gene was isolated by map based cloning of a gene responsible for normal trichome shape in the plant Arabidopsis The actin cytoskeleton in rpa1 mutants showed an increase in the number of thick actin filament bundles in developing trichome cells, culminating in an "actin knot" and numerous actin rings surrounding the cell nuc leus. Our in vitro analysis of RPA1 function showed that it interacts with ADF and inhibits actin binding to ADF. To understand how RPA1 is regulated, we conducted a yeast 2 hybrid screen to identify proteins that interact with RPA1. We found that a conser ved, plant specific protein containing a Development and Cell Death (DCD) domain interacts with RPA1. We named this protein RPA1 interactor (RPI1). Putative rpi1 T DNA insertion mutants show no obvious phenotype, and double mutants with rpa1 mutants show n o additional phenotypes beyond those seen for rpa1 single mutants. Analysis of the actin cytoskeleton in rpi1 mutants is underway. 27 Gut microbiota composition correlates with colorectal polyp prevalence Mai V Sun Y, Culpepper T Ukhanova M, Vedam Mai V, Polyak S, Valentine J Department of Microbiology and Cell Science, University of Florida, Gainesville, FL Emerging Pathogens Institute, University of Florida, Gainesville, FL Human gut microbiota has been associated with various diseases. Little is known about the contribution of gut microbiota to colorectal carcinogenesis in humans. We performed a nested case control study in human volunteers undergoing a screening colonoscopy. Data on dietary habits and medical history, a fecal sample and multip le colon biopsy samples were collected from 91 subjects. We analyzed microbiota composition by 16S rRNA sequencing in 30 individuals presenting with at least one polyp and 30 age and gender matched controls. 16S rRNA sequences covering the V2 region were i nitially analyzed using the RDP pipeline, followed by MEGABLAST against Greengenes and UNIFRAC analysis. Further, we used ESPRIT and performed DISCRIMINANT analysis. After removing low quality reads we retained 209850 sequences from stool samples, and 4215 0 sequences from biopsy samples with an average length of 211 nucleotides for the analysis. While UNIFRAC based PCA analysis suggested that microbiota in fecal samples differed in composition from that in biopsy samples, no difference in diversity was dete cted when cases were compared to controls. MEGABLAST analysis revealed various OTU's that differed in prevalence between cases and controls. ESPRIT based DISCRIMINANT analysis revealed a pattern at the 92% similarity level that distinguished cases from con trols. MEGABLAST and ESPRIT based DISCRIMINANT analysis suggest that gut microbiota composition correlates with colorectal polyp prevalence. 28 MAPit exploration of chromatin subpopulations in KSHV Darst RP 1 Haecker I 2 Pardo CE 1 Renne R 2,* Kladde MP 1 1 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL The transition between latency and lytic replication of Kaposi's sarco ma associated herpesvirus (KSHV) is regulated epigenetically. During latency, the virus persists as a multi copy episome, attached to host chromatin by the viral protein LANA. Loss of repressive chromatin, by treatment with sodium butyrate, TPA, or 5 aza d C, reactivates expression of the lytic switch RTA. Spontaneous reactivation also occurs. Whether all copies of the virus within a single cell share a common epigenetic state is unknown. We used
MAPit to measure the inherent epigenetic variability of KSHV. We have found that both the LANA and RTA promoters exist in several chromatin states within a single population of cells. Our method provides a view of single molecule chromatin dynamics. 29 Too much of a good thing: SKN 1 increases stress resistance and lifespan but inhibits growth and reproduction in C. elegans Deonarine A Empinado H, Choe KP Department of Biology, University of Florida, Gainesville, FL In animals, cap'n'collar transcription factors activate multiple cytoprotective genes that en hance stress resistance and increase lifespan. Not surprisingly, the human cap'n'collar transcription factor Nrf2 has become a tremendously popular chemopreventative target for stress related pathologies such as cancer, neurodegeneration, chronic inflammat ion, and asthma. However, recent studies have found that Nrf2 promotes tumor growth in humans. In mice, mutations that constitutively activate Nrf2 have severe consequences on metabolism and body size. Despite the tremendous interest in their therapeutic p otential, very little is known about the molecular mechanisms by which of capn'collar transcription factors control processes other than stress resistance and ageing. Our lab is using the genetically tractable nematode C. elegans as a model to understand the molecular and physiological functions of capn'collar transcription factors. The orthologue of Nrf2 in C. elegans is SKN 1. Similar to Nrf2, we find that constitutive SKN 1 activity promotes stress resistance and increases lifespan, but profoundly decr eases body size, growth rate, and reproduction. We are performing a suite of functional genomic and genetic studies to identify genes and pathways that mediate the different physiological effects of SKN 1. Interestingly, preliminary studies suggest that th e pro survival effectors of SKN 1 may be distinct from the anti growth and reproduction effectors. 30 Phenotypic correlation of differing mutations in beta glucuronidase in mouse models of MPS VII Devanney SC 1 McD oom I 2 Le Prell CG 3 Heldermon CD 1 1 Division of Hematology and Oncology, Department of Medicine, University of Florida, Gainesville, FL 2 Department of Ophthalmology, University of Florida, Gainesville, FL 3 Department of Speech, Language, and Hearing Sciences University of Florida, Gainesvil le, FL The original mouse model for deficiencies in the enzyme beta glucuronidase (GUSB) has been well characterized for many experimental applications. The original mouse model has only a single base pair mutation that prevents quantitative PCR study met hods. Another GUSB mouse model on a C3H background strain exists with a transposon insertion into the gene (MPSVII2J). On the C3H strain the disease phenotype is attenuated. The transposon insertion mutation now exists on the BL/6 background. We have initi ated a study to determine whether the attenuation of disease is due to strain or to the different gene mutation. We are currently comparing vision though electroretinography (ERG), hearing through auditory evoked brainstem response (ABR), femur lengths and life span of the two models. The 2J model would be more amenable to quantitative cell number determinations. Our preliminary data comparing ERG, ABR, and femur length show a similar pattern of disease manifestation between the two models with the homozygo us mutants of both groups exhibiting roughly the same deficiencies in hearing, sight and femur length. Life span studies are still ongoing with an obvious trend of high morbidity for the mutants of both models early o n. The preliminary data suggest that bo th the MPS VII and MPS 2J models seem to follow very similar patters of disease manifestation and can be used to effectively study the human lysosomal storage disease MPS VII. 31 Identification of novel components regulating salicylic acid accumulation i n plant immunity Ding Y Mou Z Department of Microbiology and Cell Science, University of Florida, Gainesville, FL Salicylic acid i s one of the most important signal molecules that regulate plant defense against pathogens. However, our understanding of SA biosynthesis and its regulation remains rudimentary due to the lack of a high throughput method to quantify SA levels. The methods currently used, including HPLC and GC/MS, are expensive and time consuming, therefore not suitable for large scale screen of SA metabolic mutants. Recently, our laboratory developed a rapid biosensor based method for quantification of SA and established a high throughput strategy for isolation of SA metabolic mutants. Using this method, we performed a pilot genetic screen f or suppressors of npr1 a mutant hyper accumulates SA during pathogen infection. We ha ve identified 25 putative npr1 suppressors that accumulate lower levels of SA than npr1 upon pathogen infection. SA levels in six of them have been confirmed by HPLC, an d all six npr1 suppressors are more susceptible than npr1 to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. We are trying to screen a total of ~100,000 EMS induced M 2 plants to saturate this screen. Molecular characterization of the np r1 suppressors will help identify novel components regulating SA accumulation in plant immune responses. Since SA is both necessary and sufficient for resistance against a broad spectrum of pathogens, the new components indentified could be applied to pla nts for increasing their resistance to a wide variety of pathogens.
32 A novel regulator of xenobiotic detoxification genes identified in C. elegans Empinado H Deonarine A, Choe KP Department of Biology, University of Florida, Gainesville, FL Glutat hione S transferases (GSTs) detoxify xenobiotics and promote cellular redox balance. The C. elegans genome encodes over 30 GSTs, many of which are transcriptionally induced by xenobiotics and oxidative stress. Using a transgenic reporter, we previously per formed a genome wide RNAi screen for regulators of gst 4 expression. The WD40 repeat protein WDR 23 was among the strongest repressors of gst 4 and we reported that WDR 23 functions with the CUL 4/DDB 1 ubiquitin ligase to repress the transcription factor SKN 1. RNAi for most of the other repressors does not have an additive effect on gst 4 expression when combined with a null allele of wdr 23 suggesting that they function in common pathways. One exception is WDR 46, a second WD40 repeat family member. WDR 46 is a homologue of the yeast protein Utp7, which functions in ribosome biogenesis, kinetochore organization, and chromosome segregation. Nothing is known about WDR 46 or its orthologues in animals. Real time PCR data from a deletion mutant confirm that WDR 46 represses expression of endogenous gst 4 Preliminary data suggest that WDR 46 protein is expressed in the nucleus of many cells in C. elegans and that loss of function increases resistance to xenobiotic stress. Loss of WDR 46 also dramatically redu ces brood size suggesting a role in reproduction. We are currently determining if WDR 46 regulates lifespan and designing screens to identify interacting genes and transcription factors. 33 Epigenetic evidence of pluripotency gene derepression in clinica l osteosarcoma Esparragoza PA 1 Gilbert JL 1 Rosenbluth AL 1 Hankowski KE 1 Ghivizzani SC 2,* Gibbs CP 2 Terada N 1,* 1 Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 2 Department of Orthopaedics and Reha bilitation, University of Florida, Gainesville, FL Aberrant expression of pluripotency specific genes such as Nanog and Oct4 has been found in many clinical cancers including osteosarcoma. Moreover, it has been implicated that the cells expressing pluripo tency markers represent a cancer stem cell like sub population with higher tumor initiating capacities. However, most of the previous studies lack true evidence of epigenetic derepression in cancer, leaving some skeptical of the claims. In order to elucida te whether epigenetic derepression indeed occurs in pluripotency genes in osteosarcoma, we performed bisulfite sequencing analysis of DNA methylation in multiple osteosarcoma cell lines established from patients. In two out of four osteosarcoma lines, the Nanog gene promoter was largely demethylated as seen in control ES cells. This is the first evidence, to our knowledge, that a pluripotency gene is indeed epigenetically altered in osteosarcoma. In contrast to Nanog, the Oct4 gene promoter was largely meth ylated in all osteosarcoma cell lines as seen in somatic cell control. The study provides a novel insight as to how molecular reprogramming occurs in cancer. Understanding how cancer cells gain abnormal reprogramming and express Nanog can lead to the devel opment of novel therapeutic targets against cancer. 3 4 Acid alpha glucosidase delivery using adeno associated virus 2/9 in adult animals improves respiratory and cardiac f unction in Pompe d isease Falk DJ Soustek MS, Mah CS Cloutier DA, Germain SA, Ke lley JS, Erger KE, Conlon TJ, Clement N, Byrne BJ Powell Gene Therapy Center, University of Florida, Gainesville, FL Department of Pediatrics, University of Florida, Gainesville, FL Pompe disease is an autosomal recessive disorder caused by a lack of e nzyme responsible for degradation of lysosomal glycogen, acid glucosidase (GAA). Respiratory muscle and cardiac dysfunction are primary features of this disorder and we have incorporated an animal model of Pompe disease ( Gaa / mouse) to characterize the se aspects. This study focuses on the efficacy of rAAV9 DES hGAA (AAV9) vs. hGAA enzyme replacement therapy (ERT) for treatment of Pompe disease. Adult Gaa / mice (n=6/group) were assigned to one of the following groups; A) single systemic injection of la ctated ringers ( Gaa / ), B) single systemic injection of AAV9, or C) semi monthly injection of ERT (duration 3 mon ths). Assessment of contractile function 3 mo post treatment revealed a significant increase in specific force measured #60 Hz in AAV9 and ERT groups when compared to untreated Gaa / animals. AAV9 or ERT groups demonstrated significant improvement in ejec tion fraction at three months ( Gaa / 62.651.22%; AAV9 73.513.13%; ERT 73.293.59%). AAV9 and ERT groups show significant reduction in left ventricular mass ( Gaa / 4.960.16 mg/g; AAV9 3.750.41mg/g; ERT 4.110.11mg/g). This is the first study to indic ate a single systemic administration of rAAV2/9 DES hGAA vector is as effective as lifelong semi monthly ERT regimen and can significantly augment respiratory muscle and cardiac function while improving cardiac morphology at 3 mo post treatment in a murine model of Pompe disease.
35 Escape from s iRNA induced s ilencing in an o rthobunyavirus, T ensaw virus Fitzpatrick DM Maruniak JE Department of Entomology and Nematology, University of Florida, Gainesville, FL The infectivity of populations of sho rt interfering RNA (siRNA) induced virus esc ape mutants from Vero cells was assessed in both vertebrate and mosquito cell culture s using a model orthobunyavirus, Tensaw virus (TSV). RNA interference is a major component of antiviral immunity in dipteran i nsects, including mosquitoes. Virus specific siRNAs are being considered as a potential therapy to provide resistance to viral infection in vivo However, due to the high mutation rate of viral RNA polymerases, siRNA induced silencing of RNA viruses often induces the emergence of escape mutants and a delayed reestablishment of infection. However, host alternation during arboviral transmission cycles constrains major genomic shifts, because only a minority of emerging virions f rom infection in vertebrates is viable in mosquitoes, and vice versa. Still, few studies have examined the infectivity profiles of viral escape populations, especially in arboviruses. SiRNAs targeting the overlapping nucleocapsid/nonstructural protein coding regions of TSV significantly reduce viral titer in Vero cells for 48 h. However, viral escape is seen at 72 h, and this viral population establishes infection. Exposing escape populations to a second round of siRNA selection in Vero cells results in sustained reduction in viral titer for up to 120 h. Further experiments will examine escape mechanisms, changes to particle to infectivity ratio, and genomic mutations associated with siRNA induced selection. 36 Xanthomonas albilineans needs an OmpA family outer protein for disease sympt om development and multiplication in the sugarcane stalk Rott PC 1,2 Fleites LA 2 Marlow G 2 Royer M 1 Gabriel 2,* 1 CIRAD, UMR BGPI (Biologie et G n tique des Interactions Plante Parasite), Montpellier, France 2 Plant Pathology Department, University of Florida, Gainesville, FL Xanthomonas albilineans (Xa) is a systemic, xylem invading pathogen that causes sugarcane leaf scald. Xa produces albicidin, a potent antibiotic and phytotoxin which blocks chloroplast differentiation, thus causing the foliar symp toms of the disease. Albicidin is the only known pathogenicity factor in Xa, yet albicidin deficient mutants are still able to colonize the sugarcane plant. In an attempt to identify other major pathogenicity factors, we screened 1,216 independent Tn5 inse rtions in Xa strain XaFL07 1 by single inoculation onto sugarcane cultivar CP80 1743. Mutants were screened for reduced pathogenicity (i.e., capacity to induce leaf symptoms and to multiply in the sugarcane stalk). Five independent Tn5 insertions were foun d in gene XALc_0557, which is predicted to encode an OmpA family outer membrane protein. Each of these insertions resulted in a mutant strain that elicited very slight to no symptoms and was not able to move as efficiently within the sugarcane stalk, both spatially and in intensity, as wild type XaFL07 1. Additional phenotypic studies showed that these mutants: 1) produced albicidin, 2) were less motile and 3) were slower growing than the wild type Xa in vitro However, these OmpA mutants were able to multi ply in sugarcane leaf tissue to levels similar to the wild type strain XaFL07 1. Complementation analyses are currently underway. 37 Maize rough endosperm3 mutant exhibits altered regulation of genes involved in cell cycle and starch biosynthesis Gault C 1 Fouquet R 1 Walts B 1 Sandford M 1 Martin F 1 Barbazuk WB 1,2,* Settles AM 1,3,* 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 Department of Biology, University of Florida, Gainesville, FL 3 Horticultural Scienc es Department, University of Florida, Gainesville, FL The rough endosperm3 mutant can give insight into maize endosperm development and the factors underlying kernel quality. The rgh3 mutation causes a pitted kernel phenotype, decreased endoreduplication, and abnormal cellular differentiation in the endosperm. Rgh3 likely codes for ZmURP, a splicing factor associated with the minor spliceosome, which acts on U12 introns. We proposed that RGH3 promotes cellular differentiation by downregulating cell cycle g enes to promote endoreduplication. To test this, we compared rgh3 and wildtype transcript levels obtained from a SOLiD RNA Sequencing run. About eight million reads were aligned uniquely to the genome, mapping to 37% of predicted exons in the genome. Cuffl inks analysis revealed that rgh3 had upregulated cell cycle genes and downregulated starch biosynthesis genes. Our data indicate that rgh3 cells may synthesize less DNA than wildtype because they are mitotically cycling. 38 Molecular mapping of maize see d mutants Gay BH Bagadion A, Martin F, Spielbauer G, Tseung CW, Settles AM Horticultural Sciences Department, University of Florida, Gainesville, FL Corn represents a major crop in United States' agriculture with last year's value of the field corn cr op estimated at $48.5 billion. The primary product of corn is the seed. Understanding seed development is therefore of paramount importance for future crop improvement.
Molecular genetics or mutant gene cloning is needed to identify the genes important for seed development. A map based approach will be used to clone transposon tagged mutations affecting seed development in maize. Map based approach begins with crossing seed mutant lines to diverse corn inbreds. A mapping population was generated to score ge netic linkage between the seed mutant phenotype and molecular markers, such as Single nucleotide polymorphisms (SNPs) and Simple Sequence Repeats (SSRs). The goal of this project is to map each mutant to a <1 centimorgan (cM) genetic interval. Currently, 1 44 seed mutants have been sequenced for genomic DNA flanking the transposons. Existing Mu transposon flanking sequence tags (FSTs) will be used to identify transposon insertions that co localize with rgh mutant map positions. We expect to correlate transpo son insertions with several seed mutants over the next year. Genes disrupted in the mutant loci will help point to important pathways required for seed metabolism and development. A long term impact of this study is expected to bring new approaches towards improving the overall production of maize and other cereal grains. 39 RNA seq in Drosophila hybrids reveals species specific gene regulation Graze RM 1 Novelo LL 2 Amin V 1 Casella G 2,* Nuzhdin SV 3,4 McIntyre LM 1,2,* 1 Department of Molecular Geneti cs and Microbiology, University of Florida, Gainesville, FL 2 Department of Statistics, University of Florida, Gainesville, FL 3 Section of Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles CA 4 Department of Evolution and Ecology, University of California, Davis, CA Isoform diversity is a fundamental component of the biology of sexual dimorphism, immune response and neurological function. Regulation of multi transcript genes is complex, di fferent regulatory mechanisms can impact subsets of alternative transcripts depending on promoter structure, splicing, and shared regulatory regions within transcripts. Thus, isoforms can be linked in their regulation, but can also be independently regula ted with independent impacts on phenotype. The aim of this project is to understand how species differences in gene regulation result in dependent and independent expression differences among transcript isoforms. Short read sequencing was used to estimate cis differences in gene expression between species by measuring allele specific expression in D. melanogaster D. simulans F1 hybrid female head tissues. Technical and mapping error, as well as copy number differences, can result in bias of allele specific expression estimates toward a specific allele or overall toward a specific parental genome. To control for bias in exon level estimates of allele specific expression, a novel Bayesian model was implemented in which allele frequencies in F1 hybrid DNA sequ ence reads are used to correct systematic bias in allele specific expression estimates from RNA seq data. Using exon specific estimates and sequence variation near splicing junctions, species differences in cis regulation were surveyed across all exons in single and in multi transcript genes. 40 Tissue specific roles of FgfR2 in urethral tube closure Gredler ML 1 Seifert AW 1 Cohn MJ 1 3, 1 Department of Biology, University of Florida, Gainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 3 Howard Hughes Medical Institute, University of Florida, Gainesville, FL Hypospadias is a malformation of the penis that is characterized by incomplete closure of the urethral tube and affects approximately 1 in 125 live male births. Patterning of the mammalian external genitalia requires coordination of proximal to distal outgrowth with urethral tubulogenesis. Reciprocal signaling between the urethral epithelium and mesenchyme of the genital tubercle the embryonic precursor of the penis and clitoris maintains the expression of key genes necessary for development of the external genitalia. Fibroblast growth factor 10 ( Fgf10 ) is expressed in the genital tubercle mesenchyme and signals through the receptor FgfR2 whi ch is expressed in two adjacent cell populations, the urethral epithelium and the preputial ectoderm. Null mutations in FgfR2 result in failure of urethral tube closure (hypospadias) and loss of mature urethral epithelium. In this study, we explore the dis tinct roles of FgfR2 in the urethra and ectoderm using tissue specific knockouts in mice. We find that the loss of FgfR2 from the ectoderm results in hypospadias, although the resulting urethral epithelium maintains relatively normal character. In contrast deletion of FgfR2 from urethral endoderm inhibits maturation into a complex epithelium although gross hypospadias does not occur. Our results demonstrate that FgfR2 plays distinct roles in these two cell populations, and that both are necessary for prope r patterning of the genital tubercle and maturation of the urethra. 41 Altered LKB1 CRTC1 signaling promotes esophageal cancer cell migration and invasion Gu Y 1 Lin S 1 Li J L 2 Nakagawa H 3 Chen Z 1 Tian L 1 Ucar DA 4 Shen H 1 Lu J 5 Hochwald SN 4 Kay e FJ 6 Wu L 1 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Sanford Bur nham Medical Research Institute at Lake Nona, Orlando, FL
3 Division of Gastroenterology, Department of Medicine, Abramson Cancer Center, University of Pen nsylvania, Philadelphia, PA 4 Department of Surgery, University of Florida, Gainesville, FL 5 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 6 University of Florida Shands Cancer Center, Gainesville, FL LKB1 is a tumor suppressor gene whose mutations cause Peutz Jeghers syndrome (PJS) with an increased cancer risk and is a target for mutational inactivation in human malignancies. LKB1 encodes a serine/threonine kinase that plays critical roles in cel l growth, polarity and metabolism. A novel and important function of LKB1 is its ability to regulate the phosphorylation of CREB regulated transcriptional co activators (CRTC) whose aberrant activation is linked with oncogenic activities. However, the role s and mechanisms of LKB1 and CRTC in the pathogenesis of esophageal cancer have not been previously investigated. In this study, we observed altered LKB1 CRTC1 signaling in human esophageal cancer cell lines and patient samples. LKB1 negatively regulates e sophageal cancer cell migration and invasion in vitro. Mechanistically, we determined that CRTC1 signaling becomes activated due to LKB1 loss, which results in the transcriptional activation of specific downstream targets including LYPD3, a critical mediat or for LKB1 loss of function. Our data indicate that de regulated LKB1 CRTC signaling might represent a crucial mechanism for esophageal cancer progression. 4 2 Role of strigolactones on maize growth and development Guan JC Masaharu S, Klee HJ McCarty DR Horticultural Sciences Department, University of Florida, Gainesville, FL Strigolactone (SL) is a new plant hormone known to be crucial for control of branching in many plant species. Selection for altered apical dominance conferred by the Teosinte Branched1 ( TB1 ) gene played a major role in domestication of maize. To discover the role of SL in regulation of apical dominance in maize, a putative SL biosynthesis mutant was characterized SLs are derived from carotenoids through the action of two diffe rent plastid localized carotenoid cleavage dioxygenases (CCDs) i.e. CCD7 and CCD8. A Ds6 like element was found to be inserted exactly at the boundary between 2 nd intron and 3 rd exon of ZmCCD8 gene The ZmCCD8 transcripts are disrupted by the Ds6 like eleme nt. Unlike other plant species, the zmccd8 mutant shows only a subtle increase in branching number However, the length of lateral branches is increased in comparison to non mutant siblings. The zmccd8 mutants are about 10% shorter in stature than wild typ e siblings. In addition, the zmccd8 phenotype includes alterations in root, leaf, and ear development. The phenotype of the tb1, zmccd8 double mutant is similar to the tb1 single mutant with respect to branching, but also includes zmccd8 specific phenotype s, indicating that TB1 may act in a branched pathway downstream of SL signaling. The expression levels of TB1 were not affected in zmccd8 mutants, suggesting SLs may act through post transcriptional regulation of TB1 Taken together, our results expand the roles of SL in plant growth and development. 43 The role of Nlr1 in plant development Gustin JL Fajardo DS, Tseung CW, Black J, Settles AM Horticultural Sciences Department, University of Florida, Gainesville, FL The developmental program that prod uces a flat leaf requires establishment of the adaxial and abaxial (top/bottom) axis. We recovered a maize mutant that impacts development in multiple tissues including distinct narrow leaf and rough endosperm (nlr1) phenotypes. In nlr1 mutants, characteri stic markers of adaxial/abaxial domain specification are altered including reduced blade expansion, reduced macrohair number, irregular vasculature. In addition, nlr1 alters transcript accumulation of adaxial leaf polarity genes in developing leaves. These data suggest Nlr1 is involved in adaxial domain specification in maize. To place nlr1 within the leaf development pathway, we generated a double mutant between nlr1 and a dominant mutant, Rld1 O. Rld1 O causes partial switching of the polar domains leadin g to adaxial cell types on the bottom of the leaf, and vice versa. Rld1 O nlr1 double mutants suppress the ectopic adaxial epidermal cell types, but do not impact the domain switching phenotype of Rld1 O. These data place Nlr1 downstream of polarity establ ishment. We identified a Robertson's Mutator (Mu) transposon insertion that is tightly linked to nlr1. The transposon disrupts a J domain containing protein. J domains activate Hsp70 ATPase activity and proteins containing these domains have diverse functi ons in the cell (s ee pos ter no. 8 ). Evidence suggests Nlr1 impacts leaf and seed morphology through a hypothesized role in developmental gene expression. 44 The proximal Nanog promoter is sufficient for Nanog repression during primitive endoderm cell fat e specification in murine embryonic stem cells Hankowski KE Hamazaki T, Terada N Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL Pluripotency of murine embryonic stem cells is maintained by the preci se coordination of signaling cascades and transcriptional networks promoting proliferation and preventing differentiation. During mouse preimplantation
development, pluripotent stem cells give rise to the primitive endoderm layer on the surface of the inne r cell mass. This first differentiation step occurs through the loss of Nanog expression in these cells, and is accompanied by an increase in the expression of Gata6 Recent research from our lab has identified the importance of the FGFR/Grb2/Ras/Mek/Erk s ignaling pathway in the repression of Nanog and subsequent differentiation to primitive endoderm. The major focus of this work is to understand the mechanism for FGFR mediated Nanog repression in murine embryonic stem cells. FGFR2 stimulation results in ra pid Nanog repression. We demonstrate the importance of the proximal promoter region in the repression of Nanog following FGFR2 stimulation. Interestingly, using chromatin immunoprecipitation, we found Oct4 and Sox2 transcription factors remain bound to the proximal promoter region. The importance of this region containing Oct4/Sox2 binding sites and the binding persistence of these transcription factors aft er Nanog is repressed suggests Oct and Sox2 may mediate Nanog repression as well as transcriptional ac tivation. These findings provide insight into the regulation of a critical gene involved in self renewal and pluripotency. 45 Functional characterization of Arabidopsis isopropylmalate dehydrogenases reveals their important roles in gametophyte developme nt He Y 1 Chen L 1, 2 Zhou Y 1 Mawhinney T P 3 Chen B 1 Kang B H 4 Hauser BA 1,* Chen S 1,5,* 1 Department of Biology, University of Florida, Gainesville, FL 2 State Key Laboratory of Plant Physiology and Biochemistry, China Agricultural University, Beijing, China 3 Department s of Biochemistry and Child Health, Agricultural Experiment Station, University of Missouri, Columbia, MO 4 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 5 Proteomics Division, Interdiscipli nary Center for Biotechnology Research, University of Florida, Gainesville, FL Isopropylmalate dehydrogenases (IPMDHs) catalyze the oxidative decarboxylation of 3 isopropylmalate (3 IPM) in leucine biosynthesis. The Arabidopsis genome contains three putative IPMDH g enes. Here we describe the role of IPMDH2 and IPMDH3 in leucine biosynthesis and gametophyte development. Arabidopsis IPMDH2 and IPMDH3 proteins exhibit significantly higher affinity toward 3 IPM than IPMDH1, which is indicative of pivotal role in leucine biosynthesis. Single mutants of IPMDH2 or IPMDH3 lacked a discernible phenotype. Genetic analysis showed that the ipmdh2 ipmdh3 was lethal in male gametophytes and had reduced transmission through the female gametophytes. Microscopic analysis indicated tha t the aborted pollen grains were small, abnormal in cellular structure, and arrested in germination. In addition, half of the double mutant embryo sacs exhibited slowed development. The IPMDH2/ipmdh2 ipmdh3/ipmdh3 genotype exhibited abnormal vegetative phe notypes, suggesting haplo insufficiency of IPMDH2 in the ipmdh3 background. The mutant and a triple mutant containing one allele of IPMDH2 or IPMDH3 had decreased leucine biosynthetic enzyme activities and lower free leucine levels. The latter showed chan ges in glucosinolate profiles that were significantly different from those in the ipmdh1 mutant. These results demonstrate that IPMDH2 and IPMDH3 primarily function in leucine biosynthesis, are essential for pollen development and needed for embryo sac dev elopment. 46 Effects of neonatal intracranial AAV and systemic lentivi ral gene therapy in Sanfilippo s yndrome type B mice Heldermon CD 1 Qin E 2 Ohlemiller K 3 Herzog E 4 Vogler C 5 Wozniak DF 6 Sands MS 2 1 Department of Medicine, University of Florida, Gainesville, FL 2 Department of Internal Medicine Washington University School of Medicine in St. Louis, St. Louis, MO 3 Department of Oto laryngology Washington University School of Medicine in St. Louis, St. Louis, MO 4 Department of Biology Washingt on University in St. Louis St. Louis, MO 5 Department of Pathology, Saint Louis University, St. Louis, MO 6 Department of Psychiatry, Washington University School of Medicine in St. Louis, St. Louis, MO Sanfilippo s yndrome type B (MPS IIIB) is a lyso somal storage disease resulting from the deficiency of N acetyl glucosaminidase (NAGLU). We have previously shown that intracranial adeno associated virus (AAV) based gene therapy improves several aspects of the disease such as lifespan. Others have demons trated benefit to systemic lentiviral gene therapy. Our objective was to compare the two methods alone and in combination to determine differences in effect and effectiveness. MPS IIIB mice were treated at 2 4 days of age with intracranial AAV2/5 NAGLU (AA V), intravenous lentiviral NAGLU (MND), both (AAV/MND) or neither (no tx). Normal control mice of the same age were either treated with both AAV and lentiviral NAGLU or were untreated as toxicity control and comparator arms respectively. Compared to untre ated MPS IIIB animals, preliminary results indicate all treatments resulted in some improvement in the clinical phenotype. AAV, MND and AAV/MND treatment results in improved motor function compared to untreated controls using a rocking rotarod paradigm. Ea rly results from Auditory evoked Brainstem Response measurements of hearing also demonstrate improvement in all treatment groups. Circadian rhythm analysis reveals an improvement in time to activity onset and percent of activity during
daylight in the AAV group, little improvement with MND alone and near normalization with AAV/MND treatment. All three MPS IIIB treatment groups' median lifespans are lengthened. 47. How the chicken lost its penis: developmental basis of genital reduction in avian evolution Herrera A 1 Shuster SG 2 Cohn MJ 1 3,* 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Department of Biology, University of Florida, Gainesville, FL 3 Howard Hughes Medical Institute, University of Florida, Gaines ville, FL The external genitalia of modern vertebrates exhibit an impressive degree of anatomical variation, ranging from rather primitive pattern found in turtles and crocodilians, to the paired phalluses (hemipenes) of squamate reptiles, to the complex penis of mammals, which has a closed urethral tube an d a penian bone (or baculum). While some birds have evolved elaborate external genitalia, others have reduced or completely lost their genitalia. Phylogenetic analysis of genital anatomy in birds and cr ocodilians indicates that the reduced genitalia of galliformes represents a derived condition. In order to understand the developmental mechanisms that led to evolutionary loss of external genitalia, we have performed a comparative study of three avian cl ades; Galliformes (e.g., chickens, quails,) in which the phallus is severely truncated or absent, their sister group, the Anseriformes (e.g., ducks, geese) which have extremely long, coiled phalluses, and the most basal group of birds, the Paleognaths (e. g., ostriches, emus), which have well developed genitalia similar to the anseriformes. Embryos from all three clades undergo initiation of a genital tubercle and show similar expression patterns of developmental control genes, but chick and quail genitali a fail to develop beyond the tubercle stage despite strong expression of outgrowth genes like Sonic hedgehog, Hoxd13 and Wnt5a. We have identified an ectopic domain of Bmp4, a pro apoptotic gene, in the distal tip of chicken genitalia, and found that they undergo extensive cell death after initial outgrowth. Antagonism of Bmp4 in chick genital tubercles rescues cells from apoptosis and promotes further outgrowth, suggesting that evolution of this novel gene expression domain in galliformes underlies the f ailure of genital development. We are now investigating the mechanisms of Bmp4 regulation in avian external genital development to identify how this novel domain arose during galliform evolution. These studies are helping us to understand how the evolut ion of developmental mechanisms underlies morphological diversification of genital organs. 48. Variations in cell cycle kinetics in the dentate gyrus of adult mice might explain light:dark cyc le fluctuations in neurogenesis Hoang Minh LB Ormerod BK J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL Previous work suggests that hippocampal neurogenesis in adult mice increases at nighttime, when they are most active, relative to daytime. We directly tested whether hippocampal neurogenesis is increased at night and examined whether increased cell proliferation at night is due to: 1) an increase in the number of NPCs entering the cell cycle or 2) an increase in the length of DNA synthetic (S) phase duri ng the dark phase. To measure neurogenesis at nighttime compared to daytime, adult female C57Bl/6 mice (7 wks old upon arrival; Taconic farms) were exposed to a freely moving or immobilized running wheel for one wk before being injected i.p., either once o r 6x daily with the cell synthesis marker BrdU (50mg/kg), either 2h after lights on or lights off. Mice receiving a single BrdU injection were perfused 2h later to measure cell proliferation, and mice receiving 6 daily or nightly injections were perfused 1 wk or 4 wks after the first injection to measure neurogenesis and new cell survival (n=5 per group). To compare cell cycle kinetics between daytime and nighttime, mice were injected with BrdU (50 mg/kg) in the middle of the light or dark phase (n=5 per g roup), sacrificed 4h later, and their brains processed for immunofluorescence and confocal microscopy using monoclonal antibodies directed against BrdU (S phase), Ki67 (all phases of cell cycle), and phosphohistone H3 (G2 M phase) to estimate total length of cell cycle (T c ) and length of S phase (T s ) of proliferative cells, as well as proportion of cells in G2 M phase in the dentate gyrus. 49 Live cell imaging of the endoplasmic reticulum in plant cells with altered actin dynamics Hwang A Grey PH, Cudd y K, Oppenheimer DG Department of Biology, University of Florida, Gainesville, FL The endoplasmic reticulum (ER) is the first step in the secretory membrane system, and plays an important role in membrane trafficking. In plants, the secretary membrane s ystem is responsible for secreting the needed materials for cell expansion during development. Previous results from analyzing plant epidermal hair cells (trichomes) in our lab showed that disruptions in actin dynamics leads to problems in membrane traffic king. Since the ER is the entry point for all material controlling cell expansion, we hypothesized that the ER will show defects in mutants that have altered actin dynamics. In this study, we used an ER targeted Green Fluorescent Protein (GFP) to observe t he ER in living Arabidopsis thaliana mutants
that have aberrantly shaped trichomes due to the loss of a regulator of actin depolymerization. Confocal microscopy will be used to view the ER in living mutants and we will quantify the differences in the ER be tween wildtype and mutants in terms of organization and dynamics. Because altered actin dynamics is implicated in many human diseases, a better understanding of the relationship between actin dynamics and membrane trafficking may have important implication s for human health by providing new possibilities for drug targets. 50 RNAi suppression of lignin biosynthetic genes 3 O methyltransferase (COMT) and 4 coumarate CoA ligase (4CL) in sugarcane Jung JH Kim JY, Fouad W, Vermerris W Gallo M Altpeter F Agronomy Department, University of Florida, Gainesville, FL Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Sugarcane ( Saccharum sp hybrids) is a highly productive C4 grass used as the main source of sugar and more recently to produce ethanol. Down regulation of lignin biosynthesis pathway enzymes is a promising strategy to increase the efficiency of bio ethanol production from hemicellulosic sugarcane residues. In the lignin pathway, 4 coumarate CoA ligase (4CL) and Caffeic acid 3 O methyltransferase (COMT) are key enzymes that catalyze the formation of CoA thiol esters of 4 coumarate and other hydroxycinnamates or the methylation of 5 hydroxyconiferaldehyde to sinapaldehyde, respectively. In this study, COMT and 4CL family genes were isolated from the commercially important sugarcane cultivar CP 88 1762 by a combination of cDNA library screening and PCR based approaches. Expression analysis allowed identification of candidate genes with differential expression in dif ferent tissues. For further characterization RNAi suppression vectors were generated and stably introduced into the CP 88 1762 by biolistic gene transfer. PCR and NPTII ELISA results confirmed that 38 COMT RNAi and 23 COMT+4CL RNAi transgenic lines were ge nerated. Several transgenic lines displayed drastic suppression of the target genes when evaluated by quantitative PCR. Northern blot analysis of low molecular weight RNA confirmed the accumulation of siRNAs in these transgenic sugarcane lines. Plants are currently grown to maturity in a replicated and randomized design and will be analyzed for lignin determination. 51 Development of a drought tolerant, non invasive, high biomas s crop by interspecific hybridiz ation between elephantgrass ( Pennisetum purpur eum Schum.) and pearl millet ( Pennisetum glaucum L.) Kannan B Sollenberger L, Altpeter F Agronomy Department, University of Florida, Gainesville, FL Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Elephantgrass ( Pe nnisetum purpureum Schum.) is considered one of the best adapted perennial feedstocks for biofuel production in the southern US. Elephantgrass causes less environmental problems than several other potential biomass crops. However, elephantgrass is listed a s invasive in Southern Florida by the Florida Exotic Pest Plant Council. Plant propagation and establishment of new elephantgrass plantings occurs through vegetative plant parts. Therefore, unlike most seeded crops, seed production is not necessary for ele phantgrass production and its suppression will significantly reduce its potential for invasiveness. Pearl millet ( Pennisetum glaucum L.) is one of the most drought tolerant grasses. Interspecific hybridization between elephantgrass (tetraploid) and pearl m illet (diploid) may result in drought tolerant, high biomass triploid hybrids with male and female sterility which will eliminate production of wind dispersed seeds. We produced more than 3000 triploid, interspecific hybrids between elephantgrass (tetraplo id) and pearl millet (diploid). We will present data describing the phenotypic variability in these hybrids which allowed selecting lines with excellent vigor and sterility. Current experiments are evaluating the drought tolerance and biomass production of the interspecific hybrids in comparison to 15 genetically diverse elephantgrass accessions from the world collection. 52 Investigating the number of valid species named in the genus Opheodesoma (Echinodermata: Holothuroidea: Synaptidae) using phylogene tic information combined with morphological data Kenyon LJ Michonneau F Malaco logy, Florida Museum of Natural History, University of Florida, Gainesville, FL Opheodesoma is one of the genera belonging to the sea cucumber family Synaptidae, found in shal low tropical waters; and exhibit a lot of variation in their live appearance. There are currently 11 valid species names recognized in this genus. The species described were differentiated based on morphological characters including webbing between the dig its and tentacles, the shape of the anchor plates, the shape and color the calcareous ring, the presence of rods in the tentacles and oral disk, and the distribution of miliary granules. Due to the extent of the variation observed, and because the species were described from a limited number of individuals, it is probable that the currently described species represent intra specific variation. We thus hypothesize that there are fewer species than presently recognized. A phylogenetic tree based on a portion of the mitochondrial marker CO1 sequenced from 42 specimens reveals three Evolutionary Significant Units (ESUs). We tested several morphological
characters with the hypothesis that they will co vary with the mtDNA sequence data. We did not find diagnostic morphological characters to distinguish between the two closest related ESUs. However, the third genetically distinct group did have morphological characters that co varied with the mtDNA sequence data and appears to be an un described species. Thus, no mo rphological characters have yet been found to differentiate the two closest related ESUs suggesting that if further investigations yield the same outcome, these two ESUs actually represent a single species. Including more genetic markers in our study may h elp to better understand the evolutionary history of this genus. 53 Power calculations for symptom counts in genetic association analysis Kertes DA 1,* Winner L 2 1 Department of Psychology, University of Florida, Gainesville, FL 2 Department of Statisti cs, University of Florida, Gainesville, FL Quantitative traits such as symptom counts preserve greater information about disease severity and thus may in some cases be more informative than case/control status for genetic association analysis. However, sy mptom counts typically violate assumptions of normality required for analysis conducted under the General Linear Model and are thus more appropriately treated as ordinal data. Standard power calculations for genetic association do not readily accommodate o rdinal data with multiple phenotype levels. We developed a procedure to conduct power analysis for the generalized Cochran Mantel Haenszel test for contingency tables with ordinal rows and columns. We further demonstrate that the sample size required to ac hieve satisfactory power is reduced for the ordinal phenotype compared to a case/control analysis. We tested the model using simulated data with varying non normal distributions of symptoms. The results showed the procedure was effective in calculating pow er for an ordinal phenotype with an expected sample distribution at varying allele frequencies and effect sizes. Our method can be a valuable analytic tool for genetic association analysis where phenotypes are expected to be non normally distributed but re searchers aim to preserve information about disease severity. 54 Hyperthermostable xylanase Xyl10B produced in transgenic sugarcane converts hemicellulose to fermentable sugar for fuel ethanol production Kim JY 1 ,2 Fouad WM 1,2 Gallo M 1 2, Nong G 3 Pr eston JF 3 Altpeter F 1,2, 1 Agronomy Department, University of Florida, Gainesville, FL 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 3 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL Both sugarcane leaves and bagasse represent an inexpensive and abundant cellulosic feedstock for fuel ethanol production. Xylan is after cellulose, the major polysaccharide in sugarcane residues. This polymer must be hydrolyzed to its component sugar s (xylose or xylobiose) before fermentation to ethanol. Endoxylanases are the main enzymes involved in xylan hydrolysis. Constitutive, apoplast or chloroplast targeted expression cassettes of the codon optimized, hyperthermostable GH10 xylanase from Thermo toga maritima (xyl10B) were generated and introduced into sugarcane for in planta expression. The fluorogenic xylanase activity assay detected xylanase activity in 17 transgenic lines with up to 1% of the crude protein extract being xyl10B. Lines with inte gration of the chloroplast targeted expression cassette displayed higher xylanase activity than the lines with apoplast targeted expression cassette. Mature leaves exhibited higher expression than immature leaves, expanding leaves, and internodes. The suga r release assay demonstrated that Xyl10B produced in transgenic sugarcane plants was heat stable and converted the majority of xylan to fermentable xylobiose and xylose. Improving the level of xyl10B expression in transgenic sugarcane will likely enhance t he conversion of lignocellulosic biomass to fuel ethanol. 55 Analysis of resistance mutations to hepatitis C virus protease inhibitors by deep sequencing Kirst ME 1 Li EC 1 Wang CX 1 Fried M 2 Liu C 3 Nelson D 4 Wang GP 1,* 1 Division of Infectious Disea ses, Department of Medicine, University of Florida, Gainesville, FL 2 Division of Gastroenterology and Hepatology, Department of Medicine, University of North Carolina, Chapel Hill, NC 3 Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 4 Division of Gastroenterology, Hepatology and Nutrition, Department of Medicine, University of Florida, Gainesville, FL Hepatitis C virus (HCV) encodes a nonstructural protein (NS3) protease required for virus replication. Sev eral small molecular inhibitors against NS3 protease are currently in development and appear promising in clinical trials. However, recent data suggest that naturally occurring resistance mutations to NS3 protease inhibitors are common in treatment native patients. Furthermore, due to the high replication rate and error prone nature of HCV encoded RNA polymerase, HCV circulates as viral quasispecies in those infected, raising the possibility that resistance mutations may be present in low abundance in the v iral swarm and potentially affect treatment outcome. The prevalence of these mutations is unknown. Using 454/Roche pyrosequencing, we examined ~45,000 high
quality NS3 sequences from 11 cross sectional samples of subjects infected with HCV and ~690,000 hig h quality NS3 sequences from 61 longitudinal samples of 13 HCV infected subjects undergoing liver transplant. The abundance and the dynamics of drug resistance mutations were quantified using a custom analysis pipeline. Our results indicate that resistance mutations to NS3 protease not detectable by cloning or population sequencing can be detected at low levels using pyrosequencing. Their impact on treatment outcome remains to be elucidated. Our methods should provide a framework for longitudinal analyses o f the dynamics of HCV resistance mutations in infected subjects treated with small molecular inhibitors. 5 6 Role of auxin in arsenite induced oxidative stress tolerance in Arabidopsis thaliana Krishnamurthy A Teo S, Rathinasabapathi B Horticultural S ciences Department, University of Florida, Gainesville, FL Arsenic, a widespread toxic metalloid in the earth's crust is a major public health hazard because of arsenic's carcinogenic potential. Arsenic is known to induce oxidative stress by producing rea ctive oxygen species. To understand how plants adapt to arsenic stress, we have screened a number of Arabidopsis genotypes for arsenite (AsIII) tolerance using a root bioassay In all the genotypes, AsIII significantly reduced the primary root growth but n ot the initiation of lateral roots. An auxin transport mutant (aux 1) was highly sensitive to AsIII compared to the wild type (WT). Pre treatment of WT plants with auxin transport inhibitors made plants more sensitive to AsIII and the pretreatment of aux1 mutant with IAA improved its tolerance to AsIII, consistent with a role for auxin transport in AsIII stress tolerance. Further investigations are underway to test whether AUX 1 protein is the target of AsIII inhibition and whether auxin induced genes have roles in arsenite tolerance. 57 Use of optical mapping in bacterial genome finishing Kumar D Farmerie WG Interdiscipli nary Center for Biotechnology Research, University of Florida, Gainesville, FL The cost efficiency of modern DNA sequencing technolo gy, such as the Roche 454 GS FLX, allows individual investigators to undertake bacterial genome projects that were not affordable only a few years ago. Our core laboratory has several ongoing bacterial genome projects presenting a variety of challenges to genome assembly and closure. Several factors contribute to these challenges; including sequence repeats versus read length, intrinsic sequencing errors, and dynamic genome rearrangements. Together these factors complicate genome closure when using shotgun DNA sequencing alone. The g enome finisher may experience difficulty validating their assembly in the absence of a physical map. To address this problem, we adopted whole genome optical mapping as a tool to validate bacterial genome assemblies. OpGen, Inc. (Gaithersburg, Maryland) prepared the optical maps used in this project. Briefly, an optical map is a complete genome restriction map deduced from a number of partial restriction maps. Optical maps are generated by spreading carefully extracted genomic DNA onto a treated glass surface containing many narrow channels, followed by digestion in situ with restriction enzymes. About 50 100 contiguous restriction fragments with sizes approaching up to one third of the whole genome are selected and optically measu red. The overlapping partial optical contigs are combined by alignment software to produce a contiguous whole genome restriction map. The contiguous optical map can be aligned and compared with the in silico restriction map determined for the partially com plete whole genome assembly. We successfully used optical mapping for guiding the closure of four closely related bacterial genomes. The optical map allowed us to identify assembly errors not possible using shotgun DNA sequencing data alone. Thus, we concl ude that, in order to ensure the accuracy of a finished bacterial genome, optical mapping is an important tool to validate de novo assemblies generated by next generation DNA sequencing. 58 Inhibition of type 1 diabetes by a Lactobacillus johnsonii media ted Th17 bias Lau KK 1 Benitez P 1 Ardisone A 1 Lorca G 1, Li N 1 Sankar D 1 Neu J 1 Atkinson M A 2, Larkin J 1 1 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 2 Department of Pathology, Immunology and Laboratory Medic ine, University of Florida, Gainesville, FL Although it is known that resident gut flora contribute to immune system function and homeostasis, the role of gut flora in the progression of autoimmune disease is poorly understood. It has recently been shown that distinct bacterial populations are present within rodent models prone (DP) and resistant (DR) to Type 1 Diabetes (T1D). Oral Transfer of Lactobacillus johnsonii (N62) from DR rodent to DP rodents conferred T1D resistance to DP rodents. Diabetes resist ance in N62 fed DP rodents was correlated to a TH17 bias within the mesenteric lymph nodes which was associated with high lev els of IL6 and IL23. Moreover, in vitro assays show that Lactobacillus Johnsonii mediates high IL6 and IL10 levels in antigen prese nting cells which can mediate TH17 differentiation in the presence of sufficient TCR stimulation. Together, these data suggest an interesting paradigm whereby autoimmunity can be circumvented by gut flora mediated, T cell differentiation.
5 9 Epigenetic s ilencing of germ cell specific genes in primed pluripotent stem cells Leseva M Rosenbluth A, Hankowski K, Hamazaki T, Terada N Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL During development, the cells of the mammalian embryo gradually acquire different cell fates. One of the earliest developmental decisions to be made is to permanently silence germ cell specific genes in those cells that are destined to belong to somatic lineages. We have previous ly shown that an E2F transcription factor family member, E2f6, is essential to establish repression in a subset of germ cell specific genes. Further, we have demonstrated that this repression is initiated in epiblast stem cells (EpiSCs), an in vitro model system for the post implantation epiblast, as observed by DNA promoter methylation. Here we will show that E2f6, although required, is not sufficient to establish repression both in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs), in vitro model systems for the pre implantation epiblast and differentiated somatic tissues, respectively. Also, that expression of E2f6 and its recruitment to the proximal promoters of E2f6 target genes peaks in EpiSCs. In addition, repression is associat ed with establishment of H3K9me3 and H3K27me3 marks. Recently, E2f6 has been shown to be a component of Polycomb repressive complexes (PRC). Thus, we hypothesize that repression of meiosis specific genes is established at a particular moment in time by a d evelopmental stage specific PRC repressive complex, either assembled and/or functionally activated at the transition point from pre to post implantation epiblast. 60 Chromatin boundaries require the functional cooperation between hSET1 and NURF complex es Li X 1 Wang S 1 Li Y 1 Qiu Y 2 Xiao H 3 Wu C 3 Huang S 1, 4, 1 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2 Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 3 Laboratory of Bio chemistry and Molecular Biology, National Cancer Institute, National Institutes of Health Bethesda, MD 4 University of Florida Shands Cancer Center, Gainesville, FL Chromatin modification and remodeling activities play a central role in organizing chromat in domains with epigenetic characteristics. We showed previously that USF bound 5'HS4 insulator mediates chromatin barrier activity by recruiting and organizing active histone modifications in the chicken b globin locus. To further understand the physical function of USF1, we purified USF1 associated protein complexes. We found that USF1 forms a multiprotein complex with hSET1 and NURF exhibiting histone H3K4 methyltransferase and ATP dependent chromatin remodeling activities, respectively. Both hSET1 and N URF complexes are specifically recruited by USF1 to the 5'HS4 insulator. Knocking down BPTF, a component of NURF complex, caused a rapid loss of barrier activity that protects a transgene from chromatin silencing. This is accompanied by an alteration of nu cleosome positioning that expands the nucleosome to the nucleosome free linker region in the 5'HS4 insulator and increases repressive H3K9 dimethylation. Furthermore, suppression of hSET1, a H3K4 methyltransferase, leads to not only a loss of barrier activ ity, but also a loss of H3K4 dimethylation and H3K9/K14 acetylation, as well as the recruitment of BPTF at insulator sites. Thus, our data reveal a molecular mechanism in which histone modifying enzyme hSET1 and chromatin remodeling complex NURF collaborat e together to maintain the 5'HS4 chromatin barrier activity and to prevent the encroachment of adjacent heterochromatin. 61 Evolutionary genomics implies a specific function of Ant4 in mammalian and anole lizard germ cells Lim CH 1 Hamazaki T 1 Braun E 2 ,* Wade J 3 ,4 Terada N 1,* 1 Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 2 Department of Biology, University of Florida, Gainesville, FL 3 Department of Psychology, Michigan State University East Lansi ng, MI 4 Departme nt of Zoology, Michigan State University East Lansing, MI Among Adenine nucleotide translocase (Ant) family, the latest known Ant4 is exclusively expressed in testis and is essential for spermatogenesis. Until now, Ant4 has been identifie d only in mammal and we have hypothesized that Ant4 may be a complementary gene to compensate the silencing of X linked Ant2 during male meiosis. Of interest, a recent progress in comparative genomics has revealed that the Ant4 ortholog also exists in Anol e lizard ( Anolis carolinensis ), one of the reptiles having a male heterogametic (XY) sex determination system. However, Ant2 was not likely on the sex chromosome in anole based on a copy number analysis of the ge ne between male and female. These observatio ns indicate that conservation of Ant4 is independent of linkage of the Ant2 gene to sex chromosome. Ant4 may not simply compensate the loss of Ant2 gene expression during male meiosis. There might be a specific advantage for mammals and some reptiles to ha ve Ant4 during their spermatogenesis and/or in their sperm.
62 A novel chromatin barrier element delimits the formation of facultative heterochromatin without blocking enhancer function Lin NW 1 Li GY 2 Zhao KJ 3 Zhou L 1, 1 Department of Molecular G enetics and Microbiology, University of Florida, Gainesville, FL 2 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 3 Laboratory of Molecular Immunology, National Heart Lung, and Blood Institute, National Institutes of Healt h Bethesda, MD Formation of facultative heterochromatin at specific genomic loci is fundamentally important in defining cellular properties such as differentiation potentials and responsiveness to environmental stimuli. By the nature of its formation, he terochromatin and repressive histone marks will propagate until the chain reaction is broken. While certain active promoters can block propagation of heterochromatin, there are also specialized DNA elements, referred to as barrier insulators, serve to dema rcate the boundary of facultative heterochromatin formation. In this study we identified a chromatin barrier that specifically limits the epigenetic regulation to a distal enhancer region so that repressive histone modification cannot reach the promoter an d proximal enhancer regions of reaper. Unlike all of the known insulators identified from Drosophila, the IRER (irradiation responsive enhancer region) left barrier (ILB) does not contain enhancer blocking activity. This is in accordance with the fact that epigenetic regulation of IRER is dynamic and reversible following certain stresses, under which circumstances the enhancer function of IRER is required for stress induced pro apoptotic gene expression. 63 The histone demethylase LSD1 is a novel transcri ptional co regulator of Notch signaling and maintains myogenic progenitor cells pluripotency Lin S 1 ,2 Jin B 1,2 Li J L 3 Shen H 1,2 Wu L 1,2 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 University of Florida Shands Cancer Center, Gainesville, FL 3 Sanford Burnham Medical Research Institute at Lake Nona, Orlando, FL Notch signaling plays crucial roles in cell proliferation, differentiation and survival, regulating various developmental processes and tissue ho meostasis such as myogenesis. Molecular mechanisms underlying the stringent regulation of Notch signaling remain unclear. In this study, we report the identification of the histone demethylase LSD1 as a novel component of the Notch transcription factor CSL protein complex. We showed that there is a specific and direct LSD1 and CSL interaction. The depletion of LSD1 or CSL resulted in impaired proliferative and differentiative potentials in myogenic precursor cells and led to the transcriptional de repressio n of a common set of Notch target genes. Also, LSD1 demethylated Notch target loci via CSL binding, and repressed target gene expression. Together, our data indicate that LSD1 is an important regulator of Notch signaling and is required for proper myoblast proliferation and differentiation. 64 Histone demethylase LSD1 is required for SNAI1 mediated transcriptional repression of E cadherin during epithelial mesenchymal transition Lin T Ponn A, Lu J Department of Biochemistry and Molecular Biology, Uni versity of Florida, Gainesville, FL Epithelial mesenchymal transition (EMT) has pivotal roles during embryonic development and carcinoma progression. Members of the Snai1 family of zinc finger transcription factors are central mediators of EMT and induce EMT in part by directly repressing epithelial markers such as E cadherin, a gatekeeper of the epithelial phenotype and a suppressor of tumor invasion. However, the molecular mechanism underlying Snai1 mediated transcriptional repression remains incomplete ly understood. Here we show that Snai1 physically interacts with and recruits the histone demethylase LSD1 (KDM1A) to epithelial gene promoters. LSD1 removes dimethylation of lysine 4 on histone H3 (H3K4m2), a covalent histone modification associated with active chromatin. Importantly, LSD1 is essential for Snai1 mediated transcriptional repression and for maintenance of the silenced state of Snai1 target genes in invasive cancer cells. In the absence of LSD1, Snai1 fails to repress E cadherin. In cancer ce lls in which E cadherin is silenced, depletion of LSD1 results in partial de repression of epithelial genes and elevated H3K4m2 levels at the E cadherin promoter. These results underline the critical role of LSD1 in Snai1 dependent transcriptional repressi on of epithelial markers and suggest that the LSD1 complex could be a potential therapeutic target for prevention of EMT associated tumor invasion. 65 Human hepatocyte growth factor receptor is a cellular co receptor for AAV3 Ling C 1 3 Lu Y 1,3 Kalsi JK 1 Jayandharan GR 1,3 Li B 1,3 Ma W 1,3 Cheng B 1,3 Gee SWY 1,3,4 McGoogan KE 1,3,5 Zhong L 6 ,7 Govindasamy L 8 Agbandje McKenna M 8 Srivastava, A 1 3,8 9 ,* 1 Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida, G ainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 3 Powell Gene Therapy Center, University of Florida, Gainesville, FL
4 Division of Critical Care Department of Pediatrics, University of Florida, Gaine sville, FL 5 Division of Gastroenterology Department of Pediatrics, University of Florida, Gainesville, FL 6 Gene Therapy Center University of Massachusetts, Worcester, MA 7 Hematology/Oncology Division Department of Medicine, University of Massachusetts Worcester, MA 8 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 9 University of Florida Shands Cancer Center, Gainesville, FL Transduction by AAV3 serotype vectors has been reported to be inhibited by heparin, hepara n sulfate, and fibroblast growth factor receptor 1. However, the true identity of a cellular receptor/co receptor for AAV3 remains unclear. We recently reported that of the 10 serotypes, AAV3 vectors were by far the most efficient in transducing human hepa toblastoma (HB) and human hepatocellular carcinoma (HCC) cell lines as well as primary human hepatocytes ( Glushakova et al ., 2009, Mol Genet Metab 98 (3) :289 99). Since HB and HCC cells express elevated levels of hepatocyte growth factor receptor (HGFR), we hypothesized that AAV3 uses HGFR as a putative receptor/co receptor for entry into cells. A human HCC cell line, Huh7, was transduced with scAAV3 EGFP vectors in the absence or presence of increasing concentrations of hepatocyte growth factor (HGF), the l igand for HGFR. HGF reduced AAV3 vector mediated transgene expression in a dose dependent manner. Wherea s pre incubation with HGF led t o significant inhibition of transduction, post incubation with HGF had no significant effect. AAV3 vector mediated transd uction was also strongly inhibited by pre treatment of cells with anti HGFR antibodies. In in vivo experiments, whereas efficient transduction of primary murine hepatocytes occurred by AAV8, followed by AAV2 vectors, transduction by AAV3 vectors was largel y inefficient. These data suggest that AAV3 utilizes human HGFR as a receptor/co receptor for entry into human liver specific cells. 66 Utilization of cross taxa SSRs to assess the genetic variability in sixteen Florida elephant grass ( Pennisetum purpure um S.) entries Lpez Y 1 Seib J 1 Chase CD 2,* Sollenberger LE 1 Woodard KR 1 Gallo M 1,* 1 Agronomy Department, University of Florida, Gainesville, FL 2 Horticultural Sciences Department, University of Florida, Gainesville, FL Elephant grass ( Pennisetum p urpureum S.) is an abundant, fast growing plant with significant potential as a carbohydrate source that could be farmed in the United States. Microsatellites (SSR, simple sequence repeat) allow a fine scale genetic characterization of a germplasm individu als or groups. This study addressed the transferability and utilization of microsatellite markers developed for pearl millet ( Pennisetum glaucum (L.) R. Br.) to assess the genetic variability among sixteen elephant grass entries currently growing in Florid a. One pearl millet entry was included for comparison purposes among the two pennisetum species. Two sets of pearl millet SSRs markers were used. One developed from a small insert genomic library and the other from EST data. Sixteen elephant grass entries were analyzed. Out of 18 genomic SSRs primers screened, 87% amplified with 78% showing polymorphism; and out the 16 SSR's of EST origin, 88% amplified with 81% showing polymorphism; six is the average number of alleles per locus. Nei and Li Genetic Similar ity Coefficients for the 16 elephant grass entries averaged 0.6 4 (range 0.42 0.86). A distance based dendrogram grouped the 16 elephant grass entries in six clusters, pearl millet branching independently from all of the 16 elephant grass entries. This st udy showed a high degree of primer transferability between elephant grass and pearl millet and revealed genetic variability among the elephant grasses. 67 Involvement of glucocorticoid receptor signaling in AAV2 vector mediated transgene expression Lu Y 1 3 Ling C 1 3 Jayandharan GR 1,3,4 Srivastava A 1 3,5, 1 Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida, Gainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 3 Powell Gene Therapy Center, University of Florida, Gainesville, FL 4 Department of Haematology and Centre for Stem Cell Research, Christian Medical College, Vellore, India 5 University of Florida Shands Cancer Center, Gainesville, FL We have reported th at successful entry of AAV2 into cells requires fibroblast growth factor receptor 1 (FGFR1) as a cellular co receptor ( Qing et al., 1999, Nat Med 5(1):71 7 ). Phosphorylation of F GFR1, following ligand binding, leads to activation of glucocorticoid receptor (GR) signaling. GR is present in its inactive form, and is activated by phosphorylation following receptor binding. Since in our more recent studies, we observed that AAV infection can trigger activation of the NF $! B pathway, we hypothesized that GR media ted signaling might also be activated following AAV entry into the cytosol. HeLa cells were treated for 12 h rs with various concentrations of dexamethasone, a known inducer of GR activation, prior to transduction with a scAAV2 EGFP vector. Up to ~5 fold in crease in transgene expression could be achieved following tr e atment with 10 M dexamethasone suggesting that GR activation is a positive regulator in the AAV life cycle. U2OS cells, which lack the endogenous GR expression, could not be transduced efficiently by AAV
vectors, and dexamethasone treatment failed to increase transduct ion in these cells. Transient transfection of GR expression cassette into U2OS cells led to a significant increase in AAV mediated transduction. The AAV inverted terminal repeats contain the sequence, GTTCCT, which shares partial homology to the consensus GR response element (GRE) site TGTTCT, but it remains to be investigated whether this sequence constitutes a functional GRE. 68 Foxa genes in intervertebral disk formation Maier J A Harfe B D Department of Molecular Genetics and Microbiology, Universit y of Florida, Gainesville, FL The intervertebral disk (IVD) is composed of a tough, outer annulus fibrosus, and an inner, gel like nucleus pulposus (NP). The NP is derived from the notochord in mice. Degeneration of the NP results in back pain, for which effective treatments are limited. Little is known about the mechanisms of IVD development and degeneration; this information could lead to improved treatments. The forkhead box ( Fox ) genes are expressed in many tissues and function in development and post natal life. Foxa1 and Foxa2 are expressed in all three germ layers of the early embryo and have been well characterized in endoderm but not the notochord. Foxa2 null mice die in utero lacking a notochord. Cre alleles have been used to ablate Foxa2 in the e ndoderm. These conditional alleles have also been used with a Foxa1 null allele to make double knockouts. We used these alleles with an inducible ShhERT2cre line to remove Foxa2 in tissues where Sonic hedgehog is expressed in E7.5 mouse embryos. Histology and fate mapping with the Rosa26 reporter allele were done. Mice null for Foxa1 and lacking Foxa2 in Shh expressing cells appear to have a severely deformed NP and a shortened tail. Fate mapping in these mice suggests defects in the migration of notochord cells to the NP in mice heterozygous for Foxa1 and missing Foxa2 Study of the role of Foxa family action in IVD development may provide insight into new treatments for disk degeneration. 69 Adeno associated virus vectored gene therapy for treatment of autosomal dominant retinitis pigmentosa (ADRP) suppression and replacement strategy Mao H 1 Gorbatyuk M S 2 ,3 Hauswirth WW 1,4 ,* Lewin AS 1,* 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Department of Cel l Biology and Anatomy, University of Nor th Texas Health Science Center at Fort Worth, Fort Worth, TX 3 North Texas Eye Research Institute, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 4 Department of Ophthalmology, University of Florida, Gainesville, FL Autosomal dominant retinitis pigmentosa (ADRP) is frequently caused by mutations in the RHO gene. We are engaged in development of gene therapies for ADRP using adeno associated virus (AAV), which efficiently infects and trans duces photoreceptor cells. In the current study, we designed and constructed a "hardened" form (RHO301) of the rhodopsin (RHO) gene that is specifically resistant to degradation by the siRNA 301. We had previously demonstrated that siRNA301 degrades both m utant and wild type mouse and human RHO mRNA. RHO301 was generated by introducing silent mutations to eliminate the siRNA cleavage site. In order to suppress the mutant gene and express the normal gene at an appropriate level, we developed suppression and replacement strategy by making a combination construct with both RHO301 and siRNA301 in a single virus. We are testing in a P23H mouse model of ADRP that expresses a human transgene with a prevalent ADRP mutation. With delivery of RHO301 siRNA301 in AAV5 t o P23H transgenic mice, the retinal degeneration of injected eyes was much slower compared with that of control GFP virus injected eyes. At 3 months post AAV injection, the retinal outer nuclear layer in the RHO301 siRNA301 injected eyes was thicker than t hat of control virus injected eyes, indicating better survival of photoreceptors. The sequence of siRNA makes it useable in humans with ADRP caused by RHO mutations, and we would like to develop this approach for use in human patients. 70 Mapping of a ge netic modifier of the rough endosperm3 seedling phenotype Martin F Fouquet R, Fajardo D Settles AM Horticultural Sciences Department, University of Florida, Gainesville, FL Plant Molecular and Cellular Biology Program, University of Florida, Gainesvil le, FL The rough endosperm3 ( rgh3 ) mutant causes developmental defects that are either seed or seedling lethal. Rgh3 is likely to encode a U2AF 35 Related Protein (ZmURP), which is a predicted RNA splicing factor. U2AF 35 proteins identify splice acceptor s ites during RNA processing and function through protein protein interactions. Genetic modifiers of rgh3 can identify genes that act within the same pathway as rgh3 and we investigated whether natural variation could modify the rgh3 phenotype. The rgh3 mut ation was originally isolated from the UniformMu population in the W22 inbred background. We crossed rgh3 to the B73 and Mo17 inbreds. Mutant seedlings from the B73 F2 population showed a distinct, open leaf phenotype in comparison to a hooked, adherent le af phenotype for rgh3 mutants in the W22 background. Mutant seedlings from the Mo17 F2 population seedlings were partially suppressed. To develop
a mapping population for the B73 modifier(s), we backcrossed F1 plants to a rgh3 /+ tester in the W22 backgroun d. The backcross from the B73 F1 segregated for the B73 like seedling phenotype in a 1:1 ratio. These data are consistent with a single dominant modifier. We are employing a map based cloning approach to identify the modifier gene. Using a set of 85 SSR ma rkers that are spaced throughout the genome and polymorphic between B73 and W22 we analyzed a BC3 mapping population in the W22 background. Preliminary results suggest that the B73 modifier maps to chromosome arm 9L. 71 Empirical estimates of migration r ate: case study of Yemen Miro Herrans A 1 ,2 Al Meeri A 3 Mulligan C 2 ,* 1 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2 Department of Anthropology, University of Florida, Gainesville, FL 3 Faculty of Medicine, Sana'a Uni versity, Sana'a, Yemen To understand human demographic changes, it is necessary to identify an appropriate value that describes the amount of human movement. Currently there is no empirical value that represents the amount of migration that occurs in deve loping countries. We collected five hundred (n=500) saliva samples with geographic information of the individuals, their parents and grandparents in Yemen and sequenced HVR1 of the mitochondrial DNA. We calculated the number of individuals whose birthplace was different from their mother's birthplace (n= 110) and father's birthplace (n=104), obtaining similar values for both. We calculated the number of haplotypes for the individuals (n=86) and the number of haplotypes for all samples (n=287). We determined the proportion of individuals who moved and the proportion of haplotypes that had moved based on those whose birthplace was different from their mother's birthplace. 72 Nerve dependent gene expression in the epidermis of regenerating salamander limbs M onaghan J R 1 ,2 Seifert AW 2 Voss SR 3 Gardiner VM 4 Maden M 1 2,* 1 The Regeneration Project, Evelyn F. and William L. McKnight Brain Institute, University of Florida, Gainesville, FL 2 Department of Biology, University of Florida, Gainesville, FL 3 Departmen t of Biology, University of Kentucky, Lexington, KY 4 Departme nt of Developmental and Cell Biology, University of California Irvine, CA Salamander limb regeneration is dependent upon an intact nerve supply and a specialized wound epidermis (WE) termed th e apical epidermal cap (AEC). Regeneration fails if either of these structures is removed from the injured limb as both structures are thought to supply growth cues to the regenerating tissue. Following limb amputation, nerves innervate the WE and induce g ene expression. To identify downstream targets of the nerve in the WE, we used microarray analysis to compare mRNA transcript abundances between three different types of WE: 1) the WE of regenerating forelimbs, 2) the WE of denervated regenerating forelimb s, and 3) the WE of healing flank wounds (which do not require nerves for repair). Tissues were collected from Mexican axolotls prior to injury and at 1, 3, and 7 days following injury. Using the new generation of axolotl microarrays gave us the potential to interrogate ~20,000 transcripts and as a result, many differences between the three types of WE were identified. Specifically, we found genes unique to the innervated WE of the regenerating limb, which make them prime candidates for signaling the mainte nance of proliferation in blastemal cells by the AEC. 73 Stress induced assembly of Daxx/ATRX complex at centromeres is SUMO 2 dependent Morozov VM Ishov AM Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL University of Florida Shands Cancer Center, Gainesville, FL Protein Daxx that is mostly resided at PML NB's/ND10 in normal conditions becomes accumulated at centromeres upon application of mild heat shock (HS) (42C). Mechanism of Daxx deposition requires functional Da xx SIM's and SUMO 2, but not SUMO 1, suggesting potential interaction of Daxx at centromeres with SUMOylated partner. This accumulation can be observed already at 30 minutes of HS, is reversed 1 hour after HS release and does not involve any changes in Dax x protein stability. Centromere distribution of Daxx can be also induced upon the inhibition of the proteosome dependent protein degradation by MG132 treatment suggesting degradation or de SUMOylation of putative Daxx interacting centromere partner as the potential mechanism of post stress release of Daxx. While dynamics of Daxx accumulation is similar throughout interphase upon short HS application, the extension of HS results in longer retention of Daxx at centromeres in S/G2 compared to G1 phase, suggest ing cell cycle dependent HS response. Among other tested PML NB's associated proteins, only ATRX (alpha thalassemia/mental retardation syndrome X linked chromatin remodeler) can follow Daxx re positioning. Moreover, both SUMO 2 and Daxx are required to ATR X accumulation at centromeres, suggesting formation of stress induced Daxx dependent chromatin remodeling complex at these intranuclear structures.
74 Identifying overlapping regulatory networks Morse AM McIntyre LM Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Assays for transcriptome profiling generate lists of significantly differentially expressed genes. One means to extract biological knowledge from these lists utilizes Gene Ontology ( GO ) enrichment. Along with biological knowledge, enrichment analyses can lead to hypotheses about underlying mechanism. However, genes connected in a regulatory pathway often do not share GO terms as the underlying premise of the GO is to describe function not interactio ns. Transcriptional pathways can be reconstructed algorithmically, but these exercises are often underpowered and lack incorporation of biochemical knowledge. Protein interactomes derived from biochemical pathways and experiments identifying protein intera ctions have been expanded into large interactomes. These structures are unwieldy and hard to use effectively for biological hypothesis generation. This study presents a method to identify smaller groups of genes involved in transcriptional regulatory proce sses. By integrating gene expression data with interactomes, sub networks responsible for transcriptional regulation can be identified. These specific sub networks are starting points for understanding the fundamental mechanisms involved in regulation of p athways. We demonstrate the utility of this approach by integrating published and newly generated gene expression data with an interactome network to inform our understanding of the role of jasmonic acid biosynthesis and signaling in the response of Arabid opsis to root knot nematodes. 75 Immune modulation of CRIM negative patients in enzyme replacement therapy for Pompe disease Nayak S 1 Kelley JS 1 Falk L 1 Lawson L 1 Harfe K 1 Collins S 1 Wasserfall C 2 Atkinson M 2 ,* Elder M 1 Byrne B 1 ,* 1 Department of Pediatrics, University of Florida, Gainesville, FL 2 Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL Infantile Pompe disease is due to the deficiency of enzyme % glucosidase (GAA). It is characterized by severe cardiac, skeletal muscle weakness and respiratory insufficiency resulting in death within one year of birth without treatment. Recombinant human GAA protein (rhGAA, Myozyme) is approved for ERT and prolongs life however a complication is the pro duction of high titer antibody (Ab) responses against the recombinant therapeutic protein occasionally threatening anaphylactic shock. Rituximab (an anti human CD20 Ab causing CD20+ B cell depletion), Rapamycin (an immune suppressant known to cause Teff de pletion and Treg induction) and immunosuppressant MMF have been used to minimize anti rhGAA Ab responses. Subject A received Rituximab induction followed by daily MMF accompanying twice a month rhGAA infusions. Anti rhGAA antibodies were initially at basel ine but appeared at 4 months and re ached a maximum titre of 1:8x10 6 by 11 months after treatment initiation, accompanied by elevated CD20 (2 55% CD20+) and CD19 (2 54%) levels. In contrast, Subject B and C receiving once in 3 months Rituximab and daily rap amycin accompanying the twice a month rhGAA infusions did not have detectable CD20 B cell levels and anti rhGAA antibodies remained at baseline for >18 months of treatment. 19 % CD19 B cells were observed at one timepoint in Subject B. NT proBNP and LV mas s improved in all patients. Immune modulation strategies improve patient outcome by inhibiting anti GAA antibodies. 76 Genome wide changes in inflammatory gene expression following lower extremity vein bypass O'Malley KA 1 Lopez M C 2 Hong MS 1 Coons EA 1 Baker HV 2,* Moldawer LL 1,* Nelson PR 1 ,2 1 Department of Surgery, University of Florida, Gainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Background: We studied systemic inflammation following v ein bypass surgery for peripheral arterial disease using a genome wide approach to gain insight into failure mechanisms. Methods: Blood was drawn pre operatively (PO), then two hours (2H) one day (1D), one week (1W) and one month (1M) in patients following vein bypass (N=20). Gene expression was analyzed using the Affymetrix GGH2 array platform. Unsupervised and supervised analyses were performed and genes with expression differences at p<.001 considered significant. Cross validation was performed using a l eave one out method. Changes in gene expression over time were analyzed using ANOVA, and Ingenuity Pathway Analysis (IPA) was used to investigate inflammatory pathways. Results: Unsupervised analysis of the dataset (34,834 probesets total) showed no signif icant overall clustering of gene expression. Supervised analysis based on time showed significant differences in 182 probesets between PO and 2H, and 462 probesets between PO and 1D. There were no significant differences between the PO and 1W and 1M timepo ints. ANOVA revealed 647 significant prob e sets overall (161 with FDR < .001) with a peak in differential expression centered around the 1D timepoint. IPA demonstrated significant changes in a variety of inflammatory pathways. Conclusion: Significant tempor al alterations in inflammatory gene expression are seen following lower extremity vein bypass surgery and may form the basis for future class prediction models.
77 Toward s genome wide association studies in forest trees Neves LG 1 Chamala S 2 Davis JM 1 ,3,* Barbazuk WB 1,2,* Kirst M 1,3,* 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 Department of Biology, University of Florida, Gainesville, FL 3 School of Forest Resources and Conservation, University of Florida, Gainesville, FL In contrast to previous association genetics approaches that focused on a limited number of candidate genes, advances in DNA sequencing are now beginning to allow genome wide association analysis in highly polymorphic species, such as lobl olly pine ( Pinus taeda ). We report our efforts to develop methods of genotyping based on sequence capture (Agilent's SureSelect) followed by high throughput sequencing (Illumina's GAIIx). To capture a defined genic portion of the pine genome, 55,000 120 me r sequence capture oligonucleotides were designed for 14,729 unigenes. Barcoded cDNA samples from 8 individuals were pooled and hybridized to the sequence capture oligonucleotides and sequenced in a lane of Illumina's GAIIx sequencer. An initial analysis o f the data separated two genotypes based on the barcode sequence to be further analyzed, with 7.6 and 3.6 million quality filtered reads each. Sequences were aligned to the reference EST dataset using MosaikAligner and SNPs were identified using GigaBayes. Mean depth of coverage was 93 and 55" for 9,463 and 9,080 unigenes, while 8,782 unigenes were captured in both genotypes, suggesting that capture is reproducible. A minimum of 1,917 SNPs segregated between the two genotypes, but the number increased consi derably when comparing to the EST consensus. We are currently expanding this approach to genomic DNA in pine and also poplar, where a new set of probes is being designed to capture the promoter region of the genes as well. 78 Molecular detection of rAAV genomes in blood of non human primates : development of a reliable blood test for the detection of gene doping Ni W 1 Le Guiner C 2 ,3 Penaud Budloo M 2 Moullier P 1 3 Snyder RO 1,2 ,4,* 1 Department of Molecular Genetics and Microbiology, University of Flor ida, Gainesville, FL 2 INSERM UMR649, Nantes Cedex, France 3 GENETHON, Evry, France 4 Department of Pediatrics, University of Florida, Gainesville, FL The goal of our project is to develop a test for the detection of the presence of a vector delivered trans gene encoding a protein capable of enhancing athletic performance. Several studies have shown that vectors derived from AAV are able to provide long term expression of a transgene after a single administration. It has been demonstrated especially after one intramuscular injection of a recombinant AAV vector that vector genomes are detectable in blood cells for several months up to 5 years. These results suggested that a sensitive PCR technology can be used to detect rAAV mediated gene doping from blood samp les. Our project consisted of a single intramuscular injection of macaques with two rAAV serotypes known for their efficiency to transduce skeletal muscle cells: rAAV1 and rAAV8. Various clinically relevant doses were tested. Each vector carried the expres sion cassette CMV cmEpo SV40 polyA that codes the cynomologous Erythropoietin gene. Our initial goal was to establish the minimal dose of vector that can cause an increase of approximately 15% of the hematocrit compared to the normal level. Once it was det ermined, an optimized PCR method was used to highlight the presence of the transgene in blood. We validated Taqman real time PCR sensitive conditions that allow us to detect at least 5 copies of the rAAV Epo transgene in the background of endogenous genomi c DNA. Data generated in our nonhuman primates will be the basis for developing a legally defensible commercial assay. 79 FGF2 targeted cationic lipid formulations for siRNA delivery Niu G 1 Hughes JA 2 Derendorf H 1,* 1 Department of Pharmaceutics, Univ ersity of Florida, Gainesville, FL 2 Roche, Nutley, NJ RNA interference (RNAi) based technologies offer an attractive strategy for the sequence specific silencing of disease causing genes. The application of small interference (si)RNAs as potential therape utic agents requires safe and effective methods for their delivery to the cytoplasm of the target cells. An FGF2 peptide ( MGLPLATC) targeted cationic lipid ( cholesteryl hemisuccinyl tris(aminoethyl) amine, CHSTAEA) system was developed for anti tumor siRN A therapy. The membrane fusion inhibitor, lyso phospholipid (ELPC) was included to increase the stability of siRNA lipoplex; hexagonal formers 1, 2 dioleoyl sn glycero 3 phosphoethanolamine (DOPE) and glycerol monooleate (GMO) were included for enhancing e ndosome escape, and FGF2 PEG DSPE could provide the long circle life a nd tumor targeting. The resultant non targeted formulation CDEG has a comparable gene silencing (96.32% 0.48% at 30 nM siRNA with charge ratio of 4:1) as the commercial reagent DharmaF ect01, while targeted FGF2 CDEG was even significantly higher than that. The cellular uptake and stability of siRNA in CDEG complex was higher than that in CHSTAEA/DOPE. The in vivo anti tumor study demonstrated that CDEG KIF11 (cell killing siRNA) and FGF 2 CDEG KIF11 treatment groups inhibited tumor growth compared to the PBS group. In conclusion, FGF2
targeted cationic lipid (CHSTAEA) system containing helper lipids ELPC, DOPE and GMO is an effective siRNA delivery system and provides a promising siRNA an ti tumor method. 80 VIVO: researcher networking and discovery across disciplines Norton HF 1 Garcia Milian R 1 Tennant MR 1,* Henning S 1 Conlon M 2 VIVO Collaboration 1 Health Science Center Libraries, University of Florida, Gainesville, FL 2 Clinical and Translational Science Institute, University of Florida, Gainesville, FL As research has become increasingly interdisciplinary, discovery of common research interests and complementary skills among potential collaborators across fields and institutions has become a priority. VIVO is a tool that facilitates this discovery process by offering detailed faculty profiles and revealing existing connections between researchers. Funded by the National Institutes of Health, VIVO is a semantic web tool for facult y profiles, research discovery, and researcher networking being developed by the University of Florida and six partner institutions. Profiles include publication and grant information, research and teaching interests, professional associations, and contact information; much of this data is ingested directly from authoritative data sources, such as institutional databases and publishers, requiring minimal effort on the part of researchers to fully populate the VIVO network. Although the VIVO platform is adop ted and implemented at an institutional level, it is designed to optimize discovery both at local and national levels. By highlighting particular use case scenarios, we aim to introduce genetics researchers to the features and benefits of VIVO and encourag e their use of this tool. In addition to demonstrating the potential of a fully developed VIVO platform, we will address the functionalities of the existing product. 81 The coding potential of the human genome: new insights from sequence compositional co ntrasts Oden SM Brocchieri L Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Gene coding genomic regions generally exhibit contrasting global compositional properties in the three codon positions, depending on the overall base composition of the sequence. General rules on the base content at the three codon positions as a function of the overall base content can be identified and exploited to score sequence regions for their coding potential. More generally, th e period three structure of coding regions imposes compositional periodicity to the sequence that, irrespective of the specific type of contrasts that we may expect to see, result in a significantly non random distribution of bases. Applying these principl es we have devised two algorithms to detect potential coding regions in sequences of any composition, one based on overall compositional expectations and one based on overall contrasts. We have applied our procedure to the human genome. We have detected in the human genome a plethora of regions, not overlapping with any of the currently annotated gene sequences, that display with high statistical significance a periodic structure often conforming to expectations for coding regions in terms of base type comp osition. The frequency of these regions is far higher than the random frequency observed in corresponding scrambled sequences. Most of these regions also show levels of complexity that distinguish them from repetitive elements and that are consistent with the complexity of known genes. 82 EASAL (efficient analysis, search and visualization of assembly landscapes) Ozkan A Sitharam M Pence J, Wang R, Peters J Department of Computer and Information Science and Engineering, University of Florida, Gainesv ille, FL It is a longstanding problem to efficiently and intuitively describe and predict the geometric structure and properties of high dimensional molecular assembly or packing configuration spaces. A satisfactory answer to this problem would: (i) deter mine the configurational entropy which determines free energy (ii) isolate those intermolecular interactions that are crucial for successful assembly pathways, which are usually heavily influenced by entropy considerations. A satisfactory method should sa tisfy the following requirements. (a) It should provide intuitive and explicit relationships between the input molecular data and the geometric properties of the configuration space; (b) It should provide quantitative accuracy guarantees derivable from the input data, including running time estimates; (c) Different modeling goals related to assembly require different levels of refinement during analysis, searching, sampling and visualization of configuration spaces. The effort spent should flexibly scale do wn at a lower refinement, but key features of the configuration space structure such as lower dimensional boundaries should not be missed: these often include highly probable regions of the configuration space. (d) Computational efficiency. EASAL software (to be opensource) is based on new theory and algorithms, and has been designed to satisfy (a), (b), (c), (d) and answer (i) and (ii). It is being tested on AAV virus assembly.
83 Lvis853 transcriptional regulator from Lactobacillus brevis participate s in sulfur compounds homeostasis Pagliai FA Fridman L, Brown SM, Lorca GL Department of Microbiology and Cell Science, University of Florida, Gainesville, FL In prokaryotes, gene regulation is primarily controlled by one component regulatory systems. A high throughput screening assay allowed the identification of small molecules that modulated the expression of Lvis853, a MarR transcriptional regulator. Lvis853 interacted with molecules that contain a common sulfur scaffold. Microbial oxidation of ino rganic sulfur compounds is governed by chemical and enzymatic reactions. In Lactobacillus brevis at least two of these enzymatic reactions may be catalyzed by Lvis852 and Lvis855 genes located in the genomic context of Lvis853. The modulation of Lvis853 a ctivity with sulfur related compounds was analyzed in vitro and in vivo By Electrophoretic Shift Mobility Assay (EMSA) we identified a binding region in the promoter of Lvis853 (PLvis853). Interestingly, EMSA results showed that some sulfur compounds impr oved Lvis853:PLvis853 interaction suggesting a novel co repression activity for this family of regulators. qRT PCR showed high expression of Lvis852, Lvis853, and Lvis855 transcripts at low concentrations of sulfate in the media. These combined results sug gest that Lvis853 may participate in the regulation of the sulfur related compounds levels in this bacterium. Currently, structure guided site directed mutagenesis is being performed. Based on a model of Lvis853 constructed using 3CDH from Silicibacter pom eroyi as a template, the residues involved in protein ligand interaction will be identified. ! 84 Effect of calcium on peanut ( Arachis hypogaea L.) pod and seed development under field conditions Pathak BP 1 Jain M 1 Gallo M 1,* Tillman B 1 Harmon AC 2,* Grusak MA 3 McKinney J 1 1 Agronomy Department, University of Florida, Gainesville, FL 2 Department of Biology, University of Florida, Gainesville, FL 3 USDA/ ARS Children's Nutrition Research Center Houston, TX Calcium (Ca) plays a significant role in peanu t seed development as an essential plant nutrient and a signaling molecule. Ca being xylem immobile, successful geocarpic development of peanut fruit is vulnerable to soil calcium levels. Previous studies have examined the effect of Ca on peanut seed devel opment during harvest. However, the stage at which developing seeds are most affected by a lack of Ca remains unclear. The effect of Ca on seed development of two varieties, C99R and Georgia Green, was studied under field conditions with low calcium soils. In half of the test plots, gypsum was applied at 30 and 60 days after planting for sufficient levels. Data were collected on pod length, seed and pod stage, fruit development, number of segments and number of seeds on each fruit. Pod length was not affect ed by calcium levels. However, Ca deficiency resulted in fewer two segmented pods, fewer fruit with two seeds, and more immature and aborted seeds. Ca is secondary messenger, coupling physiological responses to different stimuli. Several lines of evidence reiterate the important role for Ca and Ca dependent protein kinases (CDPKs) during seed development. Hence, CDPK expression was explored as a candidate sensor during peanut seed development. Quantitative RT PCR and Western blot showed spatiotemporal regul ation of CDPK expression during immature stages of seed development in both pod, as well as seed tissues. Seeds showed higher CDPK transcript and protein than pods in low soil Ca. 85 Identification of the enzymes involved in the last steps of archaeosine biosynthesis Phillips G 1 Chikwana MV 2 Maxwell A 1 El Yacoubi B 1 Swairjo M 3 Iwata Reuyl D 2 de Crcy Lagard V 1,* 1 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 2 Department of Chemistry, Portland State University, Portland, OR 3 Department of Basic Medical Sciences, Western University of Health Sciences, Pomona, CA Guanosines at position 15 of most archaeal tRNAs are modified to the 7 deazaguanosine derivative archaeosine (G + ). Although archaeosine modification h as been found in all branches of the archaeal phylogenetic tree, its biosynthetic steps have remained elusive. Comparative genomic analysis revealed that most archaeal genomes sequenced to date contain two gene families annotated as tgtA1 and tgtA2 and th at these genes cluster together in a number of genomes. tgtA1 encodes the experimentally characterized TGT enzyme, which catalyzes the insertion of the precursor 7 cyano 7 deazaguanine (preQ 0 ) into tRNA. It has been demonstrated that tgtA2 encodes a gluta mine dependent amidotransferase that catalyzes the conversion of preQ 0 modified tRNA to G + However, while the Crenoarchaeota possess a TGT homolog and have been shown to make G + some lacked TGTA2 homologs. Additional comparative genomic analysis revealed that these organisms possess either a QueC enzyme fused to a glutamine amidotransferase type II protein domain or a QueF like enzyme, suggesting that GAT QueC and QueF like enzymes might provide an alternate route for archaeosine biosynthesis. Genetic stu dies involved GAT QueC and QueF like showed that there are alternate routes in the synthesis of archaeosine in organisms lacking tgtA2
86 Plastid transformation supports high level expression of hyperthermostable GH10 xylanase Xyl10B for efficient conv ersion of hemicellulose to fermentable sugars Kim JY 1,2 Kavas M 1,2 Nong G 3 Fouad WM 1,2 Preston JF 3 Altpeter F 1,2,* 1 Agronomy Department, University of Florida, Gainesville, FL 2 Plant Molecular and Cellular Biology Program, University of Florida, Ga inesville, FL 3 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL Biofuel production from lignocellulosic biomass depends on technology that efficiently and economically releases fermentable sugars from multi polymeric cel l wall components. Xylan is after cellulose, the most abundant polysaccharide in grass and wood biomass and must be hydrolyzed to its component sugars (xylose or xylobiose) before fermentation to ethanol. Endoxylanases are the main enzymes involved in xyla n hydrolysis. Transplastomic production of cell wall degrading enzymes will reduce costs of enzyme production. The coding sequence of the hyperthermophilic xylanase xyl10B was isolated from genomic DNA of Thermotaga maritima and subcloned under regulatory elements supporting expression in the chloroplast genome. The partial rbcL and accD gene sequences flanked the xyl10B expression cassette and the selectable aadA expression cassette for site specific integration into the chloroplast genome, which was confi rmed by PCR using primer combinations annealing to the chloroplast genome and the transgene. Xyl10B represented 12% of the crude plant protein from transplastomic plants based on SDS PAGE, Western blot and enzyme activity assays. Sugar release was assayed from sweetgum xylan incubated with in planta produced recombinant Xyl0B at 85¡C by TLC and revealed that the majority of xylan was converted to fermentable sugars, suggesting that in planta expression of hyperthermostable Xyl10B will enhance conversion of hemicellulose to ethanol. 87 Genomic selection and next generation genotyping to hyper accelerate pine breeding Resende M 1 Jaramillo JJA 2 Resende MDV 3 Kirst M 1,2,* 1 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2 S chool of Forest Resources and Conservation, University of Florida, Gainesville, FL 3 EMBRAPA National Center for Forest Research Colombo, Para n Brazil Genomic Selection (GS) is a molecular based breeding method that uses information of widely distribu ted markers to track multiple associations that predict the genetic value of an individual. GS has immense value to hyper accelerate breeding of species with long generation times and that express traits of interest late in the breeding cycle. We performed a GS analysis in three clonal populations of loblolly pine ( Pinus taeda ) composed of 64 full sib families, genotyped for 3700 polymorphic SNPs and phenotyped for diameter at breast height (DBH) and total height. Markers significantly associated with those traits were used to estimate the genomic breeding value of each individual using a Genomic BLUP (Best Linear Unbias ed Prediction) procedure. The average realized selecti on accuracies obtained were 0.48 for height and 0.58 for DBH. Assuming that early sele ction can reduce the breeding cycle from 20 to 7 years, the selection efficiency gain of GS compared to pheno typic selection would exceed 100 %. These results can be further improved by adding markers in linkage disequilibrium with quantitative trait loci. For that purpose, a new genotyping method based on restriction digesting the genome followed by high throughput sequencing is being developed. This method can potentially generate hundreds of thousands of markers at very low cost, allowing for GS to become commercially viable in pine breeding programs. 88 A candidate gene for the coordinate regulation of biomass growth and carbon partitioning in Populus Ribeiro C L 1 Novaes E 2 Dervinis C 3 Kirst M 1,3 ,* 1 Plant Molecular and Cellular Biology Program, Univ ersity of Florida, Gainesville, FL 2 Agricultural Department, Universidade Federal de Goi s, Goi s, Brazil 3 School of Forest Resources and Conservation, University of Florida, Gainesville, FL Understanding the genetic regulation of wood productivity and it s chemical and physical properties is critical for developin g plant feedstock that is more s uitable for bioenergy. We identified one quantitative trait locus (QTL) on chromosome XIII of Populus with pleiotropic effects in biomass growth and composition. Th e QTL explains 56% of the heritable variation in cellulose to lignin ratio, as well as 20 25% of the heritable variation of several productivity traits. A genetical genomic analysis identified a previously uncharacterized genes, carbon partitioning and gro wth on chromosome 13 (cpg13), that is highly correlated with lignin (r=0.41) and cellulose (r= 0.41) levels, and moderately correlated with shoot biomass (r = 0.18), in a segregating population. Cpg13 is cis regulated and has several cis elements commonl y found in the promoters of monolignol biosy nthe sis genes, offering a preliminary explanation for its high expression correlation with other lignin biosynthesis genes (r = 0.79). Transcriptome analysis shows that cpg13 is most highly expressed in tissues u ndergoing secondary cell wall formation, like woody stems. Poplar plants transformed with cpg13 (RNAi, o verexpression and GFP fused) are currently being
analyzed to further understand the molecular role and regulation of cpg13. 89 Computational analysis of regulatory SNPs Marinescu VD 1 Riva A 2,* 1 Department of Neurobiology and Anatomy Unive rsity of Utah Salt Lake City, UT 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL MAPPER is a modular web based applicat ion for the analysis of Transcription Factor Binding Sites, based on an innovative methodology to predict putative TFBS in genomic sequences. The MAPPER system includes a collection of over 1,000 TFBS models, a database of precomputed TFBS in the promoters of all genes in the human, mouse and D rosophila genomes, and a search engine to scan multiple sequences in real time. We have extended MAPPER with a module to predict the potential effects of Single Nucleotide Polymorphisms on TF binding sites. This modul e, called rSNPs Mapper, automatically identifies putative binding sites that contain SNPs, and computes the difference in predictive score due to the different alleles, highlighting cases in which this change is over a set threshold. SNPs can be manually e ntered by the user, detected by comparing two alternative sequences, or downloaded from dbSNP. We will present examples of the use of rSNPs Mapper, and we will discuss how it can help in the functional analysis of SNPs by generating hypotheses on the relat ionship between genetic variation and gene regulation. We believe that this tool will be extremely useful for the interpretation of the results of genome wide association studies, by identifying genotype phenotype relationships that may elucidate the mecha nisms underlying complex diseases. 90 Evaluation of hammerhead ribozymes and siRNAs using a secreted alkaline phosphatase reporter system Robinson PM 1 Hauswirth W 2,* Lewin A 3,* Schultz G 4,* 1 Interdisciplinary Program in Biomedical Sciences, Universi ty of Florida, Gainesville, FL 2 Department of Ophthalmology, University of Florida, Gainesville, FL 3 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 4 Department of Obstetrics and Gynecology, University of Florida, Gainesville, FL Purpose: Profibrotic growth factors, transforming growth factor # 1 (TGF # 1 ) and connective tissue growth factor (CTGF) stimulate the production of haze in the eye after infection, surgery and trauma. Currently, there is no clinically avail able agent to selectively reduce the activity of TGF # 1 or CTGF. In this study, we investigate the use of gene silencing technologies, ribozymes and siRNAs that target these profibrotic transcripts to reduce both mRNA levels and protein levels of these gro wth factors. Methods: Targets for the siRNAs and ribozymes were ligated into a secreted alkaline phosphatase plasmid to test efficiency. One ribozyme to TGF b1 and one to CTGF were tested. Six siRNAs targeting CTGF or TGF # 1 were tested. Results: Both TGF # 1 and CTGF ribozymes showed at least 30% decrease in secreted alkaline phosphatase expression. Out of the six siRNAs tested for TGF # 1 two produced a reduction of secreted alkaline phosphatase of at least 40%. Out of the six siRNAs tested targeting CTGF, two produced a reduction of secreted alkaline phosphatase of at least 50%. Conclusion: These data indicate that topically applied ribozymes or siRNAs may be therapeutically useful in reducing levels of profibrotic growth factors. The development of the ta rget secreted alkaline phosphatase plasmid allowed for in vitro analysis of the siRNAs without the need to upregulate or induce the endogenous genes. 91 RPA1, a novel regulator of plant actin depolymerizing factor, is required for normal actin organizati on in trichomes Roney JC Grey PH, Zhang X, Dugas SM, Cuddy KK, Oppenheimer DG Department of Biology University of Florida, Gainesville, FL In plant cells, the actin cytoskeleton plays a central role in cell expansion through its effect on membrane tra fficking. The key to this process is the rapid assembly and disassembly of actin filaments by the interactions of filaments and monomers with various other proteins. Actin depolymerizing factor/cofilins (ADFs) are a family of proteins that play a central r ole in the regulation of actin dynamics by severing actin filaments and binding actin monomers. Although ADFs are key players in actin dynamics, the mechanisms of ADF regulation in plant cells remain poorly understood. Here we show the impact a novel R egul ator of P lant A DF (RPA) has on the actin organization in plant trichome shape mutants. Fluorescence confocal microscopic analysis of the actin cytoskeleton in rpa1 mutants shows grossly aberrant F actin distribution in developing trichome cells. In the lat er stages of development, we observed the presence of an "actin knot" with many actin rings near the nucleus, suggesting that the absence of the RPA1 protein leads to an over accumulation of F actin. Our findings demonstrate the importance of the RPA1 prot ein in the actin organization in plant trichome cells.
92 Development of bioenergy sorghum for Florida Saballos A Erickson J, Vermerris W Agronomy Department, University of Florida, Gainesville, FL Due to i ts sunny and warm climate, the s tate of Florida has the potential to become a leader in bioenergy production. Sorghum can supply high yields of food, feed, and feedstocks for energy even in harsh conditions such as hot and dry climates, poor soils, and limited inputs. However, current varieti es and hybrids of sorghum are poorly adapted to the challenges presented by the sandy soils and the pervasive fungal pathogens of the area. We are working towards the improvement of sorghum as a bioenergy crop by exploiting the genetic diversity of the spe cies, including sweet and grain sorghums and high biomass sorghums with altered lignin composition. We are investigating the physiological and anatomical traits that confer enhanced drought tolerance to some genotypes, including root architecture, photosyn thetic resilience and osmoprotectant solute concentration. Genomic and genetic approa ches are being used to study the basis of sugar accumulation and its relationship with grain and biomass production. Population development for mapping of anthracnose resi stance genes present in a UF developed set of sorghum lines is underway. The combination of QTL, gene expression and physiological studies will ultimately allow us to identify genes underlying traits for successful bioenergy production. This knowledge will facilitate the creation of lines and hybrids with high yield and good bioprocessing characteristics, adapted to the stresses in their area of production. 93 Therapeutic expression of FIX at reduced doses using AAV protein phosphotase 5 helper virus Sac k BK Giridhara JR, Li Z, Herzog WR Srivastava A Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida, Gainesville, FL One obstacle for single stranded vector transduction is the need convert the ssDNA to dsDNA b efore transcription. Our work and the work of others has shown that a protein, FKBP52, is a key inhibitor of this rate limiting step and that dephosphorylation of this protein by phosphotases such as PP5 can augment gene therapy. Co injection of a self com plementary scAAV2 PP5 vectors results in a 16 fold increase in EGFP expression in murine hepatocytes in vivo We optimized this strategy to achieve transgene expression at reduced vector/helper virus doses by using hepatocyte specific promoters and alterna te AAV serotypes to achieve a further 16 fold increase in EGFP expression. We expanded this protocol to a therapeutic protein by injecting C57BL/6 mice i.v. (n=4/group) with ssAAV2 carrying hFIX (ssAAV2 hFIX) along with scAAV8 TTR PP5. Expression levels w ere measured by plasma concentration of hFIX as determined by ELISA and confirmed by immunofluorescent staining of hepatocytes for hFIX. Coadministration of 1e10 (or 1e9) vp/mouse scAAV8 PP5 helper virus and 1e10vp/mouse ssAAV2 hFIX therapeutic vector incr eased circulating hFIX by 7 10 fold 8 weeks post injection. Consequently, therapeutic levels of hFIX (4 5% of normal) were achieved but not at a higher dose of ssAAV2 hFIX. No hepatotoxicity was observed under any of these experimental conditions. This mod el is ideal for transgenes that exhibit poor expression and is currently being applied to a mouse model of hemophilia A. 94 Photomorphogenic development in strawberry ( Fragaria spp.) Salama DY 1 Folta KM 1,2,* 1 Horticultural Sciences Department, Univers ity of Florida, Gainesville, FL 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Light modulates many aspects of plant growth and development, including traits of horticultural interest such as flowering time, yield an d ripening. Studies in the model system Arabidopsis thaliana have defined a set of photosensors and their associated pathways that connect environmental signals to plant responses. Translational studies are now testing how these signaling pathways function in crop plants. Strawberry ( Fragaria spp.) is a plant of high economic value that shares many seedling attributes with Arabidopsis Similar to Arabidopsis strawberry is a small statured plant that is easily transformed and cycles rapidly from seed to see d. Strawberry is also a member of the Rosaceae Family, a valuable group of fruit, nut and ornamental crops. The goal of this work is to test the roles of light sensors in early photomorphogenic development in strawberry seedlings, and compare the responses to those of Arabidopsis The results show that strawberry seedlings are sensitive and responsive to light and maintain early stem growth inhibition and phototropic kinetics that are remarkably similar to those of Arabidopsis Unlike Arabidopsis strawberr y seedlings do not exhibit a typical far red block in greening. These findings provide a baseline for study of strawberry photomorphogenic mutants and possibly allow assessment of seedling phenotypes to predict economically important light associated behav iors in mature plants. 95 Valpr oic acid sensitive Gadd45alpha controls postmigratory neuronal differentiation Sarkisian MR Siebzehnrubl D Department of Neuroscience, University of Florida, Gainesville, FL Evelyn F. and William L. McKnight Brain Insti tute, University of Florida, Gainesville, FL
Valproic acid (VPA) is an anti epileptic and mood stabilizing drug commonly administered to pregnant women and young children. Despite an unclear mechanism of action, VPA can induce expression of growth arrest and DNA damage 45 alpha (Gadd45 % ) in cell lines. Here we show that Gadd45 % is expressed in developing mouse and human cortex and that either increases or decreases in the levels of this protein significantly impair neuronal morphology. Notably, exposure of neonatal mice to VPA significa ntly increases Gadd45 % levels in developing cortex that are accompanied by abnormal outgrowth of neuronal dendrites. Our results show that Gadd45 % plays an essential role in neuronal differentiation and that drug induced changes in the expression of this p rotein may cause undesirable changes in neuronal circuitry. 96 Skin regeneration in the Mexican axolotl Seifert AW 1 Monaghan J 1,2 Maden M 1,2,* 1 Department of Biology, University of Florida, Gainesville, FL 2 The Regeneration Project, Evelyn F. and Wil liam L. McKnight Brain Institute, University of Florida, Gainesville FL A major goal of wound healing research has been to develop therapies that replace wound repair and scarring with perfect regeneration of injured skin. At present, the only vertebrate models for scarless healing are late term fetal rodents and chick embryos. In vertebrates capable of epimorphic regeneration, an outstanding question is whether excisional wounds made outside of epimorphic regeneration fields heal perfectly or result in a fibrotic scar. In order to develop a model of adult skin regeneration in vertebrates we investigated this question in Ambystoma mexicanum (the axolotl) and show that adult axolotls are capable of completely regenerating all layers of the integument and ep idermally derived organs in full thickness flank wounds. We address the effects of an aquatic habitat on wound healing by analyzing wound repair in metamorphic axolotls (which are fully terrestrial) and show that they are also capable of complete skin rege neration. We indentify the classically defined phases established for wound repair in mammals and demonstrate that provisional matrix deposition is delayed in salamanders, which contributes to regeneration in favor of scarring. Given the emergence of the a xolotl as an emerging model organism in development and regeneration we believe our model of adult vertebrate skin regeneration will be a useful tool in developing novel therapeutic strategies to address scarring in mammals. 97 Lentiviral vectors car rying dual promoters and 2A cleavage peptides permit targeted delivery of multiple proteins to neural retina Semple Rowland SL 1 ,* Coggin WE 1 Geesey M 1 Hochman M 1 Abraham L 1 Walling E 1 Zorrilla A 1 Ludlow R 1 Verrier JD 1 Smith WC 2 1 Department of N euroscience, University of Florida, Gainesville, FL 2 Department of Ophthalmology, University of Florida, Gainesville, FL Treatment of inherited photoreceptor diseases will require multi protein therapies. To meet this challenge, we examined the expression of lentiviral vectors harboring dual promoters or 2A cleavage peptides to find those exhibiting reliable, targeted expression of their fluorescent protein cargos in neural retina. Lentivectors were built using either photoreceptor or glial promoters (XOPS 1.3, mOP500, mIRBP156, RK, mNRLL, mVIM, mCD44, and mGFAP), packaged, and injected into chicken embryos. The activities of the promoters alone, in duplicate, or when paired with a different promoter or 2A cleavage peptide were analyzed using fluorescent mic roscopy and western blot. IRBP156, NRLL and RK were active in cones and rods while XOPS1.3 was active only in rods. Of the glial promoters, only GFAP activity was restricted to Muller cells; both VIM and CD44 were active in neural as well as Muller cells. Dual promoter vectors carrying either IRBP156 and RK or XOPS1.3 and mOP500 exhibited robust expression of both reporter proteins in cones and rods or rods only, respectively. Expression of the upstream protein was lower than the downstream protein in vecto rs carrying two copies of either the RK or the IRBP156 promoter and in a vector carrying the CD44 and VIM promoters. Vectors carrying 2A cleavage peptides produced two proteins in targeted cells. Our results show that two therapeutic proteins can be delive red to targeted cells using dual promoter vectors or 2A cleavage peptides. 9 8 Evolving spiking neural networks for predicting transcription factor binding sites Sichtig H Riva A Department of Molecular Genetics and Microbiology, University of Florid a, Gainesville, FL Interdisciplinary problem solving initiatives of the post genomic era will require a fundamental understanding of complex biological adaptive systems, from cellular to molecular level in order to tackle intricate problems such as neurod egenerative diseases. In this work we present a description and an initial evaluation of a Spiking Neural Networ k Genetic Algorithm (SNN GA) system we are developing for the computational prediction of transcription factor binding sites (TFBS) in bioinform atics research. Th e SNN GA approach is based on modeling
information processing of biological neurons and evolutionary processes. The goal of our work is to reduce the number of false positives in the predicted TFBSs, through a more precise modeling of inf ormation contained in the alignments in the training data. We show an evaluation of four proposed network topologies to represent TFBS data and their training. We used real TFBS data from the TRANSFAC database and appropriately generated negative samples. We evaluated the network topologies for two well known models for transcription factors: ZID and RSRFC4. Benchmark performances for these models are given using MAPPER and MATCH. The results show that our method has the potential to attain very high classi fication accuracy. 99 Genetic analysis of early adventitious root development in a pseudo backcross population of Populus Silva CM 1 Novaes E 1 Boaventura Novaes CRD 1 Dervinis C 1 Drost DR 2 Acosta JJ 1 Osorio L 1 Kirst M 1, 2,* 1 School of Forest Resour ces and Conservation, University of Florida, Gainesville, FL 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL The ability to form adventitious roots (AR) from stem cuttings is c ritical for propagation of tree species t hat are cultivated for bioenergy, pulp and paper, and timber production. However, genes and molecular mechanisms that control AR formation are largely unknown. To identify quantitative trait loci (QTLs) that regulate AR development in poplar, a tree model species, we measured rooting response in a pseudo backcross population derived from a cross between a hybrid female parent of Populus trichocarpa Populus deltoides and an unrelated male parent P. deltoides Apical cuttings were collected from 234 indivi duals and grown in a hydroponic system for 18 days. The number of ARs was recorded daily. After 18 days, roots were harvested and scanned to measure length, volume, and surface area of total, primary (first order roots), and root braches (second and third order roots), as well as dry biomass and average diameter. QTLs for most of these traits mapped consistently in two regions of linkage groups II and XIV. Next, gene expression was characterized with whole transcriptome microarrays in six genotypes with alt ernative al leles in both QTL intervals, at 0, 1, 2, 4 and 8 days after establishment in hydroponic culture. This study enabled the detection of significant differences in transcript abundance across time points and between extreme genotypes, and also co lo calization of differentially expressed genes between extreme genotypes and QTL intervals, which are candidate genes that control number of ARs. 100 Exploring a replicative mechanism for rAAV vector genome maintenance Smith JK 1 Penaud Budloo M 2 Moull ier P 1 3 Snyder RO 1, 2,4,* 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 INSERM UMR649, Nantes Cedex, France 3 GENETHON, Evry, France 4 Department of Pediatrics, University of Florida, Gainesville, FL Gene therapy is quickly becoming the front runner for the treatment of many inherited genetic diseases, such as cystic fibrosis and muscular dystrophy. Several viruses have been developed as vectors for gene transfer, including Adeno Associated Virus (AAV), a small, non pathogenic ssDNA virus that infects both dividing and non dividing cells. Recombinant AAV (rAAV) vectors have been used in multiple phase I and II clinical trials; however, there are still several aspects of the biology of the vector that have y et to be investigated. It has been demonstrated in animal studies that the rAAV genome and transgene expression persists in muscle for years. Several factors could contribute to the vector genome's long term stability, including a possible "maintenance lev el" of DNA replication. To investigate this, an assay using Bromodeoxyuridine (BrdU), a synthetic thymidine analogue, to detect newly synthesized DNA will be validated in vitro by Southern blot analysis, and Immunoprecipitation and qPCR. Once validated, th e assay will then be used to determine if active replication can be detected in vivo in animals following long term transduction. It has been previously shown that rAAV vectors exist as episomal circles in muscle, and this type of analysis may give insight into the origination and generation of monomeric and concatemeric circles, and the maintenance of rAAV vector genomes in vivo 101 Tafazzin knockdown leads to mitochondrial and cardiac abnormalities Soustek M S 1 Falk DJ 1 Lewin A S 2,* Byrne B J 1,* 1 De partment of Pediatrics, University of Florida, Gainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Barth s yndrome is an X linked mitochondrial disease that is associated with mutations within the Taf azzin (TAZ) gene and is characterized by cardiomyopathy, neutropenia, and muscle weakness. Much of the molecular mechanism behind TAZ deficiencies has been gained from the study of Barth patient cells, and from manipulation of yeast, fruit fly, and zebrafi sh models of BTHS. While these models have been valuable tools in understanding the effects of tafazzin mutations on cardiolipin metabolism, the cardiovascular and immune systems of these models differ
vastly from that of mammals. Recently, Taconic Artemis generated a doxycycline inducible shRNA mediated Tafazzin knockdown mouse model; however, these mice have yet to be characterized. At 2 months of age using real time quantitative RT PCR analysis, we report that induced shRNAs result in >90% knockdown of T AZ mRNA in both heart and skeletal muscle tissue. In addition, cardiac mitochondria exhibit a reduction in cardiolipin and an accumulation of monolysocardiolipin species. Cardiac measurements made via magnetic resonance imaging revealed that TAZ deficient mice have a 20% reduction in ejection fractions when compared to control mice at 10 months of age. In conclusion, this inducible TAZ deficient murine model exhibits th e clinical phenotypes of Barth s yndrome patients and may ultimately help to improve our u nderstanding of BTHS related cardiomyopathy. 102 Dosage dependent genes affecting seed composition or weight Spielbauer G 1,2 Armstrong P 3 Baier J 1,2 Maharaj K 4 Richardson K 4 Grijalba D 1,2 Griffin M 1,2 Koscik K 1,2 Song B 5 Kahveci T 5,* Settles A M 1,2,* 1 Horticultural Sciences Department, University of Florida, Gainesville, FL 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 3 Grain Marketing and Production Research Center, Agricultural Research Service, U.S. De partment of Agriculture, Manhattan, KS 4 Department of Biology, Florida Agricultural and Mechanical University, Tallahassee, FL 5 Department of Computer and Information Science and Engineering, University of Florida, Gainesville, FL Kernel composition is an important target for developing improved grain for food, feeds, and industrial processes. In addition, genes affecting individual seed weight may have an impact on grain yield. Our goal is to identify maize mutants that have significant effects on the che mical composition or weight of seeds. We are screening the UniformMu transposon tagging population using single kernel near infrared spectroscopy (NIR) and seed weights. NIR spectroscopy determines chemical composition and seed weight phenotypes of individ ual maize kernels non destructively and at high throughput. We built an automated single kernel NIR grain analyzer and developed partial least square (PLS) calibration models to predict the individual seed starch, protein, oil, and weight. We have develope d a seed spectra and weight database to identify maize ears segregating for differences. We are focusing on phenotypes that show dosage dependent or parent of origin changes to the kernels based on the hypothesis that these genes are the best targets for m odifying the seed with transgenes. A systematic screen starting with 4,016 UniformMu families resulted in 8 candidates with differences in seed weight or composition. The candidates are phenotypically analyzed to further examine compositional differences. Finally, we have developed 454 sequencing protocols to identify the transposon insertion sites in UniformMu mutants. We are screening these insertion sites for linkage to dosage effect mutants. 103 Development of D[+] sequence deleted single stranded AAV 2 vectors: enhanced transgene expression in vitro and in vivo Ling C 1, 2 Jayandharan GR 1,3 Aslanidi GV 1,3 Li B 1,3 Lu Y 1,3 Cheng B 1,3 Ma W 1,3 Srivastava A 1 4,* 1 Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Flor ida, Gainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 3 Powell Gene Therapy Center, University of Florida, Gainesville, FL 4 University of Florida Shands Cancer Center, Gainesville, FL We have previo usly reported that a host cell protein exists, which interacts with the single stranded D[+] sequence in the right inverted terminal repeat (ITR) of the AAV2 genome (Qing et al., 1997, Proc Natl Acad Sci USA 94(20):10879 84). More recently, following purif ication and mass spectrometry, we found this protein to have partial amino acid homology to a cellular NF B repressing factor, a negative regulator of transcription. A recombinant ssAAV2 genome (ssAAV2 LC2 hrGFP) was constructed, in which the D[+] sequence was replaced by a non AAV2, substitute ( S) sequence in the right ITR. In i n vitro assays, the D[+] seque nce deleted ssAAV2 LC2 hrGFP vectors transduced human cells up to ~8 fold more efficiently than the conventional ssAAV2 hrGFP vectors. In mouse livers in vivo whereas no expression was observed in mock injected animals, and the transduction efficiency of conventional ssAAV2 hrGFP vectors was low, significantly higher numbers of hepatocytes were transduced by the D[+] sequence deleted ssAAV2 LC2 hrGFP vectors, and the extent of transgene expression was ~8 fold higher. These data support our hypothesis that a putative cellular transcription repressor factor exists, which suppresses transgene expression from ssAAV2 vectors, and suggest that removal of the D[+] sequence from ssAAV2 vectors should lead to more robust transgene expression. These studies have impl ications in the potential use of D[+] sequence deleted ssAAV vectors in human gene therapy. 104 Evolutionary dynamics of simian immunodeficiency virus (SIV) quasispecies infecting CD8 depleted Rhesus macaques Strickland S 1,2 Gray R 1,2 Nowlin B 3 Lowe A 1 Nolan D 1,2 Burdo T 3 Goodenow M 1 ,* Midkiff C 3 Alvarez X 3 Williams K 3 Salemi M 1,2 ,* 1 Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL
2 Emerging Pathogens Institute, University of Florida, Gainesvi lle, FL 3 Biology Department Boston College, Chestnut Hill, MA The progression of SIV infection in experimentally infected rhesus macaques follows a similar course to that of human immunodeficiency virus (HIV). We analyzed the phylogenetic relationship o f 354 viral sequences derived from plasma taken 21 118 days post infection from six CD8+ T lymphocyte depleted SIV infected macaques and 368 sequences derived from the inoculum (SIVmac251 viral swarm). This model was employed because of the rapid developme nt of AIDS and severe SIV encephalitis, a macrophage mediated disease. Nucleotide diversity was calculated and phylogenies were inferred for the viral swarm and each macaque. No evidence of a transmission bottleneck was observed in the peripheral virus. Th e overall genetic diversity in the monkeys was comparable to the inoculum. However, two haplotypes accounting for less than 5% of the inoculum sequences were present in at least 3 monkeys. A distinct motif in V2, previously described as macrophage tropic w as detected in the plasma at a low frequency (0.3%). Although not seen in the viral swarm, it was observed at high frequencies (>30%) in an additional 732 sequences from tissue macrophages collected at later time points from four macaques, only one of whic h showed macrophage accumulation diagnostic of SIVE. This study demonstrates the utility of the rapid AIDS macaque model for understanding the intra host evolutionary dynamics of SIV and the emergence of viral variants associated with macrophage associated inflammatory processes. 105 The role of Mini me and Grass genes of maize in establishing grass architecture Tan S 1 Koch KE 1,2,* McCarty DR 1,2,* Vermerris W 1,3,* 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 Horticultural Sciences Department, University of Florida, Gainesville, FL 3 Agronomy Department, University of Florida, Gainesville, FL A better understanding of the mechanisms controlling plant growth and development can ultimately be used to increase cr op yield, enable adaptation to variable environments, and allow novel uses for existing crops. The maize mini me and grass mutants display extreme dwarf phenotypes. We hypothesize the Mini me and Grass genes in establishing grass architecture. The mutants were identified in the Uniform Mu population and likely result from a Mutator transposon insertion. Transposon tagging is being used to clone the genes. Once cloned, identification of gene function and analysis of gene expression across a diverse set of gra sses may shed light on the role of these genes in establishing plant architecture. 106 Membrane localization of a novel regulator of actin dynamics in living plant cells Tang CW Grey PH, Oppenheimer DG Department of Biology, University of Florida, Ga inesville, FL Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Plant cell shape is dependent on membrane trafficking, which in turn, is dependent on the actin cytoskeleton. The actin cytoskeleton is made up of actin fil aments and actin binding proteins that regulate actin dynamics the polymerization and depolymerization of actin monomers. A key regulator of actin depolymerization is the aptly named actin depolymerizing factor/cofilin (ADF) family of proteins. Our lab rec ently discovered a novel, plant specific protein that regulates the function of ADF that we named RPA for R egulator of P lant A DF. A bioinformatic analysis of the RPA family revealed that some family members have a putative signal sequence at their N termin us. Usually signal sequences are found on proteins destined for the endoplasmic reticulum, and allow the proteins to be sorted to other organelles in the cell or secreted. However, since actin is usually not contained in organelles or secreted, I am testin g the hypothesis that the putative signal sequence is actually a transmembrane domain that targets RPA proteins to specific membrane compartments. Using the plant, Arabidopsis thaliana I am attaching a yellow florescent protein (YFP) to RPA11, one of the members of the family that has a putative signal sequence/ transmembrane domain. Confocal microscopy will be used to observe the YFP tag and localize the attached RPA11. Identification of the membranes to which the tagged RPA11 is localized will be done us ing co localization with organelle specific markers. 107 Direct embryogenesis from immature leaf explants supports rapid production of transgenic plants from a commercially important sugarcane cultivar Taparia Y Gallo M Altpeter F Agronomy Departme nt, University of Florida, Gainesville, FL Plant Molecular and Cellular Biology Program University of Florida, Gainesville, FL Our strategy to improve the genetic engineering protocol for a commercially important sugarcane cultivar (CP 88 1762) is aiming at reducing the time in tissue culture by eliminating a callus phase. This may positively affect the transformation efficiency and the performance of the transgenic plants. A factorial comparison of alternative auxin sources (1 naphthalenacetic acid; 2,4 dichlorophenoxyacetic acid, 4 amino 3,5,6 trichlorophyridine 2 carboxylic acid, 4 chlorophenoxy
acetic acid) and cytokinin concentrations (6 benzylaminopurine at 0.4 or 4.0 $ M) during culture initiation from immature leaf roll transverse sections, allowed the identification of a superior culture medium for induction of direct embryogenesis. Biolistic transformation of leaf roll disk explants of the commercially important cultivar CP 88 1762 was carried out 5 10 days following culture initiation with the sel ectable nptII expression cassette. The optimization of pre culture time before bombardment, particle size, acceleration pressure and subculture scheme supported a drastic reduction of the tissue culture period compared to a protocol that involves gene tran sfer to callus. Use of minimal expression cassettes and lower DNA quantities were explored to obtain simpler integration patterns. Transgenic plants were confirmed by ELISA and Southern blot analysis. We will provide details on the optimized protocol that supports producing stably transformed sugarcane plants in soil within 3 months or less time. 108 Is there a deeply conserved genetic program for cartilage development? Fibrillar collagens in invertebrates Tarazona OA 1 Cohn MJ 1 3,* 1 Department of Biolo gy, University of Florida, Gainesville, FL 2 Howard Hughes Medical Institute, University of Florida, Gainesville, FL 3 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Collagen based cartilage was long considered to be unique to jawed vertebrates, but recent work has revealed this to be a shared character of the vertebrate crown group. Phylogenetically fibrillar collagens (FCs) form three major clades (A, B and C). Clade A FCs is the most abundant protein in the extra cellular matrix (ECM) of cartilage. Clade A FC genes have been discovered in invertebrate relatives of vertebrates (i.e. amphioxus), and its domains of gene expression reveals that they contribute to the formation of the ECM of the endoskeletal tissues. Ca rtilage is also found in distantly related invertebrates, such as mollusk and arthropods, which were believed to lack FC genes due to their absence from the genomes of select model organisms. However, biochemical and ultrastructural studies suggested the p resence of FCs in the cartilage of squids. We tested the hypothesis that invertebrate and vertebrate cartilages develop using conserved developmental genetic mechanisms using degenerate RT PCR to isolate two FC clones from the cephalopod Sepia pharaonis an d the horseshoe crab Limulus polyphemus The two clones for each species correspond to the C propeptide. Bayesian phylogenetic analysis places them within the Clade A FCs. We examined their expression patterns in cephalopod and horseshoe crab embryos and f ind that they are expressed during cartilage development. The results raise the possibility of a deeply conserved genetic program for chondrogenesis in the bilateria. 109 Mechanisms of mutagenesis mediated by transcription Beckett J, Broxson C, Tornalett i S Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL Genomic DNA sequences capable to assume non canonical DNA secondary structures have been recently shown to be particularly susceptible to genetic instability, an early co ntributing factor in human disease and cancer development. Transcription appears to have a central role in the mutagenesis associated with these sites. However, little is known about how non canonical DNA structures are processed when encountered by the tr anscription machinery. G quadruplex (G4) DNA is a non canonical four stranded DNA structure that can form in G repeats by stacking of planar arrays of four hydrogen bonded guanines called G quartets. Here we show that a well characterized G4 DNA forming se quence contained in the human c myc proto oncogene is a strong block to T7 RNA polymerase (T7 RNAP) transcription under physiological concentrations of KCl. Direct evidence of G4 DNA formation was obtained by di methyl sulf ate footprinting. Consistent with the presence of G4 DNA in the transcription substrate, mutations aimed at destabilizing this G4 DNA structure correlate with a decrease in the extent of transcription arrest at the c myc sequence. The presence of an abasic site or of an 8 oxoguanine in thi s structural environment similarly affected the transition from duplex to quadruplex structure. These results will be discussed in view of current mechanisms of non B DNA induced mutagenesis. (Supported by Bankhead Coley New Investigator Grant 08BN 07) 1 10 Independent evidence for the parrot passerine clade Wang N 1,2 Braun EL 1,* Kimball RT 1,* 1 Department of Biology, University of Florida, Gainesville, FL 2 MOE Key Labo ratory for Biodiversity Science and Ecological Engineering, Beijing Normal Universit y, Beijing, China Relationships among the major groups of birds have been suggested to be resolvable based on recent large scale nu clear phylogenies. One fascinating finding is the relationship built between parrots (Psittaciformes) and passerines (Passer iformes). Since Passeriformes is the most speciose avian order, with >50% of avian species, their sister group is of major interest to evolutionary biologists. However, previous alternative hypotheses based on morphological or mitochondrial data suggest th at passerines are members of a clade that include s woodpeckers, kingfishers, and allies (Piciformes and Coraciiformes) and potentially other "higher land birds". We examine these alternatives using 30 novel nuclear
introns within 28 taxa sufficient to test the a priori plausible hypotheses. However, due to the difficulty of alignment and potential phylogenetic noise that resulted from rapidly evolving sites, we generated multiple alignments to examine such issues by using guide trees based upon alternative phylogenetic hypotheses, and by using noise reduction strategies like RY coding. We found no evidence of alignment bias, but noise reduction did have an impact on mousebirds (Coliiformes), a higher land bird group that appeared to be a "ro g u e taxon". These analyses strongly corroborated previous large scale nuclear phylogenies and provided independent evidence for the parrot passerine clade. 111 The possibility of hybridization between Terpsiphone viridis and Terpsiphone rufiventer Wildes CN Kimball RT Department of Biology, University of Florida, Gainesville, FL Males of two African species of paradise flycatchers, Terpsiphone viridis and T. rufiventer can be distinguished due to differences in tail length ( T. viridis exhibits extremely long tail s treamers, while tails of T. rufiventer are more typical) and breast color (gray in T. viridis and red in T. rufiventer ). Individuals have been found that incorporate morphological features of both T. viridis and T. rufiventer leading to hypotheses of hybri dization occurring where the two ranges overlap. However, natural plumage variation, particularly in coloration, is extensive within species of this genus making it difficult to differentiate hybridization from natural variation using plumage alone. I hypo thesize that T. viridis and T. rufiventer are hybridizing, resulting in these intermediate forms. To test this, I used microsatellite genotyping and sequencing (nuclear and mitochondrial) to identify individuals that might be the result of hybridization. 112 Discontinuous genotypic determinants in human immunodeficiency virus type 1 subtype B envelope regulate CXCR4 dependent viral entry into host cells Williams WB 1 Chang K 1 Lowe A 1 Pomeroy S 1 Appelberg S 1 Sleasman JW 2 ,3 Goodenow MM 1,* 1 Departmen t of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 2 Division of Pediatric Allergy, Immunology, and Rheumatology, Department of Pediatrics, University of South Florida St. Petersburg, Florida 3 All Children's Hospital St. Petersburg, Florida Tropism by viruses using CXCR4 coreceptor for entry into peripheral blood mononuclear cells (PBMCs) and macrophages (MDMs) (D X4) is associated with disease progression. Genotypic determinants for CXCR4 usage by D X4 viruses, par ticularly on macrophages that serve as HIV 1 viral reservoir, remain unclear. To track the genetic and functional evolution of X4 using Envelopes (Envs) during the natural history of HIV 1 infection, HIV 1 envelopes ( env ) were amplified from longitudinal PBMC proviral DNA or plasma viral RNA from seven HIV 1 infected subjects prior to and following antiretroviral therapy. Bioinformatic algorithm analysis of the Envs indicated that X4 Envs emerge prior to CD4% decline, R5 and X4 Envs coexist prior to therap y, and X4 Envs constitute the dominant viral population post therapy. Env functional studies showed that R5 or X4 viruses coexisted with R5X4 viruses, consistent with analysis based on genotype. Primary Envs entered PBMC equally well, but entered MDM less efficiently, compared to standard Envs. Site directed mutagenesis studies of specific amino acid residues that altered charge or complex architecture in env V1 domain significantly modified viral entry, indicating that residues in env V1V2 modify use of co receptor complex. Identification of discontinuous genotypic determinants for viral entry in HIV 1 env gp120 will provide novel targets for entry inhibitors, and epitopes for vaccine design to elicit neutralizing antibodies to block viral entry into host ce lls. 113 Consequences of inadequate SOCS1 expression on autoimmunity and immune homeostasis Wilson T Larkin J Department of Microbiology and Cell Science, University of Florida, Gainesville, FL The major goal of the immune system is to maintain immune homeostasis. Upon activation, naive CD4+ T cells differentiate into distinct T cell subsets, which play an essential role in maintaining homeostasis. T cell differentiation is largely dependent on the cytokine environment. In the presence of TGF beta, CD4 +CD25 (CD25 ) cells begin to express the transcription factor Foxp3, the key marker for regulatory T cells (Tregs). However, the combination of IL 6 and TGF beta induces CD25 T cells to become IL 17 producing cells (Th17). T cell differentiation is also dependent upon the cell surface and intracellular proteins that permit the cell to perceive and respond to these cytokines. Suppressor of cytokine signaling 1 (SOCS1) is a member of a family of intracellular proteins that are negative regulators of cytokin e signaling. However, the role SOCS1 plays in T cell differentiation is unknown. It has been shown that the inadequate expression of SOCS 1 leads to systemic autoimmunity. We have generated SOCS1 heterozygous (Het) mice and observed that the absence of one allele of the SOCS1 gene is sufficient to permit Treg induction from nave CD25 cells, but insufficient to allow Th17 induction. We also demonstrate that the absence of one allele of SOCS1 affects the expression of IL 17 and Foxp3 by CD25+ cells when pla ced under Treg inducing conditions. These results establish that the inadequate expression of SOCS1 may play a significant role in immune homeostasis.
114 Quinacrine modifies the interaction between transcription regulators in Francisella tularensis Wr ench AP Gonzalez C F Lorca GL Department of Microbiology and Cell Science, University of Florida, Gainesville, FL Protein protein interactions are central to many regulatory processes in living cells having a great potential as new targets for therape utics. The highly infectious intracellular pathogen, F. tularensis, encodes two s tringent s tarvation p roteins (SspA) orthologs annotated as SspA and MglA ( m acrophage g rowth l ocus A). These two transcription regulators form a complex with RNA polymerase (RN AP) and the DNA binding transcription factor, PigR (also called FevR) to positively regulate gene expression of the genes located in the F rancisella p athogenicity i sland (FPI), which is required for virulence and intracellular growth. With the increasing a cquisition of antibiotic resistance in many bacteria, there is a crucial need to identify new drugs directed to minimize and neutralize the effects of this pathogen. Using a small molecule library screening, quinacrine was identified as a thermal stabilizi ng compound for MglA and SspA of F. tularensis SCHU S4. Quinacrine was tested in vivo by using a bacterial two hybrid system. This compound was able to disrupt the MglA/SspA interaction, as shown by the decrease in # galactosidase activity by 47.5 % when compared to the control without quinacrine. The effect of quinacrine on the expression of virulence genes was assessed in Francisella novicida. The expression of iglA iglD pdpA and pdpD was assayed by qRT PCR. In a wild type strain, quinacrine decreased the expression of all four genes (2.1; 1.5; 2.2 and 5 fold, respectively) while the effect was not observed in mglA or sspA mutant strains. In addition, intramacrophage survival of F. novicida decreased by 2.5 log un its in the presence of quinacrine. In summary, using a combination of high throughput screening and functional assays, a small molecule capable of disrupting the interaction between MglA and SspA was identified. Consequently, repression of virulence factor s was observed. The results found could be applied for the identification of small molecules for other pathogenic microorganisms that use protein/protein (SspA orthologs) interactions to induce expression of virulence factors. These organisms include Yersi nia pestis Vibrio cholerae and Neisseria gonorrhoeae 115 Agronomic performance and segregation of the apomixis trait in progeny from crosses between sexual induced tetraploid and apomictic tetraploid genotypes of bahiagrass Wu H 1 ,2 Acuna C 1 Blount A 3 Altpeter F 1,2,* Quesenberry K 1,* 1 Agronomy Department, University of Florida, Gainesville, FL 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 3 Agronomy Department, North Florida Research and Education Center, Uni versity of Florida, Marianna, FL Bahiagrass, ( Paspalum notatum Flugge), is a perennial warm season grass recognized as one of the most important pasture and utility turf species of Florida. This diverse species contains germplasm with different ploidy lev els and linked reproductive characteristics. Diploids (2n = 2x = 20) reproduce sexually while tetraploids (2n = 4x = 40) reproduce asexually by apomixis. Apomixis bypasses megasporogenesis and fertilization of the egg to result in asexual seed production f ollowing apospory and pseudogamy. Bahiagrass offers the opportunity for manipulating apomixis to generate highly productive hybrids that may result in new forage or turf cultivars. Apospory in tetraploid bahiagrass is believed to be inherited as a single d ominant Mendelian factor with distorted segregation. Synteny of molecular markers was detected between the apospory controlling region of bahiagrass and specific regions of rice ( Oryza sativa L.). We generated sexual tetraploid bahiagrass by treating tissu e cultured calluses from the diploid cultivar Tifton 9 with colchicine. A segregating bahiagrass population was generated by hybridizing selected sexual and apomictic tetraploid genotypes. We will present data evaluating mode of reproduction by comparative embryo sac and molecular marker analysis and present the observed variability for growth habit, seasonal growth pattern and yield. 116 Transcriptome analyses of the Caribbean fruit fly embryonic development Xavier N 1 Schetelig MF 2 Handler AM 2,* 1 De partment of Entomology and Nematology, University of Florida, Gainesville, FL 2 Center for Medical, Agricultural, and Veterinary Entomology, Agricultural Research Service, U.S. Department of Agriculture, Gainesville, FL To isolate transcripts accumulated d uring embryogenesis we created an embryonic transcriptome of the Caribfly, Anastrepha suspensa by 454 sequencing. The cDNA library was constructed from total RNA pooled from various time points in embryogenesis using the SMART cDNA synthesis kit (BD Biosc iences), normalized using the Kamchatka crab duplex specific nuclease (Evrogen), titrated and sequenced on a 454 FLX platform. In addition, to isolate genes promoting cell death, a second transcriptome was constructed from total RNA isolated from embryos t reated with radiation to undergo cell death. In total, 58 million bases were obtained from 342,052 reads with an average length of 200 bp that formed 16,288 contigs ranging from 91 1,019 nt in length and 313,668 singletons. In vitro functional assays wer e performed on selected genes from the group of cell death genes. Proapoptotic genes hid and reaper from A. supsensa were isolated and tested. Several different versions of these genes with or without important binding and regulatory regions were generated and comparatively
tested to Drosophila homologs to verify their function. These genes could be functional studies in Tephritid species and enhance the development of new and safe applications for Sterile Insect Technique (SIT) programs. 117 Unfolded pro tein response and autophagy behave as complement pathways to dispose Z AAT in hepatocyte Xiao K 1,2 Wang L 2 McAndrew EJ 2 Wolper R 2 Rouhani FN 2 Brantly ML 2 1 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2 Divis ion of Pulmonary, Critical Care, and Sleep Medicine Department of Medicine, University of Florida, Gainesville, FL Introduction: The hereditary disorder a 1 antitrypsin (AAT) deficiency is caused by mutations in the SERPINA1 gene and is characterized by premature emphysema and severe liver disease. The most common variant, PI*Z (E342K), is characterized by improper protein folding and consequent aggregation within the endoplasmic reticulum (ER) of hepatocytes. In this study we show the activation of UPR i s dependent on both time and Z AAT dosage in human liver cells PI*ZZ. Method: Human liver cells derived from Z AAT homozygote (ATO1) were cultured at 37 ¡C. Cells were stimulated separately to observe changes in UPR and autophagy. In this study we found th at when ZAAT is over expressed in liver cell s UPR genes like ATF6, CHOP, IRE1a, PERK, Xbp1s are all down regulated. However, when ZAAT is expressed in a moderate amount, the UPR genes above will be activated on a time dependent manner. Conclusion: We conc lude that first, the UPR activation is depending on the time and dosage of ZAAT expression in the liver cell. Second, the UPR and autophagy are in a complement pathway to dispose the ZAAT in the liver cells. Understanding the relationship between these str ess responses and disease progression is critical to developing a viable therapy for liver disease in Z AAT patients. ! 118 Identification of the targets of human m iR 155 and its viral homolog miR k12 11 in lymphatic cells Yang Y 1 McIntyre L 2,* Lopez MC 2 Baker HV 2,* Boss I 2,3 Renne R 2,3,* 1 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 3 University of Florida Shands Cancer Ce nter, Gainesville, FL Short, non coding microRNAs (miRNA) are regulators of gene expression. Altered miRNA expression can be associated with cancer, but the mechanisms by which miRNAs contribute to pathogenesis are not well understood. Kaposi's Sarcoma as sociated herpesvirus (KSHV) is the etiological agent for KS and two lymphoproliferative diseases. KSHV encodes 17 microRNAs, including miR K12 11, which has been demonstrated to be a homolog of a human oncogenic miRNA, miR 155. To decipher the mechanism by which miR K12 11 contributes to the regulation of gene expression in KSHV infected tumor cells, we took a systems biology approach. By combini ng microarray experiments of miR K12 11 and miR 155 over expression in uninfected lymphatic cells, with informati on stored in public databases, novel insights were gleaned. Analysis of the microarray experiments revealed a subset of genes affected by both miR 155 and miR K12 11, and distinct targets for each miRNA.Computationally predicted targets of miR 155 were obt ained by integrating records from multiple databases, indirect targets were separated from direct targets and the regulatory pathways of the indirect targets were modeled using DNA protein and protein protein interactions. This meta analysis provides a mor e complete understanding of the common and distinct regulatory pathways that are modulated by these two orthologous miRNAs. In addition, identification of miR K12 11 targets in B lymphocytes also points to their role in KSHV lymphomagenesis. 119 Generati on and characterization of transgenic sugarcane with co integrat ion of two insect resistant genes Yao L 1,2 Wang X 1,2 Wu C 2 Yang Q 1 Li F 1 Zhu Z 3 Altpeter F 4 ,* Zeng Q 1,3 5 1 C ollege of Agronomy and Biotechnology, Yunnan Agricultural University, Kunmi ng China 2 Yunnan Academy of Agricultural Sciences, Kunming, China 3 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China 4 Agronomy Department, University of Florida, Gainesville, FL 5 Plant Molecular and Cellular B iology Program University of Florida, Gainesville, FL Sugarcane ( Saccharum spp. hybrids ) is the prime crop for production of sugar or biofuel The sugarcane stem borer causes serious yield loss in the range of 10% 25% annually Here we report enhancing s ugarcane insect resistance by Agrobacterium mediated transformation. To optimize expression of the Cowpea trypsin inhibitor (cpti) gene signal peptides for targeting and retention in the endoplasmatic reticulum were added (S C K). The expression cassette w as flanked b y a matrix attachment region ( MAR) from maize at both ends to reduce the positio n effect following transgene integration H pt or nptII selectable marker expression cassettes and genes of interest (GOI) S C K and cryIAc driven by constitutive u bi1 promoter from maize were subcloned into separate T i plasmids and co transformed into sugarcane to facilitat e marker gene removal in segregating progenies Following transformation with Agrobacterium strain LBA4404 A a total of fifty seven independent t ransgenic lines from 3 varieties
were obtained 6 0% of the transgenic lines displayed co integrat ion of the unlinked GOIs. Integration of GOIs was confirmed by PCR and Southern blot analysis. The GOIs expression in the transgenic plants w as confirmed by ei ther Northern blot or protein activity assay. Evaluation of the stem borer resistance is in progress. 120 Identify the upstream regulators of Hippo pathway in Drosophila Zhang C 1 Kim D 2 Cheng JQ 2 Zhou L 1, 1 Department of Molecular Genetics and Micr obiology, University of Florida, Gainesville, FL 2 Department of Molecular Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL Correct organ size is determined by the balance between cell death and proliferation. Perturbation of this b alance leads to cancer formation. Hippo, the Drosophila homologue of the mammalian Ste20 like kinases, MST1/2, coordinately regulates cell proliferation and apoptosis during development. The conserved Hippo signaling pathway controls organ size in not only Drosophila but also mammals. While a core kinase cascade leading from the protein kinase Hippo (Hpo) (Mst1/2 in mammals) to the transcription coactivator Yorkie (Yki) (YAP in mammals) has been established, upstream regulators of the Hippo kinase cascade are less well defined. We have identified a putative tumor suppressor NGB, a GTP binding protein that interacts with both the MST2 and its upstream regulator Merlin (Mer) in the cell culture system. A Drosophila model is established to identify its biology function and potential role in regulating Hpo/MST2 signaling pathway. Over expression of the MST2 in Drosophila eye induced the proapoptotic gene hid and caused an eye ablation or cone shape eye phenotype. The only Drosophila homolog of NGB, termed CG8801 will be either over expressed or mutated to study its function in the Hpo/MST2 pathway. 121 Ca Liberibacter asiaticus carries an excision plasmid prophage and a chromosomally integrated prophage that becomes lytic in plant infections Zhang S 1 Flores Cruz Z 1 Zhou L 2 Kang B H 3 Fleites L 1 Gooch MD 4 Wulff NA 5 Davis MJ 6 Duan YP 7 Gabriel DW 1 ,* 1 Plant Pathology Department, University of Florida, Gainesville, FL 2 Indian River Research and Education Center, University of Florida, Fort Pierce, FL 3 Dep artment of Microbiology and Cell Science, University of Florida, Gainesville, FL 4 Division of Plant Industry, Florida Department of Agriculture and Consumer Services, Gainesville, FL 5 Fundo de Defesa da Citricultura ( Fundecitrus ), Araraquara, So Paulo, Br azil 6 Citrus Research and Education Center, University of Florida, Lake Alfred, FL 7 USDA ARS, Fort Pierce, FL Huanglongbing (HLB), also known as citrus greening, is a lethal disease of citrus caused by several species of Candidatus Liberibacter, a psyllid transmitted, phloem limited, alpha proteobacteria. Ca Liberibacter asiaticus (Las) is widespread in Florida citrus. The recently published Las strain psy62 genome, derived from a psyllid, revealed a prophage like region of DNA in the genome, but phage ha ve never been associated with Las. In the present study, shotgun sequencing and a fosmid DNA library of curated Las strain UF506, originally derived from citrus symptomatic for HLB, revealed two largely homologous, circular phage genomes, SC1 and SC2 belon ging to the Podoviridae. SC2 encoded putative adhesin and peroxidase genes that had not previously been identified in Las and which may be involved in lysogenic conversion. SC2 also appeared to lack lytic cycle genes and replicated as a plasmid in both psy llids and in planta. By contrast, SC1 carried suspected lytic cycle genes and was found in nonintegrated, lytic cycle forms only in planta. Phage particles associated with Las were found in the phloem of infecte d periwinkles by transmission electron micros copy Both SC1 and SC2 were organized in tandem array as prophage in the UF506 chromosome. In psyllids, both SC1 and SC2 were found only as prophage. 122 Shade avoidance symptoms induced by green light Zhang T 1,2 Maruhnich SA 2 Folta KM 1,2,* 1 Horticul tural Sciences Department, University of Florida, Gainesville, FL 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Light quality and quantity affect plant adaptation to changing environmental conditions. Various wavele ngths in the visible and near visible spectrum have discrete effects on plant growth and development, and the effects of red, far red, blue and UV light have been well described. In this report the effect of green light on rosette architecture is tested, u sing a narrow bandwidth LED based lighting system. Upon addition of green light to a background of unchanging red and blue, plants exhibited elongation of petioles, upward leaf reorientation, and decre ased anthocyanin accumulation symptoms consistent wit h a shaded light environment. The phenotypes persist in phytochrome and cryptochrome mutant backgrounds. To further probe the molecular mechanism of the response, the accumulation of shade induced transcripts was measured in response to enriched green envi ronments. The addition of green light decreases or does not change the expression levels of genes normally induced by supplemental far red light. However, far red associated transcript accumulation patterns are observed in cryptochrome mutants when green l ight is added, indicating that the green absorbing form of cryptochrome is the active form in gating the response. The results indicate that shade symptoms can be induced
by addition of shade abundant wavebands other than far red, and that cryptochrome rec eptors assist in separating green responses from those induced by far red light. 123 Molecular development of sexually dimorphic digit length ratio (2D:4D) Zheng Z Cohn MJ Howard Hughes Medical Institute, University of Florida, Gainesville, FL Depart ment of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL In most men, the digit 2 is shorter than digit 4 whereas in most women, the second digit is equal to or longer than the fourth. The sexually dimorphic 2D:4D ratio is thoug ht to reflect testosterone exposure during development. Studies of digit ratio in adults have related it to a variety of behavioral, physiological and psychological outcomes, such as sexual orientation, and disease susceptibility. While the relationship of digit length to fetal androgen levels is widely accepted, this has not been tested in an animal model. Moreover, little is known about the developmental genetic mechanism that underpins sexually dimorphic digit development. Here we report on a mouse model with hind paws that exhibit similar 2D:4D ratio to humans. We show that antagonism of androgen receptor during early digit development causes male mice to develop an increased 2D:4D ratio. Reciprocally, either antagonism of the estrogen receptor or augmen tation of androgen levels resulted in decreased 2D:4D ratios in females. Although secondary sex characters can be affected by modulation of sex steroids at postnatal stages, only prenatal manipulations affected the digit ratio. We analyzed cell proliferati on and found that digits 2 and 4 respond differentially to sex steroids. Gene expression profiling of individual digit primordia in embryonic mice revealed that androgen and estrogen exert different effects on digit length due to quantitative and spatial d ifferences in the expression of their receptors. 124 Identification of redox sensitive proteins in guard cells: new evidence for crosstalk between guard cell ABA and MeJA signaling pathways Zhu M 1 Zhu N 1 Jin X 2 Assman n S 2 Chen S 1,3,* 1 Department of Biology, University of Florida, Gainesville, FL 2 Department of Biology, Pennsylvania State University, University Park PA 3 Proteomics Division, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL Cysteine is a v ery important amino acid particularly sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serve as redox sensors and signal transducers that regulate a variety of physiological processes, e.g., switching off photosynthetic reactions in the dark. Recently, guard cell protein components have been studied using large scale proteomics approaches. However, little is known about redox sensitive proteins and how they function in stomatal movement. Here we report cha racterization of guard cell redox proteins in abscisic acid (ABA) and methyl jasmonate (MeJA) signaling processes that lead to stomatal closure. We employed two complementary proteomics approaches, i.e., Isotope Coded Affinity Tag (ICAT) and saturation Dif ferential In Gel Electrophoresis (DIGE) to identify redox sensitive proteins and cysteines in ABA and MeJA treated guard cells. In total, 73 and 130 redox sensitive proteins were identified in ABA and MeJA treated guard cells, respectively. All the protein s contained at least one cysteine and over half of them (43/73 for ABA and 70/130 for MeJA) are predicted to form intra molecular disulfide bonds. Most of the proteins belong to energy, stress and defense and metabolism functional groups. Based on sequence s identified by mass spectrometry, 37 proteins were common to ABA and MeJA treated samples. A total of 54 redox sensitive cysteines were mapped by MS/MS. This study not only creates a comprehensive inventory of redox regulated proteins in guard cells, but also provides new evidence for crosstalk between ABA and MeJA signaling in guard cells. 1 25. In planta characterization of a strawberry ( Fragaria vesca) WD 40 protein Coleman L Chatterjee M, Folta KM Horticultural Sciences Department, University of Fl orida, Gainesville, FL An in planta study of novel WD repeat proteins revealed strong loss of function phenotypes in transgenic strawberries ( Fragaria vesca ). In particular, loss of a putative nucleolar ribosomal RNA processing protein results in a series of striking phenotypes. Studies on the orthologous protein in Mus musculus and Saccharomyces cerevisiae have suggested that it is an essential gene required for the maturation of the 28S and 5.8S rRNAs. These studies have also demonstrated the importance of this gene in rRNA synthesis, modification, processing, and ribosome biogenesis. The protein has not been characterized in plants. Loss of function lines exhibit enlarged flowers, thicker leaves, increased anthocyanin production, altered leaf forms and runne rs that produce only one leaf. Also, fruit expansion is inhibited and plant health declines after plants shift from vegetative to reproductive programs. Such analyses were not performed in Arabidopsis because of its short lifespan, and phenotypes were only observed when this gene was suppressed i n the perennial plant context. Further analysis is being conducted to better understand these effects and their relationship to ribosome biogenesis, especially as it is related to traits of horticultural intere st.