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Florida Genetics 2009 Organizing Committee Chair: Indra Vasil Members: Henry Baker, John Davis, Connie Mulligan, Diana Nolte, John Pastor Douglas Soltis, Michele Tennant, Thomas Yang FG2009 Sponsors Platinum Level Department of Molecular Genetics and Microbiology Gold Level University of Florida Genetics Institute, Center for Epigenetics, College of Liberal Arts and Sciences Graduate Program in Plant Molecular and Cellular Biology, Health Science Center Libraries, Interdisciplinary Center for Biotechnology Research Silver Level Evelyn F. & Willi am L. McKnight Brain Institute Special Thanks To Jennifer Baldwin, David Brumbaugh, Mickey Cuthbertson, Martine Horrell, Hope Parmeter Dustin Blanton, Tamar Carter, Bei Chen, Xiaoxia Han, Guangyao Li, To ng Lin, Aida Miro, Connie Philebaum, Ming Tang, Patrick Thiaville, Kai Xiao, Yuanqing Yan University of Florida Genetics Institute Director: Kenneth Berns Associate Directors: Henry Baker, Donald McCarty, Connie Mulligan, Indra Vasil Executive Board: Wi lliam Allen, Henry Baker, Kenneth Berns, John Davis, Robert Ferl, William Hauswirth, Julie Johnson, Donald McCarty, Michael Miyamoto, Connie Mulligan, Nicholas Muzyczka, Winfred Phillips, Pam ela Soltis, Douglas Soltis, Michele Tennant, Indra Vasil, Wilfred Vermerris, Marta Wayne, and Thomas Yang Scientific Advisory Board: Jeffrey Bennetzen, Ph.D., Norman and Doris Giles Professor and Georgia Research Alliance Eminent Scholar, University of Georgia, Athens, GA Ronald W. Davis, Ph.D., Director, Stanford Ge nome Technology Center, Stanford University, Stanford, CA Rebecca W. Doerge, Ph.D., Professor, Departments of Agronomy and Statistics, Purdue University, West Lafayette, IN Yoram Groner, Ph.D., The Dr. Barnet Berris Professor of Cancer Research, Depart ment of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Peter M. Howley, M.D., George Fabyan Professor of Comparative Pathology and Chair, Department of Pathology, Harvard Medical School, Boston, MA Ron Sederoff, Ph.D., Distinguished University Professor of Forestry, North Carolina State University Forest Biotechnology Group, Raleigh, NC Patricia G. Spear, Ph.D., Guy and Anne Youmans Professor and Chair, Microbiology Immunology, Northwestern University, Chicago, IL
UFGI Strategic Plan The discovery of the three dimensional double helix architecture of DNA in 1953 was not only a defining moment for biology, but arguably one of the most significant scientific discoveries of all time. It fundamentally and permanently changed the course of biology and genetics. The unraveling of DNAs structure, combined with its elegant mechanism for self replication and the existence of a universal genetic code for all living beings, have together provided the basis for the understanding of fundamental c ellular processes, mutation and genetic repair, genetic variation, the origin of life and evolution of species, and the structure/function/regulation of genes. The double helix is also proving to be of immense significance to advances in agriculture, medic ine and such other diverse fields as anthropology, criminology, computer science, engineering, immunology, nanotechnology, etc. It was the study of DNA that led to the development of tools that brought about the biotechnology revolution, the cloning of gen es, 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 better applications, which will impact directly on the quality of human life and sustainabi lity of life on earth, are yet to come. In order to attain these objectives, the digital nature of DNA and its complementarity are beginning to be exploited for the development of biology as an information based science. Indeed, a paradigm shift is already 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 biology. Indeed, biology in the 21st century will be surely dominated by this expanded visio n. 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 Epigenetics.
2009 Florida Genetics Symposium Schedule Wednesday, October 28, 2009 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 Translational Genomics Chair: Kenneth Berns 1:15 p.m. 2:00 p.m., Terry Van Dyke : Mechanistic dis covery in murine cancer models: From basic discovery to clinical translation 2:00 p.m. 2:30 p.m., Rolf Renne: KSHV encoded miRNAs and their role in viral biology 2:30 p.m. 4:30 p.m.: Poster Session and Reception (for registered attendees) *5:00 p.m 6:00 p.m., Leroy Hood: Systems biology & systems medicine catalyzing a transformation from reactive to proactive medicine *Note: All activities will be at the Cancer/Genetics Research Complex except for Dr. Hoods presentation, which will be at 5 p.m. Wednesday in the auditorium of the HPNP building on the Health Science Center campus. Thursday, October 29, 2009 8 a.m. 8:30 a.m.: Check in coffee Session II Drosophila Genetics Chair: Thomas Yang 8:30 a.m. 9:15 a.m., Mic hael Levine: Transcriptional precision in the Drosophila embryo 9:15 a.m. 9:45 a.m., Lei Zhou: Epigenetic regulation controls cellular sensitivity to stress induced apoptosis Session III Human Evolution Chair: Connie Mulligan 9:45 a.m. 10:30 a. m., Anna Di Rienzo: Adaptations to local environments in humans 10:30 a.m. 11:00 a.m., David Reed: Of lice and men: The inference of human evolutionary history from the perspective of its host specific parasites 11:15 a.m. 1:30 p.m.: Poster Session 11:30 a.m. Lunch (for registered attendees) Session IV Genome Evolution Chair: Doug Soltis 1:30 p.m. 2:00 p.m., Pam Soltis: Whole genome duplication in plant evolution: Case studies of ancient events and recent speciation 2:00 p.m. 2:30 p.m., Brad Barbazuk: A conserved alternative splicing event in plants reveals an ancient exonization of 5sRNA 2:30 p.m. 3:15 p.m., John Doebley: Darwin and d omestication 3:15 p.m. 3:45 p.m., Karen Koch: Maize domestication: Metabolic adaptations in gra in evolution
Presentation Abstracts Mechanistic discovery in murine cancer models: from basic discovery to clinical translation Van Dyke T 1, Zhang Q2, Karpinich N3, Yin C1, Miller R1, Song Y4, Pan W1, Kuslak S3 1Department of Genetics, University of North Carolina, Chapel Hill, NC 2Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 3Comprehensive Cancer Center University of North Carolina, Chapel Hill, NC 4National Cancer Institute, Frederick, MD W e have developed genetic ally engineered mice (GEM) to model many human cancers. Each model recapitulates major pathological features of the human disease. The tumor development stages in these models are associated with distinct genetic alterations in defined time windows, and as such the models provide us tools to identify potential biomarkers for therapy and early diagnosis and to develop therapeutic strategies to inform clinical trials. We have generated inducible GEM models of astrocytoma by targeting pathways (RB, KRAS and PTEN) frequently dysregulated in the human diseases. Alterations were induced specifically in adult GEM astrocytes via a tamoxifen inducible, human GFAP promoter driven CreERTAM allele. GEM with astrocyte RB pathway inactivation alone developed low grade astrocytomas (grade II). Neither constitutive KRAS activation nor PTEN inactivation alone produced detectable brain pathology. GEM harboring both inactivated RB and constitutively active KRAS in astrocytes developed high grade astrocytomas (grade III); ad dition of PTEN inactivation produced tumors with histopathological features of GBM (grade IV). These results suggest roles for RB and KRAS-PTEN pathways in astrocytoma initiation and progression, respectively. Initial studies have begun to define the rel ative contribution to disease of each disrupted pathway, including potential downstream targets for therapeutic intervention. We have also developed a strategy for modeling serous epithelial ovarian cancer (EOC) using similar approaches. Thus far, no model of this disease suitable for preclinical testing has been developed. However, several basic studies have identified genetic aberrations required to drive disease in GEM, and we have shown that the combination of pRb, p53 and BRCA1 inactivation leads to E OC characteristic of the serous type. Our results thus far in developing an inducible EOC model will be presented. Finally, we have developed a prostate cancer model that develops heterogeneous cancers dependent upon impaired pRb, Pten and p53 function, i ncluding a complex co evolution of stroma and carcinoma. Current studies are evaluating the cell of origin, the complex biology of tumor progression, specific pathways involved, and the role of inflammatory responses in disease. Biography of Terry Van Dy ke, Ph.D. Terry Van Dyke, Ph.D., studies the mechanisms and pathways to cancer development at many levels, including genetic, molecular, cell and organ biology. Because cancer can develop in more than 100 distinct mammalian cell types, her lab has utilized genetically engineered mice as the foundation of its analyses. It has established several preclinical cancer models that have facilitated analyses of the tumor suppressors, including their contribution to normal growth control and the consequences of their inactivation to multistep tumorigenesis. The approach enables a detailed examination of the molecular and cellular events in developing tumors studies that are not possible in humans. She received her Ph.D. in Medical Sciences from the University of Florida in 1981. As a postdoctoral fellow in Dr. Arnold Levine's lab in the early 1980s, she characterized one of the first transgenic cancer models. In 1993, she joined the University of North Carolina, where she is currently a Sarah Graham Kenan Distinguished Professor of Genetics with a joint appointment in Biochemistry and Biophysics. In 1998, she became the Facility Director of the UNC Animal Models Core Facility and, in 1999, she established the Carolina Mutant Mouse Regional Resource Center, one of f our in the country. In the late 1990s, Dr. Van Dyke served as co chair of an advisory committee to the Director of the National Cancer Institute on the use of mouse models to advance cancer research, which led to the establishment of the Mouse Models of Hu man Cancers Consortium (MMHCC). In this capacity she has provided significant leadership in initiatives, including the establishment of a national repository for GEM mice, an international database for mouse models of cancer, and a web site including summa ries of human cancers and mouse cancer models. She continues to serve as a MMHCC PI, leading a multi institutional collaboration on astrocytic cancers.
Systems medicine, transformational technologies and the emergence of predictive, personalized, prevent ive and participatory (P4) medicine Hood L Institute for Systems Biology, Seattle, WA The challenge for biology in the 21st century is the need to deal with its incredible complexity. One powerful way to think of biology is to view it as an informa tional science. This view leads to the conclusion that biological information is captured, mined, and integrated by biological networks and finally passed off to molecular machines for execution. Hence the challenge in understanding biological complexity is that of deciphering the operation of dynamic biological networks across the three time scales of life evolution, development and physiological responses. Systems approaches to biology are focused on delineating and deciphering dynamic biological netw orks and their interactions with simple and complex molecular machines. I will focus on our efforts at a systems approach to disease looking at prion disease in mice. We have just published a study that lays out the principles of a systems approach to d isease including dealing with the striking signal to noise problems of high throughput biological measurements and biology itself (e.g. polymorphisms). I will discuss the emerging technologies (measurement and visualization) that will transform medicine o ver the next 10 years, including next generation DNA sequencing, microfluidic protein chips and single cell analyses. I will discuss some of the computational and mathematical challenges that are fundaments to the revolution in medicine, both those that de al with medical sciences and those that deal in a general way with healthcare. It appears that systems medicine, together with pioneering changes in these emerging technologies and the development of powerful new computational and mathematical tools will transform medicine over the next five to 20 years from its currently reactive state to a mode that is predictive, personalized, preventive and participatory (P4). This will in turn lead to the digitalization of medicine, with ultimately a profound decreas e in the cost of healthcare. It will also transform the business strategies for virtually every sector of the health care industry. These considerations have led the Institute for Systems Biology to begin formulating national and international strategic pa rtnerships focused on accelerating the realization of P4 medicine. I will discuss some of these strategic partnerships and the implications for healthcare arising from P4 medicine. Biography of Leroy Hood, M.D., Ph.D. Leroy Hood M.D., Ph.D., has focuse d on fundamental biology and on bringing engineering to biology through the development of five instruments that constitute the technological foundation for modern molecular biology and genomics the DNA and protein sequencers and synthesizers and the ink jet oligonucleotide synthesizer. Early in his career, he applied these technologies to the study of molecular immunology, discovering many of the fundamental mechanisms for antibody diversity, and neurobiology; he cured the first neurological disease by gene transfer in mice. In the late 1980s, he embarked on a systems biology approach to understand immunology. In 1992, he moved to the University of Washington as founder and Chairman of the cross disciplinary Department of Molecular Biotechnology (MBT) whe re he initiated systems studies on cancer biology and prion disease. In 2000, he cofounded the Institute for Systems Biology in Seattle, Washington, to continue pioneering systems approaches to biology and medicine. Dr. Hood is now pioneering the idea tha t the systems approach to disease, the emerging technologies, and powerful new computational and mathematical tools will move medicine from its current reactive mode to a predictive, preventive, personalized and participatory mode (P4 medicine) over the ne xt five to 20 years. Dr. Hood has been awarded numerous prizes the Lasker Prize (1987), the Kyoto Prize in Advanced Technology (2002), the Lemelson MIT Prize for Innovation and Invention (2003), the Association for Molecular Pathology Award for Excellenc e in Molecular Diagnostics (2003), the Biotechnology Heritage Award (2004), the Heinz Award in Technology, the Economy and Employment (2006), election to the Inventors Hall of Fame (2007) and the Pittcon Heritage Award (2008). Dr. Hood has received 17 hono rary degrees from Institutions such as Johns Hopkins, Yale, UCLA, and Whitman College. He has published more than 650 peer reviewed papers, received 15 patents, coauthored numerous textbooks, and co authored a popular book on the human genome project (Th e Code of Codes). He is currently completing a textbook on systems biology. Dr. Hood is a member of the National Academy of Sciences, the American Philosophical Society, the American Association of Arts and Sciences, the Institute of Medicine and the Nat ional Academy of Engineering. He is one of only seven scientists elected to all three academies (NAS, NAE and IOM). Dr. Hood has also played a role in founding more than 14 biotechnology companies, including Amgen, Applied Biosystems, Systemix, Darwin and Rosetta. He has recently cofounded the company Integrated Diagnostics, which he expects to become a platform company for P4 medicine.
Transcriptional precision in the Drosophila embryo Boettiger A, Hong J W, Hendrix D, Levine M Division of Genetics, G enomics, and Development, University of California, Berkeley, CA Department of Molecular and Cell Biology, University of California, Berkeley, CA Center for Integrative Genomics, University of California, Berkeley, CA The post genome analysis of dorsal ve ntral (DV) patterning of the Drosophila embryo led to two unexpected findings. First, as many as half of the 60 70 target genes for the Dorsal regulatory gradient contain more than one enhancer for a single pattern (threshold) of gene expression. In some cases the duplicate enhancer (or shadow enhancer) maps within neighboring genes. Second, most of the Dorsal target genes contain stalled RNA Polymerase II (Pol II) prior to their induction in the early embryo. We have used a combination of comparative genome analysis and quantitative imaging methods to determine the significance of shadow enhancers and stalled Pol II in development and evolution. The identification of Dorsal target enhancers in divergent insects (flies, mosquitoes, beetles, and bees) he lped distinguish between socalled primary and shadow enhancers. In some cases (e.g., the brinker locus) the presumed shadow enhancer (located in intron 2 of the neighboring Atg5 gene in Drosophila) is the one that is conserved in evolution. Overall, DV enhancers tend to be located in fixed positions relative to the transcription units they regulate. Thus, it would appear that the evolution of novel patterns of gene expression depends on changing the sequence of old enhancers rather than inventing new one s. Some of the genes that are activated by low levels of the Dorsal gradient display erratic patterns of gene activation in dl/+ embryos. These genes tend to lack shadow enhancers, whereas those genes containing shadows seem better buffered against genetic perturbation. Previous studies on stalled (paused) Pol II at the Drosophila hsp70 heat shock locus suggested that stalling helps accelerate gene induction in response to stress. Perhaps developmental control genes, like stress genes, are poised for rapi d activation. The quantitative analysis of gene activation suggests that transcriptional synchrony is one manifestation of this poised induction. The analysis of 14 different genes in hundreds of early embryos suggests that there are two different classes of expression profiles, synchronous and stochastic. All nine genes with stalled Pol II display synchronous patterns of activation, while all five genes lacking stalled Pol II exhibit stochastic patterns of induction. Transcriptional synchrony might help en sure the orderly deployment of the complex gene networks that control embryogenesis. We propose that synchrony is a measure of population fitness. Biography of Michael Levine, Ph.D. Michael Levine, Ph.D., studies gene networks that control animal develop ment and disease. With two colleagues, he discovered the homeobox genes, which turn certain DNA segments on and off in the fruit fly to control differences in body segments and, scientists were surprised to learn, have similar functions in humans. He now works with sea squirts, whose DNA more closely resembles human genetic material. After completing his bachelors degree in genetics at the University of California Berkeley in 1976, Levine went to Yale for his Ph.D. in the Department of Molecular Biophysi cs and Biochemistry, which he received in 1981. He held postdoctoral fellowships at the University of Basel (Switzerland) and at the University of California Berkeley, before joining the faculty of Columbia University from 1984 1990, and then the Universit y of California San Diego from 1991 1996. He joined the faculty at University of CaliforniaBerkeley in 1996, where he is now F. Williams Professor and head of the Division of Genetics, Genomics, and Development and is co director of the Center for Integra tive Genomics. Levine was elected to the American Academy of Arts and Sciences in 1996 and the National Academy of Sciences in 1998. He received the Monsanto Prize in Molecular Biology from the National Academy of Sciences in 1996 and in 2009 he received t he Wilbur Cross Medal, the highest honor bestowed by the Yale University Graduate School of Arts and Sciences. Adaptations to local environments in humans Di Rienzo A Department of Human Genetics, University of Chicago Humans have adapted to a remarkab ly diverse range of environments and show striking phenotypic variation among populations. The impact of these adaptations can still be detected in the geographic distribution of genetic variants that are beneficial in one environment, but deleterious or neutral in a different habitat. We use ecological information to search the human genome for correlations between the frequency of polymorphic variants in worldwide population samples and a set of environmental variables, including climate, subsistence, e cosystem, and diet. Variants with significant correlations with these environmental variables are strong candidates for genetic adaptations to local
environments. By using this approach, we find a striking enrichment of strong environmental correlations in genic and nonsynonymous SNPs relative to non genic SNPs and for several biologically important sets of genes. This implies that climate, diet and subsistence played an important role in shaping variation in the human genome. In addition to genome wide analyses, signals of adaptation to local environments at individual loci can be used to identify SNPs that may affect gene expression and function and that may be subjected to functional analyses. Such SNPs are likely to be important in geneenvironment i nteractions. Biography for Anna Di Rienzo, Ph.D. Anna Di Rienzo, Ph.D., is a Professor of Human Genetics at the University of Chicago and a member of the Committees on Genetics, Genomics and Systems Biology, on Clinical Pharmacology and Pharmacogenomics and on Molecular Metabolism and Nutrition. She received her bachelors degree in Biological Sciences and her Ph.D. in Medical Genetics from the University of Rome La Sapienza. Her group is generally interested in characterizing the amount and patterns of variation at the DNA sequence level in human populations, and in elucidating the forces that shape and maintain this variation. She has combined empirical studies of sequence variation and modeling of population history to make inferences about human p opulation size changes. In addition, her group has worked on the action of positive natural selection at specific loci and at the genome wide level in humans. As greater attention is focused on dissecting the genetic bases of common diseases, she is inter ested in connecting signals of adaptations with genetic variants that influence human phenotypes and in developing evolutionary models of common diseases. Darwin and d omestication Doebley J Department of Genetics, University of Wisconsin, Madison, WI I n his book On the Origin of Species, Charles Darwin used plant and animal domestication as a model to inform his theory on evolution under natural selection. Artificial selection during plant domestication is thought to have been largely unconscious, th e inevitable product of a sowingreaping cycle. Selection pressures placed by humans on crops are analogous to those placed by seed dispersers such as birds on wild species. Nevertheless, Darwins use of domestication as a model for natural evolution has been controversial. Over the past 20 years, genetic and molecular research has begun to uncover the genetic basis of the changes involved in the evolution of plant form under both natural and artificial selection. In the case of domestication, approximat ely 15 genes involved in the changes in morphology have been isolated. For most of these genes, the nature of the alteration in the gene is understood. I will review what has been learned about change in form under domestication and whether any patterns a re beginning to emerge. Biography of John Doebley, Ph.D. John Doebley, Ph.D., studies how genes control changes in plant morphology during domestication with a focus on maize. He is a Professor of Genetics and a member of the Plant Breeding Faculty at t he University of Wisconsin Madison. Doebley's work has earned him several awards and honors, including election to the National Academy of Sciences in 2002. Dr. Doebly has spent the past two decades examining the genetic differences and similarities betwe en teosinte and maize (Zea mays). He and his laboratory members pioneered the use of quantitative trait locus mapping to successfully identify which regions of maize's genome are responsible for several domestication traits, features that separate maize from its undomesticated relatives. His team was one of the first to clone genes identified through QTL mapping, including a pivotal domestication gene known as teosinte branched1 ( tb1 ), which affects kernel structure and plant architecture. In his Inaugur al Article in the Proceedings of the National Academy of Sciences, he and colleagues determined that selecting for tb1 thousands of years ago did not affect genetic diversity at neighboring genes, clarifying how the maize genome was sculpted by past selective breeding and elucidating how the genomes of other organisms respond to selective pressure. He holds a bachelors degree in anthropology from West Chester State College, his master's degree in anthropology from Eastern New Mexico University and a Ph.D. in botany from the University of Wisconsin Madison.
*= UF Genetics Institute Faculty KSHV encoded miRNAs and their role in viral biology Renne R* Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL UF Shands Canc er Center, University of Florida, Gainesville, FL MicroRNAs are small, non coding RNAs that post transcriptionally regulate gene expression by binding to 3UTRs of target mRNAs. Kaposis sarcoma associated herpesvirus (KSHV), a virus linked to KS and prim ary effusion lymphoma (PEL), encodes 12 miRNA genes but only a few regulatory targets are currently known. Using ectopic expression of viral miRNAs in combination with gene expression profiling, we identified miRNA targets, including THBS, a strong anti angiogenic factor, and several genes involved in regulation of apoptosis and proliferation. In addition, we found that KSHV miR K1211 shares 100% seed sequence homology with hsa miR 155, a miRNA frequently found up regulated in lymphomas and critically impo rtant for B cell development. Based on this seed sequence homology, we hypothesized that both miRNAs regulate an overlapping set of genes. Hence, KSHVmiR K1211 may mimic hsa miR 155. To this end, we demonstrated that ectopic expression of either miRNA t argets BACH 1 in PELs (Skalsky et al., 2007, J Virol 81(23):12836 45). Using bioinformatic approaches in combination with 3UTR reporter assays, we found that CEBP/ and PU.1 are targeted by miR K1211/miR 155. Since both transcription factors play essential roles in B cell differentiation, we propose that viral miRNA mimics miR 155 in the context of B cell differentiation. Ongoing experiments to directly evaluate the effects of KSHVmiR K1211 expression on human hematopoiesis will also be discussed. Epigenetic regulation controls cellular sensitivity to stress induced apoptosis Zhou L *, Zhang C, Lin N Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL UF Shands Cancer Center, University of Florida, Gainesville, FL IAP antagonists such as reaper and hid play pivotal role in mediating cell death in response to cytotoxic stimuli. A 33kb genomic region upstream of reaper, the i rradiation r esponsive e nhancer r egion (IRER), is required for mediating the induction of both reaper and hid following irradiation. Interestingly, IRER is subject to epigenetic regulation. While it is open in undifferentiated cells during early embryogenesis, chromatins in IRER become enriched for H3K27Me3 and H3K9Me3 and form facultative het erochromatin in late embryogenesis. This epigenetic modification of IRER blocks the irradiation responsiveness of both reaper and hid and renders the cells resistant to irradiation. Several Polycomb group (PcG) proteins are required for this epigenetic reg ulation of IRER. Using an ubi DsRed reporter knocked into IRER, we found that about 2% cells in Drosophila larvae are DsRed positive, i.e. have an open IRER. These cells have higher levels of reaper expression and are much more sensitive to stress induced cell death. In addition, mosaic clone analysis showed that cells deficient for IRER developed hyperplasia, indicating that these cells are more resistant to environmental stress and/or cellular competition. Overall, our data indicated that epigenetic regul ation of pro apoptotic genes plays an important role in determining cellular sensitivity to stressinduced cell death. Of lice and men: the inference of human evolutionary history from the perspective of its hostspecific parasites Reed D* Mammalogy, Fl orida Museum of Natural History, University of Florida, Gainesville, FL Human history is written not only in our DNA, but also in the DNA of our parasites. This parascript of our history contains bits of human evolutionary history that may be obscured in host data. The ectoparasitic lice of primates have been coevolving with their primate hosts for at least 25 million years, and have already demonstrated their utility for inferring primate evolutionary history. Human and chimpanzee lice last shared a com mon ancestor 5 7 million years ago and diverged in tandem with their hosts. We also know that human head lice show a population expansion dated around 100,000 years ago that likely corresponds with the out of Africa expansion of their human hosts. Howeve r, these two examples merely confirm events in human history that are well known from human fossil and molecular data. The utility of studying the parasites is that they may shed light events that are unknown or potentially unknowable from human evidence. Research in my lab has shown that the lice on modern humans contain greater genetic diversity than can be
explained by a history restricted solely to modern Homo sapiens. DNA simulation studies show that lice arose on archaic hominids and switched to mo dern humans within the last 35,000 years, which would require direct physical contact between modern and archaic hominids. Ill provide other examples of how parasitic lice can be used to examine pivotal questions in human evolution, such as when humans be gan using rudimentary clothing. Whole genome duplication in plant evolution: case studies of ancient events and recent speciation Soltis PS* Laboratory of Molecular Systematics and Evolutionary Genetics, Florida Museum of Natural History, University of Florida, Gainesville, FL The relatively small genome of the diploid flowering plant Arabidopsis thaliana shows signatures of multiple, ancient whole genome duplications, and the genomes of other derived flowering plants are likewise duplicated. Analyses o f new genomic resources for phylogenetically early lineages of flowering plants now suggest that perhaps all flowering plants have undergone ancient whole genome duplication, explaining in part the generally large size and complexity of plant genomes. This process of whole genome duplication polyploidy has long been recognized as an important speciation mechanism in flowering plants, but its long term impact had been questioned. Studies of recently formed natural polyploid plants, such as those in the g enus Tragopogon, coupled with analysis of synthetic polyploids, are demonstrating that new polyploid genomes are dynamic. Polyploid species of Tragopogon do not simply combine the genomic complements of their diploid parental species; instead, their genom es have undergone chromosomal rearrangements, loss of genetic loci, and changes in gene regulation all in less than 80 years since their formation. Likewise, some changes are evident in early synthetic generations. The processes leading to genome duplica tion, rearrangement, and modification via gene loss and silencing have likely been occurring throughout the ~130 million years of flowering plant evolution and continue to contribute to the complexity of plant genomes. A conserved alternative splicing ev ent in plants reveals an ancient exonization of 5sRNA Barbazuk WB* Department of Biology, University of Florida, Gainesville, FL Alternative splicing (AS) creates multiple mRNA transcripts from a single gene. Recently available plant genome and transcr ipt sequence data sets are enabling a global analysis of AS in many plant species, and results are revealing differences between animals and plants in the frequency and preferred mechanisms of alternative splicing. Plant genome and transcript comparisons a re also identifying conserved alternative splicing (AS) events among evolutionarily distant species, and these analyses can prioritize AS events for functional characterization and help uncover relevant cisand transregulatory factors. A genome wide sea rch for conserved cassette exon AS events in higher plants revealed the exonization of 5S ribosomal RNA (5S rRNA) within the gene of its own transcription regulator, TFIIIA (transcription factor for polymerase III A). The 5S rRNAderived exon in TFIIIA gen e exists in all representative land plant species, but not in green algae and non plant species, suggesting it is specific to land plants. TFIIIA is essential for RNA Polymerase III based transcription of 5S rRNA in eukaryotes. Integrating comparative geno mics and molecular biology revealed that the conserved cassette exon derived from 5S rRNA is coupled with nonsense mediated mRNA decay. Most significantly, this study provides the first evidence of ancient exaptation of 5S rRNA in plants, suggesting a nove l gene regulation model mediated by the AS of an anciently exonized noncoding element. Maize domestication: metabolic adaptations in grain evolution Koch KE* Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Recent studies show that developing corn kernels and other young grains have very different interior environments than previously recognized. Little to no oxygen is detectable in their endosperms. This is centrally important, because it indicates that sites of sta rch and protein deposition in grains are likely controlled by fundamentally different metabolic processes than envisioned earlier. Consistent with this suggestion is the endosperm expression of genes for low oxygen metabolism (e.g. specific sucrose synthases, glycolytic enzymes, alcohol dehydrogenases, and sorbitol dehydrogenase). Evolution of a low oxygen, starchstoring endosperm preceded origin of grasses, but related changes in structure and
function continued within the caryopses. Unlike most grains Zea and related Andropogoneae developed a more compact zone of vascular transfer, elaborated projections at the base of the endosperm, and more extensive regions of low oxygen endosperm. Young maize kernels also differ from those of most other grains in lacking capacity for daytime oxygen production via photosynthesis in outer layers of the green grain (developing kernels are non green). Adaptations in maize and related Andropogoneae may well include additional adjustments for anaerobic metabolism and n onvascular transport.
Poster session s *= UF Genetics Institute Faculty. Presenters are underlined. 1. Generation and characterization of transgenic, fall armyworm resistant t urfgrass (Paspalum vag i natum Swartz) Altpeter F 1,2,*, Neibaur I1 ,2, Zhang H1 ,2, Meagher RL3, Gallo M1 ,2,* 1Agronomy Department, University of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Program University of Florida, Gainesville, FL 3Center for Medical, Agricultural, and Veterinary Entomol ogy, Agricultural Research Service, U.S. Department of Agriculture, Gainesville, FL Seashore paspalum (Paspalum vaginatum Swartz) is a salt tolerant, fine textured turfgrass used on golf courses in coastal, tropical and subt ropical regions. Fall armyworm [Spodoptera frugi perda (J. E. Smith)] is a devastating pest of seashore paspalum. Therefore, insect resistance is a prime target for genetic engineering of seashore pasp a lum. However, a genetic transformation protocol for seashore paspalum was lacking. Here we report the deve l opment of a genetic transformation protocol for this commercially important turfgrass species as well as the generation of fall armyworm resistant seashore paspalum expressing a synthetic, codon optimized Bt cry gene. Biolistic gene transfer of embry ogenic callus with a linea rized plasmid carrying constitutive hygromycin and Bt cry expression cassettes was followed by selection with hygromycin and regeneration of plants. Transgene integration and expression of the regenerated plants was co n firmed by P CR, Southern blot, RT PCR, and ELISA respe c tively. Resistance of transgenic plants to fall armyworm was evaluated in comparison to wildtype and correlated well to Bt cry expression levels. 2. Improving t ur f and forage properties in apomi c tic b ahiagrass (Paspalum notatum Flugge) by m u tagenesis Lomba P1,2, Altpeter F 1,2,* 1Agronomy Department, University of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Program University of Florida, Gainesville, FL Bahiagrass (Paspalum notatum Flugge) is the predom i nant forage grass and a popular turf in the southeastern United States. Bahiagrass popularity is attributed to its strong persistence under low input conditions. However, the quality of bahiagrass is limited due to its open growth habit and p rolific production of tall seedheads, coarse leaf texture, light green color and poor nutritive value. Improvement of bahiagrass cultivar Argentine by conve n tional breeding is very difficult due to its apomictic repr o duction mode. Our objective was to ex plore the potential of chemical and tissue culture derived mutagenesis for genetic improvement of apomictic bahiagrass for gener a tion of uniform mutagenized seed progeny with improved turf and forage quality. Scarified and surface sterilized bahiagrass see ds were treated with the mutagen sodium azide at various concentrations and exposure times. Callus was induced from these seeds and regenerated via so matic embryogenesis to obtain uniformly mutagenized plants. Independently mutagenized lines with reduced stem length, higher tiller density or reduced or delayed seedhead formation were established under field cond i tions for further evaluation of density, leaf texture, tiller length, color, growth pattern, biomass, seedhead and seed production. Greenhouse an d field data from s e lected mutagenized lines will be presented in comparison to non mutagenized bahiagrass. 3. Lipopolysaccharide induced activation of circu lating inflammatory molecules exert a negative influence on hippocampal progenitor cell differe n ti ation in the adult brain Asokan A Ormerod BK* J. Crayton Pruitt Family Department of Biomedical Engi neering, University of Florida, Gainesville, FL Although the function of adult neurogenesis has not been elucidated completely, a strong correlation is e merging between performance on spatial memory tasks and o p timal levels of neurogenesis. Previous reports have shown that neuroinflammation, induced by lipopolysaccharide (LPS) ablates adult hippocampal neurogenesis resulting in latent memory impairment, wh ich can be treated with non steroidal anti inflammatory drug (NSAID) treatment; however, chronic NSAID treatment is associated with adverse side effects. Here we attempt to evaluate hipp o campal neurogenesis and identify components of the neu roinflammatory response that are malicious to neuronal differentiation by quantifying cytokine levels in the blood and brain of LPS treated mice using a multiplex ELISA strategy. As expected, within 5 hr, LPS treatment increased all proinflammatory cytokines, with robus t elev a tions observed in TNF", IL 6, IL 1!, IFN #, IL17 and MCP 1 levels in se rum and hippocampus (p<0.01) and returned to baseline levels by 96 hr. However, levels of IL 12 (p40), KC and LIX overshot baseline levels and, were significantly lower in LPS versus vehicle treated mice 96 hr after treatment. Interestingly circulating levels of IL 12(p40), KC and LIX correlated negatively with new neuron (BrdU/DCX+ve) number (p <0.03). Our data suggest that these factors may modulate the pathways responsible for regul ating cell fate decisions among hippo campal progenitor cells.
4. Functional pseudogene usage in natural infe c tions with Anaplasma phagocytophilum Barbet AF 1,*, Foley JE2, Nieto NC2, Gabriel M3, Al Khedery B1 ,*, Foley P4 1 Department of Infectious Disease s and Pathology Un i versity of Florida, Gainesville, FL 2 Department of Medicine and Epidemiology, University of California, Davis, CA 3 Veterinary Genetics Laboratory, University of California, Davis, CA 4 Dep artment of Biological Sciences, California Sta te Un i versity, Sacramento CA The tickborne rickettsiales Anaplasma phagocytophilum causes persistent infections with disease severity ranging from asymptomatic to fat al. The organisms express a m a jor su rface protein, MSP2/P44 from a genomic expression s ite into which duplicated cassettes from different donor pseudogenes recombine t o generate expressed variants. There is a repertoire of ~100 pseudogenes in the g e nome, preferentially clustered close to the origin of repl i cation, although some pseudogenes a re more widely spread ove r other regions of the genome. We have d e termined the structure of expression site MSP2/P44 v a riants from diverse hosts and locations in the U.S. and Europe (n=268) and investigated if this database might give information on the us age frequency of individual g e nomic pseudogenes. The most frequently used sequence donors were located between the expression site and the origin of replication. Usage was infrequent for pseud o genes located in map positions 0 1,000,000 bp. These data suggest that genome position of pseudogenes rel a tive to the expression site influences their frequency of use as sequence donors. Many pathogens are now known to use cassette mechanisms for expression of ant i genic variants, but it is less clear what structural features influence the probability of selection of an individual sequence donor. Anaplasma phagocytophilum should pro vide a useful system to evaluate these structural features. 5. Vitamin D binding protein serum levels and g e notype in those with type I d iabetes Bierschenk L1, Blanton D 1, Han Z1, Wasserfall C1, Haller M2, Schatz D2 ,*, Atkinson M1 ,* 1Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 2Department of Pediatrics, University of Florida, Gaines ville, FL Type 1 diabetes (T1D) is a complex autoimmune disease and a role for vitamin D has come under scrutiny recently. While our own studies have not specifically associated serum 25OH vitamin D levels with T1D, others have found polymorphisms in the vitamin D hydroxylase enzyme loci to be associated with T1D. Here we sought to investigate the role of vitamin D binding p rotein (VDBP) both by ELISA measurement of circulating levels and restriction fra g ment length polymorphism (RFLP) analysis of the VDB P locus. A cross sectional study was performed utilizing serum samples collected from 153 controls, 203 T1D su b jects, and 116 first degree relatives of subjects with T1D. A subset of these samples consisting of 53 controls, 81 T1D subjects, and 38 firstd egree relat ives were gen o typed. VDBP levels (mean $g/mL; 95% CI) were highest in healthy controls (528.2; 467.3589.0), intermediate in first degree relatives (496.9; 410.3583.4), and lowest in T1D subjects (424.8; 403.6 446.0). A significant diffe r ence i n VDBP serum concentrations was found between the control and T1D groups (p=0.0028). RFLP analysis of sites at codons 416 and 420 of exon 11 revealed no ass o ciation between VDBP genotype, disease state or serum levels, implying circulating concentrations of this analyte may be controlled by other factors. A much larger pro s pective study needs to be undertaken in order to increase the power of the study and to evaluate our observed lo w er VDBP levels in relation to T1D development. 6. Spatial variation in the prevalence of sigma v i rus, a vertically transmitted pathogen that infects Drosophila melanogaster Blohm GM Regan K, Wayne M L* Department of Biology, University of Florida, Gainesville, FL Many emer ging pathogens (e.g. West Nile v irus, Dengue, equine encephalitis) are transmitted vertically. Theory suggests that dispersal among populations can maintain vertically transmitted pathogens by increasing the genetic variability of the pathogen at a local scale. Using distance as a proxy for the timing and magnitude of dispersal among populations, we aim to estimate the effect of di s persal on the persistence of sigma virus, a vertically transmitted pathogen on Drosophila melanogaster. During the summer of 2009, we collected individuals from 7 loc a tions along a 70mile transect near Athens, GA. We also estimated temporal variability by sampling at five different times. We determined the prevalence of sigma virus in males and females from each location through time using a CO2 e xposure assays. We then propagated the field collected adults in the laboratory to measure rates of transmission. Our results indicate that the prevalence of sigma virus is highly variable among populations, ranging from 0% to 60% (mean: 25%), with no spatial trends in total prevalence. T he results of this study contribute to our understanding of host pathogen coevolution while potentially informing management strategies to reduce the incidence of vertically transmitted pathogens in h u mans.
7. An evolutionary basis for hypospadias: mole c u lar development of external genitalia in the turtle Trachemys scripta elegans Boger LJ 1, Cohn MJ1,2,3,* 1Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL 2Department of Biology, University of Florida, Gainesvi lle, FL 3Howard Hughes Medical Institute, University of Florida, Gainesville, FL Hypospadias is a birth defect of the penis in which the urethral tube fails to close. From an evolutionary perspe c tive, a closed urethra is a derived character of mammals, in that other tetrapods possess genitalia with an o pen urethral groove or sulcus. The phallus of the turtle Trachemys scripta elegans (the red eared slider) has fibrovascular structures and a collagen fiber arrangement h o mologous to that of mammals; however, like other non mammalian tetrapods, the phallus has an open sulcus rather than an enclosed urethra. Comparison of external genital development in turtles and mammals has the potential to reveal a genetic basis for the origin of the uret h ra, and may uncover novel pathways involved in urethral tubulogenesis. We collected male T. scripta elegans e m bryos at various developmental stages and examined the expression patterns of genes known to be involved in the development of mammalian genitalia. Here we show e x pression patterns of such genes, including Shh, Msx2, Bmp4, and Twist. Further analysis of these genetic ne t works will not only help to answer the larger evolutionary question of which developmental processes are conserved and which are variable between turtles and mammals, but may also give a better understanding of the etiology of hypospadias. 8. KSHV miRK12 11 expression in human proge nitors during in vivo hematopoiesis induces B cell expansion in NOD/LtSz scid IL2R!null mice Boss IW 1, Nadeau PE2, Abbott JR2, Mergia A2,*, Renne R1 ,3,* 1Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2Department of Infectious Diseases and Pathology Un i versity of Florida, Gainesville, FL 3University of Florida Shands Cancer Center, Gainesville, FL MiRNAs are small non coding RNA molecules that regulate gene expression. Kaposis sarcoma associated herpesv i rus (KSHV), a lymphotropic virus, expresses 12 miRNAs during latent infection. Previous work by our lab and ot h ers found that one of these viral miRNAs, miR K1211, shares 100% seed sequence homology with hsa miR 155, an oncogenic miRNA involved in B cell activation and di f ferentiation (Skalsky et al., 2007, J Virol 81(23):12836 45; Gottwein et al., 2007, Nature 450(7172):1 0969 ). Exper i mental analysis found that both miRNAs regulate an ove r lapping set of gene targets and that KSHV infected prim a ry effusion lymphoma (PEL) cell lines do not express miR 155. These data suggested that miR K1211 may mimic the function of miR 155 in PEL cells, causing dysregulated B cell differentiation. In this study, we examined the e f fects of miR K1211 expression and its ability to mimic miR155 in human cord blood progenitors during hemat o poiesis in NOD/ LtSzscid IL2R!null mice. Using foamy virus we forced expression of miR K1211 or miR 155 in proge nitor cells transplanted into sub lethally irradiated mice. Following reconstitution, we analyzed cell lineage differe n tiation with FACS and histological staining. Results show that forced expre ssion of either miRNA leads to increased Bcell expansion in the spleen. This is the first in vivo study examining the role of a KSHV miRNA in human cells and further promotes the hypothesis that miR K1211 is a functional mimic of miR155. In summary, th ese data suggest a role for miR K1211 in KSHV lymphomagenesis. 9. Recombination of adaptive alleles in introduced populations Bouchard F 1, McBride G1, Marcus C1, Wayne ML2 ,* 1Graduate Program in Genetics and Genomics, University of Florida, Gainesville FL 2Department of Biology University of Florida, Gainesville, FL During introductions to new environments, populations have an increased probability of extinction due to a s e vere genetic bottleneck and a suite of selective pressures to which they are n ot adapted. Multiple introductions of genetically divergent populations followed by interbree d ing can produce recombinants with adaptive alleles from each source that are better adapted to the introduced environment and more likely to survive than a single i n troduction. It has been proposed that this complementary gene action may be an important evolutionary force in the establishment of exotic invasive species. Testing the e f fect of multiple introductions in the field is difficult due to problems in sampli ng, lack of replication, and lack of proper controls. Using several geographically divergent lines of Drosophila melanogaster and ethanol concentrated food as a novel environment; the effects of mu l tiple introductions were investigated in an artificial evo l u tion experiment. Egg to adult viability was used as a measure of fitness in the novel environment. Results show that having multiple introductions increases fitness for some populations. This could have implications in the study of invasive species and t heir management. 10. Tradeoff hypothesis and evolution of mycov i rus virulence Brusini J 1, Robin C2 1Department of Biology University of Florida, Gainesville, FL 2INRA, UMR1202 BIOGECO, Equipe de Pathologie F ores tire, Villenave d'Ornon Cedex, France
All viruses that infect fungi are avirulent (i.e host fitness does not decline in response to infection). These mycov i ruses can be transmitted vertically or horizontally, with horizontal transmission occurring during somatic fusion. Here we present a rare example of a virulent mycovirus, Cryphonectria hypovirus 1 (CHV 1), which infects the fu n gal pathogen Cryphonectria parasitica in Europe. In a cross inoculation experiment, we found important differ ences in the degree of host specialization between the tw o sub types studied, including a strong positive correla tion between virulence and horizontal transmission for one viral sub type. The low diversity of genes controlling fusion in European populations of C. parasitica suggests that horizontal transmission of the virus is common and, as such, our results s uggest that the virulence of at least one subtype of CHV1 is driven by the trade off hypoth e sis of virulence evolution. We therefore suggest that the general avirulence of mycoviruses is the direct cons e q uence of the high diversity of genes controlling fusion in fungalhost populations. 11. Expansion of a direct shoot organogenesis sy s tem in peanut to include U.S. varieties Burns S 1, Gallo M1,2,*, Tillman B1, 3 1Agronomy Department, University of Florid a, Gainesville, FL 2Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 3North Florida Research and Education Center, University of Florida, Marianna, FL An alternative to lengthy bombardment and regeneration protocols is Agrobacteriummediated transformation e m ploying direct shoot organogenesis, which allows for transgenic plants to be obtained quickly (3 4 months). Peanut cultivars, Florida 07 (Runner), Georgia Green (Runner), Georgia Brown (Spanish), New Mexico A (V a lencia), and VC2 (Virginia), were selected to represent all four market types. Two types of cotyledonary explants were examined, those that previously had an attached embryoaxis upon cotyledon separation (explant A) and those that were embryo axis free upon separation (e x plant B). Explants were classified as having no growth, callus like growth, adventitious bud formation or small plantlet development (1, 2, 3 and 4 respectively). Diff e rential shoot induction was observed for the cotyledon explants examined (Pr>[t]= <0.0001 ). Explant A had greater shoot induction with a visual rating of 1.75, while explant B had a rating of 1.64 (Pr>[t]= <0.0001). Cult i vars responded to the culture conditions differently with Georgia Green on 40 $ M BA producing the m ost shoot buds (31.2%) and the highest visual rating (2.22), fo l lowed by VC2 on 10 $ M BA (17.3%, 1.84), New MexicoA on 640 $ M BA (15.9%, 1.84), Georgia Brown on 80 $ M BA (9.1%, 1.73), and Florida 07 on 40 $ M BA (5.6%, 1.82). Georgia Green, New Mexico A an d VC2 appear to be the best suited for future transformation experiments based on their shoot bud production. 12. An analytical methodology for the in situ visu a lization of cellulases in maize cell wall mutants Caicedo HM 1, Ladisch M2, Mosier N2, Vermer ris W1, 1Agronomy Department, University of Florida, Gainesville, FL 2Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN The current interest in alternative fuels from renewable resources has been triggered by t he increasing global demand for energy along with environmental concerns. Corn stover, the vegetative residues remaining after the grain harvest, is an abundant feedstock (lignocellulosic biomass) for the production of fermentable sugars (gl u cose) which ca n be used for the production of ethanol. The composition and structure of the lignocellulosic biomass has a big impact on the efficiency of enzymatic sa c charification. We have studied the composition of lignin in corn stover through genetic approaches. The brown m i drib1 (bm1), bm3 and near isogenic bm1 bm3 mutations affect lignin subunit composition and each increase the yield of glucose per gram dry stover relative to the wild type control. Here, we present an analytical methodology to investigate the basi s of the enhanced hydrolysis in the bm mutants by assaying the binding of cellulases to sto v er, using recombinant proteins consisting of the cellulose binding module (CBM) isolated from Trichoderma reesei endoglucanases labeled with green fluorescent prote in (GFP). Because of lignin autofluorescence, this approach can not be performed in situ, but instead has to rely on a fluorescence subtraction assay. The optimized assay showed that the increased rate of hydrolysis in the bm mutants was due to enhanced bin ding relative to the wild type control, which effectively increases the titer of the cellulases. 13. Rapid microsatellite development in organisms without sequenced genomes using 454 s equencing Chaffee CL Braun EL*, Osenberg CW Department of Biology, University of Florida, Gainesville, FL Until recently, computational methods for identifying candidate microsatellite loci were only available for those o r ganisms with a sequenced genome. New, low cost DNA sequencing technology (454 Titanium shotgun seque n c ing), however, makes it possible to use these computa tional methods for organisms that do not have such g e nomic resources available. We used such an approach to identify candidate loci for two coral reef organisms for which little is known about their po pulation genetics: Christmas tree worms (Spirobranchus giganteus) and vermetid gastropods (Dendropoma maximum). Although superficially similar, the species are quite divergent phyl o genetically, have different larval durations (and hence dispersal potential ) and interact with corals in opposite ways. By using ICBRs 454 sequencing capabilities, we were able to identify hundreds of candidate loci, without the need to build costly and time
consuming screening libraries. Employing modern sequencing technologies a l lowed us to quickly focus our efforts on gathering data to understand the patterns of dispersal and relatedness within the S. giganteus and D. maximum clades. Theoret i cal dispersal potential is greater for S. giganteus, so we expect D. maximum to show l ess evidenc e of gene flow, but a multitude of factors may keep actual dispersal well below theoretical limits. Understanding the population dynamics of these organisms could open up exciting new links between community ecology and phylogeography. 14. Micr oarray analyses of the transcriptional re s ponses of human peripheral m o nocytes to Bacillus anthracis lethal toxin Chauncey KM 1, Lopez MC2, Szarowicz SE1, Baker HV2 ,*, Southwick FS1 1Department of Medicine, University of Florida, Gaines ville, FL 2Depa rtm ent of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL Anthrax lethal toxin (LT), produced by the Gram positive bacteria Bacillus anthracis, is a potent zinc dependent m e talloprotease that cleaves the N terminus of MAPKKs and i s known to play a major role in impairing the host i m mune system during an inhalation anthrax infection. Here, we present the transcriptional responses of lethal toxin treated human monocytes in order to further elucidate the mechanisms of LT induced host immune system collapse. Using Affymetrix Human Genome U133 Plus 2.0 Arrays, we identify over 820 probe sets differentially regulated after LT treatment at a 0.001 significance, interrupting the normal transduction of over 60 known pathways. As e x pected, th e MAPK signaling pathway was drastically af fected by LT, while the p38 pathway was also highly i m pacted. Numerous genes outside the well recognized pathways were also influenced by LT, including genes involved in actin regulation, signal transduction, tran scri p tional regulation and cytokine signaling. Using these r e sults, we can further understand how anthrax LT impairs normal human monocyte function by focusing on the unique transcriptional responses and their contribution to host immune system dysfunction 15. Proteomics and mass spectrometry applic a tions in biomedical research Diaz C, Chow M, Zheng R, Chung A, Chen S Proteomics Division, Interdiscipli nary Center for Biotec h nology Research, University of Florida, Gainesville, FL Proteomics and mass sp ectrometry have provided u n precedented tools for fast, accurate, high throughput biomolecular separation and characterization, which are indispensable towards understanding the biological and medical systems. Studying at the protein level allows r e searchers to investigate how proteins, their dynamics and modifications affect cellular processes and how cellular processes and the environment affect proteins. Here we present our capabilities in proteomics and other analytical services. The tools include a gel based 2D DIGE (Two Dimens ional Difference Gel Electrophoresis) and gel free iTRAQ (Isobaric Tags for Relative and Absolute Quantit a tion). Along with our capacity of separating thousands of proteins and characterizing differential protein expression, we hav e a suite of state of the art mass spectrometers available for biomedical sciences and advanced technol o gy research. These instruments are mainly used for pr o tein identification, posttranslational modification characterization and protein expression analys is (e.g., Mass Wes t ern). Our facility is also set up to provide Edman de novo N terminal protein sequence analysis and Biacore biom o lecule interaction analysis. We are fully set up to synthe s ize and purify peptides and have a good track record with this se rvice as well. Proteomics and mass spectrometry are useful in large scale survey of proteome for hypoth e sis generation as well as in detailed analysis of target pro teins for hypothesis testing. 16. A redox active isopropylmalate dehydrogenase functions in the biosynthesis of glucosinolates and leucine in Arabidopsis He, Y1,2, Mawhinney T3 ,4, Preuss M5, Schroeder AC5, Chen B1, Abraham L1, Jez J5, Strul J1, Chen S 1,2,6,* 1Department of Biology, University of Florida, Gainesville, FL 2Plant Molecular and C ellular Biology Program University of Florida, Gainesville, FL 3Department of Biochemistry University of Missouri, C o lumbia, MO 4Department of Child Health, University of Missouri, C o lumbia, MO 5Department of Bi ology, Washington University, St. Louis, MO 6Proteomics Division, Interdiscipli nary Center for Biotec h nology Research, University of Florida, Gainesville, FL We report a detailed functional characterization of an Arabidopsis isopropylmalate dehydrogenase (AtIPMDH1) that is involved in both gluco sinolate biosynthesis and leucine biosynthesis. AtIPMDH1 shares high homology with enzymes from bacteria and yeast known to function in leucine biosynthesis. In plants, AtIPMDH1 co expresses with nearly all known genes in aliphatic glucosinolate bi o synthes is. Mutation of AtIPMDH1 leads to a significant reduction in the levels of free leucine and glucosinolates with side chains of four carbons or longer. Complement a tion of the mutant phenotype by ectopic expression of AtIPMDH1, together with the enzymes sub strate specific i ty, implicates AtIPMDH1 in both glucosinolate and leucine biosynthesis. This functional assignment is substantiated by the subcellular localization of the protein in the chl o roplast stroma and the gene expression patterns in diffe r ent
tissu es. Interestingly, AtIPMDH1 activity is regulated by a thiol based redox modification. Collectively, this study has characterized the first enzyme in plants that catalyzes the oxidative decarboxylation step in both le u cine biosynthesis (primary metabolism) and methionine chain elongation of glucosinolates (specialized metabo l ism). It provides evidence for the hypothesis that the two pathways share a common origin and suggests a role for redox regulation of glucosinolate and leucine synthesis in plants. 1 7. A novel transcriptional pathway regulating x e nobiotic detoxification genes in Caenorhabditis elegans as a target for multidrug resistance Choe KP* Department of Biology, University of Florida, Gainesville, FL Anthelmintics have been used to control p arasitic nem a todes for decades and many species are evolving multidrug resistance. In diverse organisms, multidrug resi s tance is mediated by the increased expression of enzymes that detoxify xenobiotics. Unfortunately, the molecular and genetic mechanisms of detoxification and their roles in multidrug resistance are poorly defined in nemat odes. Transcription factors that control the expression of xenobiotic detoxification genes are promising, but largely u n explored, multidrug resistance targets. The transcr iption factor SKN 1 activates the expression of xenobiotic deto x ification genes in the nematode C. elegans. We recently used genomewide RNAi screening to identify a principal pathway regulating SKN 1. Genetic, molecular, and bi o chemical data support a model in which the WD40 repeat protein WDR 23 regulates SKN 1 abundance. Importantly, the homologous mammalian transcription factor Nrf2 is regulated by a distinct mechanism. Therefore, the WDR 23/SKN 1 pathway is a promising new target for drugs that inhibit xenobiotic detoxification and drug resistance in nematodes without affecting analogous pathways in mammals. The small size, simple culturing characteristics, and genetic tractability of C. elegans make it an ideal sy s tem in which to screen for pharmacolog ical inhibitors of SKN 1 that would provide tools for studying the role of SKN 1 in parasitic species and could greatly increase the useful life of anthelmintics. 18. Cell autonomous role of hedgehog signaling in notochord and i ntervertebra l disc d evelopm ent Choi KS Harfe BD* Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL The vertebrate notochord is a transient embryonic stru c ture 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 nuc leus pulposus, which is located in the center of each i n tervertebral disc. Although hedgehog signaling has been well studied over the past decades, the role of hedgehog s ignaling in the notochord and nucleus pulposus is still unclear. Here we show that hedgehog signaling is re quired not only for maintaining notochord structure but for forming the nucleus pulposus. Upon loss of hedgehog signaling, notochord formation was i nitiated but failed to differentiate to make the notochord sheath, which normally surrounds the notochord. Failure to form a notochord sheath resulted in aberrant nucleus pulposus formation and downregulation of T, Noto, and Foxa2 expre s sion in caudal mut ant notochords. Interestingly, Shh e x pression was also decreased in the entire notochord and floorplate during early embryonic development. In later development, Shh expression in the notochord became discontinuous in the caudal mutant notochord. Our data indicates that hedgehog signaling plays a role in maintai n ing Shh expression in both the notochord and floorplate. In addition, these results demonstrate that hedgehog si g naling is required for formation of the notochord sheath and intervertebral discs. 19. A noncoding RNA found during gonadogenesis in a turtle that expresses temperature dependent sex determination Chojnowski JL Braun EL* Department of Biology, University of Florida, Gainesville, FL Noncoding RNAs (ncRNAs) have been spotlighted in the past few years as more than just regulatory machinery and wasted space. They have been implicated in major biological processes such as cancer development, gene expression regulation, and nuclear trafficking. MALAT1 (metastasis associated lung adrenocar cinoma transcript 1) is a recently discovered long ncRNA found to be upregu lated in breast cancer. It is conserved among mammals and it may regulate gene expression, possibly after clea vage yielding a smaller tRNA like cytoplasmic RNA (~61nt). MALAT1 may have a more permanent role in development than previously thought since it accumulates at high levels in mammalian ovaries and shows high levels of accumulation in male turtles during gonadogenesis. Determining local expression patterns and establishing p atterns of hormonal regulation in the developing turtle gonad will reveal the contribution of MALAT1 to turtle g o nadogenesis and facilitate understanding the role of this ncRNA in sexual development across vertebrates. 20. Generation of early AMD mouse model by i n duction of oxidative stress Seo S1 Cohen ZP1, Yun L2, Hauswirth WW3,*, Lewin AS1,* 1Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL 2Department of Ophthalmology, University of Oklahoma Health Scienc es Center, Oklahoma City, OK
3Department of Ophthalmology, University of Florida, Ga i nesville, FL Purpose: Oxidative stress in the retinal pigment epith e lium (RPE) complex is thought to be a leading cause in the development of age related macular degeneration. Our hypothesis is that reduction of RPE specific MnSOD2 will cause increased levels of reactive oxygen species in the retina/RPE/choroid complex leading to pathogenesis of the early signs of AMD. Methods: We introduced Cre through a subretinal injec tion of VMD2 CRE virus into the floxed SOD2 mice. Alternatively, we bred mice to intr o duce a Tet On RPE65Cre+/ allele into SOD2f/f mice. The expression of MnSOD2 was measured by immunohist o chemical staining for MnSOD2 in RPE flat mounts. The disease pheno types were characterized by fundus, dih y droethidium, 4 HNE staining and histological analysis and fundus. Results: In the reporter mouse line, YFP expre s sion was detected in the RPE layer. Reduced expression of sod2 was confirmed by staining of SOD2 in RPE flat mounting, immunohistochemical staining and western blot. The increased oxidative stress in the RPE was mea s ured by DHE, DCFDA and 4 HNE staining. The histology of retinas showed shortened outer photoreceptors, thinning of outer nuclear layer and RPE damages. Fundus was pe r formed and showed significant increased number of ye l lowish deposits that appeared drusen. Conclusion: RPE specific down regulation of SOD2 leads to increased oxi d ative stress in the RPE and histological changes. 21. Functional interchangeability of rod and cone transducin subunits Deng W T 1, Sakurai K2, Liu J1, Dinculescu A1, Li J1, Pang J1, Chiodo VA1, Boye SL1, Chang B3, Kefalov VJ2, Hau s wirth WW1 ,* 1Department of Ophthalmology, University of Florida, Ga i nesville, FL 2Departme nt of Ophthalmolog y and Visual Sciences, Washington University, St. Louis MO 3The Jackson Laboratory, Bar Harbor, ME Rod and cone photoreceptors use similar but distinct sets of phototransduction proteins to achieve different fun c tional properties, suita ble for their role as dim and bright light receptors, respectively. The role of the structural differences between rod and cone transducin subunits (T ) in determining the functional differences between rods and cones is unknown. To address this question, we studied the translocation and signaling properties of rod T expressed in cones and cone T expressed in rods in three mou se strains: rod T knockout, cone T GNAT2cpfl3 mutant, and rod and cone T double mutant rd17 mouse. Surprisingly, although the rod/cone T" are only 79% identical, exogenously expressed rod or cone T" localized and translocated identically to endogenous T in each photoreceptor type. Moreover, exogenously expressed rod or cone T" rescued electroretinogram re s ponses (ERGs) in mice lacking functional cone or rod T respectively. Ex vivo transretinal ERG and single cell r e cordings from rd17 retinas treated wi th rod or cone T showed comparable rod sensitivity and response kinetics. These results demonstrate that cone T forms a function al heterotrimeric Gprotein complex in rods and that rod and cone T couple equally well to the rod phototransduc tion cascade. Thus, rod and cone transducin subunits are functionally interchangeable and their signaling pro p erties do not contribute to the intrinsic light sensitivity differences between rods and cones. 22. Genetic connectivity, phylogeography and demograp hic hi story of the West Indian t opshell Cittarium pica (Arqueogastropoda: Trochidae): impli cations for management and conservation Diaz Ferguson E 1, Haney RA2, Silliman B1, Wares J3 1Department of Biology University of Florida Gainesville, FL 2Department of Organismal Biology and Anatomy, Unive r sity of Chicago Chicago, IL 3Department of Genetics University of Georgia, Athens, GA We examined the phylogeographic structure, genetic d i versity, demographic history and connectivity patterns of the the Caribbean Top Shell, along its distributional range using mtDNA sequence variation (COI and 16S). Genetic diversity patterns determined using values of nucleotide and haplotype diversity exhibited a longitudinal gradient increase from South Western areas to Eastern and North Western regions. Tajima s DT and Fus Fs neutrality tests were significant (p<0.04) and negative on Eastern and North Western sites as evidence for population expansion. Phylogeographic structure and genetic connectivity was tested using AMOVA, Fst per wise and migration rates. Spatial distribution of haplotypes was examined using median joining haplotype networks, parsimony trees and Monmoniers algorithm. Phylogeographic structure among regions and within populations was elevated and five r e gio ns were detected: 1. North Western (BSS) 2. North Eastern (WPR and USVI) 3. South Eastern (BN) 4. Central Panama (BV) and 5. South Western (CR, PC and BO). The observed regional structure was concordant with previous regions established for corals and fish es in four out of five cases (1, 2, 4, 5). Connectivity was restricted to specific areas and coherent with current patterns also in four out of fiv e cases (<300Km of separation; 1. South Western sites (CR, PC, BO) 2. North Eastern sites (USVI and WPR) 3 B onaireSouth Western sites 4. Bahamas North Eastern 5. Bahamas Buenaventura ) 23. Reducing lignin content in bahiagrass by down regulation of 4 coumarateCoA ligase Fouad WM 1,2, Martin L1,2, Vermerris W1,2,*, Altpeter F1,2,* 1Agronomy Department, Uni versity of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Program University of Florida, Gainesville, FL
Bahiagrass is one of the most important warm season forage grasses. In Florida alone it is grown on more than 5 million acres. Howeve r, the high lignin content in the bahiagrass biomass significantly reduces its forage qual i ty. A key enzyme in the lignin biosynthetic pathway is th e 4coumarate CoA ligase (4CL); it catalyzes the formation of CoA thiol esters of 4 coumarate and other hydr oxycinn amates. We cloned four 4CL cDNA s from tetraploi d ba hiagrass cv. Argentine and an RNAi construct targeting a highly conserved domain was constructed using 200 bp of the coding sequences. The 4CL RNAi construct was intr o duced to bahiagrass callus by b iolistic gene transfer under transcriptional control of three alternative promoters: the constitutive e35S promoter, OsC4H promoter for xylem specific expression and the ZmdJ1 promoter for expre s sion in the green tissue. Following regeneration of plants their transgenic nature was confirmed using PCR and Southern blot analysis. Significant reduction of 4CL gene expression was detected in several transgenic lines by Northern blot analysis. RNAi suppression of 4CL was more effective under transcriptional cont rol of the xylem speci f ic OsC4H promoter than under control of the e35S or ZmdJ1 promoters. Data describing the effect of 4CL suppression on Klason lignin in transgenic lines will be pre sented. 24. The rough endosperm3 locus encodes a predicted splicing f actor required for plant develo p ment and regulation of cell proliferation Fouquet R Fajardo D, Martin F, Policht T, Tseung CW, Settles AM* Horticultural Sciences Department, University of Florida, Gainesville, FL Plant development requires controlled ce ll proliferation and cell differentiation. The rough endosperm3 (rgh3) mutant is essential for maize seed and seedling develo p ment. Our phenotypic characterization of rgh3 suggests that this locus is required to regulate both cell differenti a tion and proli feration. Rgh3 seedlings germinate at a low rate and seedling leaves are typically adherent. Mutant rgh3 plants are lethal within 2 4 weeks of planting; ho w ever, the Rgh3 locus is not essential for cell viability. M u tant endosperm tissues are far more prol iferative than normal endosperm tissues when grown in vitro. These data suggest that the Rgh3 locus has an essential dev e lopmental role. We cloned a Mu1 insertion that is tightly linked to the rgh3 mutant. The Mu1 element disrupts a U2AF35 Related Protein that we named ZmUrp. We iden tified a second rgh3 allele from a directed EMS tagging experiment that suggests the loss of ZmURP protein causes the rgh3 phenotype. ZmUrp produces multiple spliced products with only one variant encoding a pr e dicted protein. A GFP fusion with ZmURP is localized to the nucleolus and nuclear speckles when transiently e x pressed in Arabidopsis protoplast or in tobacco epidermal cells. These data are consistent with ZmURP having a function in RNA splicing and suggest a role for RNA spli c ing in regulating cell proliferation and cell differentiation. 25. Using Saccharomyces cerevisiae as a model to study the genetic interaction between Arabidopsis 143 3s and G box binding factor 3 (GBF3) Gokirmak T 1, Laughner BJ2, Paul AL2,*, Ferl RJ1,2,* 1Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2Horticultural Sciences Department, University of Florida, Gainesville, FL G box binding factor 3 (GBF3) is a member of a bZIP transcription factor family that recognizes a cis acting element called G box (5' CCACGTGG 3'). G box is present in many environmentally regulated plant gene promoters (Lu et al., 1996, Plant Cell 8 (5):847 57; Mallappa et al., 2008, J Biol Chem 283 (51):35772 82; Menkens et al., 1995, Tren ds Biochem Sci 20 (12):506 10; Shinozaki et al., 1997, Plant Physiol 115 (2):327 34). Microarray and RT PCR studies showed that GBF3 is regulated both dev e lopmentally and environmentally. In plants, 143 3s were first discovered to be part of transcriptional G box DNA binding complexes in Arabidopsis Adh promoter (Lu et al., 1992, Proc Natl Acad Sci U S A 89 (23):11490 4). This same study showed that the 14 3 3 proteins did not di rectly interact with G box. However, it is proposed that 143 3s can be part of t he G box multiprotein complex through interaction with GBF3. In this study, we showed that there is a genetic and direct interaction between 14 3 3s and GBF3. Expression of GBF3 in Saccharomyces cerevisiae causes cellular toxicity. This toxicity can be eli minated by deletion of N terminal proline rich domain or the C terminal uncharacterized domain. This suggests that these two domains are essential for GBF3 function. Furthermore, co expression of 14 3 3s in yeast rescues GBF3 mediated cellular toxicity thr ough direct 143 3/GBF3 interaction. 26. Comparative proteomics of redox regulated proteins Alvarez S1, Zhu M1, Goldsmith J 1, Chen S1,2,* 1Department of Biology, University o f Florida, Gainesville, FL 2Proteomics Division, Interdiscipli nary Center for Biotec h nology Research, University of Florida, Gainesville, FL We report the dynamic changes of redox proteins in r e sponse to oxidative stress induced by methyl jasmonate (MeJA) in Arabidopsis using a 2D gel based comparative proteomics approach. Monobrom obimane (mBBr), a flu o rescent dye, was used to label the thiol groups of proteins obtained after alkylation of free thiol groups and reduction of disulfide bonds. The labeled proteins were separated on 2D gels. After visualization of disulfide proteins, to tal proteins were stained with SyproRuby to compare the signal intensity of the thiol groups labeled with
mBBr and the protein expression levels. A comparative map of p o tential redox regulated proteins from MeJA treated and control Arabidopsis shoots and r oots was established. Proteins involved in the sulfur and the antioxidant metabo l isms (e.g., cysteine synthase, dehydroascorbate redu c tase), the lipid metabolism (e.g., mosaic death 1), the jasmonate biosynthesis pathway (e.g., allene oxide cy c lase 2) and several stress responsive proteins were ident i fied using LC MS/MS. Collectively, the comparative pr o teomics approach allowed us to differentiate potential redox proteins from structural disulfide proteins. Novel disulfide proteins were identified, and the cysteines i n volved in the formation of disulfide bonds were mapped. The functional significance of the redox proteins will be discussed. 27. Regulation of cysteine synthesis in soybean Gordon C 1,2, Kirst M2,*, Vyas D2,3, Harmon A1,2,* 1Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2Department of Biology, University of Florida, Gainesville, FL 3Indian Institute of Integrative Medicine (CSIR), Jammu, India Cysteine biosynthesis is the terminal step of sulfur assim i la tion, and it lies at the junction between sulfur and nitr o gen metabolism. Cysteine is synthesized from serine by the enzymes serine acetyltransferase (SAT) and O acetylserine (thiol) lyase (OASTL). In Arabidopsis, cyst e ine synthesis is regulated by metabol ites which control the reversible formation of a complex between SAT and OASTL, called the cysteine synthase complex (CSC). Our results for three SATs and two OASTLs from soybean show that, as in Arabidopsis, formation of CSC enhances SAT activity while it inhibits OASTL activity. However, the plastidic/cytosolic SAT (GmSerat2;1) was less able than the cytosolic SAT to inhibit OASTL. GmSerat2;1 has an isoform unique phosphorylation site, and the mutant GmSerat2;1S378D mimics constitutive phosphorylation in that it is insensitive to feedback inhibition by cysteine. GmSerat2;1S378D did not inhibit OASTL activity to the same degree as wild type isoforms. In addition, in assays of the ability of CSC to synthesize cysteine from serine, complexes containing GmSera t2;1S378D showed the highest activity relative to those containing wild type e n zyme. These experiments suggest that phosphorylation supports higher production of cysteine, by relieving fee d back inhibition of GmSerat2;1 and by lowering inhibition of OASTL i n the CSC. (Supported by USDA NRI CREE S Award 2006 3531817392) 28. Evolutionary patterns of HCV infection are i n dependent of clinical phenotype Gray RR 1, Santos LA2, Veras NMC3, Goodenow MM1 ,*, S a lemi M1 ,* 1Department of Pathology, Immunology and Labo ratory Medicine, University of Florida, Gainesville, FL 2Laboratorio Avancado de Saude Publica, Centro de Pes quisa, Goncalo Moniz, Fundacao Oswaldo Cruz, Salvador, BA, Brazil 3Instituto de Biologia, Universidade de Brasilia, Brasilia, DF, Brazil Backgroun d: Hepatitis C virus (HCV) infection is ass o ciated with development of hepatocellular cancer (HCC), chronic hepatitis and liver cirrhosis. The high evolutionary rate of HCV results in the rapid accumulation of mutations over time. Although previous studies have attempted to define a correlation between evolutionary patterns and in vivo viral pathogenicity, the relationship remains unclear. Methods: Previously published sequence datasets from 22 HCV infected subjects were re analyzed individually using power ful phylogenetic techniques. Datasets were derived from serially sampled plasma (n=15) or multiple tissues (n=7) and included sequences from the core gene (n=7), the hypervariable region (HVRI, n=9) and the envelope region (n=6). Results: The HVRI dataset contained too little phylogenetic signal to analyze with confidence. Inferred phylogenies for the longitudinal envelope s e quences indicated strong temporal evolution, purifying selection, and constant population diversity. Patterns were consistent between the subjects with mild and severe di s ease. In the datasets containing core sequences amplified from multiple tissues, several analyses detected signif i cant anatomical compartmentalization and population subdivision in a subset of patients with HCC. Conclus ions: Previously reported characteristics of HCV evolution p o tentially linked with liver disease stage and development of HCC could not be replicated in this study using robust phylogenetic methods. 29. Regulatory divergence in Drosophila melan o gaster an d D. simulans, a genome wide analysis of allele specific expression Graze RM 1, McIntyre LM1,2,*, Main BJ3, Wayne ML4,*, Nuzhdin SV3,5 1Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL 2Department of Statistics, Un iversity of Florida, Gaine s ville, FL 3Section of Molecular and Computational Biology, Depar t ment of Biological Sciences, University of Southern Cal i fornia, Los Angeles, CA 4Department of Biology, University of Florida, Gainesville, FL 5Department of Evolut ion and Ecology, University of Cal i fornia, Davis, CA Species specific regulation of gene expression contributes to the development and maintenance of reproductive iso lation and to species differences in ecologically important traits. A better understand ing of the evolutionary forces which shape regulatory variation and divergence can be developed by comparing expression differences among species and interspecific hybrids. Once expression diffe r ences are identified, the underlying genetics of regulatory variation or
divergence can be explored. With the goal of associating cis and/or trans components of regulatory d i vergence with differences in gene expression, overall and allele specific expression levels were assayed genomewide in female adult heads of D. melanogaster, D. simulans and their F1 hybrids. A greater proportion of cis di f ferences than trans differences were identified for genes expressed in heads and, in accordance with previous st u dies, cis differences also explained a larger number of spec ies differences in overall expression level. Regulatory divergence was found to be prevalent among genes associated with defense, olfaction, and among genes dow n stream of the Drosophila sex determination hierarchy. In addition, two genes, with critical roles in sex determin a tion and micro RNA processing, Sxl and loqs, were identi fied as misexpressed in hybrid female heads, potentially contributing to hybrid incompatibility. 30. Tissue specific roles of FgfR2 in urethral tube closure Gredler ML 1, Seifert AW1, Ornitz DM2, Cohn MJ1,3,4,* 1Department of Biology, University of Florida, Gainesville, FL 2Department of Developmental Biology, Washington Un i versity, St. Louis, MO 3Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesvil le, FL 4Howard Hughes Medical Institute, University of Florida, Gainesville, FL Hypospadias is a malformation of the penis that is chara c terized by incomplete closure of the urethral tube and affects approximat ely 1 in 125 live male births. Patterning of the mammalian external genitalia requires coordination of proximal to distal outgrowth with urethral tubulogen e sis. Reciprocal signaling between the urethral epithelium and mesenchyme of the genital tubercle the embryonic precursor of the penis and clito ris maintains the expression of key genes necessary for developm ent of the e x ternal genitalia. Fibroblast growth factor 10 (Fgf10) is e x pressed in the genital tubercle mesenchyme and signals through the receptor FgfR2 that is expressed in two adjacent ce ll populations, the urethral epithelium and the ectoderm. Null mutations in FgfR2 result in failure of ureth ral tube closure (hypospadias) and loss of mature urethral epitheli um. In this study, we explore the distinct roles of FgfR2 in the urethra and in t he 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 maintain s relatively normal character. In contrast, deletion of FgfR2 from urethral en doderm inhibits its maturation into a complex epith e lium, although gro ss hypospadias does not occur. Our results demonstrate that FgfR2 plays distinct roles in these two cell populations, and that both are necessary for proper patterning of the genital tub ercle and matur a tion of the urethra. 31. The role of Nlr1 in leaf and plant development Gustin JL Fajardo DS, Tseung CW, Black J Settles AM* Horticultural Sciences Department, University of Florida, Gainesville, FL The developmental program that produ ces a flat leaf blade requires specification of polar domains including establishment of the a daxial and abaxial (top/bottom) axi s. Several developmental mutants suggest transcript processing mechanisms are likely to have important roles in leaf morphogene sis. We recovered a maize mutation that impacts development in multiple tissues including distinct narrow leaf and rough endosperm (nlr1) phen o types. The nlr1 mutant displays characteristic markers of altered adaxial/abaxial domain specification including r e duced blade expansion, reduced number of macrohairs, and irregular lateral vein organization. In addition, nlr1 alters transcript accumulation of adaxial leaf polarity genes in developing leaves. These data suggest Nlr1 is involved in adaxial domain spec ification in maize. We identified a transposon insertion from a Robertsons Muta tor (Mu) flanking sequence tag that is tightly linked to nlr1. The transposon disrupts a Type C, J domain protein, named DjC78. J domains activate Hsp70 ATPase activity and pr oteins containing these domains have diverse fun c tions in th e cell. DjC78 also contains an arginine/serine (RS) rich domain, which is found in pre mRNA splicing factors. Transient expression of ZmDjC78 indicates local i zation to the nucleus in punctate sub domains consistent with a hypothesis that ZmDjC78 is involved in transcri p tional processing. Based on these data, we hypothesize Nlr1 has a role in developmental gene expression. 32. A subset of the diverse COG0523 family of putative metal chaperones is l inked to zinc homeost a sis in all kingdoms of life Haas CE 1, Rodionov DA2,3, Kropat J4 ,5, Malasarn D4 ,5, Me r chant SS4 ,5, de Crcy Lagard V1 ,* 1 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 2Burnham Institute for Medi cal Research, La Jolla, CA 3AA Kharkevich Institute for Information Transmission Problems, Moscow, Russia 4Department of Chemistry and Biochemistry Univers ity of California Los Angeles, CA 5Institute for Genomics and Proteomics, University of Cal i fornia, Los Angeles, CA COG0523 proteins are, like the Ni chaperones of the UreG family, part of the G3E family of GTPases linking them to metallocenter biosynthesis. Even though the first COG0523 encoding gene, cobW, was identified almost 20 years ago, little is known concerning the func tion of this ubiquitous family. Based on a combination of compar a tive genomics, literature and phylogenetic analyses and experimental validations, the COG0523 family can be separated into at
least fifteen subgroups. The CobW su b group involved in B12 synthesis represents only one small sub fraction of the family. Another, larger subgroup, is suggested to play a predominant role in the response to Zn limitation based on the presence of the corresponding COG0523encoding genes downs tream from putative Zur binding sites. We have also predicted a link between COG0523 and regulation by Zn in Archaea and show that two COG0523 genes are induced upon Zn depletion in a eukaryotic reference organism, Chlamydomonas reinhar d tii. This work lay s the foundation for the pursuit by exp e rimental methods of the specific role of COG0523 in metal trafficking. Based on phylogeny and comparative geno m ics, both the metal specificity and the protein target(s) might vary from one subgroup to another. Additi onally, Zur dependent expression of COG0523 may represent a mechanism for hierarchal Zn distribution in the face of inadequate Zn nutrition. 33. A phylogenomic survey of avian transposons Han K L Braun EB*, Kimball RT* Department of Biology, Universit y of Florida, Gainesville, FL Transposon insertions are rare genomic changes that are thought to have a low probability of multiple independent insertions at any single location, making them useful for phylogenetics. We searched for transposons and determi ned their distribution among loci and clades by exami n ing non coding sequences from a large scale dataset representing the diversity of birds. The majority of tran s posons identified were CR1 (chicken repeat 1) elements, which were even more frequent relati ve to other transp o son types than expected based upon the chicken genome. Most large insertions in the aligned intron data were attributed to transposable elements, suggesting that transpo sons have a major effect on genome size variation among species. Les s than half of the insertions were synapomo r phic, and these united clades that were also well su p ported by nucleotide analyses. Although most transposon insertions were homoplasyfree, several appeared to e x hibit homoplasy or presented other potential prob lems for phylogenetic analyses due to independent insertions at the same site or precise deletion of an insertion. These results suggest that transposons, while a valuable tool in phylogenetics, need to be examined rigorously, and co n clusions based upon th ese insertions treated with caution unless corroborated by congruence among multiple inse r tions or independent types of data. 34. Polymorphisms in KSHV microRNA sequences observed in clinical samples from KS and MCD patients cause miRNA maturation and exp ression di f ferences Han S 1, Marshall V2, Whitby D2, Renne R1 ,* 1Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL 2Viral Oncology Section, AIDS and Cancer Virus Program, SAICFrederick, NCI Frederick, Frederick, MD MicroRNAs (miRNAs) are 22 24 nt singlestranded RNA molecules that post transcriptional regulate gene expres sion. So far 12 miRNAs have been identified in Kaposis sarcomaassociated herpesvirus (KSHV), which causes Kaposis sarcoma (KS), Multicentric Ca stleman Disease (MCD), and Primary effusion lymphoma (PEL). Recently, we sequenced the KSHV miRNA encoding region in clinical samples from KS and MCD patients of different geographical origins and several PEL cell lines. Our analysis r e vealed single or mul tiple nucleotide polymorphisms within pre miRNA sequences in several miRNAs (Marshall et al., 2007, J Infect Dis 195(5):645 59) Because miRNA processing by microprocessor and Dicer depends on primiRNA secondary structure, effects of polymorphisms on miRN A maturation cannot be deduced. To ask whether naturally occurring polymorphisms affect miRNA expre s sion, we analyzed polymorphisms occurring in several pre miRNA for their expression. Using in vitro maturation a s says, we determined the maturation affect b y single and multiple polymorphisms within pre miRNAs in miR K122, 4, 5, 7, and 9. Transient transfection assays and mi R NA array were performed as further experiments. Our results demonstrate that naturally occurring polymo r phisms in KSHV miRNA genes translate into maturation differences. Since, KSHV encoded miRNAs can regulate genes in angiogenesis, apoptosis and cell survival pat h ways, mutations in miRNA genes may be associated with clinical variants or phenotypes of KS, PEL, and MCD. 35. The role o f genetics in vitiligo susceptibility Herbstman DM 1, Hou W2, Garvan CW3, McCormack WT4 ,*, Wallace MR1 ,* 1Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL 2Department of Epidemiology and Health Policy Research, Uni versity of Florida, Gainesville, FL 3Office of Educational Research, University of Florida, Ga i nesville, FL 4Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL Vitiligo is an autoimmune pigment disorder of skin, which is seen on patients as depigmented areas that may enlarge. It affects about 0.5 1% of all ethnic groups worl d wide, and is associated with an increased risk for other autoimmune diseases. The cause of vitiligo is unknown, but is thought to inv olve genetic and environmental fac tors. Our hypothesis is that vitiligo pathogenesis is caused in part by genetic susceptibility to both autoi m mune and autocytotoxic events, due to polymorphisms in genes involved in the regulation of the immune response and melanin production. Human genomic DNA samples from vitiligo patients, their family members, and healthy controls with no autoimmune diseases were genotyped for a number of different single
nucleotide polymorphisms (SNPs) in COMT, TYR, TYRP1, DCT, and PAH, genes i n volved in melanin biosynthesis, and the immunoregulatory gene AIRE. Using case/control and family based genetic association studies, as well as haplotype analysis, susce p tibility to vitiligo was linked to the AIRE gene; significant results were also found in the melanin biosynthesis genes. These results support a possible role for genes involved in immune system regulation, as well as for genes involved in melanin synthesis, in vitiligo susceptibility. Our aim was to identify genes involved in vitiligo susceptibility so that in the future, therapies that might prevent or amel i orate vitiligo may be developed based on our understan d ing of genetic causes of vitiligo. 36. Circadian cycle dependent effects on neural progenitor cell proliferation, d ifferentiation and survival in adult mice Hoang Minh LB 1, Kelly P1, Palmer TD2, Ormerod BK1,2,* 1J. Crayton Pruitt Family Department of Biomedical Engi neering, University of Florida, Gainesville, FL 2Department of Neurosurgery, Stanford University, Stan ford, CA Previous work suggests that adult hippocampal neurog e nesis increases during the dark cycle in mice, when they are most active. We tested whether hippocampal neur o genesis is increased during the dark (active) phase of the light:dark cycle, and whe ther nigral neurogenesis may become detectable if we looked during the dark cycle, when levels would potentially be increased. Adult (8 week old) female C57Bl/6 mice that were exposed to a freely moving or immobilized running wheel were injected with eith er a single i.p. injection or a series of 6 injections of the cell synthesis marker BrdU (50 mg/kg) either 2h after lights on (0700) or 2h after lights off (2100). 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 week or 4 weeks after the first BrdU injection to measure neurogenesis and new cell survival (n=5 per group). ANOVAs revealed that cell proliferation in both the hippocampus and substantia ni gra was increased in runners and during the dark cycle (p< 0.05). Although more new neurons were produced in the hippocampus of mice during the dark cycle versus light cycle (p<0.05) no new neurons were found in the substantia nigra. In addition, cell survi val was increased among cells produced during the dark versus light cycle in both regions (p<0.05). Here, we uncover an interesting model for examining the mechanisms that govern prog e nitor cell behavior. 37. Involvement of SSRP1 in latent replicat ion of Kaposis sarcoma associated h erpesvirus Hu J 1,2, Liu E1,2, Renne R1,2,* 1Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL 2University of Florida Shands Cancer Center, Gainesville, FL Kaposis sarcomaassociated herpesvirus (KSHV) is a -herpesvirus that undergoes both lytic and latent infection. During latent infection, two viral elements are required for DNA replication: the latency associated nuclear antigen (LANA), which functions as an origin binding protein, and the origin, which resides within the terminal repeats (TRs) of the viral genome. Previously, we have identified two ciselements within TR which are required for replication: two LANA binding sites (LBS1/2) and a GC rich RE el e ment upstream of LBS1/2. To further characterize RE, we constructed a 71 bp minimal replicon (MR) and performed a detailed mutational analysis. It indicated that the first 8 nts within RE are critical for replication. Changing the po sition and the distance between RE and LBS1/2 can affect ori activity, suggesting that RE may function as a loading pad for cellular proteins involved in replication. Using bi o tinylated DNA fragments of wt or mutant MR as probes, we identified several cellular origin interacting proteins putatively involved in LANA dependent replication. Thirty proteins were found to preferentially bind to MR. Among these proteins, SSRP1, a subunit of the FACT complex, and TRF2 formed complexes with LANA at the MR region. SiRNA based knockdown of SSRP1, but not dominant negativebased knock d own of TRF2, significantly d e creased the efficiency of TR replication. These results indicate SSRP1 as a novel cellular protein involved in L A NA dependent DNA replication. 38. Characterization of sugarcane caffeic acid 3 -Omethyltransferase (COMT) and 4 coumarateCoA ligase (4CL) Jung JH 1,2, Kim JY1,2, Fouad W1,2, Vermerris W1,2,*, Gallo M1,2,*, Altpeter F1,2,* 1Agronomy Department, University of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Sugarcane is the highest yielding biomass producer. Typically, farmers reduce the sugarcane postharvest leaf r e sidue by open air burning. Fuel grade ethanol can be made from sugarcane leaf litter residue following acid hydrolysis pre treatments to rem ove lignin which acts as a physical barrier to enzyme hydrolysis. Thus, down regulation of lignin biosynthesis pathway enzymes is a promising strategy to increase the efficiency of bio ethanol production from hemicellulosic sugarcane res i dues. In the ligni n pathway, 4coumarate CoA ligase (4CL) and Caffeic acid 3 -Omethyltransferase (COMT) are key enzymes that catalyze
the formation of CoA thiol e s ters of 4 coumarate and other hydroxycinnamates or the methylation of 5 hydroxyconiferaldehyde to sinapald e hyde respectively. However, sugarcane has a complex polyploid genome and these genes belong to a large gene family. Their broad substrate specificities have made it difficult to identify orthologs that are specifically involved in lignin biosynthesis. We have isolated two 4CL genes and two COMT genes from a commercially important s u garcane cultivar by a PCR based strategy. Results from RT PCR expression analysis in different tissues will be presented. In vitro substrate preferences will be determined from purified enzymes. RNAi suppression of s e lected target genes will allow validation of their relative importance in lignin biosynthesis of sugarcane. 39. Transgenic flowering locus T expression: a b y pass to long term juvenility Kamps TL Pajon M, Moore GA* Ho rticultural Sciences Department, University of Florida, Gainesville, FL Citrus plants display a very long period of growth and d e velopment (up to 10 years) prior to flowering and fruiting when grown from seed or from tissue culture regenerants of juvenile transformation. This developmentally regulated delay severely impedes utilizing genetic based strategies to improve fruit quality, disease resistance, and responses to abi otic environmental parameters. Our goal is to ove r come this barrier using genes that promote precocious flowering. Ectopic expression of certain genes behind a constitutive promoter has been shown in many species t o promote precocious flowering. One of these genes, FT (Flowering Locus T) encodes a small, globular protein which is transported from phloem companion cells in leaves to shoot apical meristems, where it functions in promoting flowering. Importantly, in some species the FT protein has been shown to be transferable across graft unions. Transgenic citrus over expressing FT will be used with grafting strategies to induce precocious flowering of breeding genotypes, and possibly for manipulating f lowe r ing of commercial citrus. To date, Agrobacteriummediated transformation of juvenile tissue of the citrus hybrid Carrizo citrange and t obacco has been carried out with genomic clones of the three citrus FT orthologs, ciFT1, ciFT2, and ciFT3. Surprisingly, flowering has been observed during tissue culture of some citrus materials transformed with the 34FMVciFT3 GUS construct. 40. Devel opmental stage, reproductive tissue, and cytotype effects on transcriptional and post transcriptional regulation of mitochondrial genes Kamps TL Siripant MN, Chamusco KC, Chase CD* Horticultural Sciences Department, University of Florida, Gainesville, F L Cytoplasmic male sterility (CMS) is maternally inherited and results from the interaction of nuclear and mitoch o drial expressed gene products. We have profiled tra n scripts and protein products of mitochondrial genes in developmentally staged pollen and d eveloping female r e productive structures (immature ears) from isogenic NB and CMSS maize cytotypes that do not have CMSS nuc lear fertility restoring alleles. Western blots showed the accumulation of mitochondrial ATP synthase and respir a tory complex subu nits were clearly reduced in both CMS S and normal microspores as compared to the immature ears. We examined RNA editing, a post transcriptional feature of plant mitochondrial gene expression, to test a possible mechanism for producing the observed tissue and developmental stage specific phenotypes. We report on the editing patterns determined from sequence anal y sis of RT PCR products of atp4, atp6, atp8, and atp9 for microspores and immature ears of our isogen ic Mo17 CMS S and NB cytotypes. In addition, th e comparison of these results to publically available data of NB editing, and results from RNA editing prediction software will be presented. 41. Generation and characterization of interspeci f ic hybrids between elephantgrass (Pennisetum purpureum Schum.) and pearl millet (Pennisetum glaucum L.) Kannan B 1,2, Sollenberger L1,2, Altpeter F1,2,* 1Agronomy Department, University of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Program University of Florida, Gainesville, FL Napiergrass (Pennisetum purpureum Schum.) has been introduced to all tropical and subtropical areas of the world because of its ability to produce large amounts of high quality forage biomass. Napiergrass is also cons i dered one of the best adapted perennial feedstocks for biofuel production in the southern U S. Napiergrass cau s es less environmental problems than several other potential biomass crops. However, napiergrass is listed as inv a sive in Southern Florida by the Florida Exotic Pest Plant Council. Plant propagation an d establishment of new na piergrass plantings occurs through vegetative plant parts. Therefore, unlike seeded crops, flowering of napiergrass is not necessary for crop production and its suppression will significantly reduce its potential for invasiveness. We produced triploid, interspecific hybrids between napie r grass (tetraploid) and pearl millet (diploid) to introduce male and female sterility. Tall, stress tolerant parents were chosen with the goal to generate interspecific hyb r ids with good productivity and persistence as well as male and female sterility. Pearl millet (AA genome) multiline population with A4 CMS represented the female parent and was crossed with allopolyploid napiergrass (AABB) genotypes Merkeron or N 51. We will present data d e scrib ing the phenotypic variability in these hybrids which allowed selecting lines with excellent vigor and sterility. Further experiments will evaluate the persistence of the interspecific hybrids.
42. Induction of a novel putative trypsin inhibitor gene in F ortunella margarita upon canker infection Khalaf A 1 ,2,3, Moore G1,2,*, Gmitter FG1,2,3 1Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2Horticultural Sciences Department, University of Florida, Gainesville, FL 3Citr us Research and Education Center, University of Flo r ida, Lake Alfred, FL Asiatic citrus canker caused by Xanthomonas axonopodis pv. citri (Xac) has been considered one of the most severe diseases of citrus species and cultivars. Previously we have shown t hat Fortunella margarita exhibits a hype r sensitive response to Xac indicative of an incompatible interaction. In addition, a microarray expression analysis was done to confirm visual results suggestive of the hypersensitive response; 352 genes were identif ied to be significantly differentially expressed after infection. The above study identified the components of the incompat i ble interaction, reactive oxygen species (ROS) production, and programmed cell death (PCD). In addition, a number of common defense mechanisms and a number of resi s tance genes were identified. In this study we show the differential expression of a novel putative protease inhibitor in F. margarita post canker infection. Further charact e rization of the gene will be presented. 43. Biolis tic gene transfer of minimal expression cassettes into sugarcane Kim JY 1,2, Gallo M1 ,2,*, Altpeter F1 ,2,* 1Agronomy Department, University of Florida, Gainesville, FL 2Plant 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 recently to produce ethanol, a renewable transportation fuel. Biolistic gene transfer is superior to alternative methods in genetic engineering, when mul tiple expression cassettes need to be co expressed. However, biolistic gene transfer is som e times associated with complex transgene integration that may cause gene silencing. This study investigated the integration complexity following biolistic gene transfer of minimal transgene expression cassettes without vector backbone and the effects on transgene expression. Cross sections of immature inflorescences from sugarcane (cv. CP 881762) were placed on callus induction medium and subcultured biweekly to ind uce embryogenic callus. E m bryogenic callus was used as a target for biolistic gene transfer of an expression cassette consisting of nptII u n der the control of the constitutive 35S promoter with HSP70 intron and NOS 3 UTR. Prior to gene transfer, the vecto r backbone was removed by restriction digestion. The minimal nptII expression cassette was precipitated on 1.0 $m gold. Transgenic plants were regenerated follo w ing selection on geneticin or paromomycin containing m e dia. PCR screening suggested that 83% o f the regen e rated plants were transgenic. Ten plants were randomly selected per experiment (total of 60 plants) and analyzed for transgene expression by NPTII ELISA and transgene integration by Southern blot analysis. 44. Epigenetic bases of neuronal d iversity and plasticity in Aplysia californica: toward single ne u ron epigenome Kohn AB 1, B obkova Y1, Moroz LL1,2,* 1Whitney Laboratory for Marine Bioscience, University of Florida, St. Augustine, FL 2Department of Neuroscience, University of Florida, G a i nesville, FL What are the genomic bases of unique neuronal pheno types? Epigenetics refers to inheritable modifications in phenotypes that do not involve changing the underlying DNA sequences. These mechanisms include DNA methyl a tion of the cytosine resi dues in CpG dinucleotides, covalent and non covalent modifications to the histone pr o teins, chromatin remodeling by the exchange of histone variants, chromatin dynamics involving the switching of active and silent chromatin, non coding RNA including siRNA, and regulation of transcription. We identified and cloned 13 canonical histones and their variants expressed in the CNS of Aplysia californica including a unique mo l luscan H3.4 as well as the H2Macro only described in ve r tebrates. We also identified 15 ma jor histone modifying enzymes as well as more than 50 other components i n volved in static and dynamic chromatin remodeling. Using 454/SOLiD sequencing from single neurons and in situ hybridization, we show that the histones, histone modifying enzymes and m any other chromatin associated proteins revealed a high level of differential expression: nea r ly all central neurons in Aplysia have their own unique expression profiles. This work also opens unprecedented opportunity to study the epigenome of individual n eurons at a resolution difficult to achieve elsewhere. 45. Population genomics using high throughput platforms in Drosophila Kulathinal RJ 1, Sackton TB2, Clark AG3, Hartl DL2, Barb a zuk WB4,*, McIntyre LM1,* 1Department of Molecular Genetics and Microbio logy, Un i versity of Florida, Gainesville, FL 2Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 3Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 4Department of Biology, University of Florida, Gainesville, FL
Genomics is transf orming the field of population genetics by offering novel approaches to estimate population d i versity at the genome level. The challenges are to identify questions that such high throughput data can successfully address i n addition to developing sound analy tical and statistical methods. Here, we evaluate the utility of two platforms that will advance population genomics inf e rence. First, we apply the Roche/454 platform to survey, at shallow coverage, natural variation in Drosophila mel a nogaster from two populations. Reads were aligned to the reference D. melanogaster genomic assembly, SNPs identified, and nucleotide variati on was quantified genome wide. Simulations and empirical results suggest that nucleotide diversity ca n be accurately estimated from sparse data with as litt le as 0.20x coverage per line. Such unbiased genomic sampling demonstrates that short read sequencing methods provide an efficient means to quant i fy variation in genome organization and content. As a s econd approach, we explore the use of a populationbased, allele specific SNP chip (Affymetrix custom array) that was recently developed in our lab. Hybridization l e vels of gDNA against array features were compared b e tween F1 hybrids and their parents fro m two divergent populations of D. simulans to evaluate the p ower to detect heterozygosity. The valuation of these two pla t forms is both instructive and c ritical as we enter a new era in population genomics. 46. Analysis of the role of transforming growth fa c torbeta (TGFb) on the proteolytic processing of connective tissue growth factor (CTGF) Kuznia PM 1, Lewin AS1,*, Schultz GS2,* 1Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL 2Department of Obstetrics and Gy necology, University of Florida, Gainesville, FL Purpose: The TGF" system which includes CTGF has been determined to play a key role in the formation of scar tissue. CTGF has the ability to stimulate two opposing functions, proliferation and differentiat ion. These d i verse functions may be associated with proteolytic processing of CTGF. Methods: Human corneal fibroblasts (HCF) were serum starved for 48 hours then stimulated with 5ng/mL of TGF At different time points, cell e x tracts and conditioned media were collected with the a d dition of a protease inhibitor cocktail. In a second expe riment, serum starved HCF cultures had differing amounts of protease inhibitor cocktails with or without EDTA and then stimulated with TGF at 48 hours. After 24 hours, cell extracts and conditioned media were removed. All collected samples were separated by SDSPAGE and an a lyzed us ing western blots. Results: The addition of TGF to the HCF increased levels of ~38kDa CTGF in both the cell extracts and conditioned media. In serum starved media, ~75 and 150kDa molecular weight bands were observed, but with the addition of TGF these bands d e creased. Also, the addition of TGF caused the level of the ~18kDa fragment to decrease, whereas the ~20kDa fragment remained constant. Finally, the addition of the any protease inhibitor cocktail reduced levels of ~20kDa fragment in serum starv ed media. Conclusion: These data indicate that TGF influences the production and prote o lytic processing of CTGF. 47. Polycomb group protei n Bmi1 binds to the l a tent HSV 1 genome and maintains repressive marks during latency Kwiatkowski DK 1, Thompson HW2, Bloom DC1,* 1Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL 2Section of Biostatistics, Louisiana State University Health Sciences Center, New Orleans, LA The mechanism by which Herpes Simplex Virus type 1 (HSV 1) establishes latency in sensory neurons is largely unknown. Recent studies indicate that epigenetic modifications of the chromatin associ ated with the latent g e nome may play a key role in the transcriptional control of lytic genes during latency. In this study, we found both constitutive and facultative types of heterochromatin to be prese nt on the latent HSV 1 genome. Deposition of faculta tive marks trimethyl H3K27 and histone variant macroH2A varied at different sites on the genome wh e reas the constitutive marker trimethyl H3K9 did not. In addition, we show that in the absence of the LAT, the latent genome shows a dramatic increase in trim ethyl H3K27, suggesting that expression of the LAT during l a tency may act to promote an appropriate heterochromatic state that represses lytic genes but is still poised for rea c tivation. Due to the presence of the mark trimethyl H3K27, we examined whether polycomb group proteins, which methylate H3K27, are present on the HSV1 ge nome during latency. Our data indicates that Bmi1, a member of the PRC1 maintenance complex, associates with specific sites in the genome, with the highest level of e nrichment at th e LAT enhancer. To our knowledge, this is the first example of polycomb mediated repression of a viral genome leading to the establishment of latency. 48. H4R3 methylation facilitates beta globin tra n scription by regulating histone acetyltransferase bindi ng and H3 acetylation Li X 1, Hu X1,2, Patel B1, Zhou Z1, Liang S1, Ybarra R1, Qiu Y3, Felsenfeld G4, Bungert J1,*, Huang S1,5* 1Department of Biochemistry and Molecular Biology, Un i versity of Florida, Gainesville, FL 2Edmond H. Fischer Signal Transduct ion Laboratory, Jilin University, Changchun, China 3Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 4Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Insti tutes of H ealth, Bethesda, MD
5University of Florida Shands Cancer Center, Gainesville, FL Histone modifications play an important role in the process of transcription. However, how certain histone modifications are functionally linked to transcriptional a c tivity remains unclear. The globin genes are regulated by a highly organized chromatin structure that juxtaposes the locus control region (LCR) with downstream globin genes. We report here that the targeted recruitment of asymmetric dimethyl H4R3 catalyzed by PRM T1 facilitates histone H3 acetylation on Lys 9/Lys14 and is important for efficient interactions between the LCR and the bmaj promoter during transcription. We show that dimethyl H4R3 provides the binding surface for PCAF and facilitates histone H3 acetyla tion in vitro. Furthermore, knock down (KD) of PRMT1 by RNA interference in erythroid progen i tor cells prevents histone acetylation, enhancer and promoter interaction, as well as the recruitment of transcri p tion complexes to the active betaglobin promoter. Rei n troducing rat PRMT1 into the PRMT1 KD MEL cells rescues PRMT1 binding and beta globin transcription. Taken t o gether, our data suggest that PRMT1 mediated dimethyl H4R3 facilitates histone acetylation and enhan c er/promoter communications, which lead t o the efficient recruitment of transcription preinitiation complexes to a c tive promoters. 49. Defective erythropoiesis in transgenic mice e x pressing dominant negative upstream stimulatory factor Liang SY 1, Moghimi B1, CrusselleDavis VJ1, Lin I J1, R o sen berg MH1, Li X1, Strouboulis J2, Huang S1,3, *, Bungert J1 ,3,4,* 1Department of Biochemistry and Molecular Biology, Un i versity of Florida, Gainesville, FL 2Institute of Molecular Oncology, BSRC Alexander Fle m ing, Varkiza, Greece 3Center for Epigenetics Un iversity of Florida, Gainesville, FL 4Powell Gene Therapy Center, University of Florida, Ga i nesville, FL Transcription factor USF is a ubiquitously expressed member of the helixloop helix family of proteins. It binds with high affinity to Ebox elements and, through interaction with coactivators, aides in the formation of transcri p tion complexes. Previous work demonstrated that USF regulates genes during erythroid differentiation, including HoxB4 and globin. Here we show that erythroid specific expression of a dominant negative mutant of USF, A USF, in transgenic mice reduces expression of all type globin genes and leads to diminished association of RNA polym e rase II with locus control region eleme nt HS2 and with the globin gene promoter. We further show that expression of A USF reduces expression of several key ery t hroid specific transcription factors, including EKLF and Tal 1. We provide evidence demonstrating that USF int e racts with known regul atory DNA elements in the EKLF and Tal 1 gene loci in erythroid cells. Furthermore, AUSF expressing transgenic mice exhibit a defect in the formation of CD71(+) progenitor and Ter 119(+) mature ery t hroid cells. In summary, the data demonstrate that USF regulates globin gene expression indirectly by enhancing expression of erythroid transcription factors and directly by mediating the recruitment of transcription complexes to the globin gene locus. 50. Identification and characterization of a barrier/insul ator flanking an enhancer region in Dr o sophila Lin NW Zhang YP, Zhang C, Zhou L* Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL In our previous work, we identified an irradiation responsive enhancer region (I RER), a ~33 kb evolution a rily conserved intergenic region upstream of the pro apoptotic gene reaper (rpr), that is required for the indu c tion of pro apoptotic genes rpr and hid in response to i r radiation. During developmental stage 12, this IRER turns into a facultative heterochromatin structure refractory to DNase I, accompanying with the enrichment of repressive chromatin marks, such as H3K27me3 and H3K9me3, as well as several PcG proteins. We have proven that this switch of chromatin structure is respons ible for the sens i tive to resistant transition of pro apoptotic genes responsiveness to irradiation during embryogenesis. The epig e netic modification in the late embryos is limited in the rpr upstream regulatory region, without affecting the rpr promoter and basic enhancer region. Using a reporter assay, we showed that a 9kb region at the IRER left boundary contains the barrier function that prevents the propagation of heterochromatin associated with PcG mediated silencing. Efforts have been taken to identify the essential barrier/insulator element and the cisfactors r e quired for the barrier activity. 51. Defensive reaperinduction of michelob_x(mx) in mosquito midgut cells following virus infection Liu B 1,2, Becnel J3, Zhang Y1,2, Zhou L1,2,* 1Depart ment of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL 2University of Florida Shands Cancer Center, Gainesville, FL 3Center for Medical, Agricultural, and Veterinary Entomol ogy, Agricultural Research Service, U.S. Department of Agriculture, Gainesville, FL Apoptosis is an important cellular response to viral infe c tion. Genetic analysis in Drosophila revealed that a group of IAP antagonists, including reaper, play a pivotal role in regulating cell death during development and in response to environmental stimuli. However, there is a lack of em pirical evidence as to whether reaper like pro apoptotic genes are involved in regulating cell death during path o gen infection of insect vectors. In this study, we cloned the reaper ortholog
mx_Cuqu, from the mosquito Culex quinquefasciatus. Like reaper and other mx orthologs in Anopheles and Aedes, mx_Cuqu induces rapid cell death when expressed in insect cells, which can be blocked/suppressed by co expression of IAP1 or the viral caspase inhibitor P35. Interestingly, mx is strongly i n duced following CuniNPV (Baculoviridae: Deltabaculovirus) infection of Cx. quinquefasciatus larvae. In situ hybridiz a tion indicated that the mRNA of mx accumulates to very high level in midgut cells infected wi th the virus. However, the infected cells fail to undergo apoptosis, rather they become necrotic at the end of the infection. To our kno w ledge, this is the first evidence that reaper like IAP antagonists are involved in mediating the pro apoptotic response against pathogen infections in mosquitoes. 52. The role of Foxa genes in intervertebral disk formation Maier J Harfe B* Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL The intervertebral disk (IVD) is compose d of a tough, outer annulus fibrosus, and an inner, g el like nucleus pu l posus (NP). The NP is deriv ed from the notochord in mice. Degeneration of the N P results in back pain. Despite its prevalence, effective treatments for chronic back pain are limited. L ittle is known about the mechanisms of IVD development and degeneration; this information coul d lead to improved treatments. The forkhead box (Fox) genes are expressed in many tissues and function in d e velopment and post natal life. Foxa1 and Foxa2 genes a re expressed in all three germ layers of the early embryo. They have been well studied in the endoderm, but not in the notochord. Foxa2 null mice die in utero lacking a n o tochord. Cre alleles have been used to ablate Foxa2 in the endoderm. These conditi onal alleles have also been used with a Foxa1 null al lele to make double knockouts. We used these alleles with an inducible ShhERT2cre line to remove Foxa2 in tissues where Sonic hedgehog is ex pressed in E7.5 mouse embryos. Histology and fatemapping 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. Fatemapping in these mice suggests defects in the migration of notochord cells to the NP in mic e heterozy g ous for Foxa1 and missing Foxa2. Study of the role of Foxa family action in IVD development may provide i n sight into new treatments for disk degeneration. 53. Adeno associated virus vectored gene therapy with wild type rhodopsin gene for treat ment of autosomal dominant retinitis pigmentosa (ADRP) Mao H 1, Gorbatyuk M1, Hauswirth WW1,2,*, Lewin AS1,* 1Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL 2Department of Ophthalmology, University of Florid a, Ga i nesville, FL Autosomal dominant retinitis pigmentosa (ADRP) is fr e quently caused by mutations in the RHO gene, which codes for the ops in of rod photoreceptor cells. We are engaged in development of gene therapies for ADRP u s ing adenoassociated virus (AAV), which efficiently infects and t ransduces photoreceptor cells. In the present set of experiments, we designed and constructed a hardened form of the rhodopsin (RHO) gene that is specifically resistant to degradation by the siRNA 301. We had previo usly demonstrated that siRNA301 degrades both m u tant and wild type mouse and human RHO mRNA. The hardened RHO gene (RHO301) was generate d by intr o ducing silent mutations to elim inate the siRNA cleavage site. We are testing gene therapy in a mouse model of ADRP that expresses a human transgene with a prevalent ADRP mutation: proline 23 substituted by histidine (P23H). With delivery of RHO301 in AAV5 to P23H tran s genic mice in a background of mouse RHO+/ the retinal degeneration of inj ected eyes was slower compared with that of uninjected eyes or of control injected eyes. The finding that a gene encoding wildtype rhodopsin could moderate the retinal degeneration in this model suggests that P23H rhodopsin causes a dominant negative effect, but not by gain of a toxic function, since increased pr o duction of normal rhodopsin can suppress the effect of the mutation. Our finding implies that some RHO mut a tions leading to ADRP can be treated by gene transfer of normal RHO. 54. Subsets of SSR markers enable rapi d bulk segregant analyses in multiple maize inbred bac k grounds Martin F 1,2, Dailey S1, Settles AM1,2,* 1Horticultural Sciences Department, University of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Map based cloning is a robust approach to identify the genetic causes of mutant phenotypes. The recent compl e tion of the maize genome has made map based cloning practical. Maize has high levels of nucleotide diversity fu r ther enabling positional cloning. The maize community has developed mutagenized populations that are primarily within a few inbred genetic backgrounds. A core set of molecular markers that can be used for cloning within these backgrounds will be useful. Simple sequence r e peats (SS Rs) are short motifs of 2 to 6 bases in length that are repeated in tandem arrays. SSRs can give s e quence length polymorphisms that are rapid and cheap to use. Currently, the maize genetic map is composed of a vast array of marker types with many markers h aving SSRs but only being characterized for allelic variation in restriction fragments or single nucleotide polymorphisms. We screened
480 public SSR markers for polymorphisms between: B73, the inbred used for sequencing the g e nome; Mo17, a heterotic inbre d with B73; and W22, used to develop multiple mutagenized populations. Less than 50% of the SSRs were polymorphic for each inbred pair comparison. Polymorphic markers were then selected to cover the genome with even spacing to allow bulk segregant analysis Twenty additional markers were tested to cover gaps within the genome, and 89 94 mar k ers were needed for each tested inbred pair. We demo n strate the robustness of these marker sets by mapping a defective kernel mutation. 55. Tolerance induction to cytop lasmic galactosidase by hepatic AAV gene transfer implications for antigen presentation and immun o toxicity Martino AT 1, Nayak S1, Hoffman BE1, Cooper M1, Liao G2, Markusic DM1, Byrne BJ1,*, Terhorst C2, Herzog RW1,* 1Department of Pediatrics, Unive rsity of Florida, Gaine s ville, FL 2Beth Israel Deaconess Medical Center, Boston, MA AAV hepatic gene transfer induces immune tolerance to several protein antigens and has been exploited in animal models for systemic delivery of therapeutic proteins. Adequate levels of transgene expression in hepatocytes i n duce an immune suppressive T cell response. We tested whether AAV gene transfer can induce tolerance to a cytoplasmic protein. AAV2 hepatic gene transfer for expres sion of galactosidase ( gal) was per formed in mice, followed by a secondary gal gene transfer with an ade noviral vector to provoke a severe immunotoxic response. Transgene expression from AAV2 in ~2% of hepatocytes almost completely protected from inflammatory T cell re s ponses against gal, eliminated antibody formation, and significantly reduced adenovirus induced hepatotoxicity. Consequently, ~10% of hepatocytes continued to express gal 45 days after secondary Ad LacZ gene transfer, while control mice had lost all Ad LacZ expression. A combination of adoptive transfer studies and flow cytom e tric analyses demonstrated induction of Tregs that actively suppressed CD8+ T cell responses to gal and that were amplified in liver and spleen upon secondary Ad LacZ gene transfer. These data demo nstrate that tolerance i n duction by hepatic AAV gene transfer does not require systemic delivery of the transgene product and that e x pression of a cytoplasmic neo antigen in few hepatocytes can induce Treg and provide longterm suppression of inflammatory responses and immunotoxicity. 56. The genetic basis of natural variation in Phy s comitrella McDaniel SF 1 ,*, von Stackelberg M2, Perroud P F3, Quatr a no RS3, Rensing SA2 1Department of Biology, University of Florida, Gainesville FL 2Faculty of Biology, Un iversity of Freiburg, Freiburg, Ge r many 3Department of Bi ology, Washington University, St. Louis, MO Genetic variation among natural populations of laboratory model systems represents a rich resource for gene di s covery. Studying natural variants, as oppos ed to those generated by traditional means of mutagenesis, requires more sophisticated statistical approaches, but this fram e work allows for the study of the interactions among mu l tiple alleles at many loc i in a single experiment. Here we describe efforts to build a foundation for studies of natural variation in developmental processes in the moss Phy s comitrella patens (Funariaceae). This species has recently emerged as an important model system for comparative genomics, in part because of the ease of gene targeting in this system. First we report genealogical analyses includ ing isolates from several populations of P. patens and r e lated species in the genus Physcomitrium, which unamb i guously indicate that the so called genus Physcomitrella arose at least thr ee times from distinct ancestors within the genus Physcomitrium. Second, we describe the use of fluorescently tagged P. patens lines for the rapid identifi cation of outcrossed diploids. Although the divergent is o lates of P. patens were not interfertile, we did generate recombinants between European isolates of P. patens. These exhibited a wide variety of dev elopmental pheno types. Third, we describe efforts to develop a set of SNPbased markers and preliminary quantitative trait locus analyses of the genetic basis of this developmental vari a tion. 57. The future of organ regeneration: re emergence of the axolotl in the genomic era Monaghan J 1, Seifert AW2, Maden M2 ,* 1The Regeneration Project, Evelyn F. and William L. McKnight Brain Institute University of Florida, Gainesville, FL 2Department of Biology University of Florida, Gainesville, FL The ultimate goal of regenerative medicine is to repair a damaged organ either by epimorphic regeneration or by promoting the functioning of endogenous stem cells. U n fortunately, the well characterized genetic models which are used for such studies Drosophila, zebrafish, mouse have extremely limited regenerative abilities. The ideal model for regeneration studies is the axolotl, Ambystoma mexicanum, which can per fectly regenerate almost every organ studied brain, spinal cord, limbs, tail, gut, gills, jaws, lung, skin, retina. However, the axolotl has, until recently, lacked the genetic tools required of a modern model organism, but now techniques for producing tran s genics are available and a nearly complete library of the axolotl transcriptome has been generated allowing the production of gene arrays for geno mic analysis. Here we
describe our recent experiments with the axolotl using arrays to investigate spina l cord and limb regeneration and tissue transplantation from transgenic animals to i n vestigate limb regeneration and wound healing. 58. Prevention and treatment of inhibitor form a tion in gene therapy for hemophilia B Nayak S 1, Cao O2, Herzog RW2 ,* 1Adva nced Concentration in Immunology and Microbiol o gy Interdisciplinary Program in Biomedical Sciences, Un i versity of Florida, Gainesville, FL 2Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida, Gainesville, FL Immun e responses to therapeutic proteins limit treatment of inherited protein deficiencies. Rapamycin (Rap)/IL 10/specific antigen epitope depletes antigenspecific e f fector T cells and induces CD4+CD25+FoxP3+Treg. This strategy prevented inhibitor formation in muscle directed gene transfer in hemophilia B mice (F9 / ), indicating the potential for development of a prophylactic immune tolerance protocol. Optimization via different routes of admin istration of the drug combination Rap/IL 10/specific pep tide in ova transgenic mice showed that Treg induction was more efficient in intraperitoneal, IP (4.7x), followed by subcutaneous, SQ (3.3x) and tail vein, IV (2.8x) del i very. Deletion of Teff was more also pronounced via IP (2.7x) followed by SQ (2.3x) and IV (1.7x reduction com pared to control mice). This data shows differential route dependant effectiveness. We are testing additional routes (such as oral) and alternatives to IL 10 in the drug coc k tail (such as IL 2). We tested the protocol for treatment of inhibit ors in an ongoing immune response. Hemophilia B mice injected IM with 1x1011vg of AAV1 CMVhFIX formed anti hF.IX (6 g IgG/ml) at 1 and 2 months after gene transfer. However, animals that received the tole r ance protocol, starting at 1 month after gene tra nsfer, showed a marked reduction in anti hF.IX titers ( 0.2 g IgG/ml). The approach is useful in controlling antibody formation, when initiated early after the onset of the i m mune response. 59. Molecular detection of rAAV in blood from non human primates Ni W 1, Le Guiner C2, Bello Roufai M3, Moullier P1,2, Snyder RO1,3,4, 1Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL 2INSERM UMR649, Nantes Cedex, France 3Center of Excellence for Regenerative Health Biotechno l ogy, University of Florida, Gainesville, FL 4Department of Pediatrics, University of Florida, Gaines ville, FL Gene transfer of therapeutic genes has shown the po te n tial to treat human diseases, however, an emerging issue of this promising technology is misuse by athletes looking for an advantage. Gene doping is the transfer of genes to enhance athletic performance. Following IM injection of rAAV vectors into non human primates, our group has reported that vector DNA can be detected in serum, urine, feces, saliva, and nasal fluid for several weeks post injection. In developing a test to screen athletes, the use of less invasive sampling and sensitive detection tec h niques is imperative to detect gene doping. We are d e termining the smallest dose of a rAAV vector injected IM that can be detected in blood from non human primates. We also aim to discern the relationship between vector dose and longevity in blood. The test we are developing involves the collection of blood and the analysis of DNA by qPCR assay s. As a first target, we have optimized qPCR conditions to detect at least 5 copies of the Epo transgene in the background of endogenous sequences. Data generated in the non human primate is the basis for developing a legally defensible commercial qPCR assay. Given that gene transfer technology encompasses a vari e ty of vectors, routes of administration, as well as injection formulations, the vector biodistributions can vary widely, thus an outcome of our work will be to better define a s says that can be util ized to elucidate vector distribution for legitimate gene therapy applications 60. Epigenetic regulation of pro apoptotic genes in Drosophila during pupae development Novo M 1,2, Zhang C1,2, Pang J1,2, Zhou L1,2,* 1Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL 2University of Florida Shands Cancer Center, Gainesville, FL IAP antagonists reaper and hid play a pivotal role in m e diating cell death during development and in response to cytotoxic stimuli. The two genes have a synergistic effect on cell death induction and are often coregulated. A 33kb genomic region upstream of reaper, the IRER, is required for mediating the induction of both reaper and hid follo w ing irradiation. Interestingly, IRER is subject to e pigenetic regulation. Although it is open in most cells during early embryogenesis, chromatin in IRER becomes enriched for H3K27Me3 and H3K9Me3 and forms facultative heteroch romatin in differentiating and differentiated cells post em bryonic stage 12. This epigenetic modification of IRER blocks the irradiation responsiveness of both reaper and hid. To monitor the epigenetic status of IRER in individual cells and live animals, we knocked into IRER a n ubiquitin DsRed reporter through homologous recombination. DsRed expression is suspected to reflect the epigenetic status of IRER. In this study, we monitored its expression during the pupae stage, when apoptosis is triggered by stage specific pulses of the steroid hormone ecdysone that activate a transcriptional cascade resulting in the e x pression reaper and hid. We are also in the process of generating a novel transgenic fly that will allow us to m a nipulate cells with an open IRER. The data provide a
more detailed understanding of stagespecific apoptosis and f uture routes for research. 61. A new method for gene prediction: application to comparative analysis of Pseudomonas aerugin o sa genomes Oden SM Brocchieri L* Department of Mole cular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL We d eveloped new scoring methods and a novel compu tational gene predictor for the identification of genes based on scoring overall nucleotide base usage asymmetries between codon positions as a function of local s e quence content. Since our scoring scheme is ba sed on global compositional propensities it does not suffer from over parametrization and can be applied without detailed knowledge or assumptions on codon or dicodon usage. We applied our method to the analysis of the genomes of different strains of P. ae ruginosa, an opportunistic path o gen of great nosocomial importance. In an effort to corr e late gene content with pathogenicity, the genomes of several different pathovars of P. aeruginosa have been sequenced since publication of the first P. aeruginosa g e nome, PAO1. Currently, among all predicted protein coding genes of P. aeruginosa PAO1, only 30% are functionally characterized and only 10% have been exper i mentally verified. For meaningful comparative genomics analyses it is highly relevant to obtain improv ed charact e rizations and measures of reliability of gene predictions. We identified several new nonannotated genes in the genomes of P. aeruginosa PAO1, PA7 and PA14. However, several hypothetical genes previously annotated in P. ae ruginosa PA7 are not ch aracterized by compositional properties that distinguish them from random sequences. Our analysis provides statistically supported sets of genes and a more reliable classification of common or strainspecific genes. 62. The relationship between mutation r ate and mating system Ostrow DG Joyner Matos JA Upadhyay A, Blanton D, Rosenbloom J, Izhar K, Hong J, Chik V Griga ltchik V, C a david F, Baer CF* Department of Biology, University of Florida, Gainesville, FL Theory predicts that the strength of natural selection to reduce the deleterious mutation rate will be much stron g er in asexual and selfing taxa than in outcrossing taxa. If asexual and/or selfing lineages can be shown to have substantially lower mutation rates than their outcrossing relatives, it w ould demonstrate differential selection on the mutation rate and cast doubt on the generality of d e leterious mutation based arguments for the evolution of sex. Rhabditid nematodes provide an opportunity to directly address the question of how mating system infl u ences the mutation rate. The common ancestor of the Rhabditidae is inferred to have been gonochoristic (ou t crossing), but there have been at least six independent evolutionary transitions to hermaphroditism (self ing). We initiated a new mutation accu mulation experiment using two gonochoristic species from the genus Caenorhabditis, C. remanei and CB5161, and a gonochoristic sister species to C. briggsae (strain JU787) as well as an inbred strain of the C. elegans mutant fog 2 as a control. Descendant p opulations were compared to unmutated ancestral co n trol stocks to assess genomic mutation rates. Compar i sons between selfing taxa and congeneric gonochoristic taxa will be discussed. 63. Long term rescue following AAVmediated cone targeting gene therapy to Cpfl5 mouse, a model of human achromatopsia with CNGA3 mutation Pang J 1, Lei B2 ,3, Mao S1, Everhart D4, Liu L1, Deng W1, Li Q1, Chang B5, Barlow R4, Hauswirth WW1,* 1Department of Ophthalmology, University of Florida, Ga i nesville, FL 2Department of Ve terinary Medicine and Surgery, Unive r sity of Missouri Columbia, MO 3Department of Ophthalmology, University of Missouri Columbia, MO 4Department of Ophthalmology SUNY Upstate Medical University, Syracuse, NY 5The Jackson Laboratory, Bar Harbor ME Pur pose: To test if cone targeted CNGA3 gene delivery can restore the cone system function in cpfl5 mice, a natural model of human a chromatopsia 2 with CNGA3 mut a tion. Methods: At postnatal day 14, 1 l of AAV5 PR2.1 CNGA3 vector (1 x 1013 genome containing viral pa r ticles/ml) was injected subretinally into one eye of 20 cpfl5 mice. The other eye was used as control. Dark and light adapted ERGs were recorded periodically starting. Ten months later, vi sual function was assessed with ro u tine ERGs and 10 Hz flicker ERGs. Behavioral tests were also tested, followed by histochemical studies. Results: In treated eyes, restored light adapted ERGs were observed at 3 weeks after injections and remained stable for at least 10 months; the amplitudes were about 50% of those of the normal eyes. The dark adapted flicker ERGs also showed significantly improved cone driven responses. No cone driven ERGs were recorded in untreated eyes. Beh a vioral tests showed nearly normal cone driven visual acu i ty and contrast sensitivity in the treated eyes, but not in the untreated eyes. In the treated cpfl5 retinas, immun o histochemistry showed CNGA3 staining in the inner and outer segments of the cones. However, no CNGA3 e x pression was observed in the untreated eye from the same mouse. Conclusions: 1. AAV mediated gene therapy corrects CNGA3 deficiency in a naturally occurrin g mouse model of human a chromatopsia 2. The genetic interve n tion restores and maintains the cone function for at least 10 months.
64. Comparative proteomics of salt tolerance between Arabidopsis thaliana and Thellungiella hal o phila Pang Q 1,2, Chen S2,3,*, Dai S1, Wang Y1, Yan X1 1College of Life Science, Northeast Forestry University, Harbin, China 2Department of Biology, University of Florida, Gainesville, FL 3Proteomics Division, Interdiscipli nary Center for Biotec h nology Research, University of Florida, Gainesville, FL The majority of plants under salt stress exhibit slow growth, severe shrinkage or even d eath. To survive the stress, plants respond and adapt with complex mecha n isms. In recent years there have been many reports on salt tolerance, most studies have been focused on Arab i dopsis thaliana. However, the outcomes of such work are limited by the fac t that A. thaliana is actually a true glycophyte. More recently, the halophytic plant species Thel lungiella halophila has been proposed as an ideal model for studying molecular mechanisms of salinity tolerance in plants because of its extremophile charac teristics man i fested by extreme tolerance to high salinity. To explore proteome changes, proteins from control and NaCl treated A. thaliana and T. halophila leaf samples were extracted and separated by two dimensional gel electrophoresis. Differentially ex pressed proteins were identified by LC Q Trap MS/MS and Mascot database searching. As expected, most of the identified proteins were involved in ion tran s port, stress response, photosynthesis, and energy met a bolism in A. thaliana and T. halophila. With the use of iTRAQ tagging and two dimensional liquid chromatogr a phy mass spectrometry, it was possible to identify diff e rentially expressed membrane proteins involved in salinity responses in A. thaliana and T. halophila. 65. C3H mutant expression study in m aize Parker J 1, Vermerris W2,* 1Plant Molecular and Cellular Biology Program University of Florida, Gainesville, FL 2Agronomy Department, University of Florida, Gainesville, FL Lignin is a hydrophobic plant cell wall polymer that pr o vides structural in tegrity to the plant but also reduces the efficiency of agroindustrial processing. Use of knockout alleles to examine the synthesis of lignin has provided insights in the relationship between cell wall chemical composition and function. The C3H 118::Mu a llele in m a ize represents an insertion event in the p coumaroyl sh i kimate/quinate 3 hydroxylase (ZmC3H1) gene (kindly provided by Pioneer Hi Bred). A preliminary analysis of lignin content in the mutant indicates a strong reduction in guaiacyl (G) and syringyl (S) residues and an increase in phydroxyphenyl (H) residues. The fact that some res i dual G and S residues are present suggests that maize has a second C3H gene. A BLAST search of the maize genome sequence indeed revealed the presence of ZmC3H2. The alignment of the two deduced amino acid sequences reveals a high degree of similarity. RT PCR will be performed to study gene expression. We predict that the expression of ZmC3H2 will be higher in the mutant than in the wild type, reflecting a compen sation for the defective ZmC3H1 gene. Cell wall compositional analyses will be performed to study overall effects of the ZmC3H1 knockout. By studying ZmC3H1 and ZmC3H2 expression and effects on lignin and cellulose in maize, we will not only further enhance the knowledge of lignin biosynthesis, but discover other ways to further engineer maize to create more viable energy sources. 66. PRMT1 mediated dimethyl H4R3 cross talks with H3K4 methylations Patel B 1, Li X1, Huang S1,2,* 1Department of Biochemi stry and Molecular Biology, Un i versity of Florida, Gainesville, FL 2University of Florida Shands Cancer Center, Gainesville, FL Covalent modifications of histones regulate a number of processes essential for normal cellular functions, including gene transcription. Whereas, each of these modification s has a specific function, how they are communicated and cross regulated remains unclear. Previous work demon strated that asymmetric dimethylation of H4R3 residues by protein arginine methyltransferase PRMT1 pot entiates histone acetylation and is essential both in vitro and in vivo for the establishment or maintenance of the active histone acetylation patterns. We report here that PRMT1 mediated dimethyl H4R3 facilitates histone H3 methyl a tion on Lys 4 in vitro. We further show that loss of PRMT1 through RNAi mediated knock down in a murine erythroid leukemia cell line prevents methylations of H3K4 at PRMT1 target genes. Reintroduction of rat PRMT1 into the PRMT1 knock down cells, rescued PRMT1 binding and histone methylation patterns. Therefore, understanding the molecular mechanism of this interplay between PRMT1 and SET1 in maintaining active chromatin config u ration will provide insight into how the combinatorial regulation of histone PTMs dictates a specific bi ological outcome. 67. FoxA1 and FoxA2 regulate the Hedgehog pathway and are required for division of the e m bryonic cloaca in mice Patterson SE 1, Seifert AW1, Gray S1, Cohn MJ1,2,3,* 1Department of Biology, University of Florida, Gainesville, FL 2Departm ent of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL 3Howard Hughes Medical Institute, University of Florida, Gainesville, FL Abnormal development of the anogenital system underlies several congenital birth defects. These inc lude hyposp a dias,
a common malformation characterized by ectopic or supernumerary urethral openings, and persistent cloaca, in which there is a single opening of the urinary and al i mentary canals. Despite this, the genetic mechanisms of anogenital develop ment remain poorly understood. Using tissue specific transcriptional profiling and in situ hybrid i zation, we identified two Forkhead box genes, FoxA1 and FoxA2, expressed in epithelial cells of the developing urethra and cloaca. To determine the roles of t hese genes, we examined the effects of inactivating FoxA1 and FoxA2 in the mouse anogenital system. Removal of either FoxA1 or FoxA2 does not disrupt anogenital development, however deletion of both genes results in persistent clo a ca, suggesting that FoxA1 and FoxA2 regulate cloacal se p tation. In addition, expression of Sonic Hedgehog (Shh) pathway members, crucial for external genital develo p ment, is reduced in mice deficient for FoxA1 and FoxA2. This suggests that FoxA1 and FoxA2 are upstream regulators of the Shh pathway and are required for normal de velopment of the anogenital system. 68. Noise in the quorum sensing system of the V i brio fischeri marine bacterium Prez PD Young J, Johnson E, Hagen SJ* Department of Physics, Univers ity of Florida, Gai nesville, FL Vibrio fischeri is a luminescent marine bacterium that n a turally colonizes several marine macroscopic life forms. Their luminescence is controlled by the LuxIR quorum sensi ng (QS) mechanism, which has an architecture similar to those of many other proteobacteria including p a thogens to humans. The objective of our research is to see how noise in the gene regulatory system affects the QS communication. QS in V. fischeri has been extensively studied in bulk cultures, but little is known about how it is affected by gene noise, or the intercell variability in gene expression. We are measuring the light output of indivi d ual cells in time and under different conditions. We are using dark field microscopy imaging to find and focus the cells, and an int ensified CCD (iCCD) to collect the biolum i nescence light of individual cells (or small clusters) in time. By acquiring single cell luminescence data, we observe individual cell signals vs. time, histogram distrib u tions of small groups of cells and clusters inhibition by rich growth media, and intercell variations under different QS autoinducer concentrations. The analysis of the ind i vidual cell luminescence signal and its noise can eventua l ly lead to a better understanding of how stochastic noise affects Q S. 69. Cloning and characterization of somatic e m bryogenesis receptor kinase1 (SERK1) gene from sugarcane (Saccharum spp. hybrid) Petefish MR 1, Jain M1, Izquierdo A1, Gallo M1,2,* 1Agronomy Department, University of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Program University of Florida, Gainesville, FL Somatic Embryogenesis Receptor Kinase1 (SERK1) is an LRR RLK proposed to output a signal transduction ca s cade implicated in early embryonic development, somatic embryogenesis, repr oductive development and immune responses. Full length cDNA and genomic clones (1869 and 4752 bp, GenBank acc. nos. GQ283907 and GQ457454, respectively) encoding a putative SERK1 were isolated from an embryogenic cell culture line of suga r cane (Saccharum s pp. hybrid cv. CP88 1762). The ScSERK1 gene shares a highly conserved exon/intron structure with other members of the SERK gene family. The deduced ScSERK1 amino acid sequence is 622 residues with a predicted mass of 68.53 kDa, and a 25 res i due signal pept ide for secretory pathway targeting. The mature peptide contains all the hallmark features of LRR RLKs, namely, a leucine zipper, five LRR regions impl i cated in extracellular protein protein interactions, SPP motif, and a transmembrane domain followed by a n intr a cellular kinase domain. Phylogenetic analyses revealed ScSERK1 protein to be closest to a Sorghum bicolor hyp o thetical protein (99% identical) and Zea mays SERK1 (97% identical) as opposed to other identified SERK1 pr o teins. Quantitative RT PCR data will be discussed address ing spatiotemporal regulation of ScSERK1 expression du r ing initiation, acquisition and maintenance of in vitro s u garcane embryogenesis, regeneration, and the transition from vegetative to reproductive growth. 70. Identification o f the enzymes involved in the last steps of archaeosine synthesis Phillips G 1, Chikwana MV2, Maxwell A1, Lyons B1, El Yacoubi B1, Swairjo M3, Iwata Reuyl D2, de Crcy Lagard V1,* 1Department of Microbiology and Cell Science, University of Florida, Gaines ville, FL 2Department of Chemistry, Portland State University, Por t land, OR 3Department of Basic Medical Sciences, Western Univers i ty of Health Sciences, Pomona, CA Guanosines at position 15 of most archaeal tRNAs are modified to the 7 deazaguanosine derivative archaeosine (G+). The archaeosine modification has been found in all branches of the archaeal phylogenetic tree, and in recent years the biosynthetic steps through the formation of the precursor base preQ0 and its incorporation into tRNA have been elucidated; however, the enzyme (or enzymes) responsible for the conversion of preQ0tRNA to archaeo sine tRNA have remained elusive. Comparative genomic analysis revealed that most archaeal genomes sequenced to date contain two gene families annotated as tgt, tgtA1 and tgtA2, and that these genes cluster together in a number of genomes. TgtA1 encodes the experimentally characterized TGT enzyme, which catalyzes the insertion of the precursor preQ0 into tRNA. Archaea kingdom is divided mainly into two main p hyla Euryarchea and Cr e noarchaea.
While Euryarchaea contains homologs of tgtA2, Crenoarchea does not. Additional comparative g e nomic analysis revealed that these organisms possess either a QueC enzyme fused to a glutamine amidotransfe rase type II protein d omain (GAT QueC) or a QueF like enzyme, suggesting that G+ might be synthesized before being inserted into tRNA. Genetic and biochemical studies were conducted to test the hypothesis that TGTA2 catalyzes a late step in archaeosine biosynthesis in eurya r chaea organisms whereas GATQueC and QueF catalyzes the last step in archaeosine formation in crenoarchaeal organisms. 71. Insights into the evolutionary history of sin g ing mice, genus Scotinomys Pino JL 1, Campbell P2, Pasch B1, Reed D3 ,*, Phelps SM1 ,* 1D epa rtment of Biology, University of Florida, Gainesville, FL 2Department of Ecol ogy and Evolutionary Biology, Unive r sity of Arizona, Tucson, AZ 3Mammalogy, Florida Museum of Natural History, Univers i ty of Florida, Gainesville, FL The genus Scotinomys is r estricted to elevations >1000m in Mesoamerica. Two species, S. teguina and S. xeramp e linus, are recognized since last revision of the genus, 35 years ago. According to the literature, S. teguina is distr i buted from southern Mexico to western Panama with a major population disjunction due to the Nicaraguan lo w lands depression and S. xerampelinus occurs only south of this depression; distribution of both species ends in western Panama where they segregate elevationally (S. xerampelinus >2100m). This research examined the rel a tionships of populations of both species throughout their distributional range by analyzing mitochondrial/nuclear DNA sequences. Samples were provided by museum and field collections. Our analyses confirm haplotypes of what was thought to be S. teguina in populations to the north and south of the Nicaraguan lake, showing geographic structure among populations. Results suggest an interes t ing demographic history with evidence of a bottleneck in the southernmost populations of S. teguina and a recent expansion in northern populations. Haplotypes of former S. xerampelinus (Costa Rica Panama) were present in populations to the north of Lake Nicaragua, suggesting the possible presence of disjunct populations that would greatly expand the previousl y suggested distributional range. The mtDNA tree generated suggests other species level relationships, but more nuclear data must be added to the existing dataset to test this prediction thoroughly. 72. Effects of sigma virus on female fecundity of Drosop hila melanogaster Regan KL Wayne ML* Department of Biology, University of Florida, Gainesville, FL Infectious diseases are a departure from a state of health due to an infectious agent that is dependent on the host for nutrients, reproduction and trans mission. The vir u lence (harm to host) of the infectious agent may be a quantitative trait, if the host does not die due to infection. In the case of vertically transmitted diseases, such as sigma virus in Drosophila melanogaster, optimal virulence should b e low enough for the host to survive and repr o duce, but still detectable due to exploitation of nutrients from the host. In this experiment we looked at the effects of sigma virus on the fecundity of female flies, the viabil i ty of eggs (egg to adult hatchability), and development time. We used four infected and four uninfected lines of D. melanogaster isolated from natural populations in Georgia. There were four replicates of each line for a total of 32 vials of flies for each of the four assays. We found t hat the infected flies produced fewer eggs than the uni n fected flies. However, both groups had the same percent of fully emerged adult offspring in the egg viability exp e riment. Development time also did not differ between i n fected and uninfected flies. 7 3. In search of genetic determinants of a bi o energy sorghum ideotype Saballos A Caicedo H, Vermerris W* Agronomy Department, University of Florida, Gainesville, FL Sorghum is a promising source of biomass that can be produced as a multi purpose crop. S orghum can supply food, feed, fodder, energy and feedstocks for novel appl i cations even in harsh conditions such as hot and dry climates, poor soils, and limited inputs. We are working t o wards the improvement of sorghum as a bioenergy crop by exploiting th e rich genetic diversity of the species, i n cluding sweet and grain sorghums and high biomass sorghums with altered lignin composition. Genomic and genetic approaches are being used to study of the basis of sugar accumulation and its relationship with grain and biomass production, the resistance to drought stress via the study of the root system, and the effects of modific a tions in the lignin biosynthesis pathway. We have recently identified two genes underlying two of the brown midrib (bmr) lignin mutants. Mutations in these genes result in increased saccharification of the plants stover, an impo r tant advantage for ethanol production. The chemical ch a racteristics of lignins from additional bmr mutants are being investigated for their potential utilization in the pr o duction of novel polymers. The combination of QTL identi fication, mutant characterization and gene expression studies will ultimately allow us to identify genes underlying traits for successful bioenergy production. This kno w ledge will facilitate t he creation of lines with high yield and good bioprocessing characteristics, adapted to the stresses of their area of production. 74. The ER stress transcription factor XBP1s pro tects against amyloid neurotoxicity
Sanchez Garcia J1 Casas Tinto S2, Zhang Y2, Rincon Limas DE2, Fernandez Funez P1 ,* 1Department of Neurology, University of Florida, Gaine s ville, FL 2Department of Neurology, University of Texas Medical Branch, Galveston, TX Background: Alzheimers disease (AD) is a neurodegene r ative brai n disorder for which there is no cure. The most prominent pathologic hallmark in the AD brain is the abnormal accumulation of the amyloid beta1 42 (A! ) pep tide, but the exact pathways mediating A neurotoxicity are virtually unknown. For instance, ER stress is activated in AD; however, mostly indirect evidence suggests that ER stress plays a role in A pathogenesis. Objectives: To e x amine the role of t he ER stress in the A neurotoxicity and to understand the mechanisms of the protective a c tivity. Methods: We used transgenic flies expressing A! to characterize XBP1 and confirmed the results in human neuroblastoma treated with A oligomers. Results: We r eport that A! activates the ER stress response factor X box binding protein 1 (XBP1) in transgenic flies and in human neuroblastoma, yielding its active form, the transcription factor XBP1s. Remarkably, XBP1s is neuroprote c tive in flies expressing A! and i n human neuroblastoma treated with A oligomers. We also demonstrate that XBP1s prevents the accumulation of free Calcium in the cytosol, thus explaining its protective activity. XBP1s seems to regulate intraluminal Ca release by downregula t ing the express ion of Ryanodine receptors that are elevated by A Conclusions: Together, these results hig h light the functional relevance of XBP1s in the ER stress pathways triggered by AD, and uncover the potential of XBP1 as a therapeutic target for AD. 75. Site spec ific recombination to improve tran s genic medfly strains Schetelig MF 1, Scolari F2, Handler AM1 ,*, Kittelmann S3, Gasperi G2, Wimmer EA3 1 1Center for Medical, Agricultural, and Veterinary Entomol ogy, Agricultural Research Service, U.S. Department of Agric ulture, Gainesville, FL 2Dipartimento di Biologia Animale, Universit di Pavia, Pavia, Italy 3Department of Developmental Biology, Georg August University Gttingen, Gttingen, Germany The Sterile Insect Technique (SIT) is an environmentally friendly bio control method used in area wide pest ma n agement of the Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann). Recently, we generated new transgenic strains to improve reproductive sterility and sex specific marking of medfly. All the DNA constru cts, which were used to generate these strains, carried an attachment P site (attP) a short DNA sequence for site specific recombination. The efficiency of the reproductive sterility and marking systems and the fitness of the trans genic flies were highly influenced by position effects of the transgenes. Strains that were successfully tested and evaluated as beneficial for the functionality of the tran s genic system were then studied for further improvements by using site specific recombination via their attP site. A two step modification was tested in these transgenic medfly strains: 1) the combination of two transgenic sy s tems at a positively evaluated genomic position and 2) the increase of transgene stability by the deletion of transp o son ends with ne wly developed medfly jumpstarter strains. Such new possibilities in modifying medfly tran s genes will enhance the development of new and safe ap plications for SIT programs as well as functional studies in Tephritid species. 76. Evolving spiking neural netw orks for the pr e diction of transcription factor binding sites Sichtig H Riva A* Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL Our understanding of complex biological adaptive sy s tems, from the cellular to th e molecular level, can be used to develop valuable computational tools for interdisciplinary research in bioinformati cs and biomedical engineer ing. We propose the use of spiking neural networks, able to realistically model the neurological system, to address challenging problems in computational biology, and of artificial evolutionary processes, such as genetic alg o rithms, to tune the network parameters. These tools can be extremely useful for interdisciplinary research because of their generality and their applicability to any complex system of interest. We will present work in progress using artificial spiking neurons applied to the well known prob lem of predicting transcription factor binding sites (TFBSs) in DNA sequences. The system is trained using rea l TFBS data from the TRANSFAC database, and uses a top down modeling approach to simulate biological information processing based on neurological coding. The goal of our work is to reduce the number of false p ositives in the predicted TFBSs through a more accurate modeling of the information contained in the alignments in the training data. We will present an evaluation of our systems pe r formance for the detection of TFBSs and compare it to alternative methods. 77. Genetic characterization of the murine Ange l man syndrome imprinting center Smith EY Futtner CR, Hallett RA, DuBose AJ, Chamberlain SJ, Resnick JL* Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville FL Prader Willi syndrome (PWS) and Angelman syndrome (AS) are distinct neurological disorders resulting from i m proper gene expression from the imprinted domain on chromosome 15q11q13, the PWS/AS locus. This locus is controlled by a
bipartite imprinting center consisting of the PWS IC and the ASIC. The most wi dely accepted model of IC function proposes that the PWS IC promotes gene expression from the paternal allele while the AS IC acts to epigenetically silence the PWS IC on the maternal allele, thus silencing t he paternally expressed genes. The PWS/AS locus is well conserved from human to mouse but a murine AS IC remains uncharacterized. As in humans, the mouse Snrpn locus includes several upstream exons postulated to function in silencing the maternal allele. We have taken a transgenic approach to study the potential regulatory ro le of these alternative exons. To do so, we utilized a bacterial artificial chromosome (BAC) containing Snrpn and three alternative upstream exons. This BAC transgene displayed proper imprinted expression and epigenetic imprinting ma rks at the Snrpn DMR, thus demo n strating the p resence of a functional ASIC. Upon deletion of the three upstream exons, Snrpn was expressed after both maternal and paternal transmission of the transgene and there was a loss of the epigenetic imprint at the Snrpn DMR. Thus, through our innovative transgenic sy s tem, we have identified a functional murine AS IC co n tained within the Snrpn upstream exons. 78. Using historical museum specimens to reco n struct the evolutionary history of the southeastern pocket gopher (Geomyidae) Barrow L1,2, Reed DL1,*, All en JM1, Soto Centeno JA 1 1Mammalogy, Florida Museum of Natural History, Univers i ty of Florida, Gainesville, FL 2Department of Biology, Florida State University, Tallaha s see, FL The evolutionary history and taxonomy of the southea s tern pocket gopher (Geomys pinetis) has been notoriously elusive. We collected mitochondrial cytochromeb sequence data from modern and historical museum spec i mens to determine whether G. pinetis subspecies occu r ring west of the Apalachicola River warrants species status relative to the eastern subspecies, whether the Apal a chicola ChattahoocheeFlint River drainage serves as a geographic barrier, and whether rare subspecies of co n servation concern are genetically distinct from wi despread subspecies of G. pinetis. Maximum likelihood and Bay e sian analyses indicate two major taxonomic units within G. pinetis (average uncorrected sequence divergence = 8.5%), possibly separated by the Apalachicola Fli nt Rivers as also evidenced by ecol ogical niche m odels. Using cyt b sequences from additional pocket gophers and a fossil calibration point, we estimate divergence between the eastern and western clades of G. pinetis at roughly 1.5 million years ago. This is consistent with the age of the A palachicola drainage system and suggests that these taxa have remained isolated for a considerable time, and certainly since the last glacial maximum. This study points out the need for revision of the taxonomy and distribution of G. pinetis, suggests that geology, climate, and habitat structure in the southeastern U.S. have influenced this taxon, and highlights the utility of museum specimens for investigating rare or extinct populations. 79. Construction of an RNAi based mouse model of Barth s yndrome S oustek M 1, Partono S2, Lewin A 2, *, Byrne B1, 1Department of Pediatrics, University of Florida, Gaines ville, FL 2Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL Barth s yndrome (BTHS) is an X linked mitochondria l dis ease that is associated with mutations within the Tafazzin (TAZ) gene. BTHS is characterized by dilated cardiomyopathy, neutropenia, muscle weakness, and growth reta r dation. Studies of BTHS pathogenesis and development of therapies have been hindered due to the lack of a ma m malian model for the disease. Here we report progress in the beginning stages of creating a tissue specific TAZ knockdown model in mouse using RNA interference (RNAi) techniques. We have screened several siRNAs that effectively k nockdown exogenous mouse Taz target in tissue cultures. In addition, we have created a retroviral siRNA construct that resulted in knockdown of endoge n ous Taz in mouse NIH 3T3 cells. Furthermore, we have packaged one of the siRNAs (siRNA9) into an AAV9 ser o type vector, and have tested the infection and knockdown efficiency of this viral vector in mouse cardiac tissue. Pr e liminary data did not demonstrate knockdown of Taz mRNA, although high infection rate was observed. Our data suggest that in future resea rch TAZspecific siRNAs can be delivered as transgenes to establish a systemic model of Barth syndrome. 80. Transcriptomics of queen conch (Strombus gi gas) testis in the Florida Keys: possible role of metals in reproductive failure Spade DJ 1, Feswick A1, Glazer RA2, Barber DS1, Denslow ND1,* 1Department of Physiological Sciences, University of Flor i da, Gainesville, FL 2Fish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission, Marathon, FL Queen conchs (Strombus gigas) in the nearshore Florida Keys fail to reproduce, while offshore conchs reproduce successfully. While gonad development is hindered near shore, the responsible stressors and their modes of action are unknown. Using a custom queen conch microarray, we observed differences in gene expression of near shore versus offshore conch gonads. 257 transcripts were differentially regulated (ANOVA, p<0.01, FDR=5%). Gene O n tology (GO) term enrichment analysis (Fishers exact test, p<0.05) found significant enrichment of the terms spe r matogenesis proton transport and mitochondrial
transport, with the majority of these genes down regulated near shore, corresponding with the observed decrease in testis development near shore. Additionally, small GTPase mediated signal t ransduction was enriched, with most genes being downregulated. This disruption indicates a possible role of Ras related signaling mol e cules in conch testis development. Inductively coupled plasma mass spectrometry (ICP MS) analysis indicates that tissue b urdens of Cu, Zn, and Sn may be elevated in near shore conch tissues, in particular Cu in the gonads and Zn and Sn in the digestive gland. Therefore, metal accumulation in near shore conch tissues may have a role in the observed reproductive failure. Under standing the factors contributing to reproductive failure of nearshore conchs could aid in management efforts toward re establishing a healthy queen conch population in the Flor ida Keys. 81. Using biomarkers to predict successful versus unsuccessful agi ng in rats Speisman RB 1, Kumar A2,3, Foster TC2,3, Ormerod BK1,* 1J. Crayton Pruitt Family Department of Biomedical Engi neering, University of Florida, Gainesville, FL 2Evelyn F. and William L. McKnight Brain Institute, Unive r sity of Florida, Gainesville FL 3Department of Neuroscience, University of Florida, Ga i nesville, FL Chronological age does not predict cognitive success across senescence, which can vary from successful with minimum impairment to unsuccessful with significant impairment despit e no identifiable pathology. Senescent rats can be characterized as memory unimpaired (MU) and memory impaired (MI) using the spatial water maze (SWM) and inhibitory avoidance tasks (IAT), which exhibit a concordance in their sensitivity to age related mem ory impairments. Interestingly, senescent rats categorized as MI begin to show impaired cognition in middle age. We use this observation to investigate whether either hypothalamicpituitary adrenal axis or inflammatory biomar k ers for unsuccessful aging emerge in middle age. To test this hypothesis young (8 mo), middle aged (14 mo), and aged (20 mo) male Fischer 344 rats were tested on the SWM and IAT, where some animals received mild foot shock. Analytes in blood serum and brain tissue samples were quanti fied using a multiplex ELISA strategy. Profiles were created for each analyte to diagram the natural influence of age. Multiple pro inflammatory and recrui t ment/trafficking cytokines showed significant correlation with memory impairment in the cortex and h ippocampus suggesting a possible mechanism relating age and cogn i tion. A significant difference was also noted in middle aged MU and MI serum levels of MIP 1" and GROKC (p=0.0163 and 0.0333 respectively) which may be poss i ble biomarkers for predicting ol d age memory impairment in middleaged animals. 82. Dosage dependent genes affecting seed co m position or weight Spielbauer G 1,2, Armstrong P3, Baier J1,2, Richardson K4, Grijalba D1,2, Griffin M1,2, Koscik K1,2, Song B5, Kahveci T5,*, Settles AM1,2,* 1Horticultural Sciences Department, University of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 3Grain Marketing and Production Research Center, Agricu l tural Research Service, U.S. Department of Agriculture, Manhattan, KS 4Department of Biology, Florida Agricultural and Mechan i cal University, Tallahassee, FL 5Department of Computer and Information Science and Engineering, University of Florida, Gainesville, FL Kernel composition is an importa nt target for developing improved grain for food, feeds, and various 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 scree n ing the UniformMu transposon tagging population using single kernel near infrared spectroscopy (NIR) and seed weights. NIR spectroscopy determines chemical compos i tion and seed weight phenotypes of individ ual maize ke r nels non destructively and at high throughput. We built an automated single kernel NIR grain analyzer and developed partial least square (PLS) calibration models to pr e dict the individual seed starch, protein, oil, and weight. We have develope d a seed spectra and weight database as well as novel statistical tools to identify maize ears se gregating for differences. We are focusing on phenotypes that show dosage dependent or parent of origin changes to the kernels based on the hypothesis that the se genes are the best targets for modifying the seed with transgenes. Finally, we have developed 454 sequencing prot o cols to identify the transposon insertion sites in Unifor m Mu mutants. We are screening these insertion sites for linkage to dosage effect m utants. 83. Spontaneous network activity of fetal and adult cortical cells is altered by the addition of adult neural progenitor cells Stephens CL DeMarse TB, Ormerod BK* J. Crayton Pruitt Family Department of Biomedical Engi neering, University of Flor ida, Gainesville, FL Research has targeted neural progenitor cell (NPC) transplantation as a promising treatment for neurodegener a tive disease. Although new neurons can potentially integrate into adult neural circuits as they do in the mamm a lian hippocampu s, we do not fully understand how their transplant would affect brain function and in turn
how local cell activity would influence NPC fate. Employing m i croelectrode arrays (MEAs) we tackled these questions in a biologically relevant in vitro model of NPC transplant onto fetal or adult cortical cells. The MEA can detect and record extracellular potentials long term to observe spontaneous network activity, a well characterized phenom e non. Action potentials appear within days of cell plating and network burst s begin in 7 10d. Bursts gradually change pattern, becoming modulated at 20 30d with rates of 0.780.08Hz intermittent with 37.56.35s of quie s cence known as an immature state. Then at 3540d the bursts mature into a steady pattern (0.250.04Hz) that r emains stable (>90d). We demonstrate that a mature neural population reverts its burst pattern to an imm a ture state about 14d after NPC addition with bursts at 0.550.04Hz and quiescent periods of 24.04.64s. Co n currently, NPCs express neural or glial ma rkers at 14d. These data suggest that NPC maturation alters the exis t ing activity and therefore could alter brain activity in transplant prompting future work inquiring how to introduce new neurons for repair without compromising fun c tion. 84. The amaize ing mini me: an evolutionary switch that led to the divergence of cane and bunch grasses and potential tool in crop modific a tion? Koch KE1,2,*, McCarty DR1,2,*, Vermerris W1,3,*, Tan S 1 1Plant Molecular and Cellular Biology Program, University of Flori da, Gainesville, FL 2Horticultural Sciences Department, University of Florida, Gainesville, FL 3Agronomy Department, University of Florida, Gainesville, FL A better understanding of the mechanisms controlling plant growth and development can ultimately be used to increase crop yield, enable adaptation to variable env i ronments, and allow novel uses for existing crops. The mini me mutant of maize (Zea mays L.) is a dwarf m u tant, that, unlike any other known maize dwarf mutants, forms many basal branches and ears (female reproductive organ). The mini me mutant hence resembles a bunch grass. We hypothesize that the Mini me gene represents a key switch that led to the evolutionary divergence of cane grasses (maize, sorghum, sugar cane, bamboo, etc.) and bunch g rasses (rye grass, fes cue grass, bahia grass, etc.). The mutant was identified in the UniformMu population and is likely the result of a Mutator transposon insertion. Using the PCR based MuTail method, we have identified the APETALA2 (AP2) transcription fa ctor gene as a candi date gene for Mini me. The AP2 transcription factor is unique to plants and plays a major role in regulating plant growth, such as formation of floral organs, regulating ep i dermal cell differentiation, and response to environmental stre ss. Once cloned and characterized, the Mini me gene may be used as a tool for crop modification, leading to the development of grasses that are storm resistant, have improved biomass yields, and are able to tolerate stressful environments. 85. Development of a computer pipeline in analy z ing transcriptome sequencing (RNA seq) data Tang S Riva A* Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL Next generation transcriptome sequencing (RNAseq) has significantly impacted biomedical and bioinformatics researches by producing high resolution poly(A) transcri p tome sequencing (Shendure, 2008, Nat Methods 5(7) :5 857). It also has gained increased popularity b e cause it addresses many shortcomings in microarray technology, such as prior knowledge dependency and problems with hybridization ( Mortazavi et al., 2008 Nat Methods 5(7):621 8; Shendure, 2008, Nat Methods 5(7) :585 7 ; Sultan et al., 2008, Science 321(5891):956 60) Further more, RNA seq will enable us to identify novel transcripts and alternative splice variants. Therefore, the application of this new technology will shed light on co m plex disease study. Currently, there are very few compu t er pipelines that systematically ana lyze RNA seq data (Denoeud et al., 2008, Genome Biol 9(12):R175; Trapnell et al., 2009 Bioinformatics 25(9):1105 11) In our study, we are interested in developing a computer pipeline to assemble RNA seq data. The development of this pipeline will automate RNA seq data analysis ranging from ide ntif i cation of splice junctions to construction of global altern a tive splicing variants. The application of this computer pipeline to alternative splicing will facilitate more in depth research of complex diseases for the biomedical and/or bioinformatics s cientists. 86. Regeneration response of sugarcane leaf roll explants to different growth regulators Taparia Y Fouad WM, Gallo M*, Altpeter F* Agronomy Department, University of Florida, Gainesville, FL In vitro culture plays a crucial role in the conse rvation, creation and utilization of genetic variability of sugarcane, including cryopreservation, in vitro selection, genetic e n gineering and commercial mass production of disease free sugarcane. Young meristematic tissues such as immature leaf, immature inflorescence or basal shoot meristems are required in sugarcane to induce regenerable tissue cu l tures. In this study, the culture response of sugarcane leaf roll cross sections from the commercially important sugarcane cultivar CP 881762 was examined on media differing in auxin type and cytokinin concentration. Auxins 1naphthalenacetic acid (NAA; 10 $ M); 2,4 dichlorophenoxyacetic acid (2,4 D; 22.6 $ M), 4amino 3,5,6trichlorophyridine 2 carboxylic acid (Picloram; 40 $ M), 4 chlorophenoxy acetic acid (CPA; 10 $ M) plus naph thalenacetic acid (NAA; 10 $ M) were evaluated alone or in combination with the cytokinin 6 benzylaminopurine (6 BAP) at 0.4 or 4.0 $ M in a 3x4 factorial design with 10
replications. Differences in callus induction, callus morphology, necr osis and regeneration pathways were o b served. Data on the quantity and quality of callus and the plant regeneration efficiency of the explants on the diffe r ent media will be reported. 87. Is there a deeply conserved genetic program for cartilage developm ent? Fibrillar collagens in the cuttlefish Sepia pharaonis Tarazona OA 1, Cohn MJ2,3,* 1Department of Biology, University of Florida, Gainesville, FL 2Howard Hughes Medical Institute, University of Florida, Gainesville, FL 3Department of Molecular Genetic s and Microbiology, Un i versity of Florida, Gainesville, FL Collagenbased cartilage was long considered to be unique to jawed vertebrates, but recent work has re vealed 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 extracellular 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 contr i bute to the formation of the ECM of the endoskeleton. Cartilage tissues are also found in distantly related inve r tebrates, such as mollusk and arthropods, which were believed to lack FC genes due to their absence from the genomes of select model organisms. However, biochem i cal and ultrastructural studies suggested the presence of FCs in the cartilage of squids. We tested the hypothesis that invertebrate and vertebrate cartilages develop using conserved development al genetic mechanisms, using degenerate RTPCR we isolate two FC clones from the c e phalopod Sepia pharaonis. The two clones of 1 Kb and 1.29 Kb (SphColAa and SphColAb) correspond to the C propeptide and part of the triple helix domain. Bayesian phylogeneti c analysis places them within the clade A FCs. We examined their expression patterns in cephalopod embryos and find that they are expressed during cartilage development. The results raise the possibility of a deeply conserved genetic program for chondrogen esis in the bil a teria. 88. 5 HTTLPR genotype and drinking in bar p a trons Thombs DL 1 ,*, OMara RJ2, Hou W3, Wagenaar AC3, Dong H J4, Merves ML4, Goldberger BA4, Weiler RM2 1Department of Behavioral Science and Community Health, University of Florida, Gain esville, FL 2Department of Health Education and Behavior, University of Florida, Gainesville, FL 3Department of Epidemiology and Health Policy Research, University of Florida, Gainesville, FL 4Department of Pathology, Immunology and Laboratory Medicine, Un iversity of Florida, Gainesville, FL The serotonin transporter promoter polymorphism (5 HTTLPR) has been linked to a number of human beh a vioral traits and disorders. The variants of 5 HTTLPR are commonly reported in three forms, L/L, S/L, and S/S, with th e latter most often associated with emotional distress and/or behavioral dysfunction. No previous research has examined event level associations between 5 HTTLPR and risk behavior in natural drinking settings. This study reports associations between 5 HTTL PR, alcohol intoxic a tion, and intention to drive among patrons exiting on premise drinking establishments. Self report measures, breath alcohol concentration (BrAC) readings, and oral fluid samples for DNA analysis were collected at night. Multivariate ana lyses were performed on 225 patrons lik e ly to be near their peak intoxication level for the night. 5 HTTLPR genotype was associated with exiting patron BrAC after adjusting for potential confounders. An intera c tion effect involving 5 HTTLPR and drink speci als had an independent association with BrAC, suggesting that sele c tion of price discounted drinks increased intoxication in patrons with an L allele. In addition, patrons with the S/S genotype were 3 times more likely to intend to drive a motor vehicle (a fter drinking on the night of study partici pation) compared to those with the L/L genotype. The 5 HTTLPR genotype may play an important role in the etiology of problems associated with onpremise drinking e s tablishments. 89. Highly stable extracellul ar archaeal glyco laccase from Haloferax volcanii Uthandi S Maupin Furlow JA* Department of Microbiology and Cell Science, University of Florida, Gainesville, FL Laccases couple the oxidation of phenolic compounds to the reduction of molecular oxygen an d, thus, span a v a riety of applications. While laccases of eukaryotes and bacteria are well characterized, these enzymes have not been described in archaea. Here we report the identific a tion and characterization of a laccase (LccA) from the h a lophilic archaeon Haloferax volcanii. H. volcanii was eng i neered to over produce LccA into the milieu of cells grown to high density with a peak of activity (2.48 U ml1) at 2.9 % 109 CFU per ml. LccA was readily purified from cu l ture broth to electrophoretic homogene ity with an overall yield of up to 96% and specific activity as high as 59.27 Umg1. The enzyme purified as a 65.4 kDa monomer with an absorbance spectrum typical of blue multicopper oxidases. The mature LccA was glycosylated to 6.9 % carbohydrate content and oxidized a variety of organic substrates including bilirubin, syringaldazine (SGZ), 2,2, azino bis (3 ethylbenzothiazoline6 sulfonic acid) (ABTS) and dimethoxyphenol (DMP). Optimal oxidation of ABTS and SGZ was at 45 C and pH 6 and pH 8.4, respectiv ely. The apparent Km values for SGZ, bilirubin and ABTS were 35, 236 and 670 $ M with corresponding kcat values of 22, 29 and 10 s1, respectively. The ability of haloarchaea to thrive
in environments of unusually low water activity (high salt, solvent and/or desiccation) has made these organisms and their enzymes ideal candidates for bi o technology advancements. 90. Altering lignin content in sugarcane by RNAi suppression of 4 coumarateCoA ligase Xiong Y Steeves C, Fouad WM, Sandhu S, Gallo M*, Ve r merri s W*, Altpeter F* Agronomy Department, University of Florida, Gainesville, FL Sugarcane (Saccharum sp. hybrids) is a highly productive C4 grass used as the main source of sugar and more r e cently to produce ethanol, a renewable transportation fuel. Typica lly, farmers reduce the sugarcane post harvest leaf residue by open air burning. Fuel grade ethanol can be made from sugarcane leaf litter residue. However, a major constraint for economic ethanol production from hemicellulosic sugarcane residues is lignin which acts as a physical barrier to enzyme hydrolysis. Thus, down regulation of lignin biosynthesis pathway enzymes is a promising strategy to increase the efficiency of bio ethanol production from hemicellulosic sugarcane res i dues. Therefore, the objecti ve of this study is to reduce lignin content in sugarcane by altering 4coumarate CoA ligase (4CL), a key enzyme in lignin biosynthesis. Two 4CL partial sequences were isolated from the genome of sugarcane. Two RNAi constructs targeting a conserved re gion in the two genes were constructed using 200 bp from each of the two genes. One or both Sc4CL RNAi constructs, under the control of the xylem specific OsC4H promoter, were introduced into sugarcane callus along with a selectable nptII expression cassette by biolistic gene transfer. Following selection on medium containing geneticin and regeneration, 49 independent transgenic lines were generated. The transgenic nature of these lines was confirmed using NPTII ELISA analysis. Data descri b ing the level of 4CL s uppression will be presented. 91. Epigenetic regulation of an enhancer region controls stress induced apoptosis in Drosophila Zhang C Wang H, Zhou L* Department of Molecular Genetics and Microbiology, Un i versity of Florida, Gainesville, FL Epigenetic regulation plays an important role in stem cell maintenance, cellular differentiation, and the aging process. Our lab recently demonstrated that during Dr o sophila embryogenesis, the cellular response to ionizing radiation (IR)induced apoptosis is also und er the control of epigenetic regulation of an irradiation responsive e n hancer region (IRER). Embryonic cells before stage 11 contain an open IRER and are extremely sensitive to DNA damage induced cell death. However, chromatin in IRER form a heterochromatin structure after stage 12 and co n sequently cells lose their sensitivity to stress induced apoptosis (Zhang et al., 2008, Dev Cell 14(4):481 93.) The goal for this study is to examine the functional sign i ficance of IRER during development and stress response. Mosaic clones deficient for IRER (IRER / ) contain more cells than simultaneously generated twin spots (IRER+/+) in the imaginal discs, and have reduced level of cell death upon IR. These results indicate that loss of func tion of IRER reduces cellular sensitivity to stress induced cell death and promotes cell survival. A newly generated transgenic fly carrying an ubi DsRed reporter within IRER allows us to monitor the accessibility of IRER in the ind i vidual cell. Following 40Gy IR, more cells in the imaginal discs exhibit increased DsRed signal, suggesting an ep i genetic response of IRER to stress. Our data indicated that epigenetic regulation plays a significant role in d e termining the cellular sensitivity to stress induced cell death. 92. Genetic basis of sexual dimorphism in external genitalia and brain development Zheng Z 1, Evans K1, Cohn MJ1,2,3,* 1Department of Biology, University of Florida, Gainesville, FL 2Department of Molecular Genetics and Microbiology, Un i versi ty of Florida, Gainesville, FL 3Howard Hughes Medical Institute, University of Florida, Gainesville, FL Sexual dimorphism is the difference in structure or phys i cal characteristics between males and females of the same species. Dimorphisms are particularl y pronounced in areas regulated by endocrine signaling, including differe n tiation of gonads, genitalia, breasts, muscle mass, hair pattern, and brain regionalization. Male and female exte r nal genitalia develop from the same embryonic precursor, the genital tubercle, starting at E10.5 in mice. The tuber cle is then masculinized to form the penis or feminized to form the clitoris. Sexual differentiation of the brain has been proposed to occur around this same period, causing dimorphism in specific brain nuclei and total size. Andr o gens have been proposed to drive differentiation of the genitalia and brain, but little is known about the genetic targets of androgen signaling during organogenesis. To address this question, we produced spatial, temporal and quantitative analysis of gene expression in the external genitalia a nd brain of mouse embryos. The bone morph o genetic p roteins Bmp4 and Bmp7, and the Wnt antagonist Dkk2 are expressed at higher levels in female external genitalia and brains at E18.5. Ptc1, which is a receptor of Hedgehog, is expressed at higher levels in male external genitalia, but in female brains. Fgf receptors are also e x pressed in dimorphic patterns in external genitalia and brains. Our results suggest that androgens may direct sexual dimorph ism of the external genitalia and brain through a common suite of genetic targets.
93. USF and NF E2 cooperate to regulate the r e cruitment and activity of RNA polymerase II in the betaglobin gene locus Zhou Z Li X, Deng C, Huang S*, Bungert J* Depart ment of Biochemistry and Molecular Biology, Un i versity of Florida, Gainesville, FL The human betaglobin gene is expressed at high levels in erythroid cells and regulated by proximal and distal cisacting DNA elements, including promoter, enhancer, and a locus control region (LCR). Transcription complexes are recruited not only to the globin gene promoters but also to the LCR. Previous studies have implicated transcription factors USF and NF E2 in the recruitment of transcription complexes to the betaglob in gene locus. Here we demonstrate that while USF is required for the efficient ass o ciation of RNA polymerase II (Pol II) with LCR hypersensi tive site 2, USF and NF E2 together regulate the associ a tion of Pol II with the adult betaglobin gene promoter. Re cruitment of Pol II to the LCR occurs in undifferentiated cells but phosphorylation of globin gene locus associated Pol II at the C terminal domain (CTD) is mediated by erythroid differentiation and requires the activity of NF E2. Furthermore, we provide e vidence showing that USF interacts with NF E2 in erythroid cells. The data demon strate that ubiquitous and tissue restricted transcription factors collaborate to regulate the recruitment and activity of transcription complexes in a tissue specific chromati n domain. 94. Identification of thiolbased redox regulated proteins in guard cell ABA signaling Zhu M1 Zhu N1, Simons B2, Chen S1,3,* 1Department of Biology, University of Florida, Gainesville, FL 2MDS Analytical Technologies (SCIEX), Ontario, Canada 3Proteomics Division, Interdiscipli nary Center for Biotec h nology Research, University of Florida, Gainesville, FL As a central control mechanism in cell metabolism, redox regulation is employed to adjust the plant antioxidant d e fense system to the preva iling environment. Cysteine is one of the most important amino acids with the capacity to be oxidized (e.g., to form disulfide bond s when ox i dized) or to keep sulfhydryl group s in reduced state s The oxidization and reduction of sulfhydryl groups has been found to be an essential regulatory switch in a spectrum of physiological processes in plants. Previous studies su g gest that ABA can induce reactive oxygen species produc tion and potentially oxidative stress of plant cells. And a large part of ABA responsi ve proteins in guard cells are redox related. However, thiol based redox switches r e main largely unknown in plant stomatal opening and clo s ing processes. Here we employ two complementary redox proteomics methods, isotope coded affinity tag (ICAT) and satur ation differential gel electrophoresis (DIGE) to identify thiol based redox regulated proteins under ABA treatment of guard cells. Most of the proteins with disu l fide bond formation or breakage in the course of the treatment belong to the energy, stress an d metabolism functional groups. This analysis not only creates a most comprehensive inventory of components in the redox re g ulation system underlying ABA function in guard cells but also highlights some interesting candidates in the ABA signal transduction for future investigation. 95. Quantification of plant metabolites by multiple reaction monitoring mass spectrometry Zhu N 1, Pang Q1, Zhu M1, Jin X2, Assmann SM2, Chen S1,3,4,* 1Department of Biology, University of Florida, Gainesville, FL 2Biology Depa rtment, Pennsylvania State University, Un i versity Park, PA 3Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 4Proteomics Division, Interdisciplinary Center for Biotec h nology Research, University of Florida, Gainesville, FL The analysis of different plant metabolites is essential for understanding the molecular networks underlying plant functions. Because of the low abundance, high complexity and dynamics of many metabolites, accurate measur e ment is often challenging. We have developed a liquid chromatography multiple reaction monitoring (MRM) mass spectrometry (MS) method to capture the small molecules in plants. Plant extracts are separated by reverse phase and hydrophilic interaction chromatographic columns to enhance c overage. MRM MS on a linear ion trap mass spectrometer is used for absolute and relative quantification. With this technology, our aim is to establish a ta r geted plant metabolite database that includes MS spectra, MRM transitions, standard curve and quanti ties for each target metabolite in plants. The database has more than 400 chemicals that are divided into seventeen groups, e.g. organic acids, hormones, carbohydrates, and speci a lized metabolites. The establishment of this platform will enhance our capabi lity in building molecular networks.