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Sample records for gene expression system

  1. The TRANSFAC system on gene expression regulation.

    Science.gov (United States)

    Wingender, E; Chen, X; Fricke, E; Geffers, R; Hehl, R; Liebich, I; Krull, M; Matys, V; Michael, H; Ohnhäuser, R; Prüss, M; Schacherer, F; Thiele, S; Urbach, S

    2001-01-01

    The TRANSFAC database on transcription factors and their DNA-binding sites and profiles (http://www.gene-regulation.de/) has been quantitatively extended and supplemented by a number of modules. These modules give information about pathologically relevant mutations in regulatory regions and transcription factor genes (PathoDB), scaffold/matrix attached regions (S/MARt DB), signal transduction (TRANSPATH) and gene expression sources (CYTOMER). Altogether, these distinct database modules constitute the TRANSFAC system. They are accompanied by a number of program routines for identifying potential transcription factor binding sites or for localizing individual components in the regulatory network of a cell.

  2. A riboswitch-based inducible gene expression system for mycobacteria.

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    Jessica C Seeliger

    Full Text Available Research on the human pathogen Mycobacterium tuberculosis (Mtb would benefit from novel tools for regulated gene expression. Here we describe the characterization and application of a synthetic riboswitch-based system, which comprises a mycobacterial promoter for transcriptional control and a riboswitch for translational control. The system was used to induce and repress heterologous protein overexpression reversibly, to create a conditional gene knockdown, and to control gene expression in a macrophage infection model. Unlike existing systems for controlling gene expression in Mtb, the riboswitch does not require the co-expression of any accessory proteins: all of the regulatory machinery is encoded by a short DNA segment directly upstream of the target gene. The inducible riboswitch platform has the potential to be a powerful general strategy for creating customized gene regulation systems in Mtb.

  3. Control of gene expression by CRISPR-Cas systems

    Science.gov (United States)

    2013-01-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) loci and their associated cas (CRISPR-associated) genes provide adaptive immunity against viruses (phages) and other mobile genetic elements in bacteria and archaea. While most of the early work has largely been dominated by examples of CRISPR-Cas systems directing the cleavage of phage or plasmid DNA, recent studies have revealed a more complex landscape where CRISPR-Cas loci might be involved in gene regulation. In this review, we summarize the role of these loci in the regulation of gene expression as well as the recent development of synthetic gene regulation using engineered CRISPR-Cas systems. PMID:24273648

  4. Relating Perturbation Magnitude to Temporal Gene Expression in Biological Systems

    Energy Technology Data Exchange (ETDEWEB)

    Callister, Stephen J.; Parnell, John J.; Pfrender, Michael E.; Hashsham, Syed

    2009-03-19

    A method to quantitatively relate stress to response at the level of gene expression is described using Saccharomyces cerevisiae as a model organism. Stress was defined as the magnitude of perturbation and strain was defined as the magnitude of cumulative response in terms of gene expression. Expression patterns of sixty genes previously reported to be significantly impacted by osmotic shock or belonging to the high-osmotic glycerol, glycerolipid metabolism, and glycolysis pathways were determined following perturbations of increasing sodium chloride concentrations (0, 0.5, 0.7, 1.0, 1.5, and 1.4 M). Expression of these genes was quantified temporally using reverse transcriptase real time polymerase chain reaction. The magnitude of cumulative response was obtained by calculating the total moment of area of the temporal response envelope for all the 60 genes, either together or for the set of genes related to each pathway. A non-linear relationship between stress and response was observed for the range of stress studied. This study examines a quantitative approach to quantify the strain at the level of gene expression to relate stress to strain in biological systems. The approach should be generally applicable to quantitatively evaluate the response of organisms to environmental change.

  5. Regulatory systems for hypoxia-inducible gene expression in ischemic heart disease gene therapy.

    Science.gov (United States)

    Kim, Hyun Ah; Rhim, Taiyoun; Lee, Minhyung

    2011-07-18

    Ischemic heart diseases are caused by narrowed coronary arteries that decrease the blood supply to the myocardium. In the ischemic myocardium, hypoxia-responsive genes are up-regulated by hypoxia-inducible factor-1 (HIF-1). Gene therapy for ischemic heart diseases uses genes encoding angiogenic growth factors and anti-apoptotic proteins as therapeutic genes. These genes increase blood supply into the myocardium by angiogenesis and protect cardiomyocytes from cell death. However, non-specific expression of these genes in normal tissues may be harmful, since growth factors and anti-apoptotic proteins may induce tumor growth. Therefore, tight gene regulation is required to limit gene expression to ischemic tissues, to avoid unwanted side effects. For this purpose, various gene expression strategies have been developed for ischemic-specific gene expression. Transcriptional, post-transcriptional, and post-translational regulatory strategies have been developed and evaluated in ischemic heart disease animal models. The regulatory systems can limit therapeutic gene expression to ischemic tissues and increase the efficiency of gene therapy. In this review, recent progresses in ischemic-specific gene expression systems are presented, and their applications to ischemic heart diseases are discussed.

  6. An optimized, chemically regulated gene expression system for Chlamydomonas.

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    Paola Ferrante

    Full Text Available BACKGROUND: Chlamydomonas reinhardtii is a model system for algal and cell biology and is used for biotechnological applications, such as molecular farming or biological hydrogen production. The Chlamydomonas metal-responsive CYC6 promoter is repressed by copper and induced by nickel ions. However, induction by nickel is weak in some strains, poorly reversible by chelating agents like EDTA, and causes, at high concentrations, toxicity side effects on Chlamydomonas growth. Removal of these bottlenecks will encourage the wide use of this promoter as a chemically regulated gene expression system. METHODOLOGY: Using a codon-optimized Renilla luciferase as a reporter gene, we explored several strategies to improve the strength and reversibility of CYC6 promoter induction. Use of the first intron of the RBCS2 gene or of a modified TAP medium increases the strength of CYC6 induction up to 20-fold. In the modified medium, induction is also obtained after addition of specific copper chelators, like TETA. At low concentrations (up to 10 microM TETA is a more efficient inducer than Ni, which becomes a very efficient inducer at higher concentrations (50 microM. Neither TETA nor Ni show toxicity effects at the concentrations used. Unlike induction by Ni, induction by TETA is completely reversible by micromolar copper concentrations, thus resulting in a transient "wave" in luciferase activity, which can be repeated in subsequent growth cycles. CONCLUSIONS: We have worked out a chemically regulated gene expression system that can be finely tuned to produce temporally controlled "waves" in gene expression. The use of cassettes containing the CYC6 promoter, and of modified growth media, is a reliable and economically sustainable system for the temporally controlled expression of foreign genes in Chlamydomonas.

  7. Using interpolation to estimate system uncertainty in gene expression experiments.

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    Lee J Falin

    Full Text Available The widespread use of high-throughput experimental assays designed to measure the entire complement of a cell's genes or gene products has led to vast stores of data that are extremely plentiful in terms of the number of items they can measure in a single sample, yet often sparse in the number of samples per experiment due to their high cost. This often leads to datasets where the number of treatment levels or time points sampled is limited, or where there are very small numbers of technical and/or biological replicates. Here we introduce a novel algorithm to quantify the uncertainty in the unmeasured intervals between biological measurements taken across a set of quantitative treatments. The algorithm provides a probabilistic distribution of possible gene expression values within unmeasured intervals, based on a plausible biological constraint. We show how quantification of this uncertainty can be used to guide researchers in further data collection by identifying which samples would likely add the most information to the system under study. Although the context for developing the algorithm was gene expression measurements taken over a time series, the approach can be readily applied to any set of quantitative systems biology measurements taken following quantitative (i.e. non-categorical treatments. In principle, the method could also be applied to combinations of treatments, in which case it could greatly simplify the task of exploring the large combinatorial space of future possible measurements.

  8. Modified gateway system for double shRNA expression and Cre/lox based gene expression

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    Leung Lisa

    2011-03-01

    Full Text Available Abstract Background The growing need for functional studies of genes has set the stage for the development of versatile tools for genetic manipulations. Results Aiming to provide tools for high throughput analysis of gene functions, we have developed a modified short hairpin RNA (shRNA and gene expression system based on Gateway Technology. The system contains a series of entry and destination vectors that enables easy transfer of shRNA or cDNA into lentiviral expression systems with a variety of selection or marker genes (i.e. puromycin, hygromycin, green fluorescent protein-EGFP, yellow fluorescent protein-YFP and red fluorescent protein-dsRed2. Our shRNA entry vector pENTR.hU6.hH1 containing two tandem human shRNA expression promoters, H1 and U6, was capable of co-expressing two shRNA sequences simultaneously. The entry vector for gene overexpression, pENTR.CMV.ON was constructed to contain CMV promoter with a multiple cloning site flanked by loxP sites allowing for subsequent Cre/lox recombination. Both shRNA and cDNA expression vectors also contained attL sites necessary for recombination with attR sites in our destination expression vectors. As proof of principle we demonstrate the functionality and efficiency of this system by testing expression of several cDNA and shRNA sequences in a number of cell lines. Conclusion Our system is a valuable addition to already existing library of Gateway based vectors and can be an essential tool for many aspects of gene functional studies.

  9. Relating perturbation magnitude to temporal gene expression in biological systems

    Directory of Open Access Journals (Sweden)

    Pfrender Michael E

    2009-03-01

    Full Text Available Abstract Background Most transcriptional activity is a result of environmental variability. This cause (environment and effect (gene expression relationship is essential to survival in any changing environment. The specific relationship between environmental perturbation and gene expression – and stability of the response – has yet to be measured in detail. We describe a method to quantitatively relate perturbation magnitude to response at the level of gene expression. We test our method using Saccharomyces cerevisiae as a model organism and osmotic stress as an environmental stress. Results Patterns of gene expression were measured in response to increasing sodium chloride concentrations (0, 0.5, 0.7, 1.0, and 1.2 M for sixty genes impacted by osmotic shock. Expression of these genes was quantified over five time points using reverse transcriptase real-time polymerase chain reaction. Magnitudes of cumulative response for specific pathways, and the set of all genes, were obtained by combining the temporal response envelopes for genes exhibiting significant changes in expression with time. A linear relationship between perturbation magnitude and response was observed for the range of concentrations studied. Conclusion This study develops a quantitative approach to describe the stability of gene response and pathways to environmental perturbation and illustrates the utility of this approach. The approach should be applicable to quantitatively evaluate the response of organisms via the magnitude of response and stability of the transcriptome to environmental change.

  10. A novel gene delivery system targeting cells expressing VEGF receptors

    Institute of Scientific and Technical Information of China (English)

    LIJUNMIN; JINGCHULUO; 等

    1999-01-01

    Two ligand oligopeptides GV1 and GV2 were designed according to the putative binding region of VEGF to its receptors.GV1,GV2 and endosome releasing oligopeptide HA20 were conjugated with poly-L-lysine or protamine and the resulting conjugates could interact with DNA in a noncovalent bond to form a complex.Using pSV2-β-galactosidase as a reporter gene,it has been demonstrated that exogenous gene was transferred into bovine aortic arch-derived endothelial cells (ABAE) and human malignant melanoma cell lines (A375) in vitro.In vivo experiments,exogenous gene was transferred into tumor vascular endothelial cells and tumor cells of subcutaneously transplanted human colon cancer LOVO,human malignant melanoma A375 and human hepatoma graft in nude mice.This system could also target gene to intrahepatically transplanted human hepatoma injected via portal vein in nude mice.These results are correlated with the relevant receptors(flt-1,flk-1/KDR) expression on the targeted cells and tissues.

  11. WeGET: predicting new genes for molecular systems by weighted co-expression

    NARCIS (Netherlands)

    Szklarczyk, R.; Megchelenbrink, W.; Cizek, P.; Ledent, M.; Velemans, G.; Szklarczyk, D.; Huynen, M.A.

    2016-01-01

    We have developed the Weighted Gene Expression Tool and database (WeGET, http://weget.cmbi.umcn.nl) for the prediction of new genes of a molecular system by correlated gene expression. WeGET utilizes a compendium of 465 human and 560 murine gene expression datasets that have been collected from

  12. 21 CFR 862.1163 - Cardiac allograft gene expression profiling test system.

    Science.gov (United States)

    2010-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1163 Cardiac allograft gene expression profiling test system....

  13. [Ribozyme riboswitch based gene expression regulation systems for gene therapy applications: progress and challenges].

    Science.gov (United States)

    Feng, Jing-Xian; Wang, Jia-wen; Lin, Jun-sheng; Diao, Yong

    2014-11-01

    Robust and efficient control of therapeutic gene expression is needed for timing and dosing of gene therapy drugs in clinical applications. Ribozyme riboswitch provides a promising building block for ligand-controlled gene-regulatory system, based on its property that exhibits tunable gene regulation, design modularity, and target specificity. Ribozyme riboswitch can be used in various gene delivery vectors. In recent years, there have been breakthroughs in extending ribozyme riboswitch's application from gene-expression control to cellular function and fate control. High throughput screening platforms were established, that allow not only rapid optimization of ribozyme riboswitch in a microbial host, but also straightforward transfer of selected devices exhibiting desired activities to mammalian cell lines in a predictable manner. Mathematical models were employed successfully to explore the performance of ribozyme riboswitch quantitively and its rational design predictably. However, to progress toward gene therapy relevant applications, both precision rational design of regulatory circuits and the biocompatibility of regulatory ligand are still of crucial importance.

  14. Molecular transformation, gene cloning, and gene expression systems for filamentous fungi

    Science.gov (United States)

    Gold, Scott E.; Duick, John W.; Redman, Regina S.; Rodriguez, Rusty J.

    2001-01-01

    This chapter discusses the molecular transformation, gene cloning, and gene expression systems for filamentous fungi. Molecular transformation involves the movement of discrete amounts of DNA into cells, the expression of genes on the transported DNA, and the sustainable replication of the transforming DNA. The ability to transform fungi is dependent on the stable replication and expression of genes located on the transforming DNA. Three phenomena observed in bacteria, that is, competence, plasmids, and restriction enzymes to facilitate cloning, were responsible for the development of molecular transformation in fungi. Initial transformation success with filamentous fungi, involving the complementation of auxotrophic mutants by exposure to sheared genomic DNA or RNA from wt isolates, occurred with low transformation efficiencies. In addition, it was difficult to retrieve complementing DNA fragments and isolate genes of interest. This prompted the development of transformation vectors and methods to increase efficiencies. The physiological studies performed with fungi indicated that the cell wall could be removed to generate protoplasts. It was evident that protoplasts could be transformed with significantly greater efficiencies than walled cells.

  15. The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems.

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    Tamer Z Salem

    Full Text Available The simian virus 40 polyadenylation signal (SV40 polyA has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS. In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV, the polyhedrin promoter (very late promoter transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt, and gp37. In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications.

  16. The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems.

    Science.gov (United States)

    Salem, Tamer Z; Seaborn, Craig P; Turney, Colin M; Xue, Jianli; Shang, Hui; Cheng, Xiao-Wen

    2015-01-01

    The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications).

  17. Controlled Gene Expression Systems for Lactic Acid Bacteria : Transferable Nisin-Inducible Expression Cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp.

    NARCIS (Netherlands)

    Kleerebezem, Michiel; Beerthuyzen, Marke M.; Vaughan, Elaine E.; Vos, Willem M. de; Kuipers, Oscar P.

    1997-01-01

    A transferable dual-plasmid inducible gene expression system for use in lactic acid bacteria that is based on the autoregulatory properties of the antimicrobial peptide nisin produced by Lactococcus lactis was developed. Introduction of the two plasmids allowed nisin-inducible gene expression in Lac

  18. Global state measures of the dentate gyrus gene expression system predict antidepressant-sensitive behaviors.

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    Benjamin A Samuels

    Full Text Available BACKGROUND: Selective serotonin reuptake inhibitors (SSRIs such as fluoxetine are the most common form of medication treatment for major depression. However, approximately 50% of depressed patients fail to achieve an effective treatment response. Understanding how gene expression systems respond to treatments may be critical for understanding antidepressant resistance. METHODS: We take a novel approach to this problem by demonstrating that the gene expression system of the dentate gyrus responds to fluoxetine (FLX, a commonly used antidepressant medication, in a stereotyped-manner involving changes in the expression levels of thousands of genes. The aggregate behavior of this large-scale systemic response was quantified with principal components analysis (PCA yielding a single quantitative measure of the global gene expression system state. RESULTS: Quantitative measures of system state were highly correlated with variability in levels of antidepressant-sensitive behaviors in a mouse model of depression treated with fluoxetine. Analysis of dorsal and ventral dentate samples in the same mice indicated that system state co-varied across these regions despite their reported functional differences. Aggregate measures of gene expression system state were very robust and remained unchanged when different microarray data processing algorithms were used and even when completely different sets of gene expression levels were used for their calculation. CONCLUSIONS: System state measures provide a robust method to quantify and relate global gene expression system state variability to behavior and treatment. State variability also suggests that the diversity of reported changes in gene expression levels in response to treatments such as fluoxetine may represent different perspectives on unified but noisy global gene expression system state level responses. Studying regulation of gene expression systems at the state level may be useful in guiding new

  19. The Cpx System Regulates Virulence Gene Expression in Vibrio cholerae

    Science.gov (United States)

    Acosta, Nicole; Pukatzki, Stefan

    2015-01-01

    Bacteria possess signal transduction pathways capable of sensing and responding to a wide variety of signals. The Cpx envelope stress response, composed of the sensor histidine kinase CpxA and the response regulator CpxR, senses and mediates adaptation to insults to the bacterial envelope. The Cpx response has been implicated in the regulation of a number of envelope-localized virulence determinants across bacterial species. Here, we show that activation of the Cpx pathway in Vibrio cholerae El Tor strain C6706 leads to a decrease in expression of the major virulence factors in this organism, cholera toxin (CT) and the toxin-coregulated pilus (TCP). Our results indicate that this occurs through the repression of production of the ToxT regulator and an additional upstream transcription factor, TcpP. The effect of the Cpx response on CT and TCP expression is mostly abrogated in a cyclic AMP receptor protein (CRP) mutant, although expression of the crp gene is unaltered. Since TcpP production is controlled by CRP, our data suggest a model whereby the Cpx response affects CRP function, which leads to diminished TcpP, ToxT, CT, and TCP production. PMID:25824837

  20. Building gene co-expression networks using transcriptomics data for systems biology investigations

    DEFF Research Database (Denmark)

    Kadarmideen, Haja; Watson-Haigh, Nathan S.

    2012-01-01

    Gene co-expression networks (GCN), built using high-throughput gene expression data are fundamental aspects of systems biology. The main aims of this study were to compare two popular approaches to building and analysing GCN. We use real ovine microarray transcriptomics datasets representing four...

  1. Effect of SNPs in protein kinase Czgene on gene expression in the reporter gene detection system

    Institute of Scientific and Technical Information of China (English)

    Zhuo Liu; Hong-Xia Sun; Yong-Wei Zhang; Yun-Feng Li; Jin Zuo; Yan Meng; Fu-De Fang

    2004-01-01

    AIM: To investigated the effects of the SNPs (rs411021,rs436045, rs427811, rs385039 and rs809912) on gene expression and further identify the susceptibility genes of type 2 diabetes.METHODS: Ten allele fragments (49 bp each) were synthesized according to the 5 SNPs mentioned above.These fragments were cloned into luciferase reporter gene vector and then transfected into HepG2 cells. The activity of the luciferase was assayed. Effects of the SNPs on RNA splicing were analyzed by bioinformatics.RESULTS: rs427811T allele and rs809912G allele enhanced the activity of the reporter gene expression. None of the 5 SNPs affected RNA splicing.CONCLUSION: SNPs in protein kinase Cz (PKCZ) gene probably play a role in the susceptibility to type 2 diabetes by affecting the expression level of the relevant genes.

  2. The Influence of Gene Expression Time Delays on Gierer–Meinhardt Pattern Formation Systems

    KAUST Repository

    Seirin Lee, S.

    2010-03-23

    There are numerous examples of morphogen gradients controlling long range signalling in developmental and cellular systems. The prospect of two such interacting morphogens instigating long range self-organisation in biological systems via a Turing bifurcation has been explored, postulated, or implicated in the context of numerous developmental processes. However, modelling investigations of cellular systems typically neglect the influence of gene expression on such dynamics, even though transcription and translation are observed to be important in morphogenetic systems. In particular, the influence of gene expression on a large class of Turing bifurcation models, namely those with pure kinetics such as the Gierer-Meinhardt system, is unexplored. Our investigations demonstrate that the behaviour of the Gierer-Meinhardt model profoundly changes on the inclusion of gene expression dynamics and is sensitive to the sub-cellular details of gene expression. Features such as concentration blow up, morphogen oscillations and radical sensitivities to the duration of gene expression are observed and, at best, severely restrict the possible parameter spaces for feasible biological behaviour. These results also indicate that the behaviour of Turing pattern formation systems on the inclusion of gene expression time delays may provide a means of distinguishing between possible forms of interaction kinetics. Finally, this study also emphasises that sub-cellular and gene expression dynamics should not be simply neglected in models of long range biological pattern formation via morphogens. © 2010 Society for Mathematical Biology.

  3. pHUSH: a single vector system for conditional gene expression

    OpenAIRE

    Eby Mike; Murray Lesley J; Gogineni Alvin; Gu Zhenyu; Peng Li; Hoeflich Klaus P; Gray Daniel C; Kljavin Noelyn; Seshagiri Somasekar; Cole Mary J; Davis David P

    2007-01-01

    Abstract Background Conditional expression vectors have become a valuable research tool to avoid artefacts that may result from traditional gene expression studies. However, most systems require multiple plasmids that must be independently engineered into the target system, resulting in experimental delay and an increased potential for selection of a cell subpopulation that differs significantly from the parental line. We have therefore developed pHUSH, an inducible expression system that all...

  4. Systematic expression analysis of Hox genes at adulthood reveals novel patterns in the central nervous system.

    Science.gov (United States)

    Hutlet, Bertrand; Theys, Nicolas; Coste, Cécile; Ahn, Marie-Thérèse; Doshishti-Agolli, Konstantin; Lizen, Benoît; Gofflot, Françoise

    2016-04-01

    Hox proteins are key regulators of animal development, providing positional identity and patterning information to cells along the rostrocaudal axis of the embryo. Although their embryonic expression and function are well characterized, their presence and biological importance in adulthood remains poorly investigated. We provide here the first detailed quantitative and neuroanatomical characterization of the expression of the 39 Hox genes in the adult mouse brain. Using RT-qPCR we determined the expression of 24 Hox genes mainly in the brainstem of the adult brain, with low expression of a few genes in the cerebellum and the forebrain. Using in situ hybridization (ISH) we have demonstrated that expression of Hox genes is maintained in territories derived from the early segmental Hox expression domains in the hindbrain. Indeed, we show that expression of genes belonging to paralogy groups PG2-8 is maintained in the hindbrain derivatives at adulthood. The spatial colinearity, which characterizes the early embryonic expression of Hox genes, is still observed in sequential antero-posterior boundaries of expression. Moreover, the main mossy and climbing fibres precerebellar nuclei express PG2-8 Hox genes according to their migration origins. Second, ISH confirms the presence of Hox gene transcripts in territories where they are not detected during development, suggesting neo-expression in these territories in adulthood. Within the forebrain, we have mapped Hoxb1, Hoxb3, Hoxb4, Hoxd3 and Hoxa5 expression in restricted areas of the sensory cerebral cortices as well as in specific thalamic relay nuclei. Our data thus suggest a requirement of Hox genes beyond their role of patterning genes, providing a new dimension to their functional relevance in the central nervous system.

  5. Construction of a transformation system for the stable expression of foreign genes in Chlorella sp.

    Institute of Scientific and Technical Information of China (English)

    Wang Yiyun; Gao Xiaorong; Wang Changhai

    2007-01-01

    A stable transformation system for the expression of foreign genes in the unicellular green marine alga (Chlorella sp. MACC/C95) was established. Using electroporation, the alga was transformed with a plasmid containing the phytase gene under the control of CaMV35S promoter and the neomycin phosphotransferase(npt)as a seleetable marker gene. The integration of the phytase gene into the Chlorella genome was revealed by PCR and Southern blotting analysis. RT-PCR analysis revealed the expression of phytasegene at the transcript level. The enhanced activity of phytase enzyme in the transformants confirmed the integration and successful expression of phytase gene. The introduced phytase gene and its protein expression were stably maintained for at least 30 generations in media devoid of selectable antibiotics G418. This is an important step toward the production of useful foreign proteins in Chlorella sp. MACC/C95.

  6. System for stable β-estradiol-inducible gene expression in the moss Physcomitrella patens.

    Science.gov (United States)

    Kubo, Minoru; Imai, Akihiro; Nishiyama, Tomoaki; Ishikawa, Masaki; Sato, Yoshikatsu; Kurata, Tetsuya; Hiwatashi, Yuji; Reski, Ralf; Hasebe, Mitsuyasu

    2013-01-01

    Inducible transgene expression provides a useful tool to analyze gene function. The moss Physcomitrellapatens is a model basal land plant with well-developed research tools, including a high efficiency of gene targeting and substantial genomics resources. However, current systems for controlled transgene expression remain limited. Here we report the development of an estrogen receptor mediated inducible gene expression system, based on the system used in flowering plants. After identifying the appropriate promoters to drive the chimeric transducer, we succeeded in inducing transcription over 1,000-fold after 24 h incubation with β-estradiol. The P. patens system was also effective for high-level long-term induction of gene expression; transcript levels of the activated gene were maintained for at least seven days on medium containing β-estradiol. We also established two potentially neutral targeting sites and a set of vectors for reproducible expression of two transgenes. This β-estradiol-dependent system will be useful to test genes individually or in combination, allowing stable, inducible transgenic expression in P. patens.

  7. A systemic lupus erythematosus gene expression array in disease diagnosis and classification: a preliminary report.

    Science.gov (United States)

    Juang, Y-T; Peoples, C; Kafri, R; Kyttaris, V C; Sunahori, K; Kis-Toth, K; Fitzgerald, L; Ergin, S; Finnell, M; Tsokos, G C

    2011-03-01

    Systemic lupus erythematosus (SLE) is a clinically heterogeneous disease diagnosed on the presence of a constellation of clinical and laboratory findings. At the pathogenetic level, multiple factors using diverse biochemical and molecular pathways have been recognized. Succinct recognition and classification of clinical disease subsets, as well as the availability of disease biomarkers, remains largely unsolved. Based on information produced by the present authors' and other laboratories, a lupus gene expression array consisting of 30 genes, previously claimed to contribute to aberrant function of T cells, was developed. An additional eight genes were included as controls. Peripheral blood was obtained from 10 patients (19 samples) with SLE and six patients with rheumatoid arthritis (RA) as well as 19 healthy controls. T cell mRNA was subjected to reverse transcription and PCR, and the gene expression levels were measured. Conventional statistical analysis was performed along with principal component analysis (PCA) to capture the contribution of all genes to disease diagnosis and clinical parameters. The lupus gene expression array faithfully informed on the expression levels of genes. The recorded changes in expression reflect those reported in the literature by using a relatively small (5 ml) amount of peripheral blood. PCA of gene expression levels placed SLE samples apart from normal and RA samples regardless of disease activity. Individual principal components tended to define specific disease manifestations such as arthritis and proteinuria. Thus, a lupus gene expression array based on genes previously claimed to contribute to immune pathogenesis of SLE may define the disease, and principal components of the expression of 30 genes may define patients with specific disease manifestations.

  8. The Gene Expression Patterns of Peripheral Blood Mononuclear Cells in Patients with Systemic Lupus Erythematosus

    Institute of Scientific and Technical Information of China (English)

    LI Shouxin; JIANG Wei; HUANG Rui; WANG Xiaohui; LIU Wen; SHEN Shouyin

    2007-01-01

    This study examined the gene expression patterns of peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus (SLE) by using serial analysis of gene expression (SAGE) technology. Following the construction of serial analysis of gene expression (SAGE) library of PBMCs collected from 3 cases of familial SLE patients, a large scale of tag sequencing was performed. The data extracted from sequencing files was analyzed with SAGE 2000 V 4.5 software.The top 30 expressed genes of SLE patients were uploaded to http://david.niaid.nih. gov/david/ease.htm and the functional classification of genes was obtained. The differences among those expressed gene were analyzed by Chi-square tests. The results showed that a total of 1286 unique SAGE tags were identified from 1814 individual SAGE tags. Among the 1286 unique tags, 86.8% had single copy, and only 0.2% tags had more than 20 copies. And 68.4% of the tags matched known expressed sequences, 41.1% of which matched more than one known expressed sequence. About 31.6% of the tags had no match and could represent potentially novel genes. Approximately one third of the top 30 genes were ribosomal protein, and the rest were genes related to metabolism or with unknown functions. Eight tags were found to express differentially in SAGE library of SLE patients. This study draws a profile of gene expression patterns of PBMCs in patients with SLE. Comparison of SAGE database from PBMCs between normal individuals and SLE patients will help us to better understand the pathogenesis of SLE.

  9. Engineering of a green-light inducible gene expression system in Synechocystis sp. PCC6803.

    Science.gov (United States)

    Abe, Koichi; Miyake, Kotone; Nakamura, Mayumi; Kojima, Katsuhiro; Ferri, Stefano; Ikebukuro, Kazunori; Sode, Koji

    2014-03-01

    In order to construct a green-light-regulated gene expression system for cyanobacteria, we characterized a green-light sensing system derived from Synechocystis sp. PCC6803, consisting of the green-light sensing histidine kinase CcaS, the cognate response regulator CcaR, and the promoter of cpcG2 (PcpcG 2 ). CcaS and CcaR act as a genetic controller and activate gene expression from PcpcG 2 with green-light illumination. The green-light induction level of the native PcpcG 2 was investigated using GFPuv as a reporter gene inserted in a broad-host-range vector. A clear induction of protein expression from native PcpcG 2 under green-light illumination was observed; however, the expression level was very low compared with Ptrc , which was reported to act as a constitutive promoter in cyanobacteria. Therefore, a Shine-Dalgarno-like sequence derived from the cpcB gene was inserted in the 5' untranslated region of the cpcG2 gene, and the expression level of CcaR was increased. Thus, constructed engineered green-light sensing system resulted in about 40-fold higher protein expression than with the wild-type promoter with a high ON/OFF ratio under green-light illumination. The engineered green-light gene expression system would be a useful genetic tool for controlling gene expression in the emergent cyanobacterial bioprocesses. © 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  10. Candidate innate immune system gene expression in the ecological model Daphnia

    Science.gov (United States)

    Decaestecker, Ellen; Labbé, Pierrick; Ellegaard, Kirsten; Allen, Judith E.; Little, Tom J.

    2011-01-01

    The last ten years have witnessed increasing interest in host–pathogen interactions involving invertebrate hosts. The invertebrate innate immune system is now relatively well characterised, but in a limited range of genetic model organisms and under a limited number of conditions. Immune systems have been little studied under real-world scenarios of environmental variation and parasitism. Thus, we have investigated expression of candidate innate immune system genes in the water flea Daphnia, a model organism for ecological genetics, and whose capacity for clonal reproduction facilitates an exceptionally rigorous control of exposure dose or the study of responses at many time points. A unique characteristic of the particular Daphnia clones and pathogen strain combinations used presently is that they have been shown to be involved in specific host–pathogen coevolutionary interactions in the wild. We choose five genes, which are strong candidates to be involved in Daphnia–pathogen interactions, given that they have been shown to code for immune effectors in related organisms. Differential expression of these genes was quantified by qRT-PCR following exposure to the bacterial pathogen Pasteuria ramosa. Constitutive expression levels differed between host genotypes, and some genes appeared to show correlated expression. However, none of the genes appeared to show a major modification of expression level in response to Pasteuria exposure. By applying knowledge from related genetic model organisms (e.g. Drosophila) to models for the study of evolutionary ecology and coevolution (i.e. Daphnia), the candidate gene approach is temptingly efficient. However, our results show that detection of only weak patterns is likely if one chooses target genes for study based on previously identified genome sequences by comparison to homologues from other related organisms. Future work on the Daphnia–Pasteuria system will need to balance a candidate gene approach with more

  11. Evolution of vertebrate central nervous system is accompanied by novel expression changes of duplicate genes.

    Science.gov (United States)

    Chen, Yuan; Ding, Yun; Zhang, Zuming; Wang, Wen; Chen, Jun-Yuan; Ueno, Naoto; Mao, Bingyu

    2011-12-20

    The evolution of the central nervous system (CNS) is one of the most striking changes during the transition from invertebrates to vertebrates. As a major source of genetic novelties, gene duplication might play an important role in the functional innovation of vertebrate CNS. In this study, we focused on a group of CNS-biased genes that duplicated during early vertebrate evolution. We investigated the tempo-spatial expression patterns of 33 duplicate gene families and their orthologs during the embryonic development of the vertebrate Xenopus laevis and the cephalochordate Brachiostoma belcheri. Almost all the identified duplicate genes are differentially expressed in the CNS in Xenopus embryos, and more than 50% and 30% duplicate genes are expressed in the telencephalon and mid-hindbrain boundary, respectively, which are mostly considered as two innovations in the vertebrate CNS. Interestingly, more than 50% of the amphioxus orthologs do not show apparent expression in the CNS in amphioxus embryos as detected by in situ hybridization, indicating that some of the vertebrate CNS-biased duplicate genes might arise from non-CNS genes in invertebrates. Our data accentuate the functional contribution of gene duplication in the CNS evolution of vertebrate and uncover an invertebrate non-CNS history for some vertebrate CNS-biased duplicate genes. Copyright © 2011. Published by Elsevier Ltd.

  12. Obtaining Relevant Genes by Analysis of Expression Arrays with a Multi-Agent System

    Directory of Open Access Journals (Sweden)

    Alfonso GONZÁLEZ

    2015-05-01

    Full Text Available Triple negative breast cancer (TNBC is an aggressive form of breast cancer. Despite treatment with chemotherapy, relapses are frequent and response to these treatments is not the same in younger women as in older women. Therefore, the identification of genes that provoke this disease is required, as well as the identification of therapeutic targets.There are currently different hybridization techniques, such as expression ar-rays, which measure the signal expression of both the genomic and tran-scriptomic levels of thousands of genes of a given sample. Probesets of Gene 1.0 ST GeneChip arrays provide the ultimate genome transcript coverage, providing a measurement of the expression level of the sample.This paper proposes a multi-agent system to manage information of expres-sion arrays, with the goal of providing an intuitive system that is also extensible to analyze and interpret the results.The roles of agent integrate different types of techniques, from statistical and data mining techniques that select a set of genes, to search techniques that find pathways in which such genes participate, and information extraction techniques that apply a CBR system to check if these genes are involved in the disease.

  13. Investigation of a common gene expression signature in gastrointestinal cancers using systems biology approaches.

    Science.gov (United States)

    Baghaei, Kaveh; Hosseinkhan, Nazanin; Asadzadeh Aghdaei, Hamid; Zali, M R

    2017-09-04

    According to GLOBOCAN 2012, the incidence and the mortality rate of colorectal, stomach and liver cancers are the highest among the total gastrointestinal (GI) cancers. Here we aimed to find the common genes and pathways that are simultaneously deregulated in these three malignancies using systems biology approaches. Here we conducted a differential expression analysis on high-quality gene expression datasets of gastric cancer (GC), colorectal cancer (CRC) and hepatocellular carcinoma (HCC). To address the inter gene correlations that were neglected in differential expression studies, we also applied differential co-expression analysis on the understudied datasets. The common significant differentially expressed genes (DEGs) among the three cancers were used for further regulatory and PPI network construction. In parallel the regulatory roles of miRNAs and lncRNAs in the common DEGs were investigated. 23 common DEGs were detected between GC, CRC and HCC. Two cases of potential feed forward loops were identified in the constructed TF-target regulatory network, indicating the probable cross-talk between biological pathways. The result of a vulnerability test on the common PPI network resulted in the finding of three candidates, the simultaneous targeting of which will disintegrate the main parts of the network. The results of the differential co-expression study led to the identification of respectively 7 and 1 common differentially co-expressed pairs of genes between GC and CRC and between CRC and HCC. The results of the differential expression study introduced new common players in CRC, GC and HCC and provided better insights into the molecular characteristics of these GI malignancies. Moreover, we concluded that differential co-expression studies are an essential complement for differential expression studies that just take single differentially expressed genes into account.

  14. Central nervous system gene expression changes in a transgenic mouse model for bovine spongiform encephalopathy

    Directory of Open Access Journals (Sweden)

    Tortosa Raül

    2011-10-01

    Full Text Available Abstract Gene expression analysis has proven to be a very useful tool to gain knowledge of the factors involved in the pathogenesis of diseases, particularly in the initial or preclinical stages. With the aim of finding new data on the events occurring in the Central Nervous System in animals affected with Bovine Spongiform Encephalopathy, a comprehensive genome wide gene expression study was conducted at different time points of the disease on mice genetically modified to model the bovine species brain in terms of cellular prion protein. An accurate analysis of the information generated by microarray technique was the key point to assess the biological relevance of the data obtained in terms of Transmissible Spongiform Encephalopathy pathogenesis. Validation of the microarray technique was achieved by RT-PCR confirming the RNA change and immunohistochemistry techniques that verified that expression changes were translated into variable levels of protein for selected genes. Our study reveals changes in the expression of genes, some of them not previously associated with prion diseases, at early stages of the disease previous to the detection of the pathological prion protein, that might have a role in neuronal degeneration and several transcriptional changes showing an important imbalance in the Central Nervous System homeostasis in advanced stages of the disease. Genes whose expression is altered at early stages of the disease should be considered as possible therapeutic targets and potential disease markers in preclinical diagnostic tool development. Genes non-previously related to prion diseases should be taken into consideration for further investigations.

  15. Central nervous system gene expression changes in a transgenic mouse model for bovine spongiform encephalopathy.

    Science.gov (United States)

    Tortosa, Raül; Castells, Xavier; Vidal, Enric; Costa, Carme; Ruiz de Villa, María del Carmen; Sánchez, Alex; Barceló, Anna; Torres, Juan María; Pumarola, Martí; Ariño, Joaquín

    2011-10-28

    Gene expression analysis has proven to be a very useful tool to gain knowledge of the factors involved in the pathogenesis of diseases, particularly in the initial or preclinical stages. With the aim of finding new data on the events occurring in the Central Nervous System in animals affected with Bovine Spongiform Encephalopathy, a comprehensive genome wide gene expression study was conducted at different time points of the disease on mice genetically modified to model the bovine species brain in terms of cellular prion protein. An accurate analysis of the information generated by microarray technique was the key point to assess the biological relevance of the data obtained in terms of Transmissible Spongiform Encephalopathy pathogenesis. Validation of the microarray technique was achieved by RT-PCR confirming the RNA change and immunohistochemistry techniques that verified that expression changes were translated into variable levels of protein for selected genes. Our study reveals changes in the expression of genes, some of them not previously associated with prion diseases, at early stages of the disease previous to the detection of the pathological prion protein, that might have a role in neuronal degeneration and several transcriptional changes showing an important imbalance in the Central Nervous System homeostasis in advanced stages of the disease. Genes whose expression is altered at early stages of the disease should be considered as possible therapeutic targets and potential disease markers in preclinical diagnostic tool development. Genes non-previously related to prion diseases should be taken into consideration for further investigations.

  16. Analysis of gene expression profiles in human systemic lupus erythematosus using oligonucleotide microarray.

    Science.gov (United States)

    Han, G-M; Chen, S-L; Shen, N; Ye, S; Bao, C-D; Gu, Y-Y

    2003-04-01

    Epidemiologic studies suggest a strong genetic component for susceptibility to systemic lupus erythematosus (SLE). To investigate the genetic mechanism of pathogenesis of SLE, we studied the difference in gene expression of peripheral blood cells between 10 SLE patients and 18 healthy controls using oligonucleotide microarray. When gene expression for patients was compared to the mean of normal controls, among the 3002 target genes, 61 genes were identified with greater than a two-fold change difference in expression level. Of these genes, 24 were upregulated and 37 downregulated in at least half of the patients. By the Welch's ANOVA/Welch's t-test, all these 61 genes were significantly different (PTSA-1/Sca-2) may play an important role in the mechanism of SLE pathogenesis. TSA-1 antigens may represent an important alternative pathway for T-cell activation that may be involved in IFN-mediated immunomodulation. Hierarchical clustering showed that patient samples were clearly separated from controls based on their gene expression profile. These results demonstrate that high-density oligonucleotide microarray has the potential to explore the mechanism of pathogenesis of systemic lupus erythematosus.

  17. A Novel Tightly Regulated Gene Expression System for the Human Intestinal Symbiont Bacteroides thetaiotaomicron

    Directory of Open Access Journals (Sweden)

    Regis Stentz

    2016-07-01

    Full Text Available There is considerable interest in studying the function of Bacteroides species resident in the human gastrointestinal (GI-tract and the contribution they make to host health. Reverse genetics and protein expression techniques, such as those developed for well-characterised Escherichia coli cannot be applied to Bacteroides species as they and other members of the Bacteriodetes phylum have unique promoter structures. The availability of useful Bacteroides-specific genetic tools is therefore limited. Here we describe the development of an effective mannan-controlled gene expression system for Bacteroides thetaiotaomicron containing the mannan-inducible promoter–region of an α-1,2-mannosidase gene (BT_3784, a ribosomal binding site designed to modulate expression, a multiple cloning site to facilitate the cloning of genes of interest, and a transcriptional terminator. Using the Lactobacillus pepI as a reporter gene, mannan induction resulted in an increase of reporter activity in a time- and concentration-dependent manner with a wide range of activity. The endogenous BtcepA cephalosporinase gene was used to demonstrate the suitability of this novel expression system, enabling the isolation of a His-tagged version of BtCepA. We have also shown with experiments performed in mice that the system can be induced in vivo in the presence of an exogenous source of mannan. By enabling the controlled expression of endogenous and exogenous genes in B. thetaiotaomicron this novel inducer-dependent expression system will aid in defining the physiological role of individual genes and the functional analyses of their products.

  18. Gene expression programming for power system static security ...

    African Journals Online (AJOL)

    user

    1*Electrical Power and Machines Department, Ain Shams University, Cairo, EGYPT. 2 ENPPI ... DOI: http://dx.doi.org/10.4314/ijest.v4i2.6. 1. .... Power system security assessment using Kohonen neural network has been illustrated by Niebor et al. ..... Learning rate of BPNN was assumed to be 0.12 and momentum constant.

  19. pHUSH: a single vector system for conditional gene expression

    Directory of Open Access Journals (Sweden)

    Eby Mike

    2007-09-01

    Full Text Available Abstract Background Conditional expression vectors have become a valuable research tool to avoid artefacts that may result from traditional gene expression studies. However, most systems require multiple plasmids that must be independently engineered into the target system, resulting in experimental delay and an increased potential for selection of a cell subpopulation that differs significantly from the parental line. We have therefore developed pHUSH, an inducible expression system that allows regulated expression of shRNA, miRNA or cDNA cassettes on a single viral vector. Results Both Pol II and Pol III promoters have been successfully combined with a second expression cassette containing a codon-optimized tetracycline repressor and selectable marker. We provide examples of how pHUSH has been successfully employed to study the function of target genes in a number of cell types within in vitro and in vivo assays, including conditional gene knockdown in a murine model of brain cancer. Conclusion We have successfully developed and employed a single vector system that enables Doxycycline regulated RNAi or transgene expression in a variety of in vitro and in vivo model systems. These studies demonstrate the broad application potential of pHUSH for conditional genetic engineering in mammalian cells.

  20. Adipose tissue endocannabinoid system gene expression: depot differences and effects of diet and exercise

    Directory of Open Access Journals (Sweden)

    Yang Rongze

    2011-10-01

    Full Text Available Abstract Background Alterations of endocannabinoid system in adipose tissue play an important role in lipid regulation and metabolic dysfunction associated with obesity. The purpose of this study was to determine whether gene expression levels of cannabinoid type 1 receptor (CB1 and fatty acid amide hydrolase (FAAH are different in subcutaneous abdominal and gluteal adipose tissue, and whether hypocaloric diet and aerobic exercise influence subcutaneous adipose tissue CB1 and FAAH gene expression in obese women. Methods Thirty overweight or obese, middle-aged women (BMI = 34.3 ± 0.8 kg/m2, age = 59 ± 1 years underwent one of three 20-week weight loss interventions: caloric restriction only (CR, N = 9, caloric restriction plus moderate-intensity aerobic exercise (CRM, 45-50% HRR, N = 13, or caloric restriction plus vigorous-intensity aerobic exercise (CRV, 70-75% HRR, N = 8. Subcutaneous abdominal and gluteal adipose tissue samples were collected before and after the interventions to measure CB1 and FAAH gene expression. Results At baseline, FAAH gene expression was higher in abdominal, compared to gluteal adipose tissue (2.08 ± 0.11 vs. 1.78 ± 0.10, expressed as target gene/β-actin mRNA ratio × 10-3, P Conclusions There are depot differences in subcutaneous adipose tissue endocannabinoid system gene expression in obese individuals. Aerobic exercise training may preferentially modulate abdominal adipose tissue endocannabinoid-related gene expression during dietary weight loss. Trial Registration ClinicalTrials.gov: NCT00664729.

  1. A highly sensitive and specific system for large-scale gene expression profiling

    Directory of Open Access Journals (Sweden)

    Wang Hui-Yun

    2008-01-01

    Full Text Available Abstract Background Rapid progress in the field of gene expression-based molecular network integration has generated strong demand on enhancing the sensitivity and data accuracy of experimental systems. To meet the need, a high-throughput gene profiling system of high specificity and sensitivity has been developed. Results By using specially designed primers, the new system amplifies sequences in neighboring exons separated by big introns so that mRNA sequences may be effectively discriminated from other highly related sequences including their genes, unprocessed transcripts, pseudogenes and pseudogene transcripts. Probes used for microarray detection consist of sequences in the two neighboring exons amplified by the primers. In conjunction with a newly developed high-throughput multiplex amplification system and highly simplified experimental procedures, the system can be used to analyze >1,000 mRNA species in a single assay. It may also be used for gene expression profiling of very few (n = 100 or single cells. Highly reproducible results were obtained from duplicate samples with the same number of cells, and from those with a small number (100 and a large number (10,000 of cells. The specificity of the system was demonstrated by comparing results from a breast cancer cell line, MCF-7, and an ovarian cancer cell line, NCI/ADR-RES, and by using genomic DNA as starting material. Conclusion Our approach may greatly facilitate the analysis of combinatorial expression of known genes in many important applications, especially when the amount of RNA is limited.

  2. Construction of a System for the Stable Expression of Foreign Genes in Dunaliella salina

    Institute of Scientific and Technical Information of China (English)

    GENGDe-Gui; HANYan; WANGYi-Qin; WANGPeng; ZHANGLi-Ming; LIWen-Bin; SUNYong-Ru

    2004-01-01

    A stable transformation system for the expression of foreign genes in the unicellular greenmarine alga (Dunaliella salina Teod.) was established. Using electroporation, the alga was transformed witha plasmid containing the hepatitis B surface antigen (HBsAg) gene and the chloramphenicol acetyltransferase(CAT) gene as a selectable gene. PCR and Southern blotting analysis indicated that the HBsAEgene wasintegrated into the D. salina genome. Northern dotting analysis showed that the HBsAg gene was expressedat the mRNA level. The stable expression of HBsAg protein in transformants was confirmed by HBsAgenzyme-linked immunosorbent assay (HBsAg EUSA) and Western blotting analysis. Also, PCR and Southernblotting analyses showed that the CA Tgene was integrated into the D, salina genome, and CAT EUSAindicated that CAT protein was stably expressed in the cells. The introduced HBsAg DNA and HBsAgprotein expression were stably maintained for at least 60 generations in media devoid of chloramphenicol.This is the first report of the stable expression of foreign genes in D. salina.

  3. Cloning of Porcine Lactoferrin Gene and Construction of Expression System in Recombinant Lactobacillus

    Institute of Scientific and Technical Information of China (English)

    ZONG Xiaolin; HA Zhuo; ZHAO Lili; LIU Diqiu; QIAO Xinyuan; JIANG Yanping; GE Junwei; LI Yijing; TANG Lijie

    2011-01-01

    Lactobacillus was selected as a bacterial carrier for expression of N-lobe of porcine lactoferrin (PLFN). A pair of primers was designed with Oligo6.0 and used to amplify PLFN gene. It was in accordance with the characters of translational fusions from gene and expression vector plasmid. A 1 077 bp fragment of the gene from PLF was cloned from mammary gland tissue of the lactating sow on the third day by RT-PCR; the gene was connected with the vector plasmid pPG612.1 and transformed into the host strain JM109. The recombinant expression vector plasmid pPG612-PLFN was created and identified by using plasmid extraction, PCR, restriction enzyme digestion and sequence analysis. The recombinant plasmid was transformed into Lactobacillus casei ATCC393, Lactobacillus plantarum KLDS 1.0344, Lactobacillus paracasei KLDS 1.0652 and Lactobacillus pentosus KLDS 1.0413 by electroporation, and produced the recombinant strains of pPG612-PLFN/L, casei, pPG612-PLFN/L, plantarum, pPG612-PLFN/ L. paracasei and pPG612-PLFN/L, pentosus, respectively. The results indicated that PLFN gene had inserted into the expression vectors and achieved multiple Laetobacillus expression systems. It electes the base for the expression and production of recombinant porcine lactoferrin in Lactobaeillus

  4. Construction of an inducible cell-communication system that amplifies Salmonella gene expression in tumor tissue.

    Science.gov (United States)

    Dai, Yumei; Toley, Bhushan J; Swofford, Charles A; Forbes, Neil S

    2013-06-01

    Bacterial therapies have the potential to overcome resistances that cause chemotherapies to fail. When using bacteria to produce anticancer agents in tumors, triggering gene expression is necessary to prevent systemic toxicity. The use of chemical triggers, however, is hampered by poor delivery of inducing molecules, which reduces the number of activated bacteria. To solve this problem, we created a cell-communication system that enables activated bacteria to induce inactive neighbors. We hypothesized that introducing cell communication into Salmonella would improve direct triggering strategies by increasing protein production, increasing sensitivity to inducer molecules, and enabling expression in tumor tissue. To test these hypotheses we integrated the PBAD promoter into the quorum-sensing machinery from Vibrio fischeri. The expression of a fluorescent reporter gene was compared to expression from non-communicating controls. Function in three-dimensional tissue was tested in a tumor-on-a-chip device. Bacterial communication increased fluorescence 40-fold and increased sensitivity to inducer molecules more than 10,000-fold. The system enabled bacteria to activate neighbors and increased the time-scale of protein production. Gene expression was controllable and tightly regulated. At the optimal inducing signal, communicating bacteria produced 350 times more protein than non-communicating bacteria. The cell-communication system created in this study has uses beyond cancer therapy, including protein manufacturing, bioremediation and biosensing. It would enable amplified induction of gene expression in any environment that limits availability of inducer molecules. Ultimately, because inducible cellular communication enables gene expression in tissue, it will be a critical component of bacterial anticancer therapies.

  5. Expanding the molecular toolbox for Lactococcus lactis: construction of an inducible thioredoxin gene fusion expression system

    LENUS (Irish Health Repository)

    Douillard, Francois P

    2011-08-09

    Abstract Background The development of the Nisin Inducible Controlled Expression (NICE) system in the food-grade bacterium Lactococcus lactis subsp. cremoris represents a cornerstone in the use of Gram-positive bacterial expression systems for biotechnological purposes. However, proteins that are subjected to such over-expression in L. lactis may suffer from improper folding, inclusion body formation and\\/or protein degradation, thereby significantly reducing the yield of soluble target protein. Although such drawbacks are not specific to L. lactis, no molecular tools have been developed to prevent or circumvent these recurrent problems of protein expression in L. lactis. Results Mimicking thioredoxin gene fusion systems available for E. coli, two nisin-inducible expression vectors were constructed to over-produce various proteins in L. lactis as thioredoxin fusion proteins. In this study, we demonstrate that our novel L. lactis fusion partner expression vectors allow high-level expression of soluble heterologous proteins Tuc2009 ORF40, Bbr_0140 and Tuc2009 BppU\\/BppL that were previously insoluble or not expressed using existing L. lactis expression vectors. Over-expressed proteins were subsequently purified by Ni-TED affinity chromatography. Intact heterologous proteins were detected by immunoblotting analyses. We also show that the thioredoxin moiety of the purified fusion protein was specifically and efficiently cleaved off by enterokinase treatment. Conclusions This study is the first description of a thioredoxin gene fusion expression system, purposely developed to circumvent problems associated with protein over-expression in L. lactis. It was shown to prevent protein insolubility and degradation, allowing sufficient production of soluble proteins for further structural and functional characterization.

  6. Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae

    Science.gov (United States)

    Rantasalo, Anssi; Czeizler, Elena; Virtanen, Riitta; Rousu, Juho; Lähdesmäki, Harri; Penttilä, Merja

    2016-01-01

    This work describes the development and characterization of a modular synthetic expression system that provides a broad range of adjustable and predictable expression levels in S. cerevisiae. The system works as a fixed-gain transcription amplifier, where the input signal is transferred via a synthetic transcription factor (sTF) onto a synthetic promoter, containing a defined core promoter, generating a transcription output signal. The system activation is based on the bacterial LexA-DNA-binding domain, a set of modified, modular LexA-binding sites and a selection of transcription activation domains. We show both experimentally and computationally that the tuning of the system is achieved through the selection of three separate modules, each of which enables an adjustable output signal: 1) the transcription-activation domain of the sTF, 2) the binding-site modules in the output promoter, and 3) the core promoter modules which define the transcription initiation site in the output promoter. The system has a novel bidirectional architecture that enables generation of compact, yet versatile expression modules for multiple genes with highly diversified expression levels ranging from negligible to very strong using one synthetic transcription factor. In contrast to most existing modular gene expression regulation systems, the present system is independent from externally added compounds. Furthermore, the established system was minimally affected by the several tested growth conditions. These features suggest that it can be highly useful in large scale biotechnology applications. PMID:26901642

  7. Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Anssi Rantasalo

    Full Text Available This work describes the development and characterization of a modular synthetic expression system that provides a broad range of adjustable and predictable expression levels in S. cerevisiae. The system works as a fixed-gain transcription amplifier, where the input signal is transferred via a synthetic transcription factor (sTF onto a synthetic promoter, containing a defined core promoter, generating a transcription output signal. The system activation is based on the bacterial LexA-DNA-binding domain, a set of modified, modular LexA-binding sites and a selection of transcription activation domains. We show both experimentally and computationally that the tuning of the system is achieved through the selection of three separate modules, each of which enables an adjustable output signal: 1 the transcription-activation domain of the sTF, 2 the binding-site modules in the output promoter, and 3 the core promoter modules which define the transcription initiation site in the output promoter. The system has a novel bidirectional architecture that enables generation of compact, yet versatile expression modules for multiple genes with highly diversified expression levels ranging from negligible to very strong using one synthetic transcription factor. In contrast to most existing modular gene expression regulation systems, the present system is independent from externally added compounds. Furthermore, the established system was minimally affected by the several tested growth conditions. These features suggest that it can be highly useful in large scale biotechnology applications.

  8. 21 CFR 866.6040 - Gene expression profiling test system for breast cancer prognosis.

    Science.gov (United States)

    2010-04-01

    ... cancer prognosis. 866.6040 Section 866.6040 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Tumor... cancer prognosis. (a) Identification. A gene expression profiling test system for breast cancer...

  9. A single-cell bioluminescence imaging system for monitoring cellular gene expression in a plant body.

    Science.gov (United States)

    Muranaka, Tomoaki; Kubota, Saya; Oyama, Tokitaka

    2013-12-01

    Gene expression is a fundamental cellular process and expression dynamics are of great interest in life science. We succeeded in monitoring cellular gene expression in a duckweed plant, Lemna gibba, using bioluminescent reporters. Using particle bombardment, epidermal and mesophyll cells were transfected with the luciferase gene (luc+) under the control of a constitutive [Cauliflower mosaic virus 35S (CaMV35S)] and a rhythmic [Arabidopsis thaliana CIRCADIAN CLOCK ASSOCIATED 1 (AtCCA1)] promoter. Bioluminescence images were captured using an EM-CCD (electron multiply charged couple device) camera. Luminescent spots of the transfected cells in the plant body were quantitatively measured at the single-cell level. Luminescence intensities varied over a 1,000-fold range among CaMV35S::luc+-transfected cells in the same plant body and showed a log-normal-like frequency distribution. We monitored cellular gene expression under light-dark conditions by capturing bioluminescence images every hour. Luminescence traces of ≥50 individual cells in a frond were successfully obtained in each monitoring procedure. Rhythmic and constitutive luminescence behaviors were observed in cells transfected with AtCCA1::luc+ and CaMV35S::luc+, respectively. Diurnal rhythms were observed in every AtCCA1::luc+-introduced cell with traceable luminescence, and slight differences were detected in their rhythmic waveforms. Thus the single-cell bioluminescence monitoring system was useful for the characterization of cellular gene expression in a plant body.

  10. Hapten-derivatized nanoparticle targeting and imaging of gene expression by multimodality imaging systems.

    Science.gov (United States)

    Cheng, C-M; Chu, P-Y; Chuang, K-H; Roffler, S R; Kao, C-H; Tseng, W-L; Shiea, J; Chang, W-D; Su, Y-C; Chen, B-M; Wang, Y-M; Cheng, T-L

    2009-01-01

    Non-invasive gene monitoring is important for most gene therapy applications to ensure selective gene transfer to specific cells or tissues. We developed a non-invasive imaging system to assess the location and persistence of gene expression by anchoring an anti-dansyl (DNS) single-chain antibody (DNS receptor) on the cell surface to trap DNS-derivatized imaging probes. DNS hapten was covalently attached to cross-linked iron oxide (CLIO) to form a 39+/-0.5 nm DNS-CLIO nanoparticle imaging probe. DNS-CLIO specifically bound to DNS receptors but not to a control single-chain antibody receptor. DNS-CLIO (100 microM Fe) was non-toxic to both B16/DNS (DNS receptor positive) and B16/phOx (control receptor positive) cells. Magnetic resonance (MR) imaging could detect as few as 10% B16/DNS cells in a mixture in vitro. Importantly, DNS-CLIO specifically bound to a B16/DNS tumor, which markedly reduced signal intensity. Similar results were also shown with DNS quantum dots, which specifically targeted CT26/DNS cells but not control CT26/phOx cells both in vitro and in vivo. These results demonstrate that DNS nanoparticles can systemically monitor the expression of DNS receptor in vivo by feasible imaging systems. This targeting strategy may provide a valuable tool to estimate the efficacy and specificity of different gene delivery systems and optimize gene therapy protocols in the clinic.

  11. Pregnancy-Induced Changes in Systemic Gene Expression among Healthy Women and Women with Rheumatoid Arthritis.

    Directory of Open Access Journals (Sweden)

    Anuradha Mittal

    Full Text Available Pregnancy induces drastic biological changes systemically, and has a beneficial effect on some autoimmune conditions such as rheumatoid arthritis (RA. However, specific systemic changes that occur as a result of pregnancy have not been thoroughly examined in healthy women or women with RA. The goal of this study was to identify genes with expression patterns associated with pregnancy, compared to pre-pregnancy as baseline and determine whether those associations are modified by presence of RA.In our RNA sequencing (RNA-seq dataset from 5 healthy women and 20 women with RA, normalized expression levels of 4,710 genes were significantly associated with pregnancy status (pre-pregnancy, first, second and third trimesters over time, irrespective of presence of RA (False Discovery Rate (FDR-adjusted p value<0.05. These genes were enriched in pathways spanning multiple systems, as would be expected during pregnancy. A subset of these genes (n = 256 showed greater than two-fold change in expression during pregnancy compared to baseline levels, with distinct temporal trends through pregnancy. Another 98 genes involved in various biological processes including immune regulation exhibited expression patterns that were differentially associated with pregnancy in the presence or absence of RA.Our findings support the hypothesis that the maternal immune system plays an active role during pregnancy, and also provide insight into other systemic changes that occur in the maternal transcriptome during pregnancy compared to the pre-pregnancy state. Only a small proportion of genes modulated by pregnancy were influenced by presence of RA in our data.

  12. Intersectional Gene Expression in Zebrafish Using the Split KalTA4 System.

    Science.gov (United States)

    Almeida, Rafael Gois; Lyons, David Anthony

    2015-12-01

    In this study, we describe the adaptation of the split Gal4 system for zebrafish. The Gal4-UAS system is widely used for expression of genes-of-interest by crossing driver lines expressing the transcription factor Gal4 (under the control of the promoter of interest) with reporter lines where upstream activating sequence (UAS) repeats (recognized by Gal4) drive expression of the genes-of-interest. In the Split Gal4 system, hemi-drivers separately encode the DNA-binding domain (DBD) and the activation domain (AD) of Gal4. When encoded under two different promoters, only those cells in the intersection of the promoters' expression pattern and in which both promoters are active reconstitute a functional Gal4 and activate expression from a UAS-driven transgene. We split the zebrafish-optimized version of Gal4, KalTA4, and generated a hemi-driver encoding the KalTA4 DBD and a hemi-driver encoding KalTA4's AD. We show that split KalTA4 domains can assemble in vivo and transactivate a UAS reporter transgene and that each hemi-driver alone cannot transactivate the reporter. Also, transactivation can happen in several cell types, with similar efficiency to intact KalTA4. Finally, in transient mosaic expression assays, we show that when hemi-drivers are preceded by two distinct promoters, they restrict the expression of an UAS-driven reporter from a broader pattern (sox10) to its constituent smaller neuronal pattern. The Split KalTA4 system should be useful for expression of genes-of-interest in an intersectional manner, allowing for more refined manipulations of cell populations in zebrafish.

  13. Pax8, a murine paired box gene expressed in the developing excretory system and thyroid gland.

    Science.gov (United States)

    Plachov, D; Chowdhury, K; Walther, C; Simon, D; Guenet, J L; Gruss, P

    1990-10-01

    Several mouse genes designated 'Pax genes' contain a highly conserved DNA sequence homologous to the paired box of Drosophila. Here we describe the isolation of Pax8, a novel paired box containing clone from an 8.5 day p.c. mouse embryo cDNA library. An open reading frame of 457 amino acids (aa) contains the 128 aa paired domain near the amino terminus. Another conserved region present in some other paired box genes, the octapeptide Tyr-Ser-Ile-Asn-Gly-Leu-Leu-Gly, is located 43 aa C-terminal to the paired domain. Using an interspecies backcross system, we have mapped the Pax8 gene within the proximal portion of mouse chromosome 2 in a close linkage to the surf locus. Several developmental mutations are located in this region. In situ hybridization was used to determine the pattern of Pax8 expression during mouse embryogenesis. Pax8 is expressed transiently between 11.5 and 12.5 days of gestation along the rostrocaudal axis extending from the myelencephalon throughout the length of the neural tube, predominantly in two parallel regions on either side of the basal plate. We also detected Pax8 expression in the developing thyroid gland beginning at 10.5 days of gestation, during the thyroid evagination. In the mesonephros and metanephros the expression of Pax8 was localized to the mesenchymal condensations, which are induced by the nephric duct and ureter, respectively. These condensations develop to functional units, the nephrons, of the kidney. These data are consistent with a role for Pax8 in the induction of kidney epithelium. The embryonic expression pattern of Pax8 is compared with that of Pax2, another recently described paired box gene expressed in the developing excretory system.

  14. Identification and Characterization of a Rat Novel Gene RSEP4 Expressed Specifically in Central Nervous System

    Institute of Scientific and Technical Information of China (English)

    Xi-Dao WANG; Ling-Wei KONG; Zhi-Qin XIE; Yu-Qiu ZHANG; Zhi-Xin LIN; Zhi-Qi ZHAO; Lei YU; Nai-He JING

    2004-01-01

    The low-abundantly expressed genes composed the majorities of the mRNAs expressed in the central nervous system (CNS), and were thought to be important for the normal brain functions. Through differential screening a low-abundance cDNA sublibrary with mRNA from neuropathic pain of chronic constriction injury (CCI) model, we have identified a novel rat gene, rat spinal-cord expression protein 4 gene (RSEP4). The total length ofRSEP4 cDNA is 2006 bp, with a 501 nucleotide open reading frame (ORF) that encodes a 167 amino acid polypeptide. Northern blot revealed that RSEP4 was expressed specifically in the CNS. In situ hybridization showed that the mRNA of RSEP4 was strongly expressed in the CA1, CA2, CA3 and DG regions of hippocampus, the Purkinje cells of cerebellum, and the small sensory neurons of dorsal horn and large motor neurons of ventral horn of spinal cord. Over-expression of RSEP4-EGFP fusion protein in the human embryonic kidney 293T cells showed that RSEP4 protein was mainly localized in the cell cytoplasm. These results suggest that RSEP4 may play some roles in the CNS.

  15. Two capa-genes are expressed in the neuroendocrine system of Rhodnius prolixus.

    Science.gov (United States)

    Neupert, Susanne; Russell, William K; Russell, David H; Predel, Reinhard

    2010-03-01

    CAPA-peptides have been isolated from a broad range of insect species and are typical of the neurosecretory system of the abdominal ventral nerve cord. In Rhodnius prolixus, which is known to transmit Chagas' disease, CAPA-peptides have potent antidiuretic effects. Recently, two capa-genes were isolated from this hemipteran insect; it was the first description of the occurrence of multiple capa-genes from a single insect species. The expression of peptides from one of these capa-genes was confirmed by mass spectrometry Paluzzi (2008) [10]. In this study, the abdominal neuroendocrine system of R. prolixus was screened for the occurrence of products of the second annotated capa-gene. Single nerve preparations of abdominal segmental nerves 1-3 of R. prolixus were analyzed by MALDI-TOF mass spectrometry and the products of both capa-genes were identified by tandem mass spectrometry. The co-occurrence of the different CAPA-peptides, including CAPA-precursor peptides, in all spectra from abdominal segmental nerves 1 to 3 suggests a common expression of both capa-genes in the same neuroendocrine cells. A comparison of mass spectra obtained from abdominal segmental nerves with those of the subesophageal ganglion (SEG) revealed an obvious differential processing of the CAPA-precursors within the CNS.

  16. Dynamic expression of leukocyte innate immune genes in whole blood from horses with lipopolysaccharide-induced acute systemic inflammation

    DEFF Research Database (Denmark)

    Vinther, Anne Mette L.; Skovgaard, Kerstin; Heegaard, Peter M. H.

    2015-01-01

    ) and sepsis. Therefore, the aim of this study was to investigate the expression of 31 selected blood leukocyte immune genes in an equine model of acute systemic inflammation to identify significantly regulated genes and to describe their expression dynamics during a 24-h experimental period. Systemic...

  17. Gene expression in the neuropeptide Y system during ethanol withdrawal kindling in rats

    DEFF Research Database (Denmark)

    Olling, Janne Damm; Ulrichsen, Jakob; Correll, Mette;

    2010-01-01

    ), and an isocalorically fed control group. Gene expression of NPY and its receptors Y1, Y2, and Y5 was studied in the hippocampal dentate gyrus (DG) and CA3/CA1, as well as piriform cortex (PirCx), and neocortex (NeoCx). RESULTS: MW+/- as well as SW groups showed decreased NPY gene expression in all hippocampal areas......+/- groups compared with the SW group. The MW+ group differed from the MW- group in the PirCx, where Y2 gene expression was significantly higher. CONCLUSION: Multiple withdrawal episodes reversibly decreased NPY and NPY receptor mRNA levels at peak withdrawal, with smaller decreases in NPY mRNA levels...... and augmented decreases in Y1/Y5 mRNA levels compared with a SW episode. Multiple withdrawal-induced seizures increased the Y2 mRNA levels in PirCx. These complex changes in NPY system gene expression could play a role in the ethanol withdrawal kindling process....

  18. Engineered Regulatory Systems Modulate Gene Expression of Human Commensals in the Gut.

    Science.gov (United States)

    Lim, Bentley; Zimmermann, Michael; Barry, Natasha A; Goodman, Andrew L

    2017-04-20

    The gut microbiota is implicated in numerous aspects of health and disease, but dissecting these connections is challenging because genetic tools for gut anaerobes are limited. Inducible promoters are particularly valuable tools because these platforms allow real-time analysis of the contribution of microbiome gene products to community assembly, host physiology, and disease. We developed a panel of tunable expression platforms for the prominent genus Bacteroides in which gene expression is controlled by a synthetic inducer. In the absence of inducer, promoter activity is fully repressed; addition of inducer rapidly increases gene expression by four to five orders of magnitude. Because the inducer is absent in mice and their diets, Bacteroides gene expression inside the gut can be modulated by providing the inducer in drinking water. We use this system to measure the dynamic relationship between commensal sialidase activity and liberation of mucosal sialic acid, a receptor and nutrient for pathogens. VIDEO ABSTRACT. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Extended function of plasmid partition genes: the Sop system of linear phage-plasmid N15 facilitates late gene expression.

    Science.gov (United States)

    Ravin, Nikolai V; Rech, Jérôme; Lane, David

    2008-05-01

    The mitotic stability of the linear plasmid-prophage N15 of Escherichia coli depends on a partition system closely related to that of the F plasmid SopABC. The two Sop systems are distinguished mainly by the arrangement of their centromeric SopB-binding sites, clustered in F (sopC) and dispersed in N15 (IR1 to IR4). Because two of the N15 inverted repeat (IR) sites are located close to elements presumed (by analogy with phage lambda) to regulate late gene expression during the lytic growth of N15, we asked whether Sop partition functions play a role in this process. In N15, a putative Q antiterminator gene is located 6 kb upstream of the probable major late promoter and two intrinsic terminator-like sequences, in contrast to lambda, where the Q gene is adjacent to the late promoter. Northern hybridization and lacZ reporter activity confirmed the identity of the N15 late promoter (p52), demonstrated antiterminator activity of the Q analogue, and located terminator sequences between p52 and the first open reading frame. Following prophage induction, N15 mutated in IR2 (downstream from gene Q) or IR3 (upstream of p52) showed a pronounced delay in lysis relative to that for wild-type N15. Expression of ir3(-)-p52::lacZ during N15 wild-type lytic growth was strongly reduced relative to the equivalent ir3(+) fusion. The provision of Q protein and the IR2 and SopAB proteins in trans to ir3(+)-p52::lacZ increased expression beyond that seen in the absence of any one of these factors. These results indicate that the N15 Sop system has a dual role: partition and regulation of late gene transcription during lytic growth.

  20. Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system.

    Science.gov (United States)

    Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C; Moon, Tae Seok

    2016-03-18

    A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA-asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions.

  1. Pregnancy-Induced Changes in Systemic Gene Expression among Healthy Women and Women with Rheumatoid Arthritis

    DEFF Research Database (Denmark)

    Mittal, Anuradha; Pachter, Lior; Nelson, J Lee

    2015-01-01

    Background Pregnancy induces drastic biological changes systemically, and has a beneficial effect on some autoimmune conditions such as rheumatoid arthritis (RA). However, specific systemic changes that occur as a result of pregnancy have not been thoroughly examined in healthy women or women...... with RA. The goal of this study was to identify genes with expression patterns associated with pregnancy, compared to pre-pregnancy as baseline and determine whether those associations are modified by presence of RA. Results In our RNA sequencing (RNA-seq) dataset from 5 healthy women and 20 women with RA......, normalized expression levels of 4,710 genes were significantly associated with pregnancy status (pre-pregnancy, first, second and third trimesters) over time, irrespective of presence of RA (False Discovery Rate (FDR)-adjusted p value

  2. Methylation of Promoter Regions of Genes of the Human Intrauterine Renin Angiotensin System and Their Expression

    Directory of Open Access Journals (Sweden)

    Shane D. Sykes

    2015-01-01

    Full Text Available The intrauterine renin angiotensin system (RAS is implicated in placentation and labour onset. Here we investigate whether promoter methylation of RAS genes changes with gestation or labour and if it affects gene expression. Early gestation amnion and placenta were studied, as were term amnion, decidua, and placenta collected before labour (at elective caesarean section or after spontaneous labour and delivery. The expression and degree of methylation of the prorenin receptor (ATP6AP2, angiotensin converting enzyme (ACE, angiotensin II type 1 receptor (AGTR1, and two proteases that can activate prorenin (kallikrein, KLK1, and cathepsin D, CTSD were measured by qPCR and a DNA methylation array. There was no effect of gestation or labour on the methylation of RAS genes and CTSD. Amnion and decidua displayed strong correlations between the percent hypermethylation of RAS genes and CTSD, suggestive of global methylation. There were no correlations between the degree of methylation and mRNA abundance of any genes studied. KLK1 was the most methylated gene and the proportion of hypermethylated KLK1 alleles was lower in placenta than decidua. The presence of intermediate methylated alleles of KLK1 in early gestation placenta and in amnion after labour suggests that KLK1 methylation is uniquely dynamic in these tissues.

  3. A score system for quality evaluation of RNA sequence tags: an improvement for gene expression profiling

    Directory of Open Access Journals (Sweden)

    Pinheiro Daniel G

    2009-06-01

    Full Text Available Abstract Background High-throughput molecular approaches for gene expression profiling, such as Serial Analysis of Gene Expression (SAGE, Massively Parallel Signature Sequencing (MPSS or Sequencing-by-Synthesis (SBS represent powerful techniques that provide global transcription profiles of different cell types through sequencing of short fragments of transcripts, denominated sequence tags. These techniques have improved our understanding about the relationships between these expression profiles and cellular phenotypes. Despite this, more reliable datasets are still necessary. In this work, we present a web-based tool named S3T: Score System for Sequence Tags, to index sequenced tags in accordance with their reliability. This is made through a series of evaluations based on a defined rule set. S3T allows the identification/selection of tags, considered more reliable for further gene expression analysis. Results This methodology was applied to a public SAGE dataset. In order to compare data before and after filtering, a hierarchical clustering analysis was performed in samples from the same type of tissue, in distinct biological conditions, using these two datasets. Our results provide evidences suggesting that it is possible to find more congruous clusters after using S3T scoring system. Conclusion These results substantiate the proposed application to generate more reliable data. This is a significant contribution for determination of global gene expression profiles. The library analysis with S3T is freely available at http://gdm.fmrp.usp.br/s3t/. S3T source code and datasets can also be downloaded from the aforementioned website.

  4. Development of a Heat-Shock Inducible Gene Expression System in the Red Alga Cyanidioschyzon merolae

    Science.gov (United States)

    Sumiya, Nobuko; Fujiwara, Takayuki; Kobayashi, Yusuke; Misumi, Osami; Miyagishima, Shin-ya

    2014-01-01

    The cell of the unicellular red alga Cyanidioschyzon merolae contains a single chloroplast and mitochondrion, the division of which is tightly synchronized by a light/dark cycle. The genome content is extremely simple, with a low level of genetic redundancy, in photosynthetic eukaryotes. In addition, transient transformation and stable transformation by homologous recombination have been reported. However, for molecular genetic analyses of phenomena that are essential for cellular growth and survival, inducible gene expression/suppression systems are needed. Here, we report the development of a heat-shock inducible gene expression system in C. merolae. CMJ101C, encoding a small heat shock protein, is transcribed only when cells are exposed to an elevated temperature. Using a superfolder GFP as a reporter protein, the 200-bp upstream region of CMJ101C orf was determined to be the optimal promoter for heat-shock induction. The optimal temperature to induce expression is 50°C, at which C. merolae cells are able to proliferate. At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression. After the heat shock, the mRNA level decreases rapidly. As an example of the system, the expression of a dominant negative form of chloroplast division DRP5B protein, which has a mutation in the GTPase domain, was induced. Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure. This result suggests that the dominant negative DRP5B inhibited the final scission of the chloroplast division site, but not the earlier stages of division site constriction. It is also suggested that cell cycle progression is not arrested by the impairment of chloroplast division at the final stage. PMID:25337786

  5. Plasmid-borne prokaryotic gene expression: Sources of variability and quantitative system characterization

    Science.gov (United States)

    Bagh, Sangram; Mazumder, Mostafizur; Velauthapillai, Tharsan; Sardana, Vandit; Dong, Guang Qiang; Movva, Ashok B.; Lim, Len H.; McMillen, David R.

    2008-02-01

    One aim of synthetic biology is to exert systematic control over cellular behavior, either for medical purposes or to “program” microorganisms. An engineering approach to the design of biological controllers demands a quantitative understanding of the dynamics of both the system to be controlled and the controllers themselves. Here we focus on a widely used method of exerting control in bacterial cells: plasmid vectors bearing gene-promoter pairs. We study two variants of the simplest such element, an unregulated promoter constitutively expressing its gene, against the varying genomic background of four Escherichia coli cell strains. Absolute protein numbers and rates of expression vary with both cell strain and plasmid type, as does the variability of expression across the population. Total variability is most strongly coupled to the cell division process, and after cell size is scaled away, plasmid copy number regulation emerges as a significant effect. We present simple models that capture the main features of the system behavior. Our results confirm that complex interactions between plasmids and their hosts can have significant effects on both expression and variability, even in deliberately simplified systems.

  6. A simple approach for estimating gene expression in Candida albicans directly from a systemic infection site.

    Science.gov (United States)

    Andes, D; Lepak, A; Pitula, A; Marchillo, K; Clark, J

    2005-09-01

    Gene expression analysis after the host-pathogen interaction is revolutionizing our understanding of the host response to infection. Numerous studies have utilized microarray analysis to follow host cell transcriptome alterations in response to interactions with infectious pathogens. However, similar analyses of pathogen transcriptional adaptation at the infection site have been limited. Understanding the nature of this interaction from the pathogen perspective at different sites and stages of infection is central to strategies for development of new anti-infective therapies. Toward this end, we developed a protocol to analyze changes in gene expression for a eukaryotic pathogen, Candida albicans, during systemic infection in mice. The experimental approach takes advantage of the resistance of the cell wall of many fungal pathogens to cell lysis, relative to mammalian cells. After lysis of mammalian cells, the tissue mixture containing fungal cells is depleted of mammalian RNA by centrifugation, followed by enzymatic digestion. RNA-digesting enzymes are then inhibited before eukaryotic cell lysis and RNA isolation. The protocol provides a reproducible quantity of RNA based on pathogen cell number. The quality of the RNA allowed reliable downstream transcriptional analysis using reverse-transcription polymerase chain reaction and microarrays. The in vivo gene expression data confirmed involvement of several putative pathogenesis genes. More importantly, the results provided a wealth of biologically interesting hypotheses to direct future investigation.

  7. Peripheral Nervous System Genes Expressed in Central Neurons Induce Growth on Inhibitory Substrates

    Science.gov (United States)

    Buchser, William J.; Smith, Robin P.; Pardinas, Jose R.; Haddox, Candace L.; Hutson, Thomas; Moon, Lawrence; Hoffman, Stanley R.; Bixby, John L.; Lemmon, Vance P.

    2012-01-01

    Trauma to the spinal cord and brain can result in irreparable loss of function. This failure of recovery is in part due to inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans (CSPGs). Peripheral nervous system (PNS) neurons exhibit increased regenerative ability compared to central nervous system neurons, even in the presence of inhibitory environments. Previously, we identified over a thousand genes differentially expressed in PNS neurons relative to CNS neurons. These genes represent intrinsic differences that may account for the PNS’s enhanced regenerative ability. Cerebellar neurons were transfected with cDNAs for each of these PNS genes to assess their ability to enhance neurite growth on inhibitory (CSPG) or permissive (laminin) substrates. Using high content analysis, we evaluated the phenotypic profile of each neuron to extract meaningful data for over 1100 genes. Several known growth associated proteins potentiated neurite growth on laminin. Most interestingly, novel genes were identified that promoted neurite growth on CSPGs (GPX3, EIF2B5, RBMX). Bioinformatic approaches also uncovered a number of novel gene families that altered neurite growth of CNS neurons. PMID:22701605

  8. Peripheral nervous system genes expressed in central neurons induce growth on inhibitory substrates.

    Directory of Open Access Journals (Sweden)

    William J Buchser

    Full Text Available Trauma to the spinal cord and brain can result in irreparable loss of function. This failure of recovery is in part due to inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans (CSPGs. Peripheral nervous system (PNS neurons exhibit increased regenerative ability compared to central nervous system neurons, even in the presence of inhibitory environments. Previously, we identified over a thousand genes differentially expressed in PNS neurons relative to CNS neurons. These genes represent intrinsic differences that may account for the PNS's enhanced regenerative ability. Cerebellar neurons were transfected with cDNAs for each of these PNS genes to assess their ability to enhance neurite growth on inhibitory (CSPG or permissive (laminin substrates. Using high content analysis, we evaluated the phenotypic profile of each neuron to extract meaningful data for over 1100 genes. Several known growth associated proteins potentiated neurite growth on laminin. Most interestingly, novel genes were identified that promoted neurite growth on CSPGs (GPX3, EIF2B5, RBMX. Bioinformatic approaches also uncovered a number of novel gene families that altered neurite growth of CNS neurons.

  9. Peripheral nervous system genes expressed in central neurons induce growth on inhibitory substrates.

    Science.gov (United States)

    Buchser, William J; Smith, Robin P; Pardinas, Jose R; Haddox, Candace L; Hutson, Thomas; Moon, Lawrence; Hoffman, Stanley R; Bixby, John L; Lemmon, Vance P

    2012-01-01

    Trauma to the spinal cord and brain can result in irreparable loss of function. This failure of recovery is in part due to inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans (CSPGs). Peripheral nervous system (PNS) neurons exhibit increased regenerative ability compared to central nervous system neurons, even in the presence of inhibitory environments. Previously, we identified over a thousand genes differentially expressed in PNS neurons relative to CNS neurons. These genes represent intrinsic differences that may account for the PNS's enhanced regenerative ability. Cerebellar neurons were transfected with cDNAs for each of these PNS genes to assess their ability to enhance neurite growth on inhibitory (CSPG) or permissive (laminin) substrates. Using high content analysis, we evaluated the phenotypic profile of each neuron to extract meaningful data for over 1100 genes. Several known growth associated proteins potentiated neurite growth on laminin. Most interestingly, novel genes were identified that promoted neurite growth on CSPGs (GPX3, EIF2B5, RBMX). Bioinformatic approaches also uncovered a number of novel gene families that altered neurite growth of CNS neurons.

  10. Identification and refinement of two strong constitutive promoters for gene expression system of Schizosaccharomyces pombe.

    Science.gov (United States)

    Wang, Hongcheng; Wang, Haiyang; Wang, Meng; Zhang, Lei; Wang, Ren; Mei, Yanzhen; Shao, Weilan

    2014-06-01

    Fission yeast Schizosaccharomyces pombe shares various important properties with higher eukaryotes and is now considered a useful host for elevated production of mammalian proteins for medicinal applications. The full-length nmt1 promoter has been widely used as a strong promoter in S. pombe expression system. In the present study, the promoters of the eno101 and gpd3 genes in S. pombe were identified as strong constitutive promoters. For convenient applications in the plasmids of S. pombe, these promoters were refined to 276-bp eno and 273-bp gpd promoters by deleting undesired sequences and examining the expression of reporter genes including lacZ and xynA. Both the refined eno and gpd promoters provided approximately 1.5-fold higher expression of LacZ than nmt1 promoter. Furthermore, gene expression under the control of the eno or gpd promoter was not repressed by the components of YES medium while nmt1 promoter was inhibited by thiamine in yeast extract. Therefore, both eno and gpd promoters offer opportunities for efficient production of recombinant proteins by S. pombe in high cell-density fermentation.

  11. Early passage bone marrow stromal cells express genes involved in nervous system development supporting their relevance for neural repair

    NARCIS (Netherlands)

    Nandoe, R.D.S.; Bossers, K.; Ritfeld, G.J.; Blits, B.; Grotenhuis, J.A.; Verhaagen, J.; Oudega, M.

    2011-01-01

    PURPOSE: The assessment of the capacity of bone marrow stromal cells (BMSC) to repair the nervous system using gene expression profiling. The evaluation of effects of long-term culturing on the gene expression profile of BMSC. METHODS: Fourty four k whole genome rat microarrays were used to study

  12. Epigenetic phenomena, chromatin dynamics, and gene expression. New theoretical approaches in the study of living systems.

    Science.gov (United States)

    Boi, Luciano

    2008-01-01

    This paper is aimed at exploring the genome at the level beyond that of DNA sequence alone. We stress the fact that the level of genes is not the sole "reality" in the living world, for there are different epigenetic processes that profoundly affect change in living systems. Moreover, epigenetics very likely influences the course of evolution and the unfolding of life. We further attempt to investigate how the genome is dynamically organized into the nuclear space within the cell. We mainly focus on analyses of higher order nuclear architecture and the dynamic interactions of chromatin with other nuclear components. We especially want to know how epigenetic phenomena influences genes expression and chromosome functions. The proper understanding of these processes require new concepts and approaches be introduced and developed. In particular, we think that research in biology has to shift from only describing molecular and local features of living systems to studying the regulatory networks of interactions among gene pathways, the folding and dynamics of chromatin structure and how environmental factors affects the behavior of organisms. There are essential components of biological information on living organisms which cannot be portrayed in the DNA sequence alone. In a post-genomic era, the importance of chromatin/epigenetic interface has become increasingly apparent. One of the purposes of current research should be to highlight the enormous impact of chromatin organization and dynamics on epigenetic phenomena, and, conversely, to emphasize the important role that epigenetic phenomena play in gene expression and cell regulation.

  13. A hormone receptor-based transactivator bridges different binary systems to precisely control spatial-temporal gene expression in Drosophila.

    Directory of Open Access Journals (Sweden)

    Shu-Yun Kuo

    Full Text Available The GAL4/UAS gene expression system is a precise means of targeted gene expression employed to study biological phenomena in Drosophila. A modified GAL4/UAS system can be conditionally regulated using a temporal and regional gene expression targeting (TARGET system that responds to heat shock induction. However heat shock-related temperature shifts sometimes cause unexpected physiological responses that confound behavioral analyses. We describe here the construction of a drug-inducible version of this system that takes advantage of tissue-specific GAL4 driver lines to yield either RU486-activated LexA-progesterone receptor chimeras (LexPR or β-estradiol-activated LexA-estrogen receptor chimeras (XVE. Upon induction, these chimeras bind to a LexA operator (LexAop and activate transgene expression. Using GFP expression as a marker for induction in fly brain cells, both approaches are capable of tightly and precisely modulating transgene expression in a temporal and dosage-dependent manner. Additionally, tissue-specific GAL4 drivers resulted in target gene expression that was restricted to those specific tissues. Constitutive expression of the active PKA catalytic subunit using these systems altered the sleep pattern of flies, demonstrating that both systems can regulate transgene expression that precisely mimics regulation that was previously engineered using the GeneSwitch/UAS system. Unlike the limited number of GeneSwitch drivers, this approach allows for the usage of the multitudinous, tissue-specific GAL4 lines for studying temporal gene regulation and tissue-specific gene expression. Together, these new inducible systems provide additional, highly valuable tools available to study gene function in Drosophila.

  14. A model of gene expression based on random dynamical systems reveals modularity properties of gene regulatory networks.

    Science.gov (United States)

    Antoneli, Fernando; Ferreira, Renata C; Briones, Marcelo R S

    2016-06-01

    Here we propose a new approach to modeling gene expression based on the theory of random dynamical systems (RDS) that provides a general coupling prescription between the nodes of any given regulatory network given the dynamics of each node is modeled by a RDS. The main virtues of this approach are the following: (i) it provides a natural way to obtain arbitrarily large networks by coupling together simple basic pieces, thus revealing the modularity of regulatory networks; (ii) the assumptions about the stochastic processes used in the modeling are fairly general, in the sense that the only requirement is stationarity; (iii) there is a well developed mathematical theory, which is a blend of smooth dynamical systems theory, ergodic theory and stochastic analysis that allows one to extract relevant dynamical and statistical information without solving the system; (iv) one may obtain the classical rate equations form the corresponding stochastic version by averaging the dynamic random variables (small noise limit). It is important to emphasize that unlike the deterministic case, where coupling two equations is a trivial matter, coupling two RDS is non-trivial, specially in our case, where the coupling is performed between a state variable of one gene and the switching stochastic process of another gene and, hence, it is not a priori true that the resulting coupled system will satisfy the definition of a random dynamical system. We shall provide the necessary arguments that ensure that our coupling prescription does indeed furnish a coupled regulatory network of random dynamical systems. Finally, the fact that classical rate equations are the small noise limit of our stochastic model ensures that any validation or prediction made on the basis of the classical theory is also a validation or prediction of our model. We illustrate our framework with some simple examples of single-gene system and network motifs.

  15. Temporal Control of Cre Recombinase-mediated in Vitro DNA Recombination by Tet-on Gene Expression System

    Institute of Scientific and Technical Information of China (English)

    Zhong-Min GUO; Kang XU; Ying YUE; Bing HUANG; Xin-Yan DENG; Nü-Qi ZHONG; Xun HONG; Xi-Gu CHEN; Dong XIAO

    2005-01-01

    Conditional gene expression and gene deletion are important experimental approaches for examining the functions of particular gene products in mouse models. These strategies exploiting Cre-mediated site-specific DNA recombination have been incorporated into transgenic and gene-targeting procedures to allow in vivo manipulation of DNA in embryonic stem cells (ES cells) or living animals. The Cre/lox P system has become widely used in conditional gene targeting, conditional gene repair and activation, inducible chromosome translocation, and chromosome engineering. In this project, we have employed the universal transgenic system and the liver-specific promoter system for tightly temporal and liver-specific control of Cre gene expression in mice that (1) integrates the advantages of the Tet-on gene expression system and Cre/lox P site-mediated gene activation, and (2) simplifies the scheme of animal crosses through a combination of two control elements in a single transgene. A liver-specific apoE promoter was inserted into the promoter cloning site upstream of the rtTA cassette of pCore construct to generate the transgene construct pApoErtTAtetO-Cre, followed by demonstrating stringent regulation of doxycycline (Dox)-induced Cre-mediated recombination in the lox P-flanked transcription STOP cassette-modified BEL-7402 cells. That is to say, in the absence of Dox, the Cre gene is not expressed and will not induce site-specific recombination between two lox P sites, whereas on exposure to Dox, the Cre gene will be expressed and the recombination will occur.Together, these data indicate that the Tet-on gene expression system is able to successfully and stringently control Cre expression in vitro, which lays a solid foundation for efficient and spatio-temporal Cre gene activation in transgenic mice.

  16. Poinsettia protoplasts - a simple, robust and efficient system for transient gene expression studies

    Directory of Open Access Journals (Sweden)

    Pitzschke Andrea

    2012-05-01

    Full Text Available Abstract Background Transient gene expression systems are indispensable tools in molecular biology. Yet, their routine application is limited to few plant species often requiring substantial equipment and facilities. High chloroplast and chlorophyll content may further impede downstream applications of transformed cells from green plant tissue. Results Here, we describe a fast and simple technique for the high-yield isolation and efficient transformation (>70% of mesophyll-derived protoplasts from red leaves of the perennial plant Poinsettia (Euphorbia pulccherrima. In this method no particular growth facilities or expensive equipments are needed. Poinsettia protoplasts display an astonishing robustness and can be employed in a variety of commonly-used downstream applications, such as subcellular localisation (multi-colour fluorescence or promoter activity studies. Due to low abundance of chloroplasts or chromoplasts, problems encountered in other mesophyll-derived protoplast systems (particularly autofluorescence are alleviated. Furthermore, the transgene expression is detectable within 90 minutes of transformation and lasts for several days. Conclusions The simplicity of the isolation and transformation procedure renders Poinsettia protoplasts an attractive system for transient gene expression experiments, including multi-colour fluorescence, subcellular localisation and promoter activity studies. In addition, they offer hitherto unknown possibilities for anthocyan research and industrial applications.

  17. Developmental gene expression of sympathetic nervous system tumors reflects their histogenesis.

    Science.gov (United States)

    Hoehner, J C; Hedborg, F; Eriksson, L; Sandstedt, B; Grimelius, L; Olsen, L; Påhlman, S

    1998-01-01

    Comparisons of the developing human sympathetic nervous system (SNS) to tumors presumed to derive from these cells may suggest tumor progenitors and predict tumor biologic behavior. Classic neuroblastoma (NB) and its more highly differentiated stroma-rich subtypes, extra-adrenal sympathetic paraganglioma, and pheochromocytoma were examined for the presence of the developmentally characterized gene products NSE, S-100, CD44, Bcl-2, HNK-1, PNMT, TrkA, IGF2, and tyrosine hydroxylase. The marker gene expression profiles of these tumors were compared with those similarly determined for a number of normal prenatal and postnatal human SNS cell types. Sympathetic paraganglioma, pheochromocytoma, and stroma-rich NB display marker expression profiles mimicking those of childhood sympathetic paraganglia, adrenal chromaffin cells, and sympathetic neurons, respectively. A selection of differentiating, extra-adrenal NB tumors with prognostically favorable features possess marker gene expression profiles paralleling that observed for fetal extra-adrenal sympathetic paraganglia/small intensely fluorescent cells. In contrast, undifferentiated, clinically aggressive NB tumors manifest characteristics mirroring that of embryonic/early fetal sympathetic neuroblasts of sympathetic ganglia and of the adrenal gland. These findings suggest that clinical features, such as primary tumor location and age at diagnosis, provide prognostic information for NB patients by virtue of the existence and biology of the presumed tumor progenitor cell type.

  18. Arabidopsis FLOWERING LOCUS D influences systemic-acquiredresistance-induced expression and histone modifications of WRKY genes

    Indian Academy of Sciences (India)

    Vijayata Singh; Shweta Roy; Deepjyoti Singh; Ashis Kumar Nandi

    2014-03-01

    A plant that is in part infected by a pathogen is more resistant throughout its whole body to subsequent infections – a phenomenon known as systemic acquired resistance (SAR). Mobile signals are synthesized at the site of infection and distributed throughout the plant through vascular tissues. Mechanism of SAR development subsequent to reaching the mobile signal in the distal tissue is largely unknown. Recently we showed that FLOWERING LOCUS D (FLD) gene of Arabidopsis thaliana is required in the distal tissue to activate SAR. FLD codes for a homologue of human-lysine-specific histone demethylase. Here we show that FLD function is required for priming (SAR induced elevated expression during challenge inoculation) of WRKY29 and WRKY6 genes. FLD also differentially influences basal and SAR-induced expression of WRKY38, WRKY65 and WRKY53 genes. In addition, we also show that FLD partly localizes in nucleus and influences histone modifications at the promoters of WRKY29 and WRKY6 genes. The results altogether indicate to the possibility of FLD’s involvement in epigenetic regulation of SAR.

  19. Stable Surface Expression of a Gene for Helicobacter pylori Toxic Porin Protein with pBAD Expression System

    Institute of Scientific and Technical Information of China (English)

    Zhixiang PENG; Xi WEI; Zhengmei LIN

    2009-01-01

    successive passages could express Hope protein, while only 1 from 5 E. coli colonies that contained lac operon-regulated plasmid encoding hopE gene could express HopE. Indi-rect immunofluorescence confirmed the expression of HopE on E. coli cell surface.

  20. A comparative study examining the cytotoxicity of inducible gene expression system ligands in different cell types.

    Science.gov (United States)

    Xie, Jinger; Nair, Ayyappan; Hermiston, Terry W

    2008-02-01

    Inducible gene expression systems are being used in many in vitro and in vivo applications for target discovery, target validation and as components in exploratory therapeutic agents. Ideally, the ligands, which activate the systems, are benign so that the effects can be strictly attributed to the induced protein. As a first step to defining the potential effects of these inducers, we tested three of them, doxycycline, muristerone A and mifepristone (for tet-, ecdysone- and progesterone antagonist-inducible systems respectively), for toxicity across a panel of normal cells and cancer cell lines. In contrast to both muristerone A and mifepristone that showed no significant toxicity on any of the tested cells, we observed that doxycycline induced cell death in selected cancer and primary cell lines. The different susceptibility of cell lines to the ligands commonly used in these inducible systems suggests that it is important to consider the effects of the inducers prior to their use in experimental in vitro cell culture systems.

  1. Estradiol upregulates calcineurin expression via overexpression of estrogen receptor alpha gene in systemic lupus erythematosus

    Directory of Open Access Journals (Sweden)

    Hui-Li Lin

    2011-04-01

    Full Text Available Systemic lupus erythematosus (SLE is an autoimmune disease primarily affecting women (9:1 compared with men. To investigate the influence of female sex hormone estrogen on the development of female-biased lupus, we compared the expression of estrogen receptor alpha (ERα gene and protein levels as well as expression of T-cell activation gene calcineurin in response to estrogen in peripheral blood lymphocytes (PBLs from SLE patients and normal controls. PBLs were isolated from 20 female SLE patients and 6 normal female controls. The amount of ERα protein in PBL was measured by flow cytometry. The expression of ERα and calcineurin messenger RNA was measured by semi-quantitative reverse transcription-polymerase chain reaction. Calcineurin phosphatase activity was measured by calcineurin assay kit. The expression of ERα messenger RNA and ERα protein was significantly increased (p=0.001 and p=0.023, respectively in PBL from SLE patients compared with that from normal controls. In addition, the basal calcineurin in PBL from SLE patients was significantly higher (p=0.000 than that from normal controls, and estrogen-induced expression of calcineurin was increased (p=0.007 in PBL from SLE patients compared with that from normal controls, a 3.15-fold increase. This increase was inhibited by the ERα antagonism ICI 182,780. The effects of ER antagonism were also found in calcineurin activity. These data suggest that overexpression of ERα gene and enhanced activation of calcineurin in response to estrogen in PBL may contribute to the pathogenesis of female dominant in SLE.

  2. Dynamics of immune system gene expression upon bacterial challenge and wounding in a social insect (Bombus terrestris).

    Science.gov (United States)

    Erler, Silvio; Popp, Mario; Lattorff, H Michael G

    2011-03-29

    The innate immune system which helps individuals to combat pathogens comprises a set of genes representing four immune system pathways (Toll, Imd, JNK and JAK/STAT). There is a lack of immune genes in social insects (e.g. honeybees) when compared to Diptera. Potentially, this might be compensated by an advanced system of social immunity (synergistic action of several individuals). The bumble bee, Bombus terrestris, is a primitively eusocial species with an annual life cycle and colonies headed by a single queen. We used this key pollinator to study the temporal dynamics of immune system gene expression in response to wounding and bacterial challenge.Antimicrobial peptides (AMP) (abaecin, defensin 1, hymenoptaecin) were strongly up-regulated by wounding and bacterial challenge, the latter showing a higher impact on the gene expression level. Sterile wounding down-regulated TEP A, an effector gene of the JAK/STAT pathway, and bacterial infection influenced genes of the Imd (relish) and JNK pathway (basket). Relish was up-regulated within the first hour after bacterial challenge, but decreased strongly afterwards. AMP expression following wounding and bacterial challenge correlates with the expression pattern of relish whereas correlated expression with dorsal was absent. Although expression of AMPs was high, continuous bacterial growth was observed throughout the experiment.Here we demonstrate for the first time the temporal dynamics of immune system gene expression in a social insect. Wounding and bacterial challenge affected the innate immune system significantly. Induction of AMP expression due to wounding might comprise a pre-adaptation to accompanying bacterial infections. Compared with solitary species this social insect exhibits reduced immune system efficiency, as bacterial growth could not be inhibited. A negative feedback loop regulating the Imd-pathway is suggested. AMPs, the end product of the Imd-pathway, inhibited the up-regulation of the transcription

  3. Dynamics of immune system gene expression upon bacterial challenge and wounding in a social insect (Bombus terrestris.

    Directory of Open Access Journals (Sweden)

    Silvio Erler

    Full Text Available The innate immune system which helps individuals to combat pathogens comprises a set of genes representing four immune system pathways (Toll, Imd, JNK and JAK/STAT. There is a lack of immune genes in social insects (e.g. honeybees when compared to Diptera. Potentially, this might be compensated by an advanced system of social immunity (synergistic action of several individuals. The bumble bee, Bombus terrestris, is a primitively eusocial species with an annual life cycle and colonies headed by a single queen. We used this key pollinator to study the temporal dynamics of immune system gene expression in response to wounding and bacterial challenge.Antimicrobial peptides (AMP (abaecin, defensin 1, hymenoptaecin were strongly up-regulated by wounding and bacterial challenge, the latter showing a higher impact on the gene expression level. Sterile wounding down-regulated TEP A, an effector gene of the JAK/STAT pathway, and bacterial infection influenced genes of the Imd (relish and JNK pathway (basket. Relish was up-regulated within the first hour after bacterial challenge, but decreased strongly afterwards. AMP expression following wounding and bacterial challenge correlates with the expression pattern of relish whereas correlated expression with dorsal was absent. Although expression of AMPs was high, continuous bacterial growth was observed throughout the experiment.Here we demonstrate for the first time the temporal dynamics of immune system gene expression in a social insect. Wounding and bacterial challenge affected the innate immune system significantly. Induction of AMP expression due to wounding might comprise a pre-adaptation to accompanying bacterial infections. Compared with solitary species this social insect exhibits reduced immune system efficiency, as bacterial growth could not be inhibited. A negative feedback loop regulating the Imd-pathway is suggested. AMPs, the end product of the Imd-pathway, inhibited the up-regulation of the

  4. The development and characterization of an exogenous green-light-regulated gene expression system in marine cyanobacteria.

    Science.gov (United States)

    Badary, Amr; Abe, Koichi; Ferri, Stefano; Kojima, Katsuhiro; Sode, Koji

    2015-06-01

    A green-light-regulated gene expression system derived from Synechocystis sp. PCC 6803 was constructed and introduced into the marine cyanobacterial strain Synechococcus sp. NKBG 15041c. The regulation system was evaluated using gfp uv as a reporter gene under red-light illumination and under simultaneous red- and green-light illumination. Expression of the reporter gene was effectively repressed under red-light illumination and increased over 10-fold by illuminating with green light. Control vectors missing either the ccaS sensor histidine kinase gene or the ccaR response regulator gene showed no detectable induction of GFPuv expression. Green-light induction of gfp uv expression was further confirmed by quantitative reverse transcription PCR. The constructed system was effective at regulating the recombinant expression of a target gene using green light in a marine cyanobacterial strain that does not naturally possess such a green-light regulation system. Thus, constructed green-light-regulated gene expression system may be used as a core platform technology for the development of marine cyanobacterial strains in which bioprocesses will be regulated by light.

  5. In situ Expression of Functional Genes Reveals Nitrogen Cycling at High Temperatures in Terrestrial Hydrothermal Systems

    Science.gov (United States)

    Loiacono, S. T.; Meyer-Dombard, D. R.

    2011-12-01

    using (reverse-transcription) polymerase chain reaction to identify the presence and expression of nifH genes, and resultant (RT-)PCR product was cloned and sequenced. Results reveal high-temperature in situ expression of nifH in select LGB features [7] which is, to the authors' knowledge, the first direct evidence of nifH transcription in the chemosynthetic zones of terrestrial hydrothermal systems. Results also indicate the presence of novel nifH sequences and allow phylogenetic comparison of nifH genes along geochemical gradients within individual hot spring features and between various thermal features in the LGB. Collectively, these results provide evidence for microbial adaptations that have led to the ability to support basic metabolic processes under "extreme" conditions. [1] Hall et al., 2008. AEM 74: 4910-4922. [2] Steunou et al., 2008. The ISME Journal 2: 364-378. [3] Hamilton et al., 2011. Microb Ecol DOI 10.1007/s00248-011-9824-9. [4] Raymond et al., 2008. EOS Trans AGU. Abstract B14A-03. [5] Havig et al., 2010. J Geophys Res-Biogeo 116: G01005. [6] Mehta & Baross, 2006. Science 314: 1783-1786. [7] Loiacono et al., 2011. Submitted FEMS Microbiol Ecol.

  6. Gene Expression Omnibus (GEO)

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene Expression Omnibus is a public functional genomics data repository supporting MIAME-compliant submissions of array- and sequence-based data. Tools are provided...

  7. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.

    Science.gov (United States)

    Gong, Shiaoching; Zheng, Chen; Doughty, Martin L; Losos, Kasia; Didkovsky, Nicholas; Schambra, Uta B; Nowak, Norma J; Joyner, Alexandra; Leblanc, Gabrielle; Hatten, Mary E; Heintz, Nathaniel

    2003-10-30

    The mammalian central nervous system (CNS) contains a remarkable array of neural cells, each with a complex pattern of connections that together generate perceptions and higher brain functions. Here we describe a large-scale screen to create an atlas of CNS gene expression at the cellular level, and to provide a library of verified bacterial artificial chromosome (BAC) vectors and transgenic mouse lines that offer experimental access to CNS regions, cell classes and pathways. We illustrate the use of this atlas to derive novel insights into gene function in neural cells, and into principal steps of CNS development. The atlas, library of BAC vectors and BAC transgenic mice generated in this screen provide a rich resource that allows a broad array of investigations not previously available to the neuroscience community.

  8. On-Orbit Quantitative Real-Time Gene Expression Analysis Using the Wetlab-2 System

    Science.gov (United States)

    Parra, Macarena; Jung, Jimmy; Almeida, Eduardo; Boone, Travis; Tran, Luan; Schonfeld, Julie

    2015-01-01

    NASA Ames Research Center's WetLab-2 Project enables on-orbit quantitative Reverse Transcriptase PCR (qRT-PCR) analysis without the need for sample return. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage of using non-toxic chemicals and does not require alcohols or other organics. The resulting RNA is dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The project selected the Cepheid SmartCycler (TradeMark), a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, because of its advantages including rugged modular design, low power consumption, rapid thermal ramp times and four-color multiplex detection. Single tube multiplex assays can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system can downlink data from the ISS to the ground after a completed run and uplink new thermal cycling programs. The ability to conduct qRT-PCR and generate results on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. Specifically, the ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. On orbit gene expression analysis can also eliminate the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples and provide on-orbit gene expression benchmarking prior to sample return. Finally, the system can also be used for analysis of

  9. Efficient activation of gene expression using a heat-shock inducible Gal4/Vp16-UAS system in medaka

    Directory of Open Access Journals (Sweden)

    Wittbrodt Joachim

    2004-10-01

    Full Text Available Abstract Background Genetic interference by DNA, mRNA or morpholino injection is a widely used approach to study gene function in developmental biology. However, the lack of temporal control over the activity of interfering molecules often hampers investigation of gene function required during later stages of embryogenesis. To elucidate the roles of genes during embryogenesis a precise temporal control of transgene expression levels in the developing organism is on demand. Results We have generated a transgenic Gal4/Vp16 activator line that is heat-shock inducible, thereby providing a tool to drive the expression of specific effector genes via Gal4/Vp16. Merging the Gal4/Vp16-UAS system with the I-SceI meganuclease and the Sleeping Beauty transposon system allows inducible gene expression in an entirely uniform manner without the need to generate transgenic effector lines. Combination of this system with fluorescent protein reporters furthermore facilitates the direct visualization of transgene expressing cells in live embryos. Conclusion The combinatorial properties of this expression system provide a powerful tool for the analysis of gene function during embryonic and larval development in fish by ectopic expression of gene products.

  10. Establishment of a Gene Expression System in Rice Chloroplast and Obtainment of PPT-Resistant Rice Plants

    Institute of Scientific and Technical Information of China (English)

    LI Yi-nü; SUN Bing-yao; SU Ning; MENG Xiang-xun; ZHANG Zhi-fang; SHEN Gui-fang

    2009-01-01

    In contrast to the situation of random integration of foreign genes in nuclear transformation,the introduction of genes via chloroplast genetic engineering is characterized by site-specific pattern via homologous recombination.To establish an expression system for alien genes in rice chloroplast,the intergenic region of ndhF and trnL was selected as target for sitespecific integration of PPT-resistant bar gene in this study.Two DNA fragments suitable for homologous recombination were cloned from rice chloroplast genome DNA using PCR technique,and the chloroplast-specific expression vector pRB was constructed by fusing a modified 16S rRNA gene promoter to bar gene together with terminator of psbA gene 3'sequence.Chloroplast transformation was carried out by biolistic bombardment of sterile rice calli with the pRB construct.Subsequently,the regenerated plantlets and seeds of progeny arising from reciprocal cross to the wild-type lines were obtained.Molecular analysis suggested that the bar gene has been integrated into rice chloroplast genome.Genetic analysis revealed that bar gene could be transmitted and expressed normally in chloroplast genome.Thus,the bar gene conferred not only selection pressure for the transformation of rice chloroplast genome,but PPT-resistant trait for rice plants as well.It is suggested that an efficient gene expression system in the rice chloroplast has been established by chloroplast transformation technique.

  11. Optimization of the Lactococcus lactis nisin-controlled gene expression system NICE for industrial applications

    Directory of Open Access Journals (Sweden)

    Mond James

    2005-05-01

    Full Text Available Abstract Background The nisin-controlled gene expression system NICE of Lactococcus lactis is one of the most widely used expression systems in Gram-positive bacteria. Despite its widespread use, no optimization of the culture conditions and nisin induction has been carried out to obtain maximum yields. As a model system induced production of lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus produced by S. simulans biovar. Staphylolyticus, was used. Three main areas need optimization for maximum yields: cell density, nisin-controlled induction and protein production, and parameters specific for the target-protein. Results In a series of pH-controlled fermentations the following parameters were optimized: pH of the culture, use of NaOH or NH4OH as neutralizing agent, the addition of zinc and phosphate, the fermentation temperature, the time point of induction (cell density of the culture, the amount of nisin added for induction and the amount of three basic medium components, i.e. yeast extract, peptone and lactose. For each culture growth and lysostaphin production was followed. Lysostaphin production yields depended on all parameters that were varied. In the course of the optimization a three-fold increase in lysostaphin yield was achieved from 100 mg/l to 300 mg/l. Conclusion Protein production with the NICE gene expression system in L. lactis strongly depends on the medium composition, the fermentation parameters and the amount of nisin added for induction. Careful optimization of key parameters lead to a significant increase in the yield of the target protein.

  12. A robust dual reporter system to visualize and quantify gene expression mediated by transcription activator-like effectors

    Directory of Open Access Journals (Sweden)

    Uhde-Stone Claudia

    2012-08-01

    Full Text Available Abstract Background Transcription activator-like effectors (TALEs are a class of naturally occurring transcription effectors that recognize specific DNA sequences and modulate gene expression. The modularity of TALEs DNA binding domain enables sequence-specific perturbation and offers broad applications in genetic and epigenetic studies. Although the efficient construction of TALEs has been established, robust functional tools to assess their functions remain lacking. Results We established a dual reporter system that was specifically designed for real-time monitoring and quantifying gene expression mediated by TALEs. We validated both sensitivity and specificity of this dual-reporter system in mammalian cells, and demonstrated that this dual reporter system is robust and potentially amenable to high throughput (HTP applications. Conclusion We have designed, constructed and validated a novel dual reporter system for assessing TALE mediated gene regulations. This system offers a robust and easy-to- use tool for real-time monitoring and quantifying gene expression in mammalian cells.

  13. Dynamic gene expression in the song system of zebra finches during the song learning period.

    Science.gov (United States)

    Olson, Christopher R; Hodges, Lisa K; Mello, Claudio V

    2015-12-01

    The brain circuitry that controls song learning and production undergoes marked changes in morphology and connectivity during the song learning period in juvenile zebra finches, in parallel to the acquisition, practice and refinement of song. Yet, the genetic programs and timing of regulatory change that establish the neuronal connectivity and plasticity during this critical learning period remain largely undetermined. To address this question, we used in situ hybridization to compare the expression patterns of a set of 30 known robust molecular markers of HVC and/or area X, major telencephalic song nuclei, between adult and juvenile male zebra finches at different ages during development (20, 35, 50 days post-hatch, dph). We found that several of the genes examined undergo substantial changes in expression within HVC or its surrounds, and/or in other song nuclei. They fit into broad patterns of regulation, including those whose expression within HVC during this period increases (COL12A1, COL 21A1, MPZL1, PVALB, and CXCR7) or decreases (e.g., KCNT2, SAP30L), as well as some that show decreased expression in the surrounding tissue with little change within song nuclei (e.g. SV2B, TAC1). These results reveal a broad range of molecular changes that occur in the song system in concert with the song learning period. Some of the genes and pathways identified are potential modulators of the developmental changes associated with the emergence of the adult properties of the song control system, and/or the acquisition of learned vocalizations in songbirds.

  14. Dual expression profile of type VI secretion system immunity genes protects pandemic Vibrio cholerae.

    Directory of Open Access Journals (Sweden)

    Sarah T Miyata

    Full Text Available The Vibrio cholerae type VI secretion system (T6SS assembles as a molecular syringe that injects toxic protein effectors into both eukaryotic and prokaryotic cells. We previously reported that the V. cholerae O37 serogroup strain V52 maintains a constitutively active T6SS to kill other Gram-negative bacteria while being immune to attack by kin bacteria. The pandemic O1 El Tor V. cholerae strain C6706 is T6SS-silent under laboratory conditions as it does not produce T6SS structural components and effectors, and fails to kill Escherichia coli prey. Yet, C6706 exhibits full resistance when approached by T6SS-active V52. These findings suggested that an active T6SS is not required for immunity against T6SS-mediated virulence. Here, we describe a dual expression profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 that provides pandemic V. cholerae strains with T6SS immunity and allows T6SS-silent strains to maintain immunity against attacks by T6SS-active bacterial neighbors. The dual expression profile allows transcription of the three genes encoding immunity proteins independently of other T6SS proteins encoded within the same operon. One of these immunity proteins, TsiV2, protects against the T6SS effector VasX which is encoded immediately upstream of tsiV2. VasX is a secreted, lipid-binding protein that we previously characterized with respect to T6SS-mediated virulence towards the social amoeba Dictyostelium discoideum. Our data suggest the presence of an internal promoter in the open reading frame of vasX that drives expression of the downstream gene tsiV2. Furthermore, VasX is shown to act in conjunction with VasW, an accessory protein to VasX, to compromise the inner membrane of prokaryotic target cells. The dual regulatory profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 permits V. cholerae to tightly control T6SS gene expression while maintaining immunity to T6SS activity.

  15. Age-related gene expression change of GABAergic system in visual cortex of rhesus macaque.

    Science.gov (United States)

    Liao, Chenghong; Han, Qian; Ma, Yuanye; Su, Bing

    2016-09-30

    Degradation of visual function is a common phenomenon during aging and likely mediated by change in the impaired central visual pathway. Treatment with GABA or its agonist could recover the ability of visual neurons in the primary visual cortex of senescent macaques. However, little is known about how GABAergic system change is related to the aged degradation of visual function in nonhuman primate. With the use of quantitative PCR method, we measured the expression change of 24 GABA related genes in the primary visual cortex (Brodmann's 17) of different age groups. In this study, both of mRNA and protein of glutamic acid decarboxylase (GAD65) were measured by real-time RT-PCR and Western blot, respectively. Results revealed that the level of GAD65 message was not significantly altered, but the proteins were significantly decreased in the aged monkey. As GAD65 plays an important role in GABA synthesis, the down-regulation of GAD65 protein was likely the key factor leading to the observed GABA reduction in the primary visual cortex of the aged macaques. In addition, 7 of 14 GABA receptor genes were up-regulated and one GABA receptor gene was significantly reduced during aging process even after Banjamini correction for multiple comparisons (Pvisual dysfunctions and most of GABA receptor genes induce a clear indication of compensatory effect for the reduced GABA release in the healthy aged monkey cortex.

  16. Computerized system for recognition of autism on the basis of gene expression microarray data.

    Science.gov (United States)

    Latkowski, Tomasz; Osowski, Stanislaw

    2015-01-01

    The aim of this paper is to provide a means to recognize a case of autism using gene expression microarrays. The crucial task is to discover the most important genes which are strictly associated with autism. The paper presents an application of different methods of gene selection, to select the most representative input attributes for an ensemble of classifiers. The set of classifiers is responsible for distinguishing autism data from the reference class. Simultaneous application of a few gene selection methods enables analysis of the ill-conditioned gene expression matrix from different points of view. The results of selection combined with a genetic algorithm and SVM classifier have shown increased accuracy of autism recognition. Early recognition of autism is extremely important for treatment of children and increases the probability of their recovery and return to normal social communication. The results of this research can find practical application in early recognition of autism on the basis of gene expression microarray analysis.

  17. FMRFamide gene and peptide expression during central nervous system development of the cephalopod mollusk, Idiosepius notoides.

    Science.gov (United States)

    Wollesen, Tim; Cummins, Scott F; Degnan, Bernard M; Wanninger, Andreas

    2010-01-01

    Mollusks are a showcase of brain evolution represented by several classes with a varying degree of nervous system centralization. Cellular and molecular processes involved in the evolution of the highly complex cephalopod brain from a simple, monoplacophoran-like ancestor are still obscure and homologies on the cellular level are poorly established. FMRFamide (Phe-Ile-Arg-Phe-NH(2))-related peptides (FaRPs) constitute an evolutionarily conserved and diverse group of neuropeptides in the central nervous system (CNS) of many metazoans. Herein, we provide a detailed description of the developing FMRFamide-like immunoreactive (Fa-lir) CNS of the pygmy squid Idiosepius notoides using gene expression analyses and immunocytochemistry. The open reading frame of the I. notoides FMRFamide gene InFMRF predicts one copy each of FIRFamide, FLRFamide (Phe-Leu-Arg-Phe-NH(2)), ALSGDAFLRFamide (Ala-Leu-Ser-Gly-Asp-Ala-Phe-Leu-Arg-Phe-NH(2)), and 11 copies of FMRFamide. Applying matrix-assisted laser desorption/ionization time-of-flight (ToF) mass spectrometry-based peptide profiling, we characterized all predicted FaRPs except ALSGDAFLRFamide. Two cell clusters express InFMRF and show FMRFamide-like-immunoreactivity within the palliovisceral ganglia, that is, the future posterior subesophageal mass, during the lobe differentiation phase. They project neurites via ventral axonal tracts, which form the scaffold of the future subesophageal mass. In the supraesophageal mass, InFMRF is first expressed during mid-embryogenesis in the superior and inferior buccal lobes. A neurite of the peduncle commissure represents the first Fa-lir element. Later, the sub- and supraesophageal mass interconnect via Fa-lir neurites and more brain lobes express InFMRF and FMRFamide-like peptides. InFMRF expression was observed in fewer brain lobes than Fa-lir elements. The early expression of InFMRF and FMRFamide-lir peptides in the visceral system and not the remaining CNS of the cephalopod I. notoides

  18. Identification and Expression Profiling of Radiation-sensitive Genes Using Plant Model System, Arabidopsis thaliana

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dong-Sub; Kang, Si-Yong; Lee, Geung-Joo; Kim, Jin-Baek

    2008-06-15

    The purpose of this study is to characterize genes specifically expressed in response to ionizing energy (gamma-rays) of acute irradiation and elucidate signalling mechanisms via functional analysis of isolated genes in Arabidopsis thaliana. Recent improvements in DNA microarray technologies and bioinformatics have made it possible to look for common features of ionizing radiation-responsive genes and their regulatory regions. It has produced massive quantities of gene expression and other functional genomics data, and its application will increase in plant genomics. In this study, we used oligonucleotide microarrays to detect the Arabidopsis genes expressed differentially by a gamma-irradiation during the vegetative (VT, 21 DAG) and reproductive (RT, 28 DAG) stages. Wild-type (Ler) Arabidopsis was irradiated with gamma-rays with 100 and 800 Gy doses. Among the 21,500 genes represented in the Agilent chip, approximately 13,500 ({sup {approx}}61.4 %) responsive genes to {nu} -irradiation were expressed with signal intensity greater than 192 when compared to the combined control (non-irradiated vegetative and reproductive pool). Expression patterns of several radiation inducible genes were confirmed by RT-PCR and Northern blotting. Our microarray results may contribute to an overall understanding of the type and quantities of genes that are expressed by an acute gamma-irradiation. In addition, to investigate the oxidative damage caused by irradiation, RT-PCR analysis for the expression of antioxidant isoenzyme genes, and a Transmission Electron Microscope (TEM) observation for visualizing the H{sub 2}O{sub 2} scavenging activity in leaves were applied.

  19. Genetic and epigenetic control of gene expression by CRISPR–Cas systems

    Science.gov (United States)

    Lo, Albert; Qi, Lei

    2017-01-01

    The discovery and adaption of bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems has revolutionized the way researchers edit genomes. Engineering of catalytically inactivated Cas variants (nuclease-deficient or nuclease-deactivated [dCas]) combined with transcriptional repressors, activators, or epigenetic modifiers enable sequence-specific regulation of gene expression and chromatin state. These CRISPR–Cas-based technologies have contributed to the rapid development of disease models and functional genomics screening approaches, which can facilitate genetic target identification and drug discovery. In this short review, we will cover recent advances of CRISPR–dCas9 systems and their use for transcriptional repression and activation, epigenome editing, and engineered synthetic circuits for complex control of the mammalian genome. PMID:28649363

  20. Blood gene expression profiling in pediatric systemic lupus erythematosus and systemic juvenile idiopathic arthritis: from bench to bedside.

    Science.gov (United States)

    Gilbert, Mileka; Punaro, Marilynn

    2014-01-01

    Blood gene expression profiling has led to major advances in the field of rheumatology over the last few decades. Specifically, DNA microarray technology has been integral in increasing our knowledge of key players in the pathogenesis of some rare pediatric rheumatic diseases. Our group, using microarray analysis, identified the interferon (IFN) gene signature in pediatric systemic lupus erythematosus (SLE) and has published data that suggest high doses of intravenous corticosteroid treatment may have benefit over strictly oral regimens. Additionally, DNA microarray technology led to our discovery that the interleukin (IL)-1 gene signature is associated with systemic juvenile idiopathic arthritis (sJIA) and to the use of IL-1 blockade with anakinra in this disease. We also reported the biologic rationale for use of anakinra early in the disease course. Anakinra is now being used as first-line treatment in sJIA in multiple centers. Herein, we review how information obtained from blood gene expression profiling has changed our clinical practice.

  1. A nitrogen source-dependent inducible and repressible gene expression system in the red alga Cyanidioschyzon merolae.

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    Takayuki eFujiwara

    2015-08-01

    Full Text Available The unicellular red alga Cyanidioschyzon merolae is a model organism for studying the basic biology of photosynthetic organisms. The C. merolae cell is composed of an extremely simple set of organelles. The genome is completely sequenced. Gene targeting and a heat-shock inducible gene expression system has been recently established. However, a conditional gene knockdown system has not been established, which is required for the examination of function of genes that are essential to cell viability and primary mutant defects. In the current study, we first evaluated the expression of a transgene from two chromosomal neutral loci located in the intergenic region between CMD184C and CMD185C, and a region upstream of the URA5.3 gene. There was no significant difference in expression between them and this result suggests that both may be used as neutral loci. We then designed an inducible and repressible gene expression by using promoters of nitrate-assimilation genes. The expression of nitrate-assimilation genes such as NR (nitrate reductase, NIR (nitrite reductase and NRT (the nitrate/nitrite transporter are reversibly regulated by their dependence on nitrogen sources. We constructed stable strains in which a cassette containing the NR, NIR or NRT promoter and sfGFP gene was inserted in a region upstream of URA5.3 and examined the efficacy of the promoters. The NR, NIR, and NRT promoters were constitutively activated in the nitrate medium, whereas their activities were extremely low in presence of ammonium. The activation of each promoter was immediately inhibited within a period of 1 hour by the addition of ammonium. Thus, a conditional knockdown system in C. merolae was successfully established. The activity varies among the promoters, and each is selectable according to the expression level of a target gene estimated by RNA-sequencing. This method is applicable to defects in genes of interest in photosynthetic organism.

  2. A nitrogen source-dependent inducible and repressible gene expression system in the red alga Cyanidioschyzon merolae.

    Science.gov (United States)

    Fujiwara, Takayuki; Kanesaki, Yu; Hirooka, Shunsuke; Era, Atsuko; Sumiya, Nobuko; Yoshikawa, Hirofumi; Tanaka, Kan; Miyagishima, Shin-Ya

    2015-01-01

    The unicellular red alga Cyanidioschyzon merolae is a model organism for studying the basic biology of photosynthetic organisms. The C. merolae cell is composed of an extremely simple set of organelles. The genome is completely sequenced. Gene targeting and a heat-shock inducible gene expression system has been recently established. However, a conditional gene knockdown system has not been established, which is required for the examination of function of genes that are essential to cell viability and primary mutant defects. In the current study, we first evaluated the expression of a transgene from two chromosomal neutral loci located in the intergenic region between CMD184C and CMD185C, and a region upstream of the URA5.3 gene. There was no significant difference in expression between them and this result suggests that both may be used as neutral loci. We then designed an inducible and repressible gene expression by using promoters of nitrate-assimilation genes. The expression of nitrate-assimilation genes such as NR (nitrate reductase), NIR (nitrite reductase), and NRT (the nitrate/nitrite transporter) are reversibly regulated by their dependence on nitrogen sources. We constructed stable strains in which a cassette containing the NR, NIR, or NRT promoter and sfGFP gene was inserted in a region upstream of URA5.3 and examined the efficacy of the promoters. The NR, NIR, and NRT promoters were constitutively activated in the nitrate medium, whereas their activities were extremely low in presence of ammonium. The activation of each promoter was immediately inhibited within a period of 1 h by the addition of ammonium. Thus, a conditional knockdown system in C. merolae was successfully established. The activity varies among the promoters, and each is selectable according to the expression level of a target gene estimated by RNA-sequencing. This method is applicable to defects in genes of interest in photosynthetic organism.

  3. Gene Expression Patterns Underlying the Reinstatement of Plasticity in the Adult Visual System

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    Ettore Tiraboschi

    2013-01-01

    Full Text Available The nervous system is highly sensitive to experience during early postnatal life, but this phase of heightened plasticity decreases with age. Recent studies have demonstrated that developmental-like plasticity can be reactivated in the visual cortex of adult animals through environmental or pharmacological manipulations. These findings provide a unique opportunity to study the cellular and molecular mechanisms of adult plasticity. Here we used the monocular deprivation paradigm to investigate large-scale gene expression patterns underlying the reinstatement of plasticity produced by fluoxetine in the adult rat visual cortex. We found changes, confirmed with RT-PCRs, in gene expression in different biological themes, such as chromatin structure remodelling, transcription factors, molecules involved in synaptic plasticity, extracellular matrix, and excitatory and inhibitory neurotransmission. Our findings reveal a key role for several molecules such as the metalloproteases Mmp2 and Mmp9 or the glycoprotein Reelin and open up new insights into the mechanisms underlying the reopening of the critical periods in the adult brain.

  4. Expression of the Wilms' tumor gene WT1 in the murine urogenital system.

    Science.gov (United States)

    Pelletier, J; Schalling, M; Buckler, A J; Rogers, A; Haber, D A; Housman, D

    1991-08-01

    The Wilms' tumor gene WT1 is a recessive oncogene that encodes a putative transcription factor implicated in nephrogenesis during kidney development. In this report we analyze expression of WT1 in the murine urogenital system. WT1 is expressed in non-germ-cell components of the testis and ovaries in both young and adult mice. In situ mRNA hybridization studies demonstrate that WT1 is expressed in the granulosa and epithelial cells of ovaries, the Sertoli cells of the testis, and in the uterine wall. In addition to the 3.1-kb WT1 transcript detected by Northern blotting of RNA from kidney, uterus, and gonads, there is an approximately 2.5-kb WT1-related mRNA species in testis. The levels of WT1 mRNA in the gonads are among the highest observed, surpassing amounts detected in the embryonic kidney. During development, these levels are differentially regulated, depending on the sexual differentiation of the gonad. Expression of WT1 mRNA in the female reproductive system does not fluctuate significantly from days 4 to 40 postpartum. In contrast, WT1 mRNA levels in the tesis increase steadily after birth, reaching their highest expression levels at day 8 postpartum and decreasing slightly as the animal matures. Expression of WT1 in the gonads is detectable as early as 12.5 days postcoitum (p.c.). As an initial step toward exploring the tissue-specific expression of WT1, DNA elements upstream of WT1 were cloned and sequenced. Three putative transcription initiation sites, utilized in testis, ovaries, and uterus, were mapped by S1 nuclease protection assays. The sequences surrounding these sites have a high G + C content, and typical upstream CCAAT and TATAA boxes are not present. These studies allowed us to identify the translation initiation site for WT1 protein synthesis. We have also used an epitope-tagging protocol to demonstrate that WT1 is a nuclear protein, consistent with its role as a transcription factor. Our results demonstrate regulation of WT1 expression

  5. Standardization of Gene Expression Quantification by Absolute Real-Time qRT-PCR System Using a Single Standard for Marker and Reference Genes

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    Yi-Hong Zhou

    2010-08-01

    Full Text Available In the last decade, genome-wide gene expression data has been collected from a large number of cancer specimens. In many studies utilizing either microarray-based or knowledge-based gene expression profiling, both the validation of candidate genes and the identification and inclusion of biomarkers in prognosis-modeling has employed real-time quantitative PCR on reverse transcribed mRNA (qRT-PCR because of its inherent sensitivity and quantitative nature. In qRT-PCR data analysis, an internal reference gene is used to normalize the variation in input sample quantity. The relative quantification method used in current real-time qRT-PCR analysis fails to ensure data comparability pivotal in identification of prognostic biomarkers. By employing an absolute qRT-PCR system that uses a single standard for marker and reference genes (SSMR to achieve absolute quantification, we showed that the normalized gene expression data is comparable and independent of variations in the quantities of sample as well as the standard used for generating standard curves. We compared two sets of normalized gene expression data with same histological diagnosis of brain tumor from two labs using relative and absolute real-time qRT-PCR. Base-10 logarithms of the gene expression ratio relative to ACTB were evaluated for statistical equivalence between tumors processed by two different labs. The results showed an approximate comparability for normalized gene expression quantified using a SSMR-based qRT-PCR. Incomparable results were seen for the gene expression data using relative real-time qRT-PCR, due to inequality in molar concentration of two standards for marker and reference genes. Overall results show that SSMR-based real-time qRT-PCR ensures comparability of gene expression data much needed in establishment of prognostic/predictive models for cancer patients—a process that requires large sample sizes by combining independent sets of data.

  6. Exploiting Gene-Expression Deconvolution to Probe the Genetics of the Immune System.

    Science.gov (United States)

    Steuerman, Yael; Gat-Viks, Irit

    2016-04-01

    Sequence variation can affect the physiological state of the immune system. Major experimental efforts targeted at understanding the genetic control of the abundance of immune cell subpopulations. However, these studies are typically focused on a limited number of immune cell types, mainly due to the use of relatively low throughput cell-sorting technologies. Here we present an algorithm that can reveal the genetic basis of inter-individual variation in the abundance of immune cell types using only gene expression and genotyping measurements as input. Our algorithm predicts the abundance of immune cell subpopulations based on the RNA levels of informative marker genes within a complex tissue, and then provides the genetic control on these predicted immune traits as output. A key feature of the approach is the integration of predictions from various sets of marker genes and refinement of these sets to avoid spurious signals. Our evaluation of both synthetic and real biological data shows the significant benefits of the new approach. Our method, VoCAL, is implemented in the freely available R package ComICS.

  7. A Double-Switch Cell Fusion-Inducible Transgene Expression System for Neural Stem Cell-Based Antiglioma Gene Therapy

    Directory of Open Access Journals (Sweden)

    Yumei Luo

    2015-01-01

    Full Text Available Recent progress in neural stem cell- (NSC- based tumor-targeted gene therapy showed that NSC vectors expressing an artificially engineered viral fusogenic protein, VSV-G H162R, could cause tumor cell death specifically under acidic tumor microenvironment by syncytia formation; however, the killing efficiency still had much room to improve. In the view that coexpression of another antitumoral gene with VSV-G can augment the bystander effect, a synthetic regulatory system that triggers transgene expression in a cell fusion-inducible manner has been proposed. Here we have developed a double-switch cell fusion-inducible transgene expression system (DoFIT to drive transgene expression upon VSV-G-mediated NSC-glioma cell fusion. In this binary system, transgene expression is coregulated by a glioma-specific promoter and targeting sequences of a microRNA (miR that is highly expressed in NSCs but lowly expressed in glioma cells. Thus, transgene expression is “switched off” by the miR in NSC vectors, but after cell fusion with glioma cells, the miR is diluted and loses its suppressive effect. Meanwhile, in the syncytia, transgene expression is “switched on” by the glioma-specific promoter. Our in vitro and in vivo experimental data show that DoFIT successfully abolishes luciferase reporter gene expression in NSC vectors but activates it specifically after VSV-G-mediated NSC-glioma cell fusion.

  8. A protein-tagging system for signal amplification in gene expression and fluorescence imaging.

    Science.gov (United States)

    Tanenbaum, Marvin E; Gilbert, Luke A; Qi, Lei S; Weissman, Jonathan S; Vale, Ronald D

    2014-10-23

    Signals in many biological processes can be amplified by recruiting multiple copies of regulatory proteins to a site of action. Harnessing this principle, we have developed a protein scaffold, a repeating peptide array termed SunTag, which can recruit multiple copies of an antibody-fusion protein. We show that the SunTag can recruit up to 24 copies of GFP, thereby enabling long-term imaging of single protein molecules in living cells. We also use the SunTag to create a potent synthetic transcription factor by recruiting multiple copies of a transcriptional activation domain to a nuclease-deficient CRISPR/Cas9 protein and demonstrate strong activation of endogenous gene expression and re-engineered cell behavior with this system. Thus, the SunTag provides a versatile platform for multimerizing proteins on a target protein scaffold and is likely to have many applications in imaging and controlling biological outputs.

  9. Peripheral blood RNA gene expression profiling in illicit methcathinone users reveals effect on immune system

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    Katrin eSikk

    2011-08-01

    Full Text Available Methcathinone (ephedrone is relatively easily accessible for abuse. Its users develop an extrapyramidal syndrome and it is not known if this is caused by methcathinone itself, by side-ingredients (manganese, or both. In the present study we aimed to clarify molecular mechanisms underlying this condition. We analyzed whole genome gene expression patterns of peripheral blood from 20 methcathinone users and 20 matched controls. Gene expression profile data was analyzed by Bayesian modelling and functional annotation. In order to verify the genechip results we performed quantitative real-time (RT PCR in selected genes. 326 out of analyzed 28,869 genes showed statistically significant differential expression with FDR adjusted p-values below 0.05. Quantitative RT-PCR confirmed differential expression for the most of selected genes. Functional annotation and network analysis indicated that most of the genes were related to activation immunological disease, cellular movement and cardiovascular disease gene network (enrichment score 42. As HIV and HCV infections were confounding factors, we performed additional stratification of patients. A similar functional activation of the immunological disease pathway was evident when we compared patients according to the injection status (past versus current users, balanced for HIV and HCV infection. However, this difference was not large therefore the major effect was related to the HIV status of the patients. Mn-methcathinone abusers have blood transcriptional patterns mostly caused by their HIV and HCV infections.

  10. Comparison of the expression levels of Fas and Apaf-1 genes in systemic sclerosis dermal fibroblasts

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    Majid Abed Khojasteh

    2016-07-01

    Full Text Available Background: Systemic sclerosis (SSc is an autoimmune rheumatic connective tissue disease. In normal wound healing process, fibroblasts are activated, proliferated and involved in tissue repair, and then removed by apoptosis. In systemic sclerosis, patient’s fibrosis occurs when fibroblasts become resistant to apoptosis and secrete a large amount of collagen and other extracellular matrixes. As the primary causes the disease are very complex and often unknown, it is necessary to consider or target the secondary causes of disease, such as the unresponsiveness of activated fibroblasts to apoptosis as the major factor in the creation and deployment of illness. In this study, we examined the expression levels of two key pro-apoptotic genes, Fas and Apaf-1, which are respectively involved in external and internal pathway of apoptosis. Methods: In a case-control study skin biopsy samples were obtained from 19 patients with diffuse SSc, and 16 healthy controls. Dermal fibroblasts were cultured and total RNA was isolated from cell populations using High Pure RNA Isolation Kit (Roche Applied Science, Mannheim, Germany, followed by cDNA synthesis using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Massachusetts, USA. Real-time PCR was performed using SYBRGreen gene expression master mix (Takara Shuzo, Co., Ltd, Shiga, Japan and specific primers for Fas and Apaf-1. Real-time data were analyzed using the (2-ΔCT×1000 method. Statistical analysis was accomplished by using the SPSS software, v22 (IBM, Armonk, NY, USA. The P value less than 0.05 were recognized as a significant threshold. All data are represented as the mean ± SEM. Results: Our results showed no significant difference in Fas (P=0.8 and Apaf-1 (P=0.17 mRNA expression levels between skin fibroblasts of systemic sclerosis patients and healthy controls. Conclusion: In this study we observed no significant change in Apaf-1 and Fas mRNA levels in systemic sclerosis

  11. A complete chitinolytic system in the atherinopsid pike silverside Chirostoma estor: gene expression and activities.

    Science.gov (United States)

    Pohls, P; González-Dávalos, L; Mora, O; Shimada, A; Varela-Echavarria, A; Toledo-Cuevas, E M; Martínez-Palacios, C A

    2016-06-01

    The expression and digestive activity of pike silverside Chirostoma estor endogenous chitinases were analysed in samples from four life stages: whole eggs; larvae; juvenile intestine and hepatopancreas and adult intestine and hepatopancreas. A chitinase cDNA was cloned and partially sequenced (GenBank accession number: FJ785521). It was highly homologous to non-acidic chitinase sequences from other fish species, suggesting that it is a chitotriosidase. Quantitative PCR showed that this chitinase was expressed throughout the life span of C. estor, with maximum expression in the hepatopancreas of juveniles. Chitotriosidase and chitobiosidase activities were found at all life stages, along with a very high level of N-acetyl glucosaminidase (NAGase). The chitotriosidase activity could be encoded by the cloned complementary (c)DNA, although additional chitinase genes may be present. The chitotriosidase activity appeared to be transcriptionally regulated only at the juvenile stage. The expression and activity of chitinases tended to increase from the early to juvenile stages, suggesting that these variables are stimulated by chitin-rich live food. Nevertheless, the feeding of juvenile and adult fish with both live food and a balanced commercial diet seemed to provoke significant reductions in pancreatic NAGase secretion and/or synthesis in the gut. Moreover, all chitinase activities were lower in adults, probably reflecting a higher intake and use of the balanced diet. The observation of chitotriosidase and chitobiosidase activities together with a very high NAGase activity suggest the presence of a complete and compensatory chitinolytic chitinase system that enables this stomachless short-gut fish species to use chitin as an energy substrate. These novel findings suggest that dietary inclusions of chitin-rich ingredients or by-products might reduce the farming costs of C. estor without impairing performance.

  12. Dynamic expression of leukocyte innate immune genes in whole blood from horses with lipopolysaccharide-induced acute systemic inflammation

    DEFF Research Database (Denmark)

    Vinther, Anne Mette L.; Skovgaard, Kerstin; Heegaard, Peter M. H.;

    2015-01-01

    Background: In horses, insights into the innate immune processes in acute systemic inflammation are limited even though these processes may be highly important for future diagnostic and therapeutic advances in high-mortality disease conditions as the systemic inflammatory response syndrome (SIRS......) and sepsis. Therefore, the aim of this study was to investigate the expression of 31 selected blood leukocyte immune genes in an equine model of acute systemic inflammation to identify significantly regulated genes and to describe their expression dynamics during a 24-h experimental period. Systemic...... expressions in blood leukocytes during equine acute LPS-induced systemic inflammation thoroughly characterized a highly regulated and dynamic innate immune response. These results provide new insights into the molecular mechanisms of equine systemic inflammation....

  13. Expression patterns of the murine LIM class homeobox gene lim1 in the developing brain and excretory system.

    Science.gov (United States)

    Fujii, T; Pichel, J G; Taira, M; Toyama, R; Dawid, I B; Westphal, H

    1994-01-01

    We report the cloning, sequence analysis, and developmental expression pattern of lim1, a member of the LIM class homeobox gene family in the mouse. lim1 cDNA encodes a predicted 406 amino acid protein that is 93% identical with the product of the Xenopus LIM class homeobox gene Xlim1. We have characterized lim1 expression from day 8.5 post coitum onward. Northern blot analysis of RNA transcripts indicates that lim1 is expressed both during embryogenesis and in the adult brain. Analysis by whole-mount and section in situ hybridization shows lim1 expression in the central nervous system from the telencephalon through the spinal cord and in the developing excretory system including pronephric region, mesonephros, nephric duct, and metanephros. In the metanephros, lim1 is strongly expressed in renal vesicles and S-shaped bodies, and transcripts are also detected in the ureteric branches.

  14. Pathogenic LRRK2 mutations do not alter gene expression in cell model systems or human brain tissue.

    Directory of Open Access Journals (Sweden)

    Michael J Devine

    Full Text Available Point mutations in LRRK2 cause autosomal dominant Parkinson's disease. Despite extensive efforts to determine the mechanism of cell death in patients with LRRK2 mutations, the aetiology of LRRK2 PD is not well understood. To examine possible alterations in gene expression linked to the presence of LRRK2 mutations, we carried out a case versus control analysis of global gene expression in three systems: fibroblasts isolated from LRRK2 mutation carriers and healthy, non-mutation carrying controls; brain tissue from G2019S mutation carriers and controls; and HEK293 inducible LRRK2 wild type and mutant cell lines. No significant alteration in gene expression was found in these systems following correction for multiple testing. These data suggest that any alterations in basal gene expression in fibroblasts or cell lines containing mutations in LRRK2 are likely to be quantitatively small. This work suggests that LRRK2 is unlikely to play a direct role in modulation of gene expression, although it remains possible that this protein can influence mRNA expression under pathogenic cicumstances.

  15. Gene Structure and Expression of the High-affinity Nitrate Transport System in Rice Roots

    Institute of Scientific and Technical Information of China (English)

    Chao Cai; Jun-Yi Wang; Yong-Guan Zhu; Qi-Rong Shen; Bin Li; Yi-Ping Tong; Zhen-Sheng Li

    2008-01-01

    Rice has a preference for uptake of ammonium over nitrate and can use ammonium-N efficiently. Consequently, transporters mediating ammonium uptake have been extensively studied, but nitrate transporters have been largely ignored. Recently,some reports have shown that rice also has high capacity to acquire nitrate from growth medium, so understanding the nitrate transport system in rice roots is very important for improving N use efficiency in rice. The present study identified four putative NRT2 and two putative NAR2 genes that encode components of the high-affinity nitrate transport system (HATS) in the rice (Oryza sativa L. subsp, japonica cv. Nipponbare) genome. OsNRT2.1 and OsNRT2.2 share an identical coding region sequence, and their deduced proteins are closely related to those from monocotyledonous plants. The two NAR2 proteins are closely related to those from mono-cotyledonous plants as well. However, OsNRT2.3 and OsNRT2.4 are more closely related to Arabidopsis NRT2 proteins. Relative quantitative reverse tranecdption-polymerase chain reaction analysis showed that all of the six genes were rapidly upregulated and then downregulated in the roots of N-starved rice plants after they were re-supplied with 0.2 mM nitrate, but the response to nitrate differed among gene members.The results from phylogenetic tree, gene structure and expression analysis implied the divergent roles for the individual members of the rice NRT2 and NAR2 families. High-affinity nitrate influx rates associated with nitrate induction in rice roots were investigated and were found to be regulated by external pH. Compared with the nitrate influx rates at pH 6.5, alkaline pH (pH 8.0) inhibited nitrate Influx, and acidic pH (pH 5.0) enhanced the nitrate influx In I h nitrate induced roots, but did not significantly affect that in 4 to 8 h nitrate induced roots.

  16. From Gene Expression to the Earth System: Isotopic Constraints on Nitrogen Cycling Across Scales

    Science.gov (United States)

    Houlton, B. Z.

    2015-12-01

    A central motivation of the Biogeosciences is to understand the cycling of biologically essential elements over multiple scales of space and time. This charge is vital to basic knowledge of Earth system functioning. It is also relevant to many of the global challenges we face, such as climate change, biodiversity conservation, and the multifaceted role of global fertilizer use in maximizing human health and well-being. Nitrogen is connected to all of these; yet it has been one of the more vexing elements to quantitatively appraise across systems and scales. Here I discuss how research in my group has been exploring the use of natural nitrogen isotope abundance (15N/14N) as a biogeochemical tracer - from the level of gene expression to nitrogen's role in global climate change. First, I present evidence for a positive correlation between the bacterial genes that encode for gaseous nitrogen production (i.e., nirS) and the 15N/14N of soil extractable nitrate pools across an array of terrestrial ecosystems. Second, I demonstrate how these local-scale results fit with our work on ecosystem-scale nitrogen isotope budgets, where we quantify a uniformly small isotope effect (i.e., ancient terrestrial plant compounds (i.e., chlorins) buried in the soil. This research aims to address the response of the nitrogen cycle to glacial-interglacial transitions over millennia, which is beyond the window of experimental testing. Together, this research highlights the utility of nitrogen isotope composition in addressing the myriad scales of this element's interaction with Earth's environment, and supports the working hypothesis that bacterial denitrification is the major fractionating pathway of nitrogen loss from the terrestrial biosphere, much like the global ocean.

  17. Burkholderia mallei and Burkholderia pseudomallei cluster 1 type VI secretion system gene expression is negatively regulated by iron and zinc.

    Directory of Open Access Journals (Sweden)

    Mary N Burtnick

    Full Text Available Burkholderia mallei is a facultative intracellular pathogen that causes glanders in humans and animals. Previous studies have demonstrated that the cluster 1 type VI secretion system (T6SS-1 expressed by this organism is essential for virulence in hamsters and is positively regulated by the VirAG two-component system. Recently, we have shown that T6SS-1 gene expression is up-regulated following internalization of this pathogen into phagocytic cells and that this system promotes multinucleated giant cell formation in infected tissue culture monolayers. In the present study, we further investigated the complex regulation of this important virulence factor. To assess T6SS-1 expression, B. mallei strains were cultured in various media conditions and Hcp1 production was analyzed by Western immunoblotting. Transcript levels of several VirAG-regulated genes (bimA, tssA, hcp1 and tssM were also determined using quantitative real time PCR. Consistent with previous observations, T6SS-1 was not expressed during growth of B. mallei in rich media. Curiously, growth of the organism in minimal media (M9G or minimal media plus casamino acids (M9CG facilitated robust expression of T6SS-1 genes whereas growth in minimal media plus tryptone (M9TG did not. Investigation of this phenomenon confirmed a regulatory role for VirAG in this process. Additionally, T6SS-1 gene expression was significantly down-regulated by the addition of iron and zinc to M9CG. Other genes under the control of VirAG did not appear to be as tightly regulated by these divalent metals. Similar results were observed for B. pseudomallei, but not for B. thailandensis. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc.

  18. Gene Expression of Type VI Secretion System Associated with Environmental Survival in Acidovorax avenae subsp. avenae by Principle Component Analysis

    Directory of Open Access Journals (Sweden)

    Zhouqi Cui

    2015-09-01

    Full Text Available Valine glycine repeat G (VgrG proteins are regarded as one of two effectors of Type VI secretion system (T6SS which is a complex multi-component secretion system. In this study, potential biological roles of T6SS structural and VgrG genes in a rice bacterial pathogen, Acidovorax avenae subsp. avenae (Aaa RS-1, were evaluated under seven stress conditions using principle component analysis of gene expression. The results showed that growth of the pathogen was reduced by H2O2 and paraquat-induced oxidative stress, high salt, low temperature, and vgrG mutation, compared to the control. However, pathogen growth was unaffected by co-culture with a rice rhizobacterium Burkholderia seminalis R456. In addition, expression of 14 T6SS structural and eight vgrG genes was significantly changed under seven conditions. Among different stress conditions, high salt, and low temperature showed a higher effect on the expression of T6SS gene compared with host infection and other environmental conditions. As a first report, this study revealed an association of T6SS gene expression of the pathogen with the host infection, gene mutation, and some common environmental stresses. The results of this research can increase understanding of the biological function of T6SS in this economically-important pathogen of rice.

  19. Gene Expression of Type VI Secretion System Associated with Environmental Survival in Acidovorax avenae subsp. avenae by Principle Component Analysis.

    Science.gov (United States)

    Cui, Zhouqi; Jin, Guoqiang; Li, Bin; Kakar, Kaleem Ullah; Ojaghian, Mohammad Reza; Wang, Yangli; Xie, Guanlin; Sun, Guochang

    2015-09-11

    Valine glycine repeat G (VgrG) proteins are regarded as one of two effectors of Type VI secretion system (T6SS) which is a complex multi-component secretion system. In this study, potential biological roles of T6SS structural and VgrG genes in a rice bacterial pathogen, Acidovorax avenae subsp. avenae (Aaa) RS-1, were evaluated under seven stress conditions using principle component analysis of gene expression. The results showed that growth of the pathogen was reduced by H₂O₂ and paraquat-induced oxidative stress, high salt, low temperature, and vgrG mutation, compared to the control. However, pathogen growth was unaffected by co-culture with a rice rhizobacterium Burkholderia seminalis R456. In addition, expression of 14 T6SS structural and eight vgrG genes was significantly changed under seven conditions. Among different stress conditions, high salt, and low temperature showed a higher effect on the expression of T6SS gene compared with host infection and other environmental conditions. As a first report, this study revealed an association of T6SS gene expression of the pathogen with the host infection, gene mutation, and some common environmental stresses. The results of this research can increase understanding of the biological function of T6SS in this economically-important pathogen of rice.

  20. Gene expression profiling of RNA extracted from FFPE tissues: NuGEN technologies' whole-transcriptome amplification system.

    Science.gov (United States)

    Turner, Leah; Heath, Joe Don; Kurn, Nurith

    2011-01-01

    Gene expression profiling of RNA isolated from formalin fixed, paraffin-embedded (FFPE) tissue samples has been historically challenging. Yet FFPE samples are sought-after because of the in-depth retrospective records typically associated with them rendering these samples a valuable resource for translational medicine studies. Extensive degradation, chemical modifications, and cross-linking have made it difficult to isolate RNA of sufficient quality required for large-scale gene expression profiling studies. NuGEN Technologies' WT-Ovation™ FFPE System linearly amplifies RNA from FFPE samples through a robust and simple whole-transcriptome approach using as little as 50 ng total RNA isolated from FFPE samples. The amplified material may be labeled with validated kits and/or protocols from NuGEN for analysis on any of the major gene expression microarray platforms, including: Affymetrix, Agilent, and Illumina gene expression arrays. Results compare well with those obtained using RNA from fresh-frozen samples. RNA quality from FFPE samples varies significantly and neither sample age nor sample size analysis via gel electrophoresis or the Agilent Bioanalyzer system accurately predict materials suitable for amplification. Therefore, NuGEN has validated a correlative qPCR-based analytical method for the RNA derived from FFPE samples which effectively predicts array results. The NuGEN approach enables fast and successful analysis of samples previously thought to be too degraded for gene expression analysis.

  1. A Model System for the Study of Gene Expression in the Undergraduate Laboratory

    Science.gov (United States)

    Hargadon, Kristian M.

    2016-01-01

    The flow of genetic information from DNA to RNA to protein, otherwise known as the "central dogma" of biology, is one of the most basic and overarching concepts in the biological sciences. Nevertheless, numerous studies have reported student misconceptions at the undergraduate level of this fundamental process of gene expression. This…

  2. Expression of the Genes Encoding the Trk and Kdp Potassium Transport Systems of Mycobacterium tuberculosis during Growth In Vitro

    Directory of Open Access Journals (Sweden)

    Moloko C. Cholo

    2015-01-01

    Full Text Available Two potassium (K+-uptake systems, Trk and Kdp, are operative in Mycobacterium tuberculosis (Mtb, but the environmental factors triggering their expression have not been determined. The current study has evaluated the expression of these genes in the Mtb wild-type and a trk-gene knockout strain at various stages of logarithmic growth in relation to extracellular K+ concentrations and pH. In both strains, mRNA levels of the K+-uptake encoding genes were relatively low compared to those of the housekeeping gene, sigA, at the early- and mid-log phases, increasing during late-log. Increased gene expression coincided with decreased K+ uptake in the context of a drop in extracellular pH and sustained high extracellular K+ concentrations. In an additional series of experiments, the pH of the growth medium was manipulated by the addition of 1N HCl/NaOH. Decreasing the pH resulted in reductions in both membrane potential and K+ uptake in the setting of significant induction of genes encoding both K+ transporters. These observations are consistent with induction of the genes encoding the active K+ transporters of Mtb as a strategy to compensate for loss of membrane potential-driven uptake of K+ at low extracellular pH. Induction of these genes may promote survival in the acidic environments of the intracellular vacuole and granuloma.

  3. Properties of the Macrophomina phaseolina endoglucanase (EGL 1) gene product in bacterial and yeast expression systems.

    Science.gov (United States)

    Wang, H; Jones, R W

    1999-09-01

    Functional expression of a beta-D-1,4 glucanase-encoding gene (egl1) from a filamentous fungus was achieved in both Escherichia coli and Saccharomyces cerevisiae using a modified version of pRS413. Optimal activity of the E. coli-expressed enzyme was found at incubation temperatures of 60 degrees C, whereas the enzyme activity was optimal at 40 degrees C when expressed by S. cerevisiae. Enzyme activity at different pH levels was similar for both bacteria and yeast, being highest at 5.0. Yeast expression resulted in a highly glycosylated protein of approx 60 kDa, compared to bacterial expression, which resulted in a protein of 30 kDa. The hyperglycosylated protein had reduced enzyme activity, indicating that E. coli is a preferred vehicle for production scale-up.

  4. Expression of hygromycin B resistance in oyster culinary-medicinal mushroom, Pleurotus ostreatus (Jacq.:Fr.)P. Kumm. (higher Basidiomycetes) using three gene expression systems.

    Science.gov (United States)

    Dong, Xiaoya; Zhang, Ke; Gao, Yuqian; Qi, Yuancheng; Shen, Jinwen; Qiu, Liyou

    2012-01-01

    Three hygromycin B phosphotransferase (hph) gene expression systems for culinary-medicinal Oyster mushroom, Pleurotus ostreatus, plasmid pSHC, pAN7-1, and pBHt1 were evaluated through PEG/CaCl(2)-mediated protoplast transformation. Plasmid pSHC is a newly constructed hph gene expression system, composed of Escherichia coli hph gene, the P. ostreatus sdi promoter, and the CaMV35S terminator. The vector pAN7-1 was commonly used for integrative transformation in filamentous fungi. Plasmid pBHtl is a T-DNA binary vector, usually introduced into fungi by Agrobacterium-mediated transformation. The results showed that plasmids pSHC, pAN7-1, and pBHt1 were all integrated into the host chromosomes and expressed hygromycin B resistance in P. ostreatus. pAN7-1 had the highest transformation efficiency and hph gene expression level, pSHC the second, and pBHt1 the lowest. Growth rates of the transformants on plates containing hygromycin B were in correspondence with their hph gene expression levels. To our knowledge, this is the first report on integrated transformation of plasmid pAN7-1 and pBHt1 in P. ostreatus.

  5. Optimization of a yeast RNA interference system for controlling gene expression and enabling rapid metabolic engineering.

    Science.gov (United States)

    Crook, Nathan C; Schmitz, Alexander C; Alper, Hal S

    2014-05-16

    Reduction of endogenous gene expression is a fundamental operation of metabolic engineering, yet current methods for gene knockdown (i.e., genome editing) remain laborious and slow, especially in yeast. In contrast, RNA interference allows facile and tunable gene knockdown via a simple plasmid transformation step, enabling metabolic engineers to rapidly prototype knockdown strategies in multiple strains before expending significant cost to undertake genome editing. Although RNAi is naturally present in a myriad of eukaryotes, it has only been recently implemented in Saccharomyces cerevisiae as a heterologous pathway and so has not yet been optimized as a metabolic engineering tool. In this study, we elucidate a set of design principles for the construction of hairpin RNA expression cassettes in yeast and implement RNA interference to quickly identify routes for improvement of itaconic acid production in this organism. The approach developed here enables rapid prototyping of knockdown strategies and thus accelerates and reduces the cost of the design-build-test cycle in yeast.

  6. MRI of Transgene Expression: Correlation to Therapeutic Gene Expression

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    Tomotsugu Ichikawa

    2002-01-01

    Full Text Available Magnetic resonance imaging (MRI can provide highresolution 3D maps of structural and functional information, yet its use of mapping in vivo gene expression has only recently been explored. A potential application for this technology is to noninvasively image transgene expression. The current study explores the latter using a nonregulatable internalizing engineered transferrin receptor (ETR whose expression can be probed for with a superparamagnetic Tf-CLIO probe. Using an HSV-based amplicon vector system for transgene delivery, we demonstrate that: 1 ETR is a sensitive MR marker gene; 2 several transgenes can be efficiently expressed from a single amplicon; 3 expression of each transgene results in functional gene product; and 4 ETR gene expression correlates with expression of therapeutic genes when the latter are contained within the same amplicon. These data, taken together, suggest that MRI of ETR expression can serve as a surrogate for measuring therapeutic transgene expression.

  7. Ovariectomy modify local renin-angiotensin-aldosterone system gene expressions in the heart of ApoE (-/-) mice.

    Science.gov (United States)

    Borges, Celina Carvalho; Penna-de-Carvalho, Aline; Medeiros Junior, Jorge L; Aguila, Marcia Barbosa; Mandarim-de-Lacerda, Carlos A

    2017-10-04

    The evaluation of the local Renin-Angiotensin-Aldosterone system (RAAS) gene expressions in the heart of ovariectomized (OVX) apolipoprotein E deficient mice (ApoE). Four-months old C57BL/6 female mice (wild-type, wt, n=20), and ApoE female mice (n=20), were submitted to OVX or a surgical procedure without ovary removal (SHAM) and formed four groups (n=10/group): SHAM/wt, SHAM/ApoE, OVX/wt, and OVX/ApoE. OVX led to greater body mass, plasma triglycerides (TG) and total cholesterol, and resulted in insulin resistance and altered RAAS gene expressions in the heart tissue. The gene expression of angiotensin-converting enzyme (ACE)-2 was lower in OVX/wt than in SHAM/wt (P=0.0004), Mas receptor (MASr) was lower in OVX/wt compared to SHAM/wt (P<0.0001). Also, angiotensin II receptor type 1 (AT1r) was higher in OVX/wt than in SHAM/wt (P=0.0229), and AT2r was lower in OVX/wt than in SHAM/wt (P=0.0121). OVX and ApoE deficiency showed interaction potentializing the insulin resistance, increasing TG levels and altering ACE and MASr gene expressions. ACE gene expression was higher in OVX/ApoE than in OVX/wt (P<0.0001), and MASr gene expression was lower in OVX/ApoE than in OVX/wt (P<0.0001). The impact of OVX on local RAAS cascade in the heart of ApoE deficient animals, besides the metabolic changes culminating with insulin resistance, involves an upregulation of renin, ACE, and AT1r gene expressions. The findings may contribute to clarify the mechanisms of development of postmenopausal hypertension and the link between RAAS and apolipoprotein E. Copyright © 2017. Published by Elsevier Inc.

  8. Researchers use Modified CRISPR Systems to Modulate Gene Expression on a Genomic Scale

    Science.gov (United States)

    Cancer Target Discovery and Development Network (CTD2) researchers at the University of California, San Francisco, developed a CRISPR system that can regulate both gene repression and activation with fewer off-target effects.

  9. Adaptive Evolution of Gene Expression in Drosophila

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    Armita Nourmohammad

    2017-08-01

    Full Text Available Gene expression levels are important quantitative traits that link genotypes to molecular functions and fitness. In Drosophila, population-genetic studies have revealed substantial adaptive evolution at the genomic level, but the evolutionary modes of gene expression remain controversial. Here, we present evidence that adaptation dominates the evolution of gene expression levels in flies. We show that 64% of the observed expression divergence across seven Drosophila species are adaptive changes driven by directional selection. Our results are derived from time-resolved data of gene expression divergence across a family of related species, using a probabilistic inference method for gene-specific selection. Adaptive gene expression is stronger in specific functional classes, including regulation, sensory perception, sexual behavior, and morphology. Moreover, we identify a large group of genes with sex-specific adaptation of expression, which predominantly occurs in males. Our analysis opens an avenue to map system-wide selection on molecular quantitative traits independently of their genetic basis.

  10. New Developments in Sleep Research: Molecular Genetics, Gene Expression, and Systems Neurobiology

    OpenAIRE

    Kilduff, Thomas S.; Lein, Ed S; de la Iglesia, Horacio; Sakurai, Takeshi; Fu, Ying-Hui; Shaw, Paul

    2008-01-01

    Understanding the mechanisms that underlie the control of sleep and wakefulness is a major research area in neuroscience. This mini-symposium review highlights some recent developments at the gene, molecular, cellular, and systems level that have advanced this field. The studies discussed below utilize organisms ranging from flies to humans and focus on the interaction between the sleep homeostatic and circadian systems, the consequences of mutations in genes involved in the circadian clock o...

  11. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli

    OpenAIRE

    Thomas Esquerré; Marie Bouvier; Catherine Turlan; Carpousis, Agamemnon J.; Laurence Girbal; Muriel Cocaign-Bousquet

    2016-01-01

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype...

  12. Alterations in Gene Expression of Components of the Renin-Angiotensin System and Its Related Enzymes in Lung Cancer

    OpenAIRE

    Benjamin Goldstein; Malav Trivedi; Speth, Robert C.

    2017-01-01

    Objectives. The study assessed the existence and significance of associations between the expression of fifteen renin-angiotensin system component genes and lung adenocarcinoma. Materials and Methods. NCBI’s built-in statistical tool, GEO2R, was used to calculate Student’s t-tests for the associations found in a DNA expression study of adenocarcinoma and matched healthy lung tissue samples. The raw data was processed with GeneSpring™ and then used to generate figures with and without Sidak’s ...

  13. Expression of codon optimized genes in microbial systems: current industrial applications and perspectives.

    Directory of Open Access Journals (Sweden)

    Claudia eElena

    2014-02-01

    Full Text Available The efficient production of functional proteins in heterologous hosts is one of the major bases of modern biotechnology. Unfortunately, many genes are difficult to express outside their original context. Due to their apparent silent nature, synonymous codon substitutions have long been thought to be trivial. In recent years, this dogma has been refuted by evidence that codon replacement can have a significant impact on gene expression levels and protein folding.In the past decade, considerable advances in the speed and cost of gene synthesis have facilitated the complete redesign of entire gene sequences, dramatically improving the likelihood of high protein expression. This technology significantly impacts the economic feasibility of microbial-based biotechnological processes by, for example, increasing the volumetric productivities of recombinant proteins or facilitating the redesign of novel biosynthetic routes for the production of metabolites.This review discusses the current applications of this technology, particularly those regarding the production of small molecules and industrially-relevant recombinant enzymes. Suggestions for future research and potential uses are provided as well.

  14. Differential expression of endocannabinoid system-related genes in the dorsal hippocampus following expression and reinstatement of morphine conditioned place preference in mice.

    Science.gov (United States)

    Li, Wei; Zhang, Cong-Li; Qiu, Zheng-Guo

    2017-03-16

    The endocannabinoid signaling plays a critical role in mediating rewarding effects to morphine. The relative stability for the expression and reinstatement of morphine conditioned place preference (CPP) suggests the involvement of differential neuroadaptations in learned associations between environmental cues and morphine. Changes in gene expression in hippocampus through the endogenous cannabinoid system (eCB) may accompany and mediate the development of such neuroadaptations to repeated morphine stimulation. To test this possibility, we systematically compared the expression of eCB-related genes in the dorsal hippocampus following the expression, extinction, and reinstatement of morphine CPP using quantitative RT-PCR analyses. We found that expression of morphine CPP was associated with significant increases in mRNA expression for the primary clearance routes for anandamide (AEA) and 2-AG (fatty acid amide hydrolase [FAAH] and monoacylglycerol lipase [MAGL], respectively), but with reductions in cannabinoid 1 receptors (CB1R) and CB2R in dorsal hippocampus following the expression of CPP. However, our results indicated that decreased in MAGL and increased CB1R mRNA levels were accompanied with morphine CPP reinstatement. No significant changes in mRNA expression for enzymes involved in AEA and 2-AG biosynthesis (N-acylphosphatidylethanolamine phospholipase D [NAPEPLD] and diacylglycerol lipase-α/β [DAGLα/β], respectively) were found in all conditions. These results suggest that differential regulation of the synthesis and/or degradation of the eCB system contribute to the expression and reinstatement of morphine CPP.

  15. Positively regulated glycerol/G3P-dependent Bacillus subtilis gene expression system based on anti-termination.

    Science.gov (United States)

    Lewin, Anna; Su, Xiao-Dong; Hederstedt, Lars

    2009-01-01

    Plasmid pLALA was constructed for glycerol or glycerol-3-phosphate inducible plasmid-borne gene expression in Bacillus subtilis and closely related Gram-positive bacteria. Gene expression using pLALA is based on anti-termination of transcription and involves the B. subtilis GlpP protein that in the presence of glycerol-3-phosphate acts as an anti-terminator protein by binding to the 5'-untranslated region of glpD mRNA. Properties and the usefulness of the system, denoted LALA, were validated by inducible production in B. subtilis strains of two water-soluble proteins (beta-galactosidase and a protein phospho-tyrosine phosphatase), and one integral membrane protein (heme A synthase). Advantages with LALA is that it is based on positive control, does not involve a DNA-binding protein, and that glycerol, a cheap and stable compound, can be used as inducer of gene expression.

  16. From System-Wide Differential Gene Expression to Perturbed Regulatory Factors: A Combinatorial Approach.

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    Gaurang Mahajan

    Full Text Available High-throughput experiments such as microarrays and deep sequencing provide large scale information on the pattern of gene expression, which undergoes extensive remodeling as the cell dynamically responds to varying environmental cues or has its function disrupted under pathological conditions. An important initial step in the systematic analysis and interpretation of genome-scale expression alteration involves identification of a set of perturbed transcriptional regulators whose differential activity can provide a proximate hypothesis to account for these transcriptomic changes. In the present work, we propose an unbiased and logically natural approach to transcription factor enrichment. It involves overlaying a list of experimentally determined differentially expressed genes on a background regulatory network coming from e.g. literature curation or computational motif scanning, and identifying that subset of regulators whose aggregated target set best discriminates between the altered and the unaffected genes. In other words, our methodology entails testing of all possible regulatory subnetworks, rather than just the target sets of individual regulators as is followed in most standard approaches. We have proposed an iterative search method to efficiently find such a combination, and benchmarked it on E. coli microarray and regulatory network data available in the public domain. Comparative analysis carried out on artificially generated differential expression profiles, as well as empirical factor overexpression data for M. tuberculosis, shows that our methodology provides marked improvement in accuracy of regulatory inference relative to the standard method that involves evaluating factor enrichment in an individual manner.

  17. From System-Wide Differential Gene Expression to Perturbed Regulatory Factors: A Combinatorial Approach.

    Science.gov (United States)

    Mahajan, Gaurang; Mande, Shekhar C

    2015-01-01

    High-throughput experiments such as microarrays and deep sequencing provide large scale information on the pattern of gene expression, which undergoes extensive remodeling as the cell dynamically responds to varying environmental cues or has its function disrupted under pathological conditions. An important initial step in the systematic analysis and interpretation of genome-scale expression alteration involves identification of a set of perturbed transcriptional regulators whose differential activity can provide a proximate hypothesis to account for these transcriptomic changes. In the present work, we propose an unbiased and logically natural approach to transcription factor enrichment. It involves overlaying a list of experimentally determined differentially expressed genes on a background regulatory network coming from e.g. literature curation or computational motif scanning, and identifying that subset of regulators whose aggregated target set best discriminates between the altered and the unaffected genes. In other words, our methodology entails testing of all possible regulatory subnetworks, rather than just the target sets of individual regulators as is followed in most standard approaches. We have proposed an iterative search method to efficiently find such a combination, and benchmarked it on E. coli microarray and regulatory network data available in the public domain. Comparative analysis carried out on artificially generated differential expression profiles, as well as empirical factor overexpression data for M. tuberculosis, shows that our methodology provides marked improvement in accuracy of regulatory inference relative to the standard method that involves evaluating factor enrichment in an individual manner.

  18. Gene Expression Profiles from Disease Discordant Twins Suggest Shared Antiviral Pathways and Viral Exposures among Multiple Systemic Autoimmune Diseases.

    Science.gov (United States)

    Gan, Lu; O'Hanlon, Terrance P; Lai, Zhennan; Fannin, Rick; Weller, Melodie L; Rider, Lisa G; Chiorini, John A; Miller, Frederick W

    2015-01-01

    Viral agents are of interest as possible autoimmune triggers due to prior reported associations and widely studied molecular mechanisms of antiviral immune responses in autoimmunity. Here we examined new viral candidates for the initiation and/or promotion of systemic autoimmune diseases (SAID), as well as possible related signaling pathways shared in the pathogenesis of those disorders. RNA isolated from peripheral blood samples from 33 twins discordant for SAID and 33 matched, unrelated healthy controls was analyzed using a custom viral-human gene microarray. Paired comparisons were made among three study groups-probands with SAID, their unaffected twins, and matched, unrelated healthy controls-using statistical and molecular pathway analyses. Probands and unaffected twins differed significantly in the expression of 537 human genes, and 107 of those were associated with viral infections. These 537 differentially expressed human genes participate in overlapping networks of several canonical, biologic pathways relating to antiviral responses and inflammation. Moreover, certain viral genes were expressed at higher levels in probands compared to either unaffected twins or unrelated, healthy controls. Interestingly, viral gene expression levels in unaffected twins appeared intermediate between those of probands and the matched, unrelated healthy controls. Of the viruses with overexpressed viral genes, herpes simplex virus-2 (HSV-2) was the only human viral pathogen identified using four distinct oligonucleotide probes corresponding to three HSV-2 genes associated with different stages of viral infection. Although the effects from immunosuppressive therapy on viral gene expression remain unclear, this exploratory study suggests a new approach to evaluate shared viral agents and antiviral immune responses that may be involved in the development of SAID.

  19. Mapping acute systemic effects of inhaled particulate matter and ozone: multiorgan gene expression and glucocorticoid activity.

    Science.gov (United States)

    Thomson, Errol M; Vladisavljevic, Djordje; Mohottalage, Susantha; Kumarathasan, Prem; Vincent, Renaud

    2013-09-01

    Recent epidemiological studies have demonstrated associations between air pollution and adverse effects that extend beyond respiratory and cardiovascular disease, including low birth weight, appendicitis, stroke, and neurological/neurobehavioural outcomes (e.g., neurodegenerative disease, cognitive decline, depression, and suicide). To gain insight into mechanisms underlying such effects, we mapped gene profiles in the lungs, heart, liver, kidney, spleen, cerebral hemisphere, and pituitary of male Fischer-344 rats immediately and 24h after a 4-h exposure by inhalation to particulate matter (0, 5, and 50mg/m(3) EHC-93 urban particles) and ozone (0, 0.4, and 0.8 ppm). Pollutant exposure provoked differential expression of genes involved in a number of pathways, including antioxidant response, xenobiotic metabolism, inflammatory signalling, and endothelial dysfunction. The mRNA profiles, while exhibiting some interorgan and pollutant-specific differences, were remarkably similar across organs for a set of genes, including increased expression of redox/glucocorticoid-sensitive genes and decreased expression of inflammatory genes, suggesting a possible hormonal effect. Pollutant exposure increased plasma levels of adrenocorticotropic hormone and the glucocorticoid corticosterone, confirming activation of the hypothalamic-pituitary-adrenal axis, and there was a corresponding increase in markers of glucocorticoid activity. Although effects were transient and presumably represent an adaptive response to acute exposure in these healthy animals, chronic activation and inappropriate regulation of the hypothalamic-pituitary-adrenal axis are associated with adverse neurobehavioral, metabolic, immune, developmental, and cardiovascular effects. The experimental data are consistent with epidemiological associations of air pollutants with extrapulmonary health outcomes and suggest a mechanism through which such health effects may be induced.

  20. Effect of long-term actual spaceflight on the expression of key genes encoding serotonin and dopamine system

    Science.gov (United States)

    Popova, Nina; Shenkman, Boris; Naumenko, Vladimir; Kulikov, Alexander; Kondaurova, Elena; Tsybko, Anton; Kulikova, Elisabeth; Krasnov, I. B.; Bazhenova, Ekaterina; Sinyakova, Nadezhda

    The effect of long-term spaceflight on the central nervous system represents important but yet undeveloped problem. The aim of our work was to study the effect of 30-days spaceflight of mice on Russian biosatellite BION-M1 on the expression in the brain regions of key genes of a) serotonin (5-HT) system (main enzymes in 5-HT metabolism - tryptophan hydroxylase-2 (TPH-2), monoamine oxydase A (MAO A), 5-HT1A, 5-HT2A and 5-HT3 receptors); b) pivotal enzymes in DA metabolism (tyrosine hydroxylase, COMT, MAO A, MAO B) and D1, D2 receptors. Decreased expression of genes encoding the 5-HT catabolism (MAO A) and 5-HT2A receptor in some brain regions was shown. There were no differences between “spaceflight” and control mice in the expression of TPH-2 and 5-HT1A, 5-HT3 receptor genes. Significant changes were found in genetic control of DA system. Long-term spaceflight decreased the expression of genes encoding the enzyme in DA synthesis (tyrosine hydroxylase in s.nigra), DA metabolism (MAO B in the midbrain and COMT in the striatum), and D1 receptor in hypothalamus. These data suggested that 1) microgravity affected genetic control of 5-HT and especially the nigrostriatal DA system implicated in the central regulation of muscular tonus and movement, 2) the decrease in the expression of genes encoding key enzyme in DA synthesis, DA degradation and D1 receptor contributes to the movement impairment and dyskinesia produced by the spaceflight. The study was supported by Russian Foundation for Basic Research grant № 14-04-00173.

  1. Experimental approaches for the study of oxytocin and vasopressin gene expression in the central nervous system

    Science.gov (United States)

    Scordalakes, Elka M.; Yue, Chunmei; Gainer, Harold

    2016-01-01

    Intron-specific probes measure heteronuclear RNA (hnRNA) levels and thus approximate the transcription rates of genes, in part because of the rapid turnover of this intermediate form of RNA in the cell nucleus. Previously, we used oxytocin (Oxt)- and vasopressin (Avp)- intron-specific riboprobes to measure changes in Oxt and Avp hnRNA levels in the supraoptic nucleus (SON) by quantitative in situ hybridization (ISH) after various classical physiological perturbations, including acute and chronic salt loading, and lactation. In the present experiments, we used a novel experimental model to study the neurotransmitter regulation of Oxt and Avp gene expression in the rat SON in vivo. Bilateral cannulae connected via tubing to Alzet osmotic mini-pumps were positioned over the SON. In every experiment, one SON was infused with PBS and served as the control SON in each animal, and the contralateral SON received infusions of various neurotransmitter agonists and antagonists. Using this approach, we found that Avp but not Oxt gene expression increased after acute (2–5 h) combined excitatory amino acid agonist and GABA antagonist treatment, similar to what we found after an acute hyperosmotic stimulus. Since both OXT and AVP are known to be comparably and robustly secreted in response to acute osmotic stimuli in vivo and glutamate agonists in vitro, our results indicate a dissociation between OXT secretion and Oxt gene transcription in vivo. PMID:18655870

  2. Challenges of incorporating gene expression data to predict HCC prognosis in the age of systems biology

    Institute of Scientific and Technical Information of China (English)

    Yan Du; Guang-Wen Cao

    2012-01-01

    Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide.The recurrence of HCC after curative treatments is currently a major hurdle.Identification of subsets of patients with distinct prognosis provides an opportunity to tailor therapeutic approaches as well as to select the patients with specific sub-phenotypes for targeted therapy.Thus,the development of gene expression profiles to improve the prediction of HCC prognosis is important for HCC management.Although several gene signatures have been evaluated for the prediction of HCC prognosis,there is no consensus on the predictive power of these signatures.Using systematic approaches to evaluate these signatures and combine them with clinicopathologic information may provide more accurate prediction of HCC prognosis.Recently,Villanueva et al[13] developed a composite prognostic model incorporating gene expression patterns in both tumor and adjacent tissues to predict HCC recurrence.In this commentary,we summarize the current progress in using gene signatures to predict HCC prognosis,and discuss the importance,existing issues and future research directions in this field.

  3. Alterations in Gene Expression of Components of the Renin-Angiotensin System and Its Related Enzymes in Lung Cancer

    Directory of Open Access Journals (Sweden)

    Benjamin Goldstein

    2017-01-01

    Full Text Available Objectives. The study assessed the existence and significance of associations between the expression of fifteen renin-angiotensin system component genes and lung adenocarcinoma. Materials and Methods. NCBI’s built-in statistical tool, GEO2R, was used to calculate Student’s t-tests for the associations found in a DNA expression study of adenocarcinoma and matched healthy lung tissue samples. The raw data was processed with GeneSpring™ and then used to generate figures with and without Sidak’s multiple comparison correction. Results. Ten genes were found to be significantly associated with adenocarcinoma. Seven of these associations remained statistically significant after correction for multiple comparisons. Notably, AGTR2, which encodes the AT2 angiotensin II receptor subtype, was significantly underexpressed in adenocarcinoma tissue (p<0.01. AGTR1, ACE, ENPEP, MME, and PRCP, which encode the AT1 angiotensin II receptor, angiotensin-converting enzyme, aminopeptidase N, neprilysin, and prolylcarboxypeptidase, respectively, were also underexpressed. AGT, which encodes angiotensinogen, the angiotensin peptide precursor, was overexpressed in adenocarcinoma tissue. Conclusion. The results suggest an association between the expression of the genes for renin-angiotensin system-related proteins and adenocarcinoma. While further research is necessary to conclusively demonstrate a link between the renin-angiotensin system and lung cancers, the results suggest that the renin-angiotensin system plays a role in the pathology of adenocarcinoma.

  4. Tumor-specific gene expression patterns with gene expression profiles

    Institute of Scientific and Technical Information of China (English)

    RUAN Xiaogang; LI Yingxin; LI Jiangeng; GONG Daoxiong; WANG Jinlian

    2006-01-01

    Gene expression profiles of 14 common tumors and their counterpart normal tissues were analyzed with machine learning methods to address the problem of selection of tumor-specific genes and analysis of their differential expressions in tumor tissues. First, a variation of the Relief algorithm, "RFE_Relief algorithm" was proposed to learn the relations between genes and tissue types. Then, a support vector machine was employed to find the gene subset with the best classification performance for distinguishing cancerous tissues and their counterparts. After tissue-specific genes were removed, cross validation experiments were employed to demonstrate the common deregulated expressions of the selected gene in tumor tissues. The results indicate the existence of a specific expression fingerprint of these genes that is shared in different tumor tissues, and the hallmarks of the expression patterns of these genes in cancerous tissues are summarized at the end of this paper.

  5. Two-Component Signal Transduction Systems That Regulate the Temporal and Spatial Expression of Myxococcus xanthus Sporulation Genes.

    Science.gov (United States)

    Sarwar, Zaara; Garza, Anthony G

    2015-09-14

    When starved for nutrients, Myxococcus xanthus produces a biofilm that contains a mat of rod-shaped cells, known as peripheral rods, and aerial structures called fruiting bodies, which house thousands of dormant and stress-resistant spherical spores. Because rod-shaped cells differentiate into spherical, stress-resistant spores and spore differentiation occurs only in nascent fruiting bodies, many genes and multiple levels of regulation are required. Over the past 2 decades, many regulators of the temporal and spatial expression of M. xanthus sporulation genes have been uncovered. Of these sporulation gene regulators, two-component signal transduction circuits, which typically contain a histidine kinase sensor protein and a transcriptional regulator known as response regulator, are among the best characterized. In this review, we discuss prototypical two-component systems (Nla6S/Nla6 and Nla28S/Nla28) that regulate an early, preaggregation phase of sporulation gene expression during fruiting body development. We also discuss orphan response regulators (ActB and FruA) that regulate a later phase of sporulation gene expression, which begins during the aggregation stage of fruiting body development. In addition, we summarize the research on a complex two-component system (Esp) that is important for the spatial regulation of sporulation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  6. The utility of optical detection system (qPCR) and bioinformatics methods in reference gene expression analysis

    Science.gov (United States)

    Skarzyńska, Agnieszka; Pawełkowicz, Magdalena; PlÄ der, Wojciech; Przybecki, Zbigniew

    2016-09-01

    Real-time quantitative polymerase chain reaction is consider as the most reliable method for gene expression studies. However, the expression of target gene could be misinterpreted due to improper normalization. Therefore, the crucial step for analysing of qPCR data is selection of suitable reference genes, which should be validated experimentally. In order to choice the gene with stable expression in the designed experiment, we performed reference gene expression analysis. In this study genes described in the literature and novel genes predicted as control genes, based on the in silico analysis of transcriptome data were used. Analysis with geNorm and NormFinder algorithms allow to create the ranking of candidate genes and indicate the best reference for flower morphogenesis study. According to the results, genes CACS and CYCL were characterised the most stable expression, but the least suitable genes were TUA and EF.

  7. Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not associated with a direct effect on expression of known defense-related genes but stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge.

    Science.gov (United States)

    van Wees, S C; Luijendijk, M; Smoorenburg, I; van Loon, L C; Pieterse, C M

    1999-11-01

    Selected strains of nonpathogenic rhizobacteria from the genus Pseudomonas are capable of eliciting broad-spectrum induced systemic resistance (ISR) in plants that is phenotypically similar to pathogen-induced systemic acquired resistance (SAR). In Arabidopsis, the ISR pathway functions independently of salicylic acid (SA) but requires responsiveness to jasmonate and ethylene. Here, we demonstrate that known defense-related genes, i.e. the SA-responsive genes PR-1, PR-2, and PR-5, the ethylene-inducible gene Hel, the ethylene- and jasmonate-responsive genes ChiB and Pdf1.2, and the jasmonate-inducible genes Atvsp, Lox1, Lox2, Pall, and Pin2, are neither induced locally in the roots nor systemically in the leaves upon induction of ISR by Pseudomonas fluorescens WCS417r. In contrast, plants infected with the virulent leaf pathogen Pseudomonas syringae pv. tomato (Pst) or expressing SAR induced by preinfecting lower leaves with the avirulent pathogen Pst(avrRpt2) exhibit elevated expression levels of most of the defense-related genes studied. Upon challenge inoculation with Pst, PR gene transcripts accumulated to a higher level in SAR-expressing plants than in control-treated and ISR-expressing plants, indicating that SAR involves potentiation of SA-responsive PR gene expression. In contrast, pathogen challenge of ISR-expressing plants led to an enhanced level of Atvsp transcript accumulation. The otherjasmonate-responsive defense-related genes studied were not potentiated during ISR, indicating that ISR is associated with the potentiation of specific jasmonate-responsive genes.

  8. Characterization of gene expression regulated by human OTK18 using Drosophila melanogaster as a model system for innate immunity

    Indian Academy of Sciences (India)

    Cole R. Spresser; Sarah E. Marshall; Kimberly A. Carlson

    2008-08-01

    OTK18 is a human transcriptional suppressor implicated in the regulation of human immunodeficiency virus type-one infection of mononuclear phagocytes. It is ubiquitously expressed in all normal tissues, but its normal homeostatic function is yet to be characterized. One hypothesis is that OTK18 aids in the regulation of the innate immune system. To test this hypothesis, cDNA microarray analysis was performed on the total RNA extracted from Drosophila melanogaster embryonic Schneider 2 (S2) cells transfected with either pEGFP-OTK18 (enhanced green fluorescent protein) or empty vector controls (pEGFP-N3) for 6, 12 and 24 h. cDNA microarray analysis revealed differential expression of genes known to be important in regulation of Drosophila innate immunity. The expression levels of two genes, Metchnikowin and CG16708 were verified by quantitative real-time reverse transcription PCR. These results suggest a role for OTK18 in innate immunity.

  9. Chloroplast two-component systems: evolution of the link between photosynthesis and gene expression

    OpenAIRE

    Puthiyaveetil, Sujith; Allen, John F.

    2009-01-01

    Two-component signal transduction, consisting of sensor kinases and response regulators, is the predominant signalling mechanism in bacteria. This signalling system originated in prokaryotes and has spread throughout the eukaryotic domain of life through endosymbiotic, lateral gene transfer from the bacterial ancestors and early evolutionary precursors of eukaryotic, cytoplasmic, bioenergetic organelles—chloroplasts and mitochondria. Until recently, it was thought that two-component systems i...

  10. Comparative gene expression analysis among vocal learners (Bengalese finch and budgerigar and non-learners (quail and ring dove reveals variable cadherin expressions in the vocal system

    Directory of Open Access Journals (Sweden)

    Eiji eMatsunaga

    2011-04-01

    Full Text Available Birds use various vocalizations to communicate with one another, and some are acquired through learning. So far, three families of birds (songbirds, parrots, and hummingbirds have been identified as having vocal learning ability. Previously, we found that cadherins, a large family of cell-adhesion molecules, show vocal control-area-related expression in a songbird, the Bengalese finch. To investigate the molecular basis of evolution in avian species, we conducted comparative analysis of cadherin expressions in the vocal and other neural systems among vocal learners (Bengalese finch and budgerigar and a non-learner (quail and ring dove. The gene expression analysis revealed that cadherin expressions were more variable in vocal and auditory areas compared to vocally unrelated areas such as the visual areas among these species. Thus, it appears that such diverse cadherin expressions might have been related to generating species diversity in vocal behavior during the evolution of avian vocal learning. 

  11. The Effect of the Human Peptide GHK on Gene Expression Relevant to Nervous System Function and Cognitive Decline.

    Science.gov (United States)

    Pickart, Loren; Vasquez-Soltero, Jessica Michelle; Margolina, Anna

    2017-02-15

    Neurodegeneration, the progressive death of neurons, loss of brain function, and cognitive decline is an increasing problem for senior populations. Its causes are poorly understood and therapies are largely ineffective. Neurons, with high energy and oxygen requirements, are especially vulnerable to detrimental factors, including age-related dysregulation of biochemical pathways caused by altered expression of multiple genes. GHK (glycyl-l-histidyl-l-lysine) is a human copper-binding peptide with biological actions that appear to counter aging-associated diseases and conditions. GHK, which declines with age, has health promoting effects on many tissues such as chondrocytes, liver cells and human fibroblasts, improves wound healing and tissue regeneration (skin, hair follicles, stomach and intestinal linings, boney tissue), increases collagen, decorin, angiogenesis, and nerve outgrowth, possesses anti-oxidant, anti-inflammatory, anti-pain and anti-anxiety effects, increases cellular stemness and the secretion of trophic factors by mesenchymal stem cells. Studies using the Broad Institute Connectivity Map show that GHK peptide modulates expression of multiple genes, resetting pathological gene expression patterns back to health. GHK has been recommended as a treatment for metastatic cancer, Chronic Obstructive Lung Disease, inflammation, acute lung injury, activating stem cells, pain, and anxiety. Here, we present GHK's effects on gene expression relevant to the nervous system health and function.

  12. The Effect of the Human Peptide GHK on Gene Expression Relevant to Nervous System Function and Cognitive Decline

    Science.gov (United States)

    Pickart, Loren; Vasquez-Soltero, Jessica Michelle; Margolina, Anna

    2017-01-01

    Neurodegeneration, the progressive death of neurons, loss of brain function, and cognitive decline is an increasing problem for senior populations. Its causes are poorly understood and therapies are largely ineffective. Neurons, with high energy and oxygen requirements, are especially vulnerable to detrimental factors, including age-related dysregulation of biochemical pathways caused by altered expression of multiple genes. GHK (glycyl-l-histidyl-l-lysine) is a human copper-binding peptide with biological actions that appear to counter aging-associated diseases and conditions. GHK, which declines with age, has health promoting effects on many tissues such as chondrocytes, liver cells and human fibroblasts, improves wound healing and tissue regeneration (skin, hair follicles, stomach and intestinal linings, boney tissue), increases collagen, decorin, angiogenesis, and nerve outgrowth, possesses anti-oxidant, anti-inflammatory, anti-pain and anti-anxiety effects, increases cellular stemness and the secretion of trophic factors by mesenchymal stem cells. Studies using the Broad Institute Connectivity Map show that GHK peptide modulates expression of multiple genes, resetting pathological gene expression patterns back to health. GHK has been recommended as a treatment for metastatic cancer, Chronic Obstructive Lung Disease, inflammation, acute lung injury, activating stem cells, pain, and anxiety. Here, we present GHK’s effects on gene expression relevant to the nervous system health and function. PMID:28212278

  13. The Effect of the Human Peptide GHK on Gene Expression Relevant to Nervous System Function and Cognitive Decline

    Directory of Open Access Journals (Sweden)

    Loren Pickart

    2017-02-01

    Full Text Available Neurodegeneration, the progressive death of neurons, loss of brain function, and cognitive decline is an increasing problem for senior populations. Its causes are poorly understood and therapies are largely ineffective. Neurons, with high energy and oxygen requirements, are especially vulnerable to detrimental factors, including age-related dysregulation of biochemical pathways caused by altered expression of multiple genes. GHK (glycyl-l-histidyl-l-lysine is a human copper-binding peptide with biological actions that appear to counter aging-associated diseases and conditions. GHK, which declines with age, has health promoting effects on many tissues such as chondrocytes, liver cells and human fibroblasts, improves wound healing and tissue regeneration (skin, hair follicles, stomach and intestinal linings, boney tissue, increases collagen, decorin, angiogenesis, and nerve outgrowth, possesses anti-oxidant, anti-inflammatory, anti-pain and anti-anxiety effects, increases cellular stemness and the secretion of trophic factors by mesenchymal stem cells. Studies using the Broad Institute Connectivity Map show that GHK peptide modulates expression of multiple genes, resetting pathological gene expression patterns back to health. GHK has been recommended as a treatment for metastatic cancer, Chronic Obstructive Lung Disease, inflammation, acute lung injury, activating stem cells, pain, and anxiety. Here, we present GHK’s effects on gene expression relevant to the nervous system health and function.

  14. FunnyBase: a systems level functional annotation of Fundulus ESTs for the analysis of gene expression

    Directory of Open Access Journals (Sweden)

    Kolell Kevin J

    2004-12-01

    Full Text Available Abstract Background While studies of non-model organisms are critical for many research areas, such as evolution, development, and environmental biology, they present particular challenges for both experimental and computational genomic level research. Resources such as mass-produced microarrays and the computational tools linking these data to functional annotation at the system and pathway level are rarely available for non-model species. This type of "systems-level" analysis is critical to the understanding of patterns of gene expression that underlie biological processes. Results We describe a bioinformatics pipeline known as FunnyBase that has been used to store, annotate, and analyze 40,363 expressed sequence tags (ESTs from the heart and liver of the fish, Fundulus heteroclitus. Primary annotations based on sequence similarity are linked to networks of systematic annotation in Gene Ontology (GO and the Kyoto Encyclopedia of Genes and Genomes (KEGG and can be queried and computationally utilized in downstream analyses. Steps are taken to ensure that the annotation is self-consistent and that the structure of GO is used to identify higher level functions that may not be annotated directly. An integrated framework for cDNA library production, sequencing, quality control, expression data generation, and systems-level analysis is presented and utilized. In a case study, a set of genes, that had statistically significant regression between gene expression levels and environmental temperature along the Atlantic Coast, shows a statistically significant (P Conclusion The methods described have application for functional genomics studies, particularly among non-model organisms. The web interface for FunnyBase can be accessed at http://genomics.rsmas.miami.edu/funnybase/super_craw4/. Data and source code are available by request at jpaschall@bioinfobase.umkc.edu.

  15. Ontogeny of the carp (Cyprinus carpio L.) innate immune system: Gene expression and experimental limitations

    DEFF Research Database (Denmark)

    Schmidt, Jacob; Przybylska, Dominika Alicja; Nielsen, Michael Engelbrecht

    2012-01-01

    , 3 and 7 post-wounding and samples were stored in RNA later for isolation of RNA. The physical tissue damage was performed using a sterile needle, which penetrated the skin and the underlying musculature in an area above the lateral line of the left side of fish. Carps at the age of 10, 16 and 24...... days post-hatch were stored and processed whole, whereas just the muscle (the left (wound area) and right filet (internal control)) was sampled for the two latter time-points. mRNA was extracted from the samples, cDNA was synthesised and gene expression was quantified using real-time RT...

  16. Genes of the adaptive immune system are expressed early in zebrafish larval development following lipopolysaccharide stimulation

    Institute of Scientific and Technical Information of China (English)

    LI Fengling; ZHANG Shicui; WANG Zhiping; LI Hongyan

    2011-01-01

    Information regarding immunocompetence of the adaptive immune system (AIS) in zebrafish Danio rerio remains limited. Here, we stimulated an immune response in fish embryos,larvae and adults using lipopolysaccharide (LPS) and measured the upregulation of a number of AIS-related genes (Rag2, AID, TCRAC, IgLC-1, mIg, sIg, IgZ and DAB) 3 and 18 h later. We found that all of the genes evaluated were strongly induced following LPS stimulation, with most of them responding at 8 d post fertilization. This confirms that a functional adaptive immune response is present in D. rerio larvae, and provides a window for further functional analyses.

  17. Biological data warehousing system for identifying transcriptional regulatory sites from gene expressions of microarray data.

    Science.gov (United States)

    Tsou, Ann-Ping; Sun, Yi-Ming; Liu, Chia-Lin; Huang, Hsien-Da; Horng, Jorng-Tzong; Tsai, Meng-Feng; Liu, Baw-Juine

    2006-07-01

    Identification of transcriptional regulatory sites plays an important role in the investigation of gene regulation. For this propose, we designed and implemented a data warehouse to integrate multiple heterogeneous biological data sources with data types such as text-file, XML, image, MySQL database model, and Oracle database model. The utility of the biological data warehouse in predicting transcriptional regulatory sites of coregulated genes was explored using a synexpression group derived from a microarray study. Both of the binding sites of known transcription factors and predicted over-represented (OR) oligonucleotides were demonstrated for the gene group. The potential biological roles of both known nucleotides and one OR nucleotide were demonstrated using bioassays. Therefore, the results from the wet-lab experiments reinforce the power and utility of the data warehouse as an approach to the genome-wide search for important transcription regulatory elements that are the key to many complex biological systems.

  18. Identification of multicomponent histidine-aspartate phosphorelay system controlling flagellar and motility gene expression in Geobacter species.

    Science.gov (United States)

    Ueki, Toshiyuki; Leang, Ching; Inoue, Kengo; Lovley, Derek R

    2012-03-30

    Geobacter species play an important role in the natural biogeochemical cycles of aquatic sediments and subsurface environments as well as in subsurface bioremediation by oxidizing organic compounds with the reduction of insoluble Fe(III) oxides. Flagellum-based motility is considered to be critical for Geobacter species to locate fresh sources of Fe(III) oxides. Functional and comparative genomic approaches, coupled with genetic and biochemical methods, identified key regulators for flagellar gene expression in Geobacter species. A master transcriptional regulator, designated FgrM, is a member of the enhancer-binding protein family. The fgrM gene in the most studied strain of Geobacter species, Geobacter sulfurreducens strain DL-1, is truncated by a transposase gene, preventing flagellar biosynthesis. Integrating a functional FgrM homolog restored flagellar biosynthesis and motility in G. sulfurreducens DL-1 and enhanced the ability to reduce insoluble Fe(III) oxide. Interrupting the fgrM gene in G. sulfurreducens strain KN400, which is motile, removed the capacity for flagellar production and inhibited Fe(III) oxide reduction. FgrM, which is also a response regulator of the two-component His-Asp phosphorelay system, was phosphorylated by histidine kinase GHK4, which was essential for flagellar production and motility. GHK4, which is a hybrid kinase with a receiver domain at the N terminus, was phosphorylated by another histidine kinase, GHK3. Therefore, the multicomponent His-Asp phosphorelay system appears to control flagellar gene expression in Geobacter species.

  19. Aerosol from Tobacco Heating System 2.2 has reduced impact on mouse heart gene expression compared with cigarette smoke.

    Science.gov (United States)

    Szostak, Justyna; Boué, Stéphanie; Talikka, Marja; Guedj, Emmanuel; Martin, Florian; Phillips, Blaine; Ivanov, Nikolai V; Peitsch, Manuel C; Hoeng, Julia

    2017-03-01

    Experimental studies clearly demonstrate a causal effect of cigarette smoking on cardiovascular disease. To reduce the individual risk and population harm caused by smoking, alternative products to cigarettes are being developed. We recently reported on an apolipoprotein E-deficient (Apoe(-/-)) mouse inhalation study that compared the effects of exposure to aerosol from a candidate modified risk tobacco product, Tobacco Heating System 2.2 (THS2.2), and smoke from the reference cigarette (3R4F) on pulmonary and vascular biology. Here, we applied a transcriptomics approach to evaluate the impact of the exposure to 3R4F smoke and THS2.2 aerosol on heart tissues from the same cohort of mice. The systems response profiles demonstrated that 3R4F smoke exposure led to time-dependent transcriptomics changes (False Discovery Rate (FDR) < 0.05; 44 differentially expressed genes at 3-months; 491 at 8-months). Analysis of differentially expressed genes in the heart tissue indicated that 3R4F exposure induced the downregulation of genes involved in cytoskeleton organization and the contractile function of the heart, notably genes that encode beta actin (Actb), actinin alpha 4 (Actn4), and filamin C (Flnc). This was accompanied by the downregulation of genes related to the inflammatory response. None of these effects were observed in the group exposed to THS2.2 aerosol. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. Detection of transgenerational spermatogenic inheritance of adult male acquired CNS gene expression characteristics using a Drosophila systems model.

    Directory of Open Access Journals (Sweden)

    Abhay Sharma

    Full Text Available Available instances of inheritance of epigenetic transgenerational phenotype are limited to environmental exposures during embryonic and adult gonadal development. Adult exposures can also affect gametogenesis and thereby potentially result in reprogramming of the germline. Although examples of epigenetic effects on gametogenesis exist, it is notable that transgenerational inheritance of environment-induced adult phenotype has not yet been reported. Epigenetic codes are considered to be critical in neural plasticity. A Drosophila systems model of pentylenetetrazole (PTZ induced long-term brain plasticity has recently been described. In this model, chronic PTZ treatment of adult males causes alterations in CNS transcriptome. Here, we describe our search for transgenerational spermatogenic inheritance of PTZ induced gene expression phenotype acquired by adult Drosophila males. We generated CNS transcriptomic profiles of F(1 adults after treating F(0 adult males with PTZ and of F(2 adults resulting from a cross between F(1 males and normal females. Surprisingly, microarray clustering showed F(1 male profile as closest to F(1 female and F(0 male profile closest to F(2 male. Differentially expressed genes in F(1 males, F(1 females and F(2 males showed significant overlap with those caused by PTZ. Interestingly, microarray evidence also led to the identification of upregulated rRNA in F(2 males. Next, we generated microarray expression profiles of adult testis from F(0 and F(1 males. Further surprising, clustering of CNS and testis profiles and matching of differentially expressed genes in them provided evidence of a spermatogenic mechanism in the transgenerational effect observed. To our knowledge, we report for the first time detection of transgenerational spermatogenic inheritance of adult acquired somatic gene expression characteristic. The Drosophila systems model offers an excellent opportunity to understand the epigenetic mechanisms underlying

  1. A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Garí, E; Piedrafita, L; Aldea, M; Herrero, E

    1997-07-01

    A set of Saccharomyces cerevisiae expression vectors has been developed in which transcription is driven by a hybrid tetO-CYC1 promoter through the action of a tetR-VP16 (tTA) activator. Expression from the promoter is regulated by tetracycline or derivatives. Various modalities of promoter and activator are used in order to achieve different levels of maximal expression. In the presence of antibiotic in the growth medium at concentrations that do not affect cell growth, expression from the tetO promoter is negligible, and upon antibiotic removal induction ratios of up to 1000-fold are observed with a lacZ reporter system. With the strongest system, overexpression levels comparable with those observed with GAL1-driven promoters are reached. For each particular promoter/tTA combination, expression can be modulated by changing the tetracycline concentration in the growth medium. These vectors may be useful for the study of the function of essential genes in yeast, as well as for phenotypic analysis of genes in overexpression conditions, without restrictions imposed by growth medium composition.

  2. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

    Science.gov (United States)

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-04-26

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation.

  3. Identification of gene profiles of CD4~+ and CD8~+ T lymphocyte in systemic lupus erythematosus by generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification

    Institute of Scientific and Technical Information of China (English)

    王惠琳

    2006-01-01

    Objective To identify LongSAGE Tags in systemic lupus erythematosus (SLE) by generation of longer cDNA fragments from serial analysis of gene expression (SAGE) tags for gene identification (GLGI). Methods CD4+ and CD8+ T lymphocytes were collected from the PBMCs of 25 patients with SLE and 10 healthy controls. Then the total RNA was extracted and reversely

  4. Expression changes of dopaminergic system-related genes in PC12 cells induced by manganese, silver, or copper nanoparticles.

    Science.gov (United States)

    Wang, Jianyong; Rahman, Mohammed F; Duhart, Helen M; Newport, Glenn D; Patterson, Tucker A; Murdock, Richard C; Hussain, Saber M; Schlager, John J; Ali, Syed F

    2009-11-01

    Nanoparticles have received a great deal of attention for producing new engineering applications due to their novel physicochemical characteristics. However, the broad application of nanomaterials has also produced concern for nanoparticle toxicity due to increased exposure from large-scale industry production. This study was conducted to investigate the potential neurotoxicity of manganese (Mn), silver (Ag), and copper (Cu) nanoparticles using the dopaminergic neuronal cell line, PC12. Selective genes associated with the dopaminergic system were investigated for expression changes and their correlation with dopamine depletion. PC12 cells were treated with 10 microg/ml Mn-40 nm, Ag-15 nm, or Cu-90 nm nanoparticles for 24 h. Cu-90 nanoparticles induced dopamine depletion in PC12 cells, which is similar to the effect induced by Mn-40 shown in a previous study. The expression of 11 genes associated with the dopaminergic system was examined using real-time RT-PCR. The expression of Txnrd1 was up-regulated after the Cu-90 treatment and the expression of Gpx1 was down-regulated after Ag-15 or Cu-90 treatment. These alterations are consistent with the oxidative stress induced by metal nanoparticles. Mn-40 induced a down-regulation of the expression of Th; Cu-90 induced an up-regulation of the expression of Maoa. This indicates that besides the oxidation mechanism, enzymatic alterations may also play important roles in the induced dopamine depletion. Mn-40 also induced a down-regulation of the expression of Park2; while the expression of Snca was up-regulated after Mn-40 or Cu-90 treatment. These data suggest that Mn and Cu nanoparticles-induced dopaminergic neurotoxicity may share some common mechanisms associated with neurodegeneration.

  5. Gene Expression Profile Reveals Abnormalities of Multiple Signaling Pathways in Mesenchymal Stem Cell Derived from Patients with Systemic Lupus Erythematosus

    Directory of Open Access Journals (Sweden)

    Yu Tang

    2012-01-01

    Full Text Available We aimed to compare bone-marrow-derived mesenchymal stem cells (BMMSCs between systemic lupus erythematosus (SLE and normal controls by means of cDNA microarray, immunohistochemistry, immunofluorescence, and immunoblotting. Our results showed there were a total of 1, 905 genes which were differentially expressed by BMMSCs derived from SLE patients, of which, 652 genes were upregulated and 1, 253 were downregulated. Gene ontology (GO analysis showed that the majority of these genes were related to cell cycle and protein binding. Pathway analysis exhibited that differentially regulated signal pathways involved actin cytoskeleton, focal adhesion, tight junction, and TGF-β pathway. The high protein level of BMP-5 and low expression of Id-1 indicated that there might be dysregulation in BMP/TGF-β signaling pathway. The expression of Id-1 in SLE BMMSCs was reversely correlated with serum TNF-α levels. The protein level of cyclin E decreased in the cell cycling regulation pathway. Moreover, the MAPK signaling pathway was activated in BMMSCs from SLE patients via phosphorylation of ERK1/2 and SAPK/JNK. The actin distribution pattern of BMMSCs from SLE patients was also found disordered. Our results suggested that there were distinguished differences of BMMSCs between SLE patients and normal controls.

  6. Comparative Analysis of Renin-Angiotensin System (RAS)-Related Gene Expression Between Hypertensive and Normotensive Rats

    Science.gov (United States)

    Williamson, Chad R.; Khurana, Sandhya; Nguyen, Phong; Byrne, Collin J.; Tai, T.C.

    2017-01-01

    Background The renal renin-angiotensin system (RAS) is physiologically important for blood pressure regulation. Altered regulation of RAS-related genes has been observed in an animal model of hypertension (spontaneously hypertensive rats – SHRs). The current understanding of certain RAS-related gene expression differences between Wistar-Kyoto rats (WKYs) and SHRs is either limited or has not been compared. The purpose of this study was to compare the regulation of key RAS-related genes in the kidneys of adult WKYs and SHRs. Material/Methods Coronal sections were dissected through the hilus of kidneys from 16-week-old male WKYs and SHRs. RT-PCR analysis was performed for Ace, Ace2, Agt, Agtr1a, Agtr1b, Agtr2, Atp6ap2 (PRR), Mas1, Ren, Rnls, and Slc12a3 (NCC). Results Increased mRNA expression was observed for Ace, Ace2, Agt, Agtr1a, Agtr1b, and Atp6ap2 in SHRs compared to WKYs. Mas1, Ren, Slc12a3, and Rnls showed no difference in expression between animal types. Conclusions This study shows that the upregulation of several key RAS-related genes in the kidney may account for the increased blood pressure of adult SHRs. PMID:28138124

  7. Expression and Quorum Sensing Regulation of Type III Secretion System Genes of Vibrio harveyi during Infection of Gnotobiotic Brine Shrimp.

    Science.gov (United States)

    Ruwandeepika, H A Darshanee; Karunasagar, Indrani; Bossier, Peter; Defoirdt, Tom

    2015-01-01

    Type III secretion systems enable pathogens to inject their virulence factors directly into the cytoplasm of the host cells. The type III secretion system of Vibrio harveyi, a major pathogen of aquatic organisms and a model species in quorum sensing studies, is repressed by the quorum sensing master regulator LuxR. In this study, we found that during infection of gnotobiotic brine shrimp larvae, the expression levels of three type III secretion operons in V. harveyi increased within the first 12h after challenge and decreased again thereafter. The in vivo expression levels were highest in a mutant with a quorum sensing system that is locked in low cell density configuration (minimal LuxR levels) and lowest in a mutant with a quorum sensing system that is locked in the high cell density configuration (maximal LuxR levels), which is consistent with repression of type III secretion by LuxR. Remarkably, in vivo expression levels of the type III secretion system genes were much (> 1000 fold) higher than the in vitro expression levels, indicating that (currently unknown) host factors significantly induce the type III secretion system. Given the fact that type III secretion is energy-consuming, repression by the quorum sensing master regulators might be a mechanism to save energy under conditions where it does not provide an advantage to the cells.

  8. Integrative Gene Cloning and Expression System for Streptomyces sp. US 24 and Streptomyces sp. TN 58 Bioactive Molecule Producing Strains

    Directory of Open Access Journals (Sweden)

    Samiha Sioud

    2009-01-01

    Full Text Available Streptomyces sp. US 24 and Streptomyces sp. TN 58, two strains producing interesting bioactive molecules, were successfully transformed using E. coli ET12567 (pUZ8002, as a conjugal donor, carrying the integrative plasmid pSET152. For the Streptomyces sp. US 24 strain, two copies of this plasmid were tandemly integrated in the chromosome, whereas for Streptomyces sp. TN 58, the integration was in single copy at the attB site. Plasmid pSET152 was inherited every time for all analysed Streptomyces sp. US 24 and Streptomyces sp. TN 58 exconjugants under nonselective conditions. The growth, morphological differentiation, and active molecules production of all studied pSET152 integrated exconjugants were identical to those of wild type strains. Consequently, conjugal transfer using pSET152 integration system is a suitable means of genes transfer and expression for both studied strains. To validate the above gene transfer system, the glucose isomerase gene (xylA from Streptomyces sp. SK was expressed in strain Streptomyces sp. TN 58. Obtained results indicated that heterologous glucose isomerase could be expressed and folded effectively. Glucose isomerase activity of the constructed TN 58 recombinant strain is of about eighteenfold higher than that of the Streptomyces sp. SK strain. Such results are certainly of importance due to the potential use of improved strains in biotechnological process for the production of high-fructose syrup from starch.

  9. Expression of immune system-related genes during ontogeny in experimentally wounded common carp (Cyprinus carpio) larvae and juveniles

    DEFF Research Database (Denmark)

    Schmidt, Jacob; Nielsen, Michael Engelbrecht

    2014-01-01

    they were microscopically indistinguishable from normal tissue by day 3 post-wounding in all but the juvenile carp wounded on day 49 post-fertilization. In these juveniles the wounded area was still visible even 7days post-wounding. On the transcriptional level a very limited response was observed......We investigated the effect of full-thickness incisional wounding on expression of genes related to the immune system in larvae and juveniles of common carp (Cyprinus carpio). The wounds were inflicted by needle puncture immediately below the anterior part of the dorsal fin on days 7, 14, 28 and 49...... after fertilization. We followed the local gene expression 1, 3 and 7days after wounding by removing head and viscera before extracting RNA from the remaining part of the fish, including the wound area. In addition, we visually followed wound healing. Overall the wounds had regenerated to a point where...

  10. Associations between the expression of epigenetically regulated genes and the expression of DNMTs and MBDs in systemic lupus erythematosus.

    Directory of Open Access Journals (Sweden)

    Eva Balada

    Full Text Available OBJECTIVES: We determined the expression of ITGAL, PRF1, KIR2DL4, CD70, and CD40LG in patients with SLE and performed correlations with the global DNA methylation status and the levels of three DNA methylation enzymes and two methyl CpG-binding domain (MBD proteins. PATIENTS AND METHODS: CD4(+ T cells were isolated from 35 SLE patients and 30 healthy controls. DNA deoxymethylcytosine content was measured by an enzyme-linked immunosorbent assay (ELISA. Transcript levels of ITGAL, PRF1, KIR2DL4, CD70, CD40LG, DNMT1, DNMT3A, DNMT3B, MBD2, and MBD4 were quantified by real-time reverse-transcription polymerase chain reaction (RT-PCR. RESULTS: SLE patients had significantly elevated transcript levels of ITGAL (18.61±22.17 vs. 7.33±9.17, p = 0.042, PRF1 (21.67±26.34 vs. 10.67±11.65, p = 0.039, and CD70 (1.45±1.63 vs. 0.67±0.28, p = 0.011. A positive correlation was observed between transcript levels of CD40LG and ITGAL (r = 0.477, p = 0.004 as well as between CD40LG and PRF1 (r = 0.557, p = 0.001. Transcript levels of KIR2DL4 were higher than controls' but it did not reach statistical significance (1.36±3.52 vs. 0.22±0.79, p = 0.560. A tight relationship with global DNA hypomethylation as well as with the expression of most of the DNA methylation-related genes was observed, especially for ITGAL, PRF1, and CD40LG. CONCLUSIONS: ITGAL, PRF1, and CD70 are overexpressed in SLE CD4(+ T cells. The tight association of CD40LG with ITGAL and PRF1 leads us to infer that it probably contributes to the pathogenesis of the disease. The apparent simultaneous regulation between their expression and the global DNA hypomethylation as well as with the transcription of many DNA methylation-related enzymes, reinforces the idea that epigenetic mechanisms are responsible for the deregulation of ITGAL, PRF1, and CD40LG.

  11. Plant gene expression in the age of systems biology: integrating transcriptional and post-transcriptional events.

    Science.gov (United States)

    Belostotsky, Dmitry A; Rose, Alan B

    2005-07-01

    The extensive mechanistic and regulatory interconnections between the various events of mRNA biogenesis are now recognized as a fundamental principle of eukaryotic gene expression, yet the specific details of the coupling between the various steps of mRNA biogenesis do differ, and sometimes dramatically, between the different kingdoms. In this review, we emphasize examples where plants must differ in this respect from other eukaryotes, and highlight a recurring trend of recruiting the conserved, versatile functional modules, which have evolved to support the general mRNA biogenesis reactions, for plant-specific functions. We also argue that elucidating the inner workings of the plant 'mRNA factory' is essential for accomplishing the ambitious goal of building the 'virtual plant'.

  12. Validation of a mouse xenograft model system for gene expression analysis of human acute lymphoblastic leukaemia

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    Francis Richard W

    2010-04-01

    Full Text Available Abstract Background Pre-clinical models that effectively recapitulate human disease are critical for expanding our knowledge of cancer biology and drug resistance mechanisms. For haematological malignancies, the non-obese diabetic/severe combined immunodeficient (NOD/SCID mouse is one of the most successful models to study paediatric acute lymphoblastic leukaemia (ALL. However, for this model to be effective for studying engraftment and therapy responses at the whole genome level, careful molecular characterisation is essential. Results Here, we sought to validate species-specific gene expression profiling in the high engraftment continuous ALL NOD/SCID xenograft. Using the human Affymetrix whole transcript platform we analysed transcriptional profiles from engrafted tissues without prior cell separation of mouse cells and found it to return highly reproducible profiles in xenografts from individual mice. The model was further tested with experimental mixtures of human and mouse cells, demonstrating that the presence of mouse cells does not significantly skew expression profiles when xenografts contain 90% or more human cells. In addition, we present a novel in silico and experimental masking approach to identify probes and transcript clusters susceptible to cross-species hybridisation. Conclusions We demonstrate species-specific transcriptional profiles can be obtained from xenografts when high levels of engraftment are achieved or with the application of transcript cluster masks. Importantly, this masking approach can be applied and adapted to other xenograft models where human tissue infiltration is lower. This model provides a powerful platform for identifying genes and pathways associated with ALL disease progression and response to therapy in vivo.

  13. Correlation of gene expression and protein production rate - a system wide study

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    Arvas Mikko

    2011-12-01

    Full Text Available Abstract Background Growth rate is a major determinant of intracellular function. However its effects can only be properly dissected with technically demanding chemostat cultivations in which it can be controlled. Recent work on Saccharomyces cerevisiae chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus Trichoderma reesei (Hypocrea jecorina that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype. Results We have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of T. reesei. Use of chemostat allowed control of growth rate and exact estimation of the extracellular specific protein production rate (SPPR. We find that major biosynthetic activities are all negatively correlated with SPPR. We also find that expression of many genes of secreted proteins and secondary metabolism, as well as various lineage specific, mostly unknown genes are positively correlated with SPPR. Finally, we enumerate possible regulators and regulatory mechanisms, arising from the data, for this response. Conclusions Based on these results it appears that in low growth rate protein production energy is very efficiently used primarly for protein production. Also, we propose that flux through early glycolysis or the TCA cycle is a more fundamental determining factor than growth rate for low growth rate protein production and we propose a novel eukaryotic response to this i.e. the lineage specific response (LSR.

  14. The flow of gene expression.

    Science.gov (United States)

    Misteli, Tom

    2004-03-01

    Gene expression is a highly interconnected multistep process. A recent meeting in Iguazu Falls, Argentina, highlighted the need to uncover both the molecular details of each single step as well as the mechanisms of coordination among processes in order to fully understand the expression of genes.

  15. Identification of differentially expressed genes using an annealing control primer system in stage III serous ovarian carcinoma

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    Bae Dong-Han

    2010-10-01

    Full Text Available Abstract Background Most patients with ovarian cancer are diagnosed with advanced stage disease (i.e., stage III-IV, which is associated with a poor prognosis. Differentially expressed genes (DEGs in stage III serous ovarian carcinoma compared to normal tissue were screened by a new differential display method, the annealing control primer (ACP system. The potential targets for markers that could be used for diagnosis and prognosis, for stage III serous ovarian cancer, were found by cluster and survival analysis. Methods The ACP-based reverse transcriptase polymerase chain reaction (RT PCR technique was used to identify DEGs in patients with stage III serous ovarian carcinoma. The DEGs identified by the ACP system were confirmed by quantitative real-time PCR. Cluster analysis was performed on the basis of the expression profile produced by quantitative real-time PCR and survival analysis was carried out by the Kaplan-Meier method and Cox proportional hazards multivariate model; the results of gene expression were compared between chemo-resistant and chemo-sensitive groups. Results A total of 114 DEGs were identified by the ACP-based RT PCR technique among patients with stage III serous ovarian carcinoma. The DEGs associated with an apoptosis inhibitory process tended to be up-regulated clones while the DEGs associated with immune response tended to be down-regulated clones. Cluster analysis of the gene expression profile obtained by quantitative real-time PCR revealed two contrasting groups of DEGs. That is, a group of genes including: SSBP1, IFI6 DDT, IFI27, C11orf92, NFKBIA, TNXB, NEAT1 and TFG were up-regulated while another group of genes consisting of: LAMB2, XRCC6, MEF2C, RBM5, FOXP1, NUDCP2, LGALS3, TMEM185A, and C1S were down-regulated in most patients. Survival analysis revealed that the up-regulated genes such as DDAH2, RNase K and TCEAL2 might be associated with a poor prognosis. Furthermore, the prognosis of patients with chemo

  16. Ascidian gene-expression profiles

    OpenAIRE

    Jeffery, William R.

    2002-01-01

    With the advent of gene-expression profiling, a large number of genes can now be investigated simultaneously during critical stages of development. This approach will be particularly informative in studies of ascidians, basal chordates whose genomes and embryology are uniquely suited for mapping developmental gene networks.

  17. Characteristic Gene Expression Profiles of Human Fibroblasts and Breast Cancer Cells in a Newly Developed Bilateral Coculture System

    Directory of Open Access Journals (Sweden)

    Takayuki Ueno

    2015-01-01

    Full Text Available The microenvironment of cancer cells has been implicated in cancer development and progression. Cancer-associated fibroblast constitutes a major stromal component of the microenvironment. To analyze interaction between cancer cells and fibroblasts, we have developed a new bilateral coculture system using a two-sided microporous collagen membrane. Human normal skin fibroblasts were cocultured with three different human breast cancer cell lines: MCF-7, SK-BR-3, and HCC1937. After coculture, mRNA was extracted separately from cancer cells and fibroblasts and applied to transcriptomic analysis with microarray. Top 500 commonly up- or downregulated genes were characterized by enrichment functional analysis using MetaCore Functional Analysis. Most of the genes upregulated in cancer cells were downregulated in fibroblasts while most of the genes downregulated in cancer cells were upregulated in fibroblasts, indicating that changing patterns of mRNA expression were reciprocal between cancer cells and fibroblasts. In coculture, breast cancer cells commonly increased genes related to mitotic response and TCA pathway while fibroblasts increased genes related to carbohydrate metabolism including glycolysis, glycogenesis, and glucose transport, indicating that fibroblasts support cancer cell proliferation by supplying energy sources. We propose that the bilateral coculture system using collagen membrane is useful to study interactions between cancer cells and stromal cells by mimicking in vivo tumor microenvironment.

  18. A modular lentiviral and retroviral construction system to rapidly generate vectors for gene expression and gene knockdown in vitro and in vivo.

    Science.gov (United States)

    Geiling, Benjamin; Vandal, Guillaume; Posner, Ada R; de Bruyns, Angeline; Dutchak, Kendall L; Garnett, Samantha; Dankort, David

    2013-01-01

    The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG) and retroviral (pREG) vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs). In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems.

  19. Arabidopsis flowering locus D influences systemic-acquired-resistance- induced expression and histone modifications of WRKY genes.

    Science.gov (United States)

    Singh, Vijayata; Roy, Shweta; Singh, Deepjyoti; Nandi, Ashis Kumar

    2014-03-01

    A plant that is in part infected by a pathogen is more resistant throughout its whole body to subsequent infections--a phenomenon known as systemic acquired resistance (SAR). Mobile signals are synthesized at the site of infection and distributed throughout the plant through vascular tissues. Mechanism of SAR development subsequent to reaching the mobile signal in the distal tissue is largely unknown. Recently we showed that flowering locus D (FLD) gene of Arabidopsis thaliana is required in the distal tissue to activate SAR. FLD codes for a homologue of human-lysine-specific histone demethylase. Here we show that FLD function is required for priming (SAR induced elevated expression during challenge inoculation) of WRKY29 and WRKY6 genes. FLD also differentially influences basal and SAR-induced expression of WRKY38, WRKY65 and WRKY53 genes. In addition, we also show that FLD partly localizes in nucleus and influences histone modifications at the promoters of WRKY29 and WRKY6 genes. The results altogether indicate to the possibility of FLD's involvement in epigenetic regulation of SAR.

  20. Gene expression of a two-component regulatory system associated with sunscreen biosynthesis in the cyanobacterium Nostoc punctiforme ATCC 29133.

    Science.gov (United States)

    Janssen, Jacob; Soule, Tanya

    2016-01-01

    Long-wavelength ultraviolet radiation (UVA) can damage cells through photooxidative stress, leading to harmful photosensitized proteins and pigments in cyanobacteria. To mitigate damage, some cyanobacteria secrete the UVA-absorbing pigment scytonemin into their extracellular sheath. Comparative genomic analyses suggest that scytonemin biosynthesis is regulated by the two-component regulatory system (TCRS) proteins encoded by Npun_F1277 and Npun_F1278 in the cyanobacterium Nostoc punctiforme ATCC 29133. To understand the dynamics of these genes, their expression was measured following exposure to UVA, UVB, high visible (VIS) irradiance and oxidative stress for 20, 40 and 60 min. Overall, both genes had statistically similar patterns of expression for all four conditions and were generally upregulated, except for those exposed to UVB by 60 min and for the cells under oxidative stress. The greatest UVA response was an upregulation by 20 min, while the response to UVB was the most dramatic and persisted through 40 min. High VIS irradiance resulted in a modest upregulation, while oxidative stress caused a slight downregulation. Both genes were also found to occur on the same transcript. These results demonstrate that these genes are positively responding to several light-associated conditions, which suggests that this TCRS may regulate more than just scytonemin biosynthesis under UVA stress. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Coupling the GAL4 UAS system with alcR for versatile cell type-specific chemically inducible gene expression in Arabidopsis.

    Science.gov (United States)

    Sakvarelidze, Lali; Tao, Zheng; Bush, Max; Roberts, Gethin R; Leader, David J; Doonan, John H; Rawsthorne, Stephen

    2007-07-01

    The Aspergillus alc regulon encodes a transcription factor, ALCR, which regulates transcription from cognate promoters such as alcA(p). In the presence of suitable chemical inducers, ALCR activates gene expression from alcA(p). The alc regulon can be transferred to other species and can be used to control the expression of reporter, metabolic and developmental genes in response to low-level ethanol exposure. In this paper, we describe a versatile system for targeting the alc regulon to specific cell types in Arabidopsis by driving ALCR expression from the GAL4 upstream activator sequence (UAS). Large numbers of Arabidopsis lines are available in which GAL4 is expressed in a variety of spatial patterns and, in turn, drives the expression of any gene cloned downstream of the UAS. We have used a previously characterized line that directs gene expression to the endosperm to demonstrate spatially restricted ethanol-inducible gene expression. We also show that the domain of inducible gene expression can easily be altered by crossing the UAS::ALCR cassette into different driver lines. We conclude that this gene switch can be used to drive gene expression in a highly responsive, but spatially restricted, manner.

  2. Gene expression in the DpnI and DpnII restriction enzyme systems of Streptococcus pneumoniae

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.A.; Sabelnikov, A.G.; Chen, Jau-Der; Greenberg, B.

    1992-12-31

    Although a number of bacterial species are naturally transformable, that is, their cells are able to take up external DNA in substantial amounts and integrate it into the chromosome without artificial manipulation of the cell surface, Streptococcus pneumoniae, the first species in which this phenomenon was detected, remains a prototype of such transformation. Cells of S. pneumonias also contain potent restriction endonucleases able to severely restrict DNA introduced during viral infection. Our current understanding of the genetic basis of the complementary DpnI and DpnII restriction systems and of the biochemistry of their component enzymes are briefly reviewed. The manner in which these enzymes impinge on the transfer of chromosomal genes and of plasmeds will be examined in detail. It will be seen that far from acting against foreign DNA in general, the restriction systems seem to be designed to exclude only infecting viral DNA The presence of complementary restriction systems in different cells of S. pneumonias enhances their effectiveness in blocking viral infection and promoting species survival. This enhanced effectiveness requires the expression of alternative restriction systems. Therefore, the ability of the cells to transfer the restriction enzyme genes and to regulate their expression are important for survival of the species.

  3. Identifying early events of gene expression in breast cancer with systems biology phylogenetics.

    Science.gov (United States)

    Abu-Asab, M S; Abu-Asab, N; Loffredo, C A; Clarke, R; Amri, H

    2013-01-01

    Advanced omics technologies such as deep sequencing and spectral karyotyping are revealing more of cancer heterogeneity at the genetic, genomic, gene expression, epigenetic, proteomic, and metabolomic levels. With this increasing body of emerging data, the task of data analysis becomes critical for mining and modeling to better understand the relevant underlying biological processes. However, the multiple levels of heterogeneity evident within and among populations, healthy and diseased, complicate the mining and interpretation of biological data, especially when dealing with hundreds to tens of thousands of variables. Heterogeneity occurs in many diseases, such as cancers, autism, macular degeneration, and others. In cancer, heterogeneity has hampered the search for validated biomarkers for early detection, and it has complicated the task of finding clonal (driver) and nonclonal (nonexpanded or passenger) aberrations. We show that subtyping of cancer (classification of specimens) should be an a priori step to the identification of early events of cancers. Studying early events in oncogenesis can be done on histologically normal tissues from diseased individuals (HNTDI), since they most likely have been exposed to the same mutagenic insults that caused the cancer in their neighboring tissues. Polarity assessment of HNTDI data variables by using healthy specimens as outgroup(s), followed by the application of parsimony phylogenetic analysis, produces a hierarchical classification of specimens that reveals the early events of the disease ontogeny within its subtypes as shared derived changes (abnormal changes) or synapomorphies in phylogenetic terminology. Copyright © 2013 S. Karger AG, Basel.

  4. Evaluation and comparison of the GUS, LUC and GFP reporter system for gene expression studies in plants

    NARCIS (Netherlands)

    Ruijter, de N.C.A.; Verhees, J.A.; Leeuwen, van W.; Krol, van der A.R.

    2003-01-01

    The detailed analysis of the expression pattern of a plant gene can give important clues about its function in plant development, cell differentiation and defence reactions. Gene expression studies have been greatly facilitated by the employment of proteins like beta-glucuronidase (GUS), green fluor

  5. Expression of the zebrafish CD133/prominin1 genes in cellular proliferation zones in the embryonic central nervous system and sensory organs.

    Science.gov (United States)

    McGrail, Maura; Batz, Lindsey; Noack, Kristin; Pandey, Saumya; Huang, Yong; Gu, Xun; Essner, Jeffrey J

    2010-06-01

    The CD133/prominin1 gene encodes a pentamembrane glycoprotein cell surface marker that is expressed in stem cells from neuroepithelial, hematopoietic, and various organ tissues. Here we report the analysis of two zebrafish CD133/prominin1 orthologues, prominin1a and prominin1b. The expression patterns of the zebrafish prominin1a and b genes were analyzed during embryogenesis using whole mount in situ hybridization. prominin1a and b show novel complementary and overlapping patterns of expression in proliferating zones in the developing sensory organs and central nervous system. The expression patterns suggest functional conservation of the zebrafish prominin1 genes. Initial analyses of prominin1a and b in neoplastic tissue show increased expression of both genes in a subpopulation of cells in malignant peripheral nerve sheath tumors in tp53 mutants. Based on these analyses, the zebrafish prominin1 genes will be useful markers for examining proliferating cell populations in adult organs, tissues, and tumors.

  6. Human Lacrimal Gland Gene Expression

    Science.gov (United States)

    Aakalu, Vinay Kumar; Parameswaran, Sowmya; Maienschein-Cline, Mark; Bahroos, Neil; Shah, Dhara; Ali, Marwan; Krishnakumar, Subramanian

    2017-01-01

    Background The study of human lacrimal gland biology and development is limited. Lacrimal gland tissue is damaged or poorly functional in a number of disease states including dry eye disease. Development of cell based therapies for lacrimal gland diseases requires a better understanding of the gene expression and signaling pathways in lacrimal gland. Differential gene expression analysis between lacrimal gland and other embryologically similar tissues may be helpful in furthering our understanding of lacrimal gland development. Methods We performed global gene expression analysis of human lacrimal gland tissue using Affymetrix ® gene expression arrays. Primary data from our laboratory was compared with datasets available in the NLM GEO database for other surface ectodermal tissues including salivary gland, skin, conjunctiva and corneal epithelium. Results The analysis revealed statistically significant difference in the gene expression of lacrimal gland tissue compared to other ectodermal tissues. The lacrimal gland specific, cell surface secretory protein encoding genes and critical signaling pathways which distinguish lacrimal gland from other ectodermal tissues are described. Conclusions Differential gene expression in human lacrimal gland compared with other ectodermal tissue types revealed interesting patterns which may serve as the basis for future studies in directed differentiation among other areas. PMID:28081151

  7. A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy

    Science.gov (United States)

    Oka, Tetsuo; Kurozumi, Kazuhiko; Shimazu, Yosuke; Ichikawa, Tomotsugu; Ishida, Joji; Otani, Yoshihiro; Shimizu, Toshihiko; Tomita, Yusuke; Sakaguchi, Masakiyo; Watanabe, Masami; Nasu, Yasutomo; Kumon, Hiromi; Date, Isao

    2016-09-01

    Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87ΔEGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma.

  8. Gene expression data from acetaminophen-induced toxicity in human hepatic in vitro systems and clinical liver samples

    Directory of Open Access Journals (Sweden)

    Robim M. Rodrigues

    2016-06-01

    Full Text Available This data set is composed of transcriptomics analyses of (i liver samples from patients suffering from acetaminophen-induced acute liver failure (ALF and (ii hepatic cell systems exposed to acetaminophen and their respective controls. The in vitro systems include widely employed cell lines i.e. HepaRG and HepG2 cells as well as a novel stem cell-derived model i.e. human skin-precursors-derived hepatocyte-like cells (hSKP-HPC. Data from primary human hepatocytes was also added to the data set “Open TG-GATEs: a large-scale toxicogenomics database” (Igarashi et al., 2015 [1]. Changes in gene expression due to acetaminophen intoxication as well as comparative information between human in vivo and in vitro samples are provided. The microarray data have been deposited in NCBI׳s Gene Expression Omnibus and are accessible through GEO Series accession number GEO: GSE74000. The provided data is used to evaluate the predictive capacity of each hepatic in vitro system and can be directly compared with large-scale publically available toxicogenomics databases. Further interpretation and discussion of these data feature in the corresponding research article “Toxicogenomics-based prediction of acetaminophen-induced liver injury using human hepatic cell systems” (Rodrigues et al., 2016 [2].

  9. Interleukin-1 (IL-1 system gene expression in granulosa cells: kinetics during terminal preovulatory follicle maturation in the mare

    Directory of Open Access Journals (Sweden)

    Gérard Nadine

    2003-05-01

    Full Text Available Abstract Background A growing body of evidences suggests that the ovary is a site of inflammatory reactions, and thus, ovarian cells could represent sources and targets of the interleukin-1 (IL-1 system. The purpose of this study was to examine the IL-1 system gene expressions in equine granulosa cells, and to study the IL-1β content in follicular fluid during the follicle maturation. For this purpose, granulosa cells and follicular fluids were collected from the largest follicle at the early dominance stage (diameter 24 ± 3 mm or during the preovulatory maturation phase, at T0 h, T6 h, T12 h, T24 h and T34 h after induction of ovulation. Cells were analysed by RT-PCR and follicular fluids were studied by gel electrophoresis and immunoblotting. Results We demonstrated that interleukin-1β (IL-1β, interleukin-1 receptor 2 (IL-1R2 and interleukin-1 receptor antagonist (IL-1RA genes are expressed in equine granulosa cells. We observed that the IL-1β and IL-1RA mRNA content changed in granulosa cells during the terminal follicular maturation whereas IL-1R2 mRNA did not vary. In follicular fluid, IL-1β content fluctuated few hours after induction of ovulation. Conclusions The expression of IL-1β gene in granulosa cells and the follicular fluid IL-1β content seem to be regulated by gonadotropins suggesting that IL-1β could be an intermediate paracrine factor involved in ovulation.

  10. Construction of prokaryotic expression system of TGF-β1 epitope gene and identification of recombinant fusion protein immunity

    Institute of Scientific and Technical Information of China (English)

    Yong-Hong Guo; Zhi-Ming Hao; Jin-Yan Luo; Jun-Hong Wang

    2005-01-01

    AIM: To insert the constructed TGF-β1the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti- TGF-β1METHODS: The TGF-β1mature TGF-β1TGF-32) was amplified by polymerase chain reaction from the recombinant pGEM-7z/TGF-β1HBcAg gene fragments (encoding HBcAg from 1-71 and 89-144 amino acid residues) were amplified from PYTA1-HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-β1into restrictive endonuclease enzyme and ligated with T4ligase. The fusion gene fragments HBcAg1-71-TGF-β1HBcAg89-144 were recloned to pET28a(+) and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a (+)/CTC was transformed and expressed in E.. Coli BL21 (DE3)under induction of IPTG. After purification with Ni+2-NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope.RESULTS: Enzyme digestion analysis and sequencing showed that TGF-β1loop of C-terminus of truncated hepatitis B core antigen.SDS-PAGE analysis showed that relative molecular mass(Mr) of the expressed product by pET28a (+)/CTC was Mr 24 600.The output of the target recombinant protein was approximately 34.8% of the total bacterial protein,mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-β1not with anti-HBcAg. The purity of protein was about 90% and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-β1could be used as anti-TGF-β1CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target TGF- epitope gene was successfully established.The fusion protein is in the form of self-assembling particles

  11. Towards systems genetic analyses in barley: Integration of phenotypic, expression and genotype data into GeneNetwork

    Directory of Open Access Journals (Sweden)

    Druka Arnis

    2008-11-01

    Full Text Available Abstract Background A typical genetical genomics experiment results in four separate data sets; genotype, gene expression, higher-order phenotypic data and metadata that describe the protocols, processing and the array platform. Used in concert, these data sets provide the opportunity to perform genetic analysis at a systems level. Their predictive power is largely determined by the gene expression dataset where tens of millions of data points can be generated using currently available mRNA profiling technologies. Such large, multidimensional data sets often have value beyond that extracted during their initial analysis and interpretation, particularly if conducted on widely distributed reference genetic materials. Besides quality and scale, access to the data is of primary importance as accessibility potentially allows the extraction of considerable added value from the same primary dataset by the wider research community. Although the number of genetical genomics experiments in different plant species is rapidly increasing, none to date has been presented in a form that allows quick and efficient on-line testing for possible associations between genes, loci and traits of interest by an entire research community. Description Using a reference population of 150 recombinant doubled haploid barley lines we generated novel phenotypic, mRNA abundance and SNP-based genotyping data sets, added them to a considerable volume of legacy trait data and entered them into the GeneNetwork http://www.genenetwork.org. GeneNetwork is a unified on-line analytical environment that enables the user to test genetic hypotheses about how component traits, such as mRNA abundance, may interact to condition more complex biological phenotypes (higher-order traits. Here we describe these barley data sets and demonstrate some of the functionalities GeneNetwork provides as an easily accessible and integrated analytical environment for exploring them. Conclusion By

  12. Applicability of gene expression and systems biology to develop pharmacogenetic predictors; antipsychotic-induced extrapyramidal symptoms as an example.

    Science.gov (United States)

    Mas, Sergi; Gassó, Patricia; Lafuente, Amelia

    2015-11-01

    Pharmacogenetics has been driven by a candidate gene approach. The disadvantage of this approach is that is limited by our current understanding of the mechanisms by which drugs act. Gene expression could help to elucidate the molecular signatures of antipsychotic treatments searching for dysregulated molecular pathways and the relationships between gene products, especially protein-protein interactions. To embrace the complexity of drug response, machine learning methods could help to identify gene-gene interactions and develop pharmacogenetic predictors of drug response. The present review summarizes the applicability of the topics presented here (gene expression, network analysis and gene-gene interactions) in pharmacogenetics. In order to achieve this, we present an example of identifying genetic predictors of extrapyramidal symptoms induced by antipsychotic.

  13. Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes.

    Science.gov (United States)

    Waring, R B; May, G S; Morris, N R

    1989-06-30

    Plasmids have been constructed in which expression of a gene can be placed under the control of the inducible promoter of the alcA gene encoding alcohol dehydrogenase I in Aspergillus nidulans. Simplified shuttle vectors carrying pyr4 which complements pyrG89 mutations have also been constructed. These are based on pUC19 and retain alpha-peptide expression. The beta-tubulin genes, tubC and benA, have been placed under the control of alcA and their expression studied. Levels of expression can be assayed phenotypically because increased synthesis of beta-tubulin inhibits vegetative growth. Sensitivity of asexual spore formation to the anti-microtubule drug benomyl provides a means of detecting very low levels of expression of the chimeric genes. Glucose almost completely represses the chimeric genes. Induction is rapid and is maximal within an hour. When a strain carrying seven copies of an alcA::tubC gene fusion was grown under inducing conditions, 6.5% of total sulfate labelled protein consisted of tubC product. Cyclopentanone was the most potent inducer of the chimeric genes on solid media but it also partially inhibited growth. Chimeric alcA::tubC and alcA::benA genes were expressed to very similar levels despite the fact that tubC utilizes many rare codons.

  14. LuxCDE-luxAB-based promoter reporter system to monitor the Yersinia enterocolitica O:3 gene expression in vivo

    Science.gov (United States)

    Bozcal, Elif; Dagdeviren, Melih; Uzel, Atac

    2017-01-01

    It is crucial to understand the in vitro and in vivo regulation of the virulence factor genes of bacterial pathogens. In this study, we describe the construction of a versatile reporter system for Yersinia enterocolitica serotype O:3 (YeO3) based on the luxCDABE operon. In strain YeO3-luxCDE we integrated the luciferase substrate biosynthetic genes, luxCDE, into the genome of the bacterium so that the substrate is constitutively produced. The luxAB genes that encode the luciferase enzyme were cloned into a suicide vector to allow cloning of any promoter-containing fragment upstream the genes. When the obtained suicide-construct is mobilized into YeO3-luxCDE bacteria, it integrates into the recipient genome via homologous recombination between the cloned promoter fragment and the genomic promoter sequence and thereby generates a single-copy and stable promoter reporter. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core hexasaccharide (OC) of YeO3 are virulence factors necessary to colonization of the intestine and establishment of infection. To monitor the activities of the OC and O-ag gene cluster promoters we constructed the reporter strains YeO3-Poc::luxAB and YeO3-Pop1::luxAB, respectively. In vitro, at 37°C both promoter activities were highest during logarithmic growth and decreased when the bacteria entered stationary growth phase. At 22°C the OC gene cluster promoter activity increased during the late logarithmic phase. Both promoters were more active in late stationary phase. To monitor the promoter activities in vivo, mice were infected intragastrically and the reporter activities monitored by the IVIS technology. The mouse experiments revealed that both LPS promoters were well expressed in vivo and could be detected by IVIS, mainly from the intestinal region of orally infected mice. PMID:28235077

  15. Global gene expression and systems biology analysis of bovine monocyte-derived macrophages in response to in vitro challenge with Mycobacterium bovis.

    Directory of Open Access Journals (Sweden)

    David A Magee

    Full Text Available BACKGROUND: Mycobacterium bovis, the causative agent of bovine tuberculosis, is a major cause of mortality in global cattle populations. Macrophages are among the first cell types to encounter M. bovis following exposure and the response elicited by these cells is pivotal in determining the outcome of infection. Here, a functional genomics approach was undertaken to investigate global gene expression profiles in bovine monocyte-derived macrophages (MDM purified from seven age-matched non-related females, in response to in vitro challenge with M. bovis (multiplicity of infection 2:1. Total cellular RNA was extracted from non-challenged control and M. bovis-challenged MDM for all animals at intervals of 2 hours, 6 hours and 24 hours post-challenge and prepared for global gene expression analysis using the Affymetrix® GeneChip® Bovine Genome Array. RESULTS: Comparison of M. bovis-challenged MDM gene expression profiles with those from the non-challenged MDM controls at each time point identified 3,064 differentially expressed genes 2 hours post-challenge, with 4,451 and 5,267 differentially expressed genes detected at the 6 hour and 24 hour time points, respectively (adjusted P-value threshold ≤ 0.05. Notably, the number of downregulated genes exceeded the number of upregulated genes in the M. bovis-challenged MDM across all time points; however, the fold-change in expression for the upregulated genes was markedly higher than that for the downregulated genes. Systems analysis revealed enrichment for genes involved in: (1 the inflammatory response; (2 cell signalling pathways, including Toll-like receptors and intracellular pathogen recognition receptors; and (3 apoptosis. CONCLUSIONS: The increased number of downregulated genes is consistent with previous studies showing that M. bovis infection is associated with the repression of host gene expression. The results also support roles for MyD88-independent signalling and intracellular PRRs in

  16. Expression of polarity genes in human cancer.

    Science.gov (United States)

    Lin, Wan-Hsin; Asmann, Yan W; Anastasiadis, Panos Z

    2015-01-01

    Polarity protein complexes are crucial for epithelial apical-basal polarity and directed cell migration. Since alterations of these processes are common in cancer, polarity proteins have been proposed to function as tumor suppressors or oncogenic promoters. Here, we review the current understanding of polarity protein functions in epithelial homeostasis, as well as tumor formation and progression. As most previous studies focused on the function of single polarity proteins in simplified model systems, we used a genomics approach to systematically examine and identify the expression profiles of polarity genes in human cancer. The expression profiles of polarity genes were distinct in different human tissues and classified cancer types. Additionally, polarity expression profiles correlated with disease progression and aggressiveness, as well as with identified cancer types, where specific polarity genes were commonly altered. In the case of Scribble, gene expression analysis indicated its common amplification and upregulation in human cancer, suggesting a tumor promoting function.

  17. Docosahexaenoyl ethanolamide improves glucose uptake and alters endocannabinoid system gene expression in proliferating and differentiating C2C12 myoblasts

    Directory of Open Access Journals (Sweden)

    Jeffrey eKim

    2014-03-01

    Full Text Available Skeletal muscle is a major storage site for glycogen and a focus for understanding insulin resistance and type-2-diabetes. New evidence indicates that overactivation of the peripheral endocannabinoid system (ECS in skeletal muscle diminishes insulin sensitivity. Specific n-6 and n-3 polyunsaturated fatty acids (PUFA are precursors for the biosynthesis of ligands that bind to and activate the cannabinoid receptors. The function of the ECS and action of PUFA in skeletal muscle glucose uptake was investigated in proliferating and differentiated C2C12 myoblasts treated with either 25µM of arachidonate (AA or docosahexaenoate (DHA, 25µM of EC [anandamide (AEA, 2-arachidonoylglycerol (2-AG, docosahexaenoylethanolamide (DHEA], 1µM of CB1 antagonist NESS0327, and CB2 antagonist AM630. Compared to the BSA vehicle control cell cultures in both proliferating and differentiated myoblasts those treated with DHEA, the EC derived from the n-3 PUFA DHA, had higher 24 h glucose uptake, while AEA and 2-AG, the EC derived from the n-6 PUFA AA, had lower basal glucose uptake. Adenylyl cyclase mRNA was higher in myoblasts treated with DHA in both proliferating and differentiated states while those treated with AEA or 2-AG were lower compared to the control cell cultures. Western blot and qPCR analysis showed higher expression of the cannabinoid receptors in differentiated myoblasts treated with DHA while the opposite was observed with AA. These findings indicate a compensatory effect of DHA and DHEA compared to AA-derived ligands on the ECS and associated ECS gene expression and higher glucose uptake in myoblasts.Key Words: endocannabinoid system •C2C12 myoblasts cannabinoid receptors glucose uptake gene expression DHEA • polyunsaturated fatty acids

  18. Argudas: arguing with gene expression information

    CERN Document Server

    McLeod, Kenneth; Burger, Albert

    2010-01-01

    In situ hybridisation gene expression information helps biologists identify where a gene is expressed. However, the databases that republish the experimental information are often both incomplete and inconsistent. This paper examines a system, Argudas, designed to help tackle these issues. Argudas is an evolution of an existing system, and so that system is reviewed as a means of both explaining and justifying the behaviour of Argudas. Throughout the discussion of Argudas a number of issues will be raised including the appropriateness of argumentation in biology and the challenges faced when integrating apparently similar online biological databases.

  19. The use of an adeno-associated viral vector for efficient bicistronic expression of two genes in the central nervous system.

    Science.gov (United States)

    Hutson, Thomas Haynes; Kathe, Claudia; Menezes, Sean Christopher; Rooney, Marie-Claire; Bueler, Hansruedi; Moon, Lawrence David Falcon

    2014-01-01

    Recombinant adeno-associated viral (AAV) vectors are one of the most promising therapeutic delivery systems for gene therapy to the central nervous system (CNS). Preclinical testing of novel gene therapies requires the careful design and production of AAV vectors and their successful application in a model of CNS injury. One major limitation of AAV vectors is their limited packaging capacity (genes (e.g., from two promoters) difficult. An internal ribosomal entry site has been used to express two genes: However, the second transgene is often expressed at lower levels than the first. In addition to this, achieving high levels of transduction in the CNS can be challenging. In this chapter we describe the cloning of a bicistronic AAV vector that uses the foot-and-mouth disease virus 2A sequence to efficiently express two genes from a single promoter. Bicistronic expression of a therapeutic gene and a reporter gene is desirable so that the axons from transduced neurons can be tracked and, after CNS injury, the amount of axonal sprouting or regeneration quantified. We go on to describe how to perform a pyramidotomy model of CNS injury and the injection of AAV vectors into the sensorimotor cortex to provide efficient transduction and bicistronic gene expression in cortical neurons such that transduced axons are detectable in the dorsal columns of the spinal cord.

  20. Gene expression profiling in circulating endothelial cells from systemic sclerosis patients shows an altered control of apoptosis and angiogenesis that is modified by iloprost infusion

    Science.gov (United States)

    2010-01-01

    Introduction Circulating endothelial cells are increased in patients affected by systemic sclerosis (SSc) and their number strongly correlates with vascular damage. The effects of iloprost in systemic sclerosis are only partially known. We aimed at studying the gene expression profile of circulating endothelial cells and the effects of iloprost infusion and gene expression in patients with systemic sclerosis. Methods We enrolled 50 patients affected by systemic sclerosis, 37 patients without and 13 patients with digital ulcers. Blood samples were collected from all patients before and 72 hours after either a single day or five days eight hours iloprost infusion. Blood samples were also collected from 50 sex- and age-matched healthy controls. Circulating endothelial cells and endothelial progenitors cells were detected in the peripheral blood of patients with systemic sclerosis by flow cytometry with a four-colour panel of antibodies. Statistical analysis was performed with the SPSS 16 statistical package.Circulating endothelial cells were then isolated from peripheral blood by immunomagnetic CD45 negative selection for the gene array study. Results The number of both circulating endothelial cells and progenitors was significantly higher in patients affected by systemic sclerosis than in controls and among patients in those with digital ulcers than in patients without them. Circulating endothelial cells and progenitors number increased after iloprost infusion. Gene array analysis of endothelial cells showed a different transcriptional profile in patients compared to controls. Indeed, patients displayed an altered expression of genes involved in the control of apoptosis and angiogenesis. Iloprost infusion had a profound impact on endothelial cells gene expression since the treatment was able to modulate a very high number of transcripts. Conclusions We report here that circulating endothelial cells in patients with systemic sclerosis show an altered expression of

  1. CNS expression pattern of Lmx1b and coexpression with Ptx genes suggest functional cooperativity in the development of forebrain motor control systems

    NARCIS (Netherlands)

    Asbreuk, CHJ; Vogelaar, CF; Hellemons, A; Smidt, MP; Burbach, JPH

    2002-01-01

    In the central nervous system, acquisition of regional specification is an important developmental process. The regional specification is reflected by restricted and overlapping expression of homeobox genes, which are regulators of this event. Here, we detail the expression pattern of Lmx1b during l

  2. The Erwinia amylovora PhoPQ system is involved in resistance to antimicrobial peptide and suppresses gene expression of two novel type III secretion systems.

    Science.gov (United States)

    Nakka, Sridevi; Qi, Mingsheng; Zhao, Youfu

    2010-10-20

    The PhoPQ system is a pleiotropic two-component signal transduction system that controls many pathogenic properties in several mammalian and plant pathogens. Three different cues have been demonstrated to activate the PhoPQ system including a mild acidic pH, antimicrobial peptides, and low Mg(2+). In this study, our results showed that phoPQ mutants were more resistant to strong acidic conditions (pH 4.5 or 5) than that of the wild-type (WT) strain, suggesting that this system in Erwinia amylovora may negatively regulate acid resistance gene expression. Furthermore, the PhoPQ system negatively regulated gene expression of two novel type III secretion systems in E. amylovora. These results are in contrast to those reported for the PhoPQ system in Salmonella and Xanthomonas, where it positively regulates type III secretion system and acid resistance. In addition, survival of phoPQ mutants was about 10-fold lower than that of WT when treated with cecropin A at pH 5.5, suggesting that the PhoPQ system renders the pathogen more resistant to cecropin A.

  3. Gene expression in colorectal cancer

    DEFF Research Database (Denmark)

    Birkenkamp-Demtroder, Karin; Christensen, Lise Lotte; Olesen, Sanne Harder

    2002-01-01

    Understanding molecular alterations in colorectal cancer (CRC) is needed to define new biomarkers and treatment targets. We used oligonucleotide microarrays to monitor gene expression of about 6,800 known genes and 35,000 expressed sequence tags (ESTs) on five pools (four to six samples in each p...... with a high frequency of loss of heterozygosity. The genes and ESTs presented in this study encode new potential tumor markers as well as potential novel therapeutic targets for prevention or therapy of CRC.......Understanding molecular alterations in colorectal cancer (CRC) is needed to define new biomarkers and treatment targets. We used oligonucleotide microarrays to monitor gene expression of about 6,800 known genes and 35,000 expressed sequence tags (ESTs) on five pools (four to six samples in each...... pool) of total RNA from left-sided sporadic colorectal carcinomas. We compared normal tissue to carcinoma tissue from Dukes' stages A-D (noninvasive to distant metastasis) and identified 908 known genes and 4,155 ESTs that changed remarkably from normal to tumor tissue. Based on intensive filtering 226...

  4. Transient expression in tobacco Bright Yellow 2 cells and pollen grains: A fast, efficient and reliable system for functional promoter analysis of plant genes

    Directory of Open Access Journals (Sweden)

    Bratić Ana M.

    2010-01-01

    Full Text Available Gene expression is mediated by DNA sequences directly upstream from the coding sequences, recruited transcription factors and RNA polymerase in a spatially-defined manner. Understanding promoter strength and regulation would enhance our understanding of gene expression. The goal of this study was to develop a fast, efficient and reliable method for testing basal promoter activity and identifying core sequences within its pollen specific elements. In this paper we examined the functionality of buckwheat metallothionein promoter by a histochemical GUS assay in two transient expression systems: BY2 cells and pollen grains. Strong promoter activity was observed in both systems.

  5. Enhanced production of shikimic acid using a multi-gene co-expression system in Escherichia coli.

    Science.gov (United States)

    Liu, Xiang-Lei; Lin, Jun; Hu, Hai-Feng; Zhou, Bin; Zhu, Bao-Quan

    2016-04-01

    Shikimic acid (SA) is the key synthetic material for the chemical synthesis of Oseltamivir, which is prescribed as the front-line treatment for serious cases of influenza. Multi-gene expression vector can be used for expressing the plurality of the genes in one plasmid, so it is widely applied to increase the yield of metabolites. In the present study, on the basis of a shikimate kinase genetic defect strain Escherichia coli BL21 (ΔaroL/aroK, DE3), the key enzyme genes aroG, aroB, tktA and aroE of SA pathway were co-expressed and compared systematically by constructing a series of multi-gene expression vectors. The results showed that different gene co-expression combinations (two, three or four genes) or gene orders had different effects on the production of SA. SA production of the recombinant BL21-GBAE reached to 886.38 mg·L(-1), which was 17-fold (P < 0.05) of the parent strain BL21 (ΔaroL/aroK, DE3).

  6. Polyandry and sex-specific gene expression.

    Science.gov (United States)

    Mank, Judith E; Wedell, Nina; Hosken, David J

    2013-03-05

    Polyandry is widespread in nature, and has important evolutionary consequences for the evolution of sexual dimorphism and sexual conflict. Although many of the phenotypic consequences of polyandry have been elucidated, our understanding of the impacts of polyandry and mating systems on the genome is in its infancy. Polyandry can intensify selection on sexual characters and generate more intense sexual conflict. This has consequences for sequence evolution, but also for sex-biased gene expression, which acts as a link between mating systems, sex-specific selection and the evolution of sexual dimorphism. We discuss this and the remarkable confluence of sexual-conflict theory and patterns of gene expression, while also making predictions about transcription patterns, mating systems and sexual conflict. Gene expression is a key link in the genotype-phenotype chain, and although in its early stages, understanding the sexual selection-transcription relationship will provide significant insights into this critical association.

  7. Multi-agent System for Obtaining Relevant Genes in Expression Analysis between Young and Older Women with Triple Negative Breast Cancer.

    Science.gov (United States)

    González-Briones, Alfonso; Ramos, Juan; De Paz, Juan Francisco; Corchado, Juan Manuel

    2015-10-21

    Triple negative breast cancer is an aggressive form of breast cancer. Despite treatment with chemotherapy, relapses are frequent and response to these treatments is not the same in younger women as in older women. Therefore, the identification of genes that cause this difference is required. The identification of therapeutic targets is one of the sought after goals to develop new drugs. Within the range of different hybridization techniques, the developed system uses expression array analysis to measure the expression of the signal levels of thousands of genes in a given sample. Probesets of Gene 1.0 ST GeneChip arrays provide categorical genome transcript coverage, providing a measurement of the expression level of the sample. This paper proposes a multi-agent system to manage information of expression arrays, with the goal of providing an intuitive system that is also extensible to analyze and interpret the results. The roles of agent integrate different types of techniques, statistical and data mining methods that select a set of genes, searching techniques that find pathways in which such genes participate, and an information extraction procedure that applies a CBR system to check if these genes are involved in the disease.

  8. Systems toxicology of chemically induced liver and kidney injuries: histopathology-associated gene co-expression modules.

    Science.gov (United States)

    Te, Jerez A; AbdulHameed, Mohamed Diwan M; Wallqvist, Anders

    2016-09-01

    Organ injuries caused by environmental chemical exposures or use of pharmaceutical drugs pose a serious health risk that may be difficult to assess because of a lack of non-invasive diagnostic tests. Mapping chemical injuries to organ-specific histopathology outcomes via biomarkers will provide a foundation for designing precise and robust diagnostic tests. We identified co-expressed genes (modules) specific to injury endpoints using the Open Toxicogenomics Project-Genomics Assisted Toxicity Evaluation System (TG-GATEs) - a toxicogenomics database containing organ-specific gene expression data matched to dose- and time-dependent chemical exposures and adverse histopathology assessments in Sprague-Dawley rats. We proposed a protocol for selecting gene modules associated with chemical-induced injuries that classify 11 liver and eight kidney histopathology endpoints based on dose-dependent activation of the identified modules. We showed that the activation of the modules for a particular chemical exposure condition, i.e., chemical-time-dose combination, correlated with the severity of histopathological damage in a dose-dependent manner. Furthermore, the modules could distinguish different types of injuries caused by chemical exposures as well as determine whether the injury module activation was specific to the tissue of origin (liver and kidney). The generated modules provide a link between toxic chemical exposures, different molecular initiating events among underlying molecular pathways and resultant organ damage. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Journal of Applied Toxicology published by John Wiley & Sons, Ltd.

  9. An insight into the possible mechanism of working of two-cistronic gene expression systems and rational designing of newer systems

    Indian Academy of Sciences (India)

    Utpal Kumar Mukhopadhyay; Girish Sahni

    2002-06-01

    The initial attempts at hyper-expressing buffalo/goat growth hormone (GH)-ORFs in Escherichia coli directly under various strong promoters were not successful despite the presence of a functional gene. High level expression of GH was achieved as a fusion protein with glutathione-S-transferase (GST). To produce native GH in an unfused state, we adapted an established strategy of two-cistronic approach in our system. In this strategy, utilizing one of the highly efficient reported sequences as the first cistron led to a nearly 1000-fold enhancement in the level of expression under an E. coli promoter (trc). In search of a newer first-cistron sequence as well as to see the generality of the two-cistronic approach, we explored the ability of different lengths of a highly expressing natural gene to act as an efficient first cistron. Surprisingly, GST, which is naturally highly expressible in E. coli, could not be fitted into a successful two-cistronic construct. In addition, placement of the entire two-cistronic expression cassette (which had earlier given high-level GH expression under trc promoter) under the T7 promoter in E. coli failed to hyper-express GH. These results suggest that the successful exploitation of the two-cistron arrangement for hyper-expression of eukaryotic ORFs in bacteria is not as straightforward as was previously thought. It appears probable that factors such as the sequence context, together with the length and codons used in the first cistron are important as well.

  10. A second rhodopsin-like protein in Cyanophora paradoxa: gene sequence and protein expression in a cell-free system.

    Science.gov (United States)

    Frassanito, Anna Maria; Barsanti, Laura; Passarelli, Vincenzo; Evangelista, Valtere; Gualtieri, Paolo

    2013-08-05

    Here we report the identification and expression of a second rhodopsin-like protein in the alga Cyanophora paradoxa (Glaucophyta), named Cyanophopsin_2. This new protein was identified due to a serendipity event, since the RACE reaction performed to complete the sequence of Cyanophopsin_1, (the first rhodopsin-like protein of C. paradoxa identified in 2009 by our group), amplified a 619 bp sequence corresponding to a portion of a new gene of the same protein family. The full sequence consists of 1175 bp consisting of 849 bp coding DNA sequence and 4 introns of 326 bp. The protein is characterized by an N-terminal region of 47 amino acids, followed by a region with 7 α-helices of 213 amino acids and a C-terminal region of 22 amino acids. This protein showed high identity with Cyanophopsin_1 and other rhodopsin-like proteins of Archea, Bacteria, Fungi and Algae. Cyanophosin_2 (CpR2) was expressed in a cell-free expression system, and characterized by means of absorption spectroscopy.

  11. A new system for regulated functional gene expression for gene therapy applications: nuclear delivery of a p16INK4A-estrogen receptor carboxy terminal fusion protein only in the presence of estrogen.

    Science.gov (United States)

    Tamura, Tomohiro; Kanuma, Tatsuya; Nakazato, Tomoko; Faried, Leri S; Aoki, Hiroshi; Minegishi, Takashi

    2010-04-01

    The clinical use of gene therapy requires tight regulation of the gene of interest and functional expression only when it is needed. Thus, it is necessary to develop ways of regulating functional gene expression with exogenous stimuli. Many regulatable systems are currently under development. For example, the tetracycline-dependent transcriptional switch has been successfully employed for in vivo preclinical applications. However, there are no examples of regulatable systems that have been employed in human clinical trials. In the present study, we established an adenovirus-delivered functional gene expression system that is regulated by estrogen. This system uses p16INK4A fused at its C-terminus to the ligand-binding domain of the estrogen receptor (DeltaERalpha). We were able to establish cell lines expressing this gene wherein the functional expression of p16INK4A is estrogen-dependent and causes the arrest of several ovarian cancer cell lines. This inducible and adenovirus-mediated gene transfer system may allow gene therapy using nuclear functioning genes in postmenopausal or ovariectomized women.

  12. Foreign gene expression in Hansenula polymorpha. A system for the synthesis of small functional peptides

    NARCIS (Netherlands)

    Faber, K.N.; Westra, S.; Waterham, H.R.; Keizer-Gunnink, I.; Harder, W.; AB, G.; Veenhuis, M.

    1996-01-01

    We describe the synthesis and purification of two functional peptides, namely human insulin-like growth factor II (IGF-II) and Xenopus laevis magainin II in Hansenula polymorpha after their synthesis as hybrid proteins fused to the C terminus of endogenous amine oxidase. The hybrid genes, placed und

  13. Enhanced gene expression of systemically administered plasmid DNA inthe liver with therapeutic ultrasound and microbubbles

    NARCIS (Netherlands)

    Raju, B.I.; Leyvi, E.; Seip, R.; Sethuraman, S.; Luo, X.; Bird, A.; Li, S.; Koeberl, D.

    2012-01-01

    Ultrasound mediated delivery (USMD) of novel therapeutic agents in the presence of microbubbles is a potentially safe and effective method for gene therapy offering many desired characteristics such as low toxicity, potential for repeated treatment, and organ specificity.In this study we tested the

  14. Zipf's Law in Gene Expression

    CERN Document Server

    Furusawa, C; Furusawa, Chikara; Kaneko, Kunihiko

    2002-01-01

    Using data from gene expression databases on various organisms and tissues, including yeast, nematodes, human normal and cancer tissues, and embryonic stem cells, we found that the abundances of expressed genes exhibit a power-law distribution with an exponent close to -1, i.e., they obey Zipf's law. Furthermore, by simulations of a simple model with an intra-cellular reaction network, we found that Zipf's law of chemical abundance is a universal feature of cells where such a network optimizes the efficiency and faithfulness of self-reproduction. These findings provide novel insights into the nature of the organization of reaction dynamics in living cells.

  15. Zipf's Law in Gene Expression

    Science.gov (United States)

    Furusawa, Chikara; Kaneko, Kunihiko

    2003-02-01

    Using data from gene expression databases on various organisms and tissues, including yeast, nematodes, human normal and cancer tissues, and embryonic stem cells, we found that the abundances of expressed genes exhibit a power-law distribution with an exponent close to -1; i.e., they obey Zipf’s law. Furthermore, by simulations of a simple model with an intracellular reaction network, we found that Zipf’s law of chemical abundance is a universal feature of cells where such a network optimizes the efficiency and faithfulness of self-reproduction. These findings provide novel insights into the nature of the organization of reaction dynamics in living cells.

  16. Correction of gene expression data

    DEFF Research Database (Denmark)

    Darbani Shirvanehdeh, Behrooz; Stewart, C. Neal, Jr.; Noeparvar, Shahin;

    2014-01-01

    This report investigates for the first time the potential inter-treatment bias source of cell number for gene expression studies. Cell-number bias can affect gene expression analysis when comparing samples with unequal total cellular RNA content or with different RNA extraction efficiencies...... an analytical approach to examine the suitability of correction methods by considering the inter-treatment bias as well as the inter-replicate variance, which allows use of the best correction method with minimum residual bias. Analyses of RNA sequencing and microarray data showed that the efficiencies...

  17. Maternal diets trigger sex-specific divergent trajectories of gene expression and epigenetic systems in mouse placenta.

    Directory of Open Access Journals (Sweden)

    Anne Gabory

    Full Text Available Males and females responses to gestational overnutrition set the stage for subsequent sex-specific differences in adult onset non communicable diseases. Placenta, as a widely recognized programming agent, contibutes to the underlying processes. According to our previous findings, a high-fat diet during gestation triggers sex-specific epigenetic alterations within CpG and throughout the genome, together with the deregulation of clusters of imprinted genes. We further investigated the impact of diet and sex on placental histology, transcriptomic and epigenetic signatures in mice. Both basal gene expression and response to maternal high-fat diet were sexually dimorphic in whole placentas. Numerous genes showed sexually dimorphic expression, but only 11 genes regardless of the diet. In line with the key role of genes belonging to the sex chromosomes, 3 of these genes were Y-specific and 3 were X-specific. Amongst all the genes that were differentially expressed under a high-fat diet, only 16 genes were consistently affected in both males and females. The differences were not only quantitative but remarkably qualitative. The biological functions and networks of genes dysregulated differed markedly between the sexes. Seven genes of the epigenetic machinery were dysregulated, due to effects of diet, sex or both, including the Y- and X-linked histone demethylase paralogues Kdm5c and Kdm5d, which could mark differently male and female epigenomes. The DNA methyltransferase cofactor Dnmt3l gene expression was affected, reminiscent of our previous observation of changes in global DNA methylation. Overall, this striking sexual dimorphism of programming trajectories impose a considerable revision of the current dietary interventions protocols.

  18. Design and Implementation of Visual Dynamic Display Software of Gene Expression Based on GTK

    Institute of Scientific and Technical Information of China (English)

    JIANG Wei; MENG Fanjiang; LI Yong; YU Xiao

    2009-01-01

    The paper presented an implement method for a dynamic gene expression display software based on the GTK. This method established the dynamic presentation system of gene expression which according to gene expression data from gene chip hybridize at different time, adopted a linearity combination model and Pearson correlation coefficient algorithm. The system described the gene expression changes in graphic form, the gene expression changes with time and the changes in characteristics of the gene expression, also the changes in relations of the gene expression and regulation relationships among genes. The system also provided an integrated platform for analysis on gene chips data, especially for the research on the network of gene regulation.

  19. Systems biology analysis of gene expression during in vivo Mycobacterium avium paratuberculosis enteric colonization reveals role for immune tolerance.

    Science.gov (United States)

    Khare, Sangeeta; Lawhon, Sara D; Drake, Kenneth L; Nunes, Jairo E S; Figueiredo, Josely F; Rossetti, Carlos A; Gull, Tamara; Everts, Robin E; Lewin, Harris A; Galindo, Cristi L; Garner, Harold R; Adams, Leslie Garry

    2012-01-01

    Survival and persistence of Mycobacterium avium subsp. paratuberculosis (MAP) in the intestinal mucosa is associated with host immune tolerance. However, the initial events during MAP interaction with its host that lead to pathogen survival, granulomatous inflammation, and clinical disease progression are poorly defined. We hypothesize that immune tolerance is initiated upon initial contact of MAP with the intestinal Peyer's patch. To test our hypothesis, ligated ileal loops in neonatal calves were infected with MAP. Intestinal tissue RNAs were collected (0.5, 1, 2, 4, 8 and 12 hrs post-infection), processed, and hybridized to bovine gene expression microarrays. By comparing the gene transcription responses of calves infected with the MAP, informative complex patterns of expression were clearly visible. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis, and genes were grouped into the specific pathways and gene ontology categories to create a holistic model. This model revealed three different phases of responses: i) early (30 min and 1 hr post-infection), ii) intermediate (2, 4 and 8 hrs post-infection), and iii) late (12 hrs post-infection). We describe here the data that include expression profiles for perturbed pathways, as well as, mechanistic genes (genes predicted to have regulatory influence) that are associated with immune tolerance. In the Early Phase of MAP infection, multiple pathways were initiated in response to MAP invasion via receptor mediated endocytosis and changes in intestinal permeability. During the Intermediate Phase, perturbed pathways involved the inflammatory responses, cytokine-cytokine receptor interaction, and cell-cell signaling. During the Late Phase of infection, gene responses associated with immune tolerance were initiated at the level of T-cell signaling. Our study provides evidence that MAP infection resulted in differentially regulated genes, perturbed pathways

  20. Systems biology analysis of gene expression during in vivo Mycobacterium avium paratuberculosis enteric colonization reveals role for immune tolerance.

    Directory of Open Access Journals (Sweden)

    Sangeeta Khare

    Full Text Available Survival and persistence of Mycobacterium avium subsp. paratuberculosis (MAP in the intestinal mucosa is associated with host immune tolerance. However, the initial events during MAP interaction with its host that lead to pathogen survival, granulomatous inflammation, and clinical disease progression are poorly defined. We hypothesize that immune tolerance is initiated upon initial contact of MAP with the intestinal Peyer's patch. To test our hypothesis, ligated ileal loops in neonatal calves were infected with MAP. Intestinal tissue RNAs were collected (0.5, 1, 2, 4, 8 and 12 hrs post-infection, processed, and hybridized to bovine gene expression microarrays. By comparing the gene transcription responses of calves infected with the MAP, informative complex patterns of expression were clearly visible. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis, and genes were grouped into the specific pathways and gene ontology categories to create a holistic model. This model revealed three different phases of responses: i early (30 min and 1 hr post-infection, ii intermediate (2, 4 and 8 hrs post-infection, and iii late (12 hrs post-infection. We describe here the data that include expression profiles for perturbed pathways, as well as, mechanistic genes (genes predicted to have regulatory influence that are associated with immune tolerance. In the Early Phase of MAP infection, multiple pathways were initiated in response to MAP invasion via receptor mediated endocytosis and changes in intestinal permeability. During the Intermediate Phase, perturbed pathways involved the inflammatory responses, cytokine-cytokine receptor interaction, and cell-cell signaling. During the Late Phase of infection, gene responses associated with immune tolerance were initiated at the level of T-cell signaling. Our study provides evidence that MAP infection resulted in differentially regulated genes, perturbed

  1. Melatonin-Induced Temporal Up-Regulation of Gene Expression Related to Ubiquitin/Proteasome System (UPS in the Human Malaria Parasite Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Fernanda C. Koyama

    2014-12-01

    Full Text Available There is an increasing understanding that melatonin and the ubiquitin/ proteasome system (UPS interact to regulate multiple cellular functions. Post-translational modifications such as ubiquitination are important modulators of signaling processes, cell cycle and many other cellular functions. Previously, we reported a melatonin-induced upregulation of gene expression related to ubiquitin/proteasome system (UPS in Plasmodium falciparum, the human malaria parasite, and that P. falciparum protein kinase 7 influences this process. This implies a role of melatonin, an indolamine, in modulating intraerythrocytic development of the parasite. In this report we demonstrate by qPCR analysis, that melatonin induces gene upregulation in nine out of fourteen genes of the UPS, consisting of the same set of genes previously reported, between 4 to 5 h after melatonin treatment. We demonstrate that melatonin causes a temporally controlled gene expression of UPS members.

  2. Homeobox gene expression in Brachiopoda

    DEFF Research Database (Denmark)

    Altenburger, Andreas; Martinez, Pedro; Wanninger, Andreas

    2011-01-01

    The molecular control that underlies brachiopod ontogeny is largely unknown. In order to contribute to this issue we analyzed the expression pattern of two homeobox containing genes, Not and Cdx, during development of the rhynchonelliform (i.e., articulate) brachiopod Terebratalia transversa. Not...

  3. Dyslipidemia rather than Type 2 Diabetes Mellitus or Chronic Periodontitis Affects the Systemic Expression of Pro- and Anti-Inflammatory Genes.

    Science.gov (United States)

    Nepomuceno, Rafael; Villela, Bárbara Scoralick; Corbi, Sâmia Cruz Tfaile; Bastos, Alliny De Souza; Dos Santos, Raquel Alves; Takahashi, Catarina Satie; Orrico, Silvana Regina Perez; Scarel-Caminaga, Raquel Mantuaneli

    2017-01-01

    A high percentage of type 2 diabetes mellitus (T2D) patients are also affected by dyslipidemia and chronic periodontitis (CP), but no studies have determined the gene expression in patients that are simultaneously affected by all three diseases. We investigated the systemic expression of immune-related genes in T2D, dyslipidemia, and CP patients. One hundred and fifty patients were separated into five groups containing 30 individuals each: (G1) poorly controlled T2D with dyslipidemia and CP; (G2) well-controlled T2D with dyslipidemia and CP; (G3) normoglycemic individuals with dyslipidemia and CP; (G4) healthy individuals with CP; (G5) systemic and periodontally healthy individuals. Blood analyses of lipid and glycemic profiles were carried out. The expression of genes, including IL10, JAK1, STAT3, SOCS3, IP10, ICAM1, IFNA, IFNG, STAT1, and IRF1, was investigated by RT-qPCR. Patients with dyslipidemia demonstrated statistically higher expression of the IL10 and IFNA genes, while IFNG, IP10, IRF1, JAK1, and STAT3 were lower in comparison with nondyslipidemic patients. Anti-inflammatory genes, such as IL10, positively correlated with parameters of glucose, lipid, and periodontal profiles, while proinflammatory genes, such as IFNG, were negatively correlated with these parameters. We conclude that dyslipidemia appears to be the primary disease that is associated with gene expression of immune-related genes, while parameters of T2D and CP were correlated with the expression of these important immune genes.

  4. Dyslipidemia rather than Type 2 Diabetes Mellitus or Chronic Periodontitis Affects the Systemic Expression of Pro- and Anti-Inflammatory Genes

    Science.gov (United States)

    Corbi, Sâmia Cruz Tfaile; Bastos, Alliny De Souza; Dos Santos, Raquel Alves; Orrico, Silvana Regina Perez

    2017-01-01

    A high percentage of type 2 diabetes mellitus (T2D) patients are also affected by dyslipidemia and chronic periodontitis (CP), but no studies have determined the gene expression in patients that are simultaneously affected by all three diseases. We investigated the systemic expression of immune-related genes in T2D, dyslipidemia, and CP patients. One hundred and fifty patients were separated into five groups containing 30 individuals each: (G1) poorly controlled T2D with dyslipidemia and CP; (G2) well-controlled T2D with dyslipidemia and CP; (G3) normoglycemic individuals with dyslipidemia and CP; (G4) healthy individuals with CP; (G5) systemic and periodontally healthy individuals. Blood analyses of lipid and glycemic profiles were carried out. The expression of genes, including IL10, JAK1, STAT3, SOCS3, IP10, ICAM1, IFNA, IFNG, STAT1, and IRF1, was investigated by RT-qPCR. Patients with dyslipidemia demonstrated statistically higher expression of the IL10 and IFNA genes, while IFNG, IP10, IRF1, JAK1, and STAT3 were lower in comparison with nondyslipidemic patients. Anti-inflammatory genes, such as IL10, positively correlated with parameters of glucose, lipid, and periodontal profiles, while proinflammatory genes, such as IFNG, were negatively correlated with these parameters. We conclude that dyslipidemia appears to be the primary disease that is associated with gene expression of immune-related genes, while parameters of T2D and CP were correlated with the expression of these important immune genes. PMID:28316372

  5. Vascular Gene Expression: A Hypothesis

    Directory of Open Access Journals (Sweden)

    Angélica Concepción eMartínez-Navarro

    2013-07-01

    Full Text Available The phloem is the conduit through which photoassimilates are distributed from autotrophic to heterotrophic tissues and is involved in the distribution of signaling molecules that coordinate plant growth and responses to the environment. Phloem function depends on the coordinate expression of a large array of genes. We have previously identified conserved motifs in upstream regions of the Arabidopsis genes, encoding the homologs of pumpkin phloem sap mRNAs, displaying expression in vascular tissues. This tissue-specific expression in Arabidopsis is predicted by the overrepresentation of GA/CT-rich motifs in gene promoters. In this work we have searched for common motifs in upstream regions of the homologous genes from plants considered to possess a primitive vascular tissue (a lycophyte, as well as from others that lack a true vascular tissue (a bryophyte, and finally from chlorophytes. Both lycophyte and bryophyte display motifs similar to those found in Arabidopsis with a significantly low E-value, while the chlorophytes showed either a different conserved motif or no conserved motif at all. These results suggest that these same genes are expressed coordinately in non- vascular plants; this coordinate expression may have been one of the prerequisites for the development of conducting tissues in plants. We have also analyzed the phylogeny of conserved proteins that may be involved in phloem function and development. The presence of CmPP16, APL, FT and YDA in chlorophytes suggests the recruitment of ancient regulatory networks for the development of the vascular tissue during evolution while OPS is a novel protein specific to vascular plants.

  6. Modulation of tyrosine hydroxylase gene expression in the central nervous system visualized by in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Berod, A.; Biguet, N.F.; Dumas, S.; Bloch, B.; Mallet, J.

    1987-03-01

    cDNA probe was used for in situ hybridization studies on histological sections through the locus coeruleus, substantia nigra, and the ventral tegmental area of the rat brain. Experimental conditions were established that yielded no background and no signal when pBR322 was used as control probe. Using the tyrosine hydroxylase probe, the authors ascertained the specificity of the labeling over catecholaminergic cells by denervation experiments and comparison of the hybridization pattern with that of immunoreactivity. The use of /sup 35/S-labeled probe enabled the hybridization signal to be resolved at the cellular level. A single injection of reserpine into the rat led to an increase of the intensity of the autoradiographic signal over the locus coeruleus area, confirming an RNA gel blot analysis. The potential of in situ hybridization to analyze patterns of modulation of gene activity as a result of nervous activity is discussed.

  7. Neighboring Genes Show Correlated Evolution in Gene Expression

    Science.gov (United States)

    Ghanbarian, Avazeh T.; Hurst, Laurence D.

    2015-01-01

    When considering the evolution of a gene’s expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. PMID:25743543

  8. SYSTEMIC IMBALANCE OF ESSENTIAL METALS AND CARDIAC GENE EXPRESSION IN RATS FOLLOWING ACUTE PULMONARY ZINC EXPOSURE

    Science.gov (United States)

    We have recently demonstrated that PM containing water-soluble zinc may cause cardiac injury following pulmonary exposure. To investigate if pulmonary zinc exposure causes systemic metal imbalance and direct cardiac effects, we intratracheally (IT) instilled male Wistar Kyoto (WK...

  9. Molecular Cloning of Phytase Gene from ASUIA279 and Its Expression in Pichia pastoris System

    Directory of Open Access Journals (Sweden)

    M. H. Mahamad Maifiah

    2011-12-01

    Full Text Available Phytases catalyze the hydrolysis of phytate (myo-inositol hexakisphosphate, one of the major storage form of phosphate in plants, with subsequent release of myo-inositol, phosphate and phytate-bound minerals. Non-ruminant animals such as chicken, swine and fish can't use the organic phosphorus and minerals from their diet because there is no phytase activity in their digestive tract. Phytate degrading enzyme is added to the animal feed diet to improve phosphorus availability from the dietary phytate and at the same time this lessen the phosphate pollution level in areas of intensive animal production as the phosphate would not be excreted out to the environment. ASUIA279, a bacterial strain isolated from Malaysian soil has potentially shown good phytase activity. In the present work, the gene encoding for phytase has been amplified from the plasmid DNA of recombinant ASUIA279(5 obtained from a previous study (unpublished data by a polymerase chain reaction (PCR methodology. The amplified phytase gene was extracted, purified then cloned into the pPICZA plasmid and transformed into Pichia pastorisX-33 strain for enzyme production.ABSTRAK: Fitase pemangkinan hidrolisis fitat (myo-inositol hexakisphosphate, merupakan salah satu cara penyimpanan utama fosfat dalam tumbuhan, dengan pelepasan berturut myo-inositol , fosfat dan galian terikat fitat. Haiwan bukan ruminan seperti ayam, khinzir dan ikan tidak dapat memanfaatkan fosforus organik serta galian yang diperolehi daripada makanan kerana tidak mempunyai aktiviti fitase di dalam saluran pencernaan mereka. Enzim pengecilan fitase dicampurkan ke dalam pemakanan haiwan untuk mempertingkatkan keperolehan fosforus dari fitat diet. Pada masa yang sama ia dapat mengurangkan tahap pencemaran fosfat di kawasan yang terdapat penternakan haiwan secara intensif agar fosfat tidak dikumuhkan ke persekitaran. ASUIA279, satu strain bakteria yang diasingkan daripada tanih di Malaysiamenunjukkan aktiviti fitase

  10. Gene Expression in Trypanosomatid Parasites

    Directory of Open Access Journals (Sweden)

    Santiago Martínez-Calvillo

    2010-01-01

    Full Text Available The parasites Leishmania spp., Trypanosoma brucei, and Trypanosoma cruzi are the trypanosomatid protozoa that cause the deadly human diseases leishmaniasis, African sleeping sickness, and Chagas disease, respectively. These organisms possess unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes and trans-splicing. Little is known about either the DNA sequences or the proteins that are involved in the initiation and termination of transcription in trypanosomatids. In silico analyses of the genome databases of these parasites led to the identification of a small number of proteins involved in gene expression. However, functional studies have revealed that trypanosomatids have more general transcription factors than originally estimated. Many posttranslational histone modifications, histone variants, and chromatin modifying enzymes have been identified in trypanosomatids, and recent genome-wide studies showed that epigenetic regulation might play a very important role in gene expression in this group of parasites. Here, we review and comment on the most recent findings related to transcription initiation and termination in trypanosomatid protozoa.

  11. Synthetic promoter libraries- tuning of gene expression

    DEFF Research Database (Denmark)

    Hammer, Karin; Mijakovic, Ivan; Jensen, Peter Ruhdal

    2006-01-01

    The study of gene function often requires changing the expression of a gene and evaluating the consequences. In principle, the expression of any given gene can be modulated in a quasi-continuum of discrete expression levels but the traditional approaches are usually limited to two extremes: gene ...

  12. Optogenetics for gene expression in mammalian cells.

    Science.gov (United States)

    Müller, Konrad; Naumann, Sebastian; Weber, Wilfried; Zurbriggen, Matias D

    2015-02-01

    Molecular switches that are controlled by chemicals have evolved as central research instruments in mammalian cell biology. However, these tools are limited in terms of their spatiotemporal resolution due to freely diffusing inducers. These limitations have recently been addressed by the development of optogenetic, genetically encoded, and light-responsive tools that can be controlled with the unprecedented spatiotemporal precision of light. In this article, we first provide a brief overview of currently available optogenetic tools that have been designed to control diverse cellular processes. Then, we focus on recent developments in light-controlled gene expression technologies and provide the reader with a guideline for choosing the most suitable gene expression system.

  13. Timing of gene expression from different genetic systems in shaping leucine and isoleucine contents of rapeseed (Brassica napus L.) meal

    Indian Academy of Sciences (India)

    Guo Lin Chen; Jian Guo Wu; Murali-Tottekkaad Variath; Chun Hai Shi

    2011-12-01

    Experiments were conducted on rapeseed (Brassica napus L.) using a diallel design with nine parents: Youcai 601, Double 20-4, Huashuang 3, Gaoyou 605, Zhongyou 821, Eyouchangjia, Zhong R-888, Tower and Zheshuang 72. The seed developmental process was divided into five stages, namely initial (days 1–15 after flowering), early (days 16–22 after flowering), middle (days 23–29), late (days 30–36), and maturing (days 37–43) developmental stages. The variation of dynamic genetic effects for leucine and isoleucine contents of rapeseed meal was analysed at five developmental stages, across different environments using the genetic models with time-dependent measures. The results from unconditional and conditional analyses indicated that the expression of diploid embryo, cytoplasmic and diploid maternal plant genes were important for leucine and isoleucine contents at different developmental stages of rapeseed, particularly at the initial and early developmental stages. Among different genetic systems, nutrition quality traits were mainly controlled by the accumulative or net maternal main effects and their GE interaction effects, except at maturity when the net diploid embryo effects were larger. The expression of genes was affected by the environmental conditions on 15, 22, 29 or 36 days after flowering, but was more stable at mature stage. For the isoleucine content the narrow-sense heritabilities on 15, 22, 29, 36, and 43 days after flowering were 43.0, 65.7, 60.1, 65.5 and 78.2%, respectively, while for the leucine content the corresponding narrow-sense heritabilities were relatively smaller. The interaction heritabilities were more important than the general heritabilities at the first three developmental times. The improvement for isoleucine content could be achieved by selection based on the higher narrow-sense heritabilities. Various genetic systems exhibited genetic correlations among the developmental times or leucine and isoleucine contents. A

  14. Pim-1 kinase inhibits the activation of reporter gene expression in Elk-1 and c-Fos reporting systems but not the endogenous gene expression: an artifact of the reporter gene assay by transient co-transfection

    Directory of Open Access Journals (Sweden)

    Yan B.

    2006-01-01

    Full Text Available We have studied the molecular mechanism and signal transduction of pim-1, an oncogene encoding a serine-threonine kinase. This is a true oncogene which prolongs survival and inhibits apoptosis of hematopoietic cells. In order to determine whether the effects of Pim-1 occur by regulation of the mitogen-activated protein kinase pathway, we used a transcriptional reporter assay by transient co-transfection as a screening method. In this study, we found that Pim-1 inhibited the Elk-1 and NFkappaB transcriptional activities induced by activation of the mitogen-activated protein kinase cascade in reporter gene assays. However, Western blots showed that the induction of Elk-1-regulated expression of endogenous c-Fos was not affected by Pim-1. The phosphorylation and activation of neither Erk1/2 nor Elk-1 was influenced by Pim-1. Also, in the gel shift assay, the pattern of endogenous NFkappaB binding to its probe was not changed in any manner by Pim-1. These data indicate that Pim-1 does not regulate the activation of Erk1/2, Elk-1 or NFkappaB. These contrasting results suggest a pitfall of the transient co-transfection reporter assay in analyzing the regulation of transcription factors outside of the chromosome context. It ensures that results from reporter gene expression assay should be verified by study of endogenous gene expression.

  15. Gene polymorphism and HLA-G expression in patients with childhood-onset systemic lupus erythematosus: A pilot study.

    Science.gov (United States)

    Cavalcanti, A; Almeida, R; Mesquita, Z; Duarte, A L B P; Donadi, E A; Lucena-Silva, N

    2017-10-01

    Human leukocyte antigen-G (HLA-G) presents inhibitory functions in immune cells and is located in a chromosomal region associated with systemic lupus erythematosus (SLE) susceptibility. Polymorphisms in 3' untranslated region (3'UTR) of HLA-G gene may influence protein expression. To date, no study analyzing HLA-G polymorphism and expression in childhood-onset systemic lupus erythematosus (cSLE) has been conducted. Therefore, we investigated the influence of HLA-G 3'UTR polymorphisms in 50 cSLE patients and 144 healthy controls. For the expression analysis, the control group included 26 healthy individuals. No significant difference in allele, genotype, and haplotype frequencies was observed between patients and control group. However, both the 14 bp deletion allele (odds ratio [OR] = 2.76, 95% confidence interval [CI] = 1.17-6.52, P = .028) and the 14 bp deletion-deletion genotype (OR = 8.00, 95% CI = 1.57-40.65, P = .006) showed an association with lupus nephritis. After Bonferroni correction, none P-value remained statistically significant. Regarding HLA-G expression, no significant difference was observed between plasma levels of cSLE patients (56.02 U/mL, interquartile range [IQR] = 37.54-75.41) and control group (49.2 U/mL, IQR = 27.84-154.4, P = .952). However, when the patients were stratified according to clinical manifestations, patients with hematological manifestations showed a lower plasma concentration of soluble HLA-G (sHLA-G) (47.08 U/mL, IQR = 34.15-61.56) than patients with no hematological manifestations (65.26 U/mL, IQR = 47.69-102.60, P = .013). These results suggest that HLA-G polymorphism has small effect on cSLE susceptibility and that sHLA-G may be involved in the pathogenesis of the disease. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16.  Prokaryotic expression systems

    Directory of Open Access Journals (Sweden)

    Dorota Porowińska

    2013-03-01

    Full Text Available For overproduction of recombinant proteins both eukaryotic and prokaryotic expression systems are used. Choosing the right system depends, among other things, on the growth rate and culture of host cells, level of the target gene expression and posttranslational processing of the synthesized protein. Regardless of the type of expression system, its basic elements are the vector and the expression host.The most widely used system for protein overproduction, both on a laboratory and industrial scale, is the prokaryotic system. This system is based primarily on the bacteria E. coli, although increasingly often Bacillus species are used. The prokaryotic system allows one to obtain large quantities of recombinant proteins in a short time. A simple and inexpensive bacterial cell culture and well-known mechanisms of transcription and translation facilitate the use of these microorganisms. The simplicity of genetic modifications and the availability of many bacterial mutants are additional advantages of the prokaryotic system. In this article we characterize the structural elements of prokaryotic expression vectors. Also strategies for preparation of the target protein gene that increase productivity, facilitate detection and purification of recombinant protein and provide its activity are discussed. Bacterial strains often used as host cells in expression systems as well as the potential location of heterologous proteins are characterized.Knowledge of the basic elements of the prokaryotic expression system allows for production of biologically active proteins in a short time and in satisfactory quantities. 

  17. Killing two birds with one stone: Model plant systems as a tool to teach the fundamental concepts of gene expression while analyzing biological data.

    Science.gov (United States)

    Makarevitch, Irina; Martinez-Vaz, Betsy

    2017-01-01

    Plants are ideal systems to teach core biology concepts due to their unique physiological and developmental features. Advances in DNA sequencing technology and genomics have allowed scientists to generate genome sequences and transcriptomics data for numerous model plant species. This information is publicly available and presents a valuable tool to introduce undergraduate students to the fundamental concepts of gene expression in the context of modern quantitative biology and bioinformatics. Modern biology classrooms must provide authentic research experiences to allow developing core competencies such as scientific inquiry, critical interpretation of experimental results, and quantitative analyses of large dataset using computational approaches. Recent educational research has shown that undergraduate students struggle when connecting gene expression concepts to classic genetics, phenotypic analyses, and overall flow of biological information in living organisms, suggesting that novel approaches are necessary to enhance learning of gene expression and regulation. This review describes different strategies and resources available to instructors willing to incorporate authentic research experiences, genomic tools, and bioinformatics analyses when teaching transcriptional regulation and gene expression in undergraduate courses. A variety of laboratory exercises and pedagogy materials developed to teach gene expression using plants are discussed. This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks, edited by Dr. Erich Grotewold and Dr. Nathan Springer. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Mechanisms of information decoding in a cascade system of gene expression

    Science.gov (United States)

    Wang, Haohua; Yuan, Zhanjiang; Liu, Peijiang; Zhou, Tianshou

    2016-05-01

    Biotechnology advances have allowed investigation of heterogeneity of cellular responses to stimuli on the single-cell level. Functionally, this heterogeneity can compromise cellular responses to environmental signals, and it can also enlarge the repertoire of possible cellular responses and hence increase the adaptive nature of cellular behaviors. However, the mechanism of how this response heterogeneity is generated remains elusive. Here, by systematically analyzing a representative cellular signaling system, we show that (1) the upstream activator always amplifies the downstream burst frequency (BF) but the noiseless activator performs better than the noisy one, remarkably for small or moderate input signal strengths, and the repressor always reduces the downstream BF but the difference in the reducing effect between noiseless and noise repressors is very small; (2) both the downstream burst size and mRNA mean are a monotonically increasing function of the activator strength but a monotonically decreasing function of the repressor strength; (3) for repressor-type input, there is a noisy signal strength such that the downstream mRNA noise arrives at an optimal level, but for activator-type input, the output noise intensity is fundamentally a monotonically decreasing function of the input strength. Our results reveal the essential mechanisms of both signal information decoding and cellular response heterogeneity, whereas our analysis provides a paradigm for analyzing dynamics of noisy biochemical signaling systems.

  19. Gene Expression Commons: an open platform for absolute gene expression profiling.

    Directory of Open Access Journals (Sweden)

    Jun Seita

    Full Text Available Gene expression profiling using microarrays has been limited to comparisons of gene expression between small numbers of samples within individual experiments. However, the unknown and variable sensitivities of each probeset have rendered the absolute expression of any given gene nearly impossible to estimate. We have overcome this limitation by using a very large number (>10,000 of varied microarray data as a common reference, so that statistical attributes of each probeset, such as the dynamic range and threshold between low and high expression, can be reliably discovered through meta-analysis. This strategy is implemented in a web-based platform named "Gene Expression Commons" (https://gexc.stanford.edu/ which contains data of 39 distinct highly purified mouse hematopoietic stem/progenitor/differentiated cell populations covering almost the entire hematopoietic system. Since the Gene Expression Commons is designed as an open platform, investigators can explore the expression level of any gene, search by expression patterns of interest, submit their own microarray data, and design their own working models representing biological relationship among samples.

  20. Gene Expression Commons: an open platform for absolute gene expression profiling.

    Science.gov (United States)

    Seita, Jun; Sahoo, Debashis; Rossi, Derrick J; Bhattacharya, Deepta; Serwold, Thomas; Inlay, Matthew A; Ehrlich, Lauren I R; Fathman, John W; Dill, David L; Weissman, Irving L

    2012-01-01

    Gene expression profiling using microarrays has been limited to comparisons of gene expression between small numbers of samples within individual experiments. However, the unknown and variable sensitivities of each probeset have rendered the absolute expression of any given gene nearly impossible to estimate. We have overcome this limitation by using a very large number (>10,000) of varied microarray data as a common reference, so that statistical attributes of each probeset, such as the dynamic range and threshold between low and high expression, can be reliably discovered through meta-analysis. This strategy is implemented in a web-based platform named "Gene Expression Commons" (https://gexc.stanford.edu/) which contains data of 39 distinct highly purified mouse hematopoietic stem/progenitor/differentiated cell populations covering almost the entire hematopoietic system. Since the Gene Expression Commons is designed as an open platform, investigators can explore the expression level of any gene, search by expression patterns of interest, submit their own microarray data, and design their own working models representing biological relationship among samples.

  1. Homotypic clusters of transcription factor binding sites: A model system for understanding the physical mechanics of gene expression

    Directory of Open Access Journals (Sweden)

    Daphne Ezer

    2014-07-01

    Full Text Available The organization of binding sites in cis-regulatory elements (CREs can influence gene expression through a combination of physical mechanisms, ranging from direct interactions between TF molecules to DNA looping and transient chromatin interactions. The study of simple and common building blocks in promoters and other CREs allows us to dissect how all of these mechanisms work together. Many adjacent TF binding sites for the same TF species form homotypic clusters, and these CRE architecture building blocks serve as a prime candidate for understanding interacting transcriptional mechanisms. Homotypic clusters are prevalent in both bacterial and eukaryotic genomes, and are present in both promoters as well as more distal enhancer/silencer elements. Here, we review previous theoretical and experimental studies that show how the complexity (number of binding sites and spatial organization (distance between sites and overall distance from transcription start sites of homotypic clusters influence gene expression. In particular, we describe how homotypic clusters modulate the temporal dynamics of TF binding, a mechanism that can affect gene expression, but which has not yet been sufficiently characterized. We propose further experiments on homotypic clusters that would be useful in developing mechanistic models of gene expression.

  2. Homotypic clusters of transcription factor binding sites: A model system for understanding the physical mechanics of gene expression.

    Science.gov (United States)

    Ezer, Daphne; Zabet, Nicolae Radu; Adryan, Boris

    2014-07-01

    The organization of binding sites in cis-regulatory elements (CREs) can influence gene expression through a combination of physical mechanisms, ranging from direct interactions between TF molecules to DNA looping and transient chromatin interactions. The study of simple and common building blocks in promoters and other CREs allows us to dissect how all of these mechanisms work together. Many adjacent TF binding sites for the same TF species form homotypic clusters, and these CRE architecture building blocks serve as a prime candidate for understanding interacting transcriptional mechanisms. Homotypic clusters are prevalent in both bacterial and eukaryotic genomes, and are present in both promoters as well as more distal enhancer/silencer elements. Here, we review previous theoretical and experimental studies that show how the complexity (number of binding sites) and spatial organization (distance between sites and overall distance from transcription start sites) of homotypic clusters influence gene expression. In particular, we describe how homotypic clusters modulate the temporal dynamics of TF binding, a mechanism that can affect gene expression, but which has not yet been sufficiently characterized. We propose further experiments on homotypic clusters that would be useful in developing mechanistic models of gene expression.

  3. Global gene expression profiling in infants with acute respiratory syncytial virus broncholitis demonstrates systemic activation of interferon signaling networks

    Science.gov (United States)

    Respiratory syncytial virus (RSV) is a leading cause of pediatric lower respiratory tract infections and has a high impact on pediatric emergency department utilization. Variation in host response may influence the pathogenesis and disease severity. We evaluated global gene expression profiles to be...

  4. Expression analysis of the CLCA gene family in mouse and human with emphasis on the nervous system

    NARCIS (Netherlands)

    M. Piirsoo (Marko); D. Meijer (Daniëlle); T. Timmusk (Tnis)

    2009-01-01

    textabstractBackground. Members of the calcium-activated chloride channel (CLCA) gene family have been suggested to possess a variety of functions including cell adhesion and tumor suppression. Expression of CLCA family members has mostly been analyzed in non-neural tissues. Here we describe the exp

  5. A systems biology analysis of the changes in gene expression via silencing of HPV-18 E1 expression in HeLa cells.

    Science.gov (United States)

    Castillo, Andres; Wang, Lu; Koriyama, Chihaya; Eizuru, Yoshito; Jordan, King; Akiba, Suminori

    2014-10-01

    Previous studies have reported the detection of a truncated E1 mRNA generated from HPV-18 in HeLa cells. Although it is unclear whether a truncated E1 protein could function as a replicative helicase for viral replication, it would still retain binding sites for potential interactions with different host cell proteins. Furthermore, in this study, we found evidence in support of expression of full-length HPV-18 E1 mRNA in HeLa cells. To determine whether interactions between E1 and cellular proteins play an important role in cellular processes other than viral replication, genome-wide expression profiles of HPV-18 positive HeLa cells were compared before and after the siRNA knockdown of E1 expression. Differential expression and gene set enrichment analysis uncovered four functionally related sets of genes implicated in host defence mechanisms against viral infection. These included the toll-like receptor, interferon and apoptosis pathways, along with the antiviral interferon-stimulated gene set. In addition, we found that the transcriptional coactivator E1A-binding protein p300 (EP300) was downregulated, which is interesting given that EP300 is thought to be required for the transcription of HPV-18 genes in HeLa cells. The observed changes in gene expression produced via the silencing of HPV-18 E1 expression in HeLa cells indicate that in addition to its well-known role in viral replication, the E1 protein may also play an important role in mitigating the host's ability to defend against viral infection.

  6. Classification with binary gene expressions

    OpenAIRE

    Tuna, Salih; Niranjan, Mahesan

    2009-01-01

    Microarray gene expression measurements are reported, used and archived usually to high numerical precision. However, properties of mRNA molecules, such as their low stability and availability in small copy numbers, and the fact that measurements correspond to a population of cells, rather than a single cell, makes high precision meaningless. Recent work shows that reducing measurement precision leads to very little loss of information, right down to binary levels. In this paper we show how p...

  7. Expression of signal transduction system encoding genes of Yersinia pseudotuberculosis IP32953 at 28°C and 3°C.

    Directory of Open Access Journals (Sweden)

    Eveliina Palonen

    Full Text Available Yersinia pseudotuberculosis is a significant psychrotrophic food pathogen whose cold tolerance mechanisms are poorly understood. Signal transduction systems serve to monitor the environment, but no systematic investigation of their role at cold temperatures in Y. pseudotuberculosis has yet been undertaken. The relative expression levels of 54 genes predicted to encode proteins belonging to signal transduction systems in Y. pseudotuberculosis IP32953 were determined at 28°C and 3°C by quantitative real-time reverse transcription-PCR. The relative expression levels of 44 genes were significantly (p<0.05 higher at 3°C than at 28°C. Genes encoding the two-component system CheA/CheY had the highest relative expression levels at 3°C. Mutational analysis revealed that cheA is important for growth and motility at 3°C. The relative expression level of one gene, rssB, encoding an RpoS regulator, was significantly (p<0.05 lower at 3°C than at 28°C. The results suggest that several signal transduction systems might be used during growth at low temperature, and at least, CheA/CheY two-component system is important for low-temperature growth.

  8. The Gene Expression Omnibus database

    Science.gov (United States)

    Clough, Emily; Barrett, Tanya

    2016-01-01

    The Gene Expression Omnibus (GEO) database is an international public repository that archives and freely distributes high-throughput gene expression and other functional genomics data sets. Created in 2000 as a worldwide resource for gene expression studies, GEO has evolved with rapidly changing technologies and now accepts high-throughput data for many other data applications, including those that examine genome methylation, chromatin structure, and genome–protein interactions. GEO supports community-derived reporting standards that specify provision of several critical study elements including raw data, processed data, and descriptive metadata. The database not only provides access to data for tens of thousands of studies, but also offers various Web-based tools and strategies that enable users to locate data relevant to their specific interests, as well as to visualize and analyze the data. This chapter includes detailed descriptions of methods to query and download GEO data and use the analysis and visualization tools. The GEO homepage is at http://www.ncbi.nlm.nih.gov/geo/. PMID:27008011

  9. Global expression differences and tissue specific expression differences in rice evolution result in two contrasting types of differentially expressed genes

    KAUST Repository

    Horiuchi, Youko

    2015-12-23

    Background Since the development of transcriptome analysis systems, many expression evolution studies characterized evolutionary forces acting on gene expression, without explicit discrimination between global expression differences and tissue specific expression differences. However, different types of gene expression alteration should have different effects on an organism, the evolutionary forces that act on them might be different, and different types of genes might show different types of differential expression between species. To confirm this, we studied differentially expressed (DE) genes among closely related groups that have extensive gene expression atlases, and clarified characteristics of different types of DE genes including the identification of regulating loci for differential expression using expression quantitative loci (eQTL) analysis data. Results We detected differentially expressed (DE) genes between rice subspecies in five homologous tissues that were verified using japonica and indica transcriptome atlases in public databases. Using the transcriptome atlases, we classified DE genes into two types, global DE genes and changed-tissues DE genes. Global type DE genes were not expressed in any tissues in the atlas of one subspecies, however changed-tissues type DE genes were expressed in both subspecies with different tissue specificity. For the five tissues in the two japonica-indica combinations, 4.6 ± 0.8 and 5.9 ± 1.5 % of highly expressed genes were global and changed-tissues DE genes, respectively. Changed-tissues DE genes varied in number between tissues, increasing linearly with the abundance of tissue specifically expressed genes in the tissue. Molecular evolution of global DE genes was rapid, unlike that of changed-tissues DE genes. Based on gene ontology, global and changed-tissues DE genes were different, having no common GO terms. Expression differences of most global DE genes were regulated by cis-eQTLs. Expression

  10. Gene expression throughout a vertebrate's embryogenesis

    Directory of Open Access Journals (Sweden)

    Hinton David E

    2011-02-01

    Full Text Available Abstract Background Describing the patterns of gene expression during embryonic development has broadened our understanding of the processes and patterns that define morphogenesis. Yet gene expression patterns have not been described throughout vertebrate embryogenesis. This study presents statistical analyses of gene expression during all 40 developmental stages in the teleost Fundulus heteroclitus using four biological replicates per stage. Results Patterns of gene expression for 7,000 genes appear to be important as they recapitulate developmental timing. Among the 45% of genes with significant expression differences between pairs of temporally adjacent stages, significant differences in gene expression vary from as few as five to more than 660. Five adjacent stages have disproportionately more significant changes in gene expression (> 200 genes relative to other stages: four to eight and eight to sixteen cell stages, onset of circulation, pre and post-hatch, and during complete yolk absorption. The fewest differences among adjacent stages occur during gastrulation. Yet, at stage 16, (pre-mid-gastrulation the largest number of genes has peak expression. This stage has an over representation of genes in oxidative respiration and protein expression (ribosomes, translational genes and proteases. Unexpectedly, among all ribosomal genes, both strong positive and negative correlations occur. Similar correlated patterns of expression occur among all significant genes. Conclusions These data provide statistical support for the temporal dynamics of developmental gene expression during all stages of vertebrate development.

  11. A chromosomally encoded T7 RNA polymerase-dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single-cell level.

    Science.gov (United States)

    Kortmann, Maike; Kuhl, Vanessa; Klaffl, Simon; Bott, Michael

    2015-03-01

    Corynebacterium glutamicum has become a favourite model organism in white biotechnology. Nevertheless, only few systems for the regulatable (over)expression of homologous and heterologous genes are currently available, all of which are based on the endogenous RNA polymerase. In this study, we developed an isopropyl-β-D-1-thiogalactopyranosid (IPTG)-inducible T7 expression system in the prophage-free strain C. glutamicum MB001. For this purpose, part of the DE3 region of Escherichia coli BL21(DE3) including the T7 RNA polymerase gene 1 under control of the lacUV5 promoter was integrated into the chromosome, resulting in strain MB001(DE3). Furthermore, the expression vector pMKEx2 was constructed allowing cloning of target genes under the control of the T7lac promoter. The properties of the system were evaluated using eyfp as heterologous target gene. Without induction, the system was tightly repressed, resulting in a very low specific eYFP fluorescence (= fluorescence per cell density). After maximal induction with IPTG, the specific fluorescence increased 450-fold compared with the uninduced state and was about 3.5 times higher than in control strains expressing eyfp under control of the IPTG-induced tac promoter with the endogenous RNA polymerase. Flow cytometry revealed that T7-based eyfp expression resulted in a highly uniform population, with 99% of all cells showing high fluorescence. Besides eyfp, the functionality of the corynebacterial T7 expression system was also successfully demonstrated by overexpression of the C. glutamicum pyk gene for pyruvate kinase, which led to an increase of the specific activity from 2.6 to 135 U mg(-1). It thus presents an efficient new tool for protein overproduction, metabolic engineering and synthetic biology approaches with C. glutamicum.

  12. Salicylic acid mediates antioxidant defense system and ABA pathway related gene expression in Oryza sativa against quinclorac toxicity.

    Science.gov (United States)

    Wang, Jian; Lv, Mengting; Islam, Faisal; Gill, Rafaqat A; Yang, Chong; Ali, Basharat; Yan, Guijun; Zhou, Weijun

    2016-11-01

    The auxin herbicide quinclorac is widely used for controlling weeds in transplanted and direct-seeded rice fields. However, its phytotoxic responses on rice are still unknown. Therefore, in the present investigation we studied the effects of different concentrations (0, 0.1 and 0.5g/L) of quinclorac herbicide on the physiological and biochemical changes of two rice cultivars (XS 134 and ZJ 88) and further analyzed the ameliorating role of salicylic acid (SA) on quinclorac toxicity in rice plants. The results revealed that exogenous application of SA significantly increased plant biomass and total chlorophyll contents in herbicide stressed plants. The lipid peroxidation and ROS (H2O2, O2(-.), (-)OH) production were significantly increased in roots and leaves of both rice cultivars under quinclorac stress, demonstrating an oxidative burst in rice plants. Whereas, application of SA significantly lowered ROS contents under quinclorac stress. Further, exogenous SA treatment significantly modulated antioxidant enzymes and enhanced GSH concentration in stress plants. Anatomical observations of leaf and root revealed that herbicide affected internal structures, while SA played a vital role in protection from toxic effects. Expression analysis of stress hormone ABA genes (OsABA8oxs, OsNCEDs) revealed that quinclorac application enhanced stress condition in cultivar ZJ 88, while SA treatment downregulated ABA genes more in cultivar XS 134, which correlated with the enhanced tolerance to quinclorac induced oxidative stress in this cultivar. The present study delineated that SA played a critical role under quinclorac stress in both rice cultivars by regulating antioxidant defense system, reducing ROS formation and preventing the degradation of internal cell organelles.

  13. Antisense expression increases gene expression variability and locus interdependency

    OpenAIRE

    Xu, Zhenyu; Wei, Wu; Gagneur, Julien; Clauder-Münster, Sandra; Smolik, Miłosz; Huber, Wolfgang; Steinmetz, Lars M.

    2011-01-01

    Genome-wide transcription profiling has revealed extensive expression of non-coding RNAs antisense to genes, yet their functions, if any, remain to be understood. In this study, we perform a systematic analysis of sense–antisense expression in response to genetic and environmental changes in yeast. We find that antisense expression is associated with genes of larger expression variability. This is characterized by more ‘switching off' at low levels of expression for genes with antisense compa...

  14. Stochastic gene expression conditioned on large deviations

    Science.gov (United States)

    Horowitz, Jordan M.; Kulkarni, Rahul V.

    2017-06-01

    The intrinsic stochasticity of gene expression can give rise to large fluctuations and rare events that drive phenotypic variation in a population of genetically identical cells. Characterizing the fluctuations that give rise to such rare events motivates the analysis of large deviations in stochastic models of gene expression. Recent developments in non-equilibrium statistical mechanics have led to a framework for analyzing Markovian processes conditioned on rare events and for representing such processes by conditioning-free driven Markovian processes. We use this framework, in combination with approaches based on queueing theory, to analyze a general class of stochastic models of gene expression. Modeling gene expression as a Batch Markovian Arrival Process (BMAP), we derive exact analytical results quantifying large deviations of time-integrated random variables such as promoter activity fluctuations. We find that the conditioning-free driven process can also be represented by a BMAP that has the same form as the original process, but with renormalized parameters. The results obtained can be used to quantify the likelihood of large deviations, to characterize system fluctuations conditional on rare events and to identify combinations of model parameters that can give rise to dynamical phase transitions in system dynamics.

  15. Induced fungal resistance to insect grazing: reciprocal fitness consequences and fungal gene expression in the Drosophila-Aspergillus model system.

    Directory of Open Access Journals (Sweden)

    Silvia Caballero Ortiz

    Full Text Available BACKGROUND: Fungi are key dietary resources for many animals. Fungi, in consequence, have evolved sophisticated physical and chemical defences for repelling and impairing fungivores. Expression of such defences may entail costs, requiring diversion of energy and nutrients away from fungal growth and reproduction. Inducible resistance that is mounted after attack by fungivores may allow fungi to circumvent the potential costs of defence when not needed. However, no information exists on whether fungi display inducible resistance. We combined organism and fungal gene expression approaches to investigate whether fungivory induces resistance in fungi. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that grazing by larval fruit flies, Drosophila melanogaster, induces resistance in the filamentous mould, Aspergillus nidulans, to subsequent feeding by larvae of the same insect. Larval grazing triggered the expression of various putative fungal resistance genes, including the secondary metabolite master regulator gene laeA. Compared to the severe pathological effects of wild type A. nidulans, which led to 100% insect mortality, larval feeding on a laeA loss-of-function mutant resulted in normal insect development. Whereas the wild type fungus recovered from larval grazing, larvae eradicated the chemically deficient mutant. In contrast, mutualistic dietary yeast, Saccharomyces cerevisiae, reached higher population densities when exposed to Drosophila larval feeding. CONCLUSIONS/SIGNIFICANCE: Our study presents novel evidence that insect grazing is capable of inducing resistance to further grazing in a filamentous fungus. This phenotypic shift in resistance to fungivory is accompanied by changes in the expression of genes involved in signal transduction, epigenetic regulation and secondary metabolite biosynthesis pathways. Depending on reciprocal insect-fungus fitness consequences, fungi may be selected for inducible resistance to maintain high fitness in

  16. Industrial-scale production and purification of a heterologous protein in Lactococcus lactis using the nisin-controlled gene expression system NICE: The case of lysostaphin

    Directory of Open Access Journals (Sweden)

    Floris Esther

    2005-05-01

    Full Text Available Abstract Background The NIsin-Controlled gene Expression system NICE of Lactococcus lactis is one of the most widespread used expression systems of Gram-positive bacteria. It is used in more than 100 laboratories for laboratory-scale gene expression experiments. However, L. lactis is also a micro-organism with a large biotechnological potential. Therefore, the aim of this study was to test whether protein production in L. lactis using the NICE system can also effectively be performed at the industrial-scale of fermentation. Results Lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus from S. simulans biovar. Staphylolyticus, was used as a model system. Food-grade lysostaphin expression constructs in L. lactis were grown at 1L-, 300-L and 3000-L scale and induced with nisin for lysostaphin production. The induction process was equally effective at all scales and yields of about 100 mg/L were obtained. Up-scaling was easy and required no specific effort. Furthermore, we describe a simple and effective way of downstream processing to obtain a highly purified lysostaphin, which has been used for clinical phase I trials. Conclusion This is the first example that shows that nisin-regulated gene expression in L. lactis can be used at industrial scale to produce large amounts of a target protein, such as lysostaphin. Downstream processing was simple and in a few steps produced a highly purified and active enzyme.

  17. Regulation of methane genes and genome expression

    Energy Technology Data Exchange (ETDEWEB)

    John N. Reeve

    2009-09-09

    At the start of this project, it was known that methanogens were Archaeabacteria (now Archaea) and were therefore predicted to have gene expression and regulatory systems different from Bacteria, but few of the molecular biology details were established. The goals were then to establish the structures and organizations of genes in methanogens, and to develop the genetic technologies needed to investigate and dissect methanogen gene expression and regulation in vivo. By cloning and sequencing, we established the gene and operon structures of all of the “methane” genes that encode the enzymes that catalyze methane biosynthesis from carbon dioxide and hydrogen. This work identified unique sequences in the methane gene that we designated mcrA, that encodes the largest subunit of methyl-coenzyme M reductase, that could be used to identify methanogen DNA and establish methanogen phylogenetic relationships. McrA sequences are now the accepted standard and used extensively as hybridization probes to identify and quantify methanogens in environmental research. With the methane genes in hand, we used northern blot and then later whole-genome microarray hybridization analyses to establish how growth phase and substrate availability regulated methane gene expression in Methanobacterium thermautotrophicus ΔH (now Methanothermobacter thermautotrophicus). Isoenzymes or pairs of functionally equivalent enzymes catalyze several steps in the hydrogen-dependent reduction of carbon dioxide to methane. We established that hydrogen availability determine which of these pairs of methane genes is expressed and therefore which of the alternative enzymes is employed to catalyze methane biosynthesis under different environmental conditions. As were unable to establish a reliable genetic system for M. thermautotrophicus, we developed in vitro transcription as an alternative system to investigate methanogen gene expression and regulation. This led to the discovery that an archaeal protein

  18. Gene expression analysis identifies global gene dosage sensitivity in cancer

    DEFF Research Database (Denmark)

    Fehrmann, Rudolf S. N.; Karjalainen, Juha M.; Krajewska, Malgorzata;

    2015-01-01

    expression. We reanalyzed 77,840 expression profiles and observed a limited set of 'transcriptional components' that describe well-known biology, explain the vast majority of variation in gene expression and enable us to predict the biological function of genes. On correcting expression profiles...... for these components, we observed that the residual expression levels (in 'functional genomic mRNA' profiling) correlated strongly with copy number. DNA copy number correlated positively with expression levels for 99% of all abundantly expressed human genes, indicating global gene dosage sensitivity. By applying...

  19. Noise in eukaryotic gene expression

    Science.gov (United States)

    Blake, William J.; KÆrn, Mads; Cantor, Charles R.; Collins, J. J.

    2003-04-01

    Transcription in eukaryotic cells has been described as quantal, with pulses of messenger RNA produced in a probabilistic manner. This description reflects the inherently stochastic nature of gene expression, known to be a major factor in the heterogeneous response of individual cells within a clonal population to an inducing stimulus. Here we show in Saccharomyces cerevisiae that stochasticity (noise) arising from transcription contributes significantly to the level of heterogeneity within a eukaryotic clonal population, in contrast to observations in prokaryotes, and that such noise can be modulated at the translational level. We use a stochastic model of transcription initiation specific to eukaryotes to show that pulsatile mRNA production, through reinitiation, is crucial for the dependence of noise on transcriptional efficiency, highlighting a key difference between eukaryotic and prokaryotic sources of noise. Furthermore, we explore the propagation of noise in a gene cascade network and demonstrate experimentally that increased noise in the transcription of a regulatory protein leads to increased cell-cell variability in the target gene output, resulting in prolonged bistable expression states. This result has implications for the role of noise in phenotypic variation and cellular differentiation.

  20. Identification of four soybean reference genes for gene expression normalization

    Science.gov (United States)

    Gene expression analysis requires the use of reference genes stably expressed independently of specific tissues or environmental conditions. Housekeeping genes (e.g., actin, tubulin, ribosomal, polyubiquitin and elongation factor 1-alpha) are commonly used as reference genes with the assumption tha...

  1. The two-component system CpxR/A represses the expression of Salmonella virulence genes by affecting the stability of the transcriptional regulator HilD

    Science.gov (United States)

    De la Cruz, Miguel A.; Pérez-Morales, Deyanira; Palacios, Irene J.; Fernández-Mora, Marcos; Calva, Edmundo; Bustamante, Víctor H.

    2015-01-01

    Salmonella enterica can cause intestinal or systemic infections in humans and animals mainly by the presence of pathogenicity islands SPI-1 and SPI-2, containing 39 and 44 genes, respectively. The AraC-like regulator HilD positively controls the expression of the SPI-1 genes, as well as many other Salmonella virulence genes including those located in SPI-2. A previous report indicates that the two-component system CpxR/A regulates the SPI-1 genes: the absence of the sensor kinase CpxA, but not the absence of its cognate response regulator CpxR, reduces their expression. The presence and absence of cell envelope stress activates kinase and phosphatase activities of CpxA, respectively, which in turn controls the level of phosphorylated CpxR (CpxR-P). In this work, we further define the mechanism for the CpxR/A-mediated regulation of SPI-1 genes. The negative effect exerted by the absence of CpxA on the expression of SPI-1 genes was counteracted by the absence of CpxR or by the absence of the two enzymes, AckA and Pta, which render acetyl-phosphate that phosphorylates CpxR. Furthermore, overexpression of the lipoprotein NlpE, which activates CpxA kinase activity on CpxR, or overexpression of CpxR, repressed the expression of SPI-1 genes. Thus, our results provide several lines of evidence strongly supporting that the absence of CpxA leads to the phosphorylation of CpxR via the AckA/Pta enzymes, which represses both the SPI-1 and SPI-2 genes. Additionally, we show that in the absence of the Lon protease, which degrades HilD, the CpxR-P-mediated repression of the SPI-1 genes is mostly lost; moreover, we demonstrate that CpxR-P negatively affects the stability of HilD and thus decreases the expression of HilD-target genes, such as hilD itself and hilA, located in SPI-1. Our data further expand the insight on the different regulatory pathways for gene expression involving CpxR/A and on the complex regulatory network governing virulence in Salmonella. PMID:26300871

  2. Endothelial Cells Expressing Endothelial and Mesenchymal Cell Gene Products in Lung Tissue From Patients With Systemic Sclerosis-Associated Interstitial Lung Disease.

    Science.gov (United States)

    Mendoza, Fabian A; Piera-Velazquez, Sonsoles; Farber, John L; Feghali-Bostwick, Carol; Jiménez, Sergio A

    2016-01-01

    To examine whether lung endothelial cells (ECs) from patients with systemic sclerosis (SSc)-associated interstitial lung disease (ILD) express mesenchymal cell-specific proteins and gene transcripts, indicative of the occurrence of endothelial-to-mesenchymal phenotypic transition (EndoMT). Lung tissue from 6 patients with SSc-associated pulmonary fibrosis was examined by histopathology and immunohistochemistry. Confocal laser microscopy was utilized to assess the simultaneous expression of EC and myofibroblast molecular markers. CD31+CD102+ ECs were isolated from the lung tissue of 2 patients with SSc-associated ILD and 2 normal control subjects, and the expression of EC and mesenchymal cell markers and other relevant genes was analyzed by quantitative polymerase chain reaction, immunofluorescence microscopy, and Western blotting. Immunohistochemical staining revealed cells expressing the EC-specific marker CD31 in the subendothelial, perivascular, and parenchymal regions of the lungs from all SSc patients. Confocal microscopy identified cells displaying simultaneous expression of von Willebrand factor and α-smooth muscle actin in small and medium-sized arterioles in the SSc lung tissue but not in normal control lungs. CD31+CD102+ ECs isolated from SSc lungs expressed high levels of mesenchymal cell-specific genes (type I collagen, type III collagen, and fibronectin), EC-specific genes (type IV collagen and VE-cadherin), profibrotic genes (transforming growth factor β1 and connective tissue growth factor), and genes encoding EndoMT-related transcription factors (TWIST1 and SNAI2). Cells coexpressing EC- and mesenchymal cell-specific molecules are present in the lungs of patients with SSc-associated ILD. CD31+CD102+ ECs isolated from SSc lungs simultaneously expressed mesenchymal cell- and EC-specific transcripts and proteins. Collectively, these observations demonstrate the occurrence of EndoMT in the lungs of patients with SSc-associated ILD. © 2016, American

  3. Extracting expression modules from perturbational gene expression compendia

    Directory of Open Access Journals (Sweden)

    Van Dijck Patrick

    2008-04-01

    Full Text Available Abstract Background Compendia of gene expression profiles under chemical and genetic perturbations constitute an invaluable resource from a systems biology perspective. However, the perturbational nature of such data imposes specific challenges on the computational methods used to analyze them. In particular, traditional clustering algorithms have difficulties in handling one of the prominent features of perturbational compendia, namely partial coexpression relationships between genes. Biclustering methods on the other hand are specifically designed to capture such partial coexpression patterns, but they show a variety of other drawbacks. For instance, some biclustering methods are less suited to identify overlapping biclusters, while others generate highly redundant biclusters. Also, none of the existing biclustering tools takes advantage of the staple of perturbational expression data analysis: the identification of differentially expressed genes. Results We introduce a novel method, called ENIGMA, that addresses some of these issues. ENIGMA leverages differential expression analysis results to extract expression modules from perturbational gene expression data. The core parameters of the ENIGMA clustering procedure are automatically optimized to reduce the redundancy between modules. In contrast to the biclusters produced by most other methods, ENIGMA modules may show internal substructure, i.e. subsets of genes with distinct but significantly related expression patterns. The grouping of these (often functionally related patterns in one module greatly aids in the biological interpretation of the data. We show that ENIGMA outperforms other methods on artificial datasets, using a quality criterion that, unlike other criteria, can be used for algorithms that generate overlapping clusters and that can be modified to take redundancy between clusters into account. Finally, we apply ENIGMA to the Rosetta compendium of expression profiles for

  4. Fluid Mechanics, Arterial Disease, and Gene Expression.

    Science.gov (United States)

    Tarbell, John M; Shi, Zhong-Dong; Dunn, Jessilyn; Jo, Hanjoong

    2014-01-01

    This review places modern research developments in vascular mechanobiology in the context of hemodynamic phenomena in the cardiovascular system and the discrete localization of vascular disease. The modern origins of this field are traced, beginning in the 1960s when associations between flow characteristics, particularly blood flow-induced wall shear stress, and the localization of atherosclerotic plaques were uncovered, and continuing to fluid shear stress effects on the vascular lining endothelial) cells (ECs), including their effects on EC morphology, biochemical production, and gene expression. The earliest single-gene studies and genome-wide analyses are considered. The final section moves from the ECs lining the vessel wall to the smooth muscle cells and fibroblasts within the wall that are fluid me chanically activated by interstitial flow that imposes shear stresses on their surfaces comparable with those of flowing blood on EC surfaces. Interstitial flow stimulates biochemical production and gene expression, much like blood flow on ECs.

  5. Expression of Signal Transduction System Encoding Genes of Yersinia pseudotuberculosis IP32953 at 28°C and 3°C

    Science.gov (United States)

    Palonen, Eveliina; Lindström, Miia; Karttunen, Reija; Somervuo, Panu; Korkeala, Hannu

    2011-01-01

    Yersinia pseudotuberculosis is a significant psychrotrophic food pathogen whose cold tolerance mechanisms are poorly understood. Signal transduction systems serve to monitor the environment, but no systematic investigation of their role at cold temperatures in Y. pseudotuberculosis has yet been undertaken. The relative expression levels of 54 genes predicted to encode proteins belonging to signal transduction systems in Y. pseudotuberculosis IP32953 were determined at 28°C and 3°C by quantitative real-time reverse transcription-PCR. The relative expression levels of 44 genes were significantly (ptwo-component system CheA/CheY had the highest relative expression levels at 3°C. Mutational analysis revealed that cheA is important for growth and motility at 3°C. The relative expression level of one gene, rssB, encoding an RpoS regulator, was significantly (psignal transduction systems might be used during growth at low temperature, and at least, CheA/CheY two-component system is important for low-temperature growth. PMID:21949852

  6. Regulation of methane genes and genome expression

    Energy Technology Data Exchange (ETDEWEB)

    John N. Reeve

    2009-09-09

    At the start of this project, it was known that methanogens were Archaeabacteria (now Archaea) and were therefore predicted to have gene expression and regulatory systems different from Bacteria, but few of the molecular biology details were established. The goals were then to establish the structures and organizations of genes in methanogens, and to develop the genetic technologies needed to investigate and dissect methanogen gene expression and regulation in vivo. By cloning and sequencing, we established the gene and operon structures of all of the “methane” genes that encode the enzymes that catalyze methane biosynthesis from carbon dioxide and hydrogen. This work identified unique sequences in the methane gene that we designated mcrA, that encodes the largest subunit of methyl-coenzyme M reductase, that could be used to identify methanogen DNA and establish methanogen phylogenetic relationships. McrA sequences are now the accepted standard and used extensively as hybridization probes to identify and quantify methanogens in environmental research. With the methane genes in hand, we used northern blot and then later whole-genome microarray hybridization analyses to establish how growth phase and substrate availability regulated methane gene expression in Methanobacterium thermautotrophicus ΔH (now Methanothermobacter thermautotrophicus). Isoenzymes or pairs of functionally equivalent enzymes catalyze several steps in the hydrogen-dependent reduction of carbon dioxide to methane. We established that hydrogen availability determine which of these pairs of methane genes is expressed and therefore which of the alternative enzymes is employed to catalyze methane biosynthesis under different environmental conditions. As were unable to establish a reliable genetic system for M. thermautotrophicus, we developed in vitro transcription as an alternative system to investigate methanogen gene expression and regulation. This led to the discovery that an archaeal protein

  7. Effects of systemic administration of histone deacetylase inhibitor on memory formation and immediate early gene expression in chick brain.

    Science.gov (United States)

    Tiunova, A A; Toropova, K A; Konovalova, E V; Anokhin, K V

    2012-09-01

    We studied the effects of histone deacetylase inhibitor that stimulates transcriptional activity via histone hyperacetylation on memory formation. Sodium butyrate and sodium valproate enhanced memory in chicks following "weak" training with memory transfer into long-term state. Quantitative analysis of c-Fos and ZENK transcriptional factor gene expression in six structures of chick brain revealed induction of these genes in the structures involved in this type of learning. Sodium valproate administration did not increase this induction, but even reduced it. These findings suggest that sodium butyrate and sodium valproate exert cognitive stimulating action in the "weak" memory formation paradigm, and that this effect is not mediated via enhanced expression of transcriptional factors, which are traditionally considered as "molecular switcher" for memory transfer into long-term state.

  8. Construction of prokaryotic expression system of ureB gene from a clinical Helicobacter pylori strain and identification of the recombinant protein immunity

    Institute of Scientific and Technical Information of China (English)

    Ya-Fei Mao; Jie Yan

    2004-01-01

    AIM: To clone ureB gene from a clinical isolate ofHelicobacter pyloriand construct a prokaryotic expression system of the gene and identify immunity of the expressed recombinant protein.METHODS: ureB gene from a clinical H pyloristrain Y06 was amplified by the high fidelity polymerase chain reaction technique. The target DNA fragment amplified from ureB gene was sequenced after T-A cloning. Prokaryotic recombinant expression vector pET32a inserted with ureB gene (pET32a-ureB) was constructed. The expression of recombinant UreB protein (rUreB) in E. coliBL21DE3 induced by isopropylthio-β-D-galactoside (IPTG) at different concentrations was examined by SDS-PAGE. Western blot using commercial antibodies against whole cell of Hpylori and an immunodiffusion assay using a self-prepared rabbit anti-rUreB antibody were applied to determine immunity of the target recombinant protein. ELISA was used to detect the antibody against rUreB in sera of 125 Hpyloriinfected patients and to examine rUreB expression in 109 Hpylori isolates.RESULTS: In comparison with the reported corresponding sequences, the nucleotide sequence homology of the cloned ureBgene was from 96.88-97.82% while the homology of its putative amino acid sequence was as high as 99.65-99.82%.The rUreB output expressed by pET32a-ureB-BL21DE3 was approximate 30% of the total bacterial proteins. rUreB specifically combined with the commercial antibodies against whole cell of H pyloriand strongly induced rabbits to produce antibody with a 1:8 immunodiffusion titer after the animals were immunized with the recombinant protein.Serum samples from all H pyloriinfected patients were positive for UreB antibody and UreB expression were detectable in all tested H pyloriisolates.CONCLUSION: A prokaryotic expression system with high expression efficiency of H pylori ureB gene was successfully established. The expressed rUreB showed qualified immunoreactivity and antigenicity. High frequencies of UreB expression in different H

  9. Local and systemic gene expression responses of Atlantic salmon (Salmo salar L. to infection with the salmon louse (Lepeophtheirus salmonis

    Directory of Open Access Journals (Sweden)

    Nilsen Frank

    2008-10-01

    Full Text Available Abstract Background The salmon louse (SL is an ectoparasitic caligid crustacean infecting salmonid fishes in the marine environment. SL represents one of the major challenges for farming of salmonids, and veterinary intervention is necessary to combat infection. This study addressed gene expression responses of Atlantic salmon infected with SL, which may account for its high susceptibility. Results The effects of SL infection on gene expression in Atlantic salmon were studied throughout the infection period from copepodids at 3 days post infection (dpi to adult lice (33 dpi. Gene expression was analyzed at three developmental stages in damaged and intact skin, spleen, head kidney and liver, using real-time qPCR and a salmonid cDNA microarray (SFA2. Rapid detection of parasites was indicated by the up-regulation of immunoglobulins in the spleen and head kidney and IL-1 receptor type 1, CD4, beta-2-microglobulin, IL-12β, CD8α and arginase 1 in the intact skin of infected fish. Most immune responses decreased at 22 dpi, however, a second activation was observed at 33 dpi. The observed pattern of gene expression in damaged skin suggested the development of inflammation with signs of Th2-like responses. Involvement of T cells in responses to SL was witnessed with up-regulation of CD4, CD8α and programmed death ligand 1. Signs of hyporesponsive immune cells were seen. Cellular stress was prevalent in damaged skin as seen by highly significant up-regulation of heat shock proteins, other chaperones and mitochondrial proteins. Induction of the major components of extracellular matrix, TGF-β and IL-10 was observed only at the adult stage of SL. Taken together with up-regulation of matrix metalloproteinases (MMP, this classifies the wounds afflicted by SL as chronic. Overall, the gene expression changes suggest a combination of chronic stress, impaired healing and immunomodulation. Steady increase of MMP expression in all tissues except liver was a

  10. Development and analysis of a highly flexible multi-gene expression system for metabolic engineering in Arabidopsis seeds and other plant tissues.

    Science.gov (United States)

    Shockey, Jay; Mason, Catherine; Gilbert, Matthew; Cao, Heping; Li, Xiangjun; Cahoon, Edgar; Dyer, John

    2015-09-01

    Production of novel value-added compounds in transgenic crops has become an increasingly viable approach in recent years. However, in many cases, product yield still falls short of the levels necessary for optimal profitability. Determination of the limiting factors is thus of supreme importance for the long-term viability of this approach. A significant challenge to most metabolic engineering projects is the need for strong coordinated co-expression of multiple transgenes. Strong constitutive promoters have been well-characterized during the >30 years since plant transformation techniques were developed. However, organ- or tissue-specific promoters are poorly characterized in many cases. Oilseeds are one such example. Reports spanning at least 20 years have described the use of certain seed-specific promoters to drive expression of individual transgenes. Multi-gene engineering strategies are often hampered by sub-optimal expression levels or improper tissue-specificity of particular promoters, or rely on the use of multiple copies of the same promoter, which can result in DNA instability or transgene silencing. We describe here a flexible system of plasmids that allows for expression of 1-7 genes per binary plasmid, and up to 18 genes altogether after multiple rounds of transformation or sexual crosses. This vector system includes six seed-specific promoters and two constitutive promoters. Effective constitutive and seed-specific RNA interference gene-suppression cloning vectors were also constructed for silencing of endogenous genes. Taken together, this molecular toolkit allows combinatorial cloning for multiple transgene expression in seeds, vegetative organs, or both simultaneously, while also providing the means to coordinately overexpress some genes while silencing others.

  11. Heterologous expression of mannanase and developing a new Reporter gene system in Lactobacillus casei and Escherichia coli

    DEFF Research Database (Denmark)

    Lin, Jinzhong; Zou, Yexia; Ma, Chengjie

    2015-01-01

    from the S-layer of L. acidophilus (pELWH). The secretion of ManB was detected in the supernatant of the pELSH-ManB transformants and in the S-layer of the cell surface of the pELWH-ManB transformants. This is the first report demonstrating that the B. pumilus manB gene is a useful reporter gene in L......Reporter gene systems are useful for studying bacterial molecular biology, including the regulation of gene expression and the histochemical analysis of protein products. Here, two genes, β-1,4-mannanase (manB) from Bacillus pumilus and β-glucuronidase (gusA) from Escherichia coli K12, were cloned...... into the expression vector pELX1. The expression patterns of these reporter genes in Lactobacillus casei were investigated by measuring their enzymatic activities and estimating their recombinant protein yields using western blot analysis. Whereas mannanase activity was positively correlated with the accumulation...

  12. Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Salem, Tamer Z. [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbial Molecular Biology, AGERI, Agricultural Research Center, Giza 12619 (Egypt); Division of Biomedical Sciences, Zewail University, Zewail City of Science and Technology, Giza 12588 (Egypt); Zhang, Fengrui [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Thiem, Suzanne M., E-mail: smthiem@msu.edu [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States)

    2013-01-20

    Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

  13. Correlating Expression Data with Gene Function Using Gene Ontology

    Institute of Scientific and Technical Information of China (English)

    LIU,Qi; DENG,Yong; WANG,Chuan; SHI,Tie-Liu; LI,Yi-Xue

    2006-01-01

    Clustering is perhaps one of the most widely used tools for microarray data analysis. Proposed roles for genes of unknown function are inferred from clusters of genes similarity expressed across many biological conditions.However, whether function annotation by similarity metrics is reliable or not and to what extent the similarity in gene expression patterns is useful for annotation of gene functions, has not been evaluated. This paper made a comprehensive research on the correlation between the similarity of expression data and of gene functions using Gene Ontology. It has been found that although the similarity in expression patterns and the similarity in gene functions are significantly dependent on each other, this association is rather weak. In addition, among the three categories of Gene Ontology, the similarity of expression data is more useful for cellular component annotation than for biological process and molecular function. The results presented are interesting for the gene functions prediction research area.

  14. Construction of a prokaryotic expression system of vacA gene and detection of vatA gene,VacA protein in Helicobacter pylori isolates and ant-VacA antibody in patients'sera

    Institute of Scientific and Technical Information of China (English)

    Jie Yan; Ya-Fei Mao

    2004-01-01

    AIM: To construct a recombinant prokaryotic expression vector inserted with Helicobacter pylori vacA gene and identify the immunity of the expressed recombinant protein,and to determine prevalence of vacA-carryinglVacA expressing Hpyloriisolates and seroprevalence of specific ant-VacA antibody in H pyloriinfected patients.METHODS: Polymerase chain reaction technique was used to amplify complete vacA gene of H pyloristrain NCTC11637 and to detect vacA gene in 109 H pylori isolates. The amplification product of the complete vacA gene was sequenced after T-A cloning. A recombinant expression vector inserted with a complete vacA gene fragment, named as pET32a-vacA, was constructed. Expression of the target recombinant protein VacA (rVacA) was examined by SDSPAGE. Western blot using commercial antibodies against whole cell of H pyloriand an immunodiffusion assay using self-prepared rabbit anti-rVacA antibody were applied to determine immunoreaction and antigenicity of rVacA. Two ELISA methods were established to detect VacA expression in H pyloriisolates and the specific anti-VacA antibody in sera from 125 patients infected with H pylori.RESULTS: In comparison with the reported corresponding sequences, homologies of nucleotide and putative amino acid sequences of the cloned vacA gene were 99.82% and 100%, respectively. The constructed recombinant prokaryotic expression system efficiently produced rVacA. rVacA was able to combine with the commercial antibodies against whole cell of H pyloriand to induce the immunized rabbit to produce specific antibody with an immunodiffusion titer of 1:4. All tested H pyloriisolates carried vacA gene, but only 66.1% expressed Vac A protein. Of the serum samples tested,42.4% were positive for specific anti-VacA antibody.CONCLUSION: A prokaryotic expression system of H pylori vacA gene was successfully constructed. The expressed rVacA can be used to detect specific anti-VacA antibody in human and to prepare antiserum in animals. The high

  15. The exceptional stem cell system of Macrostomum lignano: Screening for gene expression and studying cell proliferation by hydroxyurea treatment and irradiation

    Directory of Open Access Journals (Sweden)

    Eichberger Paul

    2007-03-01

    Full Text Available Abstract Background Flatworms are characterized by an outstanding stem cell system. These stem cells (neoblasts can give rise to all cell types including germ cells and power the exceptional regenerative capacity of many flatworm species. Macrostomum lignano is an emerging model system to study stem cell biology of flatworms. It is complementary to the well-studied planarians because of its small size, transparency, simple culture maintenance, the basal taxonomic position and its less derived embryogenesis that is more closely related to spiralians. The development of cell-, tissue- and organ specific markers is necessary to further characterize the differentiation potential of flatworm stem cells. Large scale in situ hybridization is a suitable tool to identify possible markers. Distinguished genes identified in a large scale screen in combination with manipulation of neoblasts by hydroxyurea or irradiation will advance our understanding of differentiation and regulation of the flatworm stem cell system. Results We have set up a protocol for high throughput large scale whole mount in situ hybridization for the flatworm Macrostomum lignano. In the pilot screen, a number of cell-, tissue- or organ specific expression patterns were identified. We have selected two stem cell- and germ cell related genes – macvasa and macpiwi – and studied effects of hydroxyurea (HU treatment or irradiation on gene expression. In addition, we have followed cell proliferation using a mitosis marker and bromodeoxyuridine labeling of S-phase cells after various periods of HU exposure or different irradiation levels. HU mediated depletion of cell proliferation and HU induced reduction of gene expression was used to generate a cDNA library by suppressive subtractive hybridization. 147 differentially expressed genes were sequenced and assigned to different categories. Conclusion We show that Macrostomum lignano is a suitable organism to perform high throughput large

  16. Effects of turmeric (Curcuma longa) on the expression of hepatic genes associated with biotransformation, antioxidant, and immune systems in broiler chicks fed aflatoxin.

    Science.gov (United States)

    Yarru, L P; Settivari, R S; Gowda, N K S; Antoniou, E; Ledoux, D R; Rottinghaus, G E

    2009-12-01

    The objective of the present study was to evaluate the efficacy of curcumin, an antioxidant found in turmeric (Curcuma longa) powder (TMP), to ameliorate changes in gene expression in the livers of broiler chicks fed aflatoxin B(1) (AFB(1)). Four pen replicates of 5 chicks each were assigned to each of 4 dietary treatments, which included the following: A) basal diet containing no AFB(1) or TMP (control), B) basal diet supplemented with TMP (0.5%) that supplied 74 mg/kg of curcumin, C) basal diet supplemented with 1.0 mg of AFB(1)/kg of diet, and D) basal diet supplemented with TMP that supplied 74 mg/kg of curcumin and 1.0 mg of AFB(1)/kg of diet. Aflatoxin reduced (P < 0.05) feed intake and BW gain and increased (P < 0.05) relative liver weight. Addition of TMP to the AFB(1) diet ameliorated (P < 0.05) the negative effects of AFB(1) on growth performance and liver weight. At the end of the 3-wk treatment period, livers were collected (6 per treatment) to evaluate changes in the expression of genes involved in antioxidant function [catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST)], biotransformation [epoxide hydrolase (EH), cytochrome P450 1A1 and 2H1 (CYP1A1 and CYP2H1)], and the immune system [interleukins 6 and 2 (IL-6 and IL-2)]. Changes in gene expression were determined using the quantitative real-time PCR technique. There was no statistical difference in gene expression among the 4 treatment groups for CAT and IL-2 genes. Decreased expression of SOD, GST, and EH genes due to AFB(1) was alleviated by inclusion of TMP in the diet. Increased expression of IL-6, CYP1A1 and CYP2H1 genes due to AFB(1) was also alleviated by TMP. The current study demonstrates partial protective effects of TMP on changes in expression of antioxidant, biotransformation, and immune system genes in livers of chicks fed AFB(1). Practical application of the research is supplementation of TMP in diets to prevent or reduce the

  17. A lab-on-a-chip system integrating tissue sample preparation and multiplex RT-qPCR for gene expression analysis in point-of-care hepatotoxicity assessment.

    Science.gov (United States)

    Lim, Geok Soon; Chang, Joseph S; Lei, Zhang; Wu, Ruige; Wang, Zhiping; Cui, Kemi; Wong, Stephen

    2015-10-21

    A truly practical lab-on-a-chip (LOC) system for point-of-care testing (POCT) hepatotoxicity assessment necessitates the embodiment of full-automation, ease-of-use and "sample-in-answer-out" diagnostic capabilities. To date, the reported microfluidic devices for POCT hepatotoxicity assessment remain rudimentary as they largely embody only semi-quantitative or single sample/gene detection capabilities. In this paper, we describe, for the first time, an integrated LOC system that is somewhat close to a practical POCT hepatotoxicity assessment device - it embodies both tissue sample preparation and multiplex real-time RT-PCR. It features semi-automation, is relatively easy to use, and has "sample-in-answer-out" capabilities for multiplex gene expression analysis. Our tissue sample preparation module incorporating both a microhomogenizer and surface-treated paramagnetic microbeads yielded high purity mRNA extracts, considerably better than manual means of extraction. A primer preloading surface treatment procedure and the single-loading inlet on our multiplex real-time RT-PCR module simplify off-chip handling procedures for ease-of-use. To demonstrate the efficacy of our LOC system for POCT hepatotoxicity assessment, we perform a preclinical animal study with the administration of cyclophosphamide, followed by gene expression analysis of two critical protein biomarkers for liver function tests, aspartate transaminase (AST) and alanine transaminase (ALT). Our experimental results depict normalized fold changes of 1.62 and 1.31 for AST and ALT, respectively, illustrating up-regulations in their expression levels and hence validating their selection as critical genes of interest. In short, we illustrate the feasibility of multiplex gene expression analysis in an integrated LOC system as a viable POCT means for hepatotoxicity assessment.

  18. Modeling of gap gene expression in Drosophila Kruppel mutants.

    Directory of Open Access Journals (Sweden)

    Konstantin Kozlov

    Full Text Available The segmentation gene network in Drosophila embryo solves the fundamental problem of embryonic patterning: how to establish a periodic pattern of gene expression, which determines both the positions and the identities of body segments. The gap gene network constitutes the first zygotic regulatory tier in this process. Here we have applied the systems-level approach to investigate the regulatory effect of gap gene Kruppel (Kr on segmentation gene expression. We acquired a large dataset on the expression of gap genes in Kr null mutants and demonstrated that the expression levels of these genes are significantly reduced in the second half of cycle 14A. To explain this novel biological result we applied the gene circuit method which extracts regulatory information from spatial gene expression data. Previous attempts to use this formalism to correctly and quantitatively reproduce gap gene expression in mutants for a trunk gap gene failed, therefore here we constructed a revised model and showed that it correctly reproduces the expression patterns of gap genes in Kr null mutants. We found that the remarkable alteration of gap gene expression patterns in Kr mutants can be explained by the dynamic decrease of activating effect of Cad on a target gene and exclusion of Kr gene from the complex network of gap gene interactions, that makes it possible for other interactions, in particular, between hb and gt, to come into effect. The successful modeling of the quantitative aspects of gap gene expression in mutant for the trunk gap gene Kr is a significant achievement of this work. This result also clearly indicates that the oversimplified representation of transcriptional regulation in the previous models is one of the reasons for unsuccessful attempts of mutant simulations.

  19. Further EST analysis of endocrine genes that are preferentially expressed in the neural complex of Ciona intestinalis: receptor and enzyme genes associated with endocrine system in the neural complex.

    Science.gov (United States)

    Sekiguchi, Toshio; Kawashima, Takeshi; Satou, Yutaka; Satoh, Nori

    2007-01-15

    Identification of orthologs of vertebrate neuropeptides and hypothalamic hormones in the neural complex of ascidians suggests integral roles of the ascidian neural complex in the endocrine system. In the present study, we investigated endocrine-related genes expressed in the neural complex of Ciona intestinalis. Comprehensive analyses of 3'-end sequences of the neural complex cDNAs placed 10,029 clones into 4051 independent clusters or genes, 1524 of them being expressed preferentially in this organ. Comparison of the 1524 genes with the human proteome databank demonstrated that 476 matched previously identified human proteins with distinct functions. Further analyses of sequence similarity of the 476 genes demonstrated that 21 genes are candidates for those involved in the endocrine system. Although we cannot detect hormone or peptide candidates, we found 21 genes such as receptors for peptide ligands, receptor-modulating proteins, and processing enzymes. We then characterized the Ciona prohormone convertase 2 (Ci-PC2) and carboxypeptidase E (Ci-CPE), which are associated with endoproteolytic processing of peptide hormone precursors. Furthermore, genes encoding these transcripts are expressed specifically in the neural complex of young adult ascidians. These data provide the molecular basis for further functional studies of the endocrine role of the neural complex of ascidians.

  20. Brain region-specific alterations in the gene expression of cytokines, immune cell markers and cholinergic system components during peripheral endotoxin-induced inflammation.

    Science.gov (United States)

    Silverman, Harold A; Dancho, Meghan; Regnier-Golanov, Angelique; Nasim, Mansoor; Ochani, Mahendar; Olofsson, Peder S; Ahmed, Mohamed; Miller, Edmund J; Chavan, Sangeeta S; Golanov, Eugene; Metz, Christine N; Tracey, Kevin J; Pavlov, Valentin A

    2015-03-11

    Inflammatory conditions characterized by excessive peripheral immune responses are associated with diverse alterations in brain function, and brain-derived neural pathways regulate peripheral inflammation. Important aspects of this bidirectional peripheral immune-brain communication, including the impact of peripheral inflammation on brain region-specific cytokine responses, and brain cholinergic signaling (which plays a role in controlling peripheral cytokine levels), remain unclear. To provide insight, we studied gene expression of cytokines, immune cell markers and brain cholinergic system components in the cortex, cerebellum, brainstem, hippocampus, hypothalamus, striatum and thalamus in mice after an intraperitoneal lipopolysaccharide injection. Endotoxemia was accompanied by elevated serum levels of interleukin (IL)-1β, IL-6 and other cytokines and brain region-specific increases in Il1b (the highest increase, relative to basal level, was in cortex; the lowest increase was in cerebellum) and Il6 (highest increase in cerebellum; lowest increase in striatum) mRNA expression. Gene expression of brain Gfap (astrocyte marker) was also differentially increased. However, Iba1 (microglia marker) mRNA expression was decreased in the cortex, hippocampus and other brain regions in parallel with morphological changes, indicating microglia activation. Brain choline acetyltransferase (Chat ) mRNA expression was decreased in the striatum, acetylcholinesterase (Ache) mRNA expression was decreased in the cortex and increased in the hippocampus, and M1 muscarinic acetylcholine receptor (Chrm1) mRNA expression was decreased in the cortex and the brainstem. These results reveal a previously unrecognized regional specificity in brain immunoregulatory and cholinergic system gene expression in the context of peripheral inflammation and are of interest for designing future antiinflammatory approaches.

  1. Systemic resistance in Arabidopsis induced by biocontrol bacteria is independent of salicylic acid accumulation and pathogenesis-related gene expression

    NARCIS (Netherlands)

    Pieterse, C.M.J.; Wees, A.C.M. van; Hoffland, E.; Pelt, J.A. van; Loon, L.C. van

    1996-01-01

    Systemic acquired resistance is a pathogen-inducible defense mechanism in plants. The resistant state is dependent on endogenous accumulation of salicylic acid (SA) and is characterized by the activation of genes encoding pathogenesis-related (PR) proteins. Recently, selected nonpathogenic, root-col

  2. Expression of insulin-like growth factor system genes in liver tissue during embryonic and early post-hatch development in duck (Anas platyrhynchos Domestica).

    Science.gov (United States)

    Jianmin, Zou; Jingting, Shu; Yanju, Shan; Yan, Hu; Chi, Song; Wenqi, Zhu

    2014-04-01

    The IGF system is one of the most important endocrine and paracrine growth factor systems that regulate fetal and placental growth, whereas the liver is the principal source of circulation IGF-I. In the present study, expression of IGF-I, IGF type-I receptor (IGF-IR), and IGF binding protein (IGFBP)-3 genes was quantified by RT-PCR in the liver tissue on days 13, 17, 21, 25, and 27 of embryonic development, as well as at 7 days post-hatching (PH) in meat-type Gaoyou ducks and egg-type Jinding ducks. The results showed that IGF-I mRNA could be detected as early as on E 13d, but the expression level was low throughout embryonic development before increasing dramatically by E 27d and 7 days PH in both duck breeds. However, Gaoyou ducks exhibited higher IGF-I mRNA level than Jinding ducks, and the differences were significant on E 13d, E 21d, and at 7 days PH. Expression of IGF-IR in liver increased gradually in the former stages of the embryonic development, reaching its highest point on E 21d, and then declined up until 7 days PH. The expression pattern of IGFBP-3 gene was similar to that of IGF-IR gene, increasing significantly from E 17d. The expression peak appeared on E 25d, then declined significantly just prior to hatching (day 27) and was followed by an increase at 7 days PH. In general, the expression level of IGF-IR and IGFBP-3 genes in Jinding ducks was higher than that in Gaoyou ducks. Inverse relationships were observed for the expression of IGF-I and IGF-IR, and IGF-I and IGFBP-3, whereas a positive relationship was observed for the expression of IGF-IR and IGFBP-3. Our data indicate a differential expression of selected genes that comprise the IGF system in the duck liver tissue during embryonic and early PH growth and development.

  3. Stable expression of green fluorescent protein and targeted disruption of thioredoxin peroxidase-1 gene in Babesia bovis with the WR99210/dhfr selection system.

    Science.gov (United States)

    Asada, Masahito; Tanaka, Miho; Goto, Yasuyuki; Yokoyama, Naoaki; Inoue, Noboru; Kawazu, Shin-ichiro

    2012-02-01

    We have achieved stable expression of green fluorescent protein (GFP) in Babesia bovis by using the WR99210/human dihydrofolate reductase (DHFR) gene selection system. A GFP-expression plasmid with a dhfr expression cassette (DHFR-gfp) was constructed and transfected into B. bovis by nucleofection. Following WR99210 selection, a GFP-fluorescent parasite population was obtained and the fluorescent parasite was maintained for more than 7 months under WR99210 drug pressure. The DHFR-gfp was used to construct a small circular chromosome and to target gene disruption in the parasite. For construction of the small circular chromosome (DHFR-gfp-Bbcent2), the putative centromere region of B. bovis chromosome 2 (Bbcent2) was cloned and inserted into the DHFR-gfp plasmid. Addition of Bbcent2 to the DHFR-gfp plasmid improved its segregation efficiency during parasite multiplication and GFP-expressing parasites were maintained for more than 2 months without drug pressure. For targeted disruption of a B. bovis gene we attempted to knockout the thioredoxin peroxidase-1 (TPx-1) gene (a single-copy 2-Cys peroxiredoxin gene, Tbtpx-1) by homologous recombination. To generate the targeting construct (DHFR-gfp-Bbtpx1KO), 5' and 3' portions of Bbtpx-1 were cloned into the DHFR-gfp plasmid. Following nucleofection, WR99210 selection and cloning, a GFP-fluorescent parasite population was obtained. Integration of the construct into the Bbtpx-1 locus was confirmed by PCR. The absence of Bbtpx-1 mRNA and protein were verified by reverse transcription PCR and western blot analysis/indirect immunofluorescence assay, respectively. This is the first report of targeted gene disruption of a Babesia gene. These advances in the methodology of genetic manipulation in B. bovis will facilitate functional analysis of Babesia genomes and will improve our understanding of the basic biology of apicomplexan parasites.

  4. The Myxococcus xanthus Two-Component System CorSR Regulates Expression of a Gene Cluster Involved in Maintaining Copper Tolerance during Growth and Development

    Science.gov (United States)

    Sánchez-Sutil, María Celestina; Pérez, Juana; Gómez-Santos, Nuria; Shimkets, Lawrence J.; Moraleda-Muñoz, Aurelio; Muñoz-Dorado, José

    2013-01-01

    Myxococcus xanthus is a soil-dwelling member of the δ–Proteobacteria that exhibits a complex developmental cycle upon starvation. Development comprises aggregation and differentiation into environmentally resistant myxospores in an environment that includes fluctuations in metal ion concentrations. While copper is essential for M. xanthus cells because several housekeeping enzymes use it as a cofactor, high copper concentrations are toxic. These opposing effects force cells to maintain a tight copper homeostasis. A plethora of paralogous genes involved in copper detoxification, all of which are differentially regulated, have been reported in M. xanthus. The use of in-frame deletion mutants and fusions with the reporter gene lacZ has allowed the identification of a two-component system, CorSR, that modulates the expression of an operon termed curA consisting of nine genes whose expression slowly increases after metal addition, reaching a plateau. Transcriptional regulation of this operon is complex because transcription can be initiated at different promoters and by different types of regulators. These genes confer copper tolerance during growth and development. Copper induces carotenoid production in a ΔcorSR mutant at lower concentrations than with the wild-type strain due to lack of expression of a gene product resembling subunit III of cbb3-type cytochrome c oxidase. This data may explain why copper induces carotenoid biosynthesis at suboptimal rather than optimal growth conditions in wild-type strains. PMID:23874560

  5. The Myxococcus xanthus two-component system CorSR regulates expression of a gene cluster involved in maintaining copper tolerance during growth and development.

    Directory of Open Access Journals (Sweden)

    María Celestina Sánchez-Sutil

    Full Text Available Myxococcus xanthus is a soil-dwelling member of the δ-Proteobacteria that exhibits a complex developmental cycle upon starvation. Development comprises aggregation and differentiation into environmentally resistant myxospores in an environment that includes fluctuations in metal ion concentrations. While copper is essential for M. xanthus cells because several housekeeping enzymes use it as a cofactor, high copper concentrations are toxic. These opposing effects force cells to maintain a tight copper homeostasis. A plethora of paralogous genes involved in copper detoxification, all of which are differentially regulated, have been reported in M. xanthus. The use of in-frame deletion mutants and fusions with the reporter gene lacZ has allowed the identification of a two-component system, CorSR, that modulates the expression of an operon termed curA consisting of nine genes whose expression slowly increases after metal addition, reaching a plateau. Transcriptional regulation of this operon is complex because transcription can be initiated at different promoters and by different types of regulators. These genes confer copper tolerance during growth and development. Copper induces carotenoid production in a ΔcorSR mutant at lower concentrations than with the wild-type strain due to lack of expression of a gene product resembling subunit III of cbb3-type cytochrome c oxidase. This data may explain why copper induces carotenoid biosynthesis at suboptimal rather than optimal growth conditions in wild-type strains.

  6. Looking in the mouth for noninvasive gene expression-based methods to detect oral, oropharyngeal, and systemic cancer.

    Science.gov (United States)

    Adami, Guy R; Adami, Alexander J

    2012-01-01

    Noninvasive diagnosis, whether by sampling body fluids, body scans, or other technique, has the potential to simplify early cancer detection. A classic example is Pap smear screening, which has helped to reduce cervical cancer 75% over the last 50 years. No test is error-free; the real concern is sufficient accuracy combined with ease of use. This paper will discuss methods that measure gene expression or epigenetic markers in oral cells or saliva to diagnose oral and pharyngeal cancers, without requiring surgical biopsy. Evidence for lung and other distal cancer detection is also reviewed.

  7. Heteroduplex DNA mismatch repair system of Streptococcus pneumoniae: cloning and expression of the hexA gene.

    OpenAIRE

    Balganesh, T S; Lacks, S A

    1985-01-01

    Mutations affecting heteroduplex DNA mismatch repair in Streptococcus pneumoniae were localized in two genes, hexA and hexB, by fractionation of restriction fragments carrying mutant alleles. A fragment containing the hexA4 allele was cloned in the S. pneumoniae cloning system, and the hexA+ allele was introduced into the recombinant plasmid by chromosomal facilitation of plasmid transfer. Subcloning localized the functional hexA gene to a 3.5-kilobase segment of the cloned pneumococcal DNA. ...

  8. Expression of Helicobacter pylori hspA Gene in Lactococcus lactis NICE System and Experimental Study on Its Immunoreactivity

    Directory of Open Access Journals (Sweden)

    Xiao-Juan Zhang

    2015-01-01

    Full Text Available Aim. The aim of this study was to develop an oral Lactococcus lactis (L. lactis vaccine against Helicobacter pylori (H. pylori. Methods. After L. lactis NZ3900/pNZ8110-hspA was constructed, growth curves were plotted to study whether the growth of recombinant L. lactis was affected after hspA was cloned into L. lactis and whether the growth of empty bacteria, empty plasmid bacteria, and recombinant L. lactis was affected by different concentrations of Nisin; SDS-PAGE and Western blot were adopted, respectively, to detect the HspA expressed by recombinant L. lactis and its immunoreactivity. Results. There was no effect observed from the growth curve after exogenous gene hspA was cloned into L. lactis NZ3900; different concentrations of Nisin did not affect the growth of NZ3900 and NZ3900/pNZ8110, while different concentrations of Nisin inhibited the growth of NZ3900/pNZ8110-hspA except 10 ng/mL Nisin. No HspA strip was observed from SDS-PAGE. Western blot analysis showed that HspA expressed by recombinant bacteria had favorable immunoreactivity. Conclusion. The growth of recombinant L. lactis was suppressed even though a small amount of HspA had been induced to express. Therefore recombinant L. lactis only express HspA which was not suitable to be oral vaccine against Helicobacter pylori.

  9. Combinatorial engineering for heterologous gene expression.

    Science.gov (United States)

    Zwick, Friederike; Lale, Rahmi; Valla, Svein

    2013-01-01

    Tools for strain engineering with predictable outcome are of crucial importance for the nascent field of synthetic biology. The success of combining different DNA biological parts is often restricted by poorly understood factors deriving from the complexity of the systems. We have previously identified variants for different regulatory elements of the expression cassette XylS/Pm. When such elements are combined they act in a manner consistent with their individual behavior, as long as they affect different functions, such as transcription and translation. Interestingly, sequence context does not seem to influence the final outcome significantly. Expression of reporter gene bla could be increased up to 75 times at the protein level by combining three variants in one cassette. For other tested reporter genes similar results were obtained, except that the stimulatory effect was quantitatively less. Combination of individually characterized DNA parts thus stands as suitable method to achieve a desired phenotype.

  10. Characterization and differentiation of equine experimental local and early systemic inflammation by expression responses of inflammation-related genes in peripheral blood leukocytes

    DEFF Research Database (Denmark)

    Vinther, Anne Mette L; Heegaard, Peter M. H.; Skovgaard, Kerstin;

    2016-01-01

    and hematological/biochemical examinations were performed, and serial blood samples were analyzed by reverse transcription quantitative real-time PCR. Post-induction expression profiles of all genes were compared between study groups using principal component analysis (PCA) and hierarchical clustering. Moderate......, the aim of this study was to investigate the innate peripheral blood leukocyte (PBL) immune response to local inflammation in horses, and to compare this response with the PBL immune response during the early phase of acute systemic inflammation. Expression of 22 selected inflammation-related genes...... was measured in whole blood leukocytes from 6 horses in an experimental cross-over model of lipopolysaccharide- (LPS-) induced acute synovitis (3 μg LPS intraarticularly; locally inflamed [LI] horses) and endotoxemia (1 μg LPS/kg intravenously; systemically inflamed [SI] horses). Multiple clinical...

  11. Yokukansan, a kampo medicine, protects PC12 cells from glutamate-induced death by augmenting gene expression of cystine/glutamate antiporter system Xc-.

    Directory of Open Access Journals (Sweden)

    Hitomi Kanno

    Full Text Available Effects of the kampo medicine yokukansan on gene expression of the cystine/glutamate antiporter system Xc-, which protects against glutamate-induced cytotoxicity, were examined in Pheochromocytoma cells (PC12 cells. Yokukansan inhibited glutamate-induced PC12 cell death. Similar cytoprotective effects were found in Uncaria hook. Experiments to clarify the active compounds revealed that geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook, had cytoprotective effects. These components enhanced gene expressions of system Xc- subunits xCT and 4F2hc, and also ameliorated the glutamate-induced decrease in glutathione levels. These results suggest that the cytoprotective effect of yokukansan may be attributed to geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook.

  12. Yokukansan, a kampo medicine, protects PC12 cells from glutamate-induced death by augmenting gene expression of cystine/glutamate antiporter system Xc-.

    Science.gov (United States)

    Kanno, Hitomi; Kawakami, Zenji; Mizoguchi, Kazushige; Ikarashi, Yasushi; Kase, Yoshio

    2014-01-01

    Effects of the kampo medicine yokukansan on gene expression of the cystine/glutamate antiporter system Xc-, which protects against glutamate-induced cytotoxicity, were examined in Pheochromocytoma cells (PC12 cells). Yokukansan inhibited glutamate-induced PC12 cell death. Similar cytoprotective effects were found in Uncaria hook. Experiments to clarify the active compounds revealed that geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook, had cytoprotective effects. These components enhanced gene expressions of system Xc- subunits xCT and 4F2hc, and also ameliorated the glutamate-induced decrease in glutathione levels. These results suggest that the cytoprotective effect of yokukansan may be attributed to geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook.

  13. Validation of reference genes for quantifying changes in gene expression in virus-infected tobacco.

    Science.gov (United States)

    Baek, Eseul; Yoon, Ju-Yeon; Palukaitis, Peter

    2017-10-01

    To facilitate quantification of gene expression changes in virus-infected tobacco plants, eight housekeeping genes were evaluated for their stability of expression during infection by one of three systemically-infecting viruses (cucumber mosaic virus, potato virus X, potato virus Y) or a hypersensitive-response-inducing virus (tobacco mosaic virus; TMV) limited to the inoculated leaf. Five reference-gene validation programs were used to establish the order of the most stable genes for the systemically-infecting viruses as ribosomal protein L25 > β-Tubulin > Actin, and the least stable genes Ubiquitin-conjugating enzyme (UCE) genes were EF1α > Cysteine protease > Actin, and the least stable genes were GAPDH genes, three defense responsive genes were examined to compare their relative changes in gene expression caused by each virus. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Mechanisms of Geomagnetic Field Influence on Gene Expression Using Influenza as a Model System: Basics of Physical Epidemiology

    Directory of Open Access Journals (Sweden)

    Andriy Ponomarenko

    2010-03-01

    Full Text Available Recent studies demonstrate distinct changes in gene expression in cells exposed to a weak magnetic field (MF. Mechanisms of this phenomenon are not understood yet. We propose that proteins of the Cryptochrome family (CRY are "epigenetic sensors" of the MF fluctuations, i.e., magnetic field-sensitive part of the epigenetic controlling mechanism. It was shown that CRY represses activity of the major circadian transcriptional complex CLOCK/BMAL1. At the same time, function of CRY, is apparently highly responsive to weak MF because of radical pairs that periodically arise in the functionally active site of CRY and mediate the radical pair mechanism of magnetoreception. It is known that the circadian complex influences function of every organ and tissue, including modulation of both NF-κB- and glucocorticoids- dependent signaling pathways. Thus, MFs and solar cycles-dependent geomagnetic field fluctuations are capable of altering expression of genes related to function of NF-κB, hormones and other biological regulators. Notably, NF-κB, along with its significant role in immune response, also participates in differential regulation of influenza virus RNA synthesis. Presented data suggests that in the case of global application (example—geomagnetic field, MF-mediated regulation may have epidemiological and other consequences.

  15. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy (Davis, CA); Bachkirova, Elena (Davis, CA); Rey, Michael (Davis, CA)

    2012-05-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  16. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy; Bachkirova, Elena; Rey, Michael

    2013-10-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  17. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy [Davis, CA; Bachkirova, Elena [Davis, CA; Rey, Michael [Davis, CA

    2012-05-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  18. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy; Bachkirova, Elena; Rey, Michael

    2013-10-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  19. Systemic short-chain fatty acids rapidly alter gastrointestinal structure, function, and expression of early response genes.

    Science.gov (United States)

    Tappenden, K A; McBurney, M I

    1998-07-01

    Luminal and systemic short chain fatty acids (SCFA) stimulate mucosal proliferation but the mechanism(s) is unclear. This study examined acute effects of systemic SCFAs on gastrointestinal structure and function and signals potentially mediating SCFA-induced mucosal proliferation. Male Sprague-Dawley rats (246+/-2 g) received nutrients as either standard total parenteral nutrition (TPN) or an isoenergetic, isonitrogenous formulation containing SCFAs (TPN + SCFA). Animals were randomized to one of five treatments: standard TPN for 72 hr, TPN + SCFA for 72 hr, or standard TPN followed by TPN + SCFA for the final 6, 12, and 24 hr. SCFAs reduced (P SCFA groups and ileal GLUT2 protein in the 6-, 12-, and 24-hr SCFA groups (P < 0.05). SCFAs increased (P < 0.003) ileal proglucagon abundance following 6, 12, and 24 hr, and plasma GLP-2 concentration following 12 hr (P < 0.03). Jejunal c-myc expression was increased (P < 0.001) following 6, 12, and 24 hr of SCFAs. SCFAs increased ileal c-myc, c-jun, and c-fos expression following 24 hr (P < 0.02), 12 hr (P < 0.05) and 6, 12, and 24 hr (P=0.0001), respectively. In conclusion, systemic SCFAs increase plasma GLP-2 and ileal proglucagon mRNA, GLUT2 expression and protein, and c-myc, c-jun, and c-fos expression.

  20. Decreasing the stochasticity of mammalian gene expression by a synthetic gene circuit

    Science.gov (United States)

    Nevozhay, Dmitry; Zal, Tomasz; Balazsi, Gabor

    2012-02-01

    Gene therapy and functional genetic studies usually require precisely controlled and uniform gene expression in a population of cells for reliable level of protein production. Due to this requirement, stochastic gene expression is perceived as undesirable in these fields and ideally has to be minimized. The number of approaches for decreasing gene expression stochasticity in mammalian cells is limited. This creates an unmet need to develop new gene expression systems for this purpose. Based on earlier synthetic constructs in yeast, we developed and assessed a negative feedback-based mammalian gene circuit, with uniform and low level of stochasticity in gene expression at different levels of induction. In addition, this new synthetic construct enables highly precise gene expression control in mammalian cells, due to the linear dependence of gene expression on the inducer concentration applied to the system. This mammalian gene expression circuit has potential applicability for the development of new treatment modalities in gene therapy and research tools in functional genetics. In addition, this work creates a roadmap for moving synthetic gene circuits from microbes into mammalian cells.

  1. Researchers use Modified CRISPR Systems to Modulate Gene Expression on a Genomic Scale | Office of Cancer Genomics

    Science.gov (United States)

    The genetic engineering system, clustered regularly interspaced short palindromic repeats (CRISPR), has conventionally been used to inactivate genes by making targeted double stranded cuts in DNA. While CRISPR is a useful tool, it can only be used to create loss-of-function modifications and often causes off-target effects due to the disruptive mechanism by which it works. CTD2 researchers at the University of California, San Francisco recently addressed these shortcomings in a publication in Cell.

  2. Amplification of kinetic oscillations in gene expression

    Science.gov (United States)

    Zhdanov, V. P.

    2008-10-01

    Because of the feedbacks between the DNA transcription and mRNA translation, the gene expression in cells may exhibit bistability and oscillations. The deterministic and stochastic calculations presented illustrate how the bistable kinetics of expression of one gene in a cell can be influenced by the kinetic oscillations in the expression of another gene. Due to stability of the states of the bistable kinetics of gene 1 and the relatively small difference between the maximum and minimum protein amounts during the oscillations of gene 2, the induced oscillations of gene 1 are found to typically be related either to the low-or high-reactive state of this gene. The quality of the induced oscillations may be appreciably better than that of the inducing oscillations. This means that gene 1 can serve as an amplifier of the kinetic oscillations of gene 2.

  3. cis sequence effects on gene expression

    Directory of Open Access Journals (Sweden)

    Jacobs Kevin

    2007-08-01

    Full Text Available Abstract Background Sequence and transcriptional variability within and between individuals are typically studied independently. The joint analysis of sequence and gene expression variation (genetical genomics provides insight into the role of linked sequence variation in the regulation of gene expression. We investigated the role of sequence variation in cis on gene expression (cis sequence effects in a group of genes commonly studied in cancer research in lymphoblastoid cell lines. We estimated the proportion of genes exhibiting cis sequence effects and the proportion of gene expression variation explained by cis sequence effects using three different analytical approaches, and compared our results to the literature. Results We generated gene expression profiling data at N = 697 candidate genes from N = 30 lymphoblastoid cell lines for this study and used available candidate gene resequencing data at N = 552 candidate genes to identify N = 30 candidate genes with sufficient variance in both datasets for the investigation of cis sequence effects. We used two additive models and the haplotype phylogeny scanning approach of Templeton (Tree Scanning to evaluate association between individual SNPs, all SNPs at a gene, and diplotypes, with log-transformed gene expression. SNPs and diplotypes at eight candidate genes exhibited statistically significant (p cis sequence effects in our study, respectively. Conclusion Based on analysis of our results and the extant literature, one in four genes exhibits significant cis sequence effects, and for these genes, about 30% of gene expression variation is accounted for by cis sequence variation. Despite diverse experimental approaches, the presence or absence of significant cis sequence effects is largely supported by previously published studies.

  4. [The cloning, expression, purification and immunological identification of wild-type and mutant hepatitis B virus X gene in pGEX-6P-2 system].

    Science.gov (United States)

    Zhang, Xiao-Hui; Wang, Chang-Yuan; Tan, Bing-Qin; Li, Cheng; Du, Lei; Wang, Yong-Kang; Zhang, Hong-Hua; Dong, Ge-Feng

    2011-09-01

    To settle the foundation for the future research on the influence of wild and mutant (A1762T/ G1764A) HBV X gene on the progress of chronic HBV infection and hepatic tumorigenicity, wild and mutant (A1762T/G1764A) HBxAgs expression system was constructed. The wild and mutant (A1762T/ G1764A) HBV X genes were amplified with polymerase chain reaction (PCR) from HBV genome were inserted into pGEX-6P-2 and confirmed by sequencing respectively. Prokaryotic expression vectors pGEX-6P-2-hbvx(w) and pGEX-6P-2-hbvx(m) (A1762T/G1764A) were constructed and transformed to Trans1-blue; wild and mutant HBxAgs were expressed through IPTG induction respectively; after refolding of inclusion body, the wild and mutant HBxAgs were purified with GSTrap FF; and analysised by SDS-PAGE, Western blot and ELISA. SDS-PAGE analysis showed that the expression system was able to express target protein efficiently; the concentrations of purified wild HBxAg and mutant HBxAg were 4.88 mg/mL and 5.07 mg/mL respectively; Western blot analysis certified both the wild HBxAg and the mutant HBxAg could be recognized by the same monoclonal antibody against HBxAg; the two expressed fusion antigens coated in microtiter plate were able to react with the sera of HBV infected patients but not with the sera from healthy donors in ELISA. Results demonstrated that we successfully established a system for expression of hepatitis B x antigen and lay the foundation for further research on the role and molecular mechanisms of the mutant HBxAg in the progress of chronic HBV infection and hepatic tumorigenicity.

  5. Recombinant expression of placental growth factor in baculovirus expression system

    Directory of Open Access Journals (Sweden)

    Narges Arbabi

    2016-12-01

    Full Text Available Background: Angiogenesis or formation of new blood vessels is the most important factor in physiological and pathological conditions. Human Placental growth factor (hPLGF protein in is one of the most important proteins which stimulate angiogenesis. Baculovirus expression system has been used successfully to over express eukaryotic proteins in insect cells. This system uses a very strong viral promoter, AcNPV polyhedrin, for high level of protein expression. Methods: hPLGF gene cloned in pFastBac-HT vector and transformed in DH10Bac.The recombinant bacmid was extracted and used in SF9 insect cells and transfected by cellfectin method. Target protein expression was confirmed with Western blot. Results: Transferring of the recombinant vector into Bacmid was successful and the PLGF gene sequence was confirmed. PLGF and recombinant protein expression by Western blotting was confirmed. Conclusion: Baculovirus protein expression system expresses PLGF strongly and recombinant protein can be used in different tests.

  6. Overexpression of the Maize psbA Gene Enhances Drought Tolerance Through Regulating Antioxidant System, Photosynthetic Capability, and Stress Defense Gene Expression in Tobacco.

    Science.gov (United States)

    Huo, Yongjin; Wang, Meiping; Wei, Yangyang; Xia, Zongliang

    2015-01-01

    The psbA (encoding D1 protein) plays an important role in protecting photosystem II (PSII) from oxidative damage in higher plants. In our previous study, the role of the psbA from maize (Zea mays. L) in response to SO2 stress was characterized. To date, information about the involvement of the psbA gene in drought response is scarce. Here we found that overexpression (OE) of ZmpsbA showed increased D1 protein abundance and enhanced drought stress tolerance in tobacco. The drought-tolerant phenotypes of the OE lines were accompanied by increases of key antioxidant enzymes SOD, CAT, and POD activities, but decreases of hydrogen peroxide, malondialdehyde, and ion leakage. Further investigation showed that the OE plants had much less reductions than the wild-type in the net photosynthesis rate (Pn), stomatal conductance (Gs), and the maximal photochemical efficiency of PSII (Fv/Fm) during drought stress; indicating that OE of ZmpsbA may alleviate photosynthesis inhibition during drought. qRT-PCR analysis revealed that there was significantly increased expression of NtLEA5, NtERD10C, NtAREB, and NtCDPK2 in ZmpsbA-OE lines. Together, our results indicate that ZmpsbA improves drought tolerance in tobacco possibly by alleviating photosynthesis reduction, reducing reactive oxygen species accumulation and membrane damage, and modulating stress defense gene expression. ZmpsbA could be exploited for engineering drought-tolerant plants in molecular breeding of crops.

  7. Overexpression of the Maize psbA Gene Enhances Drought Tolerance Through Regulating Antioxidant System, Photosynthetic Capability, and Stress Defense Gene Expression in Tobacco

    Directory of Open Access Journals (Sweden)

    Yongjin eHuo

    2016-01-01

    Full Text Available The psbA (encoding D1 protein plays an important role in protecting photosystem II (PSII from oxidative damage in higher plants. In our previous study, the role of the psbA from maize (Zea mays. L in response to SO2 stress was characterized. To date, information about the involvement of the psbA gene in drought response is scarce. Here we found that overexpression (OE of ZmpsbA showed increased D1 protein abundance and enhanced drought stress tolerance in tobacco. The drought-tolerant phenotypes of the OE lines were accompanied by increases of key antioxidant enzymes SOD, CAT, and POD activities, but decreases of hydrogen peroxide, malondialdehyde and ion leakage. Further investigation showed that the OE plants had much less reductions than the wild-type in the net photosynthesis rate (Pn, stomatal conductance (Gs, and the maximal photochemical efficiency of PSII (Fv/Fm during drought stress; indicating that overexpression of ZmpsbA may alleviate photosynthesis inhibition during drought. qRT-PCR analysis revealed that there was significantly increased expression of NtLEA5, NtERD10C, NtAREB, and NtCDPK2 in ZmpsbA-OE lines. Together, our results indicate that ZmpsbA improves drought tolerance in tobacco possibly by alleviating photosynthesis reduction, reducing ROS accumulation and membrane damage, and modulating stress defense gene expression. ZmpsbA could be exploited for engineering drought-tolerant plants in molecular breeding of crops.

  8. Deriving Trading Rules Using Gene Expression Programming

    Directory of Open Access Journals (Sweden)

    Adrian VISOIU

    2011-01-01

    Full Text Available This paper presents how buy and sell trading rules are generated using gene expression programming with special setup. Market concepts are presented and market analysis is discussed with emphasis on technical analysis and quantitative methods. The use of genetic algorithms in deriving trading rules is presented. Gene expression programming is applied in a form where multiple types of operators and operands are used. This gives birth to multiple gene contexts and references between genes in order to keep the linear structure of the gene expression programming chromosome. The setup of multiple gene contexts is presented. The case study shows how to use the proposed gene setup to derive trading rules encoded by Boolean expressions, using a dataset with the reference exchange rates between the Euro and the Romanian leu. The conclusions highlight the positive results obtained in deriving useful trading rules.

  9. Gene Expression Profiling of Gastric Cancer

    Science.gov (United States)

    Marimuthu, Arivusudar; Jacob, Harrys K.C.; Jakharia, Aniruddha; Subbannayya, Yashwanth; Keerthikumar, Shivakumar; Kashyap, Manoj Kumar; Goel, Renu; Balakrishnan, Lavanya; Dwivedi, Sutopa; Pathare, Swapnali; Dikshit, Jyoti Bajpai; Maharudraiah, Jagadeesha; Singh, Sujay; Sameer Kumar, Ghantasala S; Vijayakumar, M.; Veerendra Kumar, Kariyanakatte Veeraiah; Premalatha, Chennagiri Shrinivasamurthy; Tata, Pramila; Hariharan, Ramesh; Roa, Juan Carlos; Prasad, T.S.K; Chaerkady, Raghothama; Kumar, Rekha Vijay; Pandey, Akhilesh

    2015-01-01

    Gastric cancer is the second leading cause of cancer death worldwide, both in men and women. A genomewide gene expression analysis was carried out to identify differentially expressed genes in gastric adenocarcinoma tissues as compared to adjacent normal tissues. We used Agilent’s whole human genome oligonucleotide microarray platform representing ~41,000 genes to carry out gene expression analysis. Two-color microarray analysis was employed to directly compare the expression of genes between tumor and normal tissues. Through this approach, we identified several previously known candidate genes along with a number of novel candidate genes in gastric cancer. Testican-1 (SPOCK1) was one of the novel molecules that was 10-fold upregulated in tumors. Using tissue microarrays, we validated the expression of testican-1 by immunohistochemical staining. It was overexpressed in 56% (160/282) of the cases tested. Pathway analysis led to the identification of several networks in which SPOCK1 was among the topmost networks of interacting genes. By gene enrichment analysis, we identified several genes involved in cell adhesion and cell proliferation to be significantly upregulated while those corresponding to metabolic pathways were significantly downregulated. The differentially expressed genes identified in this study are candidate biomarkers for gastric adenoacarcinoma. PMID:27030788

  10. Efficient expression of the yeast metallothionein gene in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Berka, T.; Shatzman, A.; Zimmerman, J.; Strickler, J.; Rosenberg, M.

    1988-01-01

    The yeast metallothionein gene CUP1 was cloned into a bacterial expression system to achieve efficient, controlled expression of the stable, unprocessed protein product. The Escherichia coli-synthesized yeast metallothionein bound copper, cadmium, zinc, indicating that the protein was functional. Furthermore, E. coli cells expressing CUP1 acquired a new, inducible ability to selectively sequester heavy metal ions from the growth medium.

  11. Modulation of gene expression made easy

    DEFF Research Database (Denmark)

    Solem, Christian; Jensen, Peter Ruhdal

    2002-01-01

    A new approach for modulating gene expression, based on randomization of promoter (spacer) sequences, was developed. The method was applied to chromosomal genes in Lactococcus lactis and shown to generate libraries of clones with broad ranges of expression levels of target genes. In one example...... beta-glucuronidase, resulting in an operon structure in which both genes are transcribed from a common promoter. We show that there is a linear correlation between the expressions of the two genes, which facilitates screening for mutants with suitable enzyme activities. In a second example, we show......, overexpression was achieved by introducing an additional gene copy into a phage attachment site on the chromosome. This resulted in a series of strains with phosphofructokinase activities from 1.4 to 11 times the wild-type activity level. In this example, the pfk gene was cloned upstream of a gusA gene encoding...

  12. Characterization of stem cells and cancer cells on the basis of gene expression profile stability, plasticity, and robustness: dynamical systems theory of gene expressions under cell-cell interaction explains mutational robustness of differentiated cells and suggests how cancer cells emerge.

    Science.gov (United States)

    Kaneko, Kunihiko

    2011-06-01

    Here I present and discuss a model that, among other things, appears able to describe the dynamics of cancer cell origin from the perspective of stable and unstable gene expression profiles. In identifying such aberrant gene expression profiles as lying outside the normal stable states attracted through development and normal cell differentiation, the hypothesis explains why cancer cells accumulate mutations, to which they are not robust, and why these mutations create a new stable state far from the normal gene expression profile space. Such cells are in strong contrast with normal cell types that appeared as an attractor state in the gene expression dynamical system under cell-cell interaction and achieved robustness to noise through evolution, which in turn also conferred robustness to mutation. In complex gene regulation networks, other aberrant cellular states lacking such high robustness are expected to remain, which would correspond to cancer cells.

  13. The application of the human beta-globin gene locus control region and murine erythroleukemia cell system to the expression and pharmacological characterization of human endothelin receptor subtypes.

    Science.gov (United States)

    Davies, A; Whiting, E; Bath, C; Tang, E; Brennand, J

    1995-06-01

    The cDNAs encoding both A and B subtypes of the human endothelin receptor have been inserted into mammalian cell expression vectors that utilize the human globin gene, locus control region. These constructs have been introduced into murine erythroleukemia cells and inducible high level expression of the receptors has been achieved (approximately 1.5-pM/mg membrane protein and approximately 13,500 binding sites/cell for both receptor subtypes). Cell lines expressing these receptors were obtained on a rapid time scale (3-4 weeks), facilitated by the need for the analysis of only small numbers of cell clones/receptor (approximately 6). Competitive binding assays with endothelin-1 gave IC50s of 130 +/- 30 pM for endothelin-A receptor and 160 +/- 30 pM for endothelin-B receptor. Similar studies with the different isoforms of endothelin, sarafatoxin-S6b and -S6c, BQ123 and BQ3020, all gave the expected selectivity profiles. The IC50s for all compounds were in close agreement with those reported for native receptors. Thus, this expression system, which has several advantages over other described expression systems, is capable of rapidly providing large quantities of receptor for detailed pharmacological analyses or drug screening. In addition, the expressed receptors display the expected pharmacological profiles in the absence of any complicating, competing interactions from other subtypes or binding sites.

  14. Approaches to systems biology. Four methods to study single-cell gene expression, cell motility, antibody reactivity, and respiratory metabolism

    DEFF Research Database (Denmark)

    Hagedorn, Peter

    : Transcript profiling of one cell type extracted from a complex tissue containing several cell types; observation and recording of cell motility; measurement of antibody reactivities using microarrays; and invivo measurement of free and bound NADH in mitochondria. Detailed statistical analysis of the data......To understand how complex systems, such as cells, function, comprehensive Measurements of their constituent parts must be made. This can be achieved by combining methods that are each optimized to measure specific parts of the system. Four such methods,each covering a different area, are presented...... from such measurements allows models of the system to be developed and tested. For each of the methods, such analysis and modelling approaches have beenapplied and are presented: Differentially regulated genes are identified and classified according to function; cell-specfic motility models...

  15. Positively regulated bacterial expression systems.

    Science.gov (United States)

    Brautaset, Trygve; Lale, Rahmi; Valla, Svein

    2009-01-01

    Regulated promoters are useful tools for many aspects related to recombinant gene expression in bacteria, including for high-level expression of heterologous proteins and for expression at physiological levels in metabolic engineering applications. In general, it is common to express the genes of interest from an inducible promoter controlled either by a positive regulator or by a repressor protein. In this review, we discuss established and potentially useful positively regulated bacterial promoter systems, with a particular emphasis on those that are controlled by the AraC-XylS family of transcriptional activators. The systems function in a wide range of microorganisms, including enterobacteria, soil bacteria, lactic bacteria and streptomycetes. The available systems that have been applied to express heterologous genes are regulated either by sugars (L-arabinose, L-rhamnose, xylose and sucrose), substituted benzenes, cyclohexanone-related compounds, ε-caprolactam, propionate, thiostrepton, alkanes or peptides. It is of applied interest that some of the inducers require the presence of transport systems, some are more prone than others to become metabolized by the host and some have been applied mainly in one or a limited number of species. Based on bioinformatics analyses, the AraC-XylS family of regulators contains a large number of different members (currently over 300), but only a small fraction of these, the XylS/Pm, AraC/P(BAD), RhaR-RhaS/rhaBAD, NitR/PnitA and ChnR/Pb regulator/promoter systems, have so far been explored for biotechnological applications.

  16. Construction of a novel cell-surface display system for heterologous gene expression in Escherichia coli by using an outer membrane protein of Zymomonas mobilis as anchor motif.

    Science.gov (United States)

    He, Ming-Xiong; Feng, Hong; Zhang, Yi-Zheng

    2008-12-01

    A novel bacterial cell-surface display system was developed in Escherichia coli using omp1, a hypothetical outer membrane protein of Zymomonas mobilis. By using this system, we successfully expressed beta-amylase gene of sweet potato in E. coli. The display of enzyme on the membrane surface was also confirmed. The recombinant beta-amylase showed to significantly increase hydrolytic activity toward soluble starch. Our results provide a basis for constructing an engineered Z. mobilis strain directly fermenting raw starch to produce ethanol.

  17. Gene expression profiling during murine tooth development

    Directory of Open Access Journals (Sweden)

    Maria A dos Santos silva Landin

    2012-07-01

    Full Text Available The aim of this study was to describe the expression of genes, including ameloblastin (Ambn, amelogenin X chromosome (Amelx and enamelin (Enam during early (pre-secretory tooth development. The expression of these genes has predominantly been studied at post-secretory stages. Deoxyoligonucleotide microarrays were used to study gene expression during development of the murine first molar tooth germ at 24h intervals, starting at the eleventh embryonic day (E11.5 and up to the seventh day after birth (P7. The profile search function of Spotfire software was used to select genes with similar expression profile as the enamel genes (Ambn, Amelx and Enam. Microarray results where validated using real-time Reverse Transcription-Polymerase Chain Reaction (real-time RT-PCR, and translated proteins identified by Western blotting. In situ localisation of the Ambn, Amelx and Enam mRNAs were monitored from E12.5 to E17.5 using deoxyoligonucleotide probes. Bioinformatics analysis was used to associate biological functions with differentially (p ≤0.05 expressed (DE genes.Microarray results showed a total of 4362 genes including Ambn, Amelx and Enam to be significant differentially expressed throughout the time-course. The expression of the three enamel genes was low at pre-natal stages (E11.5-P0 increasing after birth (P1-P7. Profile search lead to isolation of 87 genes with significantly similar expression to the three enamel proteins. The mRNAs expressed in dental epithelium and epithelium derived cells. Although expression of Ambn, Amelx and Enam were lower during early tooth development compared to secretory stages enamel proteins were detectable by Western blotting. Bioinformatic analysis associated the 87 genes with multiple biological functions. Around thirty-five genes were associated with fifteen transcription factors.

  18. Extensive neuroadaptive changes in cortical gene-transcript expressions of the glutamate system in response to repeated intermittent MDMA administration in adolescent rats

    Directory of Open Access Journals (Sweden)

    Malki Rana

    2008-04-01

    Full Text Available Abstract Background Many studies have focused on the implication of the serotonin and dopamine systems in neuroadaptive responses to the recreational drug 3,4-methylenedioxy-metamphetamine (MDMA. Less attention has been given to the major excitatory neurotransmitter glutamate known to be implicated in schizophrenia and drug addiction. The aim of the present study was to investigate the effect of repeated intermittent MDMA administration upon gene-transcript expression of the glutamate transporters (EAAT1, EAAT2-1, EAAT2-2, the glutamate receptor subunits of AMPA (GluR1, GluR2, GluR3, the glutamate receptor subunits of NMDA (NR1, NR2A and NR2B, as well as metabotropic glutamate receptors (mGluR1, mGluR2, mGluR3, mGluR5 in six different brain regions. Adolescent male Sprague Dawley rats received MDMA at the doses of 3 × 1 and 3 × 5 mg/kg/day, or 3× vehicle 3 hours apart, every 7th day for 4 weeks. The gene-transcript levels were assessed using real-time PCR validated with a range of housekeeping genes. Results The findings showed pronounced enhancements in gene-transcript expression of GluR2, mGluR1, mGluR5, NR1, NR2A, NR2B, EAAT1, and EAAT2-2 in the cortex at bregma +1.6. In the caudate putamen, mRNA levels of GluR3, NR2A, and NR2B receptor subunits were significantly increased. In contrast, the gene-transcript expression of GluR1 was reduced in the hippocampus. In the hypothalamus, there was a significant increase of GluR1, GluR3, mGluR1, and mGluR3 gene-transcript expressions. Conclusion Repeated intermittent MDMA administration induces neuroadaptive changes in gene-transcript expressions of glutamatergic NMDA and AMPA receptor subunits, metabotropic receptors and transporters in regions of the brain regulating reward-related associative learning, cognition, and memory and neuro-endocrine functions.

  19. Gene Expression Patterns in Ovarian Carcinomas

    Science.gov (United States)

    Schaner, Marci E.; Ross, Douglas T.; Ciaravino, Giuseppe; Sørlie, Therese; Troyanskaya, Olga; Diehn, Maximilian; Wang, Yan C.; Duran, George E.; Sikic, Thomas L.; Caldeira, Sandra; Skomedal, Hanne; Tu, I-Ping; Hernandez-Boussard, Tina; Johnson, Steven W.; O'Dwyer, Peter J.; Fero, Michael J.; Kristensen, Gunnar B.; Børresen-Dale, Anne-Lise; Hastie, Trevor; Tibshirani, Robert; van de Rijn, Matt; Teng, Nelson N.; Longacre, Teri A.; Botstein, David; Brown, Patrick O.; Sikic, Branimir I.

    2003-01-01

    We used DNA microarrays to characterize the global gene expression patterns in surface epithelial cancers of the ovary. We identified groups of genes that distinguished the clear cell subtype from other ovarian carcinomas, grade I and II from grade III serous papillary carcinomas, and ovarian from breast carcinomas. Six clear cell carcinomas were distinguished from 36 other ovarian carcinomas (predominantly serous papillary) based on their gene expression patterns. The differences may yield insights into the worse prognosis and therapeutic resistance associated with clear cell carcinomas. A comparison of the gene expression patterns in the ovarian cancers to published data of gene expression in breast cancers revealed a large number of differentially expressed genes. We identified a group of 62 genes that correctly classified all 125 breast and ovarian cancer specimens. Among the best discriminators more highly expressed in the ovarian carcinomas were PAX8 (paired box gene 8), mesothelin, and ephrin-B1 (EFNB1). Although estrogen receptor was expressed in both the ovarian and breast cancers, genes that are coregulated with the estrogen receptor in breast cancers, including GATA-3, LIV-1, and X-box binding protein 1, did not show a similar pattern of coexpression in the ovarian cancers. PMID:12960427

  20. Microanalysis of gene expression in cultured cells

    NARCIS (Netherlands)

    E. van der Veer (Eveliene)

    1982-01-01

    textabstractIn this thesis two aspects of gene expression in cultured cells have been studied: the heterogeneity in gene expression in relation with the development and application of microchemical techniques for the prenatal diagnosis of inborn errors of metabolism and the possibility of inducing g

  1. Arabidopsis gene expression patterns during spaceflight

    Science.gov (United States)

    Paul, A.-L.; Ferl, R. J.

    The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments resulted in the differential expression of hundreds of genes. A 5 day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the β -Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on two fronts. First, expression patterns visualized with the Adh/GUS transgene were used to address specifically the possibility that spaceflight induces a hypoxic stress response, and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. (Paul et al., Plant Physiol. 2001, 126:613). Second, genome-wide patterns of native gene expression were evaluated utilizing the Affymetrix ATH1 GeneChip? array of 8,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes identified with the arrays was further characterized with quantitative Real-Time RT PCR (ABI - TaqmanTM). Comparison of the patterns of expression for arrays of hybridized with RNA isolated from plants exposed to spaceflight compared to the control arrays revealed hundreds of genes that were differentially expressed in response to spaceflight, yet most genes that are hallmarks of hypoxic stress were unaffected. These results will be discussed in light of current models for plant responses to the spaceflight environment, and with regard to potential future flight opportunities.

  2. Long SAGE analysis of genes differentially expressed in the midgut ...

    African Journals Online (AJOL)

    USER

    Both the technology of female egg balanced-lethal system for male ... RNA isolation. All ribonucleic acid (RNAs) were prepared from two males and two females .... There were also some genes with very high expression ..... Nucleic Acids Res.,.

  3. Expression of Sox genes in tooth development.

    Science.gov (United States)

    Kawasaki, Katsushige; Kawasaki, Maiko; Watanabe, Momoko; Idrus, Erik; Nagai, Takahiro; Oommen, Shelly; Maeda, Takeyasu; Hagiwara, Nobuko; Que, Jianwen; Sharpe, Paul T; Ohazama, Atsushi

    2015-01-01

    Members of the Sox gene family play roles in many biological processes including organogenesis. We carried out comparative in situ hybridization analysis of seventeen sox genes (Sox1-14, 17, 18, 21) during murine odontogenesis from the epithelial thickening to the cytodifferentiation stages. Localized expression of five Sox genes (Sox6, 9, 13, 14 and 21) was observed in tooth bud epithelium. Sox13 showed restricted expression in the primary enamel knots. At the early bell stage, three Sox genes (Sox8, 11, 17 and 21) were expressed in pre-ameloblasts, whereas two others (Sox5 and 18) showed expression in odontoblasts. Sox genes thus showed a dynamic spatio-temporal expression during tooth development.

  4. Expression of Sox genes in tooth development

    Science.gov (United States)

    KAWASAKI, KATSUSHIGE; KAWASAKI, MAIKO; WATANABE, MOMOKO; IDRUS, ERIK; NAGAI, TAKAHIRO; OOMMEN, SHELLY; MAEDA, TAKEYASU; HAGIWARA, NOBUKO; QUE, JIANWEN; SHARPE, PAUL T.; OHAZAMA, ATSUSHI

    2017-01-01

    Members of the Sox gene family play roles in many biological processes including organogenesis. We carried out comparative in situ hybridization analysis of seventeen sox genes (Sox1-14, 17, 18, 21) during murine odontogenesis from the epithelial thickening to the cytodifferentiation stages. Localized expression of five Sox genes (Sox6, 9, 13, 14 and 21) was observed in tooth bud epithelium. Sox13 showed restricted expression in the primary enamel knots. At the early bell stage, three Sox genes (Sox8, 11, 17 and 21) were expressed in pre-ameloblasts, whereas two others (Sox5 and 18) showed expression in odontoblasts. Sox genes thus showed a dynamic spatio-temporal expression during tooth development. PMID:26864488

  5. Gene set analysis for longitudinal gene expression data

    Directory of Open Access Journals (Sweden)

    Piepho Hans-Peter

    2011-07-01

    Full Text Available Abstract Background Gene set analysis (GSA has become a successful tool to interpret gene expression profiles in terms of biological functions, molecular pathways, or genomic locations. GSA performs statistical tests for independent microarray samples at the level of gene sets rather than individual genes. Nowadays, an increasing number of microarray studies are conducted to explore the dynamic changes of gene expression in a variety of species and biological scenarios. In these longitudinal studies, gene expression is repeatedly measured over time such that a GSA needs to take into account the within-gene correlations in addition to possible between-gene correlations. Results We provide a robust nonparametric approach to compare the expressions of longitudinally measured sets of genes under multiple treatments or experimental conditions. The limiting distributions of our statistics are derived when the number of genes goes to infinity while the number of replications can be small. When the number of genes in a gene set is small, we recommend permutation tests based on our nonparametric test statistics to achieve reliable type I error and better power while incorporating unknown correlations between and within-genes. Simulation results demonstrate that the proposed method has a greater power than other methods for various data distributions and heteroscedastic correlation structures. This method was used for an IL-2 stimulation study and significantly altered gene sets were identified. Conclusions The simulation study and the real data application showed that the proposed gene set analysis provides a promising tool for longitudinal microarray analysis. R scripts for simulating longitudinal data and calculating the nonparametric statistics are posted on the North Dakota INBRE website http://ndinbre.org/programs/bioinformatics.php. Raw microarray data is available in Gene Expression Omnibus (National Center for Biotechnology Information with

  6. Estrogen-triggered delays in mammary gland gene expression during the estrous cycle: evidence for a novel timing system.

    Science.gov (United States)

    Silberstein, Gary B; Van Horn, Katharine; Hrabeta-Robinson, Eva; Compton, Jennifer

    2006-08-01

    During the estrous cycle and beginning in estrus, the mammary gland undergoes pregnancy-like development that depends on transcriptional regulation by the estrogen and progesterone receptors (ER, PR) and Pax-2 as well as the action of the growth factors Wnt-4 and RANKL. In this report, we first describe the decay and delayed expression of ERalpha, PR, and Pax-2 proteins as well as depression of Wnt-4 and RANKL mRNA coincident with the strong estrogen surge in proestrus. In time-course studies using ovari-ectomized mice, a single estrogen injection replicated these delays and caused an 18 h delay in Wnt-4 expression. Molecular time-delay systems are at the core of cellular cycles, most notably the circadian clock, and depend on proteasome degradation of transcriptional regulators that exhibit dedicated timing functions. The cytoplasmic dynamics of these regulators govern delay duration through negative transcription/translation feedback loops. A proteasome inhibitor, PS-341, blocked estrogen-stimulated ERalpha, PR, and Pax-2 decay and proteasome chymotryptic activity, assayed using a fluorogenic substrate, was elevated in proestrus correlating with the depletion of the transcription factors. The 18-h delay in Wnt-4 induction corresponded to the turnover time of Pax-2 protein in the cytoplasm and was eliminated in Pax-2 knockout mammary tissue, demonstrating that Pax-2 has a unique timing function. The patterns of estrogen-triggered ERalpha, PR, and Pax-2 turnover were consistent with a negative transcriptional feedback. Retarding the expression of ERalpha, PR, and Pax-2 may optimize preparations for pregnancy by coordinating expression of critical receptors and transcription factors with rising estrogen and progesterone levels in estrus. The estrogen surge in proestrus has no defined mammotropic function. This study provides the first evidence that it is a synchronizing signal triggering proteasome-dependent turnover of mammary gland ERalpha, PR, and Pax-2. We

  7. Construction of prokaryotic expression system of ItB-ureB fusion gene and identification of the recombinant protein immunity and adjuvanticity

    Institute of Scientific and Technical Information of China (English)

    Jie Yan; Yuan Wang; Shi-He Shao; Ya-Fei Mao; Hua-Wen Li; Yi-Hui Luo

    2004-01-01

    AIM: To construct ItB-ureB fusion gene and its prokaryotic expression system and identify immunity and adjuvanticity of the expressed recombinant protein.METHODS: The ureB gene from a clinical Helicobacterpylori(Hpylori) strain Y06 and the ItB gene from Escherichiacoli(E coli) strain 44851 were linked into ItB-ureB fusiongene by PCR. The fusion gene sequence was analyzedafter T-A cloning. A prokaryotic recombinant expressionvector pET32a inserted with ItB-ureB fusion gene (pET32aItB-ureB) was constructed. Expression of the recombinantLTB-UreB protein (rLTB-UreB)in E. coliBL21DE3 inducedby isopropylthio-β-D-galactoside (IPTG) at differentconcentrations was detected by SDS-PAGE. Western blot assays were used to examine the immunoreaction of rLTBUreB by a commercial antibody against whole cell of H pylori and a self-prepared rabbit anti-rUreB serum, respectively, and determine the antigenicity of the recombinant proteinon inducing specific antibody in rabbits. GM1-ELISA wasused to demonstrate the adjuvanticity of rLTB-UreB. Immunoreaction of rLTB-UreB to the UreB antibody positivesera from 125 gastric patients was determined by using ELISA. RESULTS: In comparison with the corresponding sequences of original genes, the nucleotide sequence homologies of the cloned ItB-ureB fusion gene were 100%. IPTG withdifferent dosages of 0.1-1.0 mmol/L could efficiently inducepET32a-ItB-ureB-E.coli BL21DE3 to express the rLTB-UreB. The output of the target recombinant protein expressed by pET32a-ureB-E. coli BL21DE3 was approximately 35%of the total bacterial proteins. rLTB-UreB mainly presented in the form of inclusion body. Western blotting results demonstrated that rLTB-UreB could combine with the commercial antibody against whole cell of H pylori andanti-rUreB serum as well as induce rabbit to produce specific antibody. The strong ability of rLTB-UreB bindingbovine GM1 indicated the existence of adjuvanticity of the recombinant protein. All the UreB antibody positive sera from

  8. Gene expression in developing watermelon fruit

    Science.gov (United States)

    Wechter, W Patrick; Levi, Amnon; Harris, Karen R; Davis, Angela R; Fei, Zhangjun; Katzir, Nurit; Giovannoni, James J; Salman-Minkov, Ayelet; Hernandez, Alvaro; Thimmapuram, Jyothi; Tadmor, Yaakov; Portnoy, Vitaly; Trebitsh, Tova

    2008-01-01

    Background Cultivated watermelon form large fruits that are highly variable in size, shape, color, and content, yet have extremely narrow genetic diversity. Whereas a plethora of genes involved in cell wall metabolism, ethylene biosynthesis, fruit softening, and secondary metabolism during fruit development and ripening have been identified in other plant species, little is known of the genes involved in these processes in watermelon. A microarray and quantitative Real-Time PCR-based study was conducted in watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] in order to elucidate the flow of events associated with fruit development and ripening in this species. RNA from three different maturation stages of watermelon fruits, as well as leaf, were collected from field grown plants during three consecutive years, and analyzed for gene expression using high-density photolithography microarrays and quantitative PCR. Results High-density photolithography arrays, composed of probes of 832 EST-unigenes from a subtracted, fruit development, cDNA library of watermelon were utilized to examine gene expression at three distinct time-points in watermelon fruit development. Analysis was performed with field-grown fruits over three consecutive growing seasons. Microarray analysis identified three hundred and thirty-five unique ESTs that are differentially regulated by at least two-fold in watermelon fruits during the early, ripening, or mature stage when compared to leaf. Of the 335 ESTs identified, 211 share significant homology with known gene products and 96 had no significant matches with any database accession. Of the modulated watermelon ESTs related to annotated genes, a significant number were found to be associated with or involved in the vascular system, carotenoid biosynthesis, transcriptional regulation, pathogen and stress response, and ethylene biosynthesis. Ethylene bioassays, performed with a closely related watermelon genotype with a similar

  9. Gene expression in developing watermelon fruit

    Directory of Open Access Journals (Sweden)

    Hernandez Alvaro

    2008-06-01

    Full Text Available Abstract Background Cultivated watermelon form large fruits that are highly variable in size, shape, color, and content, yet have extremely narrow genetic diversity. Whereas a plethora of genes involved in cell wall metabolism, ethylene biosynthesis, fruit softening, and secondary metabolism during fruit development and ripening have been identified in other plant species, little is known of the genes involved in these processes in watermelon. A microarray and quantitative Real-Time PCR-based study was conducted in watermelon [Citrullus lanatus (Thunb. Matsum. & Nakai var. lanatus] in order to elucidate the flow of events associated with fruit development and ripening in this species. RNA from three different maturation stages of watermelon fruits, as well as leaf, were collected from field grown plants during three consecutive years, and analyzed for gene expression using high-density photolithography microarrays and quantitative PCR. Results High-density photolithography arrays, composed of probes of 832 EST-unigenes from a subtracted, fruit development, cDNA library of watermelon were utilized to examine gene expression at three distinct time-points in watermelon fruit development. Analysis was performed with field-grown fruits over three consecutive growing seasons. Microarray analysis identified three hundred and thirty-five unique ESTs that are differentially regulated by at least two-fold in watermelon fruits during the early, ripening, or mature stage when compared to leaf. Of the 335 ESTs identified, 211 share significant homology with known gene products and 96 had no significant matches with any database accession. Of the modulated watermelon ESTs related to annotated genes, a significant number were found to be associated with or involved in the vascular system, carotenoid biosynthesis, transcriptional regulation, pathogen and stress response, and ethylene biosynthesis. Ethylene bioassays, performed with a closely related watermelon

  10. FARO server: Meta-analysis of gene expression by matching gene expression signatures to a compendium of public gene expression data

    DEFF Research Database (Denmark)

    Manijak, Mieszko P.; Nielsen, Henrik Bjørn

    2011-01-01

    BACKGROUND: Although, systematic analysis of gene annotation is a powerful tool for interpreting gene expression data, it sometimes is blurred by incomplete gene annotation, missing expression response of key genes and secondary gene expression responses. These shortcomings may be partially...... circumvented by instead matching gene expression signatures to signatures of other experiments. FINDINGS: To facilitate this we present the Functional Association Response by Overlap (FARO) server, that match input signatures to a compendium of 242 gene expression signatures, extracted from more than 1700...

  11. A hammerhead ribozyme inhibits ADE1 gene expression in yeast.

    Science.gov (United States)

    Ferbeyre, G; Bratty, J; Chen, H; Cedergren, R

    1995-03-21

    To study factors that affect in vivo ribozyme (Rz) activity, a model system has been devised in Saccharomyces cerevisiae based on the inhibition of ADE1 gene expression. This gene was chosen because Rz action can be evaluated visually by the Red phenotype produced when the activity of the gene product is inhibited. Different plasmid constructs allowed the expression of the Rz either in cis or in trans with respect to ADE1. Rz-related inhibition of ADE1 expression was correlated with a Red phenotype and a diminution of ADE1 mRNA levels only when the Rz gene was linked 5' to ADE1. The presence of the expected 3' cleavage fragment was demonstrated using a technique combining RNA ligation and PCR. This yeast system and detection technique are suited to the investigation of general factors affecting Rz-catalyzed inhibition of gene expression under in vivo conditions.

  12. Application of optically-induced-dielectrophoresis in microfluidic system for purification of circulating tumour cells for gene expression analysis- Cancer cell line model

    Science.gov (United States)

    Chiu, Tzu-Keng; Chou, Wen-Pin; Huang, Song-Bin; Wang, Hung-Ming; Lin, Yung-Chang; Hsieh, Chia-Hsun; Wu, Min-Hsien

    2016-09-01

    Circulating tumour cells (CTCs) in a blood circulation system are associated with cancer metastasis. The analysis of the drug-resistance gene expression of cancer patients’ CTCs holds promise for selecting a more effective therapeutic regimen for an individual patient. However, the current CTC isolation schemes might not be able to harvest CTCs with sufficiently high purity for such applications. To address this issue, this study proposed to integrate the techniques of optically induced dielectrophoretic (ODEP) force-based cell manipulation and fluorescent microscopic imaging in a microfluidic system to further purify CTCs after the conventional CTC isolation methods. In this study, the microfluidic system was developed, and its optimal operating conditions and performance for CTC isolation were evaluated. The results revealed that the presented system was able to isolate CTCs with cell purity as high as 100%, beyond what is possible using the previously existing techniques. In the analysis of CTC gene expression, therefore, this method could exclude the interference of leukocytes in a cell sample and accordingly contribute to higher analytical sensitivity, as demonstrated in this study. Overall, this study has presented an ODEP-based microfluidic system capable of simply and effectively isolating a specific cell species from a cell mixture.

  13. Expressing exogenous genes in newts by transgenesis.

    Science.gov (United States)

    Casco-Robles, Martin Miguel; Yamada, Shouta; Miura, Tomoya; Nakamura, Kenta; Haynes, Tracy; Maki, Nobuyasu; Del Rio-Tsonis, Katia; Tsonis, Panagiotis A; Chiba, Chikafumi

    2011-05-01

    The great regenerative abilities of newts provide the impetus for studies at the molecular level. However, efficient methods for gene regulation have historically been quite limited. Here we describe a protocol for transgenically expressing exogenous genes in the newt Cynops pyrrhogaster. This method is simple: a reaction mixture of I-SceI meganuclease and a plasmid DNA carrying a transgene cassette flanked by the enzyme recognition sites is directly injected into fertilized eggs. The protocol achieves a high efficiency of transgenesis, comparable to protocols used in other animal systems, and it provides a practical number of transgenic newts (∼20% of injected embryos) that survive beyond metamorphosis and that can be applied to regenerative studies. The entire protocol for obtaining transgenic adult newts takes 4-5 months.

  14. Analysis of immune system gene expression in small rheumatoid arthritis biopsies using a combination of subtractive hybridization and high-density cDNA arrays.

    Science.gov (United States)

    Zanders, E D; Goulden, M G; Kennedy, T C; Kempsell, K E

    2000-01-13

    Subtractive hybridization of cDNAs generated from synovial RNA which had been isolated from patients with rheumatoid arthritis (RA) or normal controls was used in conjunction with high-density array hybridization to identify genes of immunological interest. The method was designed to detect gene expression in small needle biopsy specimens by means of a prior amplification of nanogram amounts of total RNA to full-length cDNA using PCR. The latter was cut with Rsa I, ligated with adapters, hybridized with unmodified driver cDNA, and subjected to suppression subtraction PCR. Differentially expressed products were cloned into E. coli and picked into 384 well plates. Inserts were obtained by PCR across the multiple cloning site, and the products arrayed at high density on nylon filters. The subtracted cDNAs were also labelled by random priming for use as probes for library screening. The libraries chosen were the subtracted one described above and a set of 45,000 ESTs from the I.M. A.G.E consortium. Clones showing positive hybridization were identified by sequence analysis and homology searching. The results showed that the subtracted hybridization approach could identify many gene fragments expressed at different levels, the most abundant being immunoglobulins and HLA-DR. The expression profile was characteristic of macrophage, B cell and plasma cell infiltration with evidence of interferon induction. In addition, a significant number of sequences without matches in the nucleotide databases were obtained, this demonstrates the utility of the method in finding novel gene fragments for further characterisation as potential members of the immune system. Although RA was studied here, the technology is applicable to any disease process even in cases where amounts of tissue may be limited.

  15. Etk/Bmx mediates expression of stress-induced adaptive genes VEGF, PAI-1, and iNOS via multiple signaling cascades in different cell systems.

    Science.gov (United States)

    Chau, Cindy H; Clavijo, Carlos A; Deng, Hong-Tao; Zhang, Qunzhou; Kim, Kwang-Jin; Qiu, Yun; Le, Anh D; Ann, David K

    2005-08-01

    We recently showed that Etk/Bmx, a member of the Tec family of nonreceptor protein tyrosine kinases, promotes tight junction formation during chronic hypoxic exposure and augments normoxic VEGF expression via a feedforward mechanism. Here we further characterized Etk's role in potentiating hypoxia-induced gene expression in salivary epithelial Pa-4 cells. Using transient transfection in conditionally activated Etk (DeltaEtk:ER) cells, we demonstrated that Etk enhances hypoxia-response element-dependent reporter activation in normoxia and hypoxia. This Etk-driven reporter activation is ameliorated by treatment with wortmannin or LFM-A13. Using lentivirus-mediated gene delivery and small interfering RNA, we provided direct evidence that hypoxia leads to transient Etk and Akt activation and hypoxia-mediated Akt activation is Etk dependent. Northern blot analyses confirmed that Etk activation led to induction of steady-state mRNA levels of endogenous VEGF and plasminogen activator inhibitor (PAI)-1, a hallmark of hypoxia-mediated gene regulation. We also demonstrated that Etk utilizes a phosphatidylinositol 3-kinase/Akt pathway to promote reporter activation driven by NF-kappaB, another oxygen-sensitive transcription factor, and to augment cytokine-induced inducible nitric oxide synthase expression in endothelial cells. To establish the clinical relevance of Etk-induced, hypoxia-mediated gene regulation, we examined Etk expression in keloid, which has elevated VEGF and PAI-1. We found that Etk is overexpressed in keloid (but not normal skin) tissues. The differential steady-state Etk protein levels were further confirmed in primary fibroblast cultures derived from these tissues, suggesting an Etk role in tissue fibrosis. Our results provide further understanding of Etk function within multiple signaling cascades to govern adaptive cytoprotection against extracellular stress in different cell systems, salivary epithelial cells, brain endothelial cells, and dermal

  16. Transgene expression systems in the Triticeae cereals.

    Science.gov (United States)

    Hensel, Götz; Himmelbach, Axel; Chen, Wanxin; Douchkov, Dimitar K; Kumlehn, Jochen

    2011-01-01

    The control of transgene expression is vital both for the elucidation of gene function and for the engineering of transgenic crops. Given the dominance of the Triticeae cereals in the agricultural economy of the temperate world, the development of well-performing transgene expression systems of known functionality is of primary importance. Transgenes can be expressed either transiently or stably. Transient expression systems based on direct or virus-mediated gene transfer are particularly useful in situations where the need is to rapidly screen large numbers of genes. However, an unequivocal understanding of gene function generally requires that a transgene functions throughout the plant's life and is transmitted through the sexual cycle, since this alone allows its effect to be decoupled from the plant's response to the generally stressful gene transfer event. Temporal, spatial and quantitative control of a transgene's expression depends on its regulatory environment, which includes both its promoter and certain associated untranslated region sequences. While many transgenic approaches aim to manipulate plant phenotype via ectopic gene expression, a transgene sequence can be also configured to down-regulate the expression of its endogenous counterpart, a strategy which exploits the natural gene silencing machinery of plants. In this review, current technical opportunities for controlling transgene expression in the Triticeae species are described. Apart from protocols for transient and stable gene transfer, the choice of promoters and other untranslated regulatory elements, we also consider signal peptides, as they too govern the abundance and particularly the sub-cellular localization of transgene products.

  17. Characterization and differentiation of equine experimental local and early systemic inflammation by expression responses of inflammation-related genes in peripheral blood leukocytes

    DEFF Research Database (Denmark)

    Vinther, Anne Mette L; Heegaard, Peter M. H.; Skovgaard, Kerstin;

    2016-01-01

    Local inflammation may progress into systemic inflammation. To increase our understanding of the basic immunological processes during transition of equine local inflammation into a systemic state, investigation into the equine systemic immune response to local inflammation is warranted. Therefore......, the aim of this study was to investigate the innate peripheral blood leukocyte (PBL) immune response to local inflammation in horses, and to compare this response with the PBL immune response during the early phase of acute systemic inflammation. Expression of 22 selected inflammation-related genes...... synovitis and mild systemic inflammation of approximately 24 h duration was confirmed by clinical and paraclinical observations in LI and SI horses, respectively. In the LI group, samples obtained 3-16 h post-injection showed distinct clustering in the PCA compared with baseline levels, indicating...

  18. Regulation of amphiregulin gene expression by β-catenin signaling in human hepatocellular carcinoma cells: a novel crosstalk between FGF19 and the EGFR system.

    Science.gov (United States)

    Latasa, Maria U; Salis, Fabiana; Urtasun, Raquel; Garcia-Irigoyen, Oihane; Elizalde, Maria; Uriarte, Iker; Santamaria, Monica; Feo, Francesco; Pascale, Rosa M; Prieto, Jesús; Berasain, Carmen; Avila, Matías A

    2012-01-01

    Hepatocellular carcinoma (HCC) is the most prevalent liver tumor and a deadly disease with limited therapeutic options. Dysregulation of cell signaling pathways is a common denominator in tumorigenesis, including hepatocarcinogenesis. The epidermal growth factor receptor (EGFR) signaling system is commonly activated in HCC, and is currently being evaluated as a therapeutic target in combination therapies. We and others have identified a central role for the EGFR ligand amphiregulin (AR) in the proliferation, survival and drug resistance of HCC cells. AR expression is frequently up-regulated in HCC tissues and cells through mechanisms not completely known. Here we identify the β-catenin signaling pathway as a novel mechanism leading to transcriptional activation of the AR gene in human HCC cells. Activation of β-catenin signaling, or expression of the T41A β-catenin active mutant, led to the induction of AR expression involving three specific β-catenin-Tcf responsive elements in its proximal promoter. We demonstrate that HCC cells expressing the T41A β-catenin active mutant show enhanced proliferation that is dependent in part on AR expression and EGFR signaling. We also demonstrate here a novel cross-talk of the EGFR system with fibroblast growth factor 19 (FGF19). FGF19 is a recently identified driver gene in hepatocarcinogenesis and an activator of β-catenin signaling in HCC and colon cancer cells. We show that FGF19 induced AR gene expression through the β-catenin pathway in human HCC cells. Importantly, AR up-regulation and EGFR signaling participated in the induction of cyclin D1 and cell proliferation elicited by FGF19. Finally, we demonstrate a positive correlation between FGF19 and AR expression in human HCC tissues, therefore supporting in clinical samples our experimental observations. These findings identify the AR/EGFR system as a key mediator of FGF19 responses in HCC cells involving β-catenin signaling, and suggest that combined targeting of

  19. Regulation of amphiregulin gene expression by β-catenin signaling in human hepatocellular carcinoma cells: a novel crosstalk between FGF19 and the EGFR system.

    Directory of Open Access Journals (Sweden)

    Maria U Latasa

    Full Text Available Hepatocellular carcinoma (HCC is the most prevalent liver tumor and a deadly disease with limited therapeutic options. Dysregulation of cell signaling pathways is a common denominator in tumorigenesis, including hepatocarcinogenesis. The epidermal growth factor receptor (EGFR signaling system is commonly activated in HCC, and is currently being evaluated as a therapeutic target in combination therapies. We and others have identified a central role for the EGFR ligand amphiregulin (AR in the proliferation, survival and drug resistance of HCC cells. AR expression is frequently up-regulated in HCC tissues and cells through mechanisms not completely known. Here we identify the β-catenin signaling pathway as a novel mechanism leading to transcriptional activation of the AR gene in human HCC cells. Activation of β-catenin signaling, or expression of the T41A β-catenin active mutant, led to the induction of AR expression involving three specific β-catenin-Tcf responsive elements in its proximal promoter. We demonstrate that HCC cells expressing the T41A β-catenin active mutant show enhanced proliferation that is dependent in part on AR expression and EGFR signaling. We also demonstrate here a novel cross-talk of the EGFR system with fibroblast growth factor 19 (FGF19. FGF19 is a recently identified driver gene in hepatocarcinogenesis and an activator of β-catenin signaling in HCC and colon cancer cells. We show that FGF19 induced AR gene expression through the β-catenin pathway in human HCC cells. Importantly, AR up-regulation and EGFR signaling participated in the induction of cyclin D1 and cell proliferation elicited by FGF19. Finally, we demonstrate a positive correlation between FGF19 and AR expression in human HCC tissues, therefore supporting in clinical samples our experimental observations. These findings identify the AR/EGFR system as a key mediator of FGF19 responses in HCC cells involving β-catenin signaling, and suggest that

  20. Cell surface expression system for the display of heterologous gene products using chimeric flagellin fusions of bacillus halodurans isolate

    CSIR Research Space (South Africa)

    Du Plessis, A

    2006-10-01

    Full Text Available N-terminal sequencing gave rise to homology to flagellin protein, product of the hag gene. protein, product of the hag gene. Gene was cloned by using degenerate primers and inverse PCR. The gene sequence as well as the up- and down- stream regions...

  1. The functional landscape of mouse gene expression

    Directory of Open Access Journals (Sweden)

    Zhang Wen

    2004-12-01

    Full Text Available Abstract Background Large-scale quantitative analysis of transcriptional co-expression has been used to dissect regulatory networks and to predict the functions of new genes discovered by genome sequencing in model organisms such as yeast. Although the idea that tissue-specific expression is indicative of gene function in mammals is widely accepted, it has not been objectively tested nor compared with the related but distinct strategy of correlating gene co-expression as a means to predict gene function. Results We generated microarray expression data for nearly 40,000 known and predicted mRNAs in 55 mouse tissues, using custom-built oligonucleotide arrays. We show that quantitative transcriptional co-expression is a powerful predictor of gene function. Hundreds of functional categories, as defined by Gene Ontology 'Biological Processes', are associated with characteristic expression patterns across all tissues, including categories that bear no overt relationship to the tissue of origin. In contrast, simple tissue-specific restriction of expression is a poor predictor of which genes are in which functional categories. As an example, the highly conserved mouse gene PWP1 is widely expressed across different tissues but is co-expressed with many RNA-processing genes; we show that the uncharacterized yeast homolog of PWP1 is required for rRNA biogenesis. Conclusions We conclude that 'functional genomics' strategies based on quantitative transcriptional co-expression will be as fruitful in mammals as they have been in simpler organisms, and that transcriptional control of mammalian physiology is more modular than is generally appreciated. Our data and analyses provide a public resource for mammalian functional genomics.

  2. Integrating Gene Expression Programming and Geographic Information Systems for Solving a Multi Site Land Use Allocation Problem

    Directory of Open Access Journals (Sweden)

    Khalid A. Eldrandaly

    2009-01-01

    Full Text Available Problem statement: Land use planning may be defined as the process of allocating different activities or uses to specific units of area within a region. Multi sites Land Use Allocation Problems (MLUA refer to the problem of allocating more than one land use type in an area. MLUA problem is one of the truly NP Complete (combinatorial optimization problems. Approach: To cope with this type of problems, intelligent techniques such as genetic algorithms and simulated annealing, have been used. In this study a new approach for solving MLUA problems was proposed by integrating Gene Expression Programming (GEP and GIS. The feasibility of the proposed approach in solving MLUA problems was checked using a fictive case study. Results: The results indicated clearly that the proposed approach gives good and satisfactory results. Conclusion/Recommendation: Integrating GIS and GEP is a promising and efficient approach for solving MLUA problems. This research focused on minimizing the development costs and maximizing the compactness of the allocated land use. The optimization model can be extended in the future to maximize also the spatial contiguity of the allocated land use.

  3. On-Chip Integration of Cell-Free Gene Expression

    Science.gov (United States)

    Buxboim, Amnon; Morpurgo, Margherita; Bar-Dagan, Maya; Frydman, Veronica; Zbaida, David; Bar-Ziv, Roy

    2006-03-01

    We present a synthetic approach for the study of gene networks in vitro which is complementary to traditional in vivo methodologies. We have developed a technology for submicron integration of functional genes and on-chip protein synthesis using a cell-free transcription/translation system. The interaction between genes is facilitated by diffusion of on-chip gene expression products from `source' genes towards `acceptor' genes. Our technology is simple and inexpensive and can serve as an improved platform for a wide variety of protein and DNA biochip applications.

  4. Differential gene expression during Trypanosoma cruzi metacyclogenesis

    Directory of Open Access Journals (Sweden)

    Marco Aurelio Krieger

    1999-09-01

    Full Text Available The transformation of epimastigotes into metacyclic trypomastigotes involves changes in the pattern of expressed genes, resulting in important morphological and functional differences between these developmental forms of Trypanosoma cruzi. In order to identify and characterize genes involved in triggering the metacyclogenesis process and in conferring to metacyclic trypomastigotes their stage specific biological properties, we have developed a method allowing the isolation of genes specifically expressed when comparing two close related cell populations (representation of differential expression or RDE. The method is based on the PCR amplification of gene sequences selected by hybridizing and subtracting the populations in such a way that after some cycles of hybridization-amplification genes specific to a given population are highly enriched. The use of this method in the analysis of differential gene expression during T. cruzi metacyclogenesis (6 hr and 24 hr of differentiation and metacyclic trypomastigotes resulted in the isolation of several clones from each time point. Northern blot analysis showed that some genes are transiently expressed (6 hr and 24 hr differentiating cells, while others are present in differentiating cells and in metacyclic trypomastigotes. Nucleotide sequencing of six clones characterized so far showed that they do not display any homology to gene sequences available in the GeneBank.

  5. Multivariate search for differentially expressed gene combinations

    Directory of Open Access Journals (Sweden)

    Klebanov Lev

    2004-10-01

    Full Text Available Abstract Background To identify differentially expressed genes, it is standard practice to test a two-sample hypothesis for each gene with a proper adjustment for multiple testing. Such tests are essentially univariate and disregard the multidimensional structure of microarray data. A more general two-sample hypothesis is formulated in terms of the joint distribution of any sub-vector of expression signals. Results By building on an earlier proposed multivariate test statistic, we propose a new algorithm for identifying differentially expressed gene combinations. The algorithm includes an improved random search procedure designed to generate candidate gene combinations of a given size. Cross-validation is used to provide replication stability of the search procedure. A permutation two-sample test is used for significance testing. We design a multiple testing procedure to control the family-wise error rate (FWER when selecting significant combinations of genes that result from a successive selection procedure. A target set of genes is composed of all significant combinations selected via random search. Conclusions A new algorithm has been developed to identify differentially expressed gene combinations. The performance of the proposed search-and-testing procedure has been evaluated by computer simulations and analysis of replicated Affymetrix gene array data on age-related changes in gene expression in the inner ear of CBA mice.

  6. Gene Expression Profiling in Porcine Fetal Thymus

    Institute of Scientific and Technical Information of China (English)

    Yanjiong Chen; Shengbin Li; Lin Ye; Jianing Geng; Yajun Deng; Songnian Hu

    2003-01-01

    obtain an initial overview of gene diversity and expression pattern in porcinethymus, 11,712 ESTs (Expressed Sequence Tags) from 100-day-old porcine thymus(FTY) were sequenced and 7,071 cleaned ESTs were used for gene expressionanalysis. Clustered by the PHRAP program, 959 contigs and 3,074 singlets wereobtained. Blast search showed that 806 contigs and 1,669 singlets (totally 5,442ESTs) had homologues in GenBank and 1,629 ESTs were novel. According to theGene Ontology classification, 36.99% ESTs were cataloged into the gene expressiongroup, indicating that although the functional gene (18.78% in defense group) ofthymus is expressed in a certain degree, the 100-day-old porcine thymus still existsin a developmental stage. Comparative analysis showed that the gene expressionpattern of the 100-day-old porcine thymus is similar to that of the human infantthymus.

  7. Phytochrome-regulated Gene Expression

    Institute of Scientific and Technical Information of China (English)

    Peter H. Quail

    2007-01-01

    Identification of all genes involved in the phytochrome (phy)-mediated responses of plants to their light environment is an important goal in providing an overall understanding of light-regulated growth and development. This article highlights and integrates the central findings of two recent comprehensive studies in Arabidopsis that have identified the genome-wide set of phy-regulated genes that respond rapidly to red-light signals upon first exposure of dark-grown seedlings, and have tested the functional relevance to normal seedling photomorphogenesis of an initial subset of these genes. The data: (a) reveal considerable complexity in the channeling of the light signals through the different phy-family members (phyA to phyE) to responsive genes; (b) identify a diversity of transcription-factor-encoding genes as major early, if not primary, targets of phy signaling, and, therefore, as potentially important regulators in the transcriptional-network hierarchy; and (c) identify auxin-related genes as the dominant class among rapidly-regulated, hormone-related genes. However, reverse-genetic functional profiling of a selected subset of these genes reveals that only a limited fraction are necessary for optimal phy-induced seedling deetiolation.

  8. Identification of reference genes in human myelomonocytic cells for gene expression studies in altered gravity.

    Science.gov (United States)

    Thiel, Cora S; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Unverdorben, Felix; Buttron, Isabell; Lauber, Beatrice; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E; Ullrich, Oliver

    2015-01-01

    Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes ("housekeeping genes") are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

  9. Involvement of the HP0165-HP0166 two-component system in expression of some acidic-pH-upregulated genes of Helicobacter pylori.

    Science.gov (United States)

    Wen, Yi; Feng, Jing; Scott, David R; Marcus, Elizabeth A; Sachs, George

    2006-03-01

    About 200 genes of the gastric pathogen Helicobacter pylori increase expression at medium pHs of 6.2, 5.5, and 4.5, an increase that is abolished or much reduced by the buffering action of urease. Genes up-regulated by a low pH include the two-component system HP0165-HP0166, suggesting a role in the regulation of some of the pH-sensitive genes. To identify targets of HP0165-HP0166, the promoter regions of genes up-regulated by a low pH were grouped based on sequence similarity. Probes for promoter sequences representing each group were subjected to electrophoretic mobility shift assays (EMSA) with recombinant HP0166-His(6) or a mutated response regulator, HP0166-D52N-His(6), that can specifically determine the role of phosphorylation of HP0166 in binding (including a control EMSA with in-vitro-phosphorylated HP0166-His(6)). Nineteen of 45 promoter-regulatory regions were found to interact with HP0166-His(6). Seven promoters for genes encoding alpha-carbonic anhydrase, omp11, fecD, lpp20, hypA, and two with unknown function (pHP1397-1396 and pHP0654-0675) were clustered in gene group A, which may respond to changes in the periplasmic pH at a constant cytoplasmic pH and showed phosphorylation-dependent binding in EMSA with HP0166-D52N-His(6). Twelve promoters were clustered in groups B and C whose up-regulation likely also depends on a reduction of the cytoplasmic pH at a medium pH of 5.5 or 4.5. Most of the target promoters in groups B and C showed phosphorylation-dependent binding with HP0166-D52N-His(6), but promoters for ompR (pHP0166-0162), pHP0682-0681, and pHP1288-1289 showed phosphorylation-independent binding. These findings, combined with DNase I footprinting, suggest that HP0165-0166 is an acid-responsive signaling system affecting the expression of pH-sensitive genes. Regulation of these genes responds either to a decrease in the periplasmic pH alone (HP0165 dependent) or also to a decrease in the cytoplasmic pH (HP0165 independent).

  10. Involvement of the HP0165-HP0166 Two-Component System in Expression of Some Acidic-pH-Upregulated Genes of Helicobacter pylori†

    Science.gov (United States)

    Wen, Yi; Feng, Jing; Scott, David R.; Marcus, Elizabeth A.; Sachs, George

    2006-01-01

    About 200 genes of the gastric pathogen Helicobacter pylori increase expression at medium pHs of 6.2, 5.5, and 4.5, an increase that is abolished or much reduced by the buffering action of urease. Genes up-regulated by a low pH include the two-component system HP0165-HP0166, suggesting a role in the regulation of some of the pH-sensitive genes. To identify targets of HP0165-HP0166, the promoter regions of genes up-regulated by a low pH were grouped based on sequence similarity. Probes for promoter sequences representing each group were subjected to electrophoretic mobility shift assays (EMSA) with recombinant HP0166-His6 or a mutated response regulator, HP0166-D52N-His6, that can specifically determine the role of phosphorylation of HP0166 in binding (including a control EMSA with in-vitro-phosphorylated HP0166-His6). Nineteen of 45 promoter-regulatory regions were found to interact with HP0166-His6. Seven promoters for genes encoding α-carbonic anhydrase, omp11, fecD, lpp20, hypA, and two with unknown function (pHP1397-1396 and pHP0654-0675) were clustered in gene group A, which may respond to changes in the periplasmic pH at a constant cytoplasmic pH and showed phosphorylation-dependent binding in EMSA with HP0166-D52N-His6. Twelve promoters were clustered in groups B and C whose up-regulation likely also depends on a reduction of the cytoplasmic pH at a medium pH of 5.5 or 4.5. Most of the target promoters in groups B and C showed phosphorylation-dependent binding with HP0166-D52N-His6, but promoters for ompR (pHP0166-0162), pHP0682-0681, and pHP1288-1289 showed phosphorylation-independent binding. These findings, combined with DNase I footprinting, suggest that HP0165-0166 is an acid-responsive signaling system affecting the expression of pH-sensitive genes. Regulation of these genes responds either to a decrease in the periplasmic pH alone (HP0165 dependent) or also to a decrease in the cytoplasmic pH (HP0165 independent). PMID:16484186

  11. Nucleosome repositioning underlies dynamic gene expression.

    Science.gov (United States)

    Nocetti, Nicolas; Whitehouse, Iestyn

    2016-03-15

    Nucleosome repositioning at gene promoters is a fundamental aspect of the regulation of gene expression. However, the extent to which nucleosome repositioning is used within eukaryotic genomes is poorly understood. Here we report a comprehensive analysis of nucleosome positions as budding yeast transit through an ultradian cycle in which expression of >50% of all genes is highly synchronized. We present evidence of extensive nucleosome repositioning at thousands of gene promoters as genes are activated and repressed. During activation, nucleosomes are relocated to allow sites of general transcription factor binding and transcription initiation to become accessible. The extent of nucleosome shifting is closely related to the dynamic range of gene transcription and generally related to DNA sequence properties and use of the coactivators TFIID or SAGA. However, dynamic gene expression is not limited to SAGA-regulated promoters and is an inherent feature of most genes. While nucleosome repositioning occurs pervasively, we found that a class of genes required for growth experience acute nucleosome shifting as cells enter the cell cycle. Significantly, our data identify that the ATP-dependent chromatin-remodeling enzyme Snf2 plays a fundamental role in nucleosome repositioning and the expression of growth genes. We also reveal that nucleosome organization changes extensively in concert with phases of the cell cycle, with large, regularly spaced nucleosome arrays being established in mitosis. Collectively, our data and analysis provide a framework for understanding nucleosome dynamics in relation to fundamental DNA-dependent transactions.

  12. Gene expression-targeted isoflavone therapy.

    Science.gov (United States)

    Węgrzyn, Alicja

    2012-04-01

    Lysosomal storage diseases (LSD) form a group of inherited metabolic disorders caused by dysfunction of one of the lysosomal proteins, resulting in the accumulation of certain compounds. Although these disorders are among first genetic diseases for which specific treatments were proposed, there are still serious unsolved problems that require development of novel therapeutic procedures. An example is neuronopathy, which develops in most of LSD and cannot be treated efficiently by currently approved therapies. Recently, a new potential therapy, called gene expression-targeted isoflavone therapy (GET IT), has been proposed for a group of LSD named mucopolysaccharidoses (MPS), in which storage of incompletely degraded glycosaminoglycans (GAGs) results in severe symptoms of virtually all tissues and organs, including central nervous system. The idea of this therapy is to inhibit synthesis of GAGs by modulating expression of genes coding for enzymes involved in synthesis of these compounds. Such a modulation is possible by using isoflavones, particularly genistein, which interfere with a signal transduction process necessary for stimulation of expression of certain genes. Results of in vitro experiments and studies on animal models indicated a high efficiency of GET IT, including correction of behavior of affected mice. However, clinical trials, performed with soy isoflavone extracts, revealed only limited efficacy. This caused a controversy about GET IT as a potential, effective treatment of patients suffering from MPS, especially neuronopathic forms of these diseases. It this critical review, I present possible molecular mechanisms of therapeutic action of isoflavones (particularly genistein) and suggest that efficacy of GET IT might be sufficiently high when using relatively high doses of synthetic genistein (which was employed in experiments on cell cultures and mouse models) rather than low doses of soy isoflavone extracts (which were used in clinical trials). This

  13. Novel redox nanomedicine improves gene expression of polyion complex vector

    Science.gov (United States)

    Toh, Kazuko; Yoshitomi, Toru; Ikeda, Yutaka; Nagasaki, Yukio

    2011-12-01

    Gene therapy has generated worldwide attention as a new medical technology. While non-viral gene vectors are promising candidates as gene carriers, they have several issues such as toxicity and low transfection efficiency. We have hypothesized that the generation of reactive oxygen species (ROS) affects gene expression in polyplex supported gene delivery systems. The effect of ROS on the gene expression of polyplex was evaluated using a nitroxide radical-containing nanoparticle (RNP) as an ROS scavenger. When polyethyleneimine (PEI)/pGL3 or PEI alone was added to the HeLa cells, ROS levels increased significantly. In contrast, when (PEI)/pGL3 or PEI was added with RNP, the ROS levels were suppressed. The luciferase expression was increased by the treatment with RNP in a dose-dependent manner and the cellular uptake of pDNA was also increased. Inflammatory cytokines play an important role in ROS generation in vivo. In particular, tumor necrosis factor (TNF)-α caused intracellular ROS generation in HeLa cells and decreased gene expression. RNP treatment suppressed ROS production even in the presence of TNF-α and increased gene expression. This anti-inflammatory property of RNP suggests that it may be used as an effective adjuvant for non-viral gene delivery systems.

  14. Novel redox nanomedicine improves gene expression of polyion complex vector

    Directory of Open Access Journals (Sweden)

    Kazuko Toh, Toru Yoshitomi, Yutaka Ikeda and Yukio Nagasaki

    2011-01-01

    Full Text Available Gene therapy has generated worldwide attention as a new medical technology. While non-viral gene vectors are promising candidates as gene carriers, they have several issues such as toxicity and low transfection efficiency. We have hypothesized that the generation of reactive oxygen species (ROS affects gene expression in polyplex supported gene delivery systems. The effect of ROS on the gene expression of polyplex was evaluated using a nitroxide radical-containing nanoparticle (RNP as an ROS scavenger. When polyethyleneimine (PEI/pGL3 or PEI alone was added to the HeLa cells, ROS levels increased significantly. In contrast, when (PEI/pGL3 or PEI was added with RNP, the ROS levels were suppressed. The luciferase expression was increased by the treatment with RNP in a dose-dependent manner and the cellular uptake of pDNA was also increased. Inflammatory cytokines play an important role in ROS generation in vivo. In particular, tumor necrosis factor (TNF-α caused intracellular ROS generation in HeLa cells and decreased gene expression. RNP treatment suppressed ROS production even in the presence of TNF-α and increased gene expression. This anti-inflammatory property of RNP suggests that it may be used as an effective adjuvant for non-viral gene delivery systems.

  15. Glycinergic-Fipronil Uptake Is Mediated by an Amino Acid Carrier System and Induces the Expression of Amino Acid Transporter Genes in Ricinus communis Seedlings.

    Science.gov (United States)

    Xie, Yun; Zhao, Jun-Long; Wang, Chuan-Wei; Yu, Ai-Xin; Liu, Niu; Chen, Li; Lin, Fei; Xu, Han-Hong

    2016-05-18

    Phloem-mobile insecticides are efficient for piercing and sucking insect control. Introduction of sugar or amino acid groups to the parent compound can improve the phloem mobility of insecticides, so a glycinergic-fipronil conjugate (GlyF), 2-(3-(3-cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-((trifluoromethyl)sulfinyl)-1H-pyrazole-5-yl)ureido) acetic acid, was designed and synthesized. Although the "Kleier model" predicted that this conjugate is not phloem mobile, GlyF can be continually detected during a 5 h collection of Ricinus communis phloem sap. Furthermore, an R. communis seedling cotyledon disk uptake experiment demonstrates that the uptake of GlyF is sensitive to pH, carbonyl cyanide m-chlorophenylhydrazone (CCCP), temperature, and p-chloromercuribenzenesulfonic acid (pCMBS) and is likely mediated by amino acid carrier system. To explore the roles of amino acid transporters (AATs) in GlyF uptake, a total of 62 AAT genes were identified from the R. communis genome in silico. Phylogenetic analysis revealed that AATs in R. communis were organized into the ATF (amino acid transporter) and APC (amino acid, polyaminem and choline transporter) superfamilies, with five subfamilies in ATF and two in APC. Furthermore, the expression profiles of 20 abundantly expressed AATs (cycle threshold (Ct) values communis seedlings. On the basis of the observation that the expression profile of the four candidate genes is similar to the time course observation for GlyF foliar disk uptake, it is suggested that those four genes are possible candidates involved in the uptake of GlyF. These results contribute to a better understanding of the mechanism of GlyF uptake as well as phloem loading from a molecular biology perspective and facilitate functional characterization of candidate AAT genes in future studies.

  16. Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon

    Directory of Open Access Journals (Sweden)

    Hackett Perry B

    2006-06-01

    Full Text Available Abstract Background Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse. One such element is the Sleeping Beauty (SB transposon that has been shown in several studies to be an effective insertional mutagen in the mouse germline. SB transposon vector studies have employed different functional elements and reporter molecules to disrupt and report the expression of endogenous mouse genes. We sought to generate a transposon system that would be capable of reporting the expression pattern of a mouse gene while allowing for conditional expression of a gene of interest in a tissue- or temporal-specific pattern. Results Here we report the systematic development and testing of a transposon-based gene-trap system incorporating the doxycycline-repressible Tet-Off (tTA system that is capable of activating the expression of genes under control of a Tet response element (TRE promoter. We demonstrate that the gene trap system is fully functional in vitro by introducing the "gene-trap tTA" vector into human cells by transposition and identifying clones that activate expression of a TRE-luciferase transgene in a doxycycline-dependent manner. In transgenic mice, we mobilize gene-trap tTA vectors, discover parameters that can affect germline mobilization rates, and identify candidate gene insertions to demonstrate the in vivo functionality of the vector system. We further demonstrate that the gene-trap can act as a reporter of endogenous gene expression and it can be coupled with bioluminescent imaging to identify genes with tissue-specific expression patterns. Conclusion Akin to the GAL4/UAS system used in the fly, we have made progress developing a tool for mutating and revealing the expression of mouse genes by generating the tTA transactivator in the presence of a secondary TRE-regulated reporter molecule. A vector like the gene

  17. Digital gene expression signatures for maize development.

    Science.gov (United States)

    Eveland, Andrea L; Satoh-Nagasawa, Namiko; Goldshmidt, Alexander; Meyer, Sandra; Beatty, Mary; Sakai, Hajime; Ware, Doreen; Jackson, David

    2010-11-01

    Genome-wide expression signatures detect specific perturbations in developmental programs and contribute to functional resolution of key regulatory networks. In maize (Zea mays) inflorescences, mutations in the RAMOSA (RA) genes affect the determinacy of axillary meristems and thus alter branching patterns, an important agronomic trait. In this work, we developed and tested a framework for analysis of tag-based, digital gene expression profiles using Illumina's high-throughput sequencing technology and the newly assembled B73 maize reference genome. We also used a mutation in the RA3 gene to identify putative expression signatures specific to stem cell fate in axillary meristem determinacy. The RA3 gene encodes a trehalose-6-phosphate phosphatase and may act at the interface between developmental and metabolic processes. Deep sequencing of digital gene expression libraries, representing three biological replicate ear samples from wild-type and ra3 plants, generated 27 million 20- to 21-nucleotide reads with frequencies spanning 4 orders of magnitude. Unique sequence tags were anchored to 3'-ends of individual transcripts by DpnII and NlaIII digests, which were multiplexed during sequencing. We mapped 86% of nonredundant signature tags to the maize genome, which associated with 37,117 gene models and unannotated regions of expression. In total, 66% of genes were detected by at least nine reads in immature maize ears. We used comparative genomics to leverage existing information from Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) in functional analyses of differentially expressed maize genes. Results from this study provide a basis for the analysis of short-read expression data in maize and resolved specific expression signatures that will help define mechanisms of action for the RA3 gene.

  18. Gene expression profile of sprinter's muscle.

    Science.gov (United States)

    Yoshioka, M; Tanaka, H; Shono, N; Shindo, M; St-Amand, J

    2007-12-01

    We have characterized the global gene expression profile in left vastus lateralis muscles of sprinters and sedentary men. The gene expression profile was analyzed by using serial analysis of gene expression (SAGE) method. The abundantly expressed transcripts in the sprinter's muscle were mainly involved in contraction and energy metabolism, whereas six transcripts were corresponding to potentially novel transcripts. Thirty-eight transcripts were differentially expressed between the sprinter and sedentary individuals. Moreover, sprinters showed higher expressions of both uncharacterized and potentially novel transcripts. Sprinters also highly expressed seven transcripts, such as glycine-rich protein, myosin heavy polypeptide (MYH) 2, expressed sequence tag similar to (EST) fructose-bisphosphate aldolase 1 isoform A (ALDOA), glyceraldehyde-3-phosphate dehydrogenase and ATP synthase F0 subunit 6. On the other hand, 20 transcripts such as MYH1, tropomyosin 2 and 3, troponin C slow, C2 fast, I slow, T1 slow and T3 fast, myoglobin, creatine kinase, ALDOA, glycogen phosphorylase, cytochrome c oxidase II and III, and NADH dehydrogenase 1 and 2 showed lower expression levels in the sprinters than the sedentary controls. The current study has characterized the global gene expressions in sprinters and identified a number of transcripts that can be subjected to further mechanistic analysis.

  19. Photic stimulation of the suprachiasmatic nucleus via the non-visual optic system. A gene expression study in the blind Crx (-/-) mouse

    DEFF Research Database (Denmark)

    Rovsing, Louise; Møller, Morten

    2014-01-01

    photoreceptors. At zeitgeber time 16, the Crx (-/-) and wild-type mice were exposed to 1 h of light. This resulted in a strong up-regulation of the immediate early genes Nr4a1, Erg, and Rrad in the SCN of both genotypes. Light stimulation during the subjective night resulted in a strong up-regulation of c...... to light during the subjective night is an immediate expression of several early response genes in the SCN. We show, by quantitative real-time polymerase chain reaction, that the amount of melanopsin mRNA in the retinal ganglion cells is preserved in the blind Crx (-/-) mouse with degenerated classic......-fos in both genotypes with a significantly higher up-regulation in the blind Crx (-/-) mouse. Expression of Grp and Vip, the genes for two classic peptides located in the SCN, was not influenced by light stimulation. The data strongly indicate the involvement of the melanopsin-based non-visual optic system...

  20. Characterization of the beta amyloid precursor protein-like gene in the central nervous system of the crab Chasmagnathus. Expression during memory consolidation

    Directory of Open Access Journals (Sweden)

    Fustiñana Maria

    2010-09-01

    Full Text Available Abstract Background Human β-amyloid, the main component in the neuritic plaques found in patients with Alzheimer's disease, is generated by cleavage of the β-amyloid precursor protein. Beyond the role in pathology, members of this protein family are synaptic proteins and have been associated with synaptogenesis, neuronal plasticity and memory, both in vertebrates and in invertebrates. Consolidation is necessary to convert a short-term labile memory to a long-term and stable form. During consolidation, gene expression and de novo protein synthesis are regulated in order to produce key proteins for the maintenance of plastic changes produced during the acquisition of new information. Results Here we partially cloned and sequenced the beta-amyloid precursor protein like gene homologue in the crab Chasmagnathus (cappl, showing a 37% of identity with the fruit fly Drosophila melanogaster homologue and 23% with Homo sapiens but with much higher degree of sequence similarity in certain regions. We observed a wide distribution of cappl mRNA in the nervous system as well as in muscle and gills. The protein localized in all tissues analyzed with the exception of muscle. Immunofluorescence revealed localization of cAPPL in associative and sensory brain areas. We studied gene and protein expression during long-term memory consolidation using a well characterized memory model: the context-signal associative memory in this crab species. mRNA levels varied at different time points during long-term memory consolidation and correlated with cAPPL protein levels Conclusions cAPPL mRNA and protein is widely distributed in the central nervous system of the crab and the time course of expression suggests a role of cAPPL during long-term memory formation.

  1. The Vibrio fischeri quorum-sensing systems ain and lux sequentially induce luminescence gene expression and are important for persistence in the squid host.

    Science.gov (United States)

    Lupp, Claudia; Urbanowski, Mark; Greenberg, E Peter; Ruby, Edward G

    2003-10-01

    Bacterial quorum sensing using acyl-homoserine lactones (acyl-HSLs) as cell-density dependent signalling molecules is important for the transcriptional regulation of many genes essential in the establishment and the maintenance of bacteria-host associations. Vibrio fischeri, the symbiotic partner of the Hawaiian bobtail squid Euprymna scolopes, possesses two distinct acyl-HSL synthase proteins, LuxI and AinS. Whereas the cell density-dependent regulation of luminescence by the LuxI-produced signal is a well-described phenomenon, and its role in light organ symbiosis has been defined, little is known about the ain system. We have investigated the impact of the V. fischeri acyl-HSL synthase AinS on both luminescence and symbiotic colonization. Through phenotypic studies of V. fischeri mutants we have found that the AinS-signal is the predominant inducer of luminescence expression in culture, whereas the impact of the LuxI-signal is apparent only at the high cell densities occurring in symbiosis. Furthermore, our studies revealed that ainS regulates activities essential for successful colonization of E. scolopes, i.e. the V. fischeri ainS mutant failed to persist in the squid light organ. Mutational inactivation of the transcriptional regulator protein LuxO in the ainS mutant partially or completely reversed all the observed phenotypes, demonstrating that the AinS-signal regulates expression of downstream genes through the inactivation of LuxO. Taken together, our results suggest that the two quorum-sensing systems in V. fischeri, ain and lux, sequentially induce the expression of luminescence genes and possibly other colonization factors.

  2. Widespread ectopic expression of olfactory receptor genes

    Directory of Open Access Journals (Sweden)

    Yanai Itai

    2006-05-01

    Full Text Available Abstract Background Olfactory receptors (ORs are the largest gene family in the human genome. Although they are expected to be expressed specifically in olfactory tissues, some ectopic expression has been reported, with special emphasis on sperm and testis. The present study systematically explores the expression patterns of OR genes in a large number of tissues and assesses the potential functional implication of such ectopic expression. Results We analyzed the expression of hundreds of human and mouse OR transcripts, via EST and microarray data, in several dozens of human and mouse tissues. Different tissues had specific, relatively small OR gene subsets which had particularly high expression levels. In testis, average expression was not particularly high, and very few highly expressed genes were found, none corresponding to ORs previously implicated in sperm chemotaxis. Higher expression levels were more common for genes with a non-OR genomic neighbor. Importantly, no correlation in expression levels was detected for human-mouse orthologous pairs. Also, no significant difference in expression levels was seen between intact and pseudogenized ORs, except for the pseudogenes of subfamily 7E which has undergone a human-specific expansion. Conclusion The OR superfamily as a whole, show widespread, locus-dependent and heterogeneous expression, in agreement with a neutral or near neutral evolutionary model for transcription control. These results cannot reject the possibility that small OR subsets might play functional roles in different tissues, however considerable care should be exerted when offering a functional interpretation for ectopic OR expression based only on transcription information.

  3. Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR

    Directory of Open Access Journals (Sweden)

    Xiang Fengning

    2008-11-01

    Full Text Available Abstract Background The wild grass species Brachypodium distachyon (Brachypodium hereafter is emerging as a new model system for grass crop genomics research and biofuel grass biology. A draft nuclear genome sequence is expected to be publicly available in the near future; an explosion of gene expression studies will undoubtedly follow. Therefore, stable reference genes are necessary to normalize the gene expression data. Results A systematic exploration of suitable reference genes in Brachypodium is presented here. Nine reference gene candidates were chosen, and their gene sequences were obtained from the Brachypodium expressed sequence tag (EST databases. Their expression levels were examined by quantitative real-time PCR (qRT-PCR using 21 different Brachypodium plant samples, including those from different plant tissues and grown under various growth conditions. Effects of plant growth hormones were also visualized in the assays. The expression stability of the candidate genes was evaluated using two analysis software packages, geNorm and NormFinder. In conclusion, the ubiquitin-conjugating enzyme 18 gene (UBC18 was validated as a suitable reference gene across all the plant samples examined. While the expression of the polyubiquitin genes (Ubi4 and Ubi10 was most stable in different plant tissues and growth hormone-treated plant samples, the expression of the S-adenosylmethionine decarboxylase gene (SamDC ranked was most stable in plants grown under various environmental stresses. Conclusion This study identified the reference genes that are most suitable for normalizing the gene expression data in Brachypodium. These reference genes will be particularly useful when stress-responsive genes are analyzed in order to produce transgenic plants that exhibit enhanced stress resistance.

  4. Heterologous expression of the glucose oxidase gene in Trichoderma atroviride leads enhanced ability to attack phytopathogenic fungi and induction of plant systemic disease resistance

    Institute of Scientific and Technical Information of China (English)

    Robert L Mach; Brunner Kurt; Matteo Lorito; Susanne Zeilinger; Rosalia Ciliento; Sheridan Woo

    2004-01-01

    @@ A transgenic strain of Trichoderma atroviride that expresses the Aspergillus niger glucose oxidase gene goxA under a homologous pathogen-inducible promoter (nag1) has been constructed, with the aim of increasing the ability of this biocontrol agent (BCA) to attack phytopathogenic fungi and enhance plant systemic disease resistance. The sporulation and growth rate of the transgenic progenies were similar to the wild-type strain Pl. goxA expression occurred immediately after contact with the plant pathogen,and the glucose oxidase formed was secreted extracellularly. The transformed strain SJ3 4, containing 12-14 copies of the transgene, produced significantly less N-acetyl-glucosaminidase and endochitinase then wild type. However, the ability of its culture filtrate to inhibit the germination of Botrytis cinerea spores was increased by about 3-fold. In comparison to P1, the transgenic strain more quickly overgrew and lysed in vitro the pathogens Rhizoctonia solani and Pythium ultimum.

  5. Identification of Reference Genes in Human Myelomonocytic Cells for Gene Expression Studies in Altered Gravity

    Directory of Open Access Journals (Sweden)

    Cora S. Thiel

    2015-01-01

    Full Text Available Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes (“housekeeping genes” are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1 which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

  6. Regulation of Gene Expression in Protozoa Parasites

    Directory of Open Access Journals (Sweden)

    Consuelo Gomez

    2010-01-01

    Full Text Available Infections with protozoa parasites are associated with high burdens of morbidity and mortality across the developing world. Despite extensive efforts to control the transmission of these parasites, the spread of populations resistant to drugs and the lack of effective vaccines against them contribute to their persistence as major public health problems. Parasites should perform a strict control on the expression of genes involved in their pathogenicity, differentiation, immune evasion, or drug resistance, and the comprehension of the mechanisms implicated in that control could help to develop novel therapeutic strategies. However, until now these mechanisms are poorly understood in protozoa. Recent investigations into gene expression in protozoa parasites suggest that they possess many of the canonical machineries employed by higher eukaryotes for the control of gene expression at transcriptional, posttranscriptional, and epigenetic levels, but they also contain exclusive mechanisms. Here, we review the current understanding about the regulation of gene expression in Plasmodium sp., Trypanosomatids, Entamoeba histolytica and Trichomonas vaginalis.

  7. Regulation of meiotic gene expression in plants

    Directory of Open Access Journals (Sweden)

    Adele eZhou

    2014-08-01

    Full Text Available With the recent advances in genomics and sequencing technologies, databases of transcriptomes representing many cellular processes have been built. Meiotic transcriptomes in plants have been studied in Arabidopsis thaliana, rice (Oryza sativa, wheat (Triticum aestivum, petunia (Petunia hybrida, sunflower (Helianthus annuus, and maize (Zea mays. Studies in all organisms, but particularly in plants, indicate that a very large number of genes are expressed during meiosis, though relatively few of them seem to be required for the completion of meiosis. In this review, we focus on gene expression at the RNA level and analyze the meiotic transcriptome datasets and explore expression patterns of known meiotic genes to elucidate how gene expression could be regulated during meiosis. We also discuss mechanisms, such as chromatin organization and non-coding RNAs, that might be involved in the regulation of meiotic transcription patterns.

  8. Reference genes for gene expression studies in wheat flag leaves grown under different farming conditions

    Directory of Open Access Journals (Sweden)

    Cordeiro Raposo Fernando

    2011-09-01

    Full Text Available Abstract Background Internal control genes with highly uniform expression throughout the experimental conditions are required for accurate gene expression analysis as no universal reference genes exists. In this study, the expression stability of 24 candidate genes from Triticum aestivum cv. Cubus flag leaves grown under organic and conventional farming systems was evaluated in two locations in order to select suitable genes that can be used for normalization of real-time quantitative reverse-transcription PCR (RT-qPCR reactions. The genes were selected among the most common used reference genes as well as genes encoding proteins involved in several metabolic pathways. Findings Individual genes displayed different expression rates across all samples assayed. Applying geNorm, a set of three potential reference genes were suitable for normalization of RT-qPCR reactions in winter wheat flag leaves cv. Cubus: TaFNRII (ferredoxin-NADP(H oxidoreductase; AJ457980.1, ACT2 (actin 2; TC234027, and rrn26 (a putative homologue to RNA 26S gene; AL827977.1. In addition of these three genes that were also top-ranked by NormFinder, two extra genes: CYP18-2 (Cyclophilin A, AY456122.1 and TaWIN1 (14-3-3 like protein, AB042193 were most consistently stably expressed. Furthermore, we showed that TaFNRII, ACT2, and CYP18-2 are suitable for gene expression normalization in other two winter wheat varieties (Tommi and Centenaire grown under three treatments (organic, conventional and no nitrogen and a different environment than the one tested with cv. Cubus. Conclusions This study provides a new set of reference genes which should improve the accuracy of gene expression analyses when using wheat flag leaves as those related to the improvement of nitrogen use efficiency for cereal production.

  9. Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis.

    Science.gov (United States)

    dos Santos, Odelta; de Vargas Rigo, Graziela; Frasson, Amanda Piccoli; Macedo, Alexandre José; Tasca, Tiana

    2015-01-01

    Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and β-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis.

  10. Construction of a stepwise gene integration system by transient expression of actinophage R4 integrase in cyanobacterium Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Miura, Takamasa; Nishizawa, Akito; Nishizawa, Tomoyasu; Asayama, Munehiko; Takahashi, Hideo; Shirai, Makoto

    2014-08-01

    The integrase of actinophage R4, which belongs to the large serine-recombinase family, catalyzes site-specific recombination between two distinct attachment site sequences of the phage (attP) and actinomycete Streptomyces parvulus 2297 chromosome (attB). We previously reported that R4 integrase (Sre) catalyzed site-specific recombination both in vivo and in vitro. In the present study, a Sre-based system was developed for the stepwise site-specific integration of multiple genes into the chromosome of cyanobacterium Synechocystis sp. PCC 6803 (hereafter PCC 6803). A transgene-integrated plasmid with two attP sites and a non-replicative sre-containing plasmid were co-introduced into attB-inserted PCC 6803 cells. The transiently expressed Sre catalyzed highly efficient site-specific integration between one of the two attP sites on the integration plasmid and the attB site on the chromosome of PCC 6803. A second transgene-integrated plasmid with an attB site was integrated into the residual attP site on the chromosome by repeating site-specific recombination. The transformation frequencies (%) of the first and second integrations were approximately 5.1 × 10(-5) and 8.2 × 10(-5), respectively. Furthermore, the expression of two transgenes was detected. This study is the first to apply the multiple gene site-specific integration system based on R4 integrase to cyanobacteria.

  11. Gene expression profiling in autoimmune diseases

    DEFF Research Database (Denmark)

    Bovin, Lone Frier; Brynskov, Jørn; Hegedüs, Laszlo

    2007-01-01

    A central issue in autoimmune disease is whether the underlying inflammation is a repeated stereotypical process or whether disease specific gene expression is involved. To shed light on this, we analysed whether genes previously found to be differentially regulated in rheumatoid arthritis (RA...

  12. Bayesian modeling of differential gene expression.

    Science.gov (United States)

    Lewin, Alex; Richardson, Sylvia; Marshall, Clare; Glazier, Anne; Aitman, Tim

    2006-03-01

    We present a Bayesian hierarchical model for detecting differentially expressing genes that includes simultaneous estimation of array effects, and show how to use the output for choosing lists of genes for further investigation. We give empirical evidence that expression-level dependent array effects are needed, and explore different nonlinear functions as part of our model-based approach to normalization. The model includes gene-specific variances but imposes some necessary shrinkage through a hierarchical structure. Model criticism via posterior predictive checks is discussed. Modeling the array effects (normalization) simultaneously with differential expression gives fewer false positive results. To choose a list of genes, we propose to combine various criteria (for instance, fold change and overall expression) into a single indicator variable for each gene. The posterior distribution of these variables is used to pick the list of genes, thereby taking into account uncertainty in parameter estimates. In an application to mouse knockout data, Gene Ontology annotations over- and underrepresented among the genes on the chosen list are consistent with biological expectations.

  13. Perspectives: Gene Expression in Fisheries Management

    Science.gov (United States)

    Nielsen, Jennifer L.; Pavey, Scott A.

    2010-01-01

    Functional genes and gene expression have been connected to physiological traits linked to effective production and broodstock selection in aquaculture, selective implications of commercial fish harvest, and adaptive changes reflected in non-commercial fish populations subject to human disturbance and climate change. Gene mapping using single nucleotide polymorphisms (SNPs) to identify functional genes, gene expression (analogue microarrays and real-time PCR), and digital sequencing technologies looking at RNA transcripts present new concepts and opportunities in support of effective and sustainable fisheries. Genomic tools have been rapidly growing in aquaculture research addressing aspects of fish health, toxicology, and early development. Genomic technologies linking effects in functional genes involved in growth, maturation and life history development have been tied to selection resulting from harvest practices. Incorporating new and ever-increasing knowledge of fish genomes is opening a different perspective on local adaptation that will prove invaluable in wild fish conservation and management. Conservation of fish stocks is rapidly incorporating research on critical adaptive responses directed at the effects of human disturbance and climate change through gene expression studies. Genomic studies of fish populations can be generally grouped into three broad categories: 1) evolutionary genomics and biodiversity; 2) adaptive physiological responses to a changing environment; and 3) adaptive behavioral genomics and life history diversity. We review current genomic research in fisheries focusing on those that use microarrays to explore differences in gene expression among phenotypes and within or across populations, information that is critically important to the conservation of fish and their relationship to humans.

  14. Gene Expression Profiles of Inflammatory Myopathies

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    J Gordon Millichap

    2002-11-01

    Full Text Available The simultaneous expression of 10,000 genes was measured, using Affymetrix GeneChip microarrays, in muscle specimens from 45 patients with various myopathies (dystrophy, congenital myopathy, and inflammatory myopathy examined at Brigham and Women’s Hospital, and Children’s Hospital, Harvard Medical School, Boston, MA.

  15. Translational control of gene expression and disease

    NARCIS (Netherlands)

    Calkhoven, Cornelis F; Müller, Christine; Leutz, Achim

    2002-01-01

    In the past decade, translational control has been shown to be crucial in the regulation of gene expression. Research in this field has progressed rapidly, revealing new control mechanisms and adding constantly to the list of translationally regulated genes. There is accumulating evidence that trans

  16. Inferring gene networks from discrete expression data

    KAUST Repository

    Zhang, L.

    2013-07-18

    The modeling of gene networks from transcriptional expression data is an important tool in biomedical research to reveal signaling pathways and to identify treatment targets. Current gene network modeling is primarily based on the use of Gaussian graphical models applied to continuous data, which give a closedformmarginal likelihood. In this paper,we extend network modeling to discrete data, specifically data from serial analysis of gene expression, and RNA-sequencing experiments, both of which generate counts of mRNAtranscripts in cell samples.We propose a generalized linear model to fit the discrete gene expression data and assume that the log ratios of the mean expression levels follow a Gaussian distribution.We restrict the gene network structures to decomposable graphs and derive the graphs by selecting the covariance matrix of the Gaussian distribution with the hyper-inverse Wishart priors. Furthermore, we incorporate prior network models based on gene ontology information, which avails existing biological information on the genes of interest. We conduct simulation studies to examine the performance of our discrete graphical model and apply the method to two real datasets for gene network inference. © The Author 2013. Published by Oxford University Press. All rights reserved.

  17. Activity-Dependent NPAS4 Expression and the Regulation of Gene Programs Underlying Plasticity in the Central Nervous System

    Directory of Open Access Journals (Sweden)

    José Fernando Maya-Vetencourt

    2013-01-01

    Full Text Available The capability of the brain to change functionally in response to sensory experience is most active during early stages of development but it decreases later in life when major alterations of neuronal network structures no longer take place in response to experience. This view has been recently challenged by experimental strategies based on the enhancement of environmental stimulation levels, genetic manipulations, and pharmacological treatments, which all have demonstrated that the adult brain retains a degree of plasticity that allows for a rewiring of neuronal circuitries over the entire life course. A hot spot in the field of neuronal plasticity centres on gene programs that underlie plastic phenomena in adulthood. Here, I discuss the role of the recently discovered neuronal-specific and activity-dependent transcription factor NPAS4 as a critical mediator of plasticity in the nervous system. A better understanding of how modifications in the connectivity of neuronal networks occur may shed light on the treatment of pathological conditions such as brain damage or disease in adult life, some of which were once considered untreatable.

  18. Activity-Dependent NPAS4 Expression and the Regulation of Gene Programs Underlying Plasticity in the Central Nervous System

    Science.gov (United States)

    2013-01-01

    The capability of the brain to change functionally in response to sensory experience is most active during early stages of development but it decreases later in life when major alterations of neuronal network structures no longer take place in response to experience. This view has been recently challenged by experimental strategies based on the enhancement of environmental stimulation levels, genetic manipulations, and pharmacological treatments, which all have demonstrated that the adult brain retains a degree of plasticity that allows for a rewiring of neuronal circuitries over the entire life course. A hot spot in the field of neuronal plasticity centres on gene programs that underlie plastic phenomena in adulthood. Here, I discuss the role of the recently discovered neuronal-specific and activity-dependent transcription factor NPAS4 as a critical mediator of plasticity in the nervous system. A better understanding of how modifications in the connectivity of neuronal networks occur may shed light on the treatment of pathological conditions such as brain damage or disease in adult life, some of which were once considered untreatable. PMID:24024041

  19. [Expression of acylamidase gene in Rhodococcus erythropolis strains].

    Science.gov (United States)

    Lavrov, K V; Novikov, A D; Riabchenko, L E; Ianenko, A S

    2014-09-01

    The expression of a new acylamidase gene from R. erythropolis 37 was studied in Rhodococcus erythropolis strains. This acylamidase, as a result of its unique substrate specificity, can hydrolyse N-substituted amides (4'-nitroacetanilide, N-isopropylacrylamide, N'N-dimethylaminopropylacrylamide). A new expression system based on the use of the promoter region of nitrilhydratase genes from R. rhodochrous M8 was created to achieve constitutive synthesis of acylamidase in R. erythropolis cells. A fourfold improvement in the acylamidase activity of recombinant R. erythropolis cells as compared with the parent wild-type strain was obtained through the use of the new expression system.

  20. Gene expression studies using microarrays

    NARCIS (Netherlands)

    Burgess, Janette

    2001-01-01

    1. The rapid progression of the collaborative sequencing programmes that are unravelling the complete genome sequences of many organisms are opening pathways for new approaches to gene analysis. As the sequence data become available, the bottleneck in biological research will shift to understanding

  1. Differential systemic gene expression profile in patients with diabetic macular edema: Responders versus nonresponders to standard treatment

    Directory of Open Access Journals (Sweden)

    Supriya S Dabir

    2014-01-01

    Full Text Available Introduction: Diabetic macular edema (DME is a vision-threatening complication of diabetic retinopathy. The current practice of management is a" trial and error "method of using intravitreal antivascular endothelial growth factor (VEGF′′ or steroids to treat the patient and watch the response. However, if the patient′s genetic profile helps us choose appropriate medicine, it would help customize treatment option for each patient. This forms the basis of our study. Materials and Methods: A case-control, prospective, observational series, where DME patients were treated with bevacizumab and subclassified as treatment naοve, treatment responders, and treatment nonresponders. Blood samples of 20 subjects were studied, with five patients in each of the groups (nondiabetic- group 1, treatment naοve- group 2, treatment responder- group 3, and treatment nonresponder-group 4. Whole blood RNA extraction followed by labeling, amplification and hybridization was done, and microarray data analyzed. Genes were classified based on functional category and pathways. Results: The total number of genes upregulated among all three experimental groups was 5, whereas 105 genes were downregulated. There were no common genes upregulated between the responders and nonresponders. There was only one gene upregulated between the diabetic and diabetic responders posttreatment. There were 19 genes upregulated and 8 genes downregulated in the inflammatory pathway in group 2 versus group 1. There were no downregulated genes detected in vascular angiogenesis and transcription group. There were identical numbers of genes up- and downregulated in the inflammatory pathway. Seventeen genes were upreguated and 11 genes downregulated in receptor activity, which remained the predominant group in the group classification. Discussion: In summary, this study would provide an insight into the probable signaling mechanisms for disease pathogenesis as well as progression. This type

  2. Gene Expression Profiling of Clostridium botulinum under Heat Shock Stress

    Directory of Open Access Journals (Sweden)

    Wan-dong Liang

    2013-01-01

    Full Text Available During growth, C. botulinum is always exposed to different environmental changes, such as temperature increase, nutrient deprivation, and pH change; however, its corresponding global transcriptional profile is uncharacterized. This study is the first description of the genome-wide gene expression profile of C. botulinum in response to heat shock stress. Under heat stress (temperature shift from 37°C to 45°C over a period of 15 min, 176 C. botulinum ATCC 3502 genes were differentially expressed. The response included overexpression of heat shock protein genes (dnaK operon, groESL, hsp20, and htpG and downregulation of aminoacyl-tRNA synthetase genes (valS, queA, tyrR, and gatAB and ribosomal and cell division protein genes (ftsZ and ftsH. In parallel, several transcriptional regulators (marR, merR, and ompR families were induced, suggesting their involvement in reshuffling of the gene expression profile. In addition, many ABC transporters (oligopeptide transport system, energy production and conversion related genes (glpA and hupL, cell wall and membrane biogenesis related genes (fabZ, fabF, and fabG, flagella-associated genes (flhA, flhM, flhJ, flhS, and motAB, and hypothetical genes also showed changed expression patterns, indicating that they may play important roles in survival under high temperatures.

  3. Insulin gene: organisation, expression and regulation.

    Science.gov (United States)

    Dumonteil, E; Philippe, J

    1996-06-01

    Insulin, a major hormone of the endocrine pancreas, plays a key role in the control of glucose homeostasis. This review discusses the mechanisms of cell-specific expression and regulation of the insulin gene. Whereas expression is restricted to islet beta-cells in adults, the insulin gene is more widely expressed at several embryonic stages, although the role of extrapancreatic expression is still unclear. beta-cell-specific expression relies on the interactions of 5'-flanking sequence motifs of the promoter with a number of ubiquitous and islet-specific transcription factors. IEF1 and IPF-1, by their binding to the E and A boxes, respectively, of the insulin gene promoter, appear to be the major determinants of beta-cell-specific expression. IEF1 is a heterodimer of the basic helix-loop-helix family of transcription factors, whereas IPF-1 belongs to the homeodomain-containing family. beta-cell specific determinants are conserved throughout evolution, although the human insulin gene 5'-flanking sequence also contains a polymorphic minisatellite which is unique to primates and may play a role in insulin gene regulation. Glucose modulates insulin gene transcription, with multiple elements of the promoter involved in glucose responsiveness. Remarkably, IPF-1 and IEF1 are involved in both beta-cell-specific expression and glucose regulation of the insulin gene. cAMP also regulates insulin gene transcription through a CRE, in response to various hormonal stimuli. On the whole, recent studies have provided a better understanding of beta-cell differentiation and function.

  4. Molecular subsets in the gene expression signatures of scleroderma skin.

    Directory of Open Access Journals (Sweden)

    Ausra Milano

    Full Text Available BACKGROUND: Scleroderma is a clinically heterogeneous disease with a complex phenotype. The disease is characterized by vascular dysfunction, tissue fibrosis, internal organ dysfunction, and immune dysfunction resulting in autoantibody production. METHODOLOGY AND FINDINGS: We analyzed the genome-wide patterns of gene expression with DNA microarrays in skin biopsies from distinct scleroderma subsets including 17 patients with systemic sclerosis (SSc with diffuse scleroderma (dSSc, 7 patients with SSc with limited scleroderma (lSSc, 3 patients with morphea, and 6 healthy controls. 61 skin biopsies were analyzed in a total of 75 microarray hybridizations. Analysis by hierarchical clustering demonstrates nearly identical patterns of gene expression in 17 out of 22 of the forearm and back skin pairs of SSc patients. Using this property of the gene expression, we selected a set of 'intrinsic' genes and analyzed the inherent data-driven groupings. Distinct patterns of gene expression separate patients with dSSc from those with lSSc and both are easily distinguished from normal controls. Our data show three distinct patient groups among the patients with dSSc and two groups among patients with lSSc. Each group can be distinguished by unique gene expression signatures indicative of proliferating cells, immune infiltrates and a fibrotic program. The intrinsic groups are statistically significant (p<0.001 and each has been mapped to clinical covariates of modified Rodnan skin score, interstitial lung disease, gastrointestinal involvement, digital ulcers, Raynaud's phenomenon and disease duration. We report a 177-gene signature that is associated with severity of skin disease in dSSc. CONCLUSIONS AND SIGNIFICANCE: Genome-wide gene expression profiling of skin biopsies demonstrates that the heterogeneity in scleroderma can be measured quantitatively with DNA microarrays. The diversity in gene expression demonstrates multiple distinct gene expression programs

  5. Construction of prokaryotic expression system of 2 148-bp fragment from cagA gene and detection of cagA gene, CagA protein in Helicobacter pyloriisolates and its antibody in sera of patients

    Institute of Scientific and Technical Information of China (English)

    Jie Yan; Yuan Wang; Shi-He Shao; Ya-Fei Mao; Hua-Wen Li; Yi-Hui Luo

    2004-01-01

    AIM: To construct a prokaryotic expression system of a Helicobacter pylori ( H pylori) cagA gene fragment and establish enzyme-linked immunosorbent assays (ELISA) for detecting CagA and its antibody, so as to understand the manner in which the infection of CagA-expressing H pylori (CagA+ H pylori) isolates cause diseases.METHODS: H pylori strains in gastric biopsy specimens from 156 patients with positive results in rapid urease test were isolated. PCR was used to detect the frequency of cagA gene in the 109 H pylori isolates and to amplify a 2 148-bp fragment (cagA1) of cagA gene from a clinical strain Y06. A prokaryotic expression system of cagA1 gene was constructed,and the expression of the target recombinant protein (rCagA1) was examined by SDS-PAGE. Western blotting and immunodiffusion assay were employed to determine the immunoreactivity and antigenicity of rCagA1, respectively.Two ELISAs were established to detect CagA expression in 109 H pylori isolates and the presence of CagA antibody in the corresponding patients′ sera, and the correlations between infection with CagA+ H pylori and gastritis as well as peptic ulcer were analyzed.RESULTS: Of all the clinical specimens obtained, 80.8%(126/156) were found to have H pylori isolates and 97.2%of the isolates (106/109) were positive for caaA gene. In comparison with the reported data, the cloned cagA1fragment possessed 94.83% and 93.30% homologies with the nucleotide and putative amino acid sequences,respectively. The output of rCagA1 produced by the constructed recombinant prokaryotic expression system was approximately 30% of the total bacterial protein, rCagA1was able to bind to the commercial antibody against the whole-cells of H pylori and to induce the immunized rabbits to produce antibody with an immunodiffusion titer of 1:4. A proportion as high as 92.6% of the H pylori isolates (101/109)expressed CagA and 88.1% of the patients′ serum samples (96/109) were CagA antibody-positive. The percentage of

  6. Application of multidisciplinary analysis to gene expression.

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xuefel (University of New Mexico, Albuquerque, NM); Kang, Huining (University of New Mexico, Albuquerque, NM); Fields, Chris (New Mexico State University, Las Cruces, NM); Cowie, Jim R. (New Mexico State University, Las Cruces, NM); Davidson, George S.; Haaland, David Michael; Sibirtsev, Valeriy (New Mexico State University, Las Cruces, NM); Mosquera-Caro, Monica P. (University of New Mexico, Albuquerque, NM); Xu, Yuexian (University of New Mexico, Albuquerque, NM); Martin, Shawn Bryan; Helman, Paul (University of New Mexico, Albuquerque, NM); Andries, Erik (University of New Mexico, Albuquerque, NM); Ar, Kerem (University of New Mexico, Albuquerque, NM); Potter, Jeffrey (University of New Mexico, Albuquerque, NM); Willman, Cheryl L. (University of New Mexico, Albuquerque, NM); Murphy, Maurice H. (University of New Mexico, Albuquerque, NM)

    2004-01-01

    Molecular analysis of cancer, at the genomic level, could lead to individualized patient diagnostics and treatments. The developments to follow will signal a significant paradigm shift in the clinical management of human cancer. Despite our initial hopes, however, it seems that simple analysis of microarray data cannot elucidate clinically significant gene functions and mechanisms. Extracting biological information from microarray data requires a complicated path involving multidisciplinary teams of biomedical researchers, computer scientists, mathematicians, statisticians, and computational linguists. The integration of the diverse outputs of each team is the limiting factor in the progress to discover candidate genes and pathways associated with the molecular biology of cancer. Specifically, one must deal with sets of significant genes identified by each method and extract whatever useful information may be found by comparing these different gene lists. Here we present our experience with such comparisons, and share methods developed in the analysis of an infant leukemia cohort studied on Affymetrix HG-U95A arrays. In particular, spatial gene clustering, hyper-dimensional projections, and computational linguistics were used to compare different gene lists. In spatial gene clustering, different gene lists are grouped together and visualized on a three-dimensional expression map, where genes with similar expressions are co-located. In another approach, projections from gene expression space onto a sphere clarify how groups of genes can jointly have more predictive power than groups of individually selected genes. Finally, online literature is automatically rearranged to present information about genes common to multiple groups, or to contrast the differences between the lists. The combination of these methods has improved our understanding of infant leukemia. While the complicated reality of the biology dashed our initial, optimistic hopes for simple answers from

  7. Gene expression profiling: can we identify the right target genes?

    Directory of Open Access Journals (Sweden)

    J. E. Loyd

    2008-12-01

    Full Text Available Gene expression profiling allows the simultaneous monitoring of the transcriptional behaviour of thousands of genes, which may potentially be involved in disease development. Several studies have been performed in idiopathic pulmonary fibrosis (IPF, which aim to define genetic links to the disease in an attempt to improve the current understanding of the underlying pathogenesis of the disease and target pathways for intervention. Expression profiling has shown a clear difference in gene expression between IPF and normal lung tissue, and has identified a wide range of candidate genes, including those known to encode for proteins involved in extracellular matrix formation and degradation, growth factors and chemokines. Recently, familial pulmonary fibrosis cohorts have been examined in an attempt to detect specific genetic mutations associated with IPF. To date, these studies have identified families in which IPF is associated with mutations in the gene encoding surfactant protein C, or with mutations in genes encoding components of telomerase. Although rare and clearly not responsible for the disease in all individuals, the nature of these mutations highlight the importance of the alveolar epithelium in disease pathogenesis and demonstrate the potential for gene expression profiling in helping to advance the current understanding of idiopathic pulmonary fibrosis.

  8. Regulation of immunoglobulin gene rearrangement and expression.

    Science.gov (United States)

    Taussig, M J; Sims, M J; Krawinkel, U

    1989-05-01

    The molecular genetic events leading to Ig expression and their control formed the topic of a recent EMBO workshop. This report by Michael Taussig, Martin Sims and Ulrich Krawinkel discusses contributions dealing with genes expressed in early pre-B cells, the mechanism of rearrangement, aberrant rearrangements seen in B cells of SCID mice, the feedback control of rearrangement as studied in transgenic mice, the control of Ig expression at the transcriptional and post-transcriptional levels, and class switching.

  9. Vitamin D-mediated gene expression.

    Science.gov (United States)

    Lowe, K E; Maiyar, A C; Norman, A W

    1992-01-01

    The steroid hormone 1,25(OH)2D3 modulates the expression of a wide variety of genes in a tissue- and developmentally specific manner. It is well established that 1,25(OH)2D3 can up- or downregulate the expression of genes involved in cell proliferation, differentiation, and mineral homeostasis. The hormone exerts its genomic effects via interactions with the vitamin D receptor or VDR, a member of the superfamily of hormone-activated nuclear receptors which can regulate eukaryotic gene expression. The ligand-bound receptor acts as a transcription factor that binds to specific DNA sequences, HREs, in target gene promoters. The DNA-binding domains of the steroid hormone receptors are highly conserved and contain two zinc-finger motifs that recognize the HREs. The spacing and orientation of the HRE half-sites, as well as the HRE sequence, are critical for proper discrimination by the various receptors. Other nuclear factors such as fos and jun can influence vitamin D-mediated gene expression. A wide range of experimental techniques has been used to increase our understanding of how 1,25(OH)2D3 and its receptor play a central role in gene expression.

  10. Modulation of imprinted gene expression following superovulation.

    Science.gov (United States)

    Fortier, Amanda L; McGraw, Serge; Lopes, Flavia L; Niles, Kirsten M; Landry, Mylène; Trasler, Jacquetta M

    2014-05-05

    Although assisted reproductive technologies increase the risk of low birth weight and genomic imprinting disorders, the precise underlying causes remain unclear. Using a mouse model, we previously showed that superovulation alters the expression of imprinted genes in the placenta at 9.5days (E9.5) of gestation. Here, we investigate whether effects of superovulation on genomic imprinting persisted at later stages of development and assess the surviving fetuses for growth and morphological abnormalities. Superovulation, followed by embryo transfer at E3.5, as compared to spontaneous ovulation (controls), resulted in embryos of normal size and weight at 14.5 and 18.5days of gestation. The normal monoallelic expression of the imprinted genes H19, Snrpn and Kcnq1ot1 was unaffected in either the placentae or the embryos from the superovulated females at E14.5 or E18.5. However, for the paternally expressed imprinted gene Igf2, superovulation generated placentae with reduced production of the mature protein at E9.5 and significantly more variable mRNA levels at E14.5. We propose that superovulation results in the ovulation of abnormal oocytes with altered expression of imprinted genes, but that the coregulated genes of the imprinted gene network result in modulated expression. Copyright © 2014. Published by Elsevier Ireland Ltd.

  11. Gene expression during fruit ripening in avocado.

    Science.gov (United States)

    Christoffersen, R E; Warm, E; Laties, G G

    1982-06-01

    The poly(A) (+)RNA populations from avocado fruit (Persea americana Mill cv. Hass) at four stages of ripening were isolated by two cycles of oligo-dT-cellulose chromatography and examined by invitro translation, using the rabbit reticulocyte lysate system, followed by two-dimensional gel electrophoresis (isoelectric focusing followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis) of the resulting translation products. Three mRNAs increased dramatically with the climacteric rise in respiration and ethylene production. The molecular weights of the corresponding translation products from the ripening-related mRNAs are 80,000, 36,000, and 16,500. These results indicate that ripening may be linked to the expression of specific genes.

  12. Gene expression of the endolymphatic sac.

    Science.gov (United States)

    Friis, Morten; Martin-Bertelsen, Tomas; Friis-Hansen, Lennart; Winther, Ole; Henao, Ricardo; Sørensen, Mads Sølvsten; Qvortrup, Klaus

    2011-12-01

    The endolymphatic sac is part of the membranous inner ear and is thought to play a role in the fluid homeostasis and immune defense of the inner ear; however, the exact function of the endolymphatic sac is not fully known. Many of the detected mRNAs in this study suggest that the endolymphatic sac has multiple and diverse functions in the inner ear. The objective of this study was to provide a comprehensive review of the genes expressed in the endolymphatic sac in the rat and perform a functional characterization based on measured mRNA abundance. Microarray technology was used to investigate the gene expression of the endolymphatic sac with the surrounding dura. Characteristic and novel endolymphatic sac genes were determined by comparing with expressions in pure dura. In all, 463 genes were identified specific for the endolymphatic sac. Functional annotation clustering revealed 29 functional clusters.

  13. Bovine papillomavirus type 4 L1 gene transfection in a Drosophila S2 cell expression system: absence of L1 protein expression Transfecção em células de Drosophila S2 usando o gene L1 do Papilomavírus Bovino tipo 4: ausência de expressão da proteína L1

    Directory of Open Access Journals (Sweden)

    Luiz Gustavo Bentim Góes

    2008-03-01

    Full Text Available The development of a bovine papillomavirus (BPV vaccine is an outstanding challenge. BPV protein L1 gene transfection in the Drosophila melanogaster S2 cell expression system failed to produce L1 protein notwithstanding correct L1 gene insertion. Severe genetic inbalance in the host cell line, including cytogenetic alterations, may account for the lack of protein expression.O desenvolvimento de uma vacina para papilomavirus bovino (BPV consiste em grande desafio. A transfecção do gene codificante da proteína L1 de BPV em sistema de células S2 de Drosophila melanogaster não logrou sucesso, apesar da correta inserção da seqüência gênica em vetor apropriado.Graves alterações genéticas na linhagem celular S2, que incluem aberrações cromossômicas, provavelmente estão relacionadas à ausência da expressão da proteína desejada.

  14. Regulation of gene expression in human tendinopathy

    Science.gov (United States)

    2011-01-01

    Background Chronic tendon injuries, also known as tendinopathies, are common among professional and recreational athletes. These injuries result in a significant amount of morbidity and health care expenditure, yet little is known about the molecular mechanisms leading to tendinopathy. Methods We have used histological evaluation and molecular profiling to determine gene expression changes in 23 human patients undergoing surgical procedures for the treatment of chronic tendinopathy. Results Diseased tendons exhibit altered extracellular matrix, fiber disorientation, increased cellular content and vasculature, and the absence of inflammatory cells. Global gene expression profiling identified 983 transcripts with significantly different expression patterns in the diseased tendons. Global pathway analysis further suggested altered expression of extracellular matrix proteins and the lack of an appreciable inflammatory response. Conclusions Identification of the pathways and genes that are differentially regulated in tendinopathy samples will contribute to our understanding of the disease and the development of novel therapeutics. PMID:21539748

  15. Supplementary Material for: Global expression differences and tissue specific expression differences in rice evolution result in two contrasting types of differentially expressed genes

    KAUST Repository

    Horiuchi, Youko

    2015-01-01

    Abstract Background Since the development of transcriptome analysis systems, many expression evolution studies characterized evolutionary forces acting on gene expression, without explicit discrimination between global expression differences and tissue specific expression differences. However, different types of gene expression alteration should have different effects on an organism, the evolutionary forces that act on them might be different, and different types of genes might show different types of differential expression between species. To confirm this, we studied differentially expressed (DE) genes among closely related groups that have extensive gene expression atlases, and clarified characteristics of different types of DE genes including the identification of regulating loci for differential expression using expression quantitative loci (eQTL) analysis data. Results We detected differentially expressed (DE) genes between rice subspecies in five homologous tissues that were verified using japonica and indica transcriptome atlases in public databases. Using the transcriptome atlases, we classified DE genes into two types, global DE genes and changed-tissues DE genes. Global type DE genes were not expressed in any tissues in the atlas of one subspecies, however changed-tissues type DE genes were expressed in both subspecies with different tissue specificity. For the five tissues in the two japonica-indica combinations, 4.6 ± 0.8 and 5.9 ± 1.5 % of highly expressed genes were global and changed-tissues DE genes, respectively. Changed-tissues DE genes varied in number between tissues, increasing linearly with the abundance of tissue specifically expressed genes in the tissue. Molecular evolution of global DE genes was rapid, unlike that of changed-tissues DE genes. Based on gene ontology, global and changed-tissues DE genes were different, having no common GO terms. Expression differences of most global DE genes were regulated by cis

  16. Noise minimization in eukaryotic gene expression.

    Directory of Open Access Journals (Sweden)

    Hunter B Fraser

    2004-06-01

    Full Text Available All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or "noise." Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection.

  17. Noise minimization in eukaryotic gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Fraser, Hunter B.; Hirsh, Aaron E.; Giaever, Guri; Kumm, Jochen; Eisen, Michael B.

    2004-01-15

    All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or noise. Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection.

  18. Paternally expressed genes predominate in the placenta.

    Science.gov (United States)

    Wang, Xu; Miller, Donald C; Harman, Rebecca; Antczak, Douglas F; Clark, Andrew G

    2013-06-25

    The discovery of genomic imprinting through studies of manipulated mouse embryos indicated that the paternal genome has a major influence on placental development. However, previous research has not demonstrated paternal bias in imprinted genes. We applied RNA sequencing to trophoblast tissue from reciprocal hybrids of horse and donkey, where genotypic differences allowed parent-of-origin identification of most expressed genes. Using this approach, we identified a core group of 15 ancient imprinted genes, of which 10 were paternally expressed. An additional 78 candidate imprinted genes identified by RNA sequencing also showed paternal bias. Pyrosequencing was used to confirm the imprinting status of six of the genes, including the insulin receptor (INSR), which may play a role in growth regulation with its reciprocally imprinted ligand, histone acetyltransferase-1 (HAT1), a gene involved in chromatin modification, and lymphocyte antigen 6 complex, locus G6C, a newly identified imprinted gene in the major histocompatibility complex. The 78 candidate imprinted genes displayed parent-of-origin expression bias in placenta but not fetus, and most showed less than 100% silencing of the imprinted allele. Some displayed variability in imprinting status among individuals. This variability results in a unique epigenetic signature for each placenta that contributes to variation in the intrauterine environment and thus presents the opportunity for natural selection to operate on parent-of-origin differential regulation. Taken together, these features highlight the plasticity of imprinting in mammals and the central importance of the placenta as a target tissue for genomic imprinting.

  19. Gene expression profiling of solitary fibrous tumors.

    Directory of Open Access Journals (Sweden)

    François Bertucci

    Full Text Available BACKGROUND: Solitary fibrous tumors (SFTs are rare spindle-cell tumors. Their cell-of-origin and molecular basis are poorly known. They raise several clinical problems. Differential diagnosis may be difficult, prognosis is poorly apprehended by histoclinical features, and no effective therapy exists for advanced stages. METHODS: We profiled 16 SFT samples using whole-genome DNA microarrays and analyzed their expression profiles with publicly available profiles of 36 additional SFTs and 212 soft tissue sarcomas (STSs. Immunohistochemistry was applied to validate the expression of some discriminating genes. RESULTS: SFTs displayed whole-genome expression profiles more homogeneous and different from STSs, but closer to genetically-simple than genetically-complex STSs. The SFTs/STSs comparison identified a high percentage (∼30% of genes as differentially expressed, most of them without any DNA copy number alteration. One of the genes most overexpressed in SFTs encoded the ALDH1 stem cell marker. Several upregulated genes and associated ontologies were also related to progenitor/stem cells. SFTs also overexpressed genes encoding therapeutic targets such as kinases (EGFR, ERBB2, FGFR1, JAK2, histone deacetylases, or retinoic acid receptors. Their overexpression was found in all SFTs, regardless the anatomical location. Finally, we identified a 31-gene signature associated with the mitotic count, containing many genes related to cell cycle/mitosis, including AURKA. CONCLUSION: We established a robust repertoire of genes differentially expressed in SFTs. Certain overexpressed genes could provide new diagnostic (ALDH1A1, prognostic (AURKA and/or therapeutic targets.

  20. Soybean physiology and gene expression during drought.

    Science.gov (United States)

    Stolf-Moreira, R; Medri, M E; Neumaier, N; Lemos, N G; Pimenta, J A; Tobita, S; Brogin, R L; Marcelino-Guimarães, F C; Oliveira, M C N; Farias, J R B; Abdelnoor, R V; Nepomuceno, A L

    2010-10-05

    Soybean genotypes MG/BR46 (Conquista) and BR16, drought-tolerant and -sensitive, respectively, were compared in terms of morphophysiological and gene-expression responses to water stress during two stages of development. Gene-expression analysis showed differential responses in Gmdreb1a and Gmpip1b mRNA expression within 30 days of water-deficit initiation in MG/BR46 (Conquista) plants. Within 45 days of initiating stress, Gmp5cs and Gmpip1b had relatively higher expression. Initially, BR16 showed increased expression only for Gmdreb1a, and later (45 days) for Gmp5cs, Gmdefensin and Gmpip1b. Only BR16 presented down-regulated expression of genes, such as Gmp5cs and Gmpip1b, 30 days after the onset of moisture stress, and Gmgols after 45 days of stress. The faster perception of water stress in MG/BR46 (Conquista) and the better maintenance of up-regulated gene expression than in the sensitive BR16 genotype imply mechanisms by which the former is better adapted to tolerate moisture deficiency.

  1. Expressing PHB synthetic genes through chloroplast genetic engineering

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Chloroplast integration and expression vector containing expression cassettes for phbB, phbA, phbC and aadA genes was constructed and bombarded into the tobacco chloroplast genome. Transplastomic plants were analyzed with PCR and Southern blot. Their homoplastomy was also judged. Northern dot and RT-PCR analysis were employed to investigate transgene expression at transcriptional level. The results indicate that the chloroplast transformation system is compatible for poly-3-hydroxybutyrate (PHB) production.

  2. Age-related vascular gene expression profiling in mice.

    Science.gov (United States)

    Rammos, Christos; Hendgen-Cotta, Ulrike B; Deenen, Rene; Pohl, Julia; Stock, Pia; Hinzmann, Christian; Kelm, Malte; Rassaf, Tienush

    2014-01-01

    Increasing age involves a number of detrimental changes in the cardiovascular system and particularly on the large arteries. It deteriorates vascular integrity and leads to increased vascular stiffness entailing hypertension with increased cardiovascular morbidity and mortality. The consequences of continuous oxidative stress and damages to biomolecules include altered gene expression, genomic instability, mutations, loss of cell division and cellular responses to increased stress. Many studies have been performed in aged C57BL/6 mice; however, analyses of the age-related changes that occur at a gene expression level and transcriptional profile in vascular tissue have not been elucidated in depth. To determine the changes of the vascular transcriptome, we conducted gene expression microarray experiments on aortas of adult and old mice, in which age-related vascular dysfunction was confirmed by increased stiffness and associated systolic hypertension. Our results highlight differentially expressed genes overrepresented in Gene Ontology categories. Molecular interaction and reaction pathways involved in vascular functions and disease, within the transforming growth factor-beta (TGF-β) pathway, the renin-angiotensin system and the detoxification systems are displayed. Our results provide insight to an altered gene expression profile related to age, thus offering useful clues to counteract or prevent vascular aging and its detrimental consequences. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. Simultaneous tracking of fly movement and gene expression using GFP

    Directory of Open Access Journals (Sweden)

    Tavaré Simon

    2008-12-01

    Full Text Available Abstract Background Green Fluorescent Protein (GFP is used extensively as a reporter for transgene expression in Drosophila and other organisms. However, GFP has not generally been used as a reporter for circadian patterns of gene expression, and it has not previously been possible to correlate patterns of reporter expression with 3D movement and behavior of transgenic animals. Results We present a video tracking system that allows tissue-specific GFP expression to be quantified and correlated with 3D animal movement in real time. eyeless/Pax6 reporter expression had a 12 hr period that correlated with fly activity levels. hsp70 and hsp22 gene reporters were induced during fly aging in circadian patterns (24 hr and 18 hr periods, respectively, and spiked in the hours preceding and overlapping the death of the animal. The phase of hsp gene reporter expression relative to fly activity levels was different for each fly, and remained the same throughout the life span. Conclusion These experiments demonstrate that GFP can readily be used to assay longitudinally fly movement and tissue-specific patterns of gene expression. The hsp22-GFP and hsp70-GFP expression patterns were found to reflect accurately the endogenous gene expression patterns, including induction during aging and circadian periodicity. The combination of these new tracking methods with the hsp-GFP reporters revealed additional information, including a spike in hsp22 and hsp70 reporter expression preceding death, and an intriguing fly-to-fly variability in the phase of hsp70 and hsp22 reporter expression patterns. These methods allow specific temporal patterns of gene expression to be correlated with temporal patterns of animal activity, behavior and mortality.

  4. Early gene expression changes with rush immunotherapy

    Directory of Open Access Journals (Sweden)

    Barnett Sherry

    2011-09-01

    Full Text Available Abstract Background To examine whether whole genome expression profiling could reveal changes in mRNA expression of peripheral blood mononuclear cells (PBMC from allergic patients undergoing rush immunotherapy (RIT that might be manifest within the first few months of treatment. Methods For this study, PBMC from three allergic patients undergoing RIT were assessed at four timepoints: prior to RIT, at 1 week and 7 week post-RIT, during build-up and at 4 months, after establishment of a maintenance dose. PBMC mRNA gene expression changes over time were determined by oligonucleotide microarrays using the Illumina Human-6 BeadChip Platform, which simultaneously interrogates expression profiles of > 47,000 transcripts. Differentially expressed genes were identified using well-established statistical analysis for microarrays. In addition, we analyzed peripheral blood basophil high-affinity IgE receptor (Fc epsilon RI expression and T-regulatory cell frequency as detected by expression of CD3+CD4+CD25bright cells at each timepoint using flow cytometry. Results In comparing the initial 2 timepoints with the final 2 timepoints and analyzing for genes with ≥1.5-fold expression change (p less than or equal to 0.05, BH-FDR, we identified 507 transcripts. At a 2-fold change (p less than or equal to 0.05, BH-FDR, we found 44 transcripts. Of these, 28 were up-regulated and 16 were down-regulated genes. From these datasets, we have identified changes in immunologically relevant genes from both the innate and adaptive response with upregulation of expressed genes for molecules including IL-1β, IL-8, CD40L, BTK and BCL6. At the 4 month timepoint, we noted a downward trend in Fc epsilon RI expression in each of the three patients and increased allergen-specific IgG4 levels. No change was seen in the frequency of peripheral T-regulatory cells expressed over the four timepoints. Conclusions We observed significant changes in gene expression early in peripheral

  5. Gene Disease Diagnostic System

    Institute of Scientific and Technical Information of China (English)

    黄国亮; 张腾飞; 程京; 周玉祥; 刘诚迅; 金国藩; 邬敏贤; 严瑛白; 杨蓉

    2002-01-01

    Binary optics, where the optical element can be fabricated on a thin glass plate with micro-ion-etching film layer, has been widely applied in recent years. A novel optical scanning system for gene disease diagnostics described in this paper has four kinds of optical devices, including beam splitters, an array lens, an array filter and detection arrays. A software was developed to design the binary optics system using an iterative method. Two beam splitters were designed and fabricated, which can divide a beam into a 9×9 array or into a 13×13 array. The beam splitters have good diffraction efficiencies (>70%) and an even energy distribution. The gene disease diagnostic system is a portable biochip and binary optics technology. The binary optical devices in the non-confocal scanning system can raise the fluorescence detection sensitivity of the micro-array hybrid biochip.

  6. Characterization of the iron-regulated desA promoter of Streptomyces pilosus as a system for controlled gene expression in actinomycetes

    Directory of Open Access Journals (Sweden)

    Martín Juan F

    2003-05-01

    Full Text Available Abstract Background The bioavailability of iron is quite low since it is usually present as insoluble complexes. To solve the bioavailability problem microorganisms have developed highly efficient iron-scavenging systems based on the synthesis of siderophores that have high iron affinity. The systems of iron assimilation in microorganisms are strictly regulated to control the intracellular iron levels since at high concentrations iron is toxic for cells. Streptomyces pilosus synthesizes the siderofore desferrioxamine B. The first step in desferrioxamine biosynthesis is decarboxylation of L-lysine to form cadaverine, a desferrioxamine B precursor. This reaction is catalyzed by the lysine decarboxylase, an enzyme encoded by the desA gene that is repressed by iron. Results The binding of the DmdR (acronym for divalent metal dependent repressor to the desA promoter in presence of Fe2+ or other divalent ions has been characterized. A 51 bp DNA fragment of the desA promoter containing the 9 bp inverted repeat was sufficient for binding of the DmdR repressor, as observed by the electrophoretic mobility shift assay. The desA mobility shift was prevented by neutralizing DmdR with anti-DmdR antibodies or by chelating the divalent metal in the binding reaction with 2,2'-dipyridyl. Binding to the desA promoter was observed with purified DmdR repressors of Streptomyces coelicolor or Rhodococcus fascians suggesting that there is a common mechanism of iron-regulation in actinomycetes. The complete desA promoter region was coupled using transcriptional fusions to the amy reporter gene (encoding α-amylase in low copy or multicopy Streptomyces vectors. The iron-regulated desA promoter was induced by addition of the iron chelating agent 2,2'-dipyridyl resulting in a strong expression of the reporter gene. Conclusions The iron-regulated desA promoter can be used for inducible expression of genes in Streptomyces species, as shown by de-repression of the promoter

  7. Interdependence of cell growth and gene expression: origins and consequences.

    Science.gov (United States)

    Scott, Matthew; Gunderson, Carl W; Mateescu, Eduard M; Zhang, Zhongge; Hwa, Terence

    2010-11-19

    In bacteria, the rate of cell proliferation and the level of gene expression are intimately intertwined. Elucidating these relations is important both for understanding the physiological functions of endogenous genetic circuits and for designing robust synthetic systems. We describe a phenomenological study that reveals intrinsic constraints governing the allocation of resources toward protein synthesis and other aspects of cell growth. A theory incorporating these constraints can accurately predict how cell proliferation and gene expression affect one another, quantitatively accounting for the effect of translation-inhibiting antibiotics on gene expression and the effect of gratuitous protein expression on cell growth. The use of such empirical relations, analogous to phenomenological laws, may facilitate our understanding and manipulation of complex biological systems before underlying regulatory circuits are elucidated.

  8. Gene Expression Measurement Module (GEMM) - a fully automated, miniaturized instrument for measuring gene expression in space

    Science.gov (United States)

    Karouia, Fathi; Ricco, Antonio; Pohorille, Andrew; Peyvan, Kianoosh

    2012-07-01

    The capability to measure gene expression on board spacecrafts opens the doors to a large number of experiments on the influence of space environment on biological systems that will profoundly impact our ability to conduct safe and effective space travel, and might also shed light on terrestrial physiology or biological function and human disease and aging processes. Measurements of gene expression will help us to understand adaptation of terrestrial life to conditions beyond the planet of origin, identify deleterious effects of the space environment on a wide range of organisms from microbes to humans, develop effective countermeasures against these effects, determine metabolic basis of microbial pathogenicity and drug resistance, test our ability to sustain and grow in space organisms that can be used for life support and in situ resource utilization during long-duration space exploration, and monitor both the spacecraft environment and crew health. These and other applications hold significant potential for discoveries in space biology, biotechnology and medicine. Accordingly, supported by funding from the NASA Astrobiology Science and Technology Instrument Development Program, we are developing a fully automated, miniaturized, integrated fluidic system for small spacecraft capable of in-situ measuring microbial expression of thousands of genes from multiple samples. The instrument will be capable of (1) lysing bacterial cell walls, (2) extracting and purifying RNA released from cells, (3) hybridizing it on a microarray and (4) providing electrochemical readout, all in a microfluidics cartridge. The prototype under development is suitable for deployment on nanosatellite platforms developed by the NASA Small Spacecraft Office. The first target application is to cultivate and measure gene expression of the photosynthetic bacterium Synechococcus elongatus, i.e. a cyanobacterium known to exhibit remarkable metabolic diversity and resilience to adverse conditions

  9. Standardization and validation of an induced ovulation model system in buffalo cows: Characterization of gene expression changes in the periovulatory follicle.

    Science.gov (United States)

    Jyotsna, U R; Medhamurthy, R

    2009-07-01

    In bovines characterization of biochemical and molecular determinants of the dominant follicle before and during different time intervals after gonadotrophin surge requires precise identification of the dominant follicle from a follicular wave. The objectives of the present study were to standardize an experimental model in buffalo cows for accurately identifying the dominant follicle of the first wave of follicular growth and characterize changes in follicular fluid hormone concentrations as well as expression patterns of various genes associated with the process of ovulation. From the day of estrus (day 0), animals were subjected to blood sampling and ultrasonography for monitoring circulating progesterone levels and follicular growth. On day 7 of the cycle, animals were administered a PGF(2alpha) analogue (Tiaprost Trometamol, 750 microg i.m.) followed by an injection of hCG (2000 IU i.m.) 36 h later. Circulating progesterone levels progressively increased from day 1 of the cycle to 2.26+/-0.17 ng/ml on day 7 of the cycle, but declined significantly after PGF(2alpha) injection. A progressive increase in the size of the dominant follicle was observed by ultrasonography. The follicular fluid estradiol and progesterone concentrations in the dominant follicle were 600+/-16.7 and 38+/-7.6 ng/ml, respectively, before hCG injection and the concentration of estradiol decreased to 125.8+/-25.26 ng/ml, but concentration of progesterone increased to 195+/-24.6 ng/ml, 24h post-hCG injection. Inh-alpha and Cyp19A1 expressions in granulosa cells were maximal in the dominant follicle and declined in response to hCG treatment. Progesterone receptor, oxytocin and cycloxygenase-2 expressions in granulosa cells, regarded as markers of ovulation, were maximal at 24h post-hCG. The expressions of genes belonging to the super family of proteases were also examined; Cathepsin L expression decreased, while ADAMTS 3 and 5 expressions increased 24h post-hCG treatment. The results of the

  10. Alternative-splicing-mediated gene expression

    Science.gov (United States)

    Wang, Qianliang; Zhou, Tianshou

    2014-01-01

    Alternative splicing (AS) is a fundamental process during gene expression and has been found to be ubiquitous in eukaryotes. However, how AS impacts gene expression levels both quantitatively and qualitatively remains to be fully explored. Here, we analyze two common models of gene expression, each incorporating a simple splice mechanism that a pre-mRNA is spliced into two mature mRNA isoforms in a probabilistic manner. In the constitutive expression case, we show that the steady-state molecular numbers of two mature mRNA isoforms follow mutually independent Poisson distributions. In the bursting expression case, we demonstrate that the tail decay of the steady-state distribution for both mature mRNA isoforms that in general are not mutually independent can be characterized by the product of mean burst size and splicing probability. In both cases, we find that AS can efficiently modulate both the variability (measured by variance) and the noise level of the total mature mRNA, and in particular, the latter is always lower than the noise level of the pre-mRNA, implying that AS always reduces the noise. These results altogether reveal that AS is a mechanism of efficiently controlling the gene expression noise.

  11. Gene expression profiling for targeted cancer treatment.

    Science.gov (United States)

    Yuryev, Anton

    2015-01-01

    There is certain degree of frustration and discontent in the area of microarray gene expression data analysis of cancer datasets. It arises from the mathematical problem called 'curse of dimensionality,' which is due to the small number of samples available in training sets, used for calculating transcriptional signatures from the large number of differentially expressed (DE) genes, measured by microarrays. The new generation of causal reasoning algorithms can provide solutions to the curse of dimensionality by transforming microarray data into activity of a small number of cancer hallmark pathways. This new approach can make feature space dimensionality optimal for mathematical signature calculations. The author reviews the reasons behind the current frustration with transcriptional signatures derived from DE genes in cancer. He also provides an overview of the novel methods for signature calculations based on differentially variable genes and expression regulators. Furthermore, the authors provide perspectives on causal reasoning algorithms that use prior knowledge about regulatory events described in scientific literature to identify expression regulators responsible for the differential expression observed in cancer samples. The author advocates causal reasoning methods to calculate cancer pathway activity signatures. The current challenge for these algorithms is in ensuring quality of the knowledgebase. Indeed, the development of cancer hallmark pathway collections, together with statistical algorithms to transform activity of expression regulators into pathway activity, are necessary for causal reasoning to be used in cancer research.

  12. Spatial gene expression quantification in changing morphologies

    NARCIS (Netherlands)

    Botman, D.

    2016-01-01

    In systems biology, an organisms’ behavior is explained from the interactions among individual components such as genes and proteins. With few exceptions, interactions among genes and proteins are not measured directly and are therefore inferred from the observed output of a biological system. A net

  13. Predicting metastasized seminoma using gene expression.

    Science.gov (United States)

    Ruf, Christian G; Linbecker, Michael; Port, Matthias; Riecke, Armin; Schmelz, Hans U; Wagner, Walter; Meineke, Victor; Abend, Michael

    2012-07-01

    Treatment options for testis cancer depend on the histological subtype as well as on the clinical stage. An accurate staging is essential for correct treatment. The 'golden standard' for staging purposes is CT, but occult metastasis cannot be detected with this method. Currently, parameters such as primary tumour size, vessel invasion or invasion of the rete testis are used for predicting occult metastasis. Last year the association of these parameters with metastasis could not be validated in a new independent cohort. Gene expression analysis in testis cancer allowed discrimination between the different histological subtypes (seminoma and non-seminoma) as well as testis cancer and normal testis tissue. In a two-stage study design we (i) screened the whole genome (using human whole genome microarrays) for candidate genes associated with the metastatic stage in seminoma and (ii) validated and quantified gene expression of our candidate genes (real-time quantitative polymerase chain reaction) on another independent group. Gene expression measurements of two of our candidate genes (dopamine receptor D1 [DRD1] and family with sequence similarity 71, member F2 [FAM71F2]) examined in primary testis cancers made it possible to discriminate the metastasis status in seminoma. The discriminative ability of the genes exceeded the predictive significance of currently used histological/pathological parameters. Based on gene expression analysis the present study provides suggestions for improved individual decision making either in favour of early adjuvant therapy or increased surveillance. To evaluate the usefulness of gene expression profiling for predicting metastatic status in testicular seminoma at the time of first diagnosis compared with established clinical and pathological parameters. Total RNA was isolated from testicular tumours of metastasized patients (12 patients, clinical stage IIa-III), non-metastasized patients (40, clinical stage I) and adjacent 'normal' tissue

  14. A comparative analysis of green fluorescent protein and -glucuronidase protein-encoding genes as a reporter system for studying the temporal expression profiles of promoters

    Indian Academy of Sciences (India)

    P Kavita; Pradeep Kumar Burma

    2008-09-01

    The assessment of activity of promoters has been greatly facilitated by the use of reporter genes. However, the activity as assessed by reporter gene is a reflection of not only promoter strength, but also that of the stability of the mRNA and the protein encoded by the reporter gene. While a stable reporter gene product is an advantage in analysing activities of weak promoters, it becomes a major limitation for understanding temporal expression patterns of a promoter, as the reporter product persists even after the activity of the promoter ceases. In the present study we undertook a comparative analysis of two reporter genes, -glucuronidase (gus) and green fluorescent protein (sgfp), for studying the temporal expression pattern of tapetum-specific promoters A9 (Arabidopsis thaliana) and TA29 (Nicotiana tabacum). The activity of A9 and TA29 promoters as assessed by transcript profiles of the reporter genes (gus or sgfp) remained the same irrespective of the reporter gene used. However, while the deduced promoter activity using gus was extended temporally beyond the actual activity of the promoter, sgfp as recorded through its fluorescence correlated better with the transcription profile. Our results thus demonstrate that sgfp is a better reporter gene compared to gus for assessment of temporal activity of promoters. Although several earlier reports have commented on the possible errors in deducing temporal activities of promoters using GUS as a reporter protein, we experimentally demonstrate the advantage of using reporter genes such as gfp for analysis of temporal expression patterns.

  15. Gene expression profiles in skeletal muscle after gene electrotransfer

    DEFF Research Database (Denmark)

    Hojman, Pernille; Zibert, John R; Gissel, Hanne;

    2007-01-01

    with the control muscles. Most interestingly, no changes in the expression of proteins involved in inflammatory responses or muscle regeneration was detected, indicating limited muscle damage and regeneration. Histological analysis revealed structural changes with loss of cell integrity and striation pattern......BACKGROUND: Gene transfer by electroporation (DNA electrotransfer) to muscle results in high level long term transgenic expression, showing great promise for treatment of e.g. protein deficiency syndromes. However little is known about the effects of DNA electrotransfer on muscle fibres. We have......) followed by a long low voltage pulse (LV, 100 V/cm, 400 ms); a pulse combination optimised for efficient and safe gene transfer. Muscles were transfected with green fluorescent protein (GFP) and excised at 4 hours, 48 hours or 3 weeks after treatment. RESULTS: Differentially expressed genes were...

  16. Gene expression analysis of flax seed development

    Directory of Open Access Journals (Sweden)

    Sharpe Andrew

    2011-04-01

    Full Text Available Abstract Background Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed. Results We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages seed coats (globular and torpedo stages and endosperm (pooled globular to torpedo stages and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST (GenBank accessions LIBEST_026995 to LIBEST_027011 were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152 had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development. Conclusions We have developed a foundational database of expressed sequences and collection of plasmid

  17. Lithium ions induce prestalk-associated gene expression and inhibit prespore gene expression in Dictyostelium discoideum

    NARCIS (Netherlands)

    Peters, Dorien J.M.; Lookeren Campagne, Michiel M. van; Haastert, Peter J.M. van; Spek, Wouter; Schaap, Pauline

    1989-01-01

    We investigated the effect of Li+ on two types of cyclic AMP-regulated gene expression and on basal and cyclic AMP-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) levels. Li+ effectively inhibits cyclic AMP-induced prespore gene expression, half-maximal inhibition occurring at about 2mM-LiCl.

  18. Manipulation of gene expression in the mammalian nervous system: application in the study of neurite outgrowth and neuroregeneration related proteins

    NARCIS (Netherlands)

    Gispen, W.H.; Holtmaat, A.J.G.D.; Oestreicher, A.B.; Verhaagen, J.

    1998-01-01

    A fundamental issue in neurobiology entails the study of the formation of neuronal connections and their potential to regenerate following injury. In recent years, an expanding number of gene families has been identified involved in different aspects of neurite outgrowth and regeneration. These incl

  19. Manipulation of gene expression in the mammalian nervous system: application in the study of neurite outgrowth and neuroregeneration related proteins

    NARCIS (Netherlands)

    Gispen, W.H.; Holtmaat, A.J.G.D.; Oestreicher, A.B.; Verhaagen, J.

    1998-01-01

    A fundamental issue in neurobiology entails the study of the formation of neuronal connections and their potential to regenerate following injury. In recent years, an expanding number of gene families has been identified involved in different aspects of neurite outgrowth and regeneration. These

  20. Screening and expression of genes from metagenomes.

    Science.gov (United States)

    Leis, Benedikt; Angelov, Angel; Liebl, Wolfgang

    2013-01-01

    Microorganisms are the most abundant and widely spread organisms on earth. They colonize a huge variety of natural and anthropogenic environments, including very specialized ecological niches and even extreme habitats, which are made possible by the immense metabolic diversity and genetic adaptability of microbes. As most of the organisms from environmental samples defy cultivation, cultivation-independent metagenomics approaches have been applied since more than one decade to access and characterize the phylogenetic diversity in microbial communities as well as their metabolic potential and ecological functions. Thereby, metagenomics has fully emerged as an own scientific field for mining new biocatalysts for many industrially relevant processes in biotechnology and pharmaceutics. This review summarizes common metagenomic approaches ranging from sampling, isolation of nucleic acids, construction of metagenomic libraries and their evaluation. Sequence-based screenings implement next-generation sequencing platforms, microarrays or PCR-based methods, while function-based analysis covers heterologous expression of metagenomic libraries in diverse screening setups. Major constraints and advantages of each strategy are described. The importance of alternative host-vector systems is discussed, and in order to underline the role of phylogenetic and physiological distance from the gene donor and the expression host employed, a case study is presented that describes the screening of a genomic library from an extreme thermophilic bacterium in both Escherichia coli and Thermus thermophilus. Metatranscriptomics, metaproteomics and single-cell-based methods are expected to complement metagenomic screening efforts to identify novel biocatalysts from environmental samples.

  1. Ebola virus infection induces irregular dendritic cell gene expression.

    Science.gov (United States)

    Melanson, Vanessa R; Kalina, Warren V; Williams, Priscilla

    2015-02-01

    Filoviruses subvert the human immune system in part by infecting and replicating in dendritic cells (DCs). Using gene arrays, a phenotypic profile of filovirus infection in human monocyte-derived DCs was assessed. Monocytes from human donors were cultured in GM-CSF and IL-4 and were infected with Ebola virus Kikwit variant for up to 48 h. Extracted DC RNA was analyzed on SuperArray's Dendritic and Antigen Presenting Cell Oligo GEArray and compared to uninfected controls. Infected DCs exhibited increased expression of cytokine, chemokine, antiviral, and anti-apoptotic genes not seen in uninfected controls. Significant increases of intracellular antiviral and MHC I and II genes were also noted in EBOV-infected DCs. However, infected DCs failed to show any significant difference in co-stimulatory T-cell gene expression from uninfected DCs. Moreover, several chemokine genes were activated, but there was sparse expression of chemokine receptors that enabled activated DCs to home to lymph nodes. Overall, statistically significant expression of several intracellular antiviral genes was noted, which may limit viral load but fails to stop replication. EBOV gene expression profiling is of vital importance in understanding pathogenesis and devising novel therapeutic treatments such as small-molecule inhibitors.

  2. Paternal irradiation perturbs the expression of circadian genes in offspring

    Energy Technology Data Exchange (ETDEWEB)

    Gomes, Andre M.G.F.; Barber, Ruth C.; Dubrova, Yuri E., E-mail: yed2@le.ac.uk

    2015-05-15

    Highlights: • We have analysed gene expression in the offspring of irradiated male mice. • CBA/Ca and BALB/c male mice were used in our study. • The pattern of gene expression was established in four tissues. • Expression of genes in involved in rhythmic process/circadian rhythm is compromised. • Our data may explain the phenomenon of transgenerational genomic instability. - Abstract: The circadian system represents a complex network which influences the timing of many biological processes. Recent studies have established that circadian alterations play an important role in the susceptibility to many human diseases, including cancer. Here we report that paternal irradiation in mice significantly affects the expression of genes involved in rhythmic processes in their first-generation offspring. Using microarrays, the patterns of gene expression were established for brain, kidney, liver and spleen samples from the non-exposed offspring of irradiated CBA/Ca and BALB/c male mice. The most over-represented categories among the genes differentially expressed in the offspring of control and irradiated males were those involved in rhythmic process, circadian rhythm and DNA-dependent regulation of transcription. The results of our study therefore provide a plausible explanation for the transgenerational effects of paternal irradiation, including increased transgenerational carcinogenesis described in other studies.

  3. Visual sensitivities tuned by heterochronic shifts in opsin gene expression

    Directory of Open Access Journals (Sweden)

    McFarland William N

    2008-05-01

    Full Text Available Abstract Background Cichlid fishes have radiated into hundreds of species in the Great Lakes of Africa. Brightly colored males display on leks and vie to be chosen by females as mates. Strong discrimination by females causes differential male mating success, rapid evolution of male color patterns and, possibly, speciation. In addition to differences in color pattern, Lake Malawi cichlids also show some of the largest known shifts in visual sensitivity among closely related species. These shifts result from modulated expression of seven cone opsin genes. However, the mechanisms for this modulated expression are unknown. Results In this work, we ask whether these differences might result from changes in developmental patterning of cone opsin genes. To test this, we compared the developmental pattern of cone opsin gene expression of the Nile tilapia, Oreochromis niloticus, with that of several cichlid species from Lake Malawi. In tilapia, quantitative polymerase chain reaction showed that opsin gene expression changes dynamically from a larval gene set through a juvenile set to a final adult set. In contrast, Lake Malawi species showed one of two developmental patterns. In some species, the expressed gene set changes slowly, either retaining the larval pattern or progressing only from larval to juvenile gene sets (neoteny. In the other species, the same genes are expressed in both larvae and adults but correspond to the tilapia adult genes (direct development. Conclusion Differences in visual sensitivities among species of Lake Malawi cichlids arise through heterochronic shifts relative to the ontogenetic pattern of the tilapia outgroup. Heterochrony has previously been shown to be a powerful mechanism for change in morphological evolution. We found that altering developmental expression patterns is also an important mechanism for altering sensory systems. These resulting sensory shifts will have major impacts on visual communication and could help

  4. Visual sensitivities tuned by heterochronic shifts in opsin gene expression

    Science.gov (United States)

    Carleton, Karen L; Spady, Tyrone C; Streelman, J Todd; Kidd, Michael R; McFarland, William N; Loew, Ellis R

    2008-01-01

    Background Cichlid fishes have radiated into hundreds of species in the Great Lakes of Africa. Brightly colored males display on leks and vie to be chosen by females as mates. Strong discrimination by females causes differential male mating success, rapid evolution of male color patterns and, possibly, speciation. In addition to differences in color pattern, Lake Malawi cichlids also show some of the largest known shifts in visual sensitivity among closely related species. These shifts result from modulated expression of seven cone opsin genes. However, the mechanisms for this modulated expression are unknown. Results In this work, we ask whether these differences might result from changes in developmental patterning of cone opsin genes. To test this, we compared the developmental pattern of cone opsin gene expression of the Nile tilapia, Oreochromis niloticus, with that of several cichlid species from Lake Malawi. In tilapia, quantitative polymerase chain reaction showed that opsin gene expression changes dynamically from a larval gene set through a juvenile set to a final adult set. In contrast, Lake Malawi species showed one of two developmental patterns. In some species, the expressed gene set changes slowly, either retaining the larval pattern or progressing only from larval to juvenile gene sets (neoteny). In the other species, the same genes are expressed in both larvae and adults but correspond to the tilapia adult genes (direct development). Conclusion Differences in visual sensitivities among species of Lake Malawi cichlids arise through heterochronic shifts relative to the ontogenetic pattern of the tilapia outgroup. Heterochrony has previously been shown to be a powerful mechanism for change in morphological evolution. We found that altering developmental expression patterns is also an important mechanism for altering sensory systems. These resulting sensory shifts will have major impacts on visual communication and could help drive cichlid speciation

  5. Visualizing Gene Expression In Situ

    Energy Technology Data Exchange (ETDEWEB)

    Burlage, R.S.

    1998-11-02

    Visualizing bacterial cells and describing their responses to the environment are difficult tasks. Their small size is the chief reason for the difficulty, which means that we must often use many millions of cells in a sample in order to determine what the average response of the bacteria is. However, an average response can sometimes mask important events in bacterial physiology, which means that our understanding of these organisms will suffer. We have used a variety of instruments to visualize bacterial cells, all of which tell us something different about the sample. We use a fluorescence activated cell sorter to sort cells based on the fluorescence provided by bioreporter genes, and these can be used to select for particular genetic mutations. Cells can be visualized by epifluorescent microscopy, and sensitive photodetectors can be added that allow us to find a single bacterial cell that is fluorescent or bioluminescent. We have also used standard photomultipliers to examine cell aggregates as field bioreporter microorganisms. Examples of each of these instruments show how our understanding of bacterial physiology has changed with the technology.

  6. Gene expression profiles in irradiated cancer cells

    Science.gov (United States)

    Minafra, L.; Bravatà, V.; Russo, G.; Ripamonti, M.; Gilardi, M. C.

    2013-07-01

    Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses.

  7. Gene expression profiles in irradiated cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Minafra, L.; Bravatà, V.; Russo, G.; Ripamonti, M.; Gilardi, M. C. [IBFM CNR - LATO, Cefalù, Segrate (Italy)

    2013-07-26

    Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses.

  8. Gene Expression in the Human Endolymphatic Sac

    DEFF Research Database (Denmark)

    Møller, Martin Nue; Kirkeby, Svend; Vikeså, Jonas

    2015-01-01

    OBJECTIVES/HYPOTHESIS: The purpose of the present study is to explore, demonstrate, and describe the expression of genes related to the solute carrier (SLC) molecules of ion transporters in the human endolymphatic sac. STUDY DESIGN: cDNA microarrays and immunohistochemistry were used for analyses...... of fresh human endolymphatic sac tissue samples. METHODS: Twelve tissue samples of the human endolymphatic sac were obtained during translabyrinthine surgery for vestibular schwannoma. Microarray technology was used to investigate tissue sample expression of solute carrier family genes, using adjacent dura...... mater as control. Immunohistochemistry was used for verification of translation of selected genes, as well as localization of the specific protein within the sac. RESULTS: An extensive representation of the SLC family genes were upregulated in the human endolymphatic sac, including SLC26a4 Pendrin, SLC4...

  9. Global Effects of Catecholamines on Actinobacillus pleuropneumoniae Gene Expression

    Science.gov (United States)

    Li, Lu; Xu, Zhuofei; Zhou, Yang; Sun, Lili; Liu, Ziduo; Chen, Huanchun; Zhou, Rui

    2012-01-01

    Bacteria can use mammalian hormones to modulate pathogenic processes that play essential roles in disease development. Actinobacillus pleuropneumoniae is an important porcine respiratory pathogen causing great economic losses in the pig industry globally. Stress is known to contribute to the outcome of A. pleuropneumoniae infection. To test whether A. pleuropneumoniae could respond to stress hormone catecholamines, gene expression profiles after epinephrine (Epi) and norepinephrine (NE) treatment were compared with those from untreated bacteria. The microarray results showed that 158 and 105 genes were differentially expressed in the presence of Epi and NE, respectively. These genes were assigned to various functional categories including many virulence factors. Only 18 genes were regulated by both hormones. These genes included apxIA (the ApxI toxin structural gene), pgaB (involved in biofilm formation), APL_0443 (an autotransporter adhesin) and genes encoding potential hormone receptors such as tyrP2, the ygiY-ygiX (qseC-qseB) operon and narQ-narP (involved in nitrate metabolism). Further investigations demonstrated that cytotoxic activity was enhanced by Epi but repressed by NE in accordance with apxIA gene expression changes. Biofilm formation was not affected by either of the two hormones despite pgaB expression being affected. Adhesion to host cells was induced by NE but not by Epi, suggesting that the hormones affect other putative adhesins in addition to APL_0443. This study revealed that A. pleuropneumoniae gene expression, including those encoding virulence factors, was altered in response to both catecholamines. The differential regulation of A. pleuropneumoniae gene expression by the two hormones suggests that this pathogen may have multiple responsive systems for the two catecholamines. PMID:22347439

  10. Global effects of catecholamines on Actinobacillus pleuropneumoniae gene expression.

    Directory of Open Access Journals (Sweden)

    Lu Li

    Full Text Available Bacteria can use mammalian hormones to modulate pathogenic processes that play essential roles in disease development. Actinobacillus pleuropneumoniae is an important porcine respiratory pathogen causing great economic losses in the pig industry globally. Stress is known to contribute to the outcome of A. pleuropneumoniae infection. To test whether A. pleuropneumoniae could respond to stress hormone catecholamines, gene expression profiles after epinephrine (Epi and norepinephrine (NE treatment were compared with those from untreated bacteria. The microarray results showed that 158 and 105 genes were differentially expressed in the presence of Epi and NE, respectively. These genes were assigned to various functional categories including many virulence factors. Only 18 genes were regulated by both hormones. These genes included apxIA (the ApxI toxin structural gene, pgaB (involved in biofilm formation, APL_0443 (an autotransporter adhesin and genes encoding potential hormone receptors such as tyrP2, the ygiY-ygiX (qseC-qseB operon and narQ-narP (involved in nitrate metabolism. Further investigations demonstrated that cytotoxic activity was enhanced by Epi but repressed by NE in accordance with apxIA gene expression changes. Biofilm formation was not affected by either of the two hormones despite pgaB expression being affected. Adhesion to host cells was induced by NE but not by Epi, suggesting that the hormones affect other putative adhesins in addition to APL_0443. This study revealed that A. pleuropneumoniae gene expression, including those encoding virulence factors, was altered in response to both catecholamines. The differential regulation of A. pleuropneumoniae gene expression by the two hormones suggests that this pathogen may have multiple responsive systems for the two catecholamines.

  11. Sequencing and Gene Expression Analysis of Leishmania tropica LACK Gene.

    Directory of Open Access Journals (Sweden)

    Nour Hammoudeh

    2014-12-01

    Full Text Available Leishmania Homologue of receptors for Activated C Kinase (LACK antigen is a 36-kDa protein, which provokes a very early immune response against Leishmania infection. There are several reports on the expression of LACK through different life-cycle stages of genus Leishmania, but only a few of them have focused on L.tropica.The present study provides details of the cloning, DNA sequencing and gene expression of LACK in this parasite species. First, several local isolates of Leishmania parasites were typed in our laboratory using PCR technique to verify of Leishmania parasite species. After that, LACK gene was amplified and cloned into a vector for sequencing. Finally, the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, was evaluated by Reverse Transcription-PCR (RT-PCR technique.The typing result confirmed that all our local isolates belong to L.tropica. LACK gene sequence was determined and high similarity was observed with the sequences of other Leishmania species. Furthermore, the expression of LACK gene in both promastigotes and amastigotes forms was confirmed.Overall, the data set the stage for future studies of the properties and immune role of LACK gene products.

  12. The expression of tga1a gene from tobacco affects the expression of exogenous gene in transgenic plant

    Institute of Scientific and Technical Information of China (English)

    路子显; 常团结; 李旭刚; 徐军望; 李慧芬; 陈宛新; 冯德江; 肖桂芳; 朱祯

    2003-01-01

    The DNA-binding protein TGA1a of tobacco can specially interact with the enhancer sequence as-1 (-83 to -63) of CaMV35S promoter and show the function of transcriptional activation. In order to study the expression of exogenous gene affected by TGA1a, a trans-actingregulation system was formed by tandem connecting tga1a under the control of the phloem-specific promoter rolC with reporter gene under the control of CaMV35S. Then, the system abovewas utilized to construct a plant expression vector. Moreover, two plant expression vectors wereconstructed with the report gene controlled by CaMV35S and rolC promoter respectively as positive controls. Tobacco leaf disc transformed by Agrobacterium-mediated method and transgenic plants were regenerated. It was proved that the reporter gene existed in the genome of transgenic plants by Southern hybridization. The results of GUS activity indicated that the expression of tga1a controlled by rolC remarkably increased the expression of the reporter gene controlled by CaMV35S. GUS activity of transgenic plants containing trans-acting regulation system was higher than that of transgenic plants containing the reporter gene under the control of CaMV35S and rolC respectively, with the highest GUS activity of about tenfolds of two positive controls. Histochemical method demonstrated that GUS staining amassed mainly in phloem tissue of transgenic plantscontaining the trans-acting regulation system. A new model for arising the expression level and tissue-specific expression of exogenous gene in transgenic plant was established in this study.

  13. Regulation of gene expression by Goodwin's loop with many genes

    Science.gov (United States)

    Sielewiesiuk, Jan; Łopaciuk, Agata

    2012-01-01

    The paper presents a simple analysis of a long Goodwin's loop containing many genes. The genes form a closed series. The rate of transcription of any gene is up or down regulated by theprotein product of the preceding gene. We describe the loop with a system of ordinary differential equations of order s. Oscillatory solutions of the system are possible at the odd number of repressions and any number of inductions if the product of all Hill's coefficients, related to both repressions and inductions, is larger than:

  14. Induced Fungal Resistance to Insect Grazing : Reciprocal Fitness Consequences and Fungal Gene Expression in the Drosophila-Aspergillus Model System

    NARCIS (Netherlands)

    Ortiz, Silvia Caballero; Trienens, Monika; Rohlfs, Marko

    2013-01-01

    Background: Fungi are key dietary resources for many animals. Fungi, in consequence, have evolved sophisticated physical and chemical defences for repelling and impairing fungivores. Expression of such defences may entail costs, requiring diversion of energy and nutrients away from fungal growth and

  15. Mechanical Feedback and Arrest in Gene Expression

    Science.gov (United States)

    Sevier, Stuart; Levine, Herbert

    The ability to watch biochemical events at the single-molecule level has increasingly revealed that stochasticity plays a leading role in many biological phenomena. One important and well know example is the noisy, ``bursty'' manner of transcription. Recent experiments have revealed relationships between the level and noise in gene expressi