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To determine the mouseTBX1 consensus sequence, systematic evolution of ligands by exponential enrichment (SELEX) was performed with GST-TBX1, and oligonucleotide alignment generated 2 novel motifs. TBX1 protein binds strongly to a tandem repeat composed of 2 half-sites, AGGTGTGAAGGTGTGA, as well as a half-site partial site, AGGTGTGATCGCGTCAT. TBX1 also strongly activates transcription of reporter vectors in cell culture via these binding sites. Using this information, an in silico, genome-wide binding site analysis identified Tbx1 target genes for which a subset (NeuroD, Fgf8, Mef2c) were altered in gene expression in Tbx1-/- embryos. We also determined these to be bound by TBX1 in vitro and activated via luciferase assays. Three known TBX1 mutations (F148Y, H194Q and G310S) were generated and determined to bind to the consensus sequence but could not activate transcription in cell culture reporter assays, providing insight into their function for the first time. This work will provide a strong basis for future chromatin immunoprecipitation studies or biochemical studies to understand TBX1 protein function.
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THE PHYSIOLOGICAL ROLE AND FUNCTION OF N6-METHYLADENOSINEYogesh Saletore, Christopher Mason.
Weill Cornell Medical College, New York, NY.
Epigenetics is widely studied in DNA but recently, FTO, an obesity risk gene, implicates the methylation of the N6 position of adenosine (m6A) of posttranscriptional RNA in energy homeostasis. We devised a novel immunoprecipitation (IP) protocol, MeRIP-seq, to isolate mRNA for m6A sites, coupled with a novel computational peak-finder, MeRIPPeR, to localize these sites throughout the genome. MeRIP-seq isolates fragmented and RiboMinus-treated mRNA using an antibody specific for m6A. The IP fragments are sequenced using next-generation sequencing (NGS). MeRIPPeR uses Fisher’s exact test as its primary peak-finding metric to localize m6A sites throughout the genome. These sites are then analyzed using known RefSeq gene annotations. Using MeRIP-seq and MeRIPPeR, we identified mRNAs of 7,676 genes which contained m6A and discovered that m6A sites are especially enriched near stop codons and in the 5’ end of the 3’ UTR. We confirmed some m6A sites using biotinylated DNA probes and streptavidin Dynabeads, which demonstrates that MeRIP-seq successfully isolates mRNAs with m6A sites.
Our results indicate that m6A is a common modification of mRNA and show insight into the role of RNA epigenetics.
We are now further examining the physiological role of this modification in transcriptional and translational regulation and in the contexts of diseases, including acute myeloid leukemia, neural tube defect, and obesity.
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THE CHARACTERIZATION OF POTENTIAL SIDEROPHORE PRODUCING CAVE BACTERIA ISOLATED FROM
LECHUGUILLA AND SPIDER CAVES, CARLSBAD CAVERNS NATIONAL PARK (CCNP)Tammi R. Duncan, Diana E. Northup.
University of New Mexico, Albuquerque, NM.
Bacteria play large roles in the cycling of iron in surface environments, but below the surface, bacteria may have an essential role. Along with some eukaryotic organisms, bacteria have the ability to survive in a low nutrient and aphotic environment. Determining how bacteria acquire and cycle iron in the subsurface remains an unanswered question
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of subterranean ecology. One way that bacteria collect iron is through the secretion of low-molecular-weight proteins called siderophores, which have a high selectivity for ferric iron. In this study, we investigated Lechuguilla and Spider Caves in Carlsbad Caverns National Park (CCNP). These caves contain iron-rich deposits called ferromanganese deposits (FMD), which are found on the cave walls and ceilings. Previous investigations of FMD in these caves identified bacterial communities related to iron and manganese-oxidizers. Based on these findings, we hypothesize that cave bacteria obtain oxidized iron from FMD through the secretion of siderophores. This iron is used for critical cellular processes. We analyzed previous FMD-inoculated cultures from Spider and Lechuguilla Caves and collected 173 isolates of sub-cultured bacteria. These isolates were characterized by catalase and oxidase testing, and Gram staining, and tested for siderophore production. Fifty nine of the 173 isolates tested positive for siderophore production. The identification of isolates that produce siderophores supports our hypothesis that bacteria could be mining the cave FMD for oxidized iron and manganese cellular requirements. The identification of novel cave siderophores may be valuable in designing more effective antibiotics called sideromycins.
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DESULFOVIBRIO SPP. AND MERCURY METHYLATION IN THE GASTROINTESTINAL TRACT OF PRAIRIEVOLES David Supeck, Senait Assefa, Yue Chen, Tom Curtis, Gerwald Koehler.
Oklahoma State University Center for Health Sciences, Tulsa, OK.
Mercury is a heavy metal known to alter the social behavior in the highly social prairie voles. We are trying to correlate the effects of mercury on vole social behavior with changes in the composition of the gut microbiota (gut-brain axis).
Adult prairie voles received HgCl2 in drinking water ad libitum for ten weeks while control voles received unadulterated water. The intestinal microbiota were characterized using pyrosequencing and quantitative real-time PCR. Male prairie voles exposed to HgCl2 displayed reduced interaction with unfamiliar conspecifics while female prairie voles appeared to be unaffected. Compositional analyses of the microbiota of male/female and treated/untreated animals revealed differences in some genera/species including Desulfovibrio species which were present in higher numbers in mercurytreated voles. Because of the integration of the intestinal microbiota in the gut-brain axis, the observed effects of toxic metal exposure on vole behavior might also be based on changes in the microbiota. We have identified sexspecific differences that could play a role in the increased susceptibility of male voles to the behavior-altering effects of mercury-ingestion. Desulfovibrio spp. might increase the bioavailability of mercury through methylation. Future studies will be directed towards understanding of the molecular mechanisms of mercury toxicity and the role of the gut microbiota in social behavior.
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PRELIMINARY ASSEMBLY OF THE GENOME OF SPIROPLASMA HYD-1: AN ENDOSYMBIOTIC BACTERIUM
THAT PROTECTS DROSOPHILA FLIES AGAINST PARASITIC WASPSHumberto Martinez Montoya, Mariana Mateos, Rodolfo Aramayo.
Texas A&M University, College Station, TX.
Intimate associations between insects and maternally transmitted bacteria are pervasive and diverse, and greatly influence the ecology and evolution of the organisms involved. Recent studies have revealed that many such endosymbionts confer protection on their hosts against natural enemies. Similarly, many heritable symbionts manipulate their host’s reproduction to enhance their own transmission. The mechanisms by which endosymbionts confer defense or manipulate host reproduction remain largely unknown, partly due to the fastidious nature of most heritable endosymbionts. Our understanding of symbiont-mediated mechanisms, as well as their ecological and Graduate
of comparative and functional genomics and molecular evolution. Herein, we present a preliminary assembly of the genome Spiroplasma strain hyd-1 (class Mollicutes), a symbiont that confers protection on its host Drosophila hydei against parasitic wasps (Leptopilina heterotoma). A paired-end indexed library for Illumina sequencing was generated from fly hemolymph DNA extracts. Approximately 330 million, 100-bp reads were obtained and assembled in Velvet according to parameters selected with VelvetOptimiser. Our results indicate that the S. hyd-1 genome has a low GC content and small size according to previously reported Mollicute sequences. We also present a preliminary genomic comparison of the assembled Spiroplasma hyd-1 genome and its close relative, the pathogen of honeybees (S. melliferum), and discuss their differences. This study provides the groundwork for comparative and functional genomics of Drosophila-associated Spiroplasma strains that exert protective and reproductive phenotypes.
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MERKEL CELL POLYOMAVIRUS SMALL T ANTIGEN HYPERACTIVATES THE NF-ΚB PATHWAYChristian Berrios, James A. DeCaprio.
Dana-Farber Cancer Institute, Boston, MA.
Merkel cell polyomavirus (MCV) is the etiological agent of Merkel cell carcinoma (MCC). Classical studies using simian virus 40 (SV40), a model polyomavirus, have demonstrated that SV40 small T antigen (ST) plays a critical role in cellular transformation by binding to protein phosphatase 2A (PP2A) and affecting multiple signaling pathways.
MCV-ST was recently shown to be capable of transforming rat fibroblasts independent of PP2A binding. A largescale and systematic proteomic screen of viral proteins in normal human cells revealed a highly enriched and novel interaction between MCV-ST and the nuclear receptor SET domain-containing protein 1 (NSD1). NSD1 is a histone methyltransferase whose amplification has been associated with multiple forms of cancer. Additionally, NSD1 activates NF-κB through direct methylation of the NF-κB p65 subunit, providing a potential mechanism for the high levels of NF-κB activity seen in some MCC. Cocomplex formation between NSD1 and MCV-ST has been confirmed with epitope-tagged NSD1 as well as endogenous NSD1 in multiple cell lines. Using an NF-κB luciferase reporter assay, MCV-ST-expressing cells were found to have up to a 4-fold increase in NF-κB activity over both SV40-ST- and GFP-expressing cells. Additionally, analysis of the MCV-positive MCC cell lines MKL-1 and WaGa indicated high levels of activated p65 as well as phosphorylation of IκBα, compared to the MCV-negative UISO cell line. These results indicate that MCV-ST expression leads to heightened NF-κB activity in both MCC and normal cell lines, potentially through its association with NSD1, which may represent a putative drug target for MCC.
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GENETIC STUDIES OF PYRUVATE USE IN METHANOSARCINA BARKERI STR FUSAROMadeline Lopez Munoz, William Metcalf.
University of Illinois at Urbana-Champaign, Urbana, IL.
Methanogenic archaea are the only microorganisms to date capable of producing methane as an end product of their metabolism. Within methanogens, Methanosarcinales are known to use H2/CO2, methylamines, methylsulfides, methanol, and acetate as energy sources. Even though Methanosarcinales make use of such a wide substrate range for methanogenesis, complex organic compounds such as sugars and long-chain fatty acids are not substrates for methanogenesis. A Methanosarcina barkeri fusaro strain capable of using pyruvate as its sole energy and carbon source was previously isolated; however, the genetic basis of pyruvate use was not determined. Whole genome sequencing of 2 mutant strains revealed 2 mutations of interest. One was localized in the Mbar_A2165 locus, which encodes a potential transcription regulator, and a second one was in the Mbar_A1588, encoding the bpl subunit of the pyc operon. To assess the involvement of the mutations in the pyruvate using phenotype, they are being recreated in a WT background. Re-creation of the mutation present in the pyc operon did not confer the Pyr+ phenotype. In addition, a deletion of the pyc operon was constructed to evaluate the role of the enzyme in the Pyr+ phenotype.
RNA sequencing of the strains revealed an overexpression of genes involved in gas vesicle synthesis and pyruvate ferredoxin oxidoreductase, the latter being an enzyme involved in the reductive decarboxylation of pyruvate to acetylCoA. To further investigate pyruvate metabolism in M. barkeri, a strain containing a deletion of the por operon is under construction.
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BACTERIAL DEGRADATION OF ALIPHATIC ALKANES ACROSS ECOSYSTEMS IN PUERTO RICOYomarie Bernier, Sharon A Cantrell, José R Pérez-Jiménez.
Universidad del Turabo, Gurabo, PR.
Alkanes are organic compounds that reach the environment by different processes. Microbial degradation of alkanes contributes to bioremediation. However, most studies have been conducted on polluted and temperate sites. The objective is to isolate and characterize putative alkane-degrading bacteria (ADB) in response to hexane, 2,2,4-trimethylpentane, and hexadecane. Soil samples were collected at 7 sites having various pollution levels in Puerto Rico. Native microbiota was cultivated, as consortia, on rich media. The community was described using 16S rDNA-TRFLP profiles generated after digestion with HaeIII. ADB were isolated from samples on mineral media supplemented with a specific alkane as the sole carbon source. Bacterial prospects for aliphatic alkane degradation were subjected to 16S rDNA sequencing and an alkane degradation preference test. A total of 77 ADB have been isolated. Among the putative alkane degraders, we found isolates closely related to Aeromonas, Alcaligenes, Arthrobacter, Comamonas, Cupriavidus, Enterobacter, Klebsiella, Paenibacillus, and Ralstonia. All prospects have
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been able to grow in a 1.2% alkane concentration. Twenty-three percent (n = 18) were able to degrade the three alkanes. Hexadecane was preferred as an isolation carbon source (60% of ADB) over isooctane (21%) and hexane (19%). However, alkane preference tests revealed broader capability: hexane (34% of ADB), isooctane (71%), and hexadecane (83%). ADB prevails across neotropical ecosystems despite the pollution level and are physiologically heterogeneous. Vigorous growth was noticeable for polluted sites, which suggests adaptation to persistent exposure to the alkanes. This collection of alkane-degrading bacteria provides novel isolates to deal with pollution and possibilities for further disclosure of biodegraders in nature.
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