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Author (up) Cho, J.H.; Bandyopadhyay, J.; Lee, J.; Park, C.S.; Ahnn, J. file  url
openurl 
  Title Two isoforms of sarco/endoplasmic reticulum calcium ATPase (SERCA) are essential in Caenorhabditis elegans Type Journal Article
  Year 2000 Publication Gene Abbreviated Journal Gene  
  Volume 261 Issue 2 Pages 211-219  
  Keywords Alternative Splicing; Amino Acid Sequence; Animals; Caenorhabditis elegans/embryology/enzymology/*genetics; Calcium-Transporting ATPases/*genetics/metabolism; Embryo, Nonmammalian/drug effects/enzymology; Embryonic Development; Gene Expression Regulation, Enzymologic; Green Fluorescent Proteins; Isoenzymes/genetics/metabolism; Luminescent Proteins/genetics/metabolism; Microscopy, Fluorescence; Molecular Sequence Data; Phenotype; Promoter Regions, Genetic/genetics; RNA, Double-Stranded/administration & dosage/genetics; Recombinant Fusion Proteins/genetics/metabolism; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sequence Homology, Amino Acid; Tissue Distribution  
  Abstract SERCA (Sarco/Endoplasmic Reticulum Calcium ATPase), a membrane bound Ca(2+)- /Mg(2+)- dependent ATPase that sequesters Ca(2+) into the SR/ER lumen, is one of the essential components for the maintenance of intracellular Ca(2+) homeostasis. Here we describe the identification and functional characterization of a C. elegans SERCA gene (ser-1). ser-1 is a single gene alternatively spliced at its carboxyl terminus to form two isoforms (SER-1A and SER-1B) and displays a high homology (70% identity, 80% similarity) with mammalian SERCAs. Green fluorescent protein (GFP) and whole-mount immunostaining analyses reveal that SER-1 expresses in neuronal cells, body-wall muscles, pharyngeal and vulval muscles, excretory cells, and vulva epithelial cells. Furthermore, SER-1::GFP expresses during embryonic stages and the expression is maintained through the adult stages. Double-stranded RNA injection (also known as RNAi) targeted to each SER-1 isoform results in severe phenotypic defects: ser-1A(RNAi) animals show embryonic lethality, whereas ser-1B(RNAi) results in L1 larval arrest phenotype. These findings suggest that both isoforms of C. elegans SERCA, like in mammals, are essential for embryonic development and post-embryonic growth and survival.  
  Call Number Serial 451  
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Author (up) Downing, K.J.; Thomson, J.A. file  url
openurl 
  Title Introduction of the Serratia marcescens chiA gene into an endophytic Pseudomonas fluorescens for the biocontrol of phytopathogenic fungi Type Journal Article
  Year 2000 Publication Canadian Journal of Microbiology Abbreviated Journal Can J Microbiol  
  Volume 46 Issue 4 Pages 363-369  
  Keywords Chitinases/*genetics/metabolism; DNA-Binding Proteins/genetics/metabolism; Escherichia coli/genetics; Fabaceae/microbiology; *Pest Control, Biological; Plant Diseases/microbiology; Plants, Medicinal; Plasmids/genetics; Polymerase Chain Reaction/methods; Promoter Regions, Genetic; Pseudomonas fluorescens/*enzymology/*genetics/growth & development/isolation & purification; Repressor Proteins/genetics/metabolism; Rhizoctonia/*growth & development; *Saccharomyces cerevisiae Proteins; Serratia marcescens/enzymology/*genetics; *Telomere-Binding Proteins  
  Abstract An endophytic strain of Pseudomonas fluorescens was isolated from micropropagated apple plantlets and introduced into beans (Phaseolus vulgaris) via their root tips. It was shown to be present as an endophyte in the roots at a level of 1.2 x 10(5) CFU/g fresh weight. The gene coding for the major chitinase of Serratia marcescens, chiA, was cloned under the control of the tac promoter into the broad-host-range plasmid pKT240 and the integration vector pJFF350. Pseudomonas fluorescens carrying tacchiA either on the plasmid or integrated into the chromosome is an effective biocontrol agent of the phytopathogenic fungus Rhizoctonia solani on bean seedlings under plant growth chamber conditions.  
  Call Number Serial 1662  
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Author (up) Hellman, L.M.; Fried, M.G. file  url
openurl 
  Title Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions Type Journal Article
  Year 2007 Publication Nature Protocols Abbreviated Journal Nat Protoc  
  Volume 2 Issue 8 Pages 1849-1861  
  Keywords Bacterial Proteins/metabolism; Cyclic AMP Receptor Protein/metabolism; DNA-Binding Proteins/*analysis/metabolism; Electrophoretic Mobility Shift Assay/*methods; Escherichia coli/genetics/metabolism; Escherichia coli Proteins/metabolism; Humans; Lac Operon/genetics; Lac Repressors; O(6)-Methylguanine-DNA Methyltransferase/metabolism; Promoter Regions, Genetic; RNA-Binding Proteins/*analysis; Repressor Proteins/metabolism; Transcription Factors/metabolism  
  Abstract The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this protocol, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided.  
  Call Number Serial 1994  
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Author (up) Oraby, H.; Ahmad, R. file  url
openurl 
  Title Physiological and biochemical changes of CBF3 transgenic oat in response to salinity stress Type Journal Article
  Year 2012 Publication Plant Science : an International Journal of Experimental Plant Biology Abbreviated Journal Plant Sci  
  Volume 185-186 Issue Pages 331-339  
  Keywords Arabidopsis--genetics; Arabidopsis Proteins--genetics, metabolism; Avena sativa--drug effects, genetics, growth & development, physiology; Biomass; Droughts; Gene Expression; Gene Expression Regulation, Plant--physiology; Photosynthesis--drug effects; Plant Leaves--drug effects, genetics, growth & development, physiology; Plant Proteins--genetics, metabolism; Plant Roots--drug effects, genetics, growth & development, physiology; Plant Shoots--drug effects, genetics, growth & development, physiology; Plant Transpiration--drug effects; Plants, Genetically Modified; Promoter Regions, Genetic--genetics; Salinity; Seedling--drug effects, genetics, growth & development, physiology; Sodium Chloride--pharmacology; Stress, Physiological--physiology; Transcription Factors--genetics, metabolism  
  Abstract Salinity is a major abiotic constraint affecting oat productivity. Several physiological and biochemical traits have been found to be related to yield maintenance under salinity. The impact of introducing the Arabidopsis CBF3 gene controlled by the rd29A stress-inducible promoter in T(2) transgenic oat on salinity tolerance and associated physiological changes were studied. Compared with the non-transgenic control, transgenic T(2) plants exhibited greater growth and showed significant maintenance of leaf area, relative water content, chlorophyll content, photosynthetic and transpiration rates as well as increased levels of proline and soluble sugars under high salt stress. These physiological changes delayed leaf-wilting symptoms, increased tolerance and reduced yield loss. At a salinity stress level of 100mM, the CBF3-overexpressing transgenic oat showed a yield loss of 4-11% compared with >56% for the non-transgenic control. These results demonstrate that stress-inducible over-expression of CBF3 may have the potential to enhance abiotic stress tolerance in oat.  
  Call Number Serial 238  
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Author (up) Pickell, L.; Wu, Q.; Wang, X.-L.; Leclerc, D.; Friedman, H.; Peterson, A.C.; Rozen, R. file  url
doi  openurl
  Title Targeted insertion of two Mthfr promoters in mice reveals temporal- and tissue-specific regulation Type Journal Article
  Year 2011 Publication Mammalian Genome : Official Journal of the International Mammalian Genome Society Abbreviated Journal Mamm Genome  
  Volume 22 Issue 11-12 Pages 635-647  
  Keywords Animals; Blood Vessels/metabolism; Central Nervous System/metabolism; Embryo, Mammalian/*metabolism; Female; Genotype; Homocystinuria/*metabolism; Male; Methylenetetrahydrofolate Reductase (NADPH2)/deficiency/*genetics/*metabolism; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muscle Spasticity/*metabolism; Myocardium/metabolism; Placenta/metabolism; Polymorphism, Single Nucleotide; Pregnancy; *Promoter Regions, Genetic; Psychotic Disorders/metabolism; Testis/metabolism  
  Abstract Methylenetetrahydrofolate reductase (MTHFR), a key enzyme in folate metabolism, synthesizes 5-methyltetrahydrofolate, the main circulatory form of folate which is required for maintaining nontoxic levels of homocysteine and providing one-carbon units for methylation. A common 677C --> T variant in MTHFR confers mild MTHFR deficiency and has been associated with a number of human disorders, including neural tube defects and vascular disease. Two promoters of Mthfr, designated as upstream and downstream promoters, are located upstream of a transcription start site cluster and have previously demonstrated cell-specific activities. In this study we used a unique approach for targeted, single-copy transgene insertion to generate transgenic mice carrying a Mthfr upstream or Mthfr downstream promoter-reporter construct located 5' to the endogenous Hprt (hypoxanthine-guanine phosphoribosyltransferase) locus. The Mthfr downstream promoter demonstrated activity in the neural tube, neural crest cells, dorsal root ganglia, heart, and endothelial cells of blood vessels in 10.5-days post coitum embryos and placentas. Upstream promoter activity was absent at this developmental stage. Postnatally, both promoters demonstrated activity in the brain stem, hippocampus, and thalamus of 1-week-old brain that became stronger in the adult. The Mthfr upstream promoter also showed activity in the cerebellum and cerebral cortex. Both promoters were active in male reproductive tissues, including 1-week-old epididymides, and there was upstream promoter-specific activity in the adult testis. Our investigation of Mthfr regulation in an in vivo mouse model revealed temporal- and tissue-specific regulation that supports important roles for MTHFR in the developing embryo, and in postnatal brain and male reproductive tissues.  
  Call Number Serial 304  
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Author (up) Seaton, S.C.; Elliott, K.T.; Cuff, L.E.; Laniohan, N.S.; Patel, P.R.; Neidle, E.L. file  url
openurl 
  Title Genome-wide selection for increased copy number in Acinetobacter baylyi ADP1: locus and context-dependent variation in gene amplification Type Journal Article
  Year 2012 Publication Molecular Microbiology Abbreviated Journal Mol Microbiol  
  Volume 83 Issue 3 Pages 520-535  
  Keywords Acinetobacter--genetics; Base Sequence; DNA Mutational Analysis; DNA, Bacterial--genetics; Gene Amplification; Gene Dosage; Gene Duplication; Genome, Bacterial; Molecular Sequence Data; Promoter Regions, Genetic  
  Abstract Renewed interest in gene amplification stems from its importance in evolution and a variety of medical problems ranging from drug resistance to cancer. However, amplified DNA segments (amplicons) are not fully characterized in any organism. Here we report a novel Acinetobacter baylyi system for genome-wide studies. Amplification mutants that consume aromatic compounds were selected under conditions requiring high-level expression from three promoters in a linked set of chromosomal genes. Tools were developed to relocate these catabolic genes to any non-essential chromosomal position, and 49 amplification mutants from five genomic contexts were characterized. Amplicon size (18-271 kb) and copy number (2-105) indicated that 30% of mutants carried more than 1 Mb of amplified DNA. Amplification features depended on genomic position. For example, amplicons from one locus were similarly sized but displayed variable copy number, whereas those from another locus were differently sized but had comparable copy number. Additionally, the importance of sequence context was highlighted in one region where amplicons differed depending on the presence of a promoter mutation in the strain from which they were selected. DNA sequences at amplicon boundaries in 19 mutants reflected illegitimate recombination. Furthermore, steady-state duplication frequencies measured under non-selective conditions (10(-4) to 10(-5) ) confirmed that spontaneous gene duplication is a major source of genetic variation.  
  Call Number Serial 303  
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Author (up) Sirma, H.; Kumar, M.; Meena, J.K.; Witt, B.; Weise, J.M.; Lechel, A.; Ande, S.; Sakk, V.; Guguen-Guillouzo, C.; Zender, L.; Rudolph, K.-L.; Gunes, C. file  url
doi  openurl
  Title The promoter of human telomerase reverse transcriptase is activated during liver regeneration and hepatocyte proliferation Type Journal Article
  Year 2011 Publication Gastroenterology Abbreviated Journal Gastroenterology  
  Volume 141 Issue 1 Pages 326-37, 337.e1-3  
  Keywords Animals; Binding Sites; Cell Differentiation; *Cell Proliferation; Cells, Cultured; Chromatin Immunoprecipitation; E2F2 Transcription Factor/metabolism; E2F7 Transcription Factor/metabolism; Gene Expression Regulation, Enzymologic; Genes, Reporter; Hepatectomy; Hepatocytes/*enzymology; Humans; Lac Operon; Liver/*enzymology/surgery; *Liver Regeneration; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; *Promoter Regions, Genetic; RNA Interference; Regulatory Elements, Transcriptional; Retinoblastoma Protein/metabolism; Telomerase/*genetics/metabolism; Time Factors; *Transcriptional Activation  
  Abstract BACKGROUND & AIMS: Telomerase activity has not been detected in healthy human liver biopsy samples, but it is up-regulated in most human liver tumors. It is not clear whether telomerase is activated in response to acute or chronic liver injury. Telomerase activity is closely associated with expression of its catalytic subunit, telomerase reverse transcriptase (TERT). We analyzed the activity of the human TERT (hTERT) promoter during liver regeneration in vivo and hepatocyte proliferation in vitro. METHODS: We used hTERTp-lacZ transgenic mice, which contain an 8.0-kilobase pair fragment of the hTERT gene promoter, to study the role of TERT in liver regeneration following partial hepatectomy. As an in vitro model, we used the HepaRG cell line as a new model system for human hepatocyte proliferation and differentiation. RESULTS: Activity of the hTERT promoter increased significantly after partial hepatectomy; it was also induced in hepatocytes, based on immunohistologic analysis. Similar to the in vivo results, telomerase activity and hTERT expression were up-regulated in proliferating HepaRG cells and repressed in response to growth arrest and differentiation. Promoter mapping revealed that a proximal 0.3-kilobase pair fragment contains all elements necessary for regulation of hTERT in HepaRG cells. We identified E2F2 and E2F7 as transcription factors that control the differential expression of hTERT in proliferating hepatocytes, in vitro and in vivo. CONCLUSIONS: hTERT is induced in hepatocytes during liver regeneration, indicating a functional role for telomerase in human liver.  
  Call Number Serial 430  
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Author (up) Thongthae, N.; Payungporn, S.; Poovorawan, Y.; T-Thienprasert, N.P. file  url
openurl 
  Title A rational study for identification of highly effective siRNAs against hepatitis B virus Type Journal Article
  Year 2014 Publication Experimental and Molecular Pathology Abbreviated Journal Exp Mol Pathol  
  Volume 97 Issue 1 Pages 120-127  
  Keywords 2',5'-Oligoadenylate Synthetase/genetics; Algorithms; Base Sequence; Gene Expression Regulation; Hep G2 Cells/virology; Hepatitis B virus/*genetics; Humans; Luciferases/genetics/metabolism; Molecular Sequence Data; NF-kappa B/genetics; Promoter Regions, Genetic; RNA, Small Interfering/chemistry/*genetics; Regulatory Sequences, Nucleic Acid; STAT1 Transcription Factor/genetics; Thermodynamics; Effective siRNAs; Hbv Pre; Hepatitis B virus; RNA interference; siRNA predicting program  
  Abstract RNA interference (RNAi) is a powerful gene knockdown technique used for study gene function. It also potentially provides effective agents for inhibiting infectious and genetic diseases. Most of RNAi studies employ a single siRNA designing program and then require large-scale screening experiments to identify functional siRNAs. In this study, we demonstrate that an assembly of results generated from different siRNA designing programs could provide clusters of predicting sites that aided selection of potent siRNAs. Based on the clusters, three siRNA target sites were selected on a conserved RNA region of hepatitis B virus (HBV), known as HBV post-transcriptional regulatory element (HBV PRE) at nucleotide positions 1317-1337, 1357-1377 and 1644-1664. All three chosen siRNAs driven by H1 promoter were highly effective and could drastically decrease expression of HBV transcripts (core, surface and X) and surface protein without induction of interferon response and cell cytotoxicity in liver cancer cell line (HepG2). Based on prediction of secondary structures, the silencing effects of siRNAs were less effective against a loop sequence of the mRNA target with hairpin structure. In summary, we demonstrate an effectual approach for identification of functional siRNAs. Moreover, highly potent siRNAs identified here may serve as novel agents for development of nucleic acid-based HBV therapy.  
  Call Number Serial 1012  
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