Sample records for lacz reporter gene

  1. A lacZ reporter gene expression atlas for 313 adult KOMP mutant mouse lines.

    PubMed

    West, David B; Pasumarthi, Ravi K; Baridon, Brian; Djan, Esi; Trainor, Amanda; Griffey, Stephen M; Engelhard, Eric K; Rapp, Jared; Li, Bowen; de Jong, Pieter J; Lloyd, K C Kent

    2015-04-01

    Expression of the bacterial beta-galactosidase reporter gene (lacZ) in the vector used for the Knockout Mouse Project (KOMP) is driven by the endogenous promoter of the target gene. In tissues from KOMP mice, histochemical staining for LacZ enzyme activity can be used to determine gene expression patterns. With this technique, we have produced a comprehensive resource of gene expression using both whole mount (WM) and frozen section (FS) LacZ staining in 313 unique KOMP mutant mouse lines. Of these, ? 80% of mutants showed specific staining in one or more tissues, while ? 20% showed no specific staining, ? 13% had staining in only one tissue, and ? 25% had staining in >6 tissues. The highest frequency of specific staining occurred in the brain (? 50%), male gonads (42%), and kidney (39%). The WM method was useful for rapidly identifying whole organ and some substructure staining, while the FS method often revealed substructure and cellular staining specificity. Both staining methods had >90% repeatability in biological replicates. Nonspecific LacZ staining occurs in some tissues due to the presence of bacteria or endogenous enzyme activity. However, this can be effectively distinguished from reporter gene activity by the combination of the WM and FS methods. After careful annotation, LacZ staining patterns in a high percentage of mutants revealed a unique structure-function not previously reported for many of these genes. The validation of methods for LacZ staining, annotation, and expression analysis reported here provides unique insights into the function of genes for which little is currently known. PMID:25591789

  2. Measurement of Ligand-Induced Activation in Single Viable T Cells Using the lacZ Reporter Gene

    NASA Astrophysics Data System (ADS)

    Karttunen, Jaana; Shastri, Nilabh

    1991-05-01

    We have used the bacterial ?-galactosidase gene (lacZ) as a reporter gene for the rapid measurement of T-cell antigen receptor (TCR)-mediated activation of individual T cells. The reporter construct contained the lacZ gene under the control of the nuclear factor of activated T cells (NF-AT) element of the human interleukin 2 enhancer [Fiering, S., Northrop, J. P., Nolan, G. P., Matilla, P., Crabtree, G. R. & Herzenberg, L. A. (1990) Genes Dev. 4, 1823-1834]. The activity of the intracellular lacZ enzyme was analyzed by flow cytometric measurement of fluorescein accumulation in cells loaded with the fluorogenic ?-galactosidase substrate fluorescein di-?-D-galactopyranoside. As a model system, the T-cell hybridoma BO4H9.1, which is specific for the lysozyme peptide (amino acids 74-88)/A^b complex, was transfected with the NF-AT-lacZ construct. lacZ activity was induced in 50-100% of the transfectant cells following exposure to pharmacological agents, to the physiological peptide/major histocompatibility complex ligand, or to other TCR-specific stimuli. Interestingly, increasing concentrations of the stimulus increased the fraction of lacZ^+ cells, but not the level of lacZ activity per cell. Even under widely varying levels of stimulus, the level of lacZ activity in individual lacZ^+ cells remained within a remarkably narrow range. These results demonstrate that TCR-mediated activation can be readily measured in single T cells and strongly suggest that, once committed to activation, the level of NF-AT transcriptional activity in individual T cells is independent of the form or concentration of stimulus. This assay is likely to prove useful for the study of early activation events in individual T cells and of TCR ligands.

  3. Inactivation in vivo of metabotropic glutamate receptor 1 by specific chromosomal insertion of reporter gene lacZ.

    PubMed

    Conquet, F

    1995-08-01

    Two main classes of glutamate receptors have been characterized, the ionotropic (iGluRs) and the metabotropic (mGluRs) glutamate receptors. In order to better understand the function of the latter, we have used the technique of gene targeting to generate mice in which the mGluR1 subtype has been inactivated. The disruption was carried out by homologous recombination in embryonic stem (ES) cells at the level of the seven-membrane domain, leaving the extracellular part of the receptor untouched. In addition, the reporter gene lacZ was inserted in frame with mGluR1 coding sequence within the second intracellular loop of the receptor. The transmission of the mutation to the germ line showed first that the fusion protein was functional and second that mGluR1 was inactivated. Therefore, the way homologous recombination was performed in ES cells demonstrated that gene replacement of mGluR1 by lacZ could be a powerful technique to disrupt a gene and at the same time study its endogenous expression. PMID:8532168

  4. Isolation of lacZ fusions to Proteus mirabilis genes regulated by intercellular signaling: potential role for the sugar phosphotransferase (Pts) system in regulation.

    PubMed

    Sturgill, Gwen M; Siddiqui, Soofia; Ding, Xuedong; Pecora, Nicole D; Rather, Philip N

    2002-11-19

    Using a mini-Tn5lacZ1 reporter transposon, lacZ fusions have been identified in Proteus mirabilis that are activated by the accumulation of self-produced extracellular signals. Genes identified by this approach include putative homologs of pgm, nlpA and two genes of unknown function. The extracellular signal(s) involved in activation were resistant to the effects of acid and alkali. The signal required for activation of (nlpA) cma482::lacZ was sensitive to protease, suggesting the signal is a peptide or small protein. The signals behaved as polar molecules and were not extractable with ethyl acetate. A mini-Tn5Cm insertion was identified in a probable ptsI homolog that blocked activation of the cma134::lacZ fusion by an extracellular signal. The ptsI mutation did not alter extracellular signal production and may have a role in signal response. PMID:12445644

  5. Transgenic LacZ under control of Hec-6st regulatory sequences recapitulates endogenous gene expression on high endothelial venules

    PubMed Central

    Liao, Shan; Bentley, Kevin; Lebrun, Marielle; Lesslauer, Werner; Ruddle, Frank H.; Ruddle, Nancy H.

    2007-01-01

    Hec-6st is a highly specific high endothelial venule (HEV) gene that is crucial for regulating lymphocyte homing to lymph nodes (LN). The enzyme is also expressed in HEV-like vessels in tertiary lymphoid organs that form in chronic inflammation in autoimmunity, graft rejection, and microbial infection. Understanding the molecular nature of Hec-6st regulation is crucial for elucidating its function in development and disease. However, studies of HEV are limited because of the difficulties in isolating and maintaining the unique characteristics of these vessels in vitro. The novel pClasper yeast homologous recombination technique was used to isolate from a BAC clone a 60-kb DNA fragment that included the Hec-6st (Chst4) gene with flanking sequences. Transgenic mice were generated with the ?-galactosidase (LacZ) reporter gene inserted in-frame in the exon II of Hec-6st within the isolated BAC DNA fragment. LacZ was expressed specifically on HEV in LN, as indicated by its colocalization with peripheral node vascular addressin. LacZ was increased in nasal-associated lymphoid tissue during development and was reduced in LN and nasal-associated lymphoid tissue by LT?R-Ig (lymphotoxin-? receptor human Ig fusion protein) treatment in a manner identical to the endogenous gene. The transgene was expressed at high levels in lymphoid accumulations with characteristics of tertiary lymphoid organs in the salivary glands of aged mice. Thus, the Hec-6s-LacZ construct faithfully reproduces Hec-6st tissue-specific expression and can be used in further studies to drive expression of reporter or effector genes, which could visualize or inhibit HEV in autoimmunity. PMID:17360566

  6. p21-LacZ reporter mice reflect p53-dependent toxic insult

    SciTech Connect

    Vasey, Douglas B. [Division of Gene Function and Development, Roslin Institute, Roslin, Midlothian, EH25 9PS (United Kingdom)], E-mail: douglas.vasey@bbsrc.ac.uk; Wolf, C. Roland [University of Dundee Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee, DD1 9SY (United Kingdom); CXR Biosciences Limited, James Lindsay Place, Dundee Technopole, Dundee, DD1 5JJ (United Kingdom); MacArtney, Thomas; Brown, Ken [CXR Biosciences Limited, James Lindsay Place, Dundee Technopole, Dundee, DD1 5JJ (United Kingdom); Whitelaw, C. Bruce A. [Division of Gene Function and Development, Roslin Institute, Roslin, Midlothian, EH25 9PS (United Kingdom)

    2008-03-15

    There is an urgent need to discover less toxic and more selective drugs to treat disease. The use of transgenic mice that report on toxic insult-induced transcription can provide a valuable tool in this regard. To exemplify this strategy, we have generated transgenic mice carrying a p21-LacZ transgene. Transgene activity reflected endogenous p21 gene activation in various tissues, displayed compound-specific spatial expression signatures in the brain and immune tissues and enabled p53-dependent and p53-independent responses to be identified. We discuss the application of these mice in delineating the molecular events in normal cellular growth and disease and for the evaluation of drug toxicity.

  7. Genetic and biochemical characterization of some missense mutations in the lacZ gene of Escherichia coli K-12.

    PubMed Central

    Truman, P; Bergquist, P L

    1976-01-01

    Some preparations of beta-galactosidase from strains of Escherichia coli carrying point mutations in their lacZ genes did not precipitate with antibody as effectively as wild-type enzyme, but did not appear to be chain-terminating mutations as judged by polarity measurements and suppression. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extracts of induced Lac+ strains revealed that the monomer of beta-galactosidase ran as a band uncontaminated by other cellular proteins. This method was used to identify missense mutations in the alpha and beta portions of the lacZ gene. Six of 13 mutations investigated were judged to be missense by this criterion. Measurement of the degree of polarity, the ability to complement a nonsense mutation at the operator-distal extremity of the gene (omega-complementation), and suppressibility by 12 nonsense suppressors allowed the assignment of six other mutations as either number or ochre. The protein figments produced by these six nonsense mutations appeared to be degraded in vivo. One mutation that could not be classified was either a missense mutation whose protein product was degraded or a very leak nonsense mutation. Two lacZ alleles were suppressed by the ochre suppressors supM and supN, although they were missense by other criteria. The ability of supM to suppress both nonsense and missense mutations can be explained if it is derived from a tyrosine transfer ribonucleic acid with a modified base in the first position of the anticodon. The mutations assigned to the missense class were not suppressed by the missense suppressors supH, supQ, glyV, glyU, or glyT. Our results suggest that the criteria used in the past to distinguish between nonsense and missense mutations may not be conclusive even when used together. PMID:780338

  8. Regulation of RAD54- and RAD52-lacZ gene fusions in Saccharomyces cerevisiae in response to DNA damage

    SciTech Connect

    Cole, G.M.; Schild, D.; Lovett, S.T.; Mortimer, R.K.

    1987-03-01

    The RAD52 and RAD54 genes in the yeast Saccharomyces cerevisiae are involved in both DNA repair and DNA recombination. RAD54 has recently been shown to be inducible by X-rays, while RAD52 is not. To further investigate the regulation of these genes, we constructed gene fusions using 5' regions upstream of the RAD52 and RAD54 genes and a 3'-terminal fragment of the Escherichia coli beta-galactosidase gene. Yeast transformants with either an integrated or an autonomously replicating plasmid containing these fusions expressed beta-galactosidase activity constitutively. In addition, the RAD54 gene fusion was inducible in both haploid and diploid cells in response to the DNA-damaging agents X-rays, UV light, and methyl methanesulfonate, but not in response to heat shock. The RAD52-lacZ gene fusion showed little or no induction in response to X-ray or UV radiation nor methyl methanesulfonate. Typical induction levels for RAD54 in cells exposed to such agents were from 3- to 12-fold, in good agreement with previous mRNA analyses. When MATa cells were arrested in G1 with alpha-factor, RAD54 was still inducible after DNA damage, indicating that the observed induction is independent of the cell cycle. Using a yeast vector containing the EcoRI structural gene fused to the GAL1 promoter, we showed that double-strand breaks alone are sufficient in vivo for induction of RAD54.

  9. RpoS-Regulated Genes of Escherichia coli Identified by Random lacZ Fusion Mutagenesis

    Microsoft Academic Search

    Somalinga R. V. Vijayakumar; Mark G. Kirchhof; Cheryl L. Patten; Herb E. Schellhorn

    2004-01-01

    RpoS is a conserved alternative sigma factor that regulates the expression of many stress response genes in Escherichia coli. The RpoS regulon is large but has not yet been completely characterized. In this study, we report the identification of over 100 RpoS-dependent fusions in a genetic screen based on the differential expression of an operon-lacZ fusion bank in rpoS mutant

  10. Replacement by Homologous Recombination of the minK Gene With lacZ Reveals Restriction of minK Expression to the Mouse Cardiac Conduction System

    Microsoft Academic Search

    Sabina Kupershmidt; Tao Yang; Mark E. Anderson; Andy Wessels; Kevin D. Niswender; Mark A. Magnuson; Dan M. Roden

    The minK gene encodes a 129-amino acid peptide the expression of which modulates function of cardiac delayed rectifier currents ( IKr and IKs), and mutations in minK are now recognized as one cause of the congenital long-QT syndrome. We have generated minK-deficient mice in which the bacterial lacZ gene has been substituted for the minK coding region such that b-galactosidase

  11. Developmental Behavior of Embryonic Myogenic Progenitors Transplanted into Adult Muscle as Revealed by Desmin LacZ Recombinant Gene

    Microsoft Academic Search

    Gwenola Auda-Boucher; Thierry Rouaud; Josiane Fontaine-Pérus; Fabien Le Grand; Marie-France Gardahaut

    2003-01-01

    We studied the behavior of myogenic progenitors from donor desmin+\\/– LacZ embryos after implantation into tibialis anterior muscle of 2-month-old mouse hosts. Myogenic progenitors were collected from 10-day post-coital mouse embryo somite dermomyotomes (DMs), forelimb buds (LBs), and trunks. The replacement of desmin by the LacZ coding sequence allowed specific monitoring of ?-galactosidase expression in donor myogenic cells. Immunostaining for

  12. Effects of DNA repair deficiency on the mutational specificity in the lacZ gene of Escherichia coli.

    PubMed

    Watanabe-Akanuma, M; Ohta, T

    1994-12-01

    The mutational specificities of various chemical mutagens were compared in isogenic E. coli strains with different DNA repair capabilities (wild-type, uvrA, umuC, and uvrA umuC) in a reversion assay employing a set of mutant lacZ genes that can detect two types of transitions, four types of transversions, and five kinds of specific frameshift events. A uvrA derivative was more sensitive than the wild-type strain to 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone for +1G, -1G, -2(C-G), +1A and -1A frameshifts, G.C-->A.T transitions, and G.C-->T.A transversions. In a uvrA background, G.C-->T.A transversions and +1G, +1A, and -1A frameshifts appeared to be umuC-dependent, while G.C-->A.T transitions were not. N-Ethyl-N'-nitro-N-nitrosoguanidine was more mutagenic in a uvrA background for five kinds of frameshifts and G.C-->A.T transitions, but not for G.C-->T.A, A.T-->C.G, and A.T-->G.C base substitutions. A.T-->C.G transversions were totally dependent on umuC gene function. For the investigation of mutational specificities induced by frameshift mutagens, an rfa mutation was additionally introduced. The rfa strain responded to 2-nitrofluorene, which induced primarily -2(C-G) frameshift mutations. In an rfa uvrA background, benzo[a]pyrene induced +1G, -1G, +1A, and -1A frameshifts. 2-Aminoanthracene induced +1G, -1G, and +1A, but not -1A, frameshifts, with -1G frameshifts predominating. Ethidium bromide induced only two types of frameshifts, -1G and +1A. Frameshifts induced by ICR-170 were independent of umuC gene function, while those by induced 1-nitropyrene were partly umuC-dependent. PMID:7526195

  13. RHIZOSPHERE COLONIZATION OF OILSEED RAPE BY PSEUDOMONAS ALCALIGENES A9(LACZ)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Root colonization of oilseed rape by Pseudomonas alcaligenes A9(LacZ) in rhizosphere microcosms was investigated with the aid of the lacZ marker gene. Rape seeds were pelletized with A9(LacZ), an effective bacterial strain for promotion of rape seedling growth . Results indicated that A9(LacZ) popu...

  14. Tracking Adult Neovascularization during Ischemia and Inflammation Using Vegfr2-LacZ Reporter Mice

    Microsoft Academic Search

    Regina Heidenreich; Toshinori Murayama; Marcy Silver; Christine Essl; Takayuki Asahara; Martin Röcken; Georg Breier

    2008-01-01

    The vascular endothelial growth factor\\/vascular endothelial growth factor receptor 2 (VEGF\\/VEGFR-2) signal transduction system plays a key role during embryonic vascular development and adult neovascularization. In contrast to many endothelial genes, VEGFR-2 is expressed at low levels in most adult vessels but is strongly upregulated during neovascularization, leading to a pro-angiogenic response. Here, we analyzed the activity of regulatory sequences

  15. Herpes Simplex Virus Type 1 Thymidine Kinase Sequence Fused to the lacZ Gene Increases Levels of ?-Galactosidase Activity per Genome of High-Capacity but Not First-Generation Adenoviral Vectors In Vitro and In Vivo? †

    PubMed Central

    Puntel, M.; Barrett, R. J.; Mondkar, S.; Saxena, V.; Kroeger, K. M.; Muhammad, A. K. M.; Liu, C.; Bondale, N.; Sciascia, S.; Xiong, W.; Shi, Y.; Salem, A.; Zadmehr, A.; Huynh, P.; Palmer, D.; Ng, P.; Castro, M. G.; Lowenstein, P. R.

    2009-01-01

    Increased transgene expression per vector genome is an important goal in the optimization of viral vectors for gene therapy. Herein we demonstrate that herpes simplex virus type 1 (HSV1) thymidine kinase (TK) gene sequences (1,131 bp) fused to the 3? end of lacZ increase transgene expression from high-capacity adenoviral vectors (HCAd), but not from first-generation (Ad) vectors. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), in contrast, increased transgene expression levels from Ad but not HCAd vectors. The differential activity of the HSV1 TK gene and WPRE sequences was detected both in vitro and in vivo and suggests potentially different mechanisms of action or the interaction of these elements with vector genomic sequences. PMID:19073729

  16. Biological Sensor for Sucrose Availability: Relative Sensitivities of Various Reporter Genes

    Microsoft Academic Search

    WILLIAM G. MILLER; MARIA T. BRANDL; BEATRIZ QUINONES; STEVEN E. LINDOW

    2001-01-01

    A set of three sucrose-regulated transcriptional fusions was constructed. Fusions p61RYTIR, p61RYlac, and p61RYice contain the scrR sucrose repressor gene and the promoterless gfp, lacZ, and inaZ reporter genes, respectively, fused to the scrY promoter from Salmonella enterica serovar Typhimurium. Cells of Erwinia her- bicola containing these fusions are induced only in media amended with sucrose, fructose, or sorbose. While

  17. Transgenic mice with p53-responsive lacZ: p53 activity varies dramatically during normal development and determines radiation and drug sensitivity in vivo.

    PubMed Central

    Komarova, E A; Chernov, M V; Franks, R; Wang, K; Armin, G; Zelnick, C R; Chin, D M; Bacus, S S; Stark, G R; Gudkov, A V

    1997-01-01

    To analyze the involvement of p53-dependent transcriptional activation in normal development and in response to DNA damage in vivo, we created transgenic mice with a lacZ reporter gene under the control of a p53-responsive promoter. Five independent strains showed similar patterns of transgene expression. In untreated animals, lacZ expression was limited to the developing nervous system of embryos and newborn mice and was strongly decreased in the adult brain. gamma-irradiation or adriamycin treatment induced lacZ expression in the majority of cells of early embryos and in the spleen, thymus and small intestine in adult mice. Transgene expression was p53 dependent and coincided with the sites of strong p53 accumulation. The lacZ-expressing tissues and early embryos, unlike other adult tissues and late embryos, are characterized by high levels of p53 mRNA expression and respond to DNA damage by massive apoptotic cell death. Analysis of p53-null mice showed that this apoptosis is p53 dependent. These data suggest that p53 activity, monitored by the reporter lacZ transgene, is the determinant of radiation and drug sensitivity in vivo and indicate the importance of tissue and stage specificity of p53 regulation at the level of mRNA expression. PMID:9135154

  18. Characterization and development of two reporter gene systems for Clostridium acetobutylicum.

    PubMed

    Feustel, Lothar; Nakotte, Stephan; Dürre, Peter

    2004-02-01

    The use of lacZ from Thermoanaerobacterium thermosulfurigenes (encoding beta-galactosidase) and lucB from Photinus pyralis (encoding luciferase) as reporter genes in Clostridium acetobutylicum was analyzed with promoters of genes required for solventogenesis and acidogenesis. Both systems proved to be well suited and allowed the detection of differences in promoter strength at least up to 100-fold. The luciferase assay could be performed much faster and comes close to online measurement. Resequencing of lacZ revealed a sequence error in the original database entry, which resulted in beta-galactosidase with an additional 31 amino acids. Cutting off part of the gene encoding this C terminus resulted in decreased enzyme activity. The lacZ reporter data showed that bdhA (encoding butanol dehydrogenase A) is expressed during the early growth phase, followed by sol (encoding butyraldehyde/butanol dehydrogenase E and coenzyme A transferase) and bdhB (encoding butanol dehydrogenase B) expression. adc (encoding acetoacetate decarboxylase) was also induced early. There is about a 100-fold difference in expression between adc and bdhB (higher) and bdhA and the sol operon (lower). The lucB reporter activity could be increased 10-fold by the addition of ATP to the assay. Washing of the cells proved to be important in order to prevent a red shift of bioluminescence in an acidic environment (for reliable data). lucB reporter measurements confirmed the expression pattern of the sol and ptb-buk (encoding phosphotransbutyrylase and butyrate kinase) operons as determined by the lacZ reporter and showed that the expression level from the ptb promoter is 59-fold higher than that from the sol operon promoter. PMID:14766557

  19. Comparison of Three Nonviral Transfection Methods for Foreign Gene Expression in Early Chicken Embryos in Ovo

    Microsoft Academic Search

    Tatsuo Muramatsu; Yoshimoto Mizutani; Yasushige Ohmori; Jun-ichi Okumura

    1997-01-01

    By using three nonviral transfection methods, i.e., microparticle bombardment, lipofection and electroporation, the transfection efficiency and the expression intensity of a lacZ reporter gene were compared in developing chicken embryosin ovo.Of the three transfection methods employed, electroporation conferred the strongest expression of the bacterial lacZ gene with similar transfection efficiency. The results suggest that as far as transient gene expression

  20. 526. A new mouse reporter strain with nuclear-localized lacZ expression allows reliable detection of transplanted and donor-derived cells

    Microsoft Academic Search

    Pei-Rong Wang; Daniel G. Miller; George Vassilopoulos; David W. Russell

    2004-01-01

    Experiments involving transplantation of cells necessitates sensitive and specific identification of donor cells in recipient tissues. Proof of cell origin can be difficult due to a variety of confounding factors. The original gene trap mouse strain (ROSA26) has been a useful tool in this regard. However X-gal staining of chimeric tissues resulting from transplants with this model can be difficult

  1. White as a Reporter Gene to Detect Transcriptional Silencers Specifying Position-Specific Gene Expression during Drosophila Melanogaster Eye Development

    PubMed Central

    Sun, Y. H.; Tsai, C. J.; Green, M. M.; Chao, J. L.; Yu, C. T.; Jaw, T. J.; Yeh, J. Y.; Bolshakov, V. N.

    1995-01-01

    The white(+) gene was used as a reporter to detect transcriptional silencer activity in the Drosophila genome. Changes in the spatial expression pattern of white were scored in the adult eye as nonuniform patterns of pigmentation. Thirty-six independent P[lacW] transposant lines were collected. These represent 12 distinct pigmentation patterns and probably 21 loci. The spatial pigmentation pattern is due to cis-acting suppression of white(+) expression, and the suppression probably depends on cell position rather than cell type. The mechanism of suppression differs from inactivation by heterochromatin. In addition, activation of lacZ in P[lacW] occurs also in specific patterns in imaginal discs and embryos in many of the lines. The expression patterns of white(+) and lacZ may reflect the activity of regulatory elements belonging to an endogenous gene near each P[lacW] insertion site. We speculate that these putative POSE (position-specific expression) genes may have a role in pattern formation of the eye as well as other imaginal structures. Three of the loci identified are optomotor-blind, engrailed and invected. teashirt is also implicated as a candidate gene. We propose that this ``silencer trap'' may be an efficient way of identifying genes involved in imaginal pattern formation. PMID:8582614

  2. GATA6 reporter gene reveals myocardial phenotypic heterogeneity that is related to variations in gap junction coupling

    PubMed Central

    Rémond, Mathieu C.; Iaffaldano, Grazia; O'Quinn, Michael P.; Mezentseva, Nadejda V.; Garcia, Victor; Harris, Brett S.; Gourdie, Robert G.; Eisenberg, Carol A.

    2011-01-01

    This study examined transgenic mice whose expression of a ?-galactosidase (lacZ) reporter is driven by a GATA6 gene enhancer. Previous investigations established that transcription of the transgene was associated with precardiac mesoderm and primary heart tube myocardium, which decreased progressively, so that its expression was no longer observed within ventricular myocardium by midgestation. Expression of this reporter in the adult was investigated for insights into myocyte homeostasis and cardiovascular biology. Morphometric analysis determined that <1% of myocytes, often found in small clusters, express this GATA6-associated reporter in the adult heart. LacZ expression was also found in the ascending aorta. Myocardial expression of the transgene was not associated with a proliferative phenotype or new myocyte formation, as lacZ-positive myocytes neither labeled with cell division markers nor following 5-bromodeoxyuridine pulse-chase experimentation. Despite exhibiting normal adherens junctions, these myocytes appeared to exhibit decreased connexin 43 gap junctions. Treatment with the gap junctional blocker heptanol both in vivo and in culture elevated myocardial ?-galactosidase activity, suggesting that deficient gap junctional communication underlies expression of the transgenic reporter. LacZ expression within the myocardium was also enhanced in response to cryoinjury and isoproterenol-induced hypertrophy. These results reveal a previously uncharacterized phenotypic heterogeneity in the myocardium and suggest that decreased gap junctional coupling leads to induction of a signaling pathway that utilizes a unique GATA6 enhancer. Upregulation of lacZ reporter gene expression following cardiac injury indicates this transgenic mouse may serve as a model for examining the transition of the heart from healthy to pathological states. PMID:21908788

  3. The functional stability of the lacZ transcript is sensitive towards sequence alterations immediately downstream of the ribosome binding site

    Microsoft Academic Search

    Carsten Petersen

    1987-01-01

    Various synthetic DNA sequences were inserted downstream of the fourth codon of the Escherichia coli lacZ gene on plasmids containing a hybrid lacZ-galK operon. Several different sequences, one as short as 10 bp, reduced the functional stability of the lacZ message three- to fourfold, whereas others had little or no effect. Introduction of synthetic sequences into a plasmid containing the

  4. Insights Into Mutagenesis Using Escherichia coli Chromosomal lacZ Strains That Enable Detection of a Wide Spectrum of Mutational Events

    PubMed Central

    Seier, Tracey; Padgett, Dana R.; Zilberberg, Gal; Sutera, Vincent A.; Toha, Noor; Lovett, Susan T.

    2011-01-01

    Strand misalignments at DNA repeats during replication are implicated in mutational hotspots. To study these events, we have generated strains carrying mutations in the Escherichia coli chromosomal lacZ gene that revert via deletion of a short duplicated sequence or by template switching within imperfect inverted repeat (quasipalindrome, QP) sequences. Using these strains, we demonstrate that mutation of the distal repeat of a quasipalindrome, with respect to replication fork movement, is about 10-fold higher than the proximal repeat, consistent with more common template switching on the leading strand. The leading strand bias was lost in the absence of exonucleases I and VII, suggesting that it results from more efficient suppression of template switching by 3? exonucleases targeted to the lagging strand. The loss of 3? exonucleases has no effect on strand misalignment at direct repeats to produce deletion. To compare these events to other mutations, we have reengineered reporters (designed by Cupples and Miller 1989) that detect specific base substitutions or frameshifts in lacZ with the reverting lacZ locus on the chromosome rather than an F? element. This set allows rapid screening of potential mutagens, environmental conditions, or genetic loci for effects on a broad set of mutational events. We found that hydroxyurea (HU), which depletes dNTP pools, slightly elevated templated mutations at inverted repeats but had no effect on deletions, simple frameshifts, or base substitutions. Mutations in nucleotide diphosphate kinase, ndk, significantly elevated simple mutations but had little effect on the templated class. Zebularine, a cytosine analog, elevated all classes. PMID:21441210

  5. Progress in gene targeting and gene therapy for retinitis pigmentosa

    SciTech Connect

    Farrar, G.J.; Humphries, M.M.; Erven, A. [Trinity College, Dublin (Ireland)] [and others

    1994-09-01

    Previously, we localized disease genes involved in retinitis pigmentosa (RP), an inherited retinal degeneration, close to the rhodopsin and peripherin genes on 3q and 6p. Subsequently, we and others identified mutations in these genes in RP patients. Currently animal models for human retinopathies are being generated using gene targeting by homologous recombination in embryonic stem (ES) cells. Genomic clones for retinal genes including rhodopsin and peripherin have been obtained from a phage library carrying mouse DNA isogenic with the ES cell line (CC1.2). The peripherin clone has been sequenced to establish the genomic structure of the mouse gene. Targeting vectors for rhodopsin and peripherin including a neomycin cassette for positive selection and thymidine kinase genes enabling selection against random intergrants are under construction. Progress in vector construction will be presented. Simultaneously we are developing systems for delivery of gene therapies to retinal tissues utilizing replication-deficient adenovirus (Ad5). Efficacy of infection subsequent to various methods of intraocular injection and with varying viral titers is being assayed using an adenovirus construct containing a CMV promoter LacZ fusion as reporter and the range of tissues infected and the level of duration of LacZ expression monitored. Viral constructs with the LacZ reporter gene under the control of retinal specific promoters such as rhodopsin and IRBP cloned into pXCJL.1 are under construction. An update on developments in photoreceptor cell-directed expression of virally delivered genes will be presented.

  6. Acinetobacter baylyi starvation-induced genes identified through incubation in long-term stationary phase.

    PubMed

    Lostroh, C Phoebe; Voyles, Bruce A

    2010-07-01

    Acinetobacter species encounter cycles of feast and famine in nature. We show that populations of Acinetobacter baylyi strain ADP1 remain dynamic for 6 weeks in batch culture. We created a library of lacZ reporters inserted into SalI sites in the genome and then isolated 30 genes with lacZ insertions whose expression was induced by starvation during long-term stationary phase compared with their expression during exponential growth. The genes encode metabolic, gene expression, DNA maintenance, envelope, and conserved hypothetical proteins. PMID:20511417

  7. INDUCTION OF A MYCOPLASMA GALLISEPTICUM PMGA GENE IN THE CHICKEN TRACHEAL RING ORGAN CULTURE MODEL

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To search for Mycoplasma gallisepticum genes involved in colonization of the poultry respiratory tract, a transposon containing a promoterless lacZ gene was employed as a transcriptional reporter. The transposon was used to randomly mutagenize the chromosome of the M. gallisepticum S6 strain and a ...

  8. Functional Identification of a Putative ?-Galactosidase Gene in the Special lac Gene Cluster of Lactobacillus   acidophilus

    Microsoft Academic Search

    Qu Pan; Junmin Zhu; Lina Liu; Yanguang Cong; Fuquan Hu; Jinchuan Li; Xiaoping Yu

    2010-01-01

    The putative ?-galactosidase gene (lacZ) of Lactobacillus acidophilus has a very low degree of homology to the Escherichia coli ?-galactosidase gene (lacZ) and locates in a special lac gene cluster which contains two ?-galactosidase genes. No functional characteristic of the putative ?-galactosidase has been\\u000a described so far. In this study, the lacZ gene of L. acidophilus was hetero-expressed in E. coli and the recombinant

  9. A novel method to assay herpes simplex virus neutralizing antibodies using BHKICP6LacZ-5 (ELVIS) cells.

    PubMed

    Ashley, R L; Dalessio, J; Sekulovich, R E

    1997-01-01

    A novel method for determining neutralizing serum antibody titers to herpes simplex virus type 2 (HSV-2) was developed based on reduction of infectivity in BHKICP6LacZ-5 (ELVIS) cells; baby hamster kidney (BHK) cells that have been genetically engineered to contain the Escherichia coli LacZ gene under the control of an inducible herpes simplex virus type 1 (HSV-1) promoter. The test has a semiautomated, colorimetric readout resulting in rapid, objective readings of infectivity reduction. Extent of neutralization is calculated against a calibration curve of virus infectivity generated in each run. HSV-2 neutralizing activity can be detected with serum dilutions in excess of 1:5120. PMID:9473152

  10. Deletion of p66Shc in mice increases the frequency of size-change mutations in the lacZ transgene.

    PubMed

    Beltrami, Elena; Ruggiero, Antonella; Busuttil, Rita; Migliaccio, Enrica; Pelicci, Pier Giuseppe; Vijg, Jan; Giorgio, Marco

    2013-04-01

    Upon oxidative challenge the genome accumulates adducts and breaks that activate the DNA damage response to repair, arrest, or eliminate the damaged cell. Thus, reactive oxygen species (ROS) generated by endogenous oxygen metabolism are thought to affect mutation frequency. However, few studies determined the mutation frequency when oxidative stress is reduced. To test whether in vivo spontaneous mutation frequency is altered in mice with reduced oxidative stress and cell death rate, we crossed p66Shc knockout (p66KO) mice, characterized by reduced intracellular concentration of ROS and by impaired apoptosis, with a transgenic line harboring multiple copies of the lacZ mutation reporter gene as part of a plasmid that can be recovered from organs into Escherichia coli to measure mutation rate. Liver and small intestine from 2- to 24-month-old, lacZ (p66Shc+/+) and lacZp66KO mice, were investigated revealing no difference in overall mutation frequency but a significant increase in the frequency of size-change mutations in the intestine of lacZp66KO mice. This difference was further increased upon irradiation of mice with X-ray. In addition, we found that knocking down cyclophilin D, a gene that facilitates mitochondrial apoptosis acting downstream of p66Shc, increased the size-change mutation frequency in small intestine. Size-change mutations also accumulated in death-resistant embryonic fibroblasts from lacZp66KO mice treated with H2 O2 . These results indicate that p66Shc plays a role in the accumulation of DNA rearrangements and suggest that p66Shc functions to clear damaged cells rather than affect DNA metabolism. PMID:23237310

  11. Characterization of Platelet-Derived Growth Factor-A Expression in Mouse Tissues Using a lacZ Knock-In Approach

    PubMed Central

    Andrae, Johanna; Gouveia, Leonor; He, Liqun; Betsholtz, Christer

    2014-01-01

    Expression of the platelet-derived growth factor A-chain gene (Pdgfa) occurs widely in the developing mouse, where it is mainly localized to various epithelial and neuronal structures. Until now, in situ mRNA hybridization (ISH) has been the only reliable method to identify Pdgfa expression in tissue sections or whole mount preparations. Validated protocols for in situ detection of PDGF-A protein by immunohistochemistry is lacking. In particular, this has hampered understanding of Pdgfa expression pattern in adult tissues, where ISH is technically challenging. Here, we report a gene targeted mouse Pdgfa allele, Pdgfaex4COIN, which is a combined conditional knockout and reporter allele. Cre-mediated inversion of the COIN cassette inactivates Pdgfa coding while simultaneously activating a beta-galactosidase (lacZ) reporter under endogenous Pdgfa transcription control. The generated Pdgfaex4COIN-INV-lacZ allele can next be used to identify cells carrying a Pdgfa null allele, as well as to map endogenous Pdgfa expression. We evaluated the Pdgfaex4COIN-INV-lacZ allele as a reporter for endogenous Pdgfa expression patterns in mouse embryos and adults. We conclude that the expression pattern of Pdgfaex4COIN-INV-lacZ recapitulates known expression patterns of Pdgfa. We also report on novel embryonic and adult Pdgfa expression patterns in the mouse and discuss their implications for Pdgfa physiology. PMID:25166724

  12. Bacterial arylsulfate sulfotransferase as a reporter system.

    PubMed

    Yun, H J; Kwon, A R; Choi, E C

    2001-01-01

    To investigate whether the arylsulfate sulfotransferase (ASST) is suitable as a reporter system for monitoring gene expression, a reporter vector carrying the fragments of the astA coding region without the promoter region was constructed and designated as pSY815. As a test of the ASST reporter system's suitability, the regulatory regions of ermC and lacZ were inserted upstream of the coding region of the reporter gene to generate pSY815-EC and pSY815-LZ, respectively. In the absence of the inserted regulatory regions, the plasmids displayed very low background activities in Bacillus subtilis and Escherichia coli. The ASST activity under the control of the ermC regulatory region was increased 4.4-fold in B. subtilis when induced by 0.1 microgml(-1) of erythromycin. These results were consistent with a lacZ reporter gene assay of the ermC regulatory region. Furthermore, we confirmed that the lacZ promoter in E. coli was strongly induced to a 17.9-fold increase by 0.05 mM of isopropyl-beta-D-thiogalactopyranoside (IPTG) in this reporter system. These results indicate that the ASST is a suitable reporter system. The lack of endogenous activity, the simple detection of enzyme activity in the living cell, the commercially available non-toxic substrates, and the high sensitivity make ASST a useful genetic reporter system for monitoring gene expression and understanding gene regulation. PMID:11762749

  13. A comparison of the genotoxicity of ethylnitrosourea and ethyl methanesulfonate in lacZ transgenic mice (Muta Mouse).

    PubMed

    Suzuki, T; Hayashi, M; Wang, X; Yamamoto, K; Ono, T; Myhr, B C; Sofuni, T

    1997-12-01

    We compared the induction of gene mutations and chromosomal aberrations by ethylating agents in lacZ transgenic mice (Muta Mouse). Chromosomal aberrations were detected by the peripheral blood micronucleus assay. Gene mutations were detected in the lacZ transgene. A small amount of blood was sampled from a tail vessel during the expression time for fixation of gene mutations in vivo; this enabled us to detect and compare clastogenicity and gene mutations in the identical mouse. Single intraperitoneal injections of ENU (50-200 mg/kg) and EMS (100-400 mg/kg) strongly induced micronucleated reticulocytes (MN) detectable in peripheral blood 48 h after treatment. The maximum MN frequencies induced were 6.6% and 3.3% for ENU (100 mg/kg) and EMS (400 mg/kg), respectively (the control value was 0.3%). lacZ mutant frequency (MF) was analyzed in bone marrow and liver 7 days after treatment. Spontaneous MFs were 2.0-4.6 x 10(-6). MF in bone marrow was increased by ENU to 3.4 x 10(-5) at 200 mg/kg and induced by EMS to 1.8 x 10(-5) at 400 mg/kg. In liver, however, both chemicals at their highest doses induced only slight increases in MF. The induction of both micronuclei and lacZ mutations in bone marrow by both ENU and EMS correlated better with O6-ethylguanine adducts than with N7-ethylguanine adducts. The mutants (19 for ENU and 12 for EMS) were subjected to DNA sequence analysis. Among EMS-induced mutations, 75% were GC to AT transitions, which were probably caused by O6-ethylguanine. Among ENU-induced mutations, in contrast, 40% occurred as AT base pair substitutions (6 AT to TA transversions and 2 AT to GC transitions) (no such mutations were induced by EMS). These results, together with the known reactivity of ENU to thymine suggest that thymine adducts play a significant role in the ENU mutagenesis. PMID:9465915

  14. The color of mice: in the light of GFP-variant reporters

    Microsoft Academic Search

    Anna-Katerina Hadjantonakis; Andras Nagy

    2001-01-01

    The mouse currently represents the premier model organism for mammalian genetic studies. Over the past decade the production of targeted and transgenic lines of mice has become commonplace, with current technology allowing the creation of mutations at base pair resolution. Such genome modifications are becoming increasingly elaborate and often incorporate gene-based reporters for tagging different cellular populations. Until recently, lacZ,

  15. The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death

    Microsoft Academic Search

    Heli Putaala; Raija Soininen; Pekka Kilpeläinen; Jorma Wartiovaara; Karl Tryggvason

    2001-01-01

    A mouse model for congenital nephrotic syndrome (NPHS1) was generated by inactivating the nephrin gene (Nphs1) in embryonic stem cells by homologous recombination. The targeting construct contained the Escherichia coli lacZ gene as a reporter for the Nphs1 promoter .M ice homozygous for inactivated Nphs1 were born at an expected frequency of 25%. Although seemingly normal at birth, they immediately

  16. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector.

    PubMed Central

    Xiao, X; Li, J; Samulski, R J

    1996-01-01

    Muscle-directed gene transfer is being considered for the treatment of several metabolic diseases, including hemophilia and Duchene's muscular dystrophy. Previous efforts to target this tissue for somatic delivery with various vector systems have resulted in transient expression due to silencing of the transgene or to an immune response against the vector-transduced cells. We introduced recombinant adeno-associated virus vector (rAAV) carrying a lacZ reporter into muscle tissue of immunocompetent mice. The lacZ reporter gene was efficiently transduced and expressed with no evidence of a cellular immune response. Moreover, gene expression persisted for more than 1.5 years. Molecular characterization of rAAV vector DNA suggests a mechanism for persistence, since vector episomes convert to high-molecular-weight genomic DNA. These data provide the first report for establishing long-term gene transduction into mammalian muscle cells in vivo without the need for immune modulation of the organism. PMID:8892935

  17. 4-Hydroxytamoxifen probes for light-dependent spatiotemporal control of Cre-ER mediated reporter gene expression.

    PubMed

    Faal, Tannaz; Wong, Pamela T; Tang, Shengzhuang; Coulter, Alexa; Chen, Yumay; Tu, Christina H; Baker, James R; Choi, Seok Ki; Inlay, Matthew A

    2015-03-01

    The tamoxifen inducible Cre-ER/loxP system provides tissue specific temporal control of gene recombination events, and can be used to induce expression of reporter genes (e.g. GFP, LacZ) for lineage tracing studies. Cre enzyme fused with estrogen receptor (Cre-ER) is released upon tamoxifen binding, resulting in permanent activation of reporter genes within cells and their progeny. Tamoxifen and its active metabolite, hydroxytamoxifen (4OHT) diffuses rapidly in vivo, making it difficult to restrict labeling to specific locations. In this study, we developed a photocaged 4OHT molecule by covalently attaching 4OHT to an ortho-nitrobenzyl (ONB1) group, rendering 4OHT inactive. Exposure to UV radiation cleaves the bond between ONB1 and 4OHT, freeing the 4OHT to bind Cre-ER to result in downstream genetic recombination and reporter activation. We show that caged ONB1-4OHT crosses the cell membrane and uncages after short UV exposure, resulting in Cre-driven genetic recombination that can be localized to specific regions or tissues. ONB1-4OHT can provide spatial control of reporter activation and be adapted with any existing Cre-ER/loxP based system. PMID:25502239

  18. Efficient expression of gene variants that harbour AGA codons next to the initiation codon

    PubMed Central

    Zamora-Romo, Efraín; Cruz-Vera, Luis Rogelio; Vivanco-Domínguez, Serafín; Magos-Castro, Marco Antonio; Guarneros, Gabriel

    2007-01-01

    In an effort to improve the knowledge about the rules which direct the effect of the early ORF sequences on translation efficiency, we have analyzed the effect of pairs of the six arginine codons at the second and third positions on the expression of lacZ variants. Whereas the pairs of identical AGA or AGG codons were favorable for the gene expression, identical pairs of each of the four CGN codons were very inefficient. This result was unexpected because tandems of AGA or AGG codons located in more internal gene positions provoke deficient expression whilst internally located CGU and CGC are the most abundant and efficiently translated arginine codons. The mixed combinations of AGA and each of the CGN codons usually resulted in efficient rates of lacZ expression independently of the peptidyl-tRNA propensity to dissociate from the ribosome. Thus, the variant harboring the pair of AGA codons was expressed as efficiently as the variant carrying a pair of AAA codons in the same positions, a configuration reported as one of the most common and efficient for gene expression. We explain these results assuming that the presence of adenines in these early positions enhance gene expression. As expected, specific mRNA levels correlated with the intensity of lacZ expression for each variant. However, the induction of lacZ AGA AGA gene in pth cells accumulated peptidyl-tRNAArg4 as well as a short 5?-proximal lacZ mRNA fragment suggesting ribosome stalling due to depletion of aminoacylated-tRNAArg4. PMID:17726048

  19. [Gene knockout and knockin on the Escherichia coli lac operon loci using pBR322-red system].

    PubMed

    Chen, Wei; Yu, Mei; Li, Shan-Hu; Wang, Ming-Gang; Zhou, Jian-Guang

    2005-03-01

    pBR322-Red is a newly constructed recombineering plasmid, which contains a part of the pBR322 vector, a series of regulatory elements of lambda-prophage and Red recombination genes. In the beginning, we studied the best working conditions of pBR322-Red, and then modified lac operon in E. coli W3110 chromosome using the plasmid as follow: Firstly, we knockout the lacI gene using Red-mediated recombineering with overlapping single stranded DNA oligonucleotides. Secondly, we substituded the lacA and lacY genes with lacZ, a report gene, by Red-mediated linearized double strands DNA homologous recombination. Finally, we detected the expression of lacZ on these loci for the first time. The results suggested that pBR322-Red system is suitable for modifying W3110 chromosome with various recombination strategies. PMID:16013474

  20. PhaF, a Polyhydroxyalkanoate-Granule-Associated Protein of Pseudomonas oleovorans GPo1 Involved in the Regulatory Expression System for pha Genes

    Microsoft Academic Search

    MARIA A. PRIETO; BRUNO BUHLER; KUNO JUNG; BERNARD WITHOLT; BIRGIT KESSLER

    1999-01-01

    The phaC1 gene codes for the medium-chain-length polyhydroxyalkanoate (mcl PHA) synthase of Pseudo- monas oleovorans GPo1, which produces mcl PHA when grown in an excess of carbon source and under nitrogen limitation. In this work, we have demonstrated, by constructing a recombinant P. oleovorans strain carrying a phaC1::lacZ reporter system, that the phaC1 gene is expressed efficiently in the presence

  1. Characterization of protein binding to the psbAII promoter region in Synechococcus sp. strain PCC 7942 and its role in light regulated gene expression

    E-print Network

    Dickerson, Naoma Suzanne

    1994-01-01

    intensity. Translational gene fusions between each of the psbA genes and a truncated Escherichia coli lacZ gene were prepared as reporters for psbA gene activity (35). P-galactosidase assays of strains carrying these fusions show that as light intensity... decreases from 600 ttE m 2 s-I to 2 ttE m 2 s I, expression of the psbAI gene fusion increases 800%, whereas expression of the psbAII and psbAIII gene fusions decreases 90% (35). To determine if light availability affects the ratio of the two forms...

  2. LacZ ?-galactosidase: Structure and function of an enzyme of historical and molecular biological importance

    PubMed Central

    Juers, Douglas H; Matthews, Brian W; Huber, Reuben E

    2012-01-01

    This review provides an overview of the structure, function, and catalytic mechanism of lacZ ?-galactosidase. The protein played a central role in Jacob and Monod's development of the operon model for the regulation of gene expression. Determination of the crystal structure made it possible to understand why deletion of certain residues toward the amino-terminus not only caused the full enzyme tetramer to dissociate into dimers but also abolished activity. It was also possible to rationalize ?-complementation, in which addition to the inactive dimers of peptides containing the “missing” N-terminal residues restored catalytic activity. The enzyme is well known to signal its presence by hydrolyzing X-gal to produce a blue product. That this reaction takes place in crystals of the protein confirms that the X-ray structure represents an active conformation. Individual tetramers of ?-galactosidase have been measured to catalyze 38,500 ± 900 reactions per minute. Extensive kinetic, biochemical, mutagenic, and crystallographic analyses have made it possible to develop a presumed mechanism of action. Substrate initially binds near the top of the active site but then moves deeper for reaction. The first catalytic step (called galactosylation) is a nucleophilic displacement by Glu537 to form a covalent bond with galactose. This is initiated by proton donation by Glu461. The second displacement (degalactosylation) by water or an acceptor is initiated by proton abstraction by Glu461. Both of these displacements occur via planar oxocarbenium ion-like transition states. The acceptor reaction with glucose is important for the formation of allolactose, the natural inducer of the lac operon. PMID:23011886

  3. Use of phoA and lacZ fusions to study the membrane topology of ProW, a component of the osmoregulated ProU transport system of Escherichia coli.

    PubMed Central

    Haardt, M; Bremer, E

    1996-01-01

    The Escherichia coli ProU system is a member of the ATP-binding cassette (ABC) superfamily of transporters. ProU consists of three components (ProV, ProW, and ProX) and functions as a high-affinity, binding protein-dependent transport system for the osmoprotectants glycine betaine and proline betaine. The ProW protein is the integral inner membrane component of the ProU system. Its hydropathy profile predicts seven transmembrane spans and a hydrophilic amino terminus of approximately 100 residues, and it suggests the presence of an amphiphilic alpha-helix (L-61 to F-97) in close proximity to the first strongly hydrophobic segment of ProW. We have studied the membrane topology of the ProW protein by the phoA and lacZ gene fusion approach. A collection of 10 different proW-phoA fusions with alkaline phosphatase activity and 8 different proW-lacZ fusions with beta-galactosidase activity were isolated in vivo after TnphoAB and TnlacZ mutagenesis of a plasmid-encoded proW gene. The recovery of both enzymatically active ProW-PhoA and ProW-LacZ hybrid proteins indicates that segments of ProW are exposed on both sides of the cytoplasmic membrane. To compare the enzymatic activities of each of the indicator proteins joined at a particular site in ProW, we switched the phoA and lacZ reporter genes in vitro in each of the originally in vivo-isolated gene fusions. A mirror-like pattern in the enzyme activity of the resulting new ProW-PhoA and ProW-LacZ hybrid proteins emerged, thus providing positive signals for the location of both periplasmic and cytoplasmic domains in ProW. The protease kallikrein digests the amino-terminal tail of a ProW-LacZ hybrid protein in spheroplasts, suggesting that the amino terminus of ProW is located on the periplasmic side of the cytoplasmic membrane. From these data, a two-dimensional model for ProW was constructed; this model consists of seven transmembrane alpha-helices and an unusual amino-terminal tail of approximately 100 amino acid residues that protrudes into the periplasmic space. PMID:8808924

  4. Imaging of gene expression in vivo with photoacoustic tomography

    NASA Astrophysics Data System (ADS)

    Li, Li; Zemp, Roger J.; Lungu, Gina; Stoica, George; Wang, Lihong V.

    2006-02-01

    In the post-genomic era, there is an increasing interest in visualizing the expression of functional genes in vivo. With the assistance of the reporter gene technique, various imaging modalities have been adopted for this purpose. In vivo gene expression imaging promises to provide biologists with a powerful tool for deepening our understanding of developmental biology, expanding our knowledge of the genetic basis of disease, and advancing the development of medicine. In this paper, we demonstrate the feasibility of imaging gene expression with photoacoustic imaging, which offers unique absorption contrast with ultrasonic resolution in vivo. We mark tumors in rats with the lacZ reporter gene. The lacZ gene encodes an enzyme ?-galactosidase, which yields a dark blue product when acting on a colorimetric assay called X-gal. Photoacoustic tomography at 650nm clearly visualizes the presence of this blue product. The spectroscopic method can also potentially improve specificity. Considering how many staining methods are used in traditional biology, we believe that photoacoustic techniques will revolutionize the field of molecular imaging. The further development of reporter gene systems with high absorbing products in the NIR region is needed.

  5. A 19F NMR Approach using Reporter Molecule Pairs to Assess ?-Galactosidase in Human Xenograft Tumors in Vivo

    PubMed Central

    Yu, Jian-Xin; Kodibagkar, Vikram D.; Liu, Li; Mason, Ralph P.

    2011-01-01

    Gene therapy has emerged as a promising strategy for treatment of various diseases. However, widespread implementation is hampered by difficulties in assessing the success of transfection in the target tissue and the longevity of gene expression. Thus, there is increasing interest in the development of non-invasive in vivo reporter techniques to assay gene expression. We recently demonstrated the ability to detect ?-galactosidase activity in stably transfected human prostate tumor xenografts in mice in vivo using 19F NMR. We now extend the studies to human MCF7 breast cancer cells growing as xenografts in nude mice. Moreover, by using two spectrally resolved reporters (o-fluoro-p-nitrophenyl-?-D-galactopyranoside and an isomer) two tumors could be interrogated simultaneously revealing lacZ transgene activity in a stably transfected tumor versus no activity in a wild type tumor. Most significantly hydrolytic activity observed by 19F NMR corresponded with differential activity in lacZ expressing tumors. PMID:18288788

  6. Imaging Molecular Pathways: Reporter Genes

    PubMed Central

    Brogan, John; Li, Fang; Li, Wenrong; He, Zhimin; Huang, Qian; Li, Chuan-Yuan

    2012-01-01

    Molecular imaging is a rapidly advancing field that allows cancer biologists to look deeper into the complex inner workings of tumor cells, or whole tumors, in a non-invasive manner. In this review, we will summarize some recent advances that enable investigators to study various important biological processes in tumors in vivo. We will discuss novel imaging approaches that allow investigators to visualize and quantify molecular pathways, such as receptor tyrosine kinase activation, hypoxia signal transduction, apoptosis, and DNA double-strand breaks. Select examples of these applications will be discussed. Because of the limited scope of this review, we will only focus on natural reporters, such as bioluminescence and fluorescent proteins. PMID:22348248

  7. Msx genes define a population of mural cell precursors required for head blood vessel Miguel Lopes1

    E-print Network

    Boyer, Edmond

    1 Msx genes define a population of mural cell precursors required for head blood vessel maturation cedex Keywords Msx, BMP, neural crest pasteur-00611700,version1-27Jul2011 Author manuscript, published that, in the mouse embryo, Msx1Lacz and Msx2Lacz genes are expressed in mural and in a few endothelial

  8. Development of Inducible Systems To Engineer Conditional Mutants of Essential Genes of Helicobacter pylori

    Microsoft Academic Search

    Ivo G. Boneca; Chantal Ecobichon; Catherine Chaput; Aurelie Mathieu; Stephanie Guadagnini; Marie-Christine Prevost; Frederic Colland; Agnes Labigne; Hilde de Reuse

    2008-01-01

    polymerase. The amiE gene promoter of H. pylori was taken to constitutively express the LacIq repressor. Expression of the reporter gene lacZ was driven by either pTac (pILL2150) or a modified version of the ureI gene promoter in which one or two LacI-binding sites and\\/or mutated nucleotides between the ribosomal binding site and the ATG start codon (pILL2153 and pILL2157)

  9. Targeting gene expression to the female larval fat body of transgenic Aedes aegypti mosquitoes

    PubMed Central

    TOTTEN, Daniel C.; VUONG, Mai; LITVINOVA, Oksana V.; JINWAL, Umesh K.; GULIA-NUSS, Monika; HARRELL, Robert A.; BENEŠ, Helen

    2014-01-01

    As the fat body is a critical tissue for mosquito development, metamorphosis, immune and reproductive system function, characterization of regulatory modules targeting gene expression to the female mosquito fat body at distinct life stages is much needed for multiple, varied strategies for controlling vector-borne diseases such as dengue and malaria. The hexameric storage protein, Hexamerin-1.2, of the mosquito, Aedes atropalpus, is female-specific and uniquely expressed in the fat body of fourth-instar larvae and young adults. We have identified in the Hex-1.2 gene, a short regulatory module that directs female-, tissue-, and stage-specific lacZ reporter gene expression using a heterologous promoter in transgenic lines of the dengue vector, Aedes aegypti. Male transgenic larvae and pupae of one line expressed no E. coli ?-galactosidase or transgene product; in two other lines reporter gene activity was highly female-biased. All transgenic lines expressed the reporter only in the fat body. However, lacZ mRNA levels were no different in males and females at all stages examined, suggesting that the gene regulatory module drives female-specific expression by post-transcriptional regulation in the heterologous mosquito. This regulatory element from the Hex-1.2 gene thus provides a new molecular tool for transgenic mosquito control as well as functional genetic analysis in aedine mosquitoes. PMID:23241066

  10. Positron Emission Tomography Reporter Genes and Reporter Probes: Gene and Cell Therapy Applications

    PubMed Central

    Yaghoubi, Shahriar S.; Campbell, Dean O.; Radu, Caius G.; Czernin, Johannes

    2012-01-01

    Positron emission tomography (PET) imaging reporter genes (IRGs) and PET reporter probes (PRPs) are amongst the most valuable tools for gene and cell therapy. PET IRGs/PRPs can be used to non-invasively monitor all aspects of the kinetics of therapeutic transgenes and cells in all types of living mammals. This technology is generalizable and can allow long-term kinetics monitoring. In gene therapy, PET IRGs/PRPs can be used for whole-body imaging of therapeutic transgene expression, monitoring variations in the magnitude of transgene expression over time. In cell or cellular gene therapy, PET IRGs/PRPs can be used for whole-body monitoring of therapeutic cell locations, quantity at all locations, survival and proliferation over time and also possibly changes in characteristics or function over time. In this review, we have classified PET IRGs/PRPs into two groups based on the source from which they were derived: human or non-human. This classification addresses the important concern of potential immunogenicity in humans, which is important for expansion of PET IRG imaging in clinical trials. We have then discussed the application of this technology in gene/cell therapy and described its use in these fields, including a summary of using PET IRGs/PRPs in gene and cell therapy clinical trials. This review concludes with a discussion of the future direction of PET IRGs/PRPs and recommends cell and gene therapists collaborate with molecular imaging experts early in their investigations to choose a PET IRG/PRP system suitable for progression into clinical trials. PMID:22509201

  11. Mutational spectrum of bleomycin in lacZ mouse kidney: a possible model for mutational spectrum of reactive oxygen species.

    PubMed

    Guttenplan, Joseph B; Khmelnitsky, Michael; Haesevoets, Roderick; Kosinska, Wieslawa

    2004-10-01

    The mutational spectrum of bleomycin was compared with the spontaneous mutational spectrum in lacZ mouse kidney. Mice were treated with four 20 mg/kg of doses of bleomycin over a two-week period, leading to a mutant fraction several times greater than that of controls. The major class of bleomycin-induced mutations consisted of small deletions, in particular -1 deletions at AT base pairs and hot spots for deletions at 5'-GTC-3' sequences. Smaller, but significant fractions of GC > AT followed by GC > TA substitutions were also observed. In untreated mice, the major class of mutations consisted of GC > AT substitutions followed by GC > TA mutations, and a much smaller fraction of deletions. Other than the specificity of bleomycin for AT base pairs and the 5'-GTC-3' hotspots, the mutational spectrum of bleomycin in mice is similar to that reported for ionizing radiation. However, bleomycin initially mediates the formation of oxidized DNA via reduction of molecular oxygen, as opposed to the radiolysis of water. In this respect mutagenesis induced by bleomycin may be more similar to that induced by endogenous reactive oxygen species (ROS) than mutagenesis induced by ionizing radiation. If bleomycin-induced mutagenesis is an appropriate model for mutagenesis induced by ROS, then, based on the difference between the mutational spectrum of bleomycin and spontaneous mutagenesis, the latter appears not to result predominantly from ROS, at least in mouse kidney. PMID:15450417

  12. Perinuclear Mlp proteins downregulate gene expression in response to a defect in mRNA export.

    PubMed

    Vinciguerra, Patrizia; Iglesias, Nahid; Camblong, Jurgi; Zenklusen, Daniel; Stutz, Françoise

    2005-02-23

    The mRNA export adaptor Yra1p/REF contributes to nascent mRNP assembly and recruitment of the export receptor Mex67p. yra1 mutants exhibit mRNA export defects and a decrease in LacZ reporter and certain endogenous transcripts. The loss of Mlp1p/Mlp2p, two TPR-like proteins attached to nuclear pores, rescues LacZ mRNA levels and increases their appearance in the cytoplasm, without restoring bulk poly(A)+ RNA export. Chromatin immunoprecipitation, FISH and pulse-chase experiments indicate that Mlps downregulate LacZ mRNA synthesis in a yra1 mutant strain. Microarray analyses reveal that Mlp2p also reduces a subset of cellular transcripts in the yra1 mutant. Finally, we show that Yra1p genetically interacts with the shuttling mRNA-binding protein Nab2p and that loss of Mlps rescues the growth defect of yra1 and nab2 but not other mRNA export mutants. We propose that Nab2p and Yra1p are required for proper mRNP docking to the Mlp platform. Defects in Yra1p prevent mRNPs from crossing the Mlp gate and this block negatively feeds back on the transcription of a subset of genes, suggesting that Mlps link mRNA transcription and export. PMID:15692572

  13. Role of the Saccharomyces cerevisiae general regulatory factor CP1 in methionine biosynthetic gene transcription.

    PubMed Central

    O'Connell, K F; Surdin-Kerjan, Y; Baker, R E

    1995-01-01

    Saccharomyces cerevisiae general regulatory factor CP1 (encoded by the gene CEP1) is required for optimal chromosome segregation and methionine prototrophy. MET16-CYC1-lacZ reporter constructs were used to show that MET16 5'-flanking DNA contains a CP1-dependent upstream activation sequence (UAS). Activity of the UAS required an intact CP1-binding site, and the effects of cis-acting mutations on CP1 binding and UAS activity correlated. In most respects, MET16-CYC1-lacZ reporter gene expression mirrored that of chromosomal MET16; however, the endogenous gene was found to be activated in response to amino acid starvation (general control). The latter mechanism was both GCN4 and CP1 dependent. MET25 was also found to be activated by GCN4, albeit weakly. More importantly, MET25 transcription was strongly CP1 dependent in gcn4 backgrounds. The modulation of MET gene expression by GCN4 can explain discrepancies in the literature regarding CP1 dependence of MET gene transcription. Lastly, micrococcal nuclease digestion and indirect end labeling were used to analyze the chromatin structure of the MET16 locus in wild-type and cep1 cells. The results indicated that CP1 plays no major role in configuring chromatin structure in this region, although localized CP1-specific differences in nuclease sensitivity were detected. PMID:7891681

  14. Simultaneous gene inactivation and promoter reporting in cyanobacteria.

    PubMed

    Chen, Kangming; Xu, Xinyi; Gu, Liping; Hildreth, Michael; Zhou, Ruanbao

    2015-02-01

    Determining spatiotemporal gene expression and analyzing knockout mutant phenotypes have become powerful tools in elucidating the function of genes; however, genetic approaches for simultaneously inactivating a gene and monitoring its expression have not been reported in the literature. In this study, we designed a dual-functional gene knockout vector pZR606 that contains a multiple cloning site (MCS) for inserting the internal fragment of a target gene, with a gfp gene as its transcriptional marker located immediately downstream of the MCS. By using this gene knockout system, we inactivated ava_2679 from Anabaena variabilis ATCC 29413, as well as all2508, alr2887, alr3608, and all4388 from Anabaena sp. strain PCC 7120. The ava_2679 knockout mutant fails to grow diazotrophically. Morphological analysis of ava_2679 knockout mutant after nitrogen step-down revealed defective junctions between heterocysts and adjacent vegetative cells, and the heterocyst was 1.53-fold longer compared to wild-type heterocysts. The alr2887, all4388, and alr3608 mutant colonies turned yellow and showed lack of protracted growth when deprived of fixed nitrogen, consistent with the previous reports that alr2887, all4388, and alr3608 are Fox genes. The all2508 encodes a GTP-binding elongation factor (EF4/LepA), and its knockout mutant exhibited reduced diazotrophic growth. The heterocyst development of all2508 knockout was significantly delayed, and only about 4.0 % of vegetative cells differentiated to heterocysts after nitrogen deprivation for 72 h, decreased 49.6 % compared to wild-type. Thus, we discovered that All2508 may regulate heterocyst development spatiotemporally. Concurrently, the GFP reporter revealed that all five target gene expressions were up-regulated in response to nitrogen deprivation. We demonstrated that the pZR606-based specific gene knockout approach worked effectively for the five selected genes, including four previously identified Fox genes or Fox gene homolog, and a previously unknown function of gene all2508. Thus, gene expression and phenotypic analysis of mutants can be achieved simultaneously by targeted gene inactivation using the pZR606-based system. This combined approach for targeted gene inactivation and its promoter reporting with GFP may be broadly applicable to the study of gene function in other prokaryotic organisms. PMID:25434810

  15. pMH11, A tool for gene disruption and expression analysis in Azorhizobium caulinodans.

    PubMed

    D'Haeze, Wim; Verplancke, Christa; Mironov, Vladimir; Holsters, Marcelle

    2002-03-01

    Tools for mutagenesis and expression analyses are needed to study the role of bacterial genes. Here, we report the construction of pMH11, a small, mobilizable plasmid that replicates in Escherichia coli, but not in Azorhizobium caulinodans, a nodulating microsymbiont of Sesbania rostrata, and that contains a unique BamHI restriction site upstream of a promoterless lacZ gene. pMH11 and two derivatives with the multiple cloning site of pBluescript (KS(II)) are useful for mutagenesis by gene disruption and for expression analyses after selection for cointegration by kanamycin resistance. Weakly constitutive promoter activity from the vector allowed transcription of genes downstream of the integration site, so that no polar effects were caused by gene disruption. PMID:11982330

  16. Induced Reporter Gene Activity, Enhanced Stress Resistance, and Competitive Ability of a Genetically Modified Pseudomonas fluorescens Strain Released into a Field Plot Planted with Wheat

    PubMed Central

    Van Overbeek, L. S.; Van Veen, J. A.; Van Elsas, J. D.

    1997-01-01

    The fates of Pseudomonas fluorescens R2fR and its mutant derivative RIWE8, which contains a lacZ reporter gene responsive to wheat root exudate, were compared in a field microplot. Inoculant survival, root colonization, translocation, resistance to stress factors, and reporter gene activity were assessed in bulk and wheat rhizosphere soils. Populations of both strains declined gradually in bulk and wheat rhizosphere soils and on the wheat rhizoplane as determined by specific CFU and immunofluorescence (IF). In samples from both bulk soil and wheat rhizosphere, IF cell counts were up to 3 orders of magnitude greater than the corresponding numbers of CFU after 120 days, indicating the presence of nonculturable inoculant cells. Estimates of RIWE8-specific target DNA molecule numbers in bulk soil samples 3 and 120 days after inoculation by most-probable-number PCR coincided with the corresponding CFU values. Transport of both strains to deeper soil layers was observed by 3 days after introduction into the microplot. Both strains colonized wheat roots similarly, and cells were seen scattered on the surface of 1-month-old wheat seedling roots by immunogold labelling-scanning electron microscopy. On average, reporter gene activity was significantly higher in wheat rhizosphere soil containing RIWE8 cells than in bulk soil or in soils containing R2fR cells. For both strains, resistance to the four stress factors ethanol, high temperature, high osmotic tension, and oxidative stress increased progressively with residence in soil. Cells from the rhizosphere of 11-day-old seedlings showed similar levels of resistance to osmotic and oxidative stresses and enhanced resistance to ethanol and heat as compared to cells from bulk soil. By 37 days, populations of R2fR and RIWE8 in the rhizosphere were significantly more sensitive to osmotic stress than were populations in bulk soil, whereas differences in response to the other stress factors were less evident. Hence, except for the induction of reporter gene expression in strain RIWE8 in the wheat rhizosphere, the data indicated that there were no great differences in the ecological properties in soil between the lacZ-modified and parental strains. PMID:16535606

  17. Pleiotrophin gene therapy for peripheral ischemia: evaluation of full-length and truncated gene variants.

    PubMed

    Fang, Qizhi; Mok, Pamela Y; Thomas, Anila E; Haddad, Daniel J; Saini, Shereen A; Clifford, Brian T; Kapasi, Neel K; Danforth, Olivia M; Usui, Minako; Ye, Weisheng; Luu, Emmy; Sharma, Rikki; Bartel, Maya J; Pathmanabhan, Jeremy A; Ang, Andrew A S; Sievers, Richard E; Lee, Randall J; Springer, Matthew L

    2013-01-01

    Pleiotrophin (PTN) is a growth factor with both pro-angiogenic and limited pro-tumorigenic activity. We evaluated the potential for PTN to be used for safe angiogenic gene therapy using the full length gene and a truncated gene variant lacking the domain implicated in tumorigenesis. Mouse myoblasts were transduced to express full length or truncated PTN (PTN or T-PTN), along with a LacZ reporter gene, and injected into mouse limb muscle and myocardium. In cultured myoblasts, PTN was expressed and secreted via the Golgi apparatus, but T-PTN was not properly secreted. Nonetheless, no evidence of uncontrolled growth was observed in cells expressing either form of PTN. PTN gene delivery to myocardium, and non-ischemic skeletal muscle, did not result in a detectable change in vascularity or function. In ischemic hindlimb at 14 days post-implantation, intramuscular injection with PTN-expressing myoblasts led to a significant increase in skin perfusion and muscle arteriole density. We conclude that (1) delivery of the full length PTN gene to muscle can be accomplished without tumorigenesis, (2) the truncated PTN gene may be difficult to use in a gene therapy context due to inefficient secretion, (3) PTN gene delivery leads to functional benefit in the mouse acute ischemic hindlimb model. PMID:23630585

  18. Function of posterior HoxD genes in the morphogenesis of the anal sphincter.

    PubMed

    Kondo, T; Dollé, P; Zákány, J; Duboule, D

    1996-09-01

    Vertebrate 5'-located HoxD genes are expressed in the most caudal part of the digestive tract and their potential functions during gut development have been assessed by gene disruptions. We have inserted reporter lacZ sequences within the Hoxd-12 gene and analysed the morphology of the gut in these mice as well as in Hoxd-13 mutant animals. When homozygous, both mutations induce an important disorganization of the anorectal region. In particular, severe alterations of the smooth muscle layers of the rectum led to defective morphogenesis of the internal anal sphincter. Similarly, Hoxd-12 and Hoxd-13 functionally overlap during digit development. The function of these genes in the morphogenesis of the digestive system as well as their functional evolution are discussed. PMID:8787740

  19. A source of artifact in the lacZ reversion assay in Escherichia coli.

    PubMed

    Hoffmann, George R; Gray, Carol L; Lange, Paulina B; Marando, Christie I

    2015-06-01

    The lacZ reversion assay in Escherichia coli measures point mutations that occur by specific base substitutions and frameshift mutations. The tester strains cannot use lactose as a carbon source (Lac(-)), and revertants are easily detected by growth on lactose medium (Lac(+)). Six strains identify the six possible base substitutions, and five strains measure +G, -G, -CG, +A and -A frameshifts. Strong mutagens give dose-dependent increases in numbers of revertants per plate and revertant frequencies. Testing compounds that are arguably nonmutagens or weakly mutagenic, we often noted statistically significant dose-dependent increases in revertant frequency that were not accompanied by an absolute increase in numbers of revertants. The increase in frequency was wholly ascribable to a declining number of viable cells owing to toxicity. Analysis of the conditions revealed that the frequency of spontaneous revertants is higher when there are fewer viable cells per plate. The phenomenon resembles "adaptive" or "stress" mutagenesis, whereby lactose revertants accumulate in Lac(-) bacteria under starvation conditions in the absence of catabolite repression. Adaptive mutation is observed after long incubation and might be expected to be irrelevant in a standard assay using 48-h incubation. However, we found that elevated revertant frequencies occur under typical assay conditions when the bacterial lawn is thin, and this can cause increases in revertant frequency that mimic chemical mutagenesis when treatments are toxic but not mutagenic. Responses that resemble chemical mutagenesis were observed in the absence of mutagenic treatment in strains that revert by different frameshift mutations. The magnitude of the artifact is affected by cell density, dilution, culture age, incubation time, catabolite repression and the age and composition of media. Although the specific reversion assay is effective for quickly distinguishing classes of mutations induced by potent mutagens, its utility for discerning effects of weak mutagens may be compromised by the artifact. PMID:26046973

  20. Bioluminescence reporter gene-based detection of microRNAs.

    PubMed

    Ko, Hae Young; Lee, Young Sik; Kim, Soonhag

    2014-01-01

    MicroRNAs (miRNAs) are an abundant class of small noncoding RNA molecules that inhibit the expression of cognate genes in multicellular organisms. These small RNAs have been demonstrated to play crucial roles in a variety of biological processes including cell differentiation, proliferation, and survival. Knowledge of specific expression patterns of miRNAs is critical for functional studies. Here, we describe a bioluminescence reporter gene-based method to measure miRNA activity in cultured cells and mice using a Gaussia luciferase reporter gene controlled by miRNA binding sites in its 3'untranslated region. This method can be used to noninvasively monitor the expression patterns of functionally active miRNAs involved in different biological processes or diseases in mice. PMID:24166370

  1. ARCHIVAL REPORT Neuropeptide Y Receptor Gene Expression in the

    E-print Network

    Wisconsin at Madison, University of

    ) of the amygdala, a critical neural substrate for extreme anxiety. Methods: In 24 young rhesus monkeys, we measuredARCHIVAL REPORT Neuropeptide Y Receptor Gene Expression in the Primate Amygdala Predicts Anxious temperament (AT) is identifiable early in life and predicts the later development of anxiety disorders

  2. Nuclear localization of reporter genes activated by curved DNA.

    PubMed

    Udagawa, Koji; Kimura, Hajime; Tanabe, Hideyuki; Ohyama, Takashi

    2012-04-01

    Curved DNA structures with a left-handed superhelical conformation can activate eukaryotic transcription. Mechanistically, these structures favor binding to histone cores and can function as a docking site for sliding nucleosomes. Thus, promoters with this kind of curved DNA can adopt a more open structure, facilitating transcription initiation. However, whether the curved DNA segment can affect localization of a reporter gene is an open question. Localization of a gene in the nucleus often plays an important role in its expression and this phenomenon may also have a curved DNA-dependent mechanism. We examined this issue in transient and stable assay systems using a 180-bp synthetic curved DNA with a left-handed superhelical conformation. The results clearly showed that curved DNA of this kind does not have a property to deliver reporter constructs to nuclear positions that are preferable for transcription. We also identify the spatial location to which electroporation delivers a reporter plasmid in the nucleus. PMID:22197431

  3. Ferritin reporter used for gene expression imaging by magnetic resonance

    SciTech Connect

    Ono, Kenji; Fuma, Kazuya; Tabata, Kaori [Department of Brain Functions, Division of Stress Adaptation and Protection, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi 464-8601 (Japan)] [Department of Brain Functions, Division of Stress Adaptation and Protection, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi 464-8601 (Japan); Sawada, Makoto, E-mail: msawada@riem.nagoya-u.ac.jp [Department of Brain Functions, Division of Stress Adaptation and Protection, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi 464-8601 (Japan)] [Department of Brain Functions, Division of Stress Adaptation and Protection, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi 464-8601 (Japan)

    2009-10-23

    Magnetic resonance imaging (MRI) is a minimally invasive way to provide high spatial resolution tomograms. However, MRI has been considered to be useless for gene expression imaging compared to optical imaging. In this study, we used a ferritin reporter, binding with biogenic iron, to make it a powerful tool for gene expression imaging in MRI studies. GL261 mouse glioma cells were over-expressed with dual-reporter ferritin-DsRed under {beta}-actin promoter, then gene expression was observed by optical imaging and MRI in a brain tumor model. GL261 cells expressing ferritin-DsRed fusion protein showed enhanced visualizing effect by reducing T2-weighted signal intensity for in vitro and in vivo MRI studies, as well as DsRed fluorescence for optical imaging. Furthermore, a higher contrast was achieved on T2-weighted images when permeating the plasma membrane of ferritin-DsRed-expressing GL261. Thus, a ferritin expression vector can be used as an MRI reporter to monitor in vivo gene expression.

  4. Mutagenesis induced by targeted alpha therapy using 213Bi-cDTPA-9.2.27 in lacZ transgenic mice.

    PubMed

    Allen, Barry J; So, Trina; Abbas Rizvi, Syed M; Song, Emma Y; Fernandez, Harvey R; Lutz-Mann, Louise

    2009-05-01

    Targeted alpha therapy utilizes alpha-emitting radionuclides conjugated to monoclonal antibodies to allow specific irradiation of cancer cells whilst sparing normal, healthy tissues. The mutagenic potential of (213)Bi conjugated to a human melanoma antigen-specific antibody (9.2.27) was examined using an in vivo transgenic mouse model containing multiple copies of a lacZ target gene in every cell, allowing the quantification and comparison of mutagenesis in different organs. Mice received an ip injection of 16.65 MBq of (213)Bi-cDTPA-9.2.27, and were sacrificed at 24 h, 1 w and 4 w post-injection. Pharmacokinetic studies gave the absorbed and effective doses for each organ. The mutant frequency and mutant spectra were analysed for the brain, spleen and kidneys. The brain and spleen did not show significant increases in induced mutation frequencies compared to spontaneous background levels or changes in mutant spectra, these results being independent of p53 status. However, elevated mutation frequencies and persistent size change mutations were observed in the kidneys, but are not significant at the p = 0.05 level. The effect of p53 status was also evident, as p53 heterozygotes displayed higher mutation frequencies than their wild-type counterparts, suggesting a reduction in the p53 gene may lead to an increased susceptibility to mutagenesis. These effects were time dependent and levels returned to those of the controls at 4 w post-irradiation, albeit with a predominant residue of size mutations. These effects were observed at activities very much higher than those expected for the therapy of human patients. As such, the induction of secondary cancer with the (213)Bi-cDTPA-9.2.27 alpha immunoconjugate is not expected to be a significant problem in the clinic. PMID:19337032

  5. Ferritin as a Novel Reporter Gene for Photoacoustic Molecular Imaging

    PubMed Central

    Ha, Seung Han; Carson, Andrew R.; Kim, Kang

    2013-01-01

    Reporter genes may serve as endogenous contrast agents in the field of photoacoustic (PA) molecular imaging (PMI), enabling greater characterization of detailed cellular processes and disease progression. To demonstrate the feasibility of using ferritin as a reporter gene, human melanoma SK-24 (SK-MEL-24) cells were co-transfected with plasmid expressing human heavy chain ferritin (H-FT) and plasmid expressing enhanced green fluorescent protein (pEGFP-C1) using lipofectamine™ 2000. Non-transfected SK-MEL-24 cells served as a negative control. Fluorescent imaging of GFP confirmed transfection and transgene expression in co-transfected cells. To detect iron accumulation due to ferritin overexpression in SK-MEL-24 cells, a focused high-frequency ultrasonic transducer (60 MHz, f/1.5), synchronized to a pulsed laser (fluence < 5 mJ/cm2) was used to scan the PA signal at a wide range NIR wavelengths (850–950 nm). PA signal intensity from H-FT transfected SK-MEL-24 cells was about 5–9 dB higher than nontransfected SK-MEL-24 cells at 850–950 nm. Immunofluorescence and RT-PCR analysis both indicate high levels of ferritin expression in H-FT transfected SK-MEL24 cells, with little ferritin expression in nontransfected SK-MEL-24 cells. In this study, the feasibility of using ferritin as a reporter gene for PMI has been demonstrated in vitro. The use of ferritin as a reporter gene represents a novel concept for PMI using an endogenous contrast agent and may provide various opportunities for molecular imaging and basic science research. PMID:22949299

  6. Regulated Expression of Chromobox Homolog 5 Revealed in Tumors of ApcMin/+ ROSA11 Gene Trap Mice

    PubMed Central

    Thliveris, Andrew T.; Clipson, Linda; Sommer, Lacy L.; Schoenike, Barry A.; Hasenstein, Jason R.; Schlamp, Cassandra L.; Alexander, Caroline M.; Newton, Michael A.; Dove, William F.; Amos-Landgraf, James M.

    2012-01-01

    The gene-trap lacZ reporter insertion, ROSA11, in the Cbx5 mouse gene illuminates the regulatory complexity of this locus in ApcMin/+ mice. The insertion site of the ?-Geo gene-trap element lies in the 24-kb intron proximal to the coding region of Cbx5. Transcript analysis indicates that two promoters for Cbx5 flank this insertion site. Heterozygotes for the insertion express lacZ widely in fetal tissues but show limited expression in adult tissues. In the intestine, strong expression is limited to proliferative zones of crypts and tumors. Homozygotes for ROSA11, found at a lower than Mendelian frequency, express reduced levels of the coding region transcript in normal tissues, using a downstream promoter. Analysis via real-time polymerase chain reaction indicates that the upstream promoter is the dominant promoter in normal epithelium and tumors. Bioinformatic analysis of the Cbx5 locus indicates that WNT and its target transcription factor MYC can establish a feedback loop that may play a role in regulating the self-renewal of the normal intestinal epithelium and its tumors. PMID:22670227

  7. Tbx18 targets dermal condensates for labeling, isolation, and gene ablation during embryonic hair follicle formation.

    PubMed

    Grisanti, Laura; Clavel, Carlos; Cai, Xiaoqiang; Rezza, Amelie; Tsai, Su-Yi; Sennett, Rachel; Mumau, Melanie; Cai, Chen-Leng; Rendl, Michael

    2013-02-01

    How cell fate decisions of stem and progenitor cells are regulated by their microenvironment or niche is a central question in stem cell and regenerative biology. Although functional analysis of hair follicle epithelial stem cells by gene targeting is well established, the molecular and genetic characterization of the dermal counterpart during embryonic morphogenesis has been lacking because of the absence of cell type-specific drivers. Here, we report that T-box transcription factor Tbx18 specifically marks dermal papilla (DP) precursor cells during embryonic hair follicle morphogenesis. With Tbx18(LacZ), Tbx18(H2BGFP), and Tbx18(Cre) knock-in mouse models, we demonstrate LacZ and H2BGFP (nuclear green fluorescent protein) expression and Cre activity in dermal condensates of nascent first-wave hair follicles at E14.5. As Tbx18 expression becomes more widespread throughout the dermis at later developmental stages, we use tamoxifen-inducible Cre-expressing mice, Tbx18(MerCreMer), to exclusively target DP precursor cells and their progeny. Finally, we ablate Tbx18 in full knockout mice, but find no perturbations in hair follicle formation, suggesting that Tbx18 is dispensable for normal DP function. In summary, our study establishes Tbx18 as a genetic driver to target for the first time embryonic DP precursors for labeling, isolation, and gene ablation that will greatly enhance investigations into their molecular functions during hair follicle morphogenesis. PMID:22992803

  8. Genetics in methylotrophic bacteria: Appendix. Final report

    SciTech Connect

    Lidstrom, M.E.

    1998-09-01

    This research has focused primarily on promoters in Methylobacterium extorquens AM1 and in methanotrophic bacteria. In Methylobacterium extorquens work continued on the moxF promoter. The author constructed chromosomal lacZ fusions of this promoter to avoid the regulation problems of plasmid-borne fragments and has shown that this is regulated normally in the chromosome. She has constructed lacZ fusions to some of the mox genes involved in the synthesis of the cofactor, PQQ, in order to carry out similar analysis of transcription of PQQ genes. The author has continued to isolate mox genes in methanotrophs for the purpose of studying their promoters and transcriptional regulation.

  9. DNA adducts, mutant frequencies, and mutation spectra in various organs of lambda lacZ mice exposed to ethylating agents.

    PubMed

    Mientjes, E J; Luiten-Schuite, A; van der Wolf, E; Borsboom, Y; Bergmans, A; Berends, F; Lohman, P H; Baan, R A; van Delft, J H

    1998-01-01

    To investigate tissue-specific relations between DNA adducts and mutagenesis in vivo, lambda lacZ transgenic mice were treated i.p. with N-ethyl-N-nitrosourea (ENU), diethylnitrosamine (DEN), and ethyl methanesulphonate (EMS). In liver, bone marrow, and brain DNA from mice sacrificed at several time points after treatment O6-ethylguanine (O6-EtG) and N7-ethylguanine (N7-EtG) levels were determined as well as the mutant frequency (MF) in lacZ. In liver DNA of ENU- and DEN-treated mice, the bulk of O6-EtG was removed at 3 days after treatment, while the MF continued to increase thereafter. This suggests that O6-EtG is not the major premutagenic lesion in the liver. Indeed, sequence analysis of mutants showed only 24% GC-->AT transitions, consistent with the O6-EtG lesion, and 28% TA-->AT transversions, expected from O2-ethylthymine. In bone marrow after ENU treatment, a maximum mutation induction occurred at 3 days post-treatment, of which 43% were GC-->AT mutations and 22% were TA-->AT mutations. This suggests that in bone marrow O6-EtG may be a major premutagenic lesion at the 3-day time point. In liver and bone marrow, EMS treatment gave rise to a high level of N7-EtG and a low level of O6-EtG but no increase in MF. No adducts or mutation induction were observed in bone marrow of DEN-treated mice. No MF increase was observed in the brain of either ENU- or EMS-treated mice, although O6- and N7-adducts were present. PMID:9464312

  10. Mutations in BTD gene causing biotinidase deficiency: a regional report.

    PubMed

    Kasapkara, Çi?dem Seher; Akar, Melek; Özbek, Mehmet Nuri; Tüzün, Heybet; Aldudak, Bedri; Baran, R?za Taner; Tanyalç?n, Tijen

    2015-03-01

    Biotinidase deficiency is an autosomal recessive inborn error of biotin metabolism. Children with biotinidase deficiency cannot cleave biocytin and, therefore, cannot recycle biotin. Untreated individuals become secondarily biotin deficient, which in turn results in decreased activities of the biotin-dependent carboxylases and the subsequent accumulation of toxic metabolites causing clinical symptoms. Biotinidase deficiency is characterized by neurological, cutaneous manifestations and metabolic abnormalities. The worldwide incidence of profound biotinidase deficiency has been estimated at 1:112,271. The human biotinidase gene is located on chromosome 3p25 and consists of four exons with a total length of 1629 base pairs. To date, more than 100 mutations in the biotinidase gene known to cause biotinidase deficiency have been reported. The vast majority of mutations are homozygous or compound heterozygous. Finding known mutations can be correlated with the biochemical enzymatic results. This report summarizes the demographic features of patients identified as biotinidase deficient from August of 2012 through August of 2013 and mutation analysis results for 20 cases in the southeast region of Turkey. PMID:25423671

  11. Characterization of the mammalian DNA polymerase gene(s) and enzyme(s). Annual progress report

    SciTech Connect

    Mishra, N.C.

    1995-01-01

    Two Genes for DNA polymerase delta were identified from the wild type Chinese hamster ovary cells. These genes were cloned via RT-PCR from mRNA prepared the Chinese hamster ovary cells using primers specific to conserved sequences of the DNA polymerase {delta} gene. The first gene encodes a PCNA dependent DNA polymerase {delta} gene whereas the second gene encodes a PCNA independent DNA polymerase {delta} gene. Methods were developed to clone these genes in expression vector and host systems. The role of the two genes in DNA replication and repair was determined.

  12. Identification of genes in anonymous DNA sequences. Final report: Report period, 15 April 1993--15 April 1994

    SciTech Connect

    Fields, C.A.

    1994-09-01

    This Report concludes the DOE Human Genome Program project, ``Identification of Genes in Anonymous DNA Sequence.`` The central goals of this project have been (1) understanding the problem of identifying genes in anonymous sequences, and (2) development of tools, primarily the automated identification system gm, for identifying genes. The activities supported under the previous award are summarized here to provide a single complete report on the activities supported as part of the project from its inception to its completion.

  13. SHORT REPORT Open Access Methodology optimizing SAGE library tag-to-gene

    E-print Network

    Paris-Sud XI, Université de

    SHORT REPORT Open Access Methodology optimizing SAGE library tag-to-gene mapping: application in therapy and control. Several experimental approaches including SAGE (Serial analysis of gene expression. Usual SAGE tag-to-gene mapping techniques are inadequate because almost all tags are normally located

  14. TECHNOLOGY REPORT Case Studies of Ends-Out Gene Targeting in Drosophila

    E-print Network

    Quake, Stephen R.

    TECHNOLOGY REPORT Case Studies of Ends-Out Gene Targeting in Drosophila Haiyang Chen,1 Zhiguo Ma,1 com- plete targeting vector for use in a mass crossing ends- out gene targeting study. Importantly June 2008; Revised 13 October 2008; Accepted 11 December 2008 Summary: Ends-in and ends-out gene

  15. BAC transgenic analysis reveals enhancers sufficient for Hoxa13 and neighborhood gene expression in mouse embryonic distal limbs and genital bud.

    PubMed

    Lehoczky, Jessica A; Innis, Jeffrey W

    2008-01-01

    We previously demonstrated that a approximately 1 Mb domain of genes upstream of and including Hoxa13 is co-expressed in the developing mouse limbs and genitalia. A highly conserved non-coding sequence, mmA13CNS, was shown to be insufficient in transgenic mice to direct precise Hoxa13-like expression in the limb buds or genital bud, although some LacZ expression from the transgene was reproducibly found in these tissues. In this report, we used beta-globin minimal promoter LacZ recombinant BAC transgenes encompassing mmA13CNS to identify a single critical region involved in mouse Hoxa13-like embryonic genital bud expression. By analyzing the expression patterns of these overlapping BAC clones in transgenic mice, we show that at least two sequences remote to the HoxA cluster are required collectively to drive Hoxa13-like expression in developing distal limbs. Given that the paralogous posterior HoxD and neighboring genes have been shown to be under the influence of long-range distal limb and genital bud enhancers, we hypothesize that both long-range enhancers have one ancestral origin, which diverged in both sequence and function after the HoxA/D cluster duplication. PMID:18638319

  16. Gene disruption of Mfsd8 in mice provides the first animal model for CLN7 disease.

    PubMed

    Damme, Markus; Brandenstein, Laura; Fehr, Susanne; Jankowiak, Wanda; Bartsch, Udo; Schweizer, Michaela; Hermans-Borgmeyer, Irm; Storch, Stephan

    2014-05-01

    Mutations in the major facilitator superfamily domain containing 8 (MFSD8) gene coding for the lysosomal CLN7 membrane protein result in CLN7 disease, a lysosomal storage disease of childhood. CLN7 disease belongs to a group of inherited disorders, called neuronal ceroid lipofuscinoses (NCL), which are characterized by the accumulation of autofluorescent ceroid lipopigments, neuroinflammation, photoreceptor- and neurodegeneration. We have disrupted the Mfsd8 gene by insertion of a lacZ gene-trap cassette between exons 1 and 2 in mice and have analyzed the impact of Cln7 depletion on neuronal and visceral tissues. Analysis of lacZ reporter gene activity in heterozygous Mfsd8((wt/tm1a)) mice showed strong Mfsd8 mRNA expression in the cerebral cortex, in the hippocampus and in the kidney. Homozygous Mfsd8((tm1a/tm1a)) mice were viable and fertile and resembled biochemically the NCL-phenotype of human CLN7 patients including the accumulation of autofluorescent material in the brain and peripheral tissues and of subunit c of mitochondrial ATP synthase in the cerebellum and nuclei of distinct brain regions, and the degeneration of photoreceptor cells in the retina. Lysosomal storage was found in large neurons of the medulla, the hippocampus and in Purkinje cells of the cerebellum in mutant mice. The ultrastructure of the storage material revealed dense lamellar bodies with irregular forms within cerebellar and hippocampal neurons. In the brain loss of Cln7 was accompanied by mild reactive microgliosis and subtle astrogliosis by 10months of age, respectively. In summary we have generated a mouse model which is partly valuable as some but not all neuropathological features of human CLN7 disease are recapitulated thus representing an animal model to study CLN7-specific disease mechanisms. PMID:24423645

  17. A GAL4-HP1 fusion protein targeted near heterochromatin promotes gene silencing.

    PubMed

    Seum, C; Spierer, A; Delattre, M; Pauli, D; Spierer, P

    2000-11-01

    We have constructed a new reporter transgene, Winkelried, equipped with a synthetic binding site for the yeast GAL4 transcriptional activator. The binding site is inserted between the white and lacZ reporter genes, and is flanked by FRT sequences. These elements allow excision of the GAL4 binding site by crossing the transgenic line with an FLP recombinase producing strain. We have generated by X-ray irradiation two independent chromosomal rearrangements, Heidi and Tell, relocating Winkelried next to pericentromeric heterochromatin. These rearrangements induce variegation of both white and lacZ. Variegation of Winkelried in the rearranged transgenic lines responds to the loss and excess of doses of the dominant suppressors of position-effect variegation (PEV) Su(var)3-7 and Su(var)2-5. Winkelried therefore constitutes a unique tool to test the effect on variegation in cis of any factor fused to the GAL4 DNA binding domain. Indeed, a chimeric protein, made of the DNA binding site of GAL4 and of HP1, the modifier of PEV encoded by Su(var)2-5, is shown to enhance variegation of Heidi and Tell. Excision of the binding sites for GAL4 in the variegating rearrangements Heidi and Tell abolishes the modifier effect of the GAL4-HP1 chimera. Therefore, in the Heidi and Tell rearrangements, enhancement of position-effect variegation depends strictly both on the concentration of GAL4-HP1 and on the presence of its binding site in the vicinity of the reporter genes. PMID:11151674

  18. Cloning, nucleotide sequence, and regulatory analysis of the Nitrosomonas europaea dnaK gene.

    PubMed Central

    Iizumi, T; Nakamura, K

    1997-01-01

    The dnaK gene of an ammonia-oxidizing bacterium, Nitrosomonas europaea, was cloned and sequenced. It was found that the dnaK gene product was highly homologous to previously analyzed dnaK gene products from other organisms at the amino acid level. Two partial open reading frames located upstream and downstream of the dnaK gene were also found and identified as grpE and dnaJ genes, respectively, by the predicted amino acid homology of their gene products to other bacterial GrpE and DnaJ proteins. Transcription of the dnaK gene was strongly induced by a heat shock from 30 to 37 degrees C. An analysis of the expression of the dnaK gene fused to the lacZ translational reporter gene also showed eightfold increase in beta-galactosidase activity after the heat shock induction. Heat-inducible transcription start sites of the dnaK gene, revealed by primer extension analysis, were located 16 and 17 nucleotides upstream from the translational start codon of the dnaK gene, and the predicted promoter sequence showed a homology to the consensus sequence of sigma 32-dependent heat shock promoters of gram-negative bacteria. The upstream region of the dnaK gene did not contain the inverted repeat structure that was involved in the regulation of the heat shock gene of several gram-negative and gram-positive bacteria. Therefore, we conclude that the heat shock regulatory mechanism of the N. europaea dnaK gene may be similar to the sigma 32-dependent mechanism observed in other gram-negative bacteria. PMID:9143112

  19. A cloning vector for creation of Escherichia coli lacZ translational fusions and generation of linear template for chromosomal integrations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A novel cloning vector to aid in the construction of ß-galactosidase reporter systems for gene expression studies in lactose metabolizing strains of Shiga toxin producing Escherichia coli is described. The plasmid allows construction of translational fusions of cloned gene promoters with a short seg...

  20. Glycerophosphorylcholine regulates Haemophilus influenzae glpQ gene expression.

    PubMed

    Alrousan, Enas; Fan, Xin

    2015-05-01

    An important virulence strategy adopted by Haemophilus influenzae to establish a niche on the mucosal surface of the host is the phosphorylcholine (ChoP) decoration of its lipopolysaccharides, which promotes adherence to the host cells. Haemophilus influenzae is able to use glycerophosphorylcholine (GPC) from host for ChoP synthesis. Utilization of GPC requires glpQ, which encodes a glycerophosphodiester phosphodiesterase enzyme. In this study, we investigate the transcriptional regulation of glpQ gene using real-time PCR and transcriptional fusion of H. influenzae glpQ promoter to the Escherichia coli lacZ reporter gene. The glpQ promoter activities were examined under environmental conditions including changes in temperature, oxygen, high salt and minimal growth medium. Our data showed that under room temperature and anaerobic conditions, the glpQ gene expression levels were significantly higher than under other growth conditions. In addition, the glpQ gene expression levels were upregulated in the presence of GPC. These results suggest that H. influenzae may upregulate glpQ expression in response to different environments it encounters during infection, from the airway surfaces (room temperature) to deep tissues (anaerobic). Upregulation of glpQ by GPC may allow efficient use of abundant GPC from mammalian cells by H. influenzae as a source of nutrient and for ChoP decoration of lipopolysaccharide that facilitates bacterial adhesion to host cells and growth during infection. PMID:25837816

  1. Functional analysis of the promoter region of amphioxus ?-actin gene: a useful tool for driving gene expression in vivo.

    PubMed

    Feng, Jun; Li, Guang; Liu, Xin; Wang, Jing; Wang, Yi-Quan

    2014-10-01

    Amphioxus is a promising new animal model for developmental biology. To develop molecular tools for this model, we characterized the promoter region of a cytoplasmic ?-actin gene (Bb-actin-6-2) from the Chinese amphioxus Branchiostoma belcheri. In situ hybridization and real time-quantitative PCR analyses showed that this gene is expressed in many tissues throughout embryonic development. Cloning of cDNA revealed two isoforms with distinct transcription start sites. Isoform #1 exhibits a similar exon/intron and regulatory element organization to that of vertebrate ?-actin, whereas isoform #2 lacks the first exon of isoform #1 and recruits its first intron as a promoter. The activities of upstream promoter regions in the two isoforms were examined using the lacZ reporter system in amphioxus embryos. The proximal promoter of isoform #1 drove reporter gene expression broadly in 58.6 % of injected embryos. That of isoform #2 exhibited much higher activity (91.5 %) than that of isoform #1 or the human EF-1-? gene (38.2 %). We determined the minimal promoter regions of the two isoforms via functional analysis. These two regions, alone or inserted a random DNA fragment upstream, had no detectable activity, but when an upstream enhancer was inserted, the promoters directed reporter gene expression in 61.0 and 93.8 %, respectively, of injected embryos in a tissue-specific manner. Our study not only provides insight into the regulatory mechanism underlying amphioxus Bb-actin-6-2 gene expression, but also identifies two sets of efficient proximal and minimal promoters. These promoters could be used to construct gene expression vectors for transgenic studies using amphioxus as a model. PMID:25078982

  2. BRIEF REPORT: Ghrelin receptor gene polymorphisms and body size in children and adults

    E-print Network

    Paris-Sud XI, Université de

    Page 1 BRIEF REPORT: Ghrelin receptor gene polymorphisms and body size in children and adults Medicine, University of Bristol, 24 Tyndall Avenue, Bristol BS8 1TQ, UK Key Words: ghrelin, ghrelin receptor type 1a gene (GHSR) encodes the cognate receptor of ghrelin, a gut hormone that regulates food

  3. Effect of external and internal factors on the expression of reporter genes driven by the N resistance gene promoter

    PubMed Central

    Kathiria, Palak; Sidler, Corinne; Woycicki, Rafal; Yao, Youli; Kovalchuk, Igor

    2013-01-01

    The role of resistance (R) genes in plant pathogen interaction has been studied extensively due to its economical impact on agriculture. Interaction between tobacco mosaic virus (TMV) and the N protein from tobacco is one of the most widely used models to understand various aspects of pathogen resistance. The transcription activity governed by N gene promoter is one of the least understood elements of the model. In this study, the N gene promoter was cloned and fused with two different reporter genes, one encoding ?-glucuronidase (N::GUS) and another, luciferase (N::LUC). Tobacco plants transformed with the N::GUS or N::LUC reporter constructs were screened for homozygosity and stable expression. Histochemical analysis of N::GUS tobacco plants revealed that the expression is organ specific and developmentally regulated. Whereas two week old plants expressed GUS in midveins only, 6-wk-old plants also expressed GUS in leaf lamella. Roots did not show GUS expression at any time during development. Experiments to address effects of external stress were performed using N::LUC tobacco plants. These experiments showed that N gene promoter expression was suppressed when plants were exposed to high but not low temperatures. Expression was also upregulated in response to TMV, but no changes were observed in plants treated with SA. PMID:23656874

  4. Effect of external and internal factors on the expression of reporter genes driven by the N resistance gene promoter.

    PubMed

    Kathiria, Palak; Sidler, Corinne; Woycicki, Rafal; Yao, Youli; Kovalchuk, Igor

    2013-07-01

    The role of resistance (R) genes in plant pathogen interaction has been studied extensively due to its economical impact on agriculture. Interaction between tobacco mosaic virus (TMV) and the N protein from tobacco is one of the most widely used models to understand various aspects of pathogen resistance. The transcription activity governed by N gene promoter is one of the least understood elements of the model. In this study, the N gene promoter was cloned and fused with two different reporter genes, one encoding ?-glucuronidase (N::GUS) and another, luciferase (N::LUC). Tobacco plants transformed with the N::GUS or N::LUC reporter constructs were screened for homozygosity and stable expression. Histochemical analysis of N::GUS tobacco plants revealed that the expression is organ specific and developmentally regulated. Whereas two week old plants expressed GUS in midveins only, 6-wk-old plants also expressed GUS in leaf lamella. Roots did not show GUS expression at any time during development. Experiments to address effects of external stress were performed using N::LUC tobacco plants. These experiments showed that N gene promoter expression was suppressed when plants were exposed to high but not low temperatures. Expression was also upregulated in response to TMV, but no changes were observed in plants treated with SA. PMID:23656874

  5. Assessment of a novel, capsid-modified adenovirus with an improved vascular gene transfer profile

    PubMed Central

    2013-01-01

    Background Cardiovascular disorders, including coronary artery bypass graft failure and in-stent restenosis remain significant opportunities for the advancement of novel therapeutics that target neointimal hyperplasia, a characteristic of both pathologies. Gene therapy may provide a successful approach to improve the clinical outcome of these conditions, but would benefit from the development of more efficient vectors for vascular gene delivery. The aim of this study was to assess whether a novel genetically engineered Adenovirus could be utilised to produce enhanced levels of vascular gene expression. Methods Vascular transduction capacity was assessed in primary human saphenous vein smooth muscle and endothelial cells using vectors expressing the LacZ reporter gene. The therapeutic capacity of the vectors was compared by measuring smooth muscle cell metabolic activity and migration following infection with vectors that over-express the candidate therapeutic gene tissue inhibitor of matrix metalloproteinase-3 (TIMP-3). Results Compared to Adenovirus serotype 5 (Ad5), the novel vector Ad5T*F35++ demonstrated improved binding and transduction of human vascular cells. Ad5T*F35++ mediated expression of TIMP-3 reduced smooth muscle cell metabolic activity and migration in vitro. We also demonstrated that in human serum samples pre-existing neutralising antibodies to Ad5T*F35++ were less prevalent than Ad5 neutralising antibodies. Conclusions We have developed a novel vector with improved vascular transduction and improved resistance to human serum neutralisation. This may provide a novel vector platform for human vascular gene transfer. PMID:23937994

  6. Identification of the pifC gene and its role in negative control of F factor pif gene expression.

    PubMed Central

    Miller, J F; Malamy, M H

    1983-01-01

    The pif region of the F factor includes two genes, pifA and pifB, that lead to abortive T7 infection. We have identified a new gene in this region, pifC, by constructing an in vitro fusion of pif DNA at 41.6 kilobases on the F factor physical map to the lacZ gene. A PifC-LacZ fusion protein of 149,000 daltons has been identified by immunoprecipitation and polyacrylamide gel electrophoresis. This allows us to assign the N terminus of pifC to 42.5 kilobases on the F map. Using fusions of pifC, pifA, and pifB to lacZ, we have studied the regulation of pif gene expression and have shown that the product of pifC negatively controls its own expression and that of pifA and pifB. Images PMID:6413493

  7. Research Report Gene array profiling of large hypothalamic CNS regions in lactating and

    E-print Network

    Bronikowski, Anne

    Research Report Gene array profiling of large hypothalamic CNS regions in lactating and randomly of Wisconsin, Madison, WI 53706, USA c Department of Psychology, University of Wisconsin, Madison, WI 53706

  8. Reporter gene introduction and transient expression in protoplasts of Porphyra yezoensis

    Microsoft Academic Search

    Yuzuru Mizukami; Makoto Hado; Hitoshi Kito; Masahiko Kunimoto; Noboru Murase

    2004-01-01

    The transient expression of foreign genes in the protoplasts of Porphyrayezoensis was examined using three recombinant vectors, pYez-Rub-GUS, pYez-Rub-GFP and pYez-Rub-LUC, which were constructed with the\\u000a promoter sequence of the ribulose-bisphosphate-carboxylase \\/ oxygenase (Rubisco) gene as a promoter and the bacterial ?-glucuronidase\\u000a (GUS), mutant of green fluorescent protein (S65T-GFP) and firefly luciferase (LUC) genes, respectively, as reporter genes.\\u000a When the

  9. Regulation of Aspergillus nidulans penicillin biosynthesis and penicillin biosynthesis genes acvA and ipnA by glucose.

    PubMed Central

    Brakhage, A A; Browne, P; Turner, G

    1992-01-01

    Expression of the Aspergillus nidulans penicillin biosynthesis genes acvA and ipnA, encoding delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase and isopenicillin N synthetase, respectively, was analyzed. The intergenic region carrying the divergently oriented promoters was fused in frame in both orientations to Escherichia coli lacZ and E. coli uidA reporter genes. Each construct permits simultaneous expression studies of both genes. Transformants of A. nidulans carrying a single copy of either plasmid integrated at the chromosomal argB locus were selected for further investigations. Expression of both genes was directed by the 872-bp intergenic region. ipnA- and acvA-derived gene fusions were expressed from this region at different levels. ipnA had significantly higher expression than did acvA. Glucose specifically reduced the production of penicillin and significantly repressed the expression of ipnA but not of acvA gene fusions. The specific activities of isopenicillin N synthetase, the gene product of ipnA, and acyl coenzyme A:6-aminopenicillanic acid acyltransferase were also reduced in glucose-grown cultures. Images PMID:1592830

  10. Inflammatory bowel disease gene discovery. CRADA final report

    SciTech Connect

    NONE

    1997-09-09

    The ultimate goal of this project is to identify the human gene(s) responsible for the disorder known as IBD. The work was planned in two phases. The desired products resulting from Phase 1 were BAC clone(s) containing the genetic marker(s) identified by gene/Networks, Inc. as potentially linked to IBD, plasmid subclones of those BAC(s), and new genetic markers developed from these plasmid subclones. The newly developed markers would be genotyped by gene/Networks, Inc. to ascertain evidence for linkage or non-linkage of IBD to this region. If non-linkage was indicated, the project would move to investigation of other candidate chromosomal regions. Where linkage was indicated, the project would move to Phase 2, in which a physical map of the candidate region(s) would be developed. The products of this phase would be contig(s) of BAC clones in the region exhibiting linkage to IBD, as well as plasmic subclones of the BACs and further genetic marker development. There would also be continued genotyping with new polymorphic markers during this phase. It was anticipated that clones identified and developed during these two phases would provide the physical resources for eventual disease gene discovery.

  11. A novel binary T-vector with the GFP reporter gene for promoter characterization.

    PubMed

    Jiang, Shu-Ye; Vanitha, Jeevanandam; Bai, Yanan; Ramachandran, Srinivasan

    2014-01-01

    Several strategies have been developed to clone PCR fragments into desired vectors. However, most of commercially available T-vectors are not binary vectors and cannot be directly used for Agrobacterium-mediated plant genetic transformation. In this study, a novel binary T-vector was constructed by integrating two AhdI restriction sites into the backbone vector pCAMBIA 1300. The T-vector also contains a GFP reporter gene and thus, can be used to analyze promoter activity by monitoring the reporter gene. On the other hand, identification and characterization of various promoters not only benefit the functional annotation of their genes but also provide alternative candidates to be used to drive interesting genes for plant genetic improvement by transgenesis. More than 1,000 putative pollen-specific rice genes have been identified in a genome-wide level. Among them, 67 highly expressed genes were further characterized. One of the pollen-specific genes LOC_Os10g35930 was further surveyed in its expression patterns with more details by quantitative real-time reverse-transcription PCR (qRT-PCR) analysis. Finally, its promoter activity was further investigated by analyzing transgenic rice plants carrying the promoter::GFP cassette, which was constructed from the newly developed T-vector. The reporter GFP gene expression in these transgenic plants showed that the promoter was active only in mature but not in germinated pollens. PMID:25197968

  12. A Novel Binary T-Vector with the GFP Reporter Gene for Promoter Characterization

    PubMed Central

    Jiang, Shu-Ye; Vanitha, Jeevanandam; Bai, Yanan; Ramachandran, Srinivasan

    2014-01-01

    Several strategies have been developed to clone PCR fragments into desired vectors. However, most of commercially available T-vectors are not binary vectors and cannot be directly used for Agrobacterium-mediated plant genetic transformation. In this study, a novel binary T-vector was constructed by integrating two AhdI restriction sites into the backbone vector pCAMBIA 1300. The T-vector also contains a GFP reporter gene and thus, can be used to analyze promoter activity by monitoring the reporter gene. On the other hand, identification and characterization of various promoters not only benefit the functional annotation of their genes but also provide alternative candidates to be used to drive interesting genes for plant genetic improvement by transgenesis. More than 1,000 putative pollen-specific rice genes have been identified in a genome-wide level. Among them, 67 highly expressed genes were further characterized. One of the pollen-specific genes LOC_Os10g35930 was further surveyed in its expression patterns with more details by quantitative real-time reverse-transcription PCR (qRT-PCR) analysis. Finally, its promoter activity was further investigated by analyzing transgenic rice plants carrying the promoter::GFP cassette, which was constructed from the newly developed T-vector. The reporter GFP gene expression in these transgenic plants showed that the promoter was active only in mature but not in germinated pollens. PMID:25197968

  13. A report on single exon genes (SEG) in eukaryotes.

    PubMed

    Sakharkar, Meena Kishore; Chow, Vincent Tak Kwong; Chaturvedi, Iti; Mathura, Venkatarajan Subramanian; Shapshak, Paul; Kangueane, Pandjassarame

    2004-09-01

    Single exon genes (SEG) are archetypical of prokaryotes. Hence, their presence in intron-rich, multi-cellular eukaryotic genomes is perplexing. Consequently, a study on SEG origin and evolution is important. Towards this goal, we took the first initiative of identifying and counting SEG in nine completely sequenced eukaryotic organisms--four of which are unicellular (E. cuniculi, S. cerevisiae, S. pombe, P. falciparum) and five of which are multi-cellular (C. elegans, A. thaliana, D. melanogaster, M. musculus, H. sapiens). This exercise enabled us to compare their proportion in unicellular and multi-cellular genomes. The comparison suggests that the SEG fraction decreases with gene count (r = -0.80) and increases with gene density (r = 0.88) in these genomes. We also examined the distribution patterns of their protein lengths in different genomes. PMID:15353355

  14. Role of starvation genes in the survival of deep subsurface bacterial communities. Final report

    SciTech Connect

    Matin, A. [Stanford Univ., CA (United States). Dept. of Microbiology and Immunology; Schmidt, T. [Michigan State Univ., East Lansing, MI (United States). Dept. of Microbiology; Caldwell, D. [Univ. of Saskatchewan, Saskatoon, Saskatchewan (Canada). Dept. of Microbiology

    1998-11-01

    The investigation dealt with several aspects of subsurface bacterial survival and their nature. Mutants of Pseudomonas putida, a common environmental bacterium with counterparts in the subsurface, were isolated by transposon mutagenesis. These mutants were highly sensitive to starvation stress. Reporter gene fusions also showed that these genes were starvation genes since they were induced several fold when the cultures were started. Since the regulatory religions (promoters) of starvation genes are of interest in bioremediation and in experiments designed to understand the roles of starvation genes in the maintenance of microbial community structure, the promoter of one of these genes (pstarv1, contained in strain MK107) was characterized in detail. As a preliminary to these studies, the growth characteristics of Pseudomonas putida MK1 and MK107 were compared for cells growing in batch cultures or as an attached monolayer in microstat cultures.

  15. [CANCER RESEARCH 64, 13231330, February 15, 2004] Imaging Tri-Fusion Multimodality Reporter Gene Expression in Living Subjects

    E-print Network

    Tsien, Roger Y.

    to cancer research, gene therapy, and transgenic mod- els are rapidly expanding. We report construction[CANCER RESEARCH 64, 1323­1330, February 15, 2004] Imaging Tri-Fusion Multimodality Reporter Gene by the fusion gene in cell culture, we imaged living mice bearing 293T cells transiently expressing the hrl

  16. In 1997, the maize gene tb1 was reported as the first domestication QTL to be cloned (4). tb1

    E-print Network

    Forbes, Jeffrey

    In 1997, the maize gene tb1 was reported as the first domestication QTL to be cloned (4). tb1 grains to maize (as opposed to the covered grains of teosinte), was cloned (6).And thus far in 2006, in addition to the two rice shattering genes, cloning of the wheat Q gene was reported (7). Q controls

  17. MAMMALIAN CELL GENE MUTATION ASSAYS WORKING GROUP REPORT

    EPA Science Inventory

    Mammalian cell gene mutation assays have been used for many years and the diversity of the available systems attests to the varied methods found to grow mammalian dells and detect mutations. s part of the International Workshop on Standardization of Genotoxicity Test Procedures, ...

  18. Selection of lac gene fusions in vivo: ompR-lacZ fusions that define a functional domain of the ompR gene product.

    PubMed Central

    Berman, M L; Jackson, D E

    1984-01-01

    We describe a simple method for selecting Escherichia coli mutants that carry gene fusions between a cloned gene and lacZ. We test this technique with the ompR gene, which codes for a positive regulatory factor in porin synthesis. A number of OmpR-LacZ hybrid proteins are examined, and several unusual phenotypes associated with these protein fusions are described. Evidence is presented to support the two-domain model for ompR proposed previously (Hall and Silhavy, J. Mol. Biol. 151:1-15). In addition, one of the ompR-lacZ fusions exhibits a dominant OmpR- phenotype. The utility of isolating a series of lacZ gene fusions to any target gene is discussed. Images PMID:6086585

  19. Viral delivery of superoxide dismutase gene reduces cyclosporine A-induced nephrotoxicity

    Microsoft Academic Search

    Zhi Zhong; Henry D. Connor; Ming Yin; Michael D. Wheeler; Ronald P. Mason; Ronald G. Thurman

    2001-01-01

    Viral delivery of superoxide dismutase gene reduces cyclosporine A-induced nephrotoxicity.BackgroundCyclosporine A (CsA) increases free radical formation in the kidney. Accordingly, this study investigated whether gene delivery of superoxide dismutase (SOD) reduced radical production and nephrotoxicity caused by CsA.MethodsRats were given adenovirus (Ad) carrying lacZ or Cu\\/Zn-SOD genes three days prior to CsA treatment. Histology, glomerular filtration rates (GFRs) and free

  20. Tyrosinase as a multifunctional reporter gene for Photoacoustic/MRI/PET triple modality molecular imaging

    PubMed Central

    Qin, Chunxia; Cheng, Kai; Chen, Kai; Hu, Xiang; Liu, Yang; Lan, Xiaoli; Zhang, Yongxue; Liu, Hongguang; Xu, Yingding; Bu, Lihong; Su, Xinhui; Zhu, Xiaohua; Meng, Shuxian; Cheng, Zhen

    2013-01-01

    Development of reporter genes for multimodality molecular imaging is highly important. In contrast to the conventional strategies which have focused on fusing several reporter genes together to serve as multimodal reporters, human tyrosinase (TYR) – the key enzyme in melanin production – was evaluated in this study as a stand-alone reporter gene for in vitro and in vivo photoacoustic imaging (PAI), magnetic resonance imaging (MRI) and positron emission tomography (PET). Human breast cancer cells MCF-7 transfected with a plasmid that encodes TYR (named as MCF-7-TYR) and non-transfected MCF-7 cells were used as positive and negative controls, respectively. Melanin targeted N-(2-(diethylamino)ethyl)-18F-5-fluoropicolinamide was used as a PET reporter probe. In vivo PAI/MRI/PET imaging studies showed that MCF-7-TYR tumors achieved significant higher signals and tumor-to-background contrasts than those of MCF-7 tumor. Our study demonstrates that TYR gene can be utilized as a multifunctional reporter gene for PAI/MRI/PET both in vitro and in vivo. PMID:23508226

  1. Characterization of the Arxula adeninivorans AHOG1 gene and the encoded mitogen-activated protein kinase.

    PubMed

    Böer, Erik; Wartmann, Thomas; Dlubatz, Karen; Gellissen, Gerd; Kunze, Gotthard

    2004-11-01

    Arxula adeninivorans is an osmo-resistant yeast species that can tolerate high levels of osmolytes like NaCl, PEG400 and ethylene glycol. As in other yeast species, this tolerance is elicited by components of the high osmolarity glycerol (HOG) response pathway. In the present study, we isolated and characterized as a key component of this pathway the A. adeninivorans AHOG1 gene encoding the mitogen-activated protein (MAP) kinase Ahog1p, an enzyme of 45.9 kDa. The gene includes a coding sequence of 1,203 bp disrupted by a 57-bp intron. The identity of the gene was confirmed by complementation of a hog1 mutation in a Saccharomyces cerevisiae mutant strain and the high degree of homology of the derived amino acid sequence with that of MAP kinases from other yeasts and fungi. Under stress-free conditions, the inactive Ahoglp is present in low levels. When exposed to osmotic stress, Ahoglp is rendered active by phosphorylation. In addition, AHOG1 expression is increased. Assessment of the AHOG1 promoter activity with a lacZ reporter gene confirmed its inducibility by osmolytes, a characteristic not observed in homologous HOG1 genes of other yeast species. This specific property could account for the fast adaptation and high osmo-resistance encountered in this species. PMID:15526205

  2. The C. elegans neuronally expressed homeobox gene ceh-10 is closely related to genes expressed in the vertebrate eye.

    PubMed

    Svendsen, P C; McGhee, J D

    1995-05-01

    We describe the homeobox gene ceh-10 from the nematode Caenorhabditis elegans. The homeodomain of ceh-10 is closely related to the homeodomains of two genes recently cloned from the vertebrate retina, Chx10 from mice and Vsx-1 from goldfish. We show that the sequence conservation extends well beyond the homeodomain and includes a region (named the CVC domain) of roughly 60 amino acids immediately C-terminal to the homeodomain. As assayed in transgenic worms, the promoter region of ceh-10 directs expression of a lacZ reporter gene to a small number of neurons. We draw a parallel between the bipolar cells of the inner nuclear layer of the vertebrate retina, which express Chx10 and Vsx-1, and an interneuron in C. elegans called AIY, which expresses ceh-10. AIY receives synaptic input from a sensory cell, just as do bipolar cells of the vertebrate retina. In C. elegans, the sensory cell AFD is not known to be photosensitive but is known to be thermosensitive; moreover, a cell with similar position in the amphids of other nematodes has been suggested indeed to be photosensitive. Our results emphasize the highly conserved nature of sensory regulatory mechanisms and suggest one way in which photosensitive organelles might have originated in evolution. PMID:7789259

  3. Construction and identification of the adenoviral vector with dual reporter gene for multimodality molecular imaging.

    PubMed

    Wang, Yi-fan; Liu, Ting; Guo, Yu-lin; Gao, Fa-bao

    2013-08-01

    In this study, the recombinant adenovirus (Ad) vector containing dual reporter gene [i.e. human transferrin receptor gene (TFRC) and firefly luciferase reporter gene] was constructed to provide a novel experimental tool for magnetic resonance (MR) and bioluminescence dual-modality molecular imaging. The cDNA of TFRC was amplified by polymerase chain reaction (PCR) and cloned into the multiple cloning site of pShuttle-CMV-CMV-Luciferase vector. After identification by Sfi I digestion and sequencing, pShuttle-TFRC-Luciferase vector and the adenoviral backbone vector (pAdeno) were subjected to homologous recombination. The correct recombinant plasmid was then transfected into 293 packaging cells to produce adenoviral particles and confirmed by PCR. After infection of human colorectal cancer LOVO cells with Ad-TFRC-Luciferase, the expressions of transferrin receptor (TfR) and luciferase protein were detected respectively by Western blotting and bioluminescence imaging in vitro. The results showed that TFRC gene was successfully inserted into the adenoviral shuttle vector carrying luciferase gene. DNA sequence analysis indicated that the TFRC gene sequence in the shuttle plasmid was exactly the same as that reported in GenBank. The recombinant plasmid was identified correct by restriction digestion. Ad-TFRC-Luciferase recombinant adenovirus was constructed successfully, and the virus titer was 1.6×10(10) pfu/mL. Forty-eight h after dual reporter gene transfection, the expressions of TfR and luciferase protein were increased significantly (P<0.01). It was concluded that the recombinant adenovirus vector with dual reporter gene was successfully established, which may be used for in vivo tracing target cells in multimodality imaging. PMID:23904384

  4. Comprehensive genetic analysis of transcription factor pathways using a dual reporter gene system in budding yeast.

    PubMed

    Kainth, Pinay; Sassi, Holly Elizabeth; Peña-Castillo, Lourdes; Chua, Gordon; Hughes, Timothy R; Andrews, Brenda

    2009-07-01

    The development and application of genomic reagents and techniques has fuelled progress in our understanding of regulatory networks that control gene expression in eukaryotic cells. However, a full description of the network of regulator-gene interactions that determine global gene expression programs remains elusive and will require systematic genetic as well as biochemical assays. Here, we describe a functional genomics approach that combines reporter technology, genome-wide array-based reagents and high-throughput imaging to discover new regulators controlling gene expression patterns in Saccharomyces cerevisiae. Our strategy utilizes the synthetic genetic array (SGA) method to systematically introduce promoter-GFP (green fluorescent protein) reporter constructs along with a control promoter-RFP (red fluorescent protein) gene into the array of approximately 4500 viable yeast deletion mutants. Fluorescence intensities from each reporter are assayed from individual colonies arrayed on solid agar plates using a scanning fluorimager and the ratio of GFP to RFP intensity reveals deletion mutants that cause differential GFP expression. We are exploiting this screening approach to construct a detailed map describing the interplay of regulators controlling the eukaryotic cell cycle. The method is extensible to any transcription factor or signalling pathway for which an appropriate reporter gene can be devised. PMID:19269327

  5. The naphthalene catabolic (nag) genes of Polaromonas naphthalenivorans CJ2: Evolutionary implications for two gene clusters and novel regulatory control

    SciTech Connect

    Jeon, C.O.; Park, M.; Ro, H.S.; Park, W.; Madsen, E.L. [Cornell University, Ithaca, NY (United States). Dept. of Microbiology

    2006-02-15

    Polaromonas naphthalenivorans CJ2, found to be responsible for the degradation of naphthalene in situ at a coal tar waste-contaminated site, is able to grow on mineral salts agar media with naphthalene as the sole carbon source. Beginning from a 484-bp nagAc-like region, we used a genome walking strategy to sequence genes encoding the entire naphthalene degradation pathway and additional flanking regions. We found that the naphthalene catabolic genes in P. naphthalenivorans CJ2 were divided into one large and one small gene cluster, separated by an unknown distance. The large gene cluster is bounded by a LysR-type regulator (nagR). The small cluster is bounded by a MarR-type regulator (nagR2). The catabolic genes of P. naphthalenivorans CJ2 were homologous to many of those of Ralstonia U2, which uses the gentisate pathway to convert naphthalene to central metabolites. However, three open reading frames (nagY, nagM, and nagN), present in Ralstonia U2, were absent. Also, P. naphthalenivorans carries two copies of gentisate dioxygenase (nagI) with 77.4% DNA sequence identity to one another and 82% amino acid identity to their homologue in Ralstonia sp. strain U2. Investigation of the operons using reverse transcription PCR showed that each cluster was controlled independently by its respective promoter. Insertional inactivation and lacZ reporter assays showed that nagR2 is a negative regulator and that expression of the small cluster is not induced by naphthalene, salicylate, or gentisate. Association of two putative Azoarcus-related transposases with the large cluster and one Azoarcus-related putative salicylate 5-hydroxylase gene (ORF2) in the small cluster suggests that mobile genetic elements were likely involved in creating the novel arrangement of catabolic and regulatory genes in P. naphthalenivorans.

  6. Optimized reporter gene assays based on a synthetic multifunctional promoter and a secreted luciferase

    Microsoft Academic Search

    Knut Kotarsky; Liselotte Antonsson; Christer Owman; Björn Olde

    2003-01-01

    Efficient screening for ligands of seven-transmembrane, G-protein-coupled receptors, whether transfected or endogenously expressed, often involves cell-based reporter assays. Here we describe the development of reporter gene assays in HeLa cells. The reporter construct includes a synthetic multifunctional promoter with several different response motifs (NF-?B, STAT, and AP-1) and hence efficiently funnels several signaling pathways. The assay, performed with the resulting

  7. Isolation and characterization of Vsx1, a novel mouse CVC paired-like homeobox gene expressed during embryogenesis and in the retina.

    PubMed

    Ohtoshi, A; Justice, M J; Behringer, R R

    2001-08-10

    Gastrula stage mouse embryo RNA was screened by degenerate RT-PCR to yield a novel paired-like homeobox gene. The open reading frame encoded by the cDNA was most similar to human VSX1. Mouse Vsx1 encodes a protein of 363 amino acid residues that contains a CVC domain that was originally identified as a conserved motif among mouse CHX10, goldfish VSX-1 and C. elegans CEH-10. Linkage analysis showed that mouse Vsx1 mapped to the distal region of chromosome 2. RT-PCR analysis detected mouse Vsx1 transcripts from gastrulation and post-gastrulation stage mouse embryos, suggesting a role for Vsx1 during mouse embryogenesis. Analysis of the eyes of mouse chimeras generated with embryonic stem cells in which a lacZ reporter was targeted to the Vsx1 locus suggested that Vsx1 is expressed in the inner nuclear layer of the retina. PMID:11485319

  8. Characterization of the mammalian DNA polymerase gene(s) and enzyme(s). Annual progress report

    SciTech Connect

    Mishra, N.C.

    1994-01-01

    Consistent with the long term goal of our research to understand the nature of the key enzymes in eukaryotic DNA replication we have characterized the properties of the wild type DNA polymerases of the {alpha}-family and their mutants. We have also provided evidence for the role of aphidicolin in the elongation process of the in vivo DNA replication in eukaryotic cells. We also developed a technology for planned prep from a large numbers of clones for direct screening by size or restriction digestion in order to facilitate our goals to clone the DNA polymerase gene.

  9. Wnt11 gene therapy with adeno-associated virus 9 improves the survival of mice with myocarditis induced by coxsackievirus B3 through the suppression of the inflammatory reaction.

    PubMed

    Aoyama, Yutaka; Kobayashi, Koichi; Morishita, Yoshihiro; Maeda, Kengo; Murohara, Toyoaki

    2015-07-01

    The wnt signaling pathway plays important roles in development and in many diseases. Recently several reports suggest that non-canonical Wnt proteins contribute to the inflammatory response in adult animals. However, the effects of Wnt proteins on virus-induced myocarditis have not been explored. Here, we investigated the effect of Wnt11 protein in a model of myocarditis induced by coxsackievirus B3 (CVB3) using recombinant adeno-associated virus 9 (rAAV9). The effect of Wnt11 gene therapy on a CVB3-induced myocarditis model was examined using male BALB/c mice. Mice received a single intravenous injection of either rAAV9-Wnt11 or rAAV9-LacZ 2weeks before intraperitoneal administration of CVB3. Intravenous injection of the rAAV9 vector resulted in efficient, durable, and relatively cardiac-specific transgene expression. Survival was significantly greater among rAAV9-Wnt11 treated mice than among mice treated with rAAV9-LacZ (87.5% vs. 54.1%, P<0.05). Wnt11 expression also reduced the infiltration of inflammatory cells, necrosis of the myocardium, and suppressed the mRNA expression of inflammatory cytokines. This is the first report to show that Wnt11 expression improves the survival of mice with CVB3-induced myocarditis. AAV9-mediated Wnt11 gene therapy produces beneficial effects on cardiac function and increases the survival of mice with CVB3-induced myocarditis through the suppression of both infiltration of inflammatory cells and gene expression of inflammatory cytokines. PMID:25886696

  10. An Approach for Treating the Hepatobiliary Disease of Cystic Fibrosis by Somatic Gene Transfer

    NASA Astrophysics Data System (ADS)

    Yang, Yiping; Raper, Steven E.; Cohn, Jonathan A.; Engelhardt, John F.; Wilson, James M.

    1993-05-01

    Cystic fibrosis (CF) is an inherited disease of epithelial cell ion transport that is associated with pathology in multiple organ systems, including lung, pancreas, and liver. As treatment of the pulmonary manifestations of CF has improved, management of CF liver disease has become increasingly important in adult patients. This report describes an approach for treating CF liver disease by somatic gene transfer. In situ hybridization and immunocytochemistry analysis of rat liver sections indicated that the endogenous CFTR (cystic fibrosis transmembrane conductance regulator) gene is primarily expressed in the intrahepatic biliary epithelial cells. To specifically target recombinant genes to the biliary epithelium in vivo, recombinant adenoviruses expressing lacZ or human CFTR were infused retrograde into the biliary tract through the common bile duct. Conditions were established for achieving recombinant gene expression in virtually all cells of the intrahepatic bile ducts in vivo. Expression persisted in the smaller bile ducts for the duration of the experiment, which was 21 days. These studies suggest that it may be feasible to prevent CF liver disease by genetically reconstituting CFTR expression in the biliary tract, using an approach that is clinically feasible.

  11. An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast.

    PubMed Central

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

    1998-01-01

    We have developed an activator/repressor expression system for budding yeast in which tetracyclines control in opposite ways the ability of tetR-based activator and repressor molecules to bind tetO promoters. This combination allows tight expression of tetO- driven genes, both in a direct (tetracycline-repressible) and reverse (tetracycline-inducible) dual system. Ssn6 and Tup1, that are components of a general repressor complex in yeast, have been tested for their repressing properties in the dual system, using lacZ and CLN2 as reporter genes. Ssn6 gives better results and allows complete switching-off of the regulated genes, although increasing the levels of the Tup1-based repressor by expressing it from a stronger promoter improves repressing efficiency of the latter. Effector-mediated shifts between expression and non-expression conditions are rapid. The dual system here described may be useful for the functional analysis of essential genes whose conditional expression can be tightly controlled by tetracyclines. PMID:9461451

  12. Mutational Analysis of pre-mRNA Splicing in Saccharomyces cerarisiae Using a Sensitive New Reporter Gene, CUPl

    Microsoft Academic Search

    Cammie F. Lesser; Christine Guthrie

    1993-01-01

    We have developed a new reporter gene fusion to monitor mRNA splicing in yeast. An intron- containing fragment from the Saccharomyces cerevisiae ACTl gene has been fused to CUPl, the yeast metallothionein homolog. CUPl is a nonessential gene that allows cells to grow in the presence of copper in a dosage-dependent manner. By inserting previously characterized intron mutations into the

  13. The mkaC virulence gene of the Salmonella serovar Typhimurium 96 kb plasmid encodes a transcriptional activator

    Microsoft Academic Search

    Suvi Taira; Petri Riikonen; Hannu Saarilahti; Soila Sukupolvi; Mikael Rhen

    1991-01-01

    Summary. The intracellular growth and virulence of Salmonella serovar Typhimurium for mice is dependent on a plasmid-borne gene cluster termed mka. We studied the regulatory interactions of the genes mkaA, mkaB, mkaC and mkaD using lacZ gene fusions. Complementation experiments with cloned DNA fragments encoding each of the four Mka proteins indicated that mkaC enhances the expression of \\/3-galactosidase from

  14. Imaging Adenoviral-Directed Reporter Gene Expression in Living Animals with Positron Emission Tomography

    Microsoft Academic Search

    Sanjiv S. Gambhir; Jorge R. Barrio; Michael E. Phelps; Meera Iyer; Mohammad Namavari; Nagichettiar Satyamurthy; Lily Wu; Leeta A. Green; Eileen Bauer; Duncan C. MacLaren; Khoi Nguyen; Arnold J. Berk; Simon R. Cherry; Harvey R. Herschman

    1999-01-01

    We are developing quantitative assays to repeatedly and noninvasively image expression of reporter genes in living animals, using positron emission tomography (PET). We synthesized positron-emitting 8-[18F]fluoroganciclovir (FGCV) and demonstrated that this compound is a substrate for the herpes simplex virus 1 thymidine kinase enzyme (HSV1-TK). Using positron-emitting FGCV as a PET reporter probe, we imaged adenovirus-directed hepatic expression of the

  15. Germline retinoblastoma without inherited gene mutation: a case report.

    PubMed

    Quintero-Estades, José A; Izquierdo, Natalio J

    2014-01-01

    Retinoblastoma is the most common primary ocular malignancy in childhood and can occur as a germline or somatic mutation. Recent studies have suggested a higher incidence of retinoblastoma in Hispanic children as compared to non-Hispanic white children of the same ages. We report the ocular findings of a 20 years old Hispanic male with a history of bilateral retinoblastoma. Although screening is currently performned with the red reflex test, analysis of current literature suggests the need to reassess screening recommendations for retinoblastoma. PMID:25508534

  16. Germline retinoblastoma without inherited gene mutation: a case report.

    PubMed

    Quintero-Estades, José A; Izquierdo, Natalio J

    2014-01-01

    Retinoblastoma is the most common primary ocular malignancy in childhood and can occur as a germline or somatic mutation. Recent studies have suggested a higher incidence of retinoblastoma in Hispanic children as compared to non-Hispanic white children of the same ages. We report the ocular findings of a 20 years old Hispanic male with a history of bilateral retinoblastoma. Although screening is currently performed with the red reflex test, analysis of current literature suggests the need to reassess screening recommendations for retinoblastoma. PMID:25470907

  17. Circadian Rhythms in Prokaryotes: Luciferase as a Reporter of Circadian Gene Expression in Cyanobacteria

    Microsoft Academic Search

    Takao Kondo; Carl A. Strayer; Resham D. Kulkarni; Walter Taylor; Masahiro Ishiura; Susan S. Golden; Carl Hirschie Johnson

    1993-01-01

    We have used a luciferase reporter gene and continuous automated monitoring of bioluminescence to demonstrate unequivocally that cyanobacteria exhibit circadian behaviors that are fundamentally the same as circadian rhythms of eukaryotes. We also show that these rhythms can be studied by molecular methods in Synechococcus sp. PCC7942, a strain for which genetic transformation is well established. A promoterless segment of

  18. WUS and STM-based reporter genes for studying meristem development in poplar

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We describe the development of a reporter system for monitoring meristem initiation in poplar using promoters of poplar homologs to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). When ~3 kb of the 5’ flanking regions of close homologs were used to drive expression o...

  19. Brief Genetics Report Variation in the Calpain-10 Gene Affects Blood Glucose

    E-print Network

    Cox, Nancy J.

    Brief Genetics Report Variation in the Calpain-10 Gene Affects Blood Glucose Levels in the British resistance, and individuals with the G/G-genotype had significantly higher fasting plasma glucose and 2-h insulin concentrations after a 75-g oral glucose tolerance test (OGTT). We have ex- amined the effect

  20. Supercharged green fluorescent proteins as bimodal reporter genes for CEST MRI and optical imaging†

    PubMed Central

    Bar-Shir, Amnon; Liang, Yajie; Chan, Kannie W. Y.; Gilad, Assaf A.; Bulte, Jeff W. M.

    2015-01-01

    Superpositively charged mutants of green fluorescent protein (GFP) demonstrated a dramatically improved chemical exchange saturation transfer (CEST) MRI contrast compared to their wild type counterparts. The mutants +36 GFP and +48 GFP were successfully expressed in mammalian cells and retained part of their fluorescence, making them a new potential bimodal reporter gene. PMID:25697683

  1. Novel Replicon-Based Reporter Gene Assay for Detection of Rubella Virus in Clinical Specimens

    PubMed Central

    Tzeng, Wen-Pin; Zhou, Yumei; Icenogle, Joseph; Frey, Teryl K.

    2005-01-01

    Proof of concept for a novel diagnostic assay for rubella virus (RUB) based on RUB replicons expressing reporter genes was demonstrated. RUB replicons have the structural protein coding region replaced with a reporter gene such as green fluorescent protein or chloramphenicol acetyltransferase. Previously, it was shown that a replicon construct with a specific in-frame deletion in the nonstructural protein coding region (NotI, approximately nucleotides 1500 to 2100 of the genome) failed to replicate and express the reporter gene unless rescued by a coinfecting wild-type helper RUB (W.-P. Tzeng et al., Virology 289:63-73, 2001). In the present study, it was found that rescue of reporter gene expression by NotI replicons occurred when coinfection was done with clinical specimens containing RUB, indicating that this system could be the basis for a diagnostic assay. The assay was sensitive, using laboratory RUB strains and as low a dose as one plaque-forming unit. The assay was specific in that it was positive for RUB strains of both genotypes and was negative for a panel of human viruses. It was also possible to genetically sequence the RUB present in positive clinical specimens detected in the assay for genotypic strain determination. PMID:15695695

  2. Luciferase Reporter Gene Assay on Human 5-HT Receptor: Which Response Element Should Be Chosen?

    PubMed Central

    Chen, Yiming; Xu, Zhongyu; Wu, Dang; Li, Jian; Song, Cheng; Lu, Weiqiang; Huang, Jin

    2015-01-01

    Serotonin (5-HT) receptors are valuable molecular targets for antipsychotic drug discovery. Current reported methods for detecting 5-HT receptors, such as cAMP accumulation and calcium influx assay, are often demanding specialized instruments and inconvenient. The luciferase reporter gene assay, based on the responsible-element-regulated expression of luciferase, has been widely applied in the high-throughput functional assay for many targets because of its high sensitivity and reliability. However, 5-HT receptors couple to multiple G-proteins regulate respective downstream signalling pathways and are usually detected using different response elements. Hence, finding a suitable response element to fulfil the detection of different 5-HT receptors and make the results of luciferase reporter gene assays generalizable is very useful for active compounds screening. Here, we conducted three luciferase reporter assays using CRE, NFAT, and SRE response elements attached to 5-HT to detect the activation of different 5-HT receptors in CHO-K1 cells. The potencies and efficacies of the reported ligands (agonists and antagonists) were determined and compared. Our results indicate that CRE-luciferase reporter gene is sensitive and reliable to detect the activities of G protein-coupled 5-HT receptors. PMID:25622827

  3. The cytochrome c gene proximal enhancer drives activity-dependent reporter gene expression in hippocampal neurons

    PubMed Central

    Delgado, Jary Y.; Owens, Geoffrey C.

    2012-01-01

    The proximal enhancer of the cytochrome c gene (Cycs) contains binding sites for both cAMP response element binding proteins (CREB) and Nuclear Respiratory Factor 1 (NRF1). To investigate how neuronal activity regulates this enhancer region, a lentivirus was constructed in which a short-lived green fluorescent protein (GFP) was placed under the transcriptional control of the Cycs proximal enhancer linked to a synthetic core promoter. Primary hippocampal neurons were infected, and the synaptic strengths of individual neurons were measured by whole-cell patch clamping. On average the amplitude of miniature postsynaptic currents (mEPSCs) was higher in brighter GFP+ neurons, while the frequency of mEPSCs was not significantly different. Increasing neural activity by applying a GABAA receptor antagonist increased GFP expression in most neurons, which persisted after homeostatic synaptic scaling as evidenced by a decrease in the amplitude and frequency of mEPSCs. Removing the CREB binding sites revealed that calcium influx through L-type channels and NMDA receptors, and ERK1/2 activation played a role in NRF1-mediated transcription. CREB and NRF1, therefore, combine to regulate transcription of Cycs in response to changing neural activity. PMID:22408605

  4. Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters

    Microsoft Academic Search

    Cindy Peach; Jeff Velten

    1991-01-01

    Forty-five individually transformed clonal tobacco callus lines were simultaneously assayed for both chloramphenicol acetyltransferase (CAT) and ß-glucuronidase (GUS) activity resulting from expression of introduced reporter genes driven by the adjacent and divergent mannopine (mas) promoters. Excluding lines in which one or both of the enzyme activities was essentially zero, the activities of the reporter genes varied by as much as

  5. Construction and use of GFP reporter vectors for analysis of cell-type-specific gene expression in Nostoc punctiforme

    E-print Network

    Summers, Michael L.

    Construction and use of GFP reporter vectors for analysis of cell-type-specific gene expression of these GFP vectors to study cell-type-specific gene expression in differentiating filamentous cyanobacteria Abstract Two transcriptional reporter shuttle vectors were constructed for the filamentous cyanobacterium

  6. A Chloride-Inducible Gene Expression Cassette and Its Use in Induced Lysis of Lactococcus lactis

    Microsoft Academic Search

    Gerard Venema; Jan Willem Sanders; Jan Kok

    1997-01-01

    A chloride-inducible promoter previously isolated from the chromosome of Lactococcus lactis was exploited for the inducible expression of homologous and heterologous genes. An expression cassette consisting of the positive-regulator gene gadR, the chloride-inducible promoter Pgad, and the translation initiation signals of gadC was amplified by PCR. The cassette was cloned upstream of Escherichia coli lacZ, the holin-lysin cassette (lytPR) of

  7. Identification of a novel steroid inducible gene associated with the beta hsd locus of Comamonas testosteroni.

    PubMed

    Pruneda-Paz, José Luis; Linares, Mauricio; Cabrera, Julio E; Genti-Raimondi, Susana

    2004-01-01

    Comamonas testosteroni is a soil bacterium, which can use a variety of steroids as carbon and energy source. Even if it can be estimated that the complete degradation of the steroid nucleus requires more than 20 enzymatic reactions, the complete molecular characterization of the genes encoding these steroid degradative enzymes as well as the genetic organization of them remain to be elucidated. We have previously reported the cloning and nucleotide sequence of two steroid-inducible genes, beta hsd and stdC encoding 3 beta-17 beta-hydroxysteroid dehydrogenase and a hypothetical protein respectively, located in both ends of a 3.2kb HindIII fragment. Herein, we report the cloning and characterization of another steroid-inducible gene, called sip48 (steroid inducible protein), located between these two genes. The analysis of Sip48 amino acid sequence predicts a protein of 438 amino acids with a molecular mass of 48.5 kDa. This protein bears high homology with conserved hypothetical proteins of unknown function described in Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas putida, Burkholderia fungorum, Shewanella oneidensis, Pseudomonas fluorescens and Thauera aromatica. The predicted protein shows a typical structure of a leader peptide at its N-terminus. A 48.5 kDa protein encoded by the recombinant plasmid was detected by SDS-PAGE analysis of in vitro [35S]-methionine labeled polypeptides. Analysis of gene expression indicates that Sip48 is tightly controlled at the transcriptional level by several steroid compounds. In addition, transcriptional analysis of sip48 and beta hsd in a sip48 mutant strain, indicates that both genes are transcribed as a polycistronic mRNA. lacZ transcriptional fusions integrated into the chromosome of C. testosteroni demonstrate that a steroid-inducible promoter located upstream of sip48 regulates the expression of both genes. PMID:15026087

  8. Hyperinsulinemic hypoglycemia syndrome associated with mutations in the human insulin receptor gene: Report of two cases.

    PubMed

    Kuroda, Yohei; Iwahashi, Hiromi; Mineo, Ikuo; Fukui, Kenji; Fukuhara, Atsunori; Iwamoto, Ryuya; Imagawa, Akihisa; Shimomura, Iichiro

    2015-04-30

    Insulinoma and insulin or insulin receptor (IR) autoantibodies are the main causes of hyperinsulinemic hypoglycemia in adults, but the exact cause in other cases remains obscure. This study is to determine the genetic basis of hyperinsulinemic hypoglycemia in two cases without the above abnormalities. Sequence analysis of IR gene in two patients with adult-onset hyperinsulinemic hypoglycemia and their relatives were performed, and the mutant gene observed in one case was analyzed. Both cases had normal levels of fasting plasma glucose (FPG), fasting hyperinsulinemia, low insulin sensitivity, and hypoglycemia with excessive insulin secretion during oral glucose tolerance test (OGTT). Both reported adult-onset postprandial hypoglycemic symptoms. In one patient, a missense mutation (Arg256Cys) was detected in both alleles of the IR gene, and his parents had the same mutation in only one allele but no hypoglycemia. The other had a novel nonsense mutation (Trp1273X) followed by a mutation (Gln1274Lys) in one allele, and his 9-year old son had the same mutation in one allele, together with hyperinsulinemic hypoglycemia during OGTT. Overexpression experiments of the mutant gene found in Case 1 in mammalian cells showed abnormal processing of the IR protein and demonstrated reduced function of Akt/Erk phosphorylation by insulin in the cells. In two cases of hyperinsulinemic hypoglycemia in adults, we found novel mutations in IR gene considered to be linked to hypoglycemia. We propose a disease entity of adult-onset hyperinsulinemic hypoglycemia syndrome associated with mutations in IR gene. PMID:25753915

  9. MRI-based detection of alkaline phosphatase gene reporter activity using a porphyrin solubility switch

    PubMed Central

    Westmeyer, Gil G.; Emer, Elena G.; Lintelmann, Jutta; Jasanoff, Alan

    2014-01-01

    SUMMARY The ability to map patterns of gene expression noninvasively in living animals could have impact in many areas of biology. Reporter systems compatible with magnetic resonance imaging (MRI) could be particularly valuable, but existing strategies tend to lack sensitivity or specificity. Here we address the challenge of MRI-based gene mapping using the reporter enzyme secreted alkaline phosphatase (SEAP), in conjunction with a water soluble metalloporphyrin contrast agent. SEAP cleaves the porphyrin into an insoluble product that accumulates at sites of enzyme expression and can be visualized by MRI and optical absorbance. The contrast mechanism functions in vitro, in brain slices, and in animals. The system also provides the possibility of readout both in the living animal and by post mortem histology, and it notably does not require intracellular delivery of the contrast agent. The solubility switch mechanism used to detect SEAP could be adapted for imaging of additional reporter enzymes or endogenous targets. PMID:24613020

  10. Gene transcription and electromagnetic fields. Final progress report

    SciTech Connect

    Henderson, A.S.

    1992-12-31

    Our overall aim is to obtain sufficient information to allow us to ultimately determine whether ELF EM field exposure is an initiating factor in neoplastic transformation and/or if exposure can mimic characteristics of the second-step counterpart in neoplastic disease. This aim is based on our previous findings that levels of some transcripts are increased in cells exposed to EM fields. While the research is basic in nature, the ramifications have bearing on the general safety of exposure to EM fields in industrial and everyday life. A large array of diverse biological effects are reported to occur as the result of exposure to elf EM fields, suggesting that the cell response to EM fields is at a basic level, presumably initiated by molecular and/or biophysical events at the cell membrane. The hypothesized route is a signal transduction pathway involving membrane calcium fluxes. Information flow resulting from signal transduction can mediate the induction of regulatory factors in the cell, and directly affect how transcription is regulated.

  11. Multi-wavelength photoacoustic imaging of inducible tyrosinase reporter gene expression in xenograft tumors

    PubMed Central

    Paproski, Robert J.; Heinmiller, Andrew; Wachowicz, Keith; Zemp, Roger J.

    2014-01-01

    Photoacoustic imaging is an emerging hybrid imaging technology capable of breaking through resolution limits of pure optical imaging technologies imposed by optical-scattering to provide fine-resolution optical contrast information in deep tissues. We demonstrate the ability of multi-wavelength photoacoustic imaging to estimate relative gene expression distributions using an inducible expression system and co-register images with hemoglobin oxygen saturation estimates and micro-ultrasound data. Tyrosinase, the rate-limiting enzyme in melanin production, is used as a reporter gene owing to its strong optical absorption and enzymatic amplification mechanism. Tetracycline-inducible melanin expression is turned on via doxycycline treatment in vivo. Serial multi-wavelength imaging reveals very low estimated melanin expression in tumors prior to doxycycline treatment or in tumors with no tyrosinase gene present, but strong signals after melanin induction in tumors tagged with the tyrosinase reporter. The combination of new inducible reporters and high-resolution photoacoustic and micro-ultrasound technology is poised to bring a new dimension to the study of gene expression in vivo. PMID:24936769

  12. Quantification of transcriptional activities of reporter gene constructs in primary cultures of sympathetic neurons.

    PubMed

    Pajak, Fabrice; De Gois, Stéphanie; Houhou, Leïla; Védrine, Christophe; Mallet, Jacques; Berrard, Sylvie

    2003-02-01

    Primary cultures of sympathetic neurons provide an attractive cellular model for investigating the mechanisms of neurotransmitter phenotypic plasticity. However, it has not been possible to transfect these neurons by conventional techniques, and this has been a major impediment to molecular investigations of neuronal gene expression in this system. Here, reporter plasmids were transferred into the nuclei of cultured sympathetic neurons by microinjection. We developed and improved this procedure and were able to measure the transcriptional activities of two coinjected promoters in small groups of neurons, and even from a single neuron. Promoter activities can thus be quantified and normalized relative to that of a constitutively expressed promoter, allowing correction for variability in the injection and assay procedures. High and low promoter activities can be reliably quantified. Importantly, this method can be used not only for reporter plasmids but also for DNA fragments containing only a promoter and reporter gene without any vector sequence that might interfere with promoter. Using this approach, we measured neuronal promoter activities and found that one promoter region of the gene encoding choline acetyltransferase was up-regulated by more than sevenfold by leukemia inhibitory factor. This method thus provides the means to investigate the function of neuronal genes and the mechanisms that regulate their transcription in cultured sympathetic neurons. PMID:12526025

  13. Recurrent anencephaly: a case report and examination of the VANGL1 and FOXN1 genes.

    PubMed

    Sergi, Consolato; Gekas, Jean; Kamnasaran, Deepak

    2013-07-01

    We report a new and rare case of recurrent anencephaly in a family with no other apparent abnormalities. The karyotypes of the family and all affected subjects were normal. Thorough mutational analyses of VANGL1 of chromosome 1p13.1 and FOXN1 of chromosome 17q11-q12, genes that are associated with phenotypes of the anencephaly spectrum, unfortunately did not disclose any DNA variations in an affected fetus of this family. The etiology of recurrent anencephaly in this family is therefore due to mutations in genes yet to be discovered, perhaps of the planar cell polarity pathway, or to possible environmental gestational factors during development. PMID:23301910

  14. [Reconstruction and preparation of lentiviral vector system expressing dual-reporter genes].

    PubMed

    Li, Chen; Zhang, Bin; Wang, Jun; Kong, Wei-Xia; Wang, Rui-Ping; Liu, Ting; Chen, Hu

    2011-12-01

    This study was aimed to construct, package and purify the recombinant lentivirus vector carrying the firefly luciferase gene (FLUC) and red fluorescent protein gene (RFP) and to transfect the recombinant lentivirus into HeLa cells, so as to observe the expression levels of these two genes. The FLUC and RFP genes were amplified by RT-PCR and inserted in the lentiviral expression vector (pLenti-Bi-cistronic) to construct the lentiviral vector pLenti-FLUC-RFP. The viral particles were generated by cotransfection of 293T cells with pLenti-FLUC-RFP and three packaging vectors, and the virus titer was determined by calculating the percentage of RFP positive cells. After transfection of pLenti-FLUC-RFP into HeLa cells, the expression of RFP was observed by fluorescent microscopy, and the activity of FLUC was determined by luciferase reporter gene assay kit. The results showed that the inserting orientation of the RFP and FLUC genes in the lentiviral vector pLenti-FLUC-RFP were verified by restriction analysis. Targeted RFP and FLUC sequences were confirmed by DNA sequencing. The final titer obtained was 1×10(7)TU/ml. The expressions of RFP and FLUC were observed in the transfected HeLa cells. It is concluded that the pLenti-III-FLUC-RFP recombinant lentivirus vector carrying RFP gene and FLUC gene with high viral titer is constructed and packaged successfully, and provides experimental basis for studying dynamic distribution of mesenchymal stem cells in vivo. PMID:22169309

  15. [pBR322-Red mediated gene knockin, sites and expression in E. coli chromosome].

    PubMed

    Chen, Wei; Li, Shan-Hu; Yu, Mei; Wang, Ming-Gang; Zhou, Jian-Guang

    2006-01-01

    Genes lacZ, lacY and lacA in the lac opron of E. coli chromosome were respectively substituted with gene luc by using plasmid pBR322-Red, selection-counterselection system kan/sacB and various strategies of Red homologous recombination including Red mediated linearized double-stranded DNA homologous recombination and Red mediated recombineering with overlapping single stranded DNA oligonucleotides. Then, a series of new strains, CWL2, CWL4 and CWL6, were constructed and we found that they can express protein Luc efficiently. To further study the expression of exogenous genes at the site of lacZ, we have constructed a strain named CWD1 by knockin the cholera toxin B subunit(ctxb) gene at the lacZ site, then we found that CWD1 can express protein CTB efficiently and CTB was secreted out of the cell. So we assured that the sites of structure genes in the lac operon of Escherichia coli chromosome were suitable for expressing foreign genes. PMID:16469720

  16. Toxin gene expression by shiga toxin-producing Escherichia coli: the role of antibiotics and the bacterial SOS response.

    PubMed Central

    Kimmitt, P. T.; Harwood, C. R.; Barer, M. R.

    2000-01-01

    Toxin synthesis by Shiga toxin-producing Escherichia coli (STEC) appears to be coregulated through induction of the integrated bacteriophage that encodes the toxin gene. Phage production is linked to induction of the bacterial SOS response, a ubiquitous response to DNA damage. SOS-inducing antimicrobial agents, particularly the quinolones, trimethoprim, and furazolidone, were shown to induce toxin gene expression in studies of their effects on a reporter STEC strain carrying a chromosome-based stx2::lacZ transcriptional fusion. At antimicrobial levels above those required to inhibit bacterial replication, these agents are potent inducers (up to 140-fold) of the transcription of type 2 Shiga toxin genes (stx2); therefore, they should be avoided in treating patients with potential or confirmed STEC infections. Other agents (20 studied) and incubation conditions produced significant but less striking effects on stx2 transcription; positive and negative influences were observed. SOS-mediated induction of toxin synthesis also provides a mechanism that could exacerbate STEC infections and increase dissemination of stx genes. These features and the use of SOS-inducing antibiotics in clinical practice and animal husbandry may account for the recent emergence of STEC disease. PMID:10998375

  17. Targeted gene expression without a tissue-specific promoter: creating mosaic embryos using laser-induced single-cell heat shock

    NASA Technical Reports Server (NTRS)

    Halfon, M. S.; Kose, H.; Chiba, A.; Keshishian, H.

    1997-01-01

    We have developed a method to target gene expression in the Drosophila embryo to a specific cell without having a promoter that directs expression in that particular cell. Using a digitally enhanced imaging system to identify single cells within the living embryo, we apply a heat shock to each cell individually by using a laser microbeam. A 1- to 2-min laser treatment is sufficient to induce a heat-shock response but is not lethal to the heat-shocked cells. Induction of heat shock was measured in a variety of cell types, including neurons and somatic muscles, by the expression of beta-galactosidase from an hsp26-lacZ reporter construct or by expression of a UAS target gene after induction of hsGAL4. We discuss the applicability of this technique to ectopic gene expression studies, lineage tracing, gene inactivation studies, and studies of cells in vitro. Laser heat shock is a versatile technique that can be adapted for use in a variety of research organisms and is useful for any studies in which it is desirable to express a given gene in only a distinct cell or clone of cells, either transiently or constitutively, at a time point of choice.

  18. Post-transcriptional regulation of sex determination in Caenorhabditis elegans: widespread expression of the sex-determining gene fem-1 in both sexes.

    PubMed Central

    Gaudet, J; VanderElst, I; Spence, A M

    1996-01-01

    The fem-1 gene of C. elegans is one of three genes required for all aspects of male development in the nematode. Current models of sex determination propose that the products of the fem genes act in a novel signal-transduction pathway and that their activity is regulated primarily at the post-translational level in somatic tissues. We analyzed the expression of fem-1 to determine whether it revealed any additional levels of regulation. Both XX hermaphrodites and XO males express fem-1 at approximately constant levels throughout development. Somatic tissues in hermaphrodites adopt female fates, but they nonetheless express fem-1 mRNA and FEM-1 protein, suggesting that the regulation of fem-1 activity is post-transcriptional and probably post-translational. A compact promoter directs functional expression of fem-1 transgenes, as assayed by their masculinizing activity in fem-1 mutants. Activity also requires any two or more introns, suggesting that splicing may enhance fem-1 expression. The minimal noncoding sequences required for activity of fem-1 transgenes suffice to direct expression of a fem-1::lacZ reporter gene in all somatic tissues in both sexes. Many fem-1 transgenes, including those that rescue male somatic development in fem-1 mutants, paradoxically feminize the germline. We suggest that they do so by interfering with the germline expression of the endogenous fem-1 gene. Images PMID:8862524

  19. Aberrant regulation of imprinted gene expression in Gtl2 mice

    Microsoft Academic Search

    Y. Sekita; H. Wagatsuma; M. Irie; S. Kobayashi; T. Kohda; J. Matsuda; M. Yokoyama; A. Ogura; K. Schuster-Gossler; A. Gossler; F. Ishino; T. Kaneko-Ishino

    2006-01-01

    The imprinted region on mouse distal chromosome 12 covers about 1 Mb and contains at least three paternally expressed genes (Pegs: Peg9\\/Dlk1, Peg11\\/Rtl1, and Dio3) and four maternally expressed genes (Megs: Meg3\\/Gtl2, antiPeg11\\/antiRlt1, Meg8\\/Rian, and Meg9\\/Mirg). Gtl2lacZ (Gene trap locus 2) mice have a transgene (TG) insertion 2.3 kb upstream from the Meg3\\/Gtl2 promoter and show about 40% growth retardation

  20. Novel Mutations in the CLCN1 Gene of Myotonia Congenita: 2 Case Reports

    PubMed Central

    Lakraj, Amanda Amrita; Miller, Geoffrey; Vortmeyer, Alexander O.; Khokhar, Babar; Nowak, Richard J.; DiCapua, Daniel B.

    2013-01-01

    Introduction: Myotonia Congenita is an inherited myotonia that is due to a mutation in the skeletal muscle chloride channel CLCN1. These mutations lead to reduced sarcolemmal chloride conductance, causing delayed muscle relaxation that is evident as clinical and electrical myotonia. Methods: We report the clinical presentations of two individuals with Myotonia Congenita (MC). Results: Patient 1 has been diagnosed with the recessive form of MC, known as the Becker variant, and Patient 2 has been diagnosed with the dominant form of MC, known as the Thomsen variant. In both patients, the diagnosis was made based on the clinical presentation, EMG and CLCN1 gene sequencing. Patient 1 also had a muscle biopsy. Conclusions: Genetic testing in both patients reveals previously unidentified mutations in the CLCN1 gene specific to Myotonia Congenita. We report the salient clinical features of each patient and discuss the effects and common types of CLCN1 mutations and review the literature. PMID:23483815

  1. The first report of the vanC 1 gene in Enterococcus faecium isolated from a human clinical specimen

    PubMed Central

    Sun, Mingyue; Wang, Yue; Chen, Zhongju; Zhu, Xuhui; Tian, Lei; Sun, Ziyong

    2014-01-01

    The vanC1 gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC1 gene. Polymerase chain reaction (PCR) assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC1 and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC1 gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC1 gene. However, this study is the first to report the presence of the vanC1 gene in E. faecium of human origin. Additionally, our research showed the vanC1 gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC1 gene from different species. PMID:25317698

  2. The first report of the vanC1 gene in Enterococcus faecium isolated from a human clinical specimen.

    PubMed

    Sun, Mingyue; Wang, Yue; Chen, Zhongju; Zhu, Xuhui; Tian, Lei; Sun, Ziyong

    2014-08-13

    The vanC1 gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC1gene. Polymerase chain reaction (PCR) assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC1and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC1 gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC1gene. However, this study is the first to report the presence of the vanC1gene in E. faecium of human origin. Additionally, our research showed the vanC1gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC1gene from different species. PMID:25119395

  3. The first report of the vanC? gene in Enterococcus faecium isolated from a human clinical specimen.

    PubMed

    Sun, Mingyue; Wang, Yue; Chen, Zhongju; Zhu, Xuhui; Tian, Lei; Sun, Ziyong

    2014-09-01

    The vanC? gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC?gene. Polymerase chain reaction (PCR) assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC? and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC? gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC?gene. However, this study is the first to report the presence of the vanC?gene in E. faecium of human origin. Additionally, our research showed the vanC?gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC?gene from different species. PMID:25317698

  4. Firefly luciferase as a reporter of regulated gene expression in higher plants

    Microsoft Academic Search

    Andrew J. Millar; Sharla R. Short; Kazuyuki Hiratsuka; Nam-Hai Chua; Steve A. Kay

    1992-01-01

    The firefly luciferase, assayedin vivo with a low-light video camera, acts as a non-invasive, real-time reporter of the temporal and spatial regulation of gene\\u000a expression in single plants. Furthermore, the sensitivity of the luciferase assay in extracts of transformed plant tissue\\u000a makes it a particularly useful marker in transient or stable transformation experiments.

  5. The First Gene of the Bacillus subtilis clpC Operon, ctsR, Encodes a Negative Regulator of Its Own Operon and Other Class III Heat Shock Genes

    PubMed Central

    Krüger, Elke; Hecker, Michael

    1998-01-01

    The Bacillus subtilis clpC operon is regulated by two stress induction pathways relying on either ?B or a class III stress induction mechanism acting at a ?A-like promoter. When the clpC operon was placed under the control of the isopropyl-?-d-thiogalactopyranoside (IPTG)-inducible Pspac promoter, dramatic repression of the natural clpC promoters fused to a lacZ reporter gene was noticed after IPTG induction. This result strongly indicated negative regulation of the clpC operon by one of its gene products. Indeed, the negative regulator could be identified which is encoded by the first gene of the clpC operon, ctsR, containing a predicted helix-turn-helix DNA-binding motif. Deletion of ctsR abolished the negative regulation and resulted in high expression of both the clpC operon and the clpP gene under nonstressed conditions. Nevertheless, a further increase in clpC and clpP mRNA levels was observed after heat shock, even in the absence of ?B, suggesting a second induction mechanism at the vegetative promoter. Two-dimensional gel analysis and mRNA studies showed that the expression of other class III stress genes was at least partially influenced by the ctsR deletion. Studies with different clpC promoter fragments either fused to the reporter gene bgaB or used in gel mobility shift experiments with the purified CtsR protein revealed a possible target region where the repressor seemed to bind in vivo and in vitro. Our data demonstrate that the CtsR protein acts as a global repressor of the clpC operon, as well as other class III heat shock genes, by preventing unstressed transcription from either the ?B- or ?A-dependent promoter and might be inactivated or dissociate under inducing stress conditions. PMID:9852015

  6. The Development and Application of a Multiple Gene Co-Silencing System Using Endogenous URA3 as a Reporter Gene in Ganoderma lucidum

    PubMed Central

    Mu, Dashuai; Shi, Liang; Ren, Ang; Li, Mengjiao; Wu, Fengli; Jiang, Ailiang; Zhao, Mingwen

    2012-01-01

    Ganoderma lucidum is one of the most important medicinal mushrooms; however, molecular genetics research on this species has been limited due to a lack of reliable reverse genetic tools. In this study, the endogenous orotidine 5?-monophosphate decarboxylase gene (URA3) was cloned as a silencing reporter, and four gene-silencing methods using hairpin, sense, antisense, and dual promoter constructs, were introduced into G. lucidum through a simple electroporation procedure. A comparison and evaluation of silencing efficiency demonstrated that all of the four methods differentially suppressed the expression of URA3. Our data unequivocally indicate that the dual promoter silencing vector yields the highest rate of URA3 silencing compared with other vectors (up to 81.9%). To highlight the advantages of the dual promoter system, we constructed a co-silencing system based on the dual promoter method and succeeded in co-silencing URA3 and laccase in G. lucidum. The reduction of the mRNA levels of the two genes were correlated. Thus, the screening efficiency for RNAi knockdown of multiple genes may be improved by the co-silencing of an endogenous reporter gene. The molecular tools developed in this study should facilitate the isolation of genes and the characterization of the functions of multiple genes in this pharmaceutically important species, and these tools should be highly useful for the study of other basidiomycetes. PMID:22937087

  7. Temporal and spatial regulation of gene expression mediated by the promoter for the human tissue inhibitor of metalloproteinases-3 (TIMP-3)-encoding gene.

    PubMed

    Zeng, Y; Rosborough, R C; Li, Y; Gupta, A R; Bennett, J

    1998-03-01

    A complex interplay between enzymes involved in extracellular matrix formation and their inhibitors is thought to control organogenesis during mammalian development. Disturbance of this balance may result in a wide range of diseases, including macular degeneration, arthritis, and tumor metastases. In order to define elements which may be involved in regulating human tissue inhibitor of metalloproteinase 3 (TIMP3) expression, we isolated and sequenced a clone containing 1315 bp of the 5'-upstream region of the human TIMP-3-encoding gene. A 1.2 kb fragment of this clone, which contains multiple motifs which are binding sites for known transcription factors, was used to drive expression of the lacZ reporter gene in multiple lines of transgenic mice. TIMP3 promoter activity, detected through beta-galactosidase histochemical assay, was observed at high levels in selected tissues, the identity of which varied according to developmental stage. TIMP3 promoter activity was detected at embryonic and early postnatal stages in tissues undergoing extensive remodeling, such as developing somites, bones and joints, choroid plexus, webs between the digits, and the spongiotrophoblastic portion of the placenta. In adulthood, TIMP3 promoter activity was restricted to a few tissues which exhibit high metabolic activity or rapid turnover. These include the retinal pigment epithelium (RPE), cells of the kidney cortex, hair follicles, gingiva, ovarian follicles, and testis. The results suggest that TIMP3 expression plays an active role in developmental patterning and in the maintenance of specific differentiated tissues. PMID:9520110

  8. A convenient method for multiple insertions of desired genes into target loci on the Escherichia coli chromosome.

    PubMed

    Koma, Daisuke; Yamanaka, Hayato; Moriyoshi, Kunihiko; Ohmoto, Takashi; Sakai, Kiyofumi

    2012-01-01

    We developed a method to insert multiple desired genes into target loci on the Escherichia coli chromosome. The method was based on Red-mediated recombination, flippase and the flippase recognition target recombination, and P1 transduction. Using this method, six copies of the lacZ gene could be simultaneously inserted into different loci on the E. coli chromosome. The inserted lacZ genes were functionally expressed, and ?-galactosidase activity increased in proportion to the number of inserted lacZ genes. This method was also used for metabolic engineering to generate overproducers of aromatic compounds. Important genes of the shikimate pathway (aroF (fbr) and tyrA (fbr) or aroF (fbr) and pheA (fbr)) were introduced into the chromosome to generate a tyrosine or a phenylalanine overproducer. Moreover, a heterologous decarboxylase gene was introduced into the chromosome of the tyrosine or phenylalanine overproducer to generate a tyramine or a phenethylamine overproducer, respectively. The resultant strains selectively overproduced the target aromatic compounds. Thus, the developed method is a convenient tool for the metabolic engineering of E. coli for the production of valuable compounds. PMID:22127754

  9. Evaluating the MicroRNA Targeting Sites by Luciferase Reporter Gene Assay

    PubMed Central

    Jin, Yi; Chen, Zujian; Liu, Xiqiang; Zhou, Xiaofeng

    2013-01-01

    MicroRNAs are post-transcriptional regulators that control mRNA stability and the translation efficiency of their target genes. Mature microRNAs are approximately 22-nucleotide in length. They mediate post-transcriptional gene regulation by binding to the imperfect complementary sequences (a.k.a. microRNA regulatory elements, MRE) in the target mRNAs. It is estimated that more than one-third of the protein-coding genes in the human genome are regulated by microRNAs. The experimental methods to examine the interaction between the microRNA and its targeting site(s) in the mRNA are important for understanding microRNA functions. The luciferase reporter gene assay has recently been adapted to test the effect of microRNAs. In this chapter, we use a previously identified miR-138 targeting site in the 3?-untranslated region (3?-UTR) of the RhoC mRNA as an example to describe a quick method for testing the interaction of microRNA and mRNA. PMID:23007504

  10. The Anthrax Toxin Activator Gene atxA Is Associated with CO2Enhanced Non-Toxin Gene Expression in Bacillus anthracis

    Microsoft Academic Search

    ALEX R. HOFFMASTER; THERESA M. KOEHLER

    1997-01-01

    tions from atxA1 and atxA isolates grown in 5% CO2 and in air were examined by two-dimensional electro- phoresis. Comparison of the resulting protein patterns indicated that synthesis of non-toxin proteins is influenced by growth in elevated CO2 and the toxin gene regulator, atxA. Second, we generated random transcriptional lacZ fusions in B. anthracis with transposon Tn917-LTV3. Transposon-insertion libraries were

  11. A Percutaneous Optical Imaging System to Track Reporter Gene Expression from Vasculatures In Vivo

    PubMed Central

    Kar, S.; Kumar, A.; Gao, F.; Qiu, B.; Zhan, X.; Yang, X.

    2006-01-01

    This study was to develop a percutaneous optical imaging system for tracking fluorescent reporter gene expression in vasculatures. We built a percutaneous optical imaging system that primarily comprised a 1.5-mm, semi-rigid, two-port optical probe. The performance of the optical probe was first tested in vitro with cell phantoms, and then the feasibility of the percutaneous optical imaging system was validated in vivo in eight femoral artery segments of two pigs. Green fluorescent protein (GFP) gene was locally delivered into four arterial segments, while saline was delivered to the four contralateral arterial segments as controls. The targeted arteries were localized using color Doppler, and thereafter the optical probe was positioned to the target arterial segments under ultrasound guidance. Optical imaging captures were obtained using different exposure times from 10–60 seconds. Subsequently, the GFP- and saline-targeted arteries were harvested for fluorescent microscopy confirmation. The percutaneous optical probe was successfully positioned at a distance of approximately 2 mm from the targets in all eight arteries. The in vivo imaging showed higher average signal intensity in GFP-treated arteries than in saline-treated arteries. This study demonstrates the potential using the percutaneous optical imaging system to monitor, in vivo, reporter gene expression from vasculatures. PMID:16822058

  12. A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

    PubMed Central

    Marc, J; Granger, CL; Brincat, J; Fisher, DD; Kao, Th; McCubbin, AG; Cyr, RJ

    1998-01-01

    Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation. PMID:9811799

  13. Visualisation of chicken macrophages using transgenic reporter genes: insights into the development of the avian macrophage lineage.

    PubMed

    Balic, Adam; Garcia-Morales, Carla; Vervelde, Lonneke; Gilhooley, Hazel; Sherman, Adrian; Garceau, Valerie; Gutowska, Maria W; Burt, David W; Kaiser, Pete; Hume, David A; Sang, Helen M

    2014-08-01

    We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens. PMID:25063453

  14. Detection of transformed cells in crown gall tumors using the GUS reporter gene and correlation of GUS stained cells with T-DNA gene activity

    SciTech Connect

    Black, R.C. (Pennsylvania State Univ., Media (USA)); Labriola, J.; Binns, A.N. (Univ. of Pennsylvania, Philadelphia (USA))

    1990-05-01

    Crown gall tumors are a mixture of transformed hormone producing cells and normal cells. Until now it has not been possible to directly visualize these cell types in situ. We have constructed strains of Agrobacterium tumefaciens that carry the 35S-{beta}-glucuronidase (GUS) reporter gene in either wild type or mutant Ti plasmids. Using histochemical staining for GUS activity, blue (GUS positive) sectors are observed in tumor sections. In order to demonstrate that the blue sectors actually represent cells expressing other T-DNA genes, we have looked for T-DNA gene encoded enzyme activity in the stained and unstained sectors. The blue sectors accumulate octopine (a product of the octopine synthase gene on the T-DNA) while the white (GUS negative) sectors do not. We conclude that the use of the GUS reporter gene provides a sensitive and reliable method for visualizing transformation events in plant tissues. A comparison of the proportion of transformed and nontransformed cells in wild type tumors vs. tumors deficient in auxin or cytokinin encoding genes will be discussed.

  15. A single-vector EYFP reporter gene assay for G protein-coupled receptors.

    PubMed

    Hald, Helle; Wu, Boqian; Bouakaz, Lamine; Meldal, Morten

    2015-05-01

    We here present an improved and simplified assay to study signal transduction of the Gs class of G protein-coupled receptors (GPCRs). The assay is based on a single plasmid combining the genes for any Gs protein-coupled GPCR and the cAMP response element-related expression of enhanced yellow fluorescent protein. On transfection, stable human embryonic kidney 293 (HEK293) cell lines presented high assay sensitivity and an unprecedented signal-to-noise ratio of up to 300, even in the absence of trichostatin A. The robustness of the assay was demonstrated through the cloning of reporter gene cell lines with melanocortin 4 receptor (MC4R), the human type I pituitary adenylate cyclase-activating polypeptide receptor (hPAC1), and the two vasoactive intestinal peptide receptors (VPAC1 and VPAC2). PMID:25681566

  16. In vivo Tracking of Dendritic Cell using MRI Reporter Gene, Ferritin

    PubMed Central

    Kim, Hoe Suk; Woo, Jisu; Lee, Jae Hoon; Joo, Hyun Jung; Choi, YoonSeok; Kim, Hyeonjin; Moon, Woo Kyung; Kim, Seung Ja

    2015-01-01

    The noninvasive imaging of dendritic cells (DCs) migrated into lymph nodes (LNs) can provide helpful information on designing DCs-based immunotherapeutic strategies. This study is to investigate the influence of transduction of human ferritin heavy chain (FTH) and green fluorescence protein (GFP) genes on inherent properties of DCs, and the feasibility of FTH as a magnetic resonance imaging (MRI) reporter gene to track DCs migration into LNs. FTH-DCs were established by the introduction of FTH and GFP genes into the DC cell line (DC2.4) using lentivirus. The changes in the rate of MRI signal decay (R2*) resulting from FTH transduction were analyzed in cell phantoms as well as popliteal LN of mice after subcutaneous injection of those cells into hind limb foot pad by using a multiple gradient echo sequence on a 9.4 T MR scanner. The transduction of FTH and GFP did not influence the proliferation and migration abilities of DCs. The expression of co-stimulatory molecules (CD40, CD80 and CD86) in FTH-DCs was similar to that of DCs. FTH-DCs exhibited increased iron storage capacity, and displayed a significantly higher transverse relaxation rate (R2*) as compared to DCs in phantom. LNs with FTH-DCs exhibited negative contrast, leading to a high R2* in both in vivo and ex vivo T2*-weighted images compared to DCs. On histological analysis FTH-DCs migrated to the subcapsular sinus and the T cell zone of LN, where they highly expressed CD25 to bind and stimulate T cells. Our study addresses the feasibility of FTH as an MRI reporter gene to track DCs migration into LNs without alteration of their inherent properties. This study suggests that FTH-based MRI could be a useful technique to longitudinally monitor DCs and evaluate the therapeutic efficacy of DC-based vaccines. PMID:25993535

  17. A Mutagenesis Assay for Reporter Gene Screening Using Partially Degenerate Oligonucleotides of the Tandems NNT and NNC

    PubMed Central

    Xu, Huifen; Zhou, Cuilan; Zhang, Andy K.; Li, Wen; Zhang, Jia; Li, Kai

    2015-01-01

    Not all proteins are tolerable to mutations. Whether a specific protein can be a mutable target is of importance in the biotechnology and pharmaceutical industry. This study reported a novel mutagenesis assay using tandem NNT and NNC oligonucleotides to test the mutability of a candidate gene. These two tandem oligonucleotides avoid the risk of forming nonsense mutations and render flexibility of truncating or expanding the insertion size. As a reporter gene, ZeoR (zeocin resistance gene) was confirmed to have a high tolerance for mutagenesis by this new assay. PMID:26167508

  18. Naringenin Regulates Expression of Genes Involved in Cell Wall Synthesis in Herbaspirillum seropedicae?

    PubMed Central

    Tadra-Sfeir, M. Z.; Souza, E. M.; Faoro, H.; Müller-Santos, M.; Baura, V. A.; Tuleski, T. R.; Rigo, L. U.; Yates, M. G.; Wassem, R.; Pedrosa, F. O.; Monteiro, R. A.

    2011-01-01

    Five thousand mutants of Herbaspirillum seropedicae SmR1 carrying random insertions of transposon pTnMod-OGmKmlacZ were screened for differential expression of LacZ in the presence of naringenin. Among the 16 mutants whose expression was regulated by naringenin were genes predicted to be involved in the synthesis of exopolysaccharides, lipopolysaccharides, and auxin. These loci are probably involved in establishing interactions with host plants. PMID:21257805

  19. Isolation and characterization of the gene encoding xylose reductase from Kluyveromyces lactis

    Microsoft Academic Search

    Patrick Billard; Sandrine Ménart; Reinhard Fleer; Monique Bolotin-Fukuhara

    1995-01-01

    The identification of a xylose reductase (XR)-encoding gene (XYL1) from the xylose-assimilating yeast Kluyveromyces lactis (Kl) is described. XYL1 was isolated as a highly expressed fusion clone from a 'lacZ translational fusion library. DNA sequence analysis revealed an open reading frame (ORF) of 987 bp capable of encoding a polypeptide of 329 amino acids (aa). The deduced aa sequence displays

  20. A method to rapidly and accurately compare relative efficacies of non-invasive imaging reporter genes in a mouse model, and its application to luciferase reporters

    PubMed Central

    Gil, Jose S.; Machado, Hidevaldo B.; Herschman, Harvey R.

    2013-01-01

    Purpose Our goal is to develop a simple, quantitative, robust method to compare the efficacy of imaging reporter genes in culture and in vivo. We describe an adenoviral vector-liver transduction procedure, and compare the luciferase reporter efficacies. Procedures Alternative reporter genes are expressed in a common adenoviral vector. Vector amounts used in vivo are based on cell culture titrations, ensuring the same transduction efficacy is used for each vector. After imaging, in vivo and in vitro values are normalized to hepatic vector transduction using quantitative real-time PCR. Results We assayed standard firefly luciferase (FLuc), enhanced firefly luciferase (EFLuc), luciferase 2 (Luc2), humanized Renilla luciferase (hRLuc), Renilla luciferase 8.6-535 (RLuc8.6), and a membrane-bound Gaussia luciferase variant (extGLuc) in cell culture and in vivo. We observed a greater that 100-fold increase in bioluminescent signal for both EFLuc and Luc2 when compared to FLuc, and a greater than 106-fold increase for RLuc8.6 when compared to hRLuc. ExtGLuc was not detectable in liver. Conclusions Our findings contrast, in some cases, with conclusions drawn in prior comparisons of these reporter genes, and demonstrate the need for a standardized method to evaluate alternative reporter genes in vivo. Our procedure can be adapted for reporter genes that utilize alternative imaging modalities (fluorescence, bioluminescence, MRI, SPECT, PET). PMID:21850545

  1. amyP, a reporter gene to study strain degeneration in Clostridium acetobutylicum ATCC 824.

    PubMed

    Sabathé, Fabrice; Croux, Christian; Cornillot, Emmanuel; Soucaille, Philippe

    2002-04-23

    Clostridium acetobutylicum produces an extracellular alpha-amylase when grown on glucose as the sole carbon source. This enzyme was previously characterized from a biochemical point of view but its encoding gene was never identified. The 2283-bp amyP gene encodes a 83013-Da mature protein with an N-terminal domain that exhibits strong identity to the family 13 glycosyl hydrolases such as the Bacillus alpha-amylases. Transcriptional analysis revealed that amyP is transcribed in solventogenic but not in acidogenic chemostat cultures. These results are in agreement with the extracellular alpha-amylase activities indicating that the expression of amyP is regulated at the transcriptional level. amyP is located on the pSOL1 megaplasmid that carries all the genes involved in the final steps of solvent formation. Degeneration of C. acetobutylicum has been associated to the loss of pSOL1. We demonstrate here that amyP can be used as a reporter system to quantitatively follow this phenomenon. PMID:12023083

  2. Identification of a novel missense GLRA1 gene mutation in hyperekplexia: a case report

    PubMed Central

    2014-01-01

    Introduction Hereditary hyperekplexia is a neurological disorder characterized by excessive startle responses with violent jerking to noise or touch, stiffening of the trunk and limbs, clenching of the fists and attacks of a high-frequency trembling. Hyperekplexia has a heterogeneous genetic background with several identified causative genes and demonstrates both dominant and recessive inheritance. Mutations in the glycine receptor alpha 1 subunit gene occur in about 30 percent of hyperekplexia cases. Case presentation In this study, we report the case of a Hungarian boy whose abnormal movements, muscle stiffness and convulsions were first noted when he was 4 days old. Neurological and electrophysiological investigation suggested the clinical diagnosis of hyperekplexia. Conclusions Direct sequencing of the coding regions and the flanking introns of the glycine receptor alpha 1 subunit gene revealed a novel heterozygous missense mutation (c.211A/T, p.Ile71Phe). Genetic screening of our patient’s family revealed that the clinically unaffected parents and sister do not carry the mutation, suggesting that the identified sequence change is a de novo mutation. Since hyperekplexia can have severe consequences, including sudden infant death due to laryngospasm and cardiorespiratory failure, identification of the causative genetic alteration(s) of the disease is high priority. Such knowledge is necessary for prenatal diagnosis, which would allow informed family planning and greater parental sensitivity to hyperekplexia 1-associated risks. PMID:24969041

  3. Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of the xylose-utilization operon

    Microsoft Academic Search

    Thomas Rygus; Wolfgang Hillen

    1991-01-01

    We have constructed a shuttle plasmid for Bacillus megaterium and Escherichia coli that contains the promoter and repressor gene of the B. megaterium-borne operon for xylose utilization. A polylinker downstream of the promoter allows versatile cloning of genes under its transcriptional control. We have placed gdhA (encoding glucose dehydrogenase) from B. megaterium, lacZ (encoding ß-galactosidase) from E. coli, mro (encoding

  4. MEGDEL Syndrome in a Child From Palestine: Report of a Novel Mutation in SERAC1 Gene.

    PubMed

    Dweikat, Imad M; Abdelrazeq, Samer; Ayesh, Suhail; Jundi, Tawfeeq

    2015-07-01

    We report the first Palestinian child manifesting with 3-methylglutaconic aciduria psychomotor delay, muscle hypotonia, sensori-neural deafness, and Leigh-like lesions on brain magnetic resonance imaging (MRI), a clinical phenotype that is characteristic of MEGDEL syndrome. MEGDEL syndrome was recently found to be caused by mutations in SERAC1, encoding a protein essential for mitochondrial function, phospholipid remodeling, and intracellular cholesterol trafficking. We identified a novel homozygous mutation in SERAC1 gene (c.1018delT) that generates frame shift and premature termination of protein translation. Plasma and cerebrospinal fluid lactate, plasma alanine, and respiratory chain complexes in fresh muscle were normal. This report further expands the genetic spectrum of MEGDEL syndrome and adds to the evidence that it is associated with variable patterns of respiratory chain abnormalities. PMID:25051967

  5. Detection of coliform bacteria in water by polymerase chain reaction and gene probes.

    PubMed Central

    Bej, A K; Steffan, R J; DiCesare, J; Haff, L; Atlas, R M

    1990-01-01

    Polymerase chain reaction (PCR) amplification and gene probe detection of regions of two genes, lacZ and lamB, were tested for their abilities to detect coliform bacteria. Amplification of a segment of the coding region of Escherichia coli lacZ by using a PCR primer annealing temperature of 50 degrees C detected E. coli and other coliform bacteria (including Shigella spp.) but not Salmonella spp. and noncoliform bacteria. Amplification of a region of E. coli lamB by using a primer annealing temperature of 50 degrees C selectively detected E. coli and Salmonella and Shigella spp. PCR amplification and radiolabeled gene probes detected as little as 1 to 10 fg of genomic E. coli DNA and as a few as 1 to 5 viable E. coli cells in 100 ml of water. PCR amplification of lacZ and lamB provides a basis for a method to detect indicators of fecal contamination of water, and amplification of lamB in particular permits detection of E. coli and enteric pathogens (Salmonella and Shigella spp.) with the necessary specificity and sensitivity for monitoring the bacteriological quality of water so as to ensure the safety of water supplies. Images PMID:2306085

  6. Blastic plasmacytoid dendritic cell neoplasm with leukemic manifestation and ETV6 gene rearrangement: A case report

    PubMed Central

    GAO, NA; WANG, XUE-XIA; SUN, JIAN-RONG; YU, WEN-ZHENG; GUO, NONG-JIAN

    2015-01-01

    Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare malignant tumor of the hemopoietic system that arises from plasmacytoid dendritic cell precursors with a highly aggressive course. BPDCN frequently involves the skin, lymph nodes, peripheral blood and bone marrow. BPDCN is known to develop leukemic dissemination as a feature of myelomonocytic leukemia in the late phase of the disease, which leads to a poorer prognosis. In the present study, a case of BPDCN with leukemic manifestation without cutaneous involvement was reported. In addition, ETS variant gene 6 (ETV6) gene rearrangement was detected in the patient. The patient relapsed soon after complete remisson and had no response to further treatment. To the best of our knowledge, this is the first reported case of BPDCN with ETV6 rearrangement. Following chemotherapy treatment, the patient suffered from severe headache in the complete remission stage; however, brain CT scans showed no significant abnormalities. Several lumbar punctures and intrathecal chemotherapy were performed, and the patient recovered gradually. Therefore, the patient was considered to suffer from central nervous system leukemia. In conclusion, implementation of lumbar punctures and preventive intrathecal chemotherapy are required in BPDCN patients with leukemic manifestation during the remission stage. PMID:25780395

  7. A novel MitoTimer reporter gene for mitochondrial content, structure, stress, and damage in vivo.

    PubMed

    Laker, Rhianna C; Xu, Peng; Ryall, Karen A; Sujkowski, Alyson; Kenwood, Brandon M; Chain, Kristopher H; Zhang, Mei; Royal, Mary A; Hoehn, Kyle L; Driscoll, Monica; Adler, Paul N; Wessells, Robert J; Saucerman, Jeffrey J; Yan, Zhen

    2014-04-25

    Mitochondrial dysfunction plays important roles in many diseases, but there is no satisfactory method to assess mitochondrial health in vivo. Here, we engineered a MitoTimer reporter gene from the existing Timer reporter gene. MitoTimer encodes a mitochondria-targeted green fluorescent protein when newly synthesized, which shifts irreversibly to red fluorescence when oxidized. Confocal microscopy confirmed targeting of the MitoTimer protein to mitochondria in cultured cells, Caenorhabditis elegans touch receptor neurons, Drosophila melanogaster heart and indirect flight muscle, and mouse skeletal muscle. A ratiometric algorithm revealed that conditions that cause mitochondrial stress led to a significant shift toward red fluorescence as well as accumulation of pure red fluorescent puncta of damaged mitochondria targeted for mitophagy. Long term voluntary exercise resulted in a significant fluorescence shift toward green, in mice and D. melanogaster, as well as significantly improved structure and increased content in mouse FDB muscle. In contrast, high-fat feeding in mice resulted in a significant shift toward red fluorescence and accumulation of pure red puncta in skeletal muscle, which were completely ameliorated by voluntary wheel running. Hence, MitoTimer allows for robust analysis of multiple parameters of mitochondrial health under both physiological and pathological conditions and will be highly useful for future research of mitochondrial health in multiple disciplines in vivo. PMID:24644293

  8. A Multiplexed Fluorescent Calcium and NFAT Reporter Gene Assay to Identify GPCR Agonists

    PubMed Central

    Sheth, Heeral; Gorey, Colleen; Roush, Nicole; Smallman, Shelly; Collantes, Elizabeth; Santoro, Maxine; Olson, Barbara; Fitzgerald, Laura; Lee, Paul H; Shen, Xiqiang John

    2013-01-01

    Intracellular calcium response and resulting calcium signaling to an agonist-GPCR interaction are important for the measurement of compound activity in the GPCR drug development. The increase in cytosol calcium concentration can be measured by the fluorescent calcium indicator dye such as Fluo-4 in a quick assay (in 3-5 minutes) using the fluorescence imaging plate reader. The calcium signaling through the transcription factors such as NFAT that induces gene expression can be measured by the reporter gene assay that links to the expression of reporter enzyme such as the beta-lactamase that requires 5-hour incubation. We have evaluated a multiplexed assay that sequentially measures the calcium response to a GPCR agonist in a rapid fluorescent calcium dye assay, followed by a NFAT beta-lactamase assay, and compared them in the single assay format. We found that the agonist activity determined in the multiplexed assay were comparable with these determined in the single assay format and the Z’ factors were all >0.5. Five active compounds were identified that were active in both calcium dye assay and beta-lactamase assay. Therefore, our results demonstrated the utility of this multiplexed calcium assay for screening of GPCR compounds that can cross validate the primary hits and help to eliminate the false positive compounds. PMID:24396729

  9. A Multiplexed Fluorescent Calcium and NFAT Reporter Gene Assay to Identify GPCR Agonists.

    PubMed

    Sheth, Heeral; Gorey, Colleen; Roush, Nicole; Smallman, Shelly; Collantes, Elizabeth; Santoro, Maxine; Olson, Barbara; Fitzgerald, Laura; Lee, Paul H; Shen, Xiqiang John

    2013-01-01

    Intracellular calcium response and resulting calcium signaling to an agonist-GPCR interaction are important for the measurement of compound activity in the GPCR drug development. The increase in cytosol calcium concentration can be measured by the fluorescent calcium indicator dye such as Fluo-4 in a quick assay (in 3-5 minutes) using the fluorescence imaging plate reader. The calcium signaling through the transcription factors such as NFAT that induces gene expression can be measured by the reporter gene assay that links to the expression of reporter enzyme such as the beta-lactamase that requires 5-hour incubation. We have evaluated a multiplexed assay that sequentially measures the calcium response to a GPCR agonist in a rapid fluorescent calcium dye assay, followed by a NFAT beta-lactamase assay, and compared them in the single assay format. We found that the agonist activity determined in the multiplexed assay were comparable with these determined in the single assay format and the Z' factors were all >0.5. Five active compounds were identified that were active in both calcium dye assay and beta-lactamase assay. Therefore, our results demonstrated the utility of this multiplexed calcium assay for screening of GPCR compounds that can cross validate the primary hits and help to eliminate the false positive compounds. PMID:24396729

  10. Development of a Sensitive Gene Expression Reporter System and an Inducible Promoter-Repressor System for Clostridium acetobutylicum

    PubMed Central

    Girbal, Laurence; Mortier-Barrière, Isabelle; Raynaud, Frédéric; Rouanet, Céline; Croux, Christian; Soucaille, Philippe

    2003-01-01

    A sensitive gene expression reporter system was developed for Clostridium acetobutylicum ATCC 824 by using a customized gusA expression cassette. In discontinuous cultures, time course profiles of ?-glucuronidase specific activity reflected adequately in vivo dynamic up- and down-regulation of acidogenesis- and/or solventogenesis-associated promoter expression in C. acetobutylicum. Furthermore, a new inducible gene expression system was developed in C. acetobutylicum, based on the Staphylococcus xylosus xylose operon promoter-repressor regulatory system. PMID:12902297

  11. Development of a sensitive gene expression reporter system and an inducible promoter-repressor system for Clostridium acetobutylicum.

    PubMed

    Girbal, Laurence; Mortier-Barrière, Isabelle; Raynaud, Frédéric; Rouanet, Céline; Croux, Christian; Soucaille, Philippe

    2003-08-01

    A sensitive gene expression reporter system was developed for Clostridium acetobutylicum ATCC 824 by using a customized gusA expression cassette. In discontinuous cultures, time course profiles of beta-glucuronidase specific activity reflected adequately in vivo dynamic up- and down-regulation of acidogenesis- and/or solventogenesis-associated promoter expression in C. acetobutylicum. Furthermore, a new inducible gene expression system was developed in C. acetobutylicum, based on the Staphylococcus xylosus xylose operon promoter-repressor regulatory system. PMID:12902297

  12. Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay

    PubMed Central

    Parekh, Bhavin S.; Berger, Elaine; Sibley, Sharon; Cahya, Suntara; Xiao, Liqun; LaCerte, Melinda Ann; Vaillancourt, Peter; Wooden, Scott; Gately, Dennis

    2012-01-01

    Humanized monoclonal antibodies (mAbs) are the fastest growing class of biological therapeutics that are being developed for various medical indications, and more than 30 mAbs are already approved and in the market place. Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important biological function attributed to the mechanism of action of several therapeutic antibodies, particularly oncology targeting mAbs. The ADCC assay is a complicated and highly variable assay. Thus, the use of an ADCC assay as a lot release test or a stability test for clinical trial batches of mAbs has been a substantial challenge to install in quality control laboratories. We describe here the development and validation of an alternate approach, an ADCC-reporter gene assay that is based on the key attributes of the PBMC-based ADCC assay. We tested the biological relevance of this assay using an anti-CD20 based model and demonstrated that this ADCC-reporter assay correlated well with standard ADCC assays when induced with the drugable human isotypes [IgG1, IgG2, IgG4, IgG4S > P (S228P) and IgG4PAA (S228P, F234A, L235A)] and with IgG1 isotype variants with varying amounts of fucosylation. This data demonstrates that the ADCC-reporter gene assay has performance characteristics (accuracy, precision and robustness) to be used not only as a potency assay for lot release and stability testing for antibody therapeutics, but also as a key assay for the characterization and process development of therapeutic molecules. PMID:22531445

  13. The expression of nifA in Azorhizobium caulinodans requires a gene product homologous to Escherichia coli HF-I, an RNA-binding protein involved in the replication of phage Q beta RNA.

    PubMed Central

    Kaminski, P A; Desnoues, N; Elmerich, C

    1994-01-01

    We report the characterization of a mutant of Azorhizobium caulinodans, isolated after ethyl methanesulfonate mutagenesis. This Nod+ Nif- Fix- mutant is unable to synthesize 10 of 15 polypeptides normally induced under conditions of nitrogen fixation. By using lacZ fusions it was shown that nifA and nifA-regulated genes were not expressed in this strain. The mutation was complemented by a constitutively expressed nifA gene or by a 1.1-kb DNA fragment from the wild-type strain, whose nucleotide sequence revealed a single open reading frame of 255 bp coding for an 85-amino acid polypeptide. The deduced amino acid sequence is similar to that of HF-I, an RNA-binding protein of Escherichia coli, which is required for replication of bacteriophage Q beta RNA. The similarity can be extended to the function since hfq, the structural gene for HF-I, complemented the A. caulinodans mutant. The corresponding gene in A. caulinodans was termed nrfA (for nif regulatory factor). Inactivation of nrfA in the mutant was due to a missense mutation resulting in the replacement of a cysteine residue by arginine. A null mutant, constructed by disruption of nrfA, exhibited the same phenotype as the missense mutant. Thus, an additional factor can be added to the already complex system of nifA regulation in A. caulinodans. PMID:8197116

  14. LrhA as a new transcriptional key regulator of flagella, motility and chemotaxis genes in Escherichia coli.

    PubMed

    Lehnen, D; Blumer, C; Polen, T; Wackwitz, B; Wendisch, V F; Unden, G

    2002-07-01

    The function of the LysR-type regulator LrhA of Escherichia coli was defined by comparing whole-genome mRNA profiles from wild-type E. coli and an isogenic lrhA mutant on a DNA microarray. In the lrhA mutant, a large number (48) of genes involved in flagellation, motility and chemotaxis showed relative mRNA abundances increased by factors between 3 and 80. When a representative set of five flagellar, motility and chemotaxis genes was tested in lacZ reporter gene fusions, similar factors for derepression were found in the lrhA mutant. In gel retardation experiments, the LrhA protein bound specifically to flhD and lrhA promoter DNA (apparent K(D) approximately 20 nM), whereas the promoters of fliC, fliA and trg were not bound by LrhA. The expression of flhDC (encoding FlhD(2)C(2)) was derepressed by a factor of 3.5 in the lrhA mutant. FlhD(2)C(2) is known as the master regulator for the expression of flagellar and chemotaxis genes. By DNase I footprinting, LrhA binding sites at the flhDC and lrhA promoters were identified. The lrhA gene was under positive autoregulation by LrhA as shown by gel retardation and lrhA expression studies. It is suggested that LrhA is a key regulator controlling the transcription of flagellar, motility and chemotaxis genes by regulating the synthesis and concentration of FlhD(2)C(2). PMID:12123461

  15. Determination of effective rAAV-mediated gene transfer conditions to support chondrogenic differentiation processes in human primary bone marrow aspirates.

    PubMed

    Rey-Rico, A; Frisch, J; Venkatesan, J K; Schmitt, G; Madry, H; Cucchiarini, M

    2015-01-01

    The genetic modification of freshly aspirated bone marrow may provide convenient tools to enhance the regenerative capacities of cartilage defects compared with the complex manipulation of isolated progenitor cells. In the present study, we examined the ability and safety of recombinant adeno-associated virus (rAAV) serotype 2 vectors to deliver various reporter gene sequences in primary human bone marrow aspirates over time without altering the chondrogenic processes in the samples. The results demonstrate that successful rAAV-mediated gene transfer and expression of the lacZ and red fluorescent protein marker genes were achieved in transduced aspirates at very high efficiencies (90-94%) and over extended periods of time (up to 125 days) upon treatment with hirudin, an alternative anticoagulant that does not prevent the adsorption of the rAAV-2 particles at the surface of their targets compared with heparin. Application of rAAV was safe, displaying neither cytotoxic nor detrimental effects on the cellular and proliferative activities or on the chondrogenic processes in the aspirates especially using an optimal dose of 0.5?mg?ml(-1) hirudin, and application of the potent SOX9 transcription factor even enhanced these processes while counteracting hypertrophic differentiation. The current findings demonstrate the clinical value of this class of vector to durably and safely modify bone marrow aspirates as a means to further develop convenient therapeutic approaches to improve the healing of cartilage defects. PMID:25338919

  16. A genetic approach for finding small RNAs regulators of genes of interest identifies RybC as regulating the DpiA / DpiB two component system

    PubMed Central

    Mandin, Pierre; Gottesman, Susan

    2009-01-01

    In Escherichia coli, the largest class of small regulatory RNAs binds to the RNA chaperone Hfq and regulates the stability and/or translation of specific mRNAs. While recent studies have shown that some mRNAs could be subject to posttranscriptional regulation by sRNAs (e.g. mRNAs found by co-immunoprecipitation with Hfq), no method has yet been described to identify small RNAs that regulate them. We developed a method to easily make translational fusions of genes of interest to the lacZ reporter gene, under the control of a PBAD inducible promoter. A multicopy plasmid library of the E. coli genome can then be used to screen for small RNAs that affect the activity of the fusion. This screening method was first applied to the dpiB gene from the dpiBA operon, which encodes a two-component signal transduction system involved in the SOS response to ?-lactams. One small RNA, RybC, was found to negatively regulate the expression of dpiB. Using mutants in the dpiB-lacZ fusion and compensatory mutations in the RybC sRNA, we demonstrate that RybC directly basepairs with the dpiBA mRNA. PMID:19426207

  17. Regulation of expression of sodA and msrA genes of Corynebacterium glutamicum in response to oxidative and radiative stress.

    PubMed

    El Shafey, H M; Ghanem, S

    2015-01-01

    Promoters of genes encoding superoxide dismutase (sodA) and peptide methionine sulfoxide reductase (msrA) from Cory-nebacterium glutamicum were cloned and sequenced. Promoter region analysis of sodA-msrA was unable to identify putative sites of fixed eventual regulators except for possible sites of fixed OxyR and integra-tion host factor. A study of the regulation of these genes was performed using the lacZ gene of Escherichia coli as a reporter placed under the control of sequences downstream of sodA and msrA. In silico analysis was used to identify regulators in the genome of C. glutamicum, which revealed the absence of homologs of soxRS and arcA and the presence of inactive oxyR and putative candidates of the homologs of ahpC, ohrR, integration host factor, furA, IdeR, diphtheria toxin repressor, and mntR. PMID:25867357

  18. Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice

    NASA Astrophysics Data System (ADS)

    Lemaire-Vieille, Catherine; Schulze, Tobias; Podevin-Dimster, Valérie; Follet, Jérome; Bailly, Yannick; Blanquet-Grossard, Françoise; Decavel, Jean-Pierre; Heinen, Ernst; Cesbron, Jean-Yves

    2000-05-01

    The expression of the cellular form of the prion protein (PrPc) gene is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. The identification of the cell types expressing PrPc is necessary to understanding how the agent replicates and spreads from peripheral sites to the central nervous system. To determine the nature of the cell types expressing PrPc, a green fluorescent protein reporter gene was expressed in transgenic mice under the control of 6.9 kb of the bovine PrP gene regulatory sequences. It was shown that the bovine PrP gene is expressed as two populations of mRNA differing by alternative splicing of one 115-bp 5' untranslated exon in 17 different bovine tissues. The analysis of transgenic mice showed reporter gene expression in some cells that have been identified as expressing PrP, such as cerebellar Purkinje cells, lymphocytes, and keratinocytes. In addition, expression of green fluorescent protein was observed in the plexus of the enteric nervous system and in a restricted subset of cells not yet clearly identified as expressing PrP: the epithelial cells of the thymic medullary and the endothelial cells of both the mucosal capillaries of the intestine and the renal capillaries. These data provide valuable information on the distribution of PrPc at the cellular level and argue for roles of the epithelial and endothelial cells in the spread of infection from the periphery to the brain. Moreover, the transgenic mice described in this paper provide a model that will allow for the study of the transcriptional activity of the PrP gene promoter in response to scrapie infection.

  19. Reliable gene expression analysis by reverse transcription-quantitative PCR: reporting and minimizing the uncertainty in data accuracy.

    PubMed

    Remans, Tony; Keunen, Els; Bex, Geert Jan; Smeets, Karen; Vangronsveld, Jaco; Cuypers, Ann

    2014-10-01

    Reverse transcription-quantitative PCR (RT-qPCR) has been widely adopted to measure differences in mRNA levels; however, biological and technical variation strongly affects the accuracy of the reported differences. RT-qPCR specialists have warned that, unless researchers minimize this variability, they may report inaccurate differences and draw incorrect biological conclusions. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines describe procedures for conducting and reporting RT-qPCR experiments. The MIQE guidelines enable others to judge the reliability of reported results; however, a recent literature survey found low adherence to these guidelines. Additionally, even experiments that use appropriate procedures remain subject to individual variation that statistical methods cannot correct. For example, since ideal reference genes do not exist, the widely used method of normalizing RT-qPCR data to reference genes generates background noise that affects the accuracy of measured changes in mRNA levels. However, current RT-qPCR data reporting styles ignore this source of variation. In this commentary, we direct researchers to appropriate procedures, outline a method to present the remaining uncertainty in data accuracy, and propose an intuitive way to select reference genes to minimize uncertainty. Reporting the uncertainty in data accuracy also serves for quality assessment, enabling researchers and peer reviewers to confidently evaluate the reliability of gene expression data. PMID:25361954

  20. In situ Detection of ? -Galactosidase in Lenses of Transgenic Mice with a ? -Crystallin/lacZ Gene

    NASA Astrophysics Data System (ADS)

    Goring, D. R.; Rossant, J.; Clapoff, S.; Breitman, M. L.; Tsui, L.-C.

    1987-01-01

    Transgenic mice carrying the ? 2-crystallin promoter fused to the coding region of the bacterial lacZ gene were generated. The offspring of three founder mice expressed high levels of the enzyme solely in the central nuclear fiber cells of the lens as measured by an in situ assay for the detection of ? -galactosidase activity. These results suggest that ? 2-crystallin sequences between -759 to +45 contain essential information required for appropriate tissue-specific and temporal regulation of the mouse ? 2-crystallin gene. In a broader context, this study also demonstrates the utility of ? -galactosidase hybrid gene constructs for monitoring the activity of gene regulatory elements in transgenic mice.

  1. Analysis of ToxR-dependent transcription activation of ompU, the gene encoding a major envelope protein in Vibrio cholerae.

    PubMed

    Crawford, J A; Kaper, J B; DiRita, V J

    1998-07-01

    The membrane proteins ToxR and ToxS regulate a variety of genes associated with the virulence of Vibrio cholerae, the agent of human cholera. One of the ToxRS-regulated genes is the ompU gene, which encodes a porin that may also act as an adhesin. To begin to understand the mechanism of ompU transcription activation by ToxRS, we performed genetic and biochemical studies on the ompU promoter. Deletions with a 5' end-point at or downstream of -128, relative to the start site for transcription, did not direct expression of a lacZ reporter gene in wild-type V. cholerae, although the -128 promoter fragment did direct ToxRS-dependent reporter gene activity under conditions of ToxR overexpression in E. coli. Consistent with the activation data is that membranes containing ToxR and ToxS caused a gel electrophoretic mobility shift when mixed at low concentrations with deletion fragments whose end-point is at -211, but not with -128 or -68 fragments. ToxRS membranes did shift the -128 fragment when added at higher concentrations. DNase I footprinting analysis of ompU promoter DNA complexed with ToxRS membranes demonstrated protection of three sites: an upstream site ranging from -238 to -139, and two downstream sites ranging from -116 to -58 and -53 to -24. Within the DNA protected from DNase I digestion by ToxRS membranes, there are no elements bearing similarity to those identified previously within the promoters of two other ToxR-dependent genes, ctxA and toxT. We suggest a model for transcription activation that involves sequential ToxR-binding events to distinct regions in the ompU promoter. PMID:9701817

  2. Marker Gene Transfer and Oncolysis of Human Y79 Retinoblastoma Cells Mediated by Herpes simplex Virus Mutants

    Microsoft Academic Search

    Massimo Nicolò; E. Antonio Chiocca

    1998-01-01

    Three different herpes simplex virus (HSV) mutants, designated hrR3, MGH-1 and R3616, were used to infect Y79 retinoblastoma cells grown in suspension. Two parameters were assayed: (a) vector-mediated gene expression, measured by histochemical staining of a transferred LacZ transgene, and (b) virus-mediated oncolysis, determined by the inability of infected cells to exclude trypan blue dye. The tested HSV mutants were

  3. Long-Term Gene Delivery into the Livers of Immunocompetent Mice with E1\\/E4Defective Adenoviruses

    Microsoft Academic Search

    JEAN-FRANCOIS DEDIEU; EMMANUELLE VIGNE; CHRISTOPHE TORRENT; CAROLE JULLIEN; IRENE MAHFOUZ; JEAN-MICHEL CAILLAUD; NATHALIE AUBAILLY; CECILE ORSINI; JEAN-MARC GUILLAUME; PAULE OPOLON; PIA DELAERE; MICHEL PERRICAUDET; PATRICE YEH

    1997-01-01

    We have compared the in vitro and in vivo behaviors of a set of isogenic E1- and E1\\/E4-defective adenovi- ruses expressing the lacZ gene of Escherichia coli from the Rous sarcoma virus long terminal repeat. Infection of tumor-derived established cell lines of human origin with the doubly defective adenoviruses resulted in (i) a lower replication of the viral backbone that

  4. Analysis of expression of the alpha-toxin gene (hla) of Staphylococcus aureus by using a chromosomally encoded hla::lacZ gene fusion.

    PubMed Central

    Ohlsen, K; Koller, K P; Hacker, J

    1997-01-01

    The staphylococcal alpha-toxin (Hla) is a major virulence factor contributing to Staphylococcus aureus pathogenesis. To elucidate the conditions influencing hla expression, the determinant was fused to lacZ, the reporter gene coding for beta-galactosidase. The hla::lacZ fusion was integrated into the chromosome of the wild-type S. aureus strain Wood 46, leading to the variant Wood 46-3. Alpha-toxin expression was found to be dependent on temperature, showing a maximum at 42 degrees C. Furthermore, the indicator strain showed a growth phase-dependent hla regulation which was influenced by temperature. At 37 degrees C, induction of hla::lacZ expression occurred in the late exponential phase of growth, whereas at 42 degrees C, a strong induction was observed as early as the mid-exponential phase. These observations were verified by Northern blot analysis of hla mRNA and by Western blot (immunoblot) analysis of culture supernatants of strain Wood 46. It was additionally found that the induction of hla transcription at 42 degrees C was not coupled with higher concentrations of agr RNAIII, the effector molecule of the global regulator agr. Furthermore, expression of the alpha-toxin was repressed at a high osmolarity. It was also shown that oxygen is essential for hla expression and that cultivation of the S. aureus strain Wood 46-3 on solid medium and in the presence of carbon dioxide stimulated hla transcriptional activity. PMID:9284126

  5. KIT gene mutation analysis in solid tumours: biology, clincial applications and trends in diagnostic reporting.

    PubMed

    Tay, Clifton Ming; Ong, Chee Wee; Lee, Victor Kwan Min; Pang, Brendan

    2013-02-01

    Gain-of-function mutations involving c-kit protein, a cell-surface transmembrane receptor for stem cell factor, have been identified as a key oncogenic driver in a variety of solid tumours. Coupled with the development of tyrosine kinase inhibitors such as imatinib, c-kit has emerged as a viable drug target in what seems to be a validated therapeutic concept. This review will focus on gastrointestinal stromal tumours and melanomas, two types of solid tumours most closely associated with KIT gene mutations. The biology of KIT mutations in both conditions, as well as the value of KIT mutation testing in predicting disease and treatment outcomes are discussed. Since initial response to imatinib is largely influenced by mutation status, genotyping these tumours serves to facilitate personalised oncology. We also summarise our experience with diagnostic reporting of KIT mutation analysis over a period of 3 years, and briefly survey future developments in treatment, which indeed look very promising. PMID:23277171

  6. Reporter gene assay for fish-killing activity produced by Pfiesteria piscicida.

    PubMed Central

    Fairey, E R; Edmunds, J S; Deamer-Melia, N J; Glasgow, H; Johnson, F M; Moeller, P R; Burkholder, J M; Ramsdell, J S

    1999-01-01

    Collaborative studies were performed to develop a functional assay for fish-killing activity produced by Pfiesteria piscicida. Eight cell lines were used to screen organic fractions and residual water fraction by using a 3-[4, 5-dimethylthiazol-(2-4)]-diphenyltetrazolium bromide cytotoxicity assay. Diethyl ether and a residual water fraction were cytotoxic to several cell lines including rat pituitary (GH(4)C(1)) cells. Residual water as well as preextracted culture water containing P. piscicida cells induced c-fos-luciferase expressed in GH(4)C(1) cells with a rapid time course of induction and sensitive detection. The reporter gene assay detected activity in toxic isolates of P. piscicida from several North Carolina estuaries in 1997 and 1998 and may also be suitable for detecting toxic activity in human and animal serum. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:10464070

  7. Comparison of in vitro hormone activities of selected phthalates using reporter gene assays.

    PubMed

    Shen, Ouxi; Du, Guizhen; Sun, Hong; Wu, Wei; Jiang, Yi; Song, Ling; Wang, Xinru

    2009-12-01

    Phthalates are widely used in the plastic industry and food packaging, imparting softness and flexibility to normally rigid plastic medical devices and children's toys. Even though phthalates display low general toxicity, there is increasing concern on the effects of endocrine system induced by some of phthalate compounds. The hormone activity of dibutyl phthalate (DBP), mono-n-butyl phthalate (MBP) and di-2-ethylhexyl phthalate (DEHP) were assessed using the luciferase reporter gene assays. The results showed that DBP, MBP and DEHP, not only exhibited potent antiandrogenic activity, with IC(50) value of 1.05x10(-6), 1.22x10(-7)M and exceeding 1x10(-4)M respectively, but also showed the androgenic activity with EC(50) value of 6.17x10(-6), 1.13x10(-5)M and exceeding 1x10(-4)M. We also found that all the three related chemicals possessed thyroid receptor (TR) antagonist activity with IC(50) of 1.31x10(-5), 2.77x10(-6)M and exceeding 1x10(-4)M respectively, and none showed TR agonist activity. These results indicate that TR might be the targets of industrial chemicals. In the ER mediate reporter gene assay, three chemicals showed no agonistic activity except for DBP, which appeared weakly estrogenic at the concentration of 1.0x10(-4)M. Together, the findings demonstrate that the three phthalates could simultaneously disrupt the function of two or more hormonal receptors. Therefore, these phthalates should be considered in risk assessments for human health. PMID:19643168

  8. Aberrant DNA methylation of cancer-related genes in giant breast fibroadenoma: a case report

    PubMed Central

    2011-01-01

    Introduction Giant fibroadenoma is an uncommon variant of benign breast lesions. Aberrant methylation of CpG islands in promoter regions is known to be involved in the silencing of genes (for example, tumor-suppressor genes) and appears to be an early event in the etiology of breast carcinogenesis. Only hypermethylation of p16INK4a has been reported in non-giant breast fibroadenoma. In this particular case, there are no previously published data on epigenetic alterations in giant fibroadenomas. Our previous results, based on the analysis of 49 cancer-related CpG islands have confirmed that the aberrant methylation is specific to malignant breast tumors and that it is completely absent in normal breast tissue and breast fibroadenomas. Case presentation A 13-year-old Hispanic girl was referred after she had noted a progressive development of a mass in her left breast. On physical examination, a 10 × 10 cm lump was detected and axillary lymph nodes were not enlarged. After surgical removal the lump was diagnosed as a giant fibroadenoma. Because of the high growth rate of this benign tumor, we decided to analyze the methylation status of 49 CpG islands related to cell growth control. We have identified the methylation of five cancer-related CpG islands in the giant fibroadenoma tissue: ESR1, MGMT, WT-1, BRCA2 and CD44. Conclusion In this case report we show for the first time the methylation analysis of a giant fibroadenoma. The detection of methylation of these five cancer-related regions indicates substantial epigenomic differences with non-giant fibroadenomas. Epigenetic alterations could explain the higher growth rate of this tumor. Our data contribute to the growing knowledge of aberrant methylation in breast diseases. In this particular case, there exist no previous data regarding the role of methylation in giant fibroadenomas, considered by definition as a benign breast lesion. PMID:22011321

  9. Bioluminescence Reporter Gene Imaging Characterize Human Embryonic Stem Cell-Derived Teratoma Formation

    PubMed Central

    Su, Weijun; Zhou, Manqian; Zheng, Yizhou; Fan, Yan; Han, Zhongchao; Kong, Deling; Wu, Joseph C.; Xiang, Rong; Li, Zongjin

    2011-01-01

    Human embryonic stem (hES) cells are capable of differentiation into virtually all cell types and hold tremendous potential as cell sources for regenerative therapies. However, teratoma formation can be the main obstacle for hES cells therapy. In order to understand the biology and physiology of hES cells teratoma formation, we investigated the angiogenic process within teratomas and characterized teratoma cells. In this study, hES cells transduced with double fusion reporter gene that consists of firefly luciferase and enhanced green fluorescent protein (Fluc-eGFP) were injected into hind limbs of SCID mice and performed longitudinal bioluminescence imaging on these animals. To test angiogenic contribution of teratoma from host or hES cells, human and mouse endothelial cells marker CD31 was stained respectively. To further explore the characterization of teratoma derived cells, flow cytometry analysis was carried out and GFP+/SSEA-4+ cells were isolated and subcultured. Then, we re-injected the isolated GFP+/SSEA-4+ teratoma cells into SCID mice and observed by imaging. Our results show that the reporter gene imaging is an ideal technology for monitoring long-term stem cell viability, death, and proliferation. Teratomas contained vasculatures are from hES cells and host. hESCs derived teratomas express a high level of undifferentiated marker SSEA-4 and CD56, and subcultured GFP+/SSEA-4+ cells had similar expression pattern comparing to undifferentiated hES cells, except for a very high level of CD56 and a little lower expression of undifferentiated markers, such as SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Moreover, the SSEA-4+ teratoma cells can form teratomas in SCID mice, and this type teratomas grow at a lower rate compared to teratomas derived from hES cells, and are more differentiated. PMID:21328457

  10. Conditional Gene Targeting in Mouse High Endothelial Venules

    PubMed Central

    Kawashima, Hiroto; Hirakawa, Jotaro; Tobisawa, Yuki; Fukuda, Minoru; Saga, Yumiko

    2009-01-01

    High endothelial venules (HEVs) are specialized blood vessels of secondary lymphoid organs composed of endothelial cells with a characteristic cuboidal morphology. Lymphocytes selectively adhere to and migrate across HEVs to initiate immune responses. In this study, we established a novel transgenic mouse line expressing Cre recombinase under the transcriptional control of the gene encoding HEV-expressed sulfotransferase, N-acetylglucosamine-6-O-sulfotransferase 2 (GlcNAc6ST-2), using bacterial artificial chromosome recombineering. Crossing these transgenic mice with the ROSA26 reporter strain, which expresses lacZ following Cre-mediated recombination, and staining the resulting progeny with 5-bromo-4-chloro-5-indolyl-?-D-galactoside indicated that Cre recombinase was specifically expressed in mAb MECA79-reactive HEVs in secondary lymphoid organs but not in any other blood vessels of the transgenic mice. The expression of Cre recombinase correlated with a developmental switch, from immature, mAb MECA367-reactive HEVs to mature, mAb MECA79-reactive HEVs in neonatal lymph nodes. In addition to the HEVs, Cre recombinase was also strongly expressed in the colonic villi, which recapitulated the intrinsic expression of GlcNAc6ST-2 as confirmed in GlcNAc6ST-2GFP/GFP knock-in mice and by RT-PCR. Furthermore, treatment with an antimicrobial agent revealed that the colonic expression of Cre recombinase in the transgenic mice was regulated by commensal bacteria in the colon. In addition, Cre recombinase was expressed in a small subset of cells in the brain, testis, stomach, small intestine, and lung. In view of the restricted expression of Cre recombinase, this transgenic mouse line should be useful for elucidating tissue-specific gene functions using the Cre/loxP system. PMID:19380794

  11. Marker genes for the metabolic adaptation of Pseudomonas aeruginosa to the hypoxic cystic fibrosis lung environment.

    PubMed

    Eichner, Anja; Günther, Nicole; Arnold, Martin; Schobert, Max; Heesemann, Jürgen; Hogardt, Michael

    2014-11-01

    Pseudomonas aeruginosa is the leading pathogen of chronic cystic fibrosis (CF) lung infection. Life-long persistence in the inflamed and ever fluctuating CF lungs results in the selection of a variety of changes in P. aeruginosa physiology. Accumulating evidence suggests that especially metabolic changes support the survival and growth of P. aeruginosa within the hypoxic and nutritious CF mucus. To investigate if metabolic adaptations we described for hypermutable P. aeruginosa from late CF lung disease (Hoboth et al., 2009. J. Infect. Dis., pp. 118-130) may represent specific changes in response to the selective conditions within the oxygen-restricted CF mucus, we determined the expression of a set of genes during aerobic and hypoxic growth in LB and the artificial sputum medium ASM. We further focused on the regulation of the two isocitrate dehydrogenases Icd and Idh. Interestingly, both isoenzymes may replace each other under aerobic and hypoxic conditions. The NADPH- and RpoS-dependent Icd seems to be the leading isoenzyme under prolonged oxygen limitation and stationary growth phase. LacZ reporter analysis revealed that oxygen-restriction increased the expression levels of azu, cbb3-1, cbb3-2, ccpR, icd, idh and oprF gene, whereas himD and nuoA are increasingly expressed only during hypoxic growth in ASM. Overexpression of the anaerobic regulator Anr improved the expression of azu, ccpR, cbb3-2 and icd. In summary, expression of azu, cbb3-1, cbb3-2, ccpR, icd, idh, oprF, himD, and nuoA appeared to be beneficial for the growth of P. aeruginosa under hypoxic conditions indicating these genes may represent marker genes for the metabolic adaptation to the CF lung environment. PMID:25130702

  12. Effect of promoter modification on mosquitocidal cryIVB gene expression in Synechococcus sp. strain PCC 7942.

    PubMed Central

    Soltes-Rak, E; Kushner, D J; Williams, D D; Coleman, J R

    1993-01-01

    The impact of promoter modification on the expression of the mosquitocidal Bacillus thuringiensis subsp. israelensis cryIVB gene when used to transform the cyanobacterium Synechococcus sp. strain PCC 7942 has been examined. Maximal transcript and protein abundances were achieved by the addition of the lacZ promoter upstream of the cryIVB sequence. Replacement of the endogenous corresponding Bacillus sequences with the Synechococcus petF1 promoter, ribosome binding site, and initiation codon also resulted in increased expression of the cryIVB gene relative to the expression obtained with the Bacillus promoter alone but decreased expression relative to the expression achieved with the tandem array of the Bacillus and lacZ promoters. Synechococcus cells carrying plasmids in which the expression of the cryIVB gene was regulated by either the lacZ or the petF1 promoter were readily consumed by first-instar Culex restuans larvae and proved to be toxic for these organisms. Images PMID:7690220

  13. Position-independent expression of a human nerve growth factor-luciferase reporter gene cloned on a yeast artificial chromosome vector.

    PubMed Central

    Asselbergs, F A; Grossenbacher, R; Ortmann, R; Hengerer, B; McMaster, G K; Sutter, E; Widmer, R; Buxton, F

    1998-01-01

    Two yeast artificial chromosomes containing the entire human nerve growth factor gene were isolated and mapped. By homologous recombination a luciferase gene was precisely engineered into the coding portion of the NGF gene and a neomycin selection marker was placed adjacent to one of the YAC telomeres. Expression of the YAC-based NGF reporter gene and a plasmid-based NGF reporter gene were compared with the regulation of endogenous mouse NGF protein in mouse L929 fibroblasts. In contrast to the plasmid-based reporter gene, expression and regulation of the YAC-based reporter gene was independent of the site of integration of the transgene. Basic fibroblast growth factor and okadaic acid stimulated expression of the YAC transgene, whereas transforming growth factor-beta and dexamethasone inhibited it. Although cyclic AMP strongly stimulated production of the endogenous mouse NGF, no effect was seen on the human NGF reporter genes. Downregulation of the secretion of endogenous mouse NGF already occurred at an EC50 of 1-2 nM dexamethasone, but downregulation of the expression of NGF reporter genes occurred only at EC50 of 10 nM. This higher concentration was also required for upregulation of luciferase genes driven by the dexamethasone-inducible promoter of the mouse mammary tumor virus in L929 fibroblasts. PMID:9512559

  14. Identification of evolutionarily conserved, functional noncoding elements in the promoter region of the sodium channel gene SCN8A

    PubMed Central

    Drews, Valerie L.; Shi, Kehui; de Haan, Georgius

    2007-01-01

    SCN8A is a major neuronal sodium channel gene expressed throughout the central and peripheral nervous systems. Mutations of SCN8A result in movement disorders and impaired cognition. To investigate the basis for the tissue-specific expression of SCN8A, we located conserved, potentially regulatory sequences in the human, mouse, chicken, and fish genes by 5? RACE of brain RNA and genomic sequence comparison. A highly conserved 5? noncoding exon, exon 1c, is present in vertebrates from fish to mammals and appears to define the ancestral promoter region. The distance from exon 1c to the first coding exon increased tenfold during vertebrate evolution, largely by insertion of repetitive elements. The mammalian gene acquired three novel, mutually exclusive noncoding exons that are not represented in the lower vertebrates. Within the shared exon 1c, we identified four short sequence elements of 10-20 bp with an unusually high level of evolutionary conservation. The conserved elements are most similar to consensus sites for the transcription factors Pou6f1/Brn5, YY1, and REST/NRSF. Introduction of mutations into the predicted Pou6f1 and REST sites reduced promoter activity in transfected neuronal cells. A 470-bp promoter fragment containing all of the conserved elements directed brain-specific expression of the LacZ reporter in transgenic mice. Transgene expression was highest in hippocampal neurons and cerebellar Purkinje cells, consistent with the expression of the endogenous gene. The compact cluster of conserved regulatory elements in SCN8A provides a useful target for molecular analysis of neuronal gene expression. Electronic supplementary material The online version of this article (doi:10.1007/s00335-007-9059-8) contains supplementary material, which is available to authorized users. PMID:17924165

  15. Amplification and expression of a salivary gland DNA puff gene in the prothoracic gland of Bradysia hygida (Diptera: Sciaridae).

    PubMed

    Candido-Silva, Juliana Aparecida; Machado, Maiaro Cabral Rosa; Hartfelder, Klaus Hartmann; de Almeida, Jorge Cury; Paçó-Larson, Maria Luisa; Monesi, Nadia

    2015-03-01

    The DNA puff BhC4-1 gene, located in DNA puff C4 of Bradysiahygida, is amplified and expressed in the salivary gland at the end of the fourth larval instar as a late response to the increase in 20-hydroxyecdysone titer that triggers metamorphosis. Functional studies revealed that the mechanisms that regulate BhC4-1 expression in the salivary gland are conserved in transgenic Drosophila. These studies also led to the identification of a cis-regulatory module that drives developmentally regulated expression of BhC4-1-lacZ in the prothoracic gland cells of the ring gland, a compound organ which in Drosophila results from the fusion of the prothoracic glands, the corpus allatum and the corpus cardiacum. Here we have investigated the occurrence of BhC4-1 expression in B. hygida prothoracic glands. We report the identification of the B. hygida prothoracic gland and demonstrate that it releases ecdysone. Using RT-qPCR, western blots and immunolocalization experiments, we demonstrate that the BhC4-1 mRNA and the BhC4-1 protein are both expressed in the B. hygida prothoracic glands at the same time that DNA puff C4 is formed in the salivary gland. We also show that BhC4-1 is concomitantly amplified 4.8-fold in the prothoracic gland and 23-fold in the salivary gland. Our results reveal the occurrence of stage specific expression of a DNA puff gene in the prothoracic glands of B. hygida, and extend previous studies that have shown that DNA puff genes expression is not restricted to the salivary gland. In addition, the description of stage specific gene amplification in the prothoracic glands of B. hygida constitutes the first demonstration that gene amplification in Diptera might occur concomitantly in two different tissues in the same developmental stage. PMID:25666977

  16. McArdle Disease: Update of Reported Mutations and Polymorphisms in the PYGM Gene.

    PubMed

    Nogales-Gadea, Gisela; Brull, Astrid; Santalla, Alfredo; Andreu, Antoni L; Arenas, Joaquin; Martín, Miguel A; Lucia, Alejandro; de Luna, Noemi; Pinós, Tomàs

    2015-07-01

    McArdle disease is an autosomal-recessive disorder caused by inherited deficiency of the muscle isoform of glycogen phosphorylase (or "myophosphorylase"), which catalyzes the first step of glycogen catabolism, releasing glucose-1-phosphate from glycogen deposits. As a result, muscle metabolism is impaired, leading to different degrees of exercise intolerance. Patients range from asymptomatic to severely affected, including in some cases, limitations in activities of daily living. The PYGM gene codifies myophosphoylase and to date 147 pathogenic mutations and 39 polymorphisms have been reported. Exon 1 and 17 are mutational hot-spots in PYGM and 50% of the described mutations are missense. However, c.148C>T (commonly known as p.R50X) is the most frequent mutation in the majority of the studied populations. No genotype-phenotype correlation has been reported and no mutations have been described in the myophosphorylase domains affecting the phosphorylated Ser-15, the 280's loop, the pyridoxal 5'-phosphate, and the nucleoside inhibitor binding sites. A newly generated knock-in mouse model is now available, which renders the main clinical and molecular features of the disease. Well-established methods for diagnosing patients in laboratories around the world will shorten the frequent ?20-year period stretching from first symptoms appearance to the genetic diagnosis. PMID:25914343

  17. Ferritin as an Endogenous MRI Reporter for Noninvasive Imaging of Gene Expression in C6 Glioma Tumors1

    Microsoft Academic Search

    Batya Cohen; Hagit Dafni; Gila Meir; Alon Harmelin; Michal Neeman

    The heavy chain of murine ferritin, an iron storage molecule with ferroxidase activity, was developed as a novel endogenous reporter for the detection of gene expression by magnetic resonance imaging (MRI). Expression of both enhanced green fluorescent pro- tein (EGFP) and influenza hemagglutinin (HA)-tagged ferritin were tightly coregulated by tetracycline (TET), using a bidirectional expression vector. C6 cells stably expressing

  18. Bioluminescent whole-cell reporter gene assays as screening tools in the identification of antimicrobial natural product extracts.

    PubMed

    Nybond, Susanna; Karp, Matti; Yrjönen, Teijo; Tammela, Päivi

    2015-07-01

    We describe novel tools, bioluminescent whole-cell reporter gene assays, for facilitating the use of natural products in antimicrobial drug discovery. As proof-of-concept, a plant extract library was screened and follow-up experiments were carried out. Primary results can be obtained in 2-4h with high sensitivity, leading to significant improvements of the process. PMID:25937087

  19. Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions.

    PubMed

    Pandey, Naresh; Nobles, Christopher L; Zechiedrich, Lynn; Maresso, Anthony W; Silberg, Jonathan J

    2015-05-15

    Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein-protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein-protein interactions within whole animals. PMID:25265085

  20. Modified mariner transposons for random inducible-expression insertions and transcriptional reporter fusion insertions in Bacillus subtilis.

    PubMed

    Pozsgai, Eric R; Blair, Kris M; Kearns, Daniel B

    2012-02-01

    Transposons are mobile genetic elements bounded by insertion sequences that are recognized by a specific mobilizing transposase enzyme. The transposase may mobilize not only the insertion sequences but also intervening DNA. mariner is a particularly efficient transposon for the random chromosomal integration of genes and insertional mutagenesis. Here, we modify an existing mariner transposon, TnYLB, such that it can easily be genetically manipulated and introduced into Bacillus subtilis. We generate a series of three new mariner derivatives that mobilize spectinomycin, chloramphenicol, and kanamycin antibiotic resistance cassettes. Furthermore, we generate a series of transposons with a strong, outward-oriented, optionally isopropyl-?-D-thiogalactopyranoside (IPTG)-inducible promoter for the random overexpression of neighboring genes and a series of transposons with a promoterless lacZ gene for the random generation of transcriptional reporter fusions. We note that the modification of the base transposon is not restricted to B. subtilis and should be applicable to any mariner-compatible host organism, provided that in vitro mutagenesis or an in vivo species-specific delivery vector is employed. PMID:22113911

  1. Modified mariner Transposons for Random Inducible-Expression Insertions and Transcriptional Reporter Fusion Insertions in Bacillus subtilis

    PubMed Central

    Pozsgai, Eric R.; Blair, Kris M.

    2012-01-01

    Transposons are mobile genetic elements bounded by insertion sequences that are recognized by a specific mobilizing transposase enzyme. The transposase may mobilize not only the insertion sequences but also intervening DNA. mariner is a particularly efficient transposon for the random chromosomal integration of genes and insertional mutagenesis. Here, we modify an existing mariner transposon, TnYLB, such that it can easily be genetically manipulated and introduced into Bacillus subtilis. We generate a series of three new mariner derivatives that mobilize spectinomycin, chloramphenicol, and kanamycin antibiotic resistance cassettes. Furthermore, we generate a series of transposons with a strong, outward-oriented, optionally isopropyl-?-d-thiogalactopyranoside (IPTG)-inducible promoter for the random overexpression of neighboring genes and a series of transposons with a promoterless lacZ gene for the random generation of transcriptional reporter fusions. We note that the modification of the base transposon is not restricted to B. subtilis and should be applicable to any mariner-compatible host organism, provided that in vitro mutagenesis or an in vivo species-specific delivery vector is employed. PMID:22113911

  2. CYP27B1 null mice with LacZreporter gene display no 25-hydroxyvitamin D3-1alpha-hydroxylase promoter activity in the skin.

    PubMed

    Vanhooke, Janeen L; Prahl, Jean M; Kimmel-Jehan, Christine; Mendelsohn, Monica; Danielson, Eric W; Healy, Kevin D; DeLuca, Hector F

    2006-01-01

    The hormonally active form of vitamin D(3),1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is synthesized in the kidney through a tightly regulated reaction catalyzed by 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase), the product of the CYP27B1 gene. Through gene targeting in embryonic stem cells, we engineered a mouse strain in which the coding region of the 1alpha-hydroxylase gene is replaced by the genes for beta-galactosidase (lacZ) and neomycin resistance. Null mice produced no detectable 1alpha-hydroxylase transcript. The mice grew normally when maintained on a balanced diet containing 1,25(OH)(2)D(3) but rapidly developed rickets when phosphorus and 1,25(OH)(2)D(3) were restricted. Rickets was curable through administration of 1,25(OH)(2)D(3) but not its biological precursor, 25-hydroxyvitamin D(3). Upon administration of a diet low in calcium and devoid of any form of vitamin D(3), beta-galactosidase activity was detected in the kidneys of the -/- and +/- mice and in placentas harvested from -/- females bred with -/- males. No beta-galactosidase activity was detected in skin sections or in primary keratinocyte cultures from -/- animals. Our results demonstrate we have generated 1alpha-hydroxylase null mice that display phenotypes characteristic of vitamin D-dependency rickets type I. From the histochemical analysis of reporter gene expression in these mice, we conclude that acute 1,25(OH)(2)D(3) deficiency in otherwise healthy animals does not stimulate local production of 1,25(OH)(2)D(3) in the skin. These findings stand in contrast to previously published reports of 1,25(OH)(2)D(3) production in keratinocytes. PMID:16371465

  3. Cold Shock Genes cspA and cspB from Caulobacter crescentus Are Posttranscriptionally Regulated and Important for Cold Adaptation

    PubMed Central

    Mazzon, Ricardo R.; Lang, Elza A. S.; Silva, Carolina A. P. T.

    2012-01-01

    Cold shock proteins (CSPs) are nucleic acid binding chaperones, first described as being induced to solve the problem of mRNA stabilization after temperature downshift. Caulobacter crescentus has four CSPs: CspA and CspB, which are cold induced, and CspC and CspD, which are induced only in stationary phase. In this work we have determined that the synthesis of both CspA and CspB reaches the maximum levels early in the acclimation phase. The deletion of cspA causes a decrease in growth at low temperature, whereas the strain with a deletion of cspB has a very subtle and transient cold-related growth phenotype. The cspA cspB double mutant has a slightly more severe phenotype than that of the cspA mutant, suggesting that although CspA may be more important to cold adaptation than CspB, both proteins have a role in this process. Gene expression analyses were carried out using cspA and cspB regulatory fusions to the lacZ reporter gene and showed that both genes are regulated at the transcriptional and posttranscriptional levels. Deletion mapping of the long 5?-untranslated region (5?-UTR) of each gene identified a common region important for cold induction, probably via translation enhancement. In contrast to what was reported for other bacteria, these cold shock genes have no regulatory regions downstream from ATG that are important for cold induction. This work shows that the importance of CspA and CspB to C. crescentus cold adaptation, mechanisms of regulation, and pattern of expression during the acclimation phase apparently differs in many aspects from what has been described so far for other bacteria. PMID:23002229

  4. Subspecies-dependent regulation of Bacillus thuringiensis protoxin genes.

    PubMed

    Cheng, P; Wu, L; Ziniu, Y; Aronson, A

    1999-05-01

    Bacillus thuringiensis accumulates, primarily during sporulation, large quantities of insecticidal protoxins which are deposited as crystalline, intracellular inclusions. Most subspecies contain several plasmid-encoded cry genes, each of which has a unique specificity. The overall toxicity profile of a subspecies depends not only on the array of cry genes present but also on the relative expression of the genes. In general, transcription depends on sporulation-specific sigma factors, but little is known about regulation of expression of the individual genes. In order to determine whether expression of a particular cry gene varies in different subspecies, lacZ fusions to the cry promoters of two protoxin genes (cry1 class) were constructed. Protoxin accumulation and mRNA contents were also measured by performing immunoblotting and Northern analyses, respectively. The expression of a cry1Ab-lacZ fusion, but not the expression of a cry1C-lacZ fusion, was three to four times lower in B. thuringiensis subsp. aizawai strains than in B. thuringiensis subsp. kurstaki or B. thuringiensis subsp. tolworthi. Also, the Cry1Ab antigen and steady-state mRNA contents of B. thuringiensis subsp. aizawai were lower. The regulation of the genes must involve regions upstream of the promoters which are unique to each cry gene since (i) mutations in the upstream region of the cry1Ab gene resulted in enhanced expression in B. thuringiensis subsp. aizawai and (ii) no differences were found when the lacZ fusions contained the cry1Ab promoters but no upstream sequences. The capacity to regulate each of the protoxin genes must be a factor in the overall protoxin composition of a subspecies and thus its toxicity profile. PMID:10223968

  5. In vitro study for laser gene transfer in BHK-21 fibroblast cell line

    NASA Astrophysics Data System (ADS)

    Abdel Aziz, M.; Salem, D. S.; Salama, M. S.; Badr, Y.

    2009-02-01

    Modifications to our previously introduced system for laser microbeam cell surgery were carried out in the present work to match animal cells. These modifications included: 1- Using other laser system that used before, Excimer laser with 193 and 308 nm wavelengths. The used laser here, is He-Cd with low power and 441.5 nm wavelength in the visible region. 2- Instead of using pulsed laser, we used here CW He-Cd chopped by electrical chopper, which is synchronized with the mechanical motion of the mobile stage with step 40 microns, according to cell dimensions to avoid puncturing the same cell twice. The advantages of the modified here laser setup for gene transfer is: it is less damaging to the sensitive animal cell which has thin cell membrane. The present work aimed to: 1- Design a modified laser microbeam cell surgery, applicable to animal cells, such as fibroblast cells 2- To examine the efficiency of such system. 3- To assure gene transfer and its expression in the used cells. 4- To evaluate the ultra damages produced from using the laser beam as a modality for gene transfer. On the other wards, to introduce: safe, efficient and less damaging modality for gene transfer in animal cells. To achieve these goals, we applied the introduced here home-made laser setup with its synchronized parameters to introduce pBK-CMV phagemid, containing LacZ and neomycin resistance (neor )genes into BHK-21 fibroblast cell line. The results of the present work showed that: 1- Our modified laser microbeam cell surgery setup proved to be useful and efficient tool for gene transfer into fibroblast cells. 2- The presence and expression of LacZ gene was achieved using histochemical LacZ assay. 3- Selection of G418 antibiotic sensitivity assay confirmed the presence and expression towards stability of neor gene with time. 4- Presence of LacZ and neor genes in the genomic DNA of transfected fibroblast cells was indicated using PCR analysis. 5- Transmission electron microscopy indicated that, no ultradamages or changes for cell; membrane, organilles or any component of transfected fibroblast cell as a result of using laser microbeam compared with control cell.

  6. CYP27B1 null mice with LacZreporter gene display no 25-hydroxyvitamin D3-1-hydroxylase promoter activity in the skin

    Microsoft Academic Search

    Janeen L. Vanhooke; Jean M. Prahl; Christine Kimmel-Jehan; Monica Mendelsohn; Eric W. Danielson; Kevin D. Healy; Hector F. Deluca

    2006-01-01

    The hormonally active form of vitamin D3,1,25-dihydroxyvitamin D3 [1,25(OH)2D3], is synthesized in the kidney through a tightly regulated reaction catalyzed by 25-hydroxyvitamin D3-1-hydroxylase (1-hydroxylase), the product of the CYP27B1 gene. Through gene targeting in embryonic stem cells, we engineered a mouse strain in which the coding region of the 1-hydroxylase gene is replaced by the genes for -galactosidase (lacZ) and

  7. Brugada syndrome with a novel missense mutation in SCN5A gene: A case report from Bangladesh

    PubMed Central

    Sayeed, Md. Zahidus; Salam, Md. Abdus; Haque, Md. Zahirul; Islam, A.K.M. Monwarul

    2014-01-01

    Brugada syndrome is an inherited cardiac arrhythmia that follows autosomal dominant transmission and can cause sudden death. We report a case of Brugada syndrome in a 55-year-old male patient presented with recurrent palpitation, atypical chest pain and presyncope. ECG changes were consistent with type 1 Brugada. Gene analysis revealed a novel missense mutation in SCN5A gene with a genetic variation of D785N and a nucleotide change at 2353G-A. One of his children also had the same mutation. To our knowledge this is the first genetically proved case of Brugada syndrome in Bangladesh. PMID:24581105

  8. Brugada syndrome with a novel missense mutation in SCN5A gene: a case report from Bangladesh.

    PubMed

    Sayeed, Md Zahidus; Salam, Md Abdus; Haque, Md Zahirul; Islam, A K M Monwarul

    2014-01-01

    Brugada syndrome is an inherited cardiac arrhythmia that follows autosomal dominant transmission and can cause sudden death. We report a case of Brugada syndrome in a 55-year-old male patient presented with recurrent palpitation, atypical chest pain and presyncope. ECG changes were consistent with type 1 Brugada. Gene analysis revealed a novel missense mutation in SCN5A gene with a genetic variation of D785N and a nucleotide change at 2353G-A. One of his children also had the same mutation. To our knowledge this is the first genetically proved case of Brugada syndrome in Bangladesh. PMID:24581105

  9. Site-Specific Recombination-Based Genetic System for Reporting Transient or Low-Level Gene Expression†

    PubMed Central

    Casavant, N. Carol; Beattie, Gwyn A.; Phillips, Gregory J.; Halverson, Larry J.

    2002-01-01

    We report here the construction, characterization, and application of a plasmid-based genetic system that reports the expression of a target promoter by effecting an irreversible, heritable change in a bacterial cell. This system confers strong repression of the reporter gene gfp in the absence of target promoter expression and utilizes the site-specific recombination machinery of bacteriophage P22 to trigger high-level reporter gene expression in the original cell and its progeny after target gene induction. We demonstrate the effectiveness of this genetic system by tailoring it to indicate the availability of arabinose to the biological control agent Enterobacter cloacae JL1157 in culture and in the barley rhizosphere. The presence of bioavailable arabinose triggered the production of P22 excisionase and integrase from the reporter plasmid pAraLHB in JL1157, and this led to excision of the cI repressor gene, which is flanked by att sites, and the subsequent irreversible expression of gfp in the original cell and in its progeny. In culture, nearly 100% of an E. cloacae JL1157(pAraLHB) population expressed gfp after exposure to 6.5 to 65 ?M arabinose for 3 h. We used this biosensor to demonstrate that arabinose was released from the seeds of several legumes and grass species during germination and from roots of barley seedlings grown hydroponically or in soil. When introduced into microcosms containing barley, the biosensor permitted the localization of arabinose along the roots. Arabinose was present near the root-seed junction and on the seminal roots but was not detected at the root tips. This recombination-based reporter system should be useful for monitoring bacterial exposure to transient or low levels of specific molecules directly in the environment. PMID:12089047

  10. Selection of available suicide vectors for gene mutagenesis using chiA (a chitinase encoding gene) as a new reporter and primary functional analysis of chiA in Lysobacter enzymogenes strain OH11

    Microsoft Academic Search

    Yansheng Wang; Dongyu Qian; Jiaqin Fan; Baishi Hu; Fengquan Liu

    Here, three different suicide vectors were evaluated for the possibility of performing gene mutagenesis in strain OH11 using\\u000a the chiA gene (accession number: DQ888611) as a new reporter. Suicide vector pEX18GM was selected, and it was successfully applied\\u000a for disruption and in-frame deletions in the chiA gene in strain OH11, which was confirmed by PCR amplification and Southern hybridization. The

  11. Development of a novel vaccinia-neutralization assay based on reporter-gene expression.

    PubMed

    Manischewitz, Jody; King, Lisa R; Bleckwenn, Nicole A; Shiloach, Joseph; Taffs, Rolf; Merchlinsky, Michael; Eller, Nancy; Mikolajczyk, Malgorzata G; Clanton, David J; Monath, Thomas; Weltzin, Richard A; Scott, Dorothy E; Golding, Hana

    2003-08-01

    In anticipation of large-scale smallpox vaccination, clinical trials of new vaccine candidates with improved safety profiles, and new vaccinia immune globulin (VIG) products, there is an immediate need to develop new assays to measure vaccinia-specific immune responses. The classical assay to measure vaccinia neutralization, the plaque-reduction neutralization test (PRNT), is slow, labor intensive, and difficult to validate and transfer. Here we describe the development of a novel vaccinia-neutralization assay based on the expression of a reporter gene, beta-galactosidase (beta-Gal). Using a previously constructed vaccinia-beta-Gal recombinant virus, vSC56, we developed a neutralization assay that is rapid, sensitive, and reproducible. The readout is automated. We show that the neutralizing titers, ID(50), for several VIG products measured by our assay were similar to those obtained by PRNTs. A new Food and Drug Administration VIG standard was established for distribution to other laboratories. The new assay will serve as an important tool both for preclinical and clinical trials of new smallpox vaccines and for evaluation of therapeutic agents to treat vaccine-associated adverse reactions. PMID:12870127

  12. Inducible expression of photoacoustic reporter gene tyrosinase in cells using a single plasmid

    NASA Astrophysics Data System (ADS)

    Paproski, Robert J.; Zemp, Roger J.

    2012-02-01

    We have previously demonstrated that tyrosinase is a reporter gene for photoacoustic imaging since tyrosinase is the rate-limiting step in the synthesis of melanin, a pigment capable of producing strong photoacoustic signals. We previously created a cell line capable of inducible tyrosinase expression (important due to toxicity of melanin) by stably transfecting tyrosinase in MCF-7 Tet-OnR cell line (Clontech) which expresses a doxycycline-controlled transactivator. Unfortunately, Clontech provides few Tet-On Advanced cell lines making it difficult to have inducible tyrosinase expression in cell lines not provided by Clontech. In order to simplify the creation of cell lines with inducible expression of tyrosinase, we created a single plasmid that encodes both the transactivator as well as tyrosinase. PCR was used to amplify both the transactivator and tyrosinase from the Tet-OnR Advanced and pTRE-Tight-TYR plasmids, respectively. Both PCR products were cloned into the pEGFP-N1 plasmid and the newly created plasmid was transfected into ZR-75-1, MCF-7, and MIA PaCa-1 cells using lipofectamine. After several days, brown melanin was only observed in cells incubated with doxycycline, suggesting that the newly created single plasmid allowed inducible tyrosinase expression in many different cells lines.

  13. In vitro reporter gene transfection via plasmid DNA delivered by metered dose inhaler.

    PubMed

    Bains, Baljinder K; Birchall, James C; Toon, Richard; Taylor, Glyn

    2010-07-01

    Aerosolised DNA administration could potentially advance the treatment of inheritable lung diseases, lung malignancies and provide genetic immunisation against infection. Jet nebulisation, the current standard for introducing DNA formulations into the lung, is inherently inefficient. Pressurised metered dose inhalers (pMDIs) offer a potentially more efficacious and convenient alternative, especially for repeat administration. We aim to modify a novel low-energy nanotechnology process to prepare surfactant-coated pDNA nanoparticles for pulmonary gene delivery via a pMDI. Water-in-oil microemulsions containing green fluorescent protein reporter plasmid were snap-frozen and lyophilised. Lyophilised pDNA, in some cases following a surfactant wash, was incorporated into pMDIs with hydrofluoroalkane 134a (HFA134a) propellant and ethanol as cosolvent. To assess biological functionality, A549 human lung epithelial cells were exposed to aerosolised pDNA particles in the presence of dioleoyl-trimethylammonium propane (DOTAP). Transfection studies demonstrated that pDNA biological functionality was maintained following aerosolisation. In vitro toxicity assays (MTT) showed no significant cell viability loss following aerosolised pDNA treatment. We have demonstrated that pDNA particles can be incorporated into an HFA134a formulation and aerosolised using a standard valve and actuator. Particles prepared by this novel process have potential for stable and efficient delivery of pDNA to the lower respiratory tract via standard pMDI technology. PMID:20166201

  14. Abrupt and dynamic changes in gene expression revealed by live cell arrays.

    PubMed

    Walling, Maureen A; Shi, Hua; Shepard, Jason R E

    2012-03-20

    A description of the noise associated with gene expression is presented, based on a simplified form of the combined multistep processes of transcription and translation. These processes are influenced by numerous factors, including the accessibility of promoter regions to the transcriptional machinery, the kinetics of assembly of the transcription complexes, and the synthesis and degradation of both mRNA and proteins, among others. Ultimately, stochasticity in cellular processes results in variation in protein levels. Here we constructed a rationally designed RNA-based transcriptional activator to reduce these variables and provide a cleaner, more detailed portrayal of cellular noise. Functioning at a level comparable to natural transcription activation, this activator is isolated to a lacZ reporter gene in yeast cells to quantitatively describe the efficiency of the combined processes of transcription and translation. By employing single-cell array techniques to monitor individual cells simultaneously and in real time, a statistical approach to investigate noise inherent in gene expression is possible. Live cell arrays enabled cell populations to be characterized temporally at the individual cell level. The array platform allowed for a relative measure of protein production in real time and could characterize protein bursts with variable size and random timing, such that bursts occurred in a temporally indiscriminate fashion. The inherent variability and randomness of these processes is characterized, with almost half (47%) of cells experiencing bursting behavior at least once over the course of the experiment. We demonstrate that cells identified on the upper periphery of activity exhibit behaviors that are substantially different from the majority of the population, and such variable activities within a population will provide a more accurate characterization of the population. PMID:22324657

  15. Escherichia coli ?-galactosidase as an in vitro and in vivo reporter enzyme and stable transfection marker in the intracellular protozoan parasite Toxoplasma gondii

    Microsoft Academic Search

    Frank Seeber; John C. Boothroyd

    1996-01-01

    We have developed several protocols for the use of ?-galactosidase (?Gal) from Escherichia coli as a reporter enzyme in transfection studies of Toxoplasma gondii (Tg) and as a readily screenable marker for stable transformation. Three Tg expression vectors with different promoters driving lacZ were constructed and shown in transient transfections to differ in their relative expression levels. Using a fluorescent

  16. Pine Gene Discovery Project - Final Report - 08/31/1997 - 02/28/2001

    SciTech Connect

    Whetten, R. W.; Sederoff, R. R.; Kinlaw, C.; Retzel, E.

    2001-04-30

    Integration of pines into the large scope of plant biology research depends on study of pines in parallel with study of annual plants, and on availability of research materials from pine to plant biologists interested in comparing pine with annual plant systems. The objectives of the Pine Gene Discovery Project were to obtain 10,000 partial DNA sequences of genes expressed in loblolly pine, to determine which of those pine genes were similar to known genes from other organisms, and to make the DNA sequences and isolated pine genes available to plant researchers to stimulate integration of pines into the wider scope of plant biology research. Those objectives have been completed, and the results are available to the public. Requests for pine genes have been received from a number of laboratories that would otherwise not have included pine in their research, indicating that progress is being made toward the goal of integrating pine research into the larger molecular biology research community.

  17. Cell-based reporter gene assay for therapy-induced neutralizing antibodies to interferon-beta in multiple sclerosis.

    PubMed

    Martins, Thomas B; Rose, John W; Gardiner, Gareth L; Kusukawa, Noriko; Husebye, Dee; Hill, Harry R

    2013-02-01

    Patients with therapy-induced neutralizing antibodies (NAbs) to interferon-beta (IFN-?) have reduced responses to IFN-? treatment, resulting in higher relapse rates, increased magnetic resonance imaging activity, and a higher risk of disease progression. A functional assay was employed for both screening and titering of IFN-? NAbs utilizing a human cell line transfected with a luciferase reporter gene responsive to IFN-?. This assay demonstrated 100% sensitivity and specificity compared with the traditional cytopathic effect (CPE) assay and normal donor specimens. Additionally, 183 patients with multiple sclerosis (MS) undergoing therapy with IFN-? were tested in the reporter gene assay. Percent positivity for NAbs to the IFN-? was as follows: Avonex (1?) 26.5%, Rebif (1?) 34.1%, and Betaseron (1?) 31.8%. The IFN-? reporter gene assay showed excellent correlation with the well-established CPE assay offering clear advantages. The 50% false-positivity rate typically seen in enzyme-linked immunosorbent assays could be eliminated by using a functional assay for both screening and titering. Results can be reported within 20 h, and the cell line is cryopreserved, eliminating the need to maintain live viral and cell cultures. The use of this functional assay should be a valuable tool for detecting and monitoring the presence of NAbs in IFN-?-treated patients with MS. PMID:23153300

  18. Copper homeostasis-related genes in three separate transcriptional units regulated by CsoR in Corynebacterium glutamicum.

    PubMed

    Teramoto, Haruhiko; Yukawa, Hideaki; Inui, Masayuki

    2015-04-01

    In Corynebacterium glutamicum R, CsoR acts as a transcriptional repressor not only of the cognate copA-csoR operon but also of the copZ1-copB-cgR_0126 operon. It is predicted that copA and copB encode P-type ATPases for copper efflux and copZ1 encodes a metallochaperone. Here, a CsoR-binding motif was found upstream of another copZ-like gene, copZ2, and the in vitro binding of the CsoR protein to its promoter was confirmed. The monocistronic copZ2 transcript was upregulated by excess copper in a CsoR-dependent manner. Among the extended CsoR regulon, deletion of copA, but not of copB, copZ1, or copZ2, resulted in decreased resistance to copper, indicating a major role of the CopA copper exporter in the multilayered systems for copper homeostasis. A redundant role of copZ1 and copZ2 in copper resistance was also indicated by double deletion of these genes. The copper-dependent activation of the copA, copZ1, and copZ2 promoters was confirmed by lacZ reporter assays, consistent with the coordinated derepression of the three transcriptional units. The copZ1 promoter activity showed the highest responsiveness to copper and was also induced by excess zinc and nickel. Furthermore, zinc-inducible expression observed for the CsoR-regulated genes was independent of Zur, recently found to uniquely act as a transcriptional repressor of zinc efflux genes. These results implied complicated cross talk between homeostasis of multiple transition metals. PMID:25592736

  19. Horseradish peroxidase as a reporter gene and as a cell-organelle-specific marker in correlative light-electron microscopy.

    PubMed

    Schikorski, Thomas

    2010-01-01

    A modern electron microscopic approach to the investigation of the structural organization of proteins and subcellular structures demands the use of molecular genetic techniques. The successful implementation of genetic techniques is closely tied to a reporter gene such as the green fluorescent protein (GFP). Although GFP has been widely used for light microscopy, it has many limitations for use in electron microscopy. In the search for a reporter gene for electron microscopy, interest in the use of horseradish peroxidase (HRP) DNA has recently increased, and several studies already have proven the feasibility of HRP expression in mammalian cells. Here, we describe a protocol that uses a HRP chimera to label the endoplasmic reticulum of HEK cells. PMID:20602227

  20. RIKEN BSI BSIS Technical Report No.02-5 1 Blind Gene Classification

    E-print Network

    Hori, Gen

    belongs to the latter. ICA has advantage to PCA in that the former exploits higher order statistics and has no restriction on its transformation, whereas the latter exploits only second order statistics/clustering Abstract Motivation: To develop a gene classification method exploiting statistical struc- ture of gene

  1. Molecular analysis of the PAX6 gene in Mexican patients with congenital aniridia: report of four novel mutations

    Microsoft Academic Search

    Camilo E. Villarroel; Cristina Villanueva-Mendoza; Lorena Orozco; Miguel Angel Alcántara-Ortigoza; Diana F. Jiménez; Juan C. Ordaz; Ariadna González-del Angel

    2008-01-01

    Purpose: Paired box gene 6 (PAX6) heterozygous mutations are well known to cause congenital non-syndromic aniridia. These mutations produce primarily protein truncations and have been identified in approximately 40%-80% of all aniridia cases worldwide. In Mexico, there is only one previous report describing three intragenic deletions in five cases. In this study, we further analyze PAX6 variants in a group

  2. Generation of a variety of stable Influenza A reporter viruses by genetic engineering of the NS gene segment.

    PubMed

    Reuther, Peter; Göpfert, Kristina; Dudek, Alexandra H; Heiner, Monika; Herold, Susanne; Schwemmle, Martin

    2015-01-01

    Influenza A viruses (IAV) pose a constant threat to the human population and therefore a better understanding of their fundamental biology and identification of novel therapeutics is of upmost importance. Various reporter-encoding IAV were generated to achieve these goals, however, one recurring difficulty was the genetic instability especially of larger reporter genes. We employed the viral NS segment coding for the non-structural protein 1 (NS1) and nuclear export protein (NEP) for stable expression of diverse reporter proteins. This was achieved by converting the NS segment into a single open reading frame (ORF) coding for NS1, the respective reporter and NEP. To allow expression of individual proteins, the reporter genes were flanked by two porcine Teschovirus-1 2A peptide (PTV-1 2A)-coding sequences. The resulting viruses encoding luciferases, fluorescent proteins or a Cre recombinase are characterized by a high genetic stability in vitro and in mice and can be readily employed for antiviral compound screenings, visualization of infected cells or cells that survived acute infection. PMID:26068081

  3. Generation of a variety of stable Influenza A reporter viruses by genetic engineering of the NS gene segment

    PubMed Central

    Reuther, Peter; Göpfert, Kristina; Dudek, Alexandra H.; Heiner, Monika; Herold, Susanne; Schwemmle, Martin

    2015-01-01

    Influenza A viruses (IAV) pose a constant threat to the human population and therefore a better understanding of their fundamental biology and identification of novel therapeutics is of upmost importance. Various reporter-encoding IAV were generated to achieve these goals, however, one recurring difficulty was the genetic instability especially of larger reporter genes. We employed the viral NS segment coding for the non-structural protein 1 (NS1) and nuclear export protein (NEP) for stable expression of diverse reporter proteins. This was achieved by converting the NS segment into a single open reading frame (ORF) coding for NS1, the respective reporter and NEP. To allow expression of individual proteins, the reporter genes were flanked by two porcine Teschovirus-1 2A peptide (PTV-1 2A)-coding sequences. The resulting viruses encoding luciferases, fluorescent proteins or a Cre recombinase are characterized by a high genetic stability in vitro and in mice and can be readily employed for antiviral compound screenings, visualization of infected cells or cells that survived acute infection. PMID:26068081

  4. Familial hypercholesterolemia in Morocco: first report of mutations in the LDL receptor gene.

    PubMed

    El Messal, Mariame; Aït Chihab, Karima; Chater, Rachid; Vallvé, Joan Carles; Bennis, Faïza; Hafidi, Aïcha; Ribalta, Josep; Varret, Mathilde; Loutfi, Mohammed; Rabès, Jean Pierre; Kettani, Anass; Boileau, Catherine; Masana, Luis; Adlouni, Ahmed

    2003-01-01

    Familial hypercholesterolemia (FH) is a genetic disorder mainly caused by defects in the low-density lipoprotein receptor (LDLR) gene, although it can also be due to alterations in the gene encoding apolipoprotein B (familial defective apoB or FDB) or in other unidentified genes. In Morocco, the molecular basis of FH is unknown. To obtain information on this issue, 27 patients with FH from eight unrelated families were analyzed by screening the LDLR (PCR-SSCP and Southern blot) and apoB genes (PCR and restriction enzyme digestion analysis). None of the patients carried either the R3500Q or the R3531C mutation in the apoB gene. By contrast, seven mutations in the LDLR gene were identified, including five missense mutations on exons 4, 6, 8, and 14 (C113R, G266C, A370T, P664L, C690S) and two large deletions (FH Morocco-1 and FH Morocco-2). The two major rearrangements and the missense mutation G266C are novel mutations and could well be causative of FH in the Moroccan population. This study has yielded preliminary information on the mutation spectrum of the LDLR gene among patients with FH in Morocco. PMID:12730724

  5. A subgroup of Tourette's patients overexpress specific natural killer cell genes in blood: a preliminary report.

    PubMed

    Lit, Lisa; Gilbert, Donald L; Walker, Wynn; Sharp, Frank R

    2007-10-01

    Gilles de la Tourette Syndrome (TS) is a heritable, neurodevelopmental disorder characterized by motor and vocal tics. As no single gene or region has emerged from standard linkage approaches, TS may result from several as-yet-unidentified genetic factors, and may also occur due to infection-triggered, autoimmune processes. Etiological or pathogenic differences might result in clinically indistinguishable TS subgroups. We have previously used whole genome human oligonucleotide microarrays in an attempt to identify patterns of gene expression in blood linked with TS. In this proof-of-principle study, we applied Principal Components Analysis to a previously collected set of 16 familial TS and 16 control blood samples to identify subgroups. Fourteen genes, primarily Natural Killer Cell (NK) genes, discriminated between TS and all controls. Granzyme B and NKG7 were confirmed using RT-PCR. Five probesets (four genes) reside in chromosomal regions previously linked to familial TS or obsessive-compulsive disorder. Using the 14 genes, a Principal Components Analysis as well as a cluster analysis identified a TS subgroup (n = 10/16) that overexpressed the NK genes. 7/10 subjects within this subgroup were diagnosed with attention-deficit hyperactivity disorder (ADHD), suggesting that this expression profile might be associated with TS and co-morbid ADHD. Principal Components Analysis of gene expression in blood may be useful for identifying subgroups of other complex neurodevelopmental diseases, and the gene expression profile identified in this study may provide a biomarker for at least one subgroup of heritable TS. PMID:17503477

  6. The RR1 gene of herpes simplex virus type 1 is uniquely trans activated by ICP0 during infection.

    PubMed Central

    Desai, P; Ramakrishnan, R; Lin, Z W; Osak, B; Glorioso, J C; Levine, M

    1993-01-01

    As has been demonstrated for herpes simplex virus type 2, we show in this report that the herpes simplex virus type 1 ribonucleotide reductase large subunit (RR1) gene is trans activated in transient transfection assays by VP16 and ICP0 but not by ICP4. Deletion analysis demonstrated that responsiveness to induction to VP16 resides in an octamer/TAATGARAT sequence of the RR1 promoter and that the TATA box alone is sufficient to provide induction by ICP0. The induction of the RR1 gene by ICP0 but not by ICP4 suggested that it might be possible to identify the cis-acting element(s) responsive to ICP4 in an ICP4-inducible promoter. To this end, a series of chimeric promoters containing various portions of the regulatory sequences of the RR1 promoter and thymidine kinase (TK) promoter were constructed. The TK promoter is trans activated by both ICP0 and ICP4 in transient transfection assays and by ICP4 in infection. The data show that replacing the RR1 TATA region with the TK TATA region permits ICP4 inducibility even if the rest of the RR1 promoter elements remain intact. To test whether the RR1 gene is induced by ICP0 during infection, four mutant viruses were constructed. (i) TAATGARAT+ has the wild-type RR1 promoter driving chloramphenicol acetyltransferase (CAT) and the RR2 promoter driving the lacZ gene. The RR2 gene codes for the small subunit of the ribonucleotide reductase and is expressed as a beta gene. (ii) TAATGARAT- has a triple-base change in the octamer/TAATGARAT element which renders it unresponsive to VP16 trans activation, eliminating that portion of the activation of the RR1 gene. (iii) TAATGARAT- delta alpha 0 has a deletion of the alpha 0 gene. (iv) TAATGARAT- delta alpha 4 has a deletion of the alpha 4 gene. Infections were carried out in Vero cells at a multiplicity of infection of 10 per cell; cells were assayed for CAT and beta-galactosidase (beta-Gal) activities and for virus yields. The first two infections gave strong CAT and beta-Gal activities and high yields of progeny virus. Infection with the third virus showed no CAT activity but did produce high levels of beta-Gal activity and virus progeny. The fourth infection resulted in strong CAT activity but no beta-Gal activity or progeny virus. The data demonstrated that the RR1 promoter was activated in the absence of ICP4 but not in the absence of ICP0 in these infections.(ABSTRACT TRUNCATED AT 400 WORDS) Images PMID:8396674

  7. Scaffold attachment regions increase reporter gene expression in stably transformed plant cells.

    PubMed Central

    Allen, G C; Hall, G E; Childs, L C; Weissinger, A K; Spiker, S; Thompson, W F

    1993-01-01

    The yeast ARS-1 element contains a scaffold attachment region (SAR) that we have previously shown can bind to plant nuclear scaffolds in vitro. To test effects on expression, constructs in which a chimeric beta-glucuronidase (GUS) gene was flanked by this element were delivered into tobacco suspension cells by microprojectile bombardment. In stably transformed cell lines, GUS activity averaged 12-fold higher (24-fold on a gene copy basis) for a construct containing two flanking SARs than for a control construct lacking SARs. Expression levels were not proportional to gene copy number, as would have been predicted if the element simply reduced position effect variation. Instead, the element appeared to reduce an inhibitory effect on expression in certain transformants containing multiple gene copies. The effect on expression appears to require chromosomal integration, because SAR constructs were only twofold more active than the controls in transient assays. PMID:8329896

  8. Organization and control of genes encoding catabolic enzymes in Rhizobiaceae. Progress report, March 1993

    SciTech Connect

    Parke, D.; Ornston, L.N.

    1993-03-01

    Rhizobiaceae, a diverse bacterial group comprising rhizobia and agrobacteria, symbiotic partnership with plants form nitrogen-fixing nodules on plant roots or are plant pathogens. Phenolic compounds produced by plants serve as inducers of rhizobial nodulation genes and agrobacterial virulence genes reflect their capacity to utilize numerous aromatics, including phenolics, as a source of carbon and energy. In many microbes the aerobic degradation of numerous aromatic compounds to tricarboxylic acid cycle intermediates is achieved by the {beta}-ketoadipate pathway. Our initial studies focused on the organization and regulation of the ketoadipate pathway in Agrobacterium tumefaciens. We have cloned, identified and characterized a novel regulatory gene that modulates expression of an adjacent pca (protocatechuate) structural gene, pcaD. Regulation of pcaD is mediated by the regulatory gene, termed pcaQ, in concert with the intermediate {beta}-carboxy-cis,cis-muconate. {beta}-carboxy-cis,cismuconate is an unstable chemical, not marketed commercially, and it is unlikely to permeate Escherichia coli cells if supplied in media. Because of these factors, characterization of pcaQ in E. coli required an in vivo delivery system for {beta}-carboxycis,cis-muconate. This was accomplished by designing an E. coli strain that expressed an Acinetobacter calcoaceticus pcaA gene for conversion of protocatechuate to {beta}-carboxy-cis,cis-muconate.

  9. Noninvasive, quantitative imaging in living animals of a mutant dopamine D2 receptor reporter gene in which ligand binding is uncoupled from signal transduction

    Microsoft Academic Search

    Q Liang; N Satyamurthy; JR Barrio; T Toyokuni; MP Phelps; SS Gambhir; HR Herschman

    2001-01-01

    The dopamine D2 receptor (D2R) has been used in adenoviral delivery systems and in tumor cell xenografts as an in vivo reporter gene. D2R reporter gene expression has been non-invasively, repetitively and quantitatively imaged by positron emission tomography (PET), following systemic injection of a positron-labeled ligand (3-(2?-[18F]-fluoroethyl)-spiperone; FESP) and subsequent D2R-dependent sequestration. However, dopamine binding to the D2R can modulate

  10. The promoter of coconut foliar decay-associated circular single-stranded DNA directs phloem-specific reporter gene expression in transgenic tobacco

    Microsoft Academic Search

    Wolfgang Rohde; Dieter Becker; John W. Randles

    1995-01-01

    A full-length double-stranded DNA copy of the single-stranded circular DNA associated with coconut foliar decay virus (CFDV) was constructed. Full-length CFDV DNA and smaller fragments were transcriptionally fused to the ß-glucuronidase reporter gene and examined for promoter activity in vivo. In stably transformed tobacco plants, the CFDV DNA promoter confered a tissue-specific expression pattern in that the reporter gene was

  11. Two DNA repair and recombination genes in Saccharomyces cerevisiae, RAD52 and RAD54, are induced during meiosis

    SciTech Connect

    Cole, G.M.; Mortimer, R.K. (Univ. of California, Berkeley (United States)); Schild, D. (Lawrence Berkeley Lab., CA (United States))

    1989-07-01

    The DNA repair and recombination genes of Saccharomyces cerevisiae, RAD52 and RAD54, were transcriptionally induced approximately 10- to 15-fold in sporulating MATa/{alpha} cells. Congenic MATa/a cells, which did not sporulate, did not show similar increases. Assays of {beta}-galactosidase activity in strains harboring either a RAD52- or RAD54-lacZ gene fusion indicated that this induction occurred at a time concomitant with a commitment to meiotic recombination, as measured by prototroph formation from his1 heteroalleles.

  12. Adenovirus-mediated gene transfer of tissue factor pathway inhibitor induces apoptosis in vascular smooth muscle cells

    Microsoft Academic Search

    Yu Fu; Zhaoying Zhang; Gaigai Zhang; Yue Liu; Ying Cao; Jinfeng Yu; Jing Hu; Xinhua Yin

    2008-01-01

    Objective  To investigate the pro-apoptotic effect of tissue factor pathway inhibitor (TFPI) gene transfer mediated by adenovirus on\\u000a vascular smooth muscle cells (VSMCs).\\u000a \\u000a \\u000a \\u000a Methods  Rat VSMCs were infected with recombinant adenovirus containing either the TFPI (Ad-TFPI) or LacZ (Ad-LacZ) gene or DMEM in vitro.\\u000a TFPI expression was detected by ELISA. Apoptosis of VSMCs was determined by electron microscopy and flow cytometry. The expression

  13. Evaluation of Estrogenic Activity of Wastewater: Comparison Among In Vitro ER? Reporter Gene Assay, In Vivo Vitellogenin Induction, and Chemical Analysis.

    PubMed

    Ihara, Masaru; Kitamura, Tomokazu; Kumar, Vimal; Park, Chang-Beom; Ihara, Mariko O; Lee, Sang-Jung; Yamashita, Naoyuki; Miyagawa, Shinichi; Iguchi, Taisen; Okamoto, Seiichiro; Suzuki, Yutaka; Tanaka, Hiroaki

    2015-05-19

    The in vitro estrogen receptor (ER) reporter gene assay has long been used to measure estrogenic activity in wastewater. In a previous study, we demonstrated that the assay represents net estrogenic activity in the balance between estrogenic and antiestrogenic activities in wastewater. However, it remained unclear whether the net estrogenic activity measured by the in vitro ER? reporter gene assay can predict the in vivo estrogenic effect of wastewater. To determine this, we measured the following: estrogenic and antiestrogenic activities of wastewater and reclaimed water by the in vitro ER? reporter gene assay, expression of vitellogenin-1 (vtg1) and choriogenin-H (chgH) in male medaka (Oryzias latipes) by quantitative real-time PCR, and estrone, 17?-estradiol, estriol, and 17?-ethynylestradiol concentrations chemically to predict estrogenic activity. The net estrogenic activity measured by the in vitro medaka ER? reporter gene assay predicted the in vivo vtg1/chgH expression in male medaka more accurately than the concentrations of estrogens. These results also mean that in vivo vtg1/chgH expression in male medaka is determined by the balance between estrogenic and antiestrogenic activities. The in vitro medaka ER? reporter gene assay also predicted in vivo vtg1/chgH expression on male medaka better than the human ER? reporter gene assay. PMID:25902010

  14. PhaF, a Polyhydroxyalkanoate-Granule-Associated Protein of Pseudomonas oleovorans GPo1 Involved in the Regulatory Expression System for pha Genes

    PubMed Central

    Prieto, Maria A.; Bühler, Bruno; Jung, Kuno; Witholt, Bernard; Kessler, Birgit

    1999-01-01

    The phaC1 gene codes for the medium-chain-length polyhydroxyalkanoate (mcl PHA) synthase of Pseudomonas oleovorans GPo1, which produces mcl PHA when grown in an excess of carbon source and under nitrogen limitation. In this work, we have demonstrated, by constructing a recombinant P. oleovorans strain carrying a phaC1::lacZ reporter system, that the phaC1 gene is expressed efficiently in the presence of octanoic acid while its expression is repressed when glucose or citrate is used as the carbon source. Moreover, a P. oleovorans GPo1 mutant (strain GPG-Tc6) expressing higher levels of the reporter gene than the wild-type strain in the presence of glucose or citrate has been generated by mini-Tn5 insertional mutagenesis. Characterization of this mutant allowed us to conclude that phaF, a gene located downstream of the pha gene cluster, was knocked out in this strain. P. oleovorans GPG-Tc6 regained the ability to control phaC1 gene expression when complemented with the phaF wild-type gene. Sequencing data revealed the presence of three complete open reading frames (ORFs) in this region: ORF1 and phaI and phaF genes. The amino acid sequences of the phaI gene product and the N-terminal half of the PhaF protein showed a significant degree of similarity. Furthermore, the primary structure of the PhaF C terminus identifies this protein as a member of the histone H1-like group of proteins. Northern blot analysis showed two transcription units containing phaF, i.e., phaF and phaIF transcripts. Expression of the phaIF operon is more efficient in the presence of octanoic acid and is enhanced by the lack of the PhaF protein. In addition, it has also been demonstrated that both PhaF and PhaI proteins are bound to PHA granules produced by P. oleovorans. A model for the role of PhaF in regulating PHA synthesis is presented. PMID:9922249

  15. Structure and expression of nuclear genes encoding rubisco activase. Final technical report

    SciTech Connect

    Zielinski, R.E.

    1994-06-01

    Rubisco activase (Rca) is a soluble chloroplast protein that catalyzes the activation of rubisco, the enzyme that initiates the photosynthetic carbon reduction cycle, to catalytic competency. Rca in barley consists of three polypeptides, one of 46- and two of 42-kDa, but the quaternary structure of the protein is not known. The authors have isolated and completely sequenced 8.8 kb of barley genomic DNA containing two, tandemly oriented activase genes (RcaA and RcaB) and three different cDNAs encoding the 42- and 46-kDa Rca polypeptide isoforms. Genomic Southern blot assays indicate that these sequences represent the entire Rca gene family in barley. Pre-mRNAs transcribed from the RcaA gene are alternatively spliced to give mRNAs encoding both 46- (RcaA1) and 42-kDa (RcaA2) Rca isoforms. The RcaB gene encodes a single polypeptide of 42 kDa. Primer extension and northern blot assays indicate that RcaB mRNA is expressed at a level that is 10- to 100-fold lower than RcaA mRNA. Analyses at the mRNA and protein level showed that Rca gene expression is coordinated by that of the rubisco subunits during barley leaf development.

  16. Inference of quantitative models of bacterial promoters from time-series reporter gene data.

    PubMed

    Stefan, Diana; Pinel, Corinne; Pinhal, Stéphane; Cinquemani, Eugenio; Geiselmann, Johannes; de Jong, Hidde

    2015-01-01

    The inference of regulatory interactions and quantitative models of gene regulation from time-series transcriptomics data has been extensively studied and applied to a range of problems in drug discovery, cancer research, and biotechnology. The application of existing methods is commonly based on implicit assumptions on the biological processes under study. First, the measurements of mRNA abundance obtained in transcriptomics experiments are taken to be representative of protein concentrations. Second, the observed changes in gene expression are assumed to be solely due to transcription factors and other specific regulators, while changes in the activity of the gene expression machinery and other global physiological effects are neglected. While convenient in practice, these assumptions are often not valid and bias the reverse engineering process. Here we systematically investigate, using a combination of models and experiments, the importance of this bias and possible corrections. We measure in real time and in vivo the activity of genes involved in the FliA-FlgM module of the E. coli motility network. From these data, we estimate protein concentrations and global physiological effects by means of kinetic models of gene expression. Our results indicate that correcting for the bias of commonly-made assumptions improves the quality of the models inferred from the data. Moreover, we show by simulation that these improvements are expected to be even stronger for systems in which protein concentrations have longer half-lives and the activity of the gene expression machinery varies more strongly across conditions than in the FliA-FlgM module. The approach proposed in this study is broadly applicable when using time-series transcriptome data to learn about the structure and dynamics of regulatory networks. In the case of the FliA-FlgM module, our results demonstrate the importance of global physiological effects and the active regulation of FliA and FlgM half-lives for the dynamics of FliA-dependent promoters. PMID:25590141

  17. First Report of the Multidrug Resistance Gene cfr in Enterococcus faecalis of Animal Origin

    PubMed Central

    Liu, Yang; Wang, Yang; Wu, Congming; Shen, Zhangqi; Schwarz, Stefan; Du, Xiang-Dang; Dai, Lei; Zhang, Wanjiang

    2012-01-01

    The multiresistance gene cfr was identified for the first time in an Enterococcus faecalis isolate of animal origin. The 32,388-bp plasmid pEF-01, which carried the cfr gene, was sequenced completely. Three copies of the insertion sequence IS1216 were identified in pEF-01, and the detection of a cfr- and IS1216-containing amplicon by inverse PCR suggests that IS1216 may play a role in the dissemination of cfr by a recombination process. PMID:22203597

  18. Molecular characterization of a maize regulatory gene. Annual progress report, November 1991--October 1992

    SciTech Connect

    Wessler, S.R.

    1994-05-01

    All aspects of this year`s work have converged on the central theme of post-transcriptional control of R gene expression. Unlike transcriptional control, relatively little is known about post-transcriptional regulation, especially in plants. We believe that three levels of post-transcriptional regulation have been identified: control of translation initiation as evidenced by the maize Lc gene; control of nuclear localization as evidenced by the Ds allele r-m9 of maize; and control of nuclear localization through alternative splicing of the rice R homolog.

  19. Characterization of embryo-specific genes. Final report, April 1, 1987--March 31, 1992

    SciTech Connect

    Sung, R.

    1992-06-12

    The objective of the proposed research is to characterize the function and regulation of a set of embryonic genes which are expressed in the embryos, not in the plants. 22 cDNA clones were isolated from a cDNA library we constructed using mRNAS of -carrot somatic embryos. These cDNA clones identified mRNA species that are present in the somatic and zygotic embryos, but not in adult plants. The sequence of all 22cDNA clones were determined; genomic clones for three cDNA clones, DC8, DC59, and DC49 were isolated and gene sequences determined. DC8, DC49, and several other genes identified by the cDNA sequences belong to the category of late embryogenesis abundant protein genes, Lea. The function of these gens have not yet been determined, but they share common structural features, are regulated by ABA and are speculated to play a role in seed desiccation.

  20. Transgene-based anthocyanin hyper-pigmentation as a visual reporter of gene silencing in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    “Co-suppression” associated loss of flower pigmentation in transgenic petunia plants was one of the first clear indicators of the natural process of RNA-associated gene silencing in plants. We have been exploring the use of genetically engineered anthocyanin over-production in vegetative tissues as...

  1. REPORT AND RECOMMENDATIONS OF THE PANEL TO ASSESS THE NIH INVESTMENT IN RESEARCH ON GENE THERAPY

    Microsoft Academic Search

    Stuart H. Orkin; Arno G. Motulsky

    Dr. Harold Varmus, Director, National Institutes of Health (NIH), appointed an ad hoc committee to assess the current status and promise of gene therapy and provide recommendations regarding future NIH-sponsored research in this area. The Panel was asked specifically to comment on how funds and efforts should be distributed among various research areas and what funding mechanisms would be most

  2. Illuminating transcription pathways using fluorescent reporter genes and yeast functional genomics.

    PubMed

    Kainth, Pinay; Andrews, Brenda

    2010-01-01

    Technological advances have enabled researchers to probe gene regulatory pathways on an unprecedented scale. Here, we summarize our recent work that exploits a systematic screening approach in the budding yeast to discover regulators of a promoter of interest. We discuss future applications of our approach based on emerging themes in the literature. PMID:21326895

  3. Illuminating transcription pathways using fluorescent reporter genes and yeast functional genomics

    PubMed Central

    Kainth, Pinay

    2010-01-01

    Technological advances have enabled researchers to probe gene regulatory pathways on an unprecedented scale. Here, we summarize our recent work that exploits a systematic screening approach in the budding yeast to discover regulators of a promoter of interest. We discuss future applications of our approach based on emerging themes in the literature. PMID:21326895

  4. UMG Lenti: Novel Lentiviral Vectors for Efficient Transgene- and Reporter Gene Expression in Human Early Hematopoietic Progenitors

    PubMed Central

    Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G.; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B.; Grillone, Teresa; Giovannone, Emilia D.; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A. S.; Bond, Heather M.; Mesuraca, Maria; Morrone, Giovanni

    2014-01-01

    Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and –LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG–LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and progenitor cells, as well as in non-hematopoietic cells. PMID:25502183

  5. Hazardous effect of organophosphate compound, dichlorvos in transgenic Drosophila melanogaster (hsp70-lacZ): induction of hsp70, anti-oxidant enzymes and inhibition of acetylcholinesterase.

    PubMed

    Gupta, Subash Chandra; Siddique, Hifzur Rahman; Saxena, Daya Krishna; Chowdhuri, Debapratim Kar

    2005-08-30

    We tested a working hypothesis that stress genes and anti-oxidant enzyme machinery are induced by the organophosphate compound dichlorvos in a non-target organism. Third instar larvae of Drosophila melanogaster transgenic for hsp70 were exposed to 0.1 to 100.0 ppb dichlorvos and 5.0 mM CuSO(4) (an inducer of oxidative stress and stress genes) and hsp70, and activities of acetylcholinesterase (AchE), superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (LPO) product were measured. The study was further extended to examine tissue damage, if any, under such conditions. A concentration- and time-dependent increase in hsp70 and anti-oxidant enzymes was observed in the exposed organism as compared to control. A comparison of stress gene expression with SOD, CAT activities and LPO product under similar experimental conditions revealed that induction of hsp70 precedes the anti-oxidant enzyme activities in the exposed organism. Further, concomitant with a significant inhibition of AChE activity, significant induction of hsp70 was observed following chemical exposure. Mild tissue damage was observed in the larvae exposed to 10.0 ppb dichlorvos for 48 h when hsp70 expression reaches plateau. Dichlorvos at 0.1 ppb dietary concentration did not evoke significant hsp70 expression, anti-oxidant enzymes and LPO and AchE inhibition in the exposed organism, and thereby, was found to be non-hazardous to D. melanogaster. Conversely, 1.0 ppb of the test chemical stimulated a significant induction of hsp70 and anti-oxidant enzymes and significant inhibition of AchE; hence this concentration of test chemical was hazardous to the organism. The present study suggests that (a) both stress genes and anti-oxidant enzymes are stimulated as indices of cellular defense against xenobiotic hazard in D. melanogaster with hsp70 being proposed as first-tier bio-indicator of cellular hazard, (b) 0.1 ppb of the test chemical may be regarded as No Observed Adverse Effect Level (NOAEL), and 1.0 ppb dichlorvos as Low Observed Adverse Effect Level (LOAEL). PMID:16023296

  6. Pheromone-regulated Genes Required for Yeast Mating Differentiation

    PubMed Central

    Erdman, Scott; Lin, Li; Malczynski, Michael; Snyder, Michael

    1998-01-01

    Yeast cells mate by an inducible pathway that involves agglutination, mating projection formation, cell fusion, and nuclear fusion. To obtain insight into the mating differentiation of Saccharomyces cerevisiae, we carried out a large-scale transposon tagging screen to identify genes whose expression is regulated by mating pheromone. 91,200 transformants containing random lacZ insertions were screened for ?-galactosidase (?-gal) expression in the presence and absence of ? factor, and 189 strains containing pheromone-regulated lacZ insertions were identified. Transposon insertion alleles corresponding to 20 genes that are novel or had not previously been known to be pheromone regulated were examined for effects on the mating process. Mutations in four novel genes, FIG1, FIG2, KAR5/ FIG3, and FIG4 were found to cause mating defects. Three of the proteins encoded by these genes, Fig1p, Fig2p, and Fig4p, are dispensible for cell polarization in uniform concentrations of mating pheromone, but are required for normal cell polarization in mating mixtures, conditions that involve cell–cell communication. Fig1p and Fig2p are also important for cell fusion and conjugation bridge shape, respectively. The fourth protein, Kar5p/Fig3p, is required for nuclear fusion. Fig1p and Fig2p are likely to act at the cell surface as Fig1:: ?-gal and Fig2::?-gal fusion proteins localize to the periphery of mating cells. Fig4p is a member of a family of eukaryotic proteins that contain a domain homologous to the yeast Sac1p. Our results indicate that a variety of novel genes are expressed specifically during mating differentiation to mediate proper cell morphogenesis, cell fusion, and other steps of the mating process. PMID:9456310

  7. Development of a fish reporter gene system for the assessment of estrogenic compounds and sewage treatment plant effluents.

    PubMed

    Ackermann, Gabriele E; Brombacher, Eva; Fent, Karl

    2002-09-01

    This study reports on the development and application of a fish-specific estrogen-responsive reporter gene assay. The assay is based on the rainbow trout (Oncorhynchus mykiss) gonad cell line RTG-2 in which an acute estrogenic response is created by cotransfecting cultures with an expression vector containing rainbow trout estrogen receptor a complementary DNA (rtERalpha cDNA) in the presence of an estrogen-dependent reporter plasmid and an estrogen receptor (ER) agonist. In a further approach, RTG-2 cells were stably transfected with the rtERalpha cDNA expression vector, and clones responsive to 17beta-estradiol (E2) were selected. The estrogenic activity of E2, 17alpha-ethinylestradiol, 4-nonylphenol, nonylphenoxy acetic acid, 4-tert-octylphenol, bisphenol A, o,p'-DDT, p,p'-DDT, o,p'-2,2-bis(chlorophenyl)-1,1-dichloroethylene (o,p'-DDE), p,p'-DDE, o,p'-2,2-bis(chlorophenyl)-1,1-di-chloroethane (o,p'-DDD), p,p'-DDD, and p,p'-2,2-bis(chlorophenyl)acetic acid (p,p'-DDA) was assessed at increasing concentrations. All compounds except o,p'-DDT, p,p'-DDE, and p,p'-DDA showed logistic dose-response curves, which allowed the calculation of lowest-observed-effect concentrations and the concentrations at which half-maximal reporter gene activities were reached. To check whether estrogen-responsive RTG-2 cells may be used to detect the estrogenic activity of environmental samples, an extract from a sewage treatment plant (STP) effluent was assessed and found to have estrogenic activity corresponding to the transcriptional activity elicited by 0.05 nM of E2. Dose-response curves of nonylphenol, octylphenol, bisphenol A, and o,p'-DDD revealed that the RTG-2 reporter gene assay is more sensitive for these compounds when compared to transfection systems recombinant for mammalian ERs. These differences may have an effect on the calculation of E2 equivalents when estrogenic mixtures of known constitution, or environmental samples, such as STP effluents, are assessed. PMID:12206426

  8. Single Cell Quantification of Reporter Gene Expression in Live Adult Caenorhabditis elegans Reveals Reproducible Cell-Specific Expression Patterns and Underlying Biological Variation

    PubMed Central

    Mendenhall, Alexander R.; Tedesco, Patricia M.; Sands, Bryan; Johnson, Thomas E.; Brent, Roger

    2015-01-01

    In multicellular organisms such as Caenorhabditis elegans, differences in complex phenotypes such as lifespan correlate with the level of expression of particular engineered reporter genes. In single celled organisms, quantitative understanding of responses to extracellular signals and of cell-to-cell variation in responses has depended on precise measurement of reporter gene expression. Here, we developed microscope-based methods to quantify reporter gene expression in cells of Caenorhabditis elegans with low measurement error. We then quantified expression in strains that carried different configurations of Phsp-16.2-fluorescent-protein reporters, in whole animals, and in all 20 cells of the intestine tissue, which is responsible for most of the fluorescent signal. Some animals bore more recently developed single copy Phsp-16.2 reporters integrated at defined chromosomal sites, others, “classical” multicopy reporter gene arrays integrated at random sites. At the level of whole animals, variation in gene expression was similar: strains with single copy reporters showed the same amount of animal-to-animal variation as strains with multicopy reporters. At the level of cells, in animals with single copy reporters, the pattern of expression in cells within the tissue was highly stereotyped. In animals with multicopy reporters, the cell-specific expression pattern was also stereotyped, but distinct, and somewhat more variable. Our methods are rapid and gentle enough to allow quantification of expression in the same cells of an animal at different times during adult life. They should allow investigators to use changes in reporter expression in single cells in tissues as quantitative phenotypes, and link those to molecular differences. Moreover, by diminishing measurement error, they should make possible dissection of the causes of the remaining, real, variation in expression. Understanding such variation should help reveal its contribution to differences in complex phenotypic outcomes in multicellular organisms. PMID:25946008

  9. Transient expression of ? ? ? ?-glucuronidase reporter gene in Agrobacterium-inoculated shoots of various teak clones

    Microsoft Academic Search

    Sri Nanan Widiyanto; Arfri Sukmawan; Nancy Haro; Heni Rahmania

    Agrobacterium tumefaciens strain LBA4404 carrying the pBI.121 binary plasmid was used in transformation to introduce the gus (ß-glucuronidase\\/GUS) gene into teak shoot-tissues. In vitro regenerated shoots from various teak clones, i.e. the ITB, GT, P97, P96, P75, P20, and P108 clones were vacuum-infiltrated for 5 min in the suspension culture of A. tumefaciens. Seven days after selection period, the evidence

  10. Analysis of promoters in Borrelia burgdorferi by use of a transiently expressed reporter gene.

    PubMed Central

    Sohaskey, C D; Arnold, C; Barbour, A G

    1997-01-01

    A transient chloramphenicol acetyltransferase (CAT) expression system was developed for Borrelia burgdorferi. An Escherichia coli vector containing a promoterless Streptococcus agalactiae cat gene was constructed. Promoters for ospA, ospC, and flaB were placed upstream of this cat gene, and CAT assays were performed in E. coli from these stably maintained plasmids. The plasmids with putative promoters ospA and flaB were found to be approximately 20-fold more active than were the plasmids with ospC or no promoter. The level of activity correlated well with the resistance to chloramphenicol that each plasmid provided. Next, the nonreplicative plasmid constructs were transformed by electroporation into B. burgdorferi. CAT assays were performed by both thin-layer chromatography and the fluor diffusion method. Measurement of CAT activity demonstrated that the ospA promoter was again about 20-fold more active than the promoterless cat gene. The flaB and ospC promoters increased the activity seven- and threefold, respectively, over that with the promoterless construct. This simple transient-expression assay was shown to be an effective method to study promoter function in B. burgdorferi in the absence of a well-developed genetic system. PMID:9352937

  11. [Iron regulation of gene expression in the Bradyrhizobium japonicum/soybean symbiosis]. Progress report

    SciTech Connect

    Guerinot, M.L.

    1992-06-01

    We wish to address the question of whether iron plays a regulatory role in the Bradyrhizobium japonicum/soybeam symbiosis. Iron may be an important regulatory signal in planta as the bacteria must acquire iron from their plant hosts and iron-containing proteins figure prominently in all nitrogen-fixing symbioses. For example, the bacterial partner is believed to synthesize the heme moiety of leghemoglobin, which may represent as much as 25--30% of the total soluble protein in an infected plant cell. For this reason, we have focused our attention on the regulation by iron of the first step in the bacterial heme biosynthetic pathway. The enzyme which catalyzes this step, 5-aminolevulinic acid synthase, is encoded by the hemA gene which we had previously cloned and sequenced. Specific objectives include: to define the cis-acting sequences which confer iron regulation on the B. japonicum hemA gene; to identify trans-acting factors which regulate the expression of hemA by iron; to identify new loci which are transcriptionally responsive to changes in iron availability; and to examine the effects of mutations in various known regulatory genes for their effect on the expression of hemA.

  12. (Structure and expression of nuclear genes encoding rubisco activase): Progress report

    SciTech Connect

    Not Available

    1989-01-01

    Our first year's activities include: (1) completing a survey of the basic characteristics of activase gene expression in barley; and (2) isolating and structurally characterizing cDNA and genomic DNA sequences encoding activase from barley. Our goal was to determine whether activase mRNA and protein accumulation are coordinated with those of the rubisco subunits. We utilized the first leaves of barley as an experimental system for these studies because they can be used in two ways to study the expression of leaf genes: by following the naturally occurring differentiation of leaf cells, which occurs acropetally along the barley leaf; and by following the photomorphogenesis of etiolated barley seedlings. In the acropetal gradient of leaf cell differentiation, activase mRNA and mRNA and polypeptide expression is tightly coordinated with rubisco subunit mRNA and polypeptide expression. Although we have not measured their precise stoichiometry at each stage of leaf differentiation, activase protein is expressed at the level of about one polypeptide per rubisco holoenzyme in mature regions of the leaf. Coordination of the expression of activase mRNAs and polypeptides indicates that in the barley leaf gradient, activase gene expression is largely controlled at the level of transcription. However, translational controls may play a role in regulating activase expression on a short term basis.

  13. Suicidal gene therapy in an NF-?B-controlled tumor environment as monitored by a secreted blood reporter

    PubMed Central

    Badr, CE; Niers, JM; Morse, D; Koelen, JA; Vandertop, P; Noske, D; Wurdinger, T; Zalloua, PA; Tannous, BA

    2013-01-01

    The nuclear factor-?B (NF-?B) is known to be activated in many cancer types including lung, ovarian, astrocytomas, melanoma, prostate as well as glioblastoma, and has been shown to correlate with disease progression. We have cloned a novel NF-?B-based reporter system (five tandem repeats of NF-?B responsive genomic element (NF; 14bp each)) to drive the expression cassette for both a fusion between the yeast cytosine deaminase and uracil phosphoribosyltransferase (CU) as a therapeutic gene and the secreted Gaussia luciferase (Gluc) as a blood reporter, separated by an internal ribosomal entry site (NF-CU-IGluc). We showed that malignant tumor cells have high expression of Gluc, which correlates to high activation of NF-?B. When NF-?B was further activated by tumor necrosis factor-? in these cells, we observed up to 10-fold increase in Gluc levels and therefore transgene expression in human glioma cells served to greatly enhance the sensitization of these cells to the prodrug, 5-fluorocytosine both in cultured cells and in vivo subcutaneous tumor xenograft model. This inducible system provides a tool to enhance the expression of imaging and therapeutic genes for cancer therapy. PMID:21150937

  14. Recombinant receptor/reporter gene bioassays for assessing the estrogenic and dioxin-like activities of xenobiotics and complex mixtures

    SciTech Connect

    Zacharewski, T. [Univ. of Western Ontario, London, Ontario (Canada). Dept. of Pharmacology and Toxicology

    1995-12-31

    Exposure to naturally occurring or synthetic substances that possess sex steroid and/or dioxin-like activity may have long range effects on human health, reproductive fitness and environmental quality. Results from recent epidemiological studies have suggested that xenobiotics with sex steroid activity may contribute to the development of hormone-dependent cancers and disorders in the male reproductive tract as well as attenuate sperm production. However, most of these compounds, which are referred to as endocrine disruptors, are structurally dissimilar to sex steroids. Yet, based upon ambiguous assays, it has been conceded that the effects of these compounds are mediated by receptors. The authors have taken advantage of the mechanism of action of these compounds to develop recombinant receptor/reporter gene bioassays for environmental estrogens and dioxin-like compounds. The assays use an easily measurable enzyme activity (i.e. firefly luciferase), exhibit improved sensitivity and selectivity and are amenable to automation. Data will be presented demonstrating that phytoestrogens (e.g. genistein) and xenobiotics such as pesticides (e.g. DDT, Kepone), nonionic surfactants (e.g. p-nonylphenol), and precursors used in the manufacture of plastics (e.g. Bisphenol A) exhibit estrogenic activity. In addition, the assays have been used to detect estrogenic and dioxin-like activity in complex mixtures such as pulp and paper mill black liquor and effluent. These results demonstrate the utility of recombinant receptor/reporter gene bioassays for identifying substances or complex mixtures with estrogenic and/or dioxin-like activity.

  15. A seven-year storage report of good manufacturing practice-grade naked plasmid DNA: stability, topology, and in vitro/in vivo functional analysis.

    PubMed

    Walther, Wolfgang; Schmeer, Marco; Kobelt, Dennis; Baier, Ruth; Harder, Alexander; Walhorn, Volker; Anselmetti, Dario; Aumann, Jutta; Fichtner, Iduna; Schleef, Martin

    2013-12-01

    The great interest for naked plasmid DNA in gene therapy studies is reflected by the fact that it is currently used in 18% of all gene therapy trials. Therefore, validation of topology and functionality of DNA resulting from its long-term stability is an essential requirement for safe and effective gene transfer. To this aim, we analyzed the stability of good manufacturing practice-grade pCMV? reporter plasmid DNA by capillary gel electrophoresis, agarose gel electrophoresis, and atomic force microscopy. The plasmid DNA was produced for a clinical gene transfer study started in 2005 and was stored for meanwhile 7 years under continuously monitored conditions at -20 °C. The stability of plasmid DNA was monitored by LacZ transgene expression functional assays performed in vitro and in vivo on the 7-year-old plasmid DNA samples compared with plasmid batches newly produced in similar experimental conditions and quality standards. The analyses revealed that during the overall storage time and conditions, the proportion of open circular and supercoiled or covalently closed circular forms is conserved without linearization or degradation of the plasmid. The in vitro transfection and the in vivo jet-injection of DNA showed unaltered functionality of the long-stored plasmid. In summary, the 7-year-old and the newly produced plasmid samples showed similar topology and expression performance. Therefore, our stable storage conditions are effective to preserve the integrity of the DNA to be used in clinical studies. This is an important prerequisite for the long-term performance of gene transfer materials used in trials of long duration as well as of the reference material used in standardization procedures and assays. PMID:24067054

  16. Bordetella AlcS Transporter Functions in Alcaligin Siderophore Export and Is Central to Inducer Sensing in Positive Regulation of Alcaligin System Gene Expression

    PubMed Central

    Brickman, Timothy J.; Armstrong, Sandra K.

    2005-01-01

    Bordetella pertussis and Bordetella bronchiseptica, which are respiratory mucosal pathogens of mammals, produce and utilize the siderophore alcaligin to acquire iron in response to iron starvation. A predicted permease of the major facilitator superfamily class of membrane efflux pumps, AlcS (synonyms, OrfX and Bcr), was reported to be encoded within the alcaligin gene cluster. In this study, alcS null mutants were found to be defective in growth under iron starvation conditions, in iron source utilization, and in alcaligin export. trans complementation using cloned alcS genes of B. pertussis or B. bronchiseptica restored the wild-type phenotype to the alcS mutants. Although the levels of extracellular alcaligin measured in alcS strain culture fluids were severely reduced compared with the wild-type levels, alcS mutants had elevated levels of cell-associated alcaligin, implicating AlcS in alcaligin export. Interestingly, a ?alcA mutation that eliminated alcaligin production suppressed the growth defects of alcS mutants. This suppression and the alcaligin production defect were reversed by trans complementation of the ?alcA mutation in the double-mutant strain, confirming that the growth-defective phenotype of alcS mutants is associated with alcaligin production. In an alcA::mini-Tn5 lacZ1 operon fusion strain background, an alcS null mutation resulted in enhanced AlcR-dependent transcriptional responsiveness to alcaligin inducer; conversely, AlcS overproduction blunted the transcriptional response to alcaligin. These transcription studies indicate that the alcaligin exporter activity of AlcS is required to maintain appropriate intracellular alcaligin levels for normal inducer sensing and responsiveness necessary for positive regulation of alcaligin system gene expression. PMID:15901687

  17. A picornaviral 2Alike Sequence-based Tricistronic Vector Allowing for High-level Therapeutic Gene Expression Coupled to a Dual-Reporter System

    Microsoft Academic Search

    Mark J. Osborn; Angela Panoskaltsis-Mortari; Ron T. McElmurry; Scott K. Bell; Dario A. A. Vignali; Martin D. Ryan; Andrew C. Wilber; R. Scott McIvor; Jakub Tolar; Bruce R. Blazar

    2005-01-01

    The 2A-like sequences from members of the picornavirus family were utilized to construct a tricistronic vector bearing the human iduronidase (IDUA) gene along with the firefly luciferase and DsRed2 reporter genes. The 2A-like sequences mediate a cotranslational cleavage event resulting in the release of each individual protein product. Efficient cleavage was observed and all three proteins were functional in vitro

  18. Integrative vectors for constructing single-copy transcriptional fusions between Bacillus subtilis promoters and various reporter genes encoding heat-stable enzymes

    Microsoft Academic Search

    Axel Mogk; Richard Hayward; Wolfgang Schumann

    1996-01-01

    Here, we report on the construction of three integrative plasmids for Bacillus subtilis (Bs) allowing in vitro construction of transcriptional fusions. These plasmids contain a neomycin- or tetracycline-resistance cassette and one of three promoterless genes: bgaB (encoding ?-galactosidase), cat (chloramphenicol acetyltransferase), or xylE (catechol 2,3-dioxygenase). All cassettes are flanked by the 3?- and 5?-ends of the amyE gene (encoding f?-amylase)

  19. Connexin 47 (Cx47)Deficient Mice with Enhanced Green Fluorescent Protein Reporter Gene Reveal Predominant Oligodendrocytic Expression of Cx47 and Display Vacuolized Myelin in the CNS

    Microsoft Academic Search

    Benjamin Odermatt; Kerstin Wellershaus; Anke Wallraff; Gerald Seifert; Joachim Degen; Carsten Euwens; Babette Fuss; Heinrich Bussow; Karl Schilling; Christian Steinhauser; Klaus Willecke

    To further characterize the recently described gap junction gene connexin 47 (Cx47), we generated Cx47-null mice by replacing the Cx47 coding DNA with an enhanced green fluorescent protein (EGFP) reporter gene, which was thus placed under control of the endogenous Cx47 promoter. Homozygous mutant mice were fertile and showed no obvious morphological or behavioral abnormalities. Colocaliza- tion of EGFP fluorescence

  20. Agrobacterium rhizogenes lacZ-rolC gene expression in Escherichia coli: detection of the product in transgenic plants using RolC-specific antibodies.

    PubMed

    Oono, Y; Satomi, T; Uchimiya, H

    1991-07-31

    The rolC sequence of the Agrobacterium rhizogenes Ri plasmid was fused in-frame to the 3' end of the lacZ gene in plasmid pEX3. The fusion protein RolC-beta-galactosidase was accumulated as insoluble inclusion bodies in Escherichia coli. Antibodies were raised in rabbits against the fusion protein. After affinity purification, RolC-specific antibodies were found to react with a 22-kDa polypeptide prepared from roots of transgenic tobacco plants possessing a rolC gene. The result of differential centrifugation suggested that RolC is present in the soluble fraction of transformed cells. PMID:1916283

  1. Eukaryotic transcription and processing: regulation of gene expression. Final report, November 1, 1978-April 30, 1980

    SciTech Connect

    Mans, R.J.

    1980-01-01

    During this contract period, two major research objectives were accomplished: (1) the diverse activities of the maize poly A polymerase purified from seedlings were resolved and the proteins responsible were identified, and (2) maize DNA sequences, derived from mitochondria and incorporated into a circular template were transcribed by the RNA polymerase II purified from maize seedlings. The transition in research emphasis from characterization of enzymes and other cellular components involved in gene expression generally to a definitive characterization of molecules and molecular events underlying cytoplasmic male sterility (cms) in maize has been accomplished.

  2. Codon-optimized Human Sodium Iodide Symporter (opt-hNIS) as a Sensitive Reporter and Efficient Therapeutic Gene

    PubMed Central

    Kim, Young-Hwa; Youn, Hyewon; Na, Juri; Hong, Kee-Jong; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key

    2015-01-01

    To generate a more efficient in vivo reporter and therapeutic gene, we optimized the coding sequence of the human sodium/iodide symporter (NIS) gene by replacing NIS DNA codons from wild type to new codons having the highest usage in human gene translation. The Codon Adaptation Index (CAI), representing the number of codons effective for human expression, was much improved (0.79 for hNIS, 0.97 for opt-hNIS). Both wild-type (hNIS) and optimized human NIS (opt-hNIS) were cloned into pcDNA3.1 and pMSCV vectors for transfection. Various cancer cell lines such as thyroid (TPC-1, FRO, B-CPAP), breast (MDA-MB-231), liver (Hep3B), cervical (HeLa), and glioma (U87MG) were transfected with pcDNA3.1/hNIS or pcDNA3.1/opt-hNIS. 125I uptake by opt-hNIS-expressing cells was 1.6 ~ 2.1 times higher than uptake by wild-type hNIS-expressing cells. Stable cell lines were also established by retroviral transduction using pMSCV/hNIS or pMSCV/opt-hNIS, revealing higher NIS protein levels and 125I uptake in opt-hNIS-expressing cells than in hNIS-expressing cells. Moreover, scintigraphic images from cell plates and mouse xenografts showed stronger signals from opt-hNIS-expressing cells than hNIS-expressing cells, and radioactivity uptake by opt-hNIS-expressing tumors was 2.3-fold greater than that by hNIS-expressing tumors. To test the efficacy of radioiodine therapy, mouse xenograft models were established with cancer cells expressing hNIS or opt-hNIS. 131I treatment reduced tumor sizes of hNIS- and opt-hNIS-expressing tumors to 0.57- and 0.27- fold, respectively, compared to their sizes before therapy, suggesting an improved therapeutic effect of opt-hNIS. In summary, this study shows that codon optimization strongly increases hNIS protein levels and radioiodine uptake, thus supporting opt-hNIS as a more sensitive reporter and efficient therapeutic gene. PMID:25553100

  3. Codon-optimized human sodium iodide symporter (opt-hNIS) as a sensitive reporter and efficient therapeutic gene.

    PubMed

    Kim, Young-Hwa; Youn, Hyewon; Na, Juri; Hong, Kee-Jong; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key

    2015-01-01

    To generate a more efficient in vivo reporter and therapeutic gene, we optimized the coding sequence of the human sodium/iodide symporter (NIS) gene by replacing NIS DNA codons from wild type to new codons having the highest usage in human gene translation. The Codon Adaptation Index (CAI), representing the number of codons effective for human expression, was much improved (0.79 for hNIS, 0.97 for opt-hNIS). Both wild-type (hNIS) and optimized human NIS (opt-hNIS) were cloned into pcDNA3.1 and pMSCV vectors for transfection. Various cancer cell lines such as thyroid (TPC-1, FRO, B-CPAP), breast (MDA-MB-231), liver (Hep3B), cervical (HeLa), and glioma (U87MG) were transfected with pcDNA3.1/hNIS or pcDNA3.1/opt-hNIS. 125I uptake by opt-hNIS-expressing cells was 1.6~2.1 times higher than uptake by wild-type hNIS-expressing cells. Stable cell lines were also established by retroviral transduction using pMSCV/hNIS or pMSCV/opt-hNIS, revealing higher NIS protein levels and 125I uptake in opt-hNIS-expressing cells than in hNIS-expressing cells. Moreover, scintigraphic images from cell plates and mouse xenografts showed stronger signals from opt-hNIS-expressing cells than hNIS-expressing cells, and radioactivity uptake by opt-hNIS-expressing tumors was 2.3-fold greater than that by hNIS-expressing tumors. To test the efficacy of radioiodine therapy, mouse xenograft models were established with cancer cells expressing hNIS or opt-hNIS. 131I treatment reduced tumor sizes of hNIS- and opt-hNIS-expressing tumors to 0.57- and 0.27- fold, respectively, compared to their sizes before therapy, suggesting an improved therapeutic effect of opt-hNIS. In summary, this study shows that codon optimization strongly increases hNIS protein levels and radioiodine uptake, thus supporting opt-hNIS as a more sensitive reporter and efficient therapeutic gene. PMID:25553100

  4. Differential gene expression in Neurospora crassa cell types: Ribosomal RNA genes. Comprehensive final terminal report of the overall activities for past 20 years

    SciTech Connect

    Dutta, S.K.

    1988-03-01

    This paper summarizes the accomplishments over the past 20 years of DOE support to the author`s research program. Highlights include molecular analysis of genome of Neurospora crassa, RNase sensitive RNA-dependent DNA polymerase, gene amplification of rRNA genes, molecular cloning of r-DNA`s, restriction fragment length polymorphism of rDNA in N. crassa., and ribosomal RNA processing genes of N. crassa.

  5. In vivo pattern of lipopolysaccharide and anti-CD3-induced NF-kappa B activation using a novel gene-targeted enhanced GFP reporter gene mouse.

    PubMed

    Magness, Scott T; Jijon, Humberto; Van Houten Fisher, Nancy; Sharpless, Ned E; Brenner, David A; Jobin, Christian

    2004-08-01

    NF-kappa B is a family of transcription factors involved in regulating cell death/survival, differentiation, and inflammation. Although the transactivation ability of NF-kappa B has been extensively studied in vitro, limited information is available on the spatial and temporal transactivation pattern in vivo. To investigate the kinetics and cellular localization of NF-kappa B-induced transcription, we created a transgenic mouse expressing the enhanced GFP (EGFP) under the transcriptional control of NF-kappa B cis elements (cis-NF-kappa B(EGFP)). A gene-targeting approach was used to insert a single copy of a NF-kappa B-dependent EGFP reporter gene 5' of the X-linked hypoxanthine phosphoribosyltransferase locus in mouse embryonic stem cells. Embryonic fibroblasts, hepatic stellate cells, splenocytes, and dendritic cells isolated from cis-NF-kappa B(EGFP) mice demonstrated a strong induction of EGFP in response to LPS, anti-CD3, or TNF-alpha that was blocked by the NF-kappa B inhibitors BAY 11-7082 and NEMO-binding peptide. Chromatin immunoprecipitation analysis demonstrated RelA binding to the cis-NF-kappa B(EGFP) promoter. Adenoviral delivery of NF-kappa B-inducing kinase strongly induced EGFP expression in the liver of cis-NF-kappa B(EGFP) mice. Similarly, mice injected with anti-CD3 or LPS showed increased EGFP expression in mononuclear cells, lymph node, spleen, and liver as measured by flow cytometry and/or fluorescence microscopy. Using whole organ imaging, LPS selectively induced EGFP expression in the duodenum and proximal jejunum, but not in the ileum and colon. Confocal analysis indicated EGFP expression was primarily found in lamina propria mononuclear cells. In summary, the cis-NF-kappa B(EGFP) mouse will serve as a valuable tool to address multiple questions regarding the cell-specific and real-time activation of NF-kappa B during normal and diseased states. PMID:15265883

  6. Reliable Gene Expression Analysis by Reverse Transcription-Quantitative PCR: Reporting and Minimizing the Uncertainty in Data Accuracy[W][OPEN

    PubMed Central

    Remans, Tony; Keunen, Els; Bex, Geert Jan; Smeets, Karen; Vangronsveld, Jaco; Cuypers, Ann

    2014-01-01

    Reverse transcription-quantitative PCR (RT-qPCR) has been widely adopted to measure differences in mRNA levels; however, biological and technical variation strongly affects the accuracy of the reported differences. RT-qPCR specialists have warned that, unless researchers minimize this variability, they may report inaccurate differences and draw incorrect biological conclusions. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines describe procedures for conducting and reporting RT-qPCR experiments. The MIQE guidelines enable others to judge the reliability of reported results; however, a recent literature survey found low adherence to these guidelines. Additionally, even experiments that use appropriate procedures remain subject to individual variation that statistical methods cannot correct. For example, since ideal reference genes do not exist, the widely used method of normalizing RT-qPCR data to reference genes generates background noise that affects the accuracy of measured changes in mRNA levels. However, current RT-qPCR data reporting styles ignore this source of variation. In this commentary, we direct researchers to appropriate procedures, outline a method to present the remaining uncertainty in data accuracy, and propose an intuitive way to select reference genes to minimize uncertainty. Reporting the uncertainty in data accuracy also serves for quality assessment, enabling researchers and peer reviewers to confidently evaluate the reliability of gene expression data. PMID:25361954

  7. A report of a national mutation testing service for the MEN1 gene: clinical presentations and implications for mutation testing

    PubMed Central

    Cardinal, J; Bergman, L; Hayward, N; Sweet, A; Warner, J; Marks, L; Learoyd, D; Dwight, T; Robinson, B; Epstein, M; Smith, M; Teh, B; Cameron, D; Prins, J

    2005-01-01

    Introduction: Mutation testing for the MEN1 gene is a useful method to diagnose and predict individuals who either have or will develop multiple endocrine neoplasia type 1 (MEN 1). Clinical selection criteria to identify patients who should be tested are needed, as mutation analysis is costly and time consuming. This study is a report of an Australian national mutation testing service for the MEN1 gene from referred patients with classical MEN 1 and various MEN 1-like conditions. Results: All 55 MEN1 mutation positive patients had a family history of hyperparathyroidism, had hyperparathyroidism with one other MEN1 related tumour, or had hyperparathyroidism with multiglandular hyperplasia at a young age. We found 42 separate mutations and six recurring mutations from unrelated families, and evidence for a founder effect in five families with the same mutation. Discussion: Our results indicate that mutations in genes other than MEN1 may cause familial isolated hyperparathyroidism and familial isolated pituitary tumours. Conclusions: We therefore suggest that routine germline MEN1 mutation testing of all cases of "classical" MEN1, familial hyperparathyroidism, and sporadic hyperparathyroidism with one other MEN1 related condition is justified by national testing services. We do not recommend routine sequencing of the promoter region between nucleotides 1234 and 1758 (Genbank accession no. U93237) as we could not detect any sequence variations within this region in any familial or sporadic cases of MEN1 related conditions lacking a MEN1 mutation. We also suggest that testing be considered for patients <30 years old with sporadic hyperparathyroidism and multigland hyperplasia. PMID:15635078

  8. Imprinted genes and transpositions: epigenomic targets for low dose radiation effects. Final report

    SciTech Connect

    Jirtle, Randy L.

    2012-10-11

    The overall hypothesis of this grant application is that low dose ionizing radiation (LDIR) elicits adaptive responses in part by causing heritable DNA methylation changes in the epigenome. This novel postulate was tested by determining if the level of DNA methylation at the Agouti viable yellow (A{sup vy}) metastable locus is altered, in a dose-dependent manner, by low dose radiation exposure (<10 cGy) during early gestation. This information is particularly important to ascertain given the increased use of CT scans in disease diagnosis, increased number of people predicted to live and work in space, and the present concern about radiological terrorism. We showed for the first time that LDIR significantly increased DNA methylation at the A{sup vy} locus in a sex-specific manner (p=0.004). Average DNA methylation was significantly increased in male offspring exposed to doses between 0.7 cGy and 7.6 cGy with maximum effects at 1.4 cGy and 3.0 cGy (p<0.01). Offspring coat color was concomitantly shifted towards pseudoagouti (p<0.01). Maternal dietary antioxidant supplementation mitigated both the DNA methylation changes and coat color shift in the irradiated offspring (p<0.05). Thus, LDIR exposure during gestation elicits epigenetic alterations that lead to positive adaptive phenotypic changes that are negated with antioxidants, indicating they are mediated in part by oxidative stress. These findings provide evidence that in the isogenic Avy mouse model epigenetic alterations resulting from LDIR play a role in radiation hormesis, bringing into question the assumption that every dose of radiation is harmful. Our findings not only have significant implications concerning the mechanism of hormesis, but they also emphasize the potential importance of this phenomenon in determining human risk at low radiation doses. Since the epigenetic regulation of genes varies markedly between species, the effect of LDIR on other epigenetically labile genes (e.g. imprinted genes) in animals and humans needs to be defined.

  9. 716. The Human Norepinephrine Transporter and [11C]-mHED, a Novel Reporter Gene-Tracer Combination for PET Imaging of Gene Therapy

    Microsoft Academic Search

    Antoine M. J. Beerens; Anne Rixt Buursma; Marianne G. Rots; Aren van Waarde; Hidde J. Haisma; Erik F. J. de Vries

    2004-01-01

    Currently many clinical protocols for gene therapy are being evaluated for use in the treatment of human disease. In the majority of these protocols however, it is difficult to determine the exact fate of the vector, or to determine the location and extend of expression of the introduced gene. Data about the expression of transgenes is not only invaluable in

  10. ASSOCIATIONS BETWEEN CYTOKINE GENE VARIATIONS AND SELF-REPORTED SLEEP DISTURBANCE IN WOMEN FOLLOWING BREAST CANCER SURGERY

    PubMed Central

    Alfaro, Emily; Dhruva, Anand; Langford, Dale J.; Koetters, Theresa; Merriman, John D.; West, Claudia; Dunn, Laura B.; Paul, Steven M.; Cooper, Bruce; Cataldo, Janine; Hamolsky, Deborah; Elboim, Charles; Kober, Kord; Aouizerat, Bradley E.; Miaskowski, Christine

    2013-01-01

    Purpose of the research To attempt to replicate the associations found in our previous study of patients and family caregivers between interleukin 6 (IL6) and nuclear factor kappa beta 2 (NFKB2) and sleep disturbance and to identify additional genetic associations in a larger sample of patients with breast cancer. Methods and sample Patients with breast cancer (n=398) were recruited prior to surgery and followed for six months. Patients completed a self-report measure of sleep disturbance and provided a blood sample for genomic analyses. Growth mixture modeling was used to identify distinct latent classes of patients with higher and lower levels of sleep disturbance. Key results Patients who were younger and who had higher comorbidity and lower functional status were more likely to be in the high sustained sleep disturbance class. Variations in three cytokine genes (i.e., IL1 receptor 2 (IL1R2), IL13, NFKB2) predicted latent class membership. Conclusions Polymorphisms in cytokine genes may partially explain inter-individual variability in sleep disturbance. Determination of high risk phenotypes and associated molecular markers may allow for earlier identification of patients at higher risk for developing sleep disturbance and lead to the development of more targeted clinical interventions. PMID:24012192

  11. Analysis of 60 reported glioma risk SNPs replicates published GWAS findings but fails to replicate associations from published candidate-gene studies.

    PubMed

    Walsh, Kyle M; Anderson, Erik; Hansen, Helen M; Decker, Paul A; Kosel, Matt L; Kollmeyer, Thomas; Rice, Terri; Zheng, Shichun; Xiao, Yuanyuan; Chang, Jeffrey S; McCoy, Lucie S; Bracci, Paige M; Wiemels, Joe L; Pico, Alexander R; Smirnov, Ivan; Lachance, Daniel H; Sicotte, Hugues; Eckel-Passow, Jeanette E; Wiencke, John K; Jenkins, Robert B; Wrensch, Margaret R

    2013-02-01

    Genomewide association studies (GWAS) and candidate-gene studies have implicated single-nucleotide polymorphisms (SNPs) in at least 45 different genes as putative glioma risk factors. Attempts to validate these associations have yielded variable results and few genetic risk factors have been consistently replicated. We conducted a case-control study of Caucasian glioma cases and controls from the University of California San Francisco (810 cases, 512 controls) and the Mayo Clinic (852 cases, 789 controls) in an attempt to replicate previously reported genetic risk factors for glioma. Sixty SNPs selected from the literature (eight from GWAS and 52 from candidate-gene studies) were successfully genotyped on an Illumina custom genotyping panel. Eight SNPs in/near seven different genes (TERT, EGFR, CCDC26, CDKN2A, PHLDB1, RTEL1, TP53) were significantly associated with glioma risk in the combined dataset (P < 0.05), with all associations in the same direction as in previous reports. Several SNP associations showed considerable differences across histologic subtype. All eight successfully replicated associations were first identified by GWAS, although none of the putative risk SNPs from candidate-gene studies was associated in the full case-control sample (all P values > 0.05). Although several confirmed associations are located near genes long known to be involved in gliomagenesis (e.g., EGFR, CDKN2A, TP53), these associations were first discovered by the GWAS approach and are in noncoding regions. These results highlight that the deficiencies of the candidate-gene approach lay in selecting both appropriate genes and relevant SNPs within these genes. PMID:23280628

  12. Analysis of 60 Reported Glioma Risk SNPs Replicates Published GWAS Findings but Fails to Replicate Associations From Published Candidate-Gene Studies

    PubMed Central

    Walsh, Kyle M.; Anderson, Erik; Hansen, Helen M.; Decker, Paul A.; Kosel, Matt L.; Kollmeyer, Thomas; Rice, Terri; Zheng, Shichun; Xiao, Yuanyuan; Chang, Jeffrey S.; McCoy, Lucie S.; Bracci, Paige M.; Wiemels, Joe L.; Pico, Alexander R.; Smirnov, Ivan; Lachance, Daniel H.; Sicotte, Hugues; Eckel-Passow, Jeanette E.; Wiencke, John K.; Jenkins, Robert B.; Wrensch, Margaret R.

    2013-01-01

    Genomewide association studies (GWAS) and candidate-gene studies have implicated single-nucleotide polymorphisms (SNPs) in at least 45 different genes as putative glioma risk factors. Attempts to validate these associations have yielded variable results and few genetic risk factors have been consistently replicated. We conducted a case-control study of Caucasian glioma cases and controls from the University of California San Francisco (810 cases, 512 controls) and the Mayo Clinic (852 cases, 789 controls) in an attempt to replicate previously reported genetic risk factors for glioma. Sixty SNPs selected from the literature (eight from GWAS and 52 from candidate-gene studies) were successfully genotyped on an Illumina custom genotyping panel. Eight SNPs in/near seven different genes (TERT, EGFR, CCDC26, CDKN2A, PHLDB1, RTEL1, TP53) were significantly associated with glioma risk in the combined dataset (P < 0.05), with all associations in the same direction as in previous reports. Several SNP associations showed considerable differences across histologic subtype. All eight successfully replicated associations were first identified by GWAS, although none of the putative risk SNPs from candidate-gene studies was associated in the full case-control sample (all P values > 0.05). Although several confirmed associations are located near genes long known to be involved in gliomagenesis (e.g., EGFR, CDKN2A, TP53), these associations were first discovered by the GWAS approach and are in noncoding regions. These results highlight that the deficiencies of the candidate-gene approach lay in selecting both appropriate genes and relevant SNPs within these genes. PMID:23280628

  13. [Double mutant alleles in the EXT1 gene not previously reported in a teenager with hereditary multiple exostoses].

    PubMed

    Cammarata-Scalisi, Francisco; Cozar, Mónica; Grinberg, Daniel; Balcells, Susana; Asteggiano, Carla G; Martínez-Domenech, Gustavo; Bracho, Ana; Sánchez, Yanira; Stock, Frances; Delgado-Luengo, Wilmer; Zara-Chirinos, Carmen; Chacín, José Antonio

    2015-04-01

    Hereditary forms of multiple exostoses, now called EXT1/EXT2-CDG within Congenital Disorders of Glycosylation, are the most common benign bone tumors in humans and clinical description consists of the formation of several cartilage-capped bone tumors, usually benign and localized in the juxta-epiphyseal region of long bones, although wide body dissemination in severe cases is not uncommon. Onset of the disease is variable ranging from 2-3 years up to 13-15 years with an estimated incidence ranging from 1/18,000 to 1/50,000 cases in European countries. We present a double mutant alleles in the EXT1 gene not previously reported in a teenager and her family with hereditary multiple exostoses. PMID:25727835

  14. Application of p21 and klf2 reporter gene assays to identify selective histone deacetylase inhibitors for cancer therapy.

    PubMed

    Wong, Jason C; Guo, Lei; Peng, Zhenghong; Zhang, Weixing; Zhang, Nan; Lai, Wayne; Zhang, Zhenshan; Zhang, Chao; Zhang, Xiongwen; Song, Shan; Pan, Desi; Xie, Chuanming; Li, Jia; Ning, Zhiqing; Lu, Xianping; He, Yun; Chen, Li

    2011-01-01

    Novel 2-aminoanilide histone deacetylase (HDAC) inhibitors were designed to increase their contact with surface residues surrounding the HDAC active site compared to the contacts made by existing clinical 2-aminoanilides such as SNDX-275, MGCD0103, and Chidamide. Their HDAC selectivity was assessed using p21 and klf2 reporter gene assays in HeLa and A204 cells, respectively, which provide a cell-based readout for the inhibition of HDACs associated either with the p21 or klf2 promoter. A subset of the designed compounds selectively induced p21 over klf2 relative to the clinical reference compound SNDX-275. A representative lead compound from this subset had antiproliferative effects in cancer cells associated with induction of acetylated histone H4, endogenous p21, cell cycle arrest, and apoptosis. The p21- versus klf2-selective compounds described herein may provide a chemical starting point for developing clinically-differentiated HDAC inhibitors for cancer therapy. PMID:21145737

  15. Food flavonoid aryl hydrocarbon receptor-mediated agonistic/antagonistic/synergic activities in human and rat reporter gene assays.

    PubMed

    Van der Heiden, Edwige; Bechoux, Nathalie; Muller, Marc; Sergent, Thérèse; Schneider, Yves-Jacques; Larondelle, Yvan; Maghuin-Rogister, Guy; Scippo, Marie-Louise

    2009-04-01

    Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor mediating the adverse effects of dioxins and polycyclic aromatic hydrocarbons (PAHs). In this study, we investigated the genetic-, time-, dose-, species- and tissue-dependent AhR-mediated agonistic/antagonistic activities of three food flavonoids: quercetin, chrysin and genistein. To that end, four stably transfected cell lines were used in cell-based luciferase reporter gene assays: three lines were transformed with the ptKLuc vector harbouring four dioxin-responsive elements (DREs) upstream of the thymidine kinase promoter and the luciferase gene (HepG2-Luc, T-47D-Luc and H4IIE-ULg). The fourth is a patented cell line transformed with a different construct: H4IIE DR-CALUX((R)). Both H4IIE cells were compared for their genetic construction. Human hepatoma (HepG2-Luc) and human breast tumour (T-47D-Luc) cells were compared for tissue-dependent effects. Rat hepatoma (H4IIE-ULg) and human hepatoma (HepG2-Luc) cells were compared for species-dependent activities. We concluded that quercetin, chrysin and genistein act in a time-, dose-, species- and tissue-specific way. For example, genistein displayed agonistic activities when exposed to rat hepatoma cells during 6h but not after 24h. Flavonoids displayed agonistic/antagonistic activities in human breast tumour cells, depending on the exposure time, while in human hepatoma cells, only antagonistic activities of flavonoids were measured. In addition, we report, in all the cells, a synergy between an isoflavone and two food contaminants; the 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene, a PAH. In rat cells, this synergy occurred when cells were exposed to flavonoids and contaminant for 6h, while it was observed in human cells only after 24h. PMID:19286049

  16. A New Pyrimidine-Specific Reporter Gene: A Mutated Human Deoxycytidine Kinase Suitable for PET During Treatment with Acycloguanosine-Based Cytotoxic Drugs

    PubMed Central

    Likar, Yury; Zurita, Juan; Dobrenkov, Konstantin; Shenker, Larissa; Cai, Shangde; Neschadim, Anton; Medin, Jeffrey A.; Sadelain, Michel; Hricak, Hedvig; Ponomarev, Vladimir

    2015-01-01

    In this article, we describe a series of new human-derived reporter genes based on human deoxycytidine kinase (dCK) suitable for clinical PET. Methods Native dCK and its mutant reporter genes were tested in vitro and in vivo for their phosphorylation of pyrimidine- and acycloguanosine-based radiotracers including 2?-deoxy-2?-fluoroarabinofuranosylcytosine, 2?-fluoro-2?-deoxyarabinofuranosyl-5-ethyluracil (FEAU), penciclovir, and 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG) and clinically applied antiviral and anticancer drugs. Results Cells transduced with dCK mutant reporter genes showed high in vitro and in vivo uptake of pyrimidine-based radiopharmaceuticals (18F-FEAU) comparable to that of herpes simplex virus type-1 thymidine kinase (HSV1-tk)–transduced cells. These mutants did not phosphorylate acycloguanosine-based radiotracers (18F-FHBG) or antiviral drugs (ganciclovir). Furthermore, the mutants displayed suicidal activation of clinically used pyrimidine-based prodrugs (cytarabine, gemcitabine). Conclusion The mutants of human dCK can be used as pyrimidine-specific PET reporter genes for imaging with 18F-FEAU during treatment with acycloguanosine-based antiviral drugs. Additionally, the prosuicidal activity of these reporters with pyrimidine-based analogs will allow for the safe elimination of transduced cells. PMID:20810757

  17. A dynamic FRET reporter of gene expression improved by functional screening.

    PubMed

    Schifferer, Martina; Griesbeck, Oliver

    2012-09-19

    Here, we describe a reporter system that consists of a FRET biosensor and its corresponding aptamer. The FRET biosensor employs the synthetic aptamer binding peptide Rsg1.2 sandwiched between mutants of the Green Fluorescent Protein and undergoes FRET when binding its corresponding Rev Responsive Element (RRE) RNA aptamer. We developed a novel approach to engineer FRET biosensors by linker extension and screening to improve signal strength of the biosensor which we called VAmPIRe (Viral Aptamer binding Peptide based Indicator for RNA detection). We demonstrate that the system is quantitative, reversible and works with high specificity in vitro and in vivo in living bacteria and mammalian cells. Thus, VAmPIRe may become valuable for RNA localizations and as a dynamic RNA-based reporter for live cell imaging. Moreover, functional screening of large libraries as demonstrated here may become applicable to optimize some of the many FRET biosensors of cellular signaling. PMID:22946509

  18. Versatile Dual Reporter Gene Systems for Investigating Stop Codon Readthrough in Plants

    PubMed Central

    Lao, Nga T.; Maloney, Alan P.; Atkins, John F.; Kavanagh, Tony A.

    2009-01-01

    Background Translation is most often terminated when a ribosome encounters the first in-frame stop codon (UAA, UAG or UGA) in an mRNA. However, many viruses (and some cellular mRNAs) contain “stop” codons that cause a proportion of ribosomes to terminate and others to incorporate an amino acid and continue to synthesize a “readthrough”, or C-terminally extended, protein. This dynamic redefinition of codon meaning is dependent on specific sequence context. Methodology We describe two versatile dual reporter systems which facilitate investigation of stop codon readthrough in vivo in intact plants, and identification of the amino acid incorporated at the decoded stop codon. The first is based on the reporter enzymes NAN and GUS for which sensitive fluorogenic and histochemical substrates are available; the second on GST and GFP. Conclusions We show that the NAN-GUS system can be used for direct in planta measurements of readthrough efficiency following transient expression of reporter constructs in leaves, and moreover, that the system is sufficiently sensitive to permit measurement of readthrough in stably transformed plants. We further show that the GST-GFP system can be used to affinity purify readthrough products for mass spectrometric analysis and provide the first definitive evidence that tyrosine alone is specified in vivo by a ‘leaky’ UAG codon, and tyrosine and tryptophan, respectively, at decoded UAA, and UGA codons in the Tobacco mosaic virus (TMV) readthrough context. PMID:19816579

  19. Expression of the yeast PHR1 gene is induced by DNA-damaging agents

    SciTech Connect

    Sebastian, J.; Kraus, B.; Sancar, G.B. (Univ. of North Carolina, Chapel Hill (USA))

    1990-09-01

    The PHR1 gene of Saccharomyces cerevisiae encodes a photolyase which repairs specifically and exclusively pyrimidine dimers, the most frequent lesions induced in DNA by far-UV radiation. We have asked whether expression of PHR1 is modulated in response to UV-induced DNA damage and to DNA-damaging agents that induce lesions structurally dissimilar to pyrimidine dimers. Using a PHR1-lacZ fusion gene in which expression of beta-galactosidase is regulated by PHR1 5' regulatory elements, we found that exposure of cells to 254-nm light, 4-nitroquinoline-N-oxide, methyl methanesulfonate, and N-methyl-N'-nitro-N-nitrosoguanidine induced synthesis of increased amounts of fusion protein. In contrast to these DNA-damaging agents, neither heat shock nor exposure to photoreactivating light elicited a response. Induction by far-UV radiation was evident both when the fusion gene was carried on a multicopy plasmid and when it replaced the endogenous chromosomal copy of PHR1, and it was accompanied by an increase in the steady-state concentration of PHR1-lacZ mRNA. Northern (RNA) blot analysis of PHR1 mRNA encoded by the chromosomal locus was consistent with either enhanced transcription of PHR1 after DNA damage or stabilization of the transcripts. Neither the intact PHR1 or RAD2 gene was required for induction. Comparison of the region of PHR1 implicated in regulation of its expression with other damage-inducible genes from yeast cells revealed a common conserved sequence that is present in the PHR1, RAD2, and RNR2 genes and is required for damage inducibility of the latter two genes. These sequences may constitute elements of a damage-responsive regulon in S. cerevisiae.

  20. Utilization of a reporter system based on the blue pigment indigoidine biosynthetic gene bpsA for detection of promoter activity and deletion of genes in Streptomyces.

    PubMed

    Knirschova, Renata; Novakova, Renata; Mingyar, Erik; Bekeova, Carmen; Homerova, Dagmar; Kormanec, Jan

    2015-06-01

    The integrative promoter-probe plasmid pBPSA1 was constructed using a promoterless Streptomyces aureofaciens CCM3239 bpsA gene encoding a non-ribosomal peptide synthase for the biosynthesis of a blue pigment, indigoidine. bpsA was also used to prepare pAMR4 plasmid for the deletion of genes in Streptomyces with facile identification of double crossover recombination. PMID:25801098

  1. Estrogen-Responsive Transient Expression Assay Using a Brain Aromatase-Based Reporter Gene in Zebrafish (Danio rerio)

    PubMed Central

    Kim, Dong-Jae; Seok, Seung-Hyeok; Baek, Min-Won; Lee, Hui-Young; Na, Yi-Rang; Park, Sung-Hoon; Lee, Hyun-Kyoung; Dutta, Noton Kumar; Kawakami, Koichi; Park, Jae-Hak

    2009-01-01

    Whereas endogenous estrogens play an important role in the development, maintenance, and function of female and male reproductive organs, xenoestrogens present in the environment disrupt normal endocrine function in humans and wildlife. Various in vivo and in vitro assays have been developed to screen these xenoestrogens. However, traditional in vivo assays are laborious and unsuitable for large-scale screening, and in vitro assays do not necessarily replicate in vivo functioning. To overcome these limitations, we developed a transient expression assay in zebrafish, into which a brain aromatase (cyp19a1b)-based estrogen-responsive reporter gene was introduced. In response to 17?-estradiol (10?6 M) and heptachlor (10?6 M), zebrafish embryos carrying the reporter construct expressed enhanced green fluorescent protein in the olfactory bulb, telencephalon, preoptic area, and mediobasal hypothalamus. This system will serve to model the in vivo conversion and breakdown of estrogenic compounds and thus provide a rapid preliminary screening method to estimate their estrogenicity. PMID:19887024

  2. Affinity labelling with a deaminatively generated carbonium ion. Kinetics and stoicheiometry of the alkylation of methionine-500 of the lacZ beta-galactosidase of Escherichia coli by beta-D-galactopyranosylmethyl-p-nitrophenyltriazene.

    PubMed Central

    Sinnott, M L; Smith, P J

    1978-01-01

    1. beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene is an active-site-directed irreversible inhibitor of Mg2+-bound and Mg2+-free lacZ beta-galactosidase from Escherichia coli. 2. The Mg2+-enzyme binds the inhibitor more tightly but the complex then decomposes less rapidly than is the case with Mg2+-free enzyme. 3. Loss of enzyme activity is a linear function of the fraction of enzyme protomers to which are attached beta-D-galactopranosyl[14C]methyl residues: complete inactivation of fully active enzyme results in incorporation of 0.91 equivalent of carbohydrate label per enzyme protomer. 4. When the beta-galactopyranosylmethyl cation is generated in the active site of Mg2+-enzyme, it is captured essentially completely by the protein, but in the active site of Mg2+-free enzyme it is only captured with an efficiency of 25%. 5. Labelled enzyme was carboxymethylated and digested with trypsin; acidic hydrolysis of the isolated tryptic peptide, and field-desorption mass spectrometry of the isolated radioactive derivative, showed it to be 2,5-dioxo-3[2-(beta-D-galactopyranosylmethylthio)ethyl]-1,6-trimethylenepiperazine. 6. This is considered to have arisen from labelling of the sulphur atom of a methionine residue adjacent to a proline residue. 7. The complete amino acid sequence of the molecule [Fowler & Zabin (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1507-1510] enables the labelled methionine residue to be identified as either Met-421 or Met-500. 8. Sequence data [Fowler, Zabin, Sinnott & Smith (1978) J. Biol. Chem. in the press] show the site of attack to be Met-500. PMID:105721

  3. Brief Report: High Frequency of Biochemical Markers for Mitochondrial Dysfunction in Autism: No Association with the Mitochondrial Aspartate/Glutamate Carrier "SLC25A12" Gene

    ERIC Educational Resources Information Center

    Correia, Catarina; Coutinho, Ana M.; Diogo, Luisa; Grazina, Manuela; Marques, Carla; Miguel, Teresa; Ataide, Assuncao; Almeida, Joana; Borges, Luis; Oliveira, Catarina; Oliveira, Guiomar; Vicente, Astrid M.

    2006-01-01

    In the present study we confirm the previously reported high frequency of biochemical markers of mitochondrial dysfunction, namely hyperlactacidemia and increased lactate/pyruvate ratio, in a significant fraction of 210 autistic patients. We further examine the involvement of the mitochondrial aspartate/glutamate carrier gene ("SLC25A12") in…

  4. New luminescence-based approach to measurement of luciferase gene expression reporter activity and adenosine triphosphate-based determination of cell viability.

    PubMed

    Konopka, R; Hýzdalová, M; Kubala, L; Pacherník, J

    2010-01-01

    The assay employing firefly luciferase as the end-point reporter is one of the most popular gene reporter systems. However, the physiological conditions of cells may affect the reporter gene expression, which makes an assessment of cell viability desirable. Estimates of cell viability may be based on different principles. We tested for correlations between various cell viability assessments, including luminescent determination of adenosine triphosphate in whole-cell lysate, and the reporter luciferase activity in pluripotent embryonic and colon adenocarcinoma cells. Luciferase activity in cell lysate from both cell lines cultured under different conditions correlated with the amount of viable cells assessed by all of the methods employed. Importantly, it was also possible to carry out adenosine triphosphate determination in cell lysates prepared in the buffer originally designed for determining luciferase activity; it correlated significantly with adenosine triphosphate determination in cells lysed in the buffer originally designed for adenosine triphosphate determination. The results suggest that the assessment of live cells by determining adenosine triphosphate can be multiplexed with a luciferase reporter gene assay, which allows independent monitoring of both reporter expression and cell viability. PMID:20492758

  5. Novel Mutation of the GNE Gene Presenting Atypical Mild Clinical Feature: A Korean Case Report

    PubMed Central

    Choi, Young-Ah; Park, Sung-Hye; Yi, Youbin

    2015-01-01

    Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE) myopathy is caused by mutations in GNE, a key enzyme in sialic acid biosynthesis. Here, we reported a case of GNE that presented with atypical mild clinical feature and slow progression. A 48-year-old female had a complaint of left foot drop since the age of 46 years. Electromyography (EMG) and muscle biopsy from left tibialis anterior muscle were compatible with myopathy. Genetic analysis led to the identification of c.1714G>C/c.527A>T compound heterozygous mutation, which is the second most frequent mutation in Japan as far as we know. Previous research has revealed that c.1714G>C/c.527A>T compound heterozygous mutation is a mild mutation as the onset of the disease is much later than the usual age of onset of GNE myopathy and the clinical course is slowly progressive. This was the first case report in Korea of the clinicopathological characteristics of GNE myopathy with GNE (c.1714G>C/c.527A>T compound heterozygous) mutation.

  6. An efficient plasmid vector for constitutive high-level expression of foreign genes in Escherichia coli.

    PubMed

    Seo, Jeong-Woo; Hong, Won-kyung; Rairakhwada, Dina; Seo, Pil-Soo; Choi, Min Ho; Song, Ki-Bang; Rhee, Sang-Ki; Kim, Chul Ho

    2009-06-01

    The levansucrase gene (lsrA) from Rahnella aquatilis was strongly expressed in a constitutive manner in Escherichia coli when cloned into a pBluescript KS-based pRL1CP plasmid vector. The native promoter upstream of lsrA and the lacZ promoter cooperatively enhanced the expression of lsrA to a level that was comparable to that of the T7 promoter, which is used in commercial pET expression vector system. A putative rho-independent transcription termination signal downstream of lsrA was crucial for gene expression. This plasmid vector also proved to be applicable for efficient expression of other foreign genes in E. coli. PMID:19214389

  7. The sweet potato RbcS gene (IbRbcS1) promoter confers high-level and green tissue-specific expression of the GUS reporter gene in transgenic Arabidopsis.

    PubMed

    Tanabe, Noriaki; Tamoi, Masahiro; Shigeoka, Shigeru

    2015-08-10

    Sweet potato is an important crop because of its high yield and biomass production. We herein investigated the potential of the promoter activity of a small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcS) from sweet potato (Ipomoea batatas) in order to develop the high expression system of exogenous DNA in Arabidopsis. We isolated two different cDNAs (IbRbcS1 and IbRbcS2) encoding RbcS from sweet potato. Their predicted amino acid sequences were well conserved with the mature RbcS protein of other plants. The tissue-specific expression patterns of these two genes revealed that expression of IbRbcS1 was specific to green tissue, whereas that of IbRbcS2 was non-photosynthetic tissues such as roots and tubers. These results suggested that IbRbcS1 was predominantly expressed in the green tissue-specific of sweet potato over IbRbcS2. Therefore, the IbRbcS1 promoter was transformed into Arabidopsis along with ?-glucuronidase (GUS) as a reporter gene. GUS staining and semi-quantitative RT-PCR showed that the IbRbcS1 promoter conferred the expression of the GUS reporter gene in green tissue-specific and light-inducible manners. Furthermore, qPCR showed that the expression levels of GUS reporter gene in IbRbcS1 pro:GUS were same as those in CaMV 35S pro:GUS plants. These results suggest that the IbRbcS1 promoter is a potentially strong foreign gene expression system for genetic transformation in plants. PMID:25958348

  8. Neonatal Lethality, Dwarfism, and Abnormal Brain Development in Dmbx1 Mutant Mice

    Microsoft Academic Search

    Akihira Ohtoshi; Richard R. Behringer

    2004-01-01

    Dmbx1 encodes a paired-like homeodomain protein that is expressed in developing neural tissues during mouse embryogenesis. To elucidate the in vivo role of Dmbx1, we generated two Dmbx1 mutant alleles. Dmbx1 lacks the homeobox and Dmbx1z is an insertion of a lacZ reporter gene. Dmbx1z appears to be a faithful reporter of Dmbx1 expression during embryogenesis and after birth. Dmbx1-lacZ

  9. Regulation of the Alternative Oxidase Aox1 Gene in Chlamydomonas reinhardtii. Role of the Nitrogen Source on the Expression of a Reporter Gene under the Control of the Aox1 Promoter1

    PubMed Central

    Baurain, Denis; Dinant, Monique; Coosemans, Nadine; Matagne, René F.

    2003-01-01

    In higher plants, various developmental and environmental conditions enhance expression of the alternative oxidase (AOX), whereas its induction in fungi is mainly dependent on cytochrome pathway restriction and triggering by reactive oxygen species. The AOX of the unicellular green alga Chlamydomonas reinhardtii is encoded by two different genes, the Aox1 gene being much more transcribed than Aox2. To analyze the transcriptional regulation of Aox1, we have fused its 1.4-kb promoter region to the promoterless arylsulfatase (Ars) reporter gene and measured ARS enzyme activities in transformants carrying the chimeric construct. We show that the Aox1 promoter is generally unresponsive to a number of known AOX inducers, including stress agents, respiratory inhibitors, and metabolites, possibly because the AOX activity is constitutively high in the alga. In contrast, the Aox1 expression is strongly dependent on the nitrogen source, being down-regulated by ammonium and stimulated by nitrate. Inactivation of nitrate reductase leads to a further increase of expression. The stimulation by nitrate also occurs at the AOX protein and respiratory levels. A deletion analysis of the Aox1 promoter region demonstrates that a short upstream segment (?253 to +59 with respect to the transcription start site) is sufficient to ensure gene expression and regulation, but that distal elements are required for full gene expression. The observed pattern of AOX regulation points to the possible interaction between chloroplast and mitochondria in relation to a potential increase of photogenerated ATP when nitrate is used as a nitrogen source. PMID:12644691

  10. Disruption of the pdhB pyruvate dehyrogenase gene affects colony morphology, in vitro growth and cell invasiveness of Mycoplasma agalactiae.

    PubMed

    Hegde, Shivanand; Rosengarten, Renate; Chopra-Dewasthaly, Rohini

    2015-01-01

    The utilization of available substrates, the metabolic potential and the growth rates of bacteria can play significant roles in their pathogenicity. This study concentrates on Mycoplasma agalactiae, which causes significant economic losses through its contribution to contagious agalactia in small ruminants by as yet unknown mechanisms. This lack of knowledge is primarily due to its fastidious growth requirements and the scarcity of genetic tools available for its manipulation and analysis. Transposon mutagenesis of M. agalactiae type strain PG2 resulted in several disruptions throughout the genome. A mutant defective in growth in vitro was found to have a transposon insertion in the pdhB gene, which encodes a component of the pyruvate dehydrogenase complex. This growth difference was quite significant during the actively dividing logarithmic phase but a gradual recovery was observed as the cells approached stationary phase. The mutant also exhibited a different and smaller colony morphology compared to the wild type strain PG2. For complementation, pdhAB was cloned downstream of a strong vpma promoter and upstream of a lacZ reporter gene in a newly constructed complementation vector. When transformed with this vector the pdhB mutant recovered its normal growth and colony morphology. Interestingly, the pdhB mutant also had significantly reduced invasiveness in HeLa cells, as revealed by double immunofluorescence staining. This deficiency was recovered in the complemented strain, which had invasiveness comparable to that of PG2. Taken together, these data indicate that pyruvate dehydrogenase might be an important player in infection with and colonization by M. agalactiae. PMID:25799063

  11. Disruption of the pdhB Pyruvate Dehydrogenase Gene Affects Colony Morphology, In Vitro Growth and Cell Invasiveness of Mycoplasma agalactiae

    PubMed Central

    Hegde, Shivanand; Rosengarten, Renate; Chopra-Dewasthaly, Rohini

    2015-01-01

    The utilization of available substrates, the metabolic potential and the growth rates of bacteria can play significant roles in their pathogenicity. This study concentrates on Mycoplasma agalactiae, which causes significant economic losses through its contribution to contagious agalactia in small ruminants by as yet unknown mechanisms. This lack of knowledge is primarily due to its fastidious growth requirements and the scarcity of genetic tools available for its manipulation and analysis. Transposon mutagenesis of M. agalactiae type strain PG2 resulted in several disruptions throughout the genome. A mutant defective in growth in vitro was found to have a transposon insertion in the pdhB gene, which encodes a component of the pyruvate dehydrogenase complex. This growth difference was quite significant during the actively dividing logarithmic phase but a gradual recovery was observed as the cells approached stationary phase. The mutant also exhibited a different and smaller colony morphology compared to the wild type strain PG2. For complementation, pdhAB was cloned downstream of a strong vpma promoter and upstream of a lacZ reporter gene in a newly constructed complementation vector. When transformed with this vector the pdhB mutant recovered its normal growth and colony morphology. Interestingly, the pdhB mutant also had significantly reduced invasiveness in HeLa cells, as revealed by double immunofluorescence staining. This deficiency was recovered in the complemented strain, which had invasiveness comparable to that of PG2. Taken together, these data indicate that pyruvate dehydrogenase might be an important player in infection with and colonization by M. agalactiae. PMID:25799063

  12. Bioreactor mixing efficiency modulates the activity of a prpoS::GFP reporter gene in E. coli

    PubMed Central

    Delvigne, Frank; Boxus, Mathieu; Ingels, Sophie; Thonart, Philippe

    2009-01-01

    Background Extensive studies have shown that up-scaling of bioprocesses has a significant impact on the physiology of the microorganisms. Among the factors associated with the fluid dynamics of the bioreactor, concentration gradients induced by loss of the global mixing efficiency associated with the increasing scale is the main phenomena leading to strong physiological modifications at the level of the microbial population. These changes are not fully understood since they involve complex physiological mechanisms. In this work, we intend to investigate, at the single cell level, the expression of the rpoS gene associated with the stress response of E. coli. The cultures of the reporter strain have been performed in a small scale reactor as well as in a series of scaled-down bioreactors able to induce extracellular perturbations with increasing level of magnitude. Results The rpoS level has been monitored by the aim of a transcriptional reporter gene based on the synthesis of the green fluorescent protein (GFP). It has been observed that the level of GFP increases during the transition from batch to fed-batch phase. After this initial increase, the GFP content of the cell drops, primarily due to the dilution by cell division. However, a significant drop of the GFP content has been observed if using a partitioned bioreactor, for which the mixing conditions are very bad, leading to the exposure of the cells to cyclic and stochastic extracellular fluctuations. If considering the flow cytometric profile of the cell to cell GFP content, this drop has to be attributed to the appearance of segregation at the level of the GFP content among the microbial population. Conclusion The generation of extracellular perturbations (in the present case, at the level of the sugar concentration and the dissolved oxygen level) has led to a drop at the level of the rpoS expression level. This drop has to be attributed to a segregation phenomenon in microbial population, with a major sub-population exhibiting a low expression level and a minor sub-population keeping its initial elevated expression level. The intensity of the segregation, as well as its time of appearance during the culture can be related to the bioreactor mixing efficiency. PMID:19243588

  13. Gene Expression Analysis of Immunostained Endothelial Cells Isolated from Formaldehyde-fixated Paraffin Embedded Tumors Using Laser Capture Microdissection – a Technical Report

    PubMed Central

    Kaneko, Tomoatsu; Okiji, Takashi; Kaneko, Reika; Suda, Hideaki; Nör, Jacques E.

    2009-01-01

    Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. In conventional LCM, frozen tissues stained with hematoxylin are normally used to the molecular analysis. Recent studies suggested that it is possible to carry out gene expression analysis of formaldehyde-fixated paraffin embedded (FFPE) tissues that were stained with hematoxylin. However, it is still unclear if quantitative gene expression analyses can be performed from LCM cells from FFPE tissues that were subjected to immunostaining to enhance identification of target cells. In this proof-of-principle study, we analyzed by RT-PCR and real time PCR the expression of genes in factor VIII immunostained human endothelial cells that were dissected from FFPE tissues by LCM. We observed that immunostaining should be performed at 4°C to preserve the mRNA from the cells. The expression of Bcl-2 in the endothelial cells was evaluated by RT-PCR and by real time PCR. GAPDH and 18S were used as house keeping genes for RT-PCR and real time PCR, respectively. This report unveils a method for quantitative gene expression analysis in cells that were identified by immunostaining and retrieved by LCM from FFPE tissues. This method is ideally suited for the analysis of relatively rare cell types within a tissue, and should improve on our ability to perform differential diagnosis of pathologies as compared to conventional LCM. PMID:19425073

  14. Visualization of gene expression in the live subject using the Na/I symporter as a reporter gene: applications in biotherapy

    PubMed Central

    Baril, Patrick; Martin-Duque, Pilar; Vassaux, Georges

    2010-01-01

    Biotherapies involve the utilization of antibodies, genetically modified viruses, bacteria or cells for therapeutic purposes. Molecular imaging has the potential to provide unique information that will guarantee their biosafety in humans and provide a rationale for the future development of new generations of reagents. In this context, non-invasive imaging of gene expression is an attractive prospect, allowing precise, spacio-temporal measurements of gene expression in longitudinal studies involving gene transfer vectors. With the emergence of cell therapies in regenerative medicine, it is also possible to track cells injected into subjects. In this context, the Na/I symporter (NIS) has been used in preclinical studies. Associated with a relevant radiotracer (123I-, 124I-, 99mTcO4-), NIS can be used to monitor gene transfer and the spread of selectively replicative viruses in tumours as well as in cells with a therapeutic potential. In addition to its imaging potential, NIS can be used as a therapeutic transgene through its ability to concentrate therapeutic doses of radionuclides in target cells. This dual property has applications in cancer treatment and could also be used to eradicate cells with therapeutic potential in the case of adverse events. Through experience acquired in preclinical studies, we can expect that non-invasive molecular imaging using NIS as a transgene will be pivotal for monitoring in vivo the exact distribution and pharmacodynamics of gene expression in a precise and quantitative way. This review highlights the applications of NIS in biotherapy, with a particular emphasis on image-guided radiotherapy, monitoring of gene and vector biodistribution and trafficking of stem cells. This article is part of a themed section on Imaging in Pharmacology. To view the editorial for this themed section visit http://dx.doi.org/10.1111/j.1476-5381.2010.00685.x PMID:19814733

  15. Validating tyrosinase homologue MelA as a photoacoustic reporter gene for imaging Escherichia coli

    NASA Astrophysics Data System (ADS)

    Paproski, Robert J.; Li, Yan; Barber, Quinn; Lewis, John D.; Campbell, Robert; Zemp, Roger

    2015-03-01

    Antibiotic drug resistance is a major worldwide issue. Development of new therapies against pathogenic bacteria requires appropriate research tools for replicating and characterizing infections. Previously fluorescence and bioluminescence modalities have been used to image infectious burden in animal models but scattering significantly limits imaging depth and resolution. We hypothesize that photoacoustic imaging, which has improved depth-toresolution ratio, could be useful for visualizing MelA-expressing bacteria since MelA is a bacterial tyrosinase homologue involved in melanin production. Using an inducible expression system, E. coli expressing MelA were visibly black in liquid culture. Phosphate buffered saline (PBS), MelA-expressing bacteria (at different dilutions in PBS), and chicken embryo blood were injected in plastic tubes which were imaged using a VisualSonics Vevo LAZR system. Photoacoustic imaging at 6 different wavelengths (680, 700, 750, 800, 850 and 900nm) enabled spectral de-mixing to distinguish melanin signals from blood. The signal to noise ratio of 9x diluted MelA bacteria was 55, suggesting that ~20 bacteria cells could be detected with our system. When MelA bacteria were injected as a 100 ?L bolus into a chicken embryo, photoacoustic signals from deoxy- and oxy- hemoglobin as well as MelA-expressing bacteria could be separated and overlaid on an ultrasound image, allowing visualization of the bacterial location. Photoacoustic imaging may be a useful tool for visualizing bacterial infections and further work incorporating photoacoustic reporters into infectious bacterial strains is warranted.

  16. De novo exon 1 deletion of AUTS2 gene in a patient with autism spectrum disorder and developmental delay: A case report and a brief literature review.

    PubMed

    Liu, Yi; Zhao, Dongmei; Dong, Rui; Yang, Xiaomeng; Zhang, Yanqing; Tammimies, Kristiina; Uddin, Mohammed; Scherer, Stephen W; Gai, Zhongtao

    2015-06-01

    Exonic deletions disrupting the autism susceptibility candidate 2 (AUTS2) gene have been demonstrated as causal variants leading to neurodevelopmental disorders (NDDs) such as autism spectrum disorder (ASD) and developmental delay (DD). Here, we report on 830?kb de novo deletion at chromosome 7q11.22 in a 4-year-old male patient with ASD and DD. This deletion disrupts the promoter region and exon 1 of AUTS2, potentially leading to complete haploinsuffiency of the gene. In addition, we discuss the clinical presentation of the de novo deletion in the light of the previous studies describing deletions of AUTS2 in NDDs. © 2015 Wiley Periodicals, Inc. PMID:25851617

  17. Validation and application of a reporter gene assay for the determination of estrogenic endocrine disruptor activity in milk.

    PubMed

    Wielogorska, E; Elliott, C T; Danaher, M; Connolly, L

    2014-07-01

    Endocrine disruptors (EDs) are compounds known to interfere with the endocrine system by disturbing the action or pathways of natural hormones which may lead to infertility or cancer. Our diet is considered to be one of the main exposure routes to EDs. Since milk and dairy products are major components of our diet they should be monitored for ED contamination. Most assays developed to date utilise targeted, chromatography based methods which lack information on the biological activity and mixture effects of the monitored compounds. A biological reporter gene assay (RGA) was developed to assess the total estrogen hormonal load in milk. It has been validated according to EU decision 2002/657/EC. Analytes were extracted by liquid-liquid extraction with acetonitrile followed by clean up on a HLB column which yielded good recovery and small matrix effects. The method has been shown to be estrogen specific, repeatable and reproducible, with covariance values below 20%. In conclusion, this method enables the detection of low levels of estrogen hormonal activity in milk with a detection capability of 36 pg g(-)(1) EEQ and has been successfully applied in testing a range of milk samples. PMID:24769019

  18. The Vibrio cholerae Fatty Acid Regulatory Protein, FadR, Represses Transcription of plsB, the Gene Encoding the First Enzyme of Membrane Phospholipid Biosynthesis

    PubMed Central

    Feng, Youjun; Cronan, John E.

    2011-01-01

    SUMMARY Glycerol-3-phosphate (sn-glycerol-3-P, G3P) acyltransferase catalyzes the first committed step in the biosynthesis of membrane phospholipids, the acylation of G3P to form 1-acyl G3P (lysophosphatidic acid). The paradigm G3P acyltransferase is the Escherichia coli plsB gene product which acylates position-1 of G3P using fatty acids in thioester linkage to either acyl carrier protein (ACP) or CoA as acyl-donors. Although the Escherichia coli plsB gene was discovered about 30 years ago, no evidence for transcriptional control of its expression has been reported. However Kazakov and coworkers (Kazakov, A. E. et al. (2009) J Bacteriol, 191, 52–64) reported the presence of a putative FadR-binding site upstream of the candidate plsB genes of V. cholerae and three other Vibrio species suggesting that plsB might be regulated by FadR, a GntR-family transcription factor thus far known only to regulate fatty acid synthesis and degradation. We report that the V. cholerae plsB homologue restored growth of E. coli strain BB26-36 which is a G3P auxotroph due to an altered G3P acyltransferase activity. The plsB promoter was also mapped and the predicted FadR-binding palindrome was found to span positions -19 to -35, upstream of the transcription start site. Gel shift assays confirmed that both V. cholerae FadR and E. coli FadR bound the V. cholerae plsB promoter region and binding was reversed upon addition of long chain fatty acyl-CoA thioesters. The expression level of the V. cholerae plsB gene was elevated 2–3 fold in an E. coli fadR null mutant strain indicating that FadR acts as a repressor of V. cholerae plsB expression. In both E. coli and V. cholerae the ?-galactosidase activity of transcriptional fusions of the V. cholerae plsB promoter to lacZ increased 2–3 fold upon supplementation of growth media with oleic acid. Therefore, V. cholerae coordinates fatty acid metabolism with 1-acyl G3P synthesis. PMID:21771112

  19. Focally folded myelin in Charcot-Marie-Tooth type 1B disease is associated with Asn131Lys mutation in myelin protein zero gene: short report.

    PubMed

    Kocha?ski, A; Drac, H; Jedrzejowska, H; Hausmanowa-Petrusewicz, I

    2003-09-01

    Charcot-Marie-Tooth disease type 1B (CMT1B) is a demyelinating neuropathy inherited as an autosomal dominant trait. The majority of CMT1B cases are caused by mutations in the myelin protein zero (P0) gene (MPZ). Only a few mutations in MPZ gene have been reported to be associated with focally folded myelin sheaths. We have studied five patients from one family with five generations, affected by CMT1B disease. The morphological studies of sural nerve biopsy performed in the proband revealed fibers with focally folded myelin. DNA sequencing analysis showed the Asn131Lys mutation in the MPZ gene in three members of the affected family. PMID:12940837

  20. Overexpression of the MRI Reporter Genes Ferritin and Transferrin Receptor Affect Iron Homeostasis and Produce Limited Contrast in Mesenchymal Stem Cells.

    PubMed

    Pereira, Sofia M; Moss, Diana; Williams, Steve R; Murray, Patricia; Taylor, Arthur

    2015-01-01

    Imaging technologies that allow the non-invasive monitoring of stem cells in vivo play a vital role in cell-based regenerative therapies. Recently, much interest has been generated in reporter genes that enable simultaneous monitoring of the anatomical location and viability of cells using magnetic resonance imaging (MRI). Here, we investigate the efficacy of ferritin heavy chain-1 (Fth1) and transferrin receptor-1 (TfR1) as reporters for tracking mesenchymal stem cells. The overexpression of TfR1 was well tolerated by the cells but Fth1 was found to affect the cell's iron homeostasis, leading to phenotypic changes in the absence of iron supplementation and an upregulation in transcript and protein levels of the cell's endogenous transferrin receptor. Neither the sole overexpression of Fth1 nor TfR1 resulted in significant increases in intracellular iron content, although significant differences were seen when the two reporter genes were used in combination, in the presence of high concentrations of iron. The supplementation of the culture medium with iron sources was a more efficient means to obtain contrast than the use of reporter genes, where high levels of intracellular iron were reflected in transverse (T2) relaxation. The feasibility of imaging iron-supplemented cells by MRI is shown using a 3R-compliant chick embryo model. PMID:26184159

  1. Differential molecular profiling between skin carcinomas reveals four newly reported genes potentially implicated in squamous cell carcinoma development.

    PubMed

    Marionnet, Claire; Lalou, Claude; Mollier, Karine; Chazal, Marjorie; Delestaing, Gisele; Compan, Delphine; Verola, Olivier; Vilmer, Catherine; Cuminet, Jerome; Dubertret, Louis; Basset-Séguin, Nicole

    2003-05-29

    Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are skin tumors with different invasive potential. In this work, we analysed mRNA differential expression between seven BCC and five SCC and their normal skin counterparts using 1176 cDNA macroarrays and verification by RT-PCR to identify genes modulated in each tumor type. We identified 37 genes commonly modulated in both tumors and four genes specifically modulated in SCC. Among these latter RhoC and EMMPRIN genes seem to be of particular interest and could participate in SCC aggressivity. PMID:12776202

  2. Spectrum of MECP2 gene mutations in a cohort of Indian patients with Rett syndrome: report of two novel mutations.

    PubMed

    Das, Dhanjit Kumar; Raha, Sarbani; Sanghavi, Daksha; Maitra, Anurupa; Udani, Vrajesh

    2013-02-15

    Rett syndrome (RTT) is an X-linked neurodevelopmental disorder, primarily affecting females and characterized by developmental regression, epilepsy, stereotypical hand movements, and motor abnormalities. Its prevalence is about 1 in 10,000 female births. Rett syndrome is caused by mutations within methyl CpG-binding protein 2 (MECP2) gene. Over 270 individual nucleotide changes which cause pathogenic mutations have been reported. However, eight most commonly occurring missense and nonsense mutations account for almost 70% of all patients. We screened 90 individuals with Rett syndrome phenotype. A total of 19 different MECP2 mutations and polymorphisms were identified in 27 patients. Of the 19 mutations, we identified 7 (37%) frameshift, 6 (31%) nonsense, 14 (74%) missense mutations and one duplication (5%). The most frequent pathogenic changes were: missense p.T158M (11%), p.R133C (7.4%), and p.R306C (7.4%) and nonsense p.R168X (11%), p.R255X (7.4%) mutations. We have identified two novel mutations namely p.385-388delPLPP present in atypical patients and p.Glu290AlafsX38 present in a classical patient of Rett syndrome. Sequence homology for p.385-388delPLPP mutation revealed that these 4 amino acids were conserved across mammalian species. This indicated the importance of these 4 amino acids in structure and function of the protein. A novel variant p.T479T has also been identified in a patient with atypical Rett syndrome. A total of 62 (69%) patients remained without molecular genetics diagnosis that necessitates further search for mutations in other genes like CDKL5 and FOXG1 that are known to cause Rett phenotype. The majority of mutations are detected in exon 4 and only one mutation was present in exon 3. Therefore, our study suggests the need for screening exon 4 of MECP2 as first line of diagnosis in these patients. PMID:23262346

  3. Functional Consequences of Substitution Mutations in MepR, a Repressor of the Staphylococcus aureus mepA Multidrug Efflux Pump Gene

    PubMed Central

    Schindler, Bryan D.; Seo, Susan M.; Jacinto, Pauline L.; Kumaraswami, Muthiah; Birukou, Ivan; Brennan, Richard G.

    2013-01-01

    The expression of mepA, encoding the Staphylococcus aureus MepA multidrug efflux protein, is repressed by the MarR homologue MepR. MepR dimers bind differently to operators upstream of mepR and mepA, with affinity being greatest at the mepA operator. MepR substitution mutations may result in mepA overexpression, with A103V most common in clinical strains. Evaluation of the functional consequences of this and other MepR substitutions using a lacZ reporter gene assay revealed markedly reduced repressor activity in the presence of Q18P, F27L, G97E, and A103V substitutions. Reporter data were generally supported by susceptibility and efflux assays, and electrophoretic mobility shift assays (EMSAs) confirmed compromised affinities of MepR F27L and A103V for the mepR and mepA operators. One mutant protein contained two substitutions (T94P and T132M); T132M compensated for the functional defect incurred by T94P and also rescued that of A103V but not F27L, establishing it as a limited-range suppressor. The function of another derivative with 10 substitutions was minimally affected, and this may be an extreme example of suppression involving interactions among several residues. Structural correlations for the observed functional effects were ascertained by modeling mutations onto apo-MepR. It is likely that F27L and A103V affect the protein-DNA interaction by repositioning of DNA recognition helices. Negative functional consequences of MepR substitution mutations may result from interference with structural plasticity, alteration of helical arrangements, reduced protein-cognate DNA affinity, or possibly association of MepR protomers. Structural determinations will provide further insight into the consequences of these and other mutations that affect MepR function, especially the T132M suppressor. PMID:23749979

  4. Primary human cells differ in their susceptibility to rAAV-2-mediated gene transfer and duration of reporter gene expression

    Microsoft Academic Search

    Ulrich-Peter Rohr; Ralf Kronenwett; Dirk Grimm; Jürgen Kleinschmidt; Rainer Haas

    2002-01-01

    The susceptibility of a variety of different primary tissues was examined to long-term transduction with recombinant adeno-associated virus type 2 (rAAV-2) and factors influencing the transduction efficiency. In contrast to others using cell lines and animal models, emphasis was placed on the use of primary human cells. Enhanced green fluorescent protein (EGFP) marker gene expression was examined using fluorescence-activated cell

  5. Passage of Autographa californica Nuclear Polyhedrosis Virus through the Midgut Epithelium of Spodoptera exigua Larvae

    Microsoft Academic Search

    J. T. M. Flipsen; J. W. M. Martens; M. M. Van Oers; J. M. Vlak; J. W. M. Van LENT

    1995-01-01

    A special recombinant of Autographa californica multicapsid nuclear polyhedrosis virus (AcNPV) was designed to study the early histopathological events of baculovirus infection in Spodoptera exigua larvae. This recombinant contained a Drosophila melanogaster heat shock 70 promoter driving an Escherichia coli ?-galactosidase (Lac-Z) reporter gene to monitor the presence of early viral gene expression and a second reporter gene, the E.

  6. Isolated duodenal myeloid sarcoma associated with the CBF?/MYH11 fusion gene followed by acute myeloid leukemia progression: A case report and literature review

    PubMed Central

    HUANG, BO; YOU, PENG; ZHU, PING; DU, ZUNGUO; WU, BEIQIAN; XU, XIAOPING; CHEN, ZI

    2014-01-01

    Myeloid sarcoma (MS) is a rare disease that presents as an extramedullary tumorous mass of immature myeloid precursors. The majority of MS are identified in acute myeloid leukemia (AML) patients and rarely present as a primary isolated MS without AML. In addition, inversion of chromosome 16 [inv(16)] and the CBF?/MYH11 fusion gene are rarely associated with MS. The current study reports a female patient with an isolated duodenal MS, who developed AML-M4 associated with the CBF?/MYH11 fusion gene and 48,XX,inv(16),+13,+22. A review of previously reported cases of isolated MS with the CBF?/MYH11 fusion gene was also performed. Isolated MS with the CBF?/MYH11 fusion gene was often observed in abdominal lesions, with the intestinal tract being the predominantly involved site. In addition, patients with isolated MS with the CBF?/MYH11 fusion exhibited a high risk of developing systemic AML. The diagnosis of isolated MS may be particularly challenging and, therefore, determining the optimal standard treatment for isolated MS is required. PMID:25120702

  7. Construction and utilization of carotenoid reporter systems: identification of chromosomal integration sites that support suitable expression of biosynthetic genes and pathways.

    PubMed

    Sharpe, Pamela L; DiCosimo, Deana J

    2012-01-01

    In order to metabolically engineer microorganisms to produce compounds of interest, it is often desirable to integrate foreign genes into the chromosome of the host. However, the consequences of these genetic alterations are not always predictable. The use of a reporter system can often assist in determining chromosomal locations for optimal expression of foreign biosynthetic genes. The method described here involves the construction and utilization of promoterless carotenoid transposons, which provides a colorimetric screen for identifying the best chromosomal integration sites for the expression of the genes of interest. The transposons (pUTmTn5::392W and pUTmTn5::392) contain the carotenoid genes required for the production of canthaxanthin and astaxanthin, respectively. Thus, when promoterless transposons insert into the host's genome, the color of the colonies will vary based on their chromosomal location. There is a correlation between the color intensity of the colonies and the expression of the carotenoid transposon. The transposon insertion site can be determined via direct chromosomal sequencing. This sequence information is used to guide the site-specific integration of biosynthetic genes and pathways of interest. PMID:22623306

  8. Use of bgaH as a reporter gene for studying translation initiation in the archaeon Haloferax volcanii

    E-print Network

    Sullivan, Eric L., S.M. Massachusetts Institute of Technology

    2008-01-01

    The bgaH gene isolated from Haloferax lucentensis codes for P-galactosidase. To study the function of initiator tRNAs in translation initiation in Haloferax volcanii, the initiator AUG codon of the bgaH gene was mutated ...

  9. Identification of Promoter Regions in the Human Genome by Using a Retroviral Plasmid Library-Based Functional Reporter Gene Assay

    Microsoft Academic Search

    Shirin Khambata-Ford; Yueyi Liu; Christopher Gleason; Mark Dickson; Russ B. Altman; Serafim Batzoglou; Richard M. Myers

    2003-01-01

    Attempts to identify regulatory sequences in the human genome have involved experimental and computational methods such as cross-species sequence comparisons and the detection of transcription factor binding-site motifs in coexpressed genes. Although these strategies provide information on which genomic regions are likely to be involved in gene regulation, they do not give information on their functions. We have developed a

  10. Comparative Pathogenesis of Autographa californica M Nuclear Polyhedrosis Virus in Larvae of Trichoplusia ni and Heliothis virescens

    Microsoft Academic Search

    Jan O. Washburn; Bruce A. Kirkpatrick; Loy E. Volkman

    1995-01-01

    We compared early viral pathogenesis and dose mortality relationships for larvae of two highly susceptible hosts, Trichoplusia ni and Heliothis virescens, using a construct of AcMNPV containing the lacZ reporter gene. Larvae were inoculated either as newly molted fourth instars (40) or 15 hr after the molt (415). In 40-inoculated larvae, first lacZ expression was detected in the midgut epithelium

  11. Evaluation of an hPXR reporter gene assay for the detection of aquatic emerging pollutants: screening of chemicals and application to water samples

    Microsoft Academic Search

    Nicolas Creusot; Saïd Kinani; Patrick Balaguer; Nathalie Tapie; Karyn LeMenach; Emmanuelle Maillot-Maréchal; Jean-Marc Porcher; Hélène Budzinski; Sélim Aït-Aïssa

    2010-01-01

    Many environmental endocrine-disrupting compounds act as ligands for nuclear receptors. Among these receptors, the human pregnane\\u000a X receptor (hPXR) is well described as a xenobiotic sensor to various classes of chemicals, including pharmaceuticals, pesticides,\\u000a and steroids. To assess the potential use of PXR as a sensor for aquatic emerging pollutants, we employed an in vitro reporter\\u000a gene assay (HG5LN-hPXR cells)

  12. Schizophrenia and the androgen receptor gene: Report of a sibship showing co-segregation with Reifenstein Syndrome but no evidence for linkage in 23 multiply affected families

    SciTech Connect

    Arranz, M.; Sharma, T.; Sham, P.; Kerwin, R. [Institute of Psychiatry, London (United Kingdom)] [and others

    1995-10-09

    Crow et al. have reported excess sharing of alleles by male sibling pairs with schizophrenia, at a triplet repeat marker within the androgen receptor gene, indicating that mutations at or near this gene may be a risk factor for males. In this report, we describe a pair of male siblings concordant for both schizophrenia and Reifenstein syndrome, which is caused by a mutation in this gene. This provides support for the hypothesis that the androgen receptor may contribute to liability to develop schizophrenia. Because of this, we have examined a collection of 23 pedigrees multiply affected by schizophrenia for linkage to the androgen receptor. We have found no evidence for linkage by both the LOD score and affected sibling-pair methods, under a range of genetic models with a broad and narrow definition of phenotype, and when families with male-to-male transmission are excluded. However, because of the small number of informative male-male pairs in our sample, we cannot confirm or refute the excess allele sharing for males reported by Crow. 35 refs., 1 fig., 2 tabs.

  13. First Report of a Clinical, Multidrug-Resistant Enterobacteriaceae Isolate Coharboring Fosfomycin Resistance Gene fosA3 and Carbapenemase Gene blaKPC-2 on the Same Transposon, Tn1721

    PubMed Central

    Li, Gang; Zhang, Ying; Bi, Dexi; Shen, Pinghua; Ai, Fuqi; Liu, Hong; Tian, Yueru; Ma, Yiming; Wang, Bei; Rajakumar, Kumar

    2014-01-01

    In order to understand the genetic background and dissemination mechanism of carbapenem resistance and fosfomycin resistance in Enterobacteriaceae isolates, we studied a clinical Escherichia coli strain HS102707 isolate and an Enterobacter aerogenes strain HS112625 isolate, both of which were resistant to carbapenem and fosfomycin and positive for the blaKPC-2 and fosA3 genes. In addition, a clinical Klebsiella pneumoniae strain HS092839 isolate which was resistant to carbapenem was also studied. A 70-kb plasmid was successfully transferred to recipient E. coli J53 by a conjugation test. PCR and Southern blot analysis showed that blaKPC-2 was located on this plasmid. The complete sequence of pHS102707 showed that this plasmid belongs to the P11 subfamily (IncP1) and has a replication gene, several plasmid-stable genes, an intact type IV secretion system gene cluster, and a composite transposon Tn1721-Tn3 that harbored blaKPC-2. Interestingly, a composite IS26 transposon carrying fosA3 was inserted in the Tn1721-tnpA gene in pHS102707 and pHS112625, leading to the disruption of Tn1721-tnpA and the deletion of Tn1721-tnpR. However, only IS26 with a truncated Tn21-tnpR was inserted in pHS092839 at the same position. To our knowledge, this is the first report of fosA3 and blaKPC-2 colocated in the same Tn1721-Tn3–like composite transposon on a novel IncP group plasmid. PMID:25367902

  14. Transposon-induced nuclear mutations that alter chloroplast gene expression. Annual report, September 1, 1991--August 31, 1992

    SciTech Connect

    Barkan, A.

    1992-12-31

    The goal of this project is to use mutant phenotypes as a guide to nuclear genes that determine the timing and localization of chloroplast development The immediate goals are to identify nuclear mutants with defects in chloroplast gene expression from maize lines harboring active Mu transposons; characterize their phenotypes to determine the precise defect in gene expression; clone several of the most interesting mutations by exploiting the transposon tag; and use the clones to further define the roles of these genes in modulating chloroplast gene expression. Three mutants were described earlier that had global defects in chloroplast gene expression. We have found that two of these mutations are allelic. Both alleles have global defects in chloroplast translation initiation, as revealed by the failure to assemble chloroplast mRNAs into polysomes. We have isolated and characterized three new mutants from Mu lines that have novel defects in chloroplast RNA metabolism. We are now ready to begin the task of cloning several of these genes, by using the Mu transposon tag.

  15. Mapping of the fate of cell lineages generated from cells that express the Wnt4 gene by time-lapse during kidney development.

    PubMed

    Shan, Jingdong; Jokela, Tiina; Skovorodkin, Ilya; Vainio, Seppo

    2010-01-01

    The Wnt4 gene encodes a secreted signaling molecule controlling the development of several organs, such as the kidney, adrenal gland, ovary, mammary gland and pituitary gland. It is thought to act in the embryonic kidney as an auto-inducer of nephrogenesis controlling mesenchyme-to-epithelium transition, and Wnt4-deficient mice die soon after birth, probably of kidney failure. Given the requirement for Wnt4 signaling in the control of organogenesis, the targeting of Cre recombinase under the control of the Wnt4 promoter would provide a valuable tool for fate mapping and functional genomics. We report here on the generation and characterization of a Wnt4(EGFPCre) knock-in allele where the EGFPCre fusion cDNA and Neo selection cassette were targeted into the Wnt4 locus. EGFP-derived fluorescence was observed in the pretubular aggregates of the E14.5 embryonic kidney that normally express Wnt4 mRNA. Characterization of the pattern of recombination of the floxed Rosa26(LacZ) reporter with the Wnt4(EGFPCre) allele revealed that in addition to the embryonic kidney, reporter-derived staining was observed in the embryonic gonad, spinal cord, lung and adrenal gland, i.e. the sites of Wnt4 gene expression. Time-lapse fate mapping of the Wnt4(EGFPCre)-activated yellow fluorescent protein (YFP) from the Rosa26 locus in organ culture revealed that the cells that had expressed the Wnt4 gene contributed to the nephrons, some of the cells around the stalk of the developing ureter and also certain presumptive medullary stromal cells. Moreover, the time-lapse movies suggested that the first few pretubular cell aggregates may not mature into nephrons but instead appear to disintegrate. In association with this, Rosa26(YFP)-positive stromal cells emerge around these disintegrating structures. Such cells may be transient, since their derivatives are neither detected later in the more mature kidney nor is there an overlap of the Wnt4(EGFPCre); Rosa26(LacZ)-marked cells with those of the endothelial cells, the smooth muscle cells or the macrophages. The Wnt4(EGFPCre) allele provides a useful new tool for conditional mutagenesis and provides the first time-lapse-based map of the fate of nephron precursor cells. PMID:19740593

  16. The homeobox gene NTH23 of tobacco is expressed in the basal region of leaf primordia 1 The nucleotide sequence reported in this paper has been submitted to the DDBJ\\/EMBL\\/GeneBank Nucleotide Sequence Databases under accession number, AB004797. 1

    Microsoft Academic Search

    Naoki Sentoku; Masanori Tamaoki; Asuka Nishimura; Makoto Matsuoka

    1998-01-01

    We reported isolation and characterization of a homeobox gene from tobacco, NTH23. The homeodomain structure of NTH23 was highly homologous to the same regions of class 2 genes of the KN1-type homeobox (sharing more than 85% amino acid identity), but was less similar to class 1 genes of KN1-type. RNA gel blot analysis revealed that NTH23 was expressed in all

  17. Differential molecular profiling between skin carcinomas reveals four newly reported genes potentially implicated in squamous cell carcinoma development

    Microsoft Academic Search

    Claire Marionnet; Claude Lalou; Karine Mollier; Marjorie Chazal; Gisele Delestaing; Delphine Compan; Olivier Verola; Catherine Vilmer; Jerome Cuminet; Louis Dubertret; Nicole Basset-Séguin

    2003-01-01

    Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are skin tumors with different invasive potential. In this work, we analysed mRNA differential expression between seven BCC and five SCC and their normal skin counterparts using 1176 cDNA macroarrays and verification by RT–PCR to identify genes modulated in each tumor type. We identified 37 genes commonly modulated in both tumors

  18. First report of a pathogenic mutation on exon 24 of the NOTCH3 gene in a CADASIL family

    Microsoft Academic Search

    Raffaella Valenti; Silvia Bianchi; Francesca Pescini; Camilla D’Eramo; Domenico Inzitari; Maria Teresa Dotti; Leonardo Pantoni

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a genetically transmitted\\u000a small vessel disease clinically characterized by migraine, recurrent subcortical strokes, and cognitive and mood disorders.\\u000a Pathogenic mutations are located on any of the exons of the NOTCH3 gene coding for epidermal-growth factor (EGF)-like repeats of the extracellular domain of the NOTCH3 receptor. Because the\\u000a gene is

  19. Microarray analysis of genes differentially expressed in HepG2 cells cultured in simulated microgravity: preliminary report

    NASA Technical Reports Server (NTRS)

    Khaoustov, V. I.; Risin, D.; Pellis, N. R.; Yoffe, B.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.

  20. Structure and molecular characterization of barley nudix hydrolase genes.

    PubMed

    Tanaka, Sayuri; Kihara, Makoto; Sugimoto, Manabu

    2015-01-01

    Putative nudix hydrolase (NUDX) genes, which encode amino acid sequences showing homology with those of Arabidopsis NUDXs and conserve nudix motif, were identified from barley. The 14 deduced barley NUDXs (HvNUDX1-14) were classified into established subfamilies, except for 8-oxo-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) pyrophosphohydrolase and mRNA decapping enzyme subfamilies, and three substrate-unknown subfamilies. Drought and UV-C stresses, respectively, up-regulated 7 and 4 HvNUDX genes, but some homologs of Arabidopsis NUDXs showed different responses to abiotic stress. HvNUDX12 gene, belonging to diadenosine tetraphosphates (Ap?A) pyrophosphohydrolase subfamily gene and up-regulated by UV-C, was expressed in Escherichia coli cells. The recombinant protein showed 8-oxo-dGTP, Ap?A, and guanosine-3',5'-tetraphosphate (ppGpp) pyrophosphohydrolase activities, and the suppression of the lacZ amber mutation in a mutT-deficient E. coli cells caused by the incorporation of 8-oxo-GTP into mRNA was prevented to a significant degree. These results suggest that barley NUDXs have unique constitution and response of NUDX to abiotic stress. PMID:25379607

  1. Use of DEAD-box polypeptide-4 (Ddx4) gene promoter-driven fluorescent reporter mice to identify mitotically active germ cells in post-natal mouse ovaries.

    PubMed

    Park, Eun-Sil; Tilly, Jonathan L

    2015-01-01

    Several laboratories have independently isolated mitotically active germ cells, termed female germline stem cells or oogonial stem cells (OSCs), from adult mouse ovaries. However, a recent study using Ddx4-Cre;Rosa26 reporter mice concluded that such germ cells do not exist. Given the disparity in conclusions drawn in this study compared with others, we felt it was important to re-assess the utility of Ddx4-Cre;Rosa26 reporter mice for identification of OSCs in adult mouse ovaries. Transgenic Ddx4-Cre mice were crossed with Rosa26(tdTm/tdTm) mice to drive restricted tomato red (tdTm) gene expression in cells in which the Ddx4 gene promoter has been activated. Crude dispersion of ovaries from recombined offspring generated cell fractions containing tdTm-positive immature oocytes, which are incapable of proliferation and thus probably represent the uncharacterized reporter-positive ovarian cells identified in the paper Zhang et al. (2012) as being mitotically inactive. Dispersed ovaries further subjected to fluorescence-activated cell sorting yielded a large population of non-germline tdTm-positive cells, indicative of promoter 'leakiness' in the Ddx4-Cre mouse line. Nonetheless, a small percentage of these tdTm-positive cells exhibited externalized (extracellular, ec) expression of Ddx4 protein (ecDdx4-positive), expressed markers of primitive germ cells but not of oocytes, and actively proliferated in culture, all of which are characteristic features of OSCs. Thus, crude dispersion of ovaries collected from Ddx4 gene promoter-driven reporter mice is not, by itself, a reliable approach to identify OSCs, whereas the same ovarian dispersates further subjected to cell sorting strategies yield purified OSCs that can be expanded in culture. PMID:25147160

  2. An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level

    PubMed Central

    Kojima, Shin-ichiro

    2014-01-01

    RNA interference (RNAi) is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA) in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA) together with an enhanced green fluorescent protein (EGFP); the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry). Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls) is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3) and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi. To further facilitate testing of RNAi efficacy, we developed a negative selection marker ( ccdB) method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification. PMID:24741441

  3. An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level.

    PubMed

    Kojima, Shin-Ichiro; Borisy, Gary G

    2014-01-01

    RNA interference (RNAi) is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA) in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA) together with an enhanced green fluorescent protein (EGFP); the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry). Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls) is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3) and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi. To further facilitate testing of RNAi efficacy, we developed a negative selection marker ( ccdB) method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification. PMID:24741441

  4. [Metatropic dysplasia in a girl with c.1811_1812delinsAT mutation in exon 11 of the TRPV4 gene not previously reported].

    PubMed

    Cammarata-Scalisi, Francisco; Matysiak-Scholze, Uta; Heinze, Jessica; Barrera, Albaro; Lacruz-Rengel, María Angelina; Bracho, Ana; Guerrero, Yudith

    2015-01-01

    Metatropic dysplasia is a skeletal disorder with clinical heterogeneity, characterized by craniofacial dysmorphy including frontal bossing and midface hypoplasia, short trunk,progressive kyphoscoliosis and shortened limbs. The TRPV4 gene is located on 12q24.11, coding a cation channel with nonselective permeability to calcium; it is expressed and involved in many physiological processes through responses to different stimuli. Over 50 mutations in TRPV4 have been described. We present a seven months old girl with heterozygous mutation c.1811_1812delinsAT; p.I604N in intron 11 not previously reported in the TRPV4 gene and with clinical findings compatible with metatropic dysplasia. PMID:25622169

  5. Differential gene expression in neurospora crassa cell types: amplification of rRNA genes. Progress report, July 1979-30 June 1980

    SciTech Connect

    Dutta, S.K.

    1980-01-01

    The significant results obtained during 1979 to 1980 of the current research program are as follows: (1) the differential rRNA gene amplification in germinated conidia of N.crassa was confirmed. N.crassa rDNAs showed differences in degrees of homology with isolated DNAs from other Neurospora species which could be due to heterogeneity in internal spacers. Studies with N.crassa rDNA clones were initiated to study their heterogeneities. The organization of the Institutional Biohazard Committee (IBC) for Recombinant DNA research was completed and necessary certifications for the laboratory and the workers were obtained in accordance with the P/sub 2/EK/sub 1/ containment regulation of N.I.H. Known 17S and 26S N.crassa rDNA probes are being used to detect differences, if any, in restriction cleavage sites in rDNAs of different cell types and developmental mutants of N.crassa. DNAs from these N.crassa cells are restricted with EcoR/sub 1/ and Hind III and cleaved fragments separated by gel electrophoresis are transferred into nitrocellulose papers. Experiments are underway now to see if there are any changes in cleavage sites by annealing with /sup 32/P or /sup 3/H-17S or 26S rDNA probes followed by autoradiography.

  6. Evaluation of transformation in peach Prunus persica explants using green fluorescent protein (GFP) and beta-glucuronidase (GUS) reporter genes

    Microsoft Academic Search

    Isabel M. G. Padilla; Agnieszka Golis; Adele Gentile; Carmine Damiano; Ralph Scorza

    2006-01-01

    To determine the optimum conditions for Agrobacterium-mediated gene transfer, peach explants including cotyledons, embryonic axes and hypocotyl slices from non-germinated seeds\\u000a and epicotyl internode slices from germinating seeds were exposed to Agrobacterium-mediated transformation treatments. The GUS (uidA) marker gene was tested using two different A. tumefaciens strains, three plasmids and four promoters [CaMV35s, (Aocs)3AmasPmas (“super-promoter”), mas-CaMV35s, and CAB]. GFP was

  7. New ex vivo reporter assay system reveals that ? factors of an unculturable pathogen control gene regulation involved in the host switching between insects and plants

    PubMed Central

    Ishii, Yoshiko; Kakizawa, Shigeyuki; Oshima, Kenro

    2013-01-01

    Abstract Analysis of the environmental regulation of bacterial gene expression is important for understanding the nature, pathogenicity, and infection route of many pathogens. “Candidatus Phytoplasma asteris”, onion yellows strain M (OY-M), is a phytopathogenic bacterium that is able to adapt to quite different host environments, including plants and insects, with a relatively small ?850 kb genome. The OY-M genome encodes two sigma (?) factors, RpoD and FliA, that are homologous to Escherichia coli ?70 and ?28, respectively. Previous studies show that gene expression of OY-M dramatically changes upon the response to insect and plant hosts. However, very little is known about the relationship between the two ? factors and gene regulatory systems in OY-M, because phytoplasma cannot currently be cultured in vitro. Here, we developed an Escherichia coli-based ex vivo reporter assay (EcERA) system to evaluate the transcriptional induction of phytoplasmal genes by the OY-M-derived ? factors. EcERA revealed that highly expressed genes in insect and plant hosts were regulated by RpoD and FliA, respectively. We also demonstrated that rpoD expression was significantly higher in insect than in plant hosts and fliA expression was similar between the hosts. These data indicate that phytoplasma-derived RpoD and FliA play key roles in the transcriptional switching mechanism during host switching between insects and plants. Our study will be invaluable to understand phytoplasmal transmission, virulence expression in plants, and the effect of infection on insect fitness. In addition, the novel EcERA system could be broadly applied to reveal transcriptional regulation mechanisms in other unculturable bacteria. Phytoplasma, an unculturable plant pathogen, could infect plant and insect cells, and dramatically changes their genes upon the response to these hosts. By a new system developed in this study, interactions between phytoplasma promoters and sigma factors were analyzed, and overall gene expression regulation mechanism could be revealed. This model illustrates the RpoD and FliA regulatory network in phytoplasma cells during host switching. PMID:23723081

  8. Peripheral precocious puberty in a male caused by Leydig cell adenoma harboring a somatic mutation of the LHR gene: report of a case.

    PubMed

    Sangkhathat, Surasak; Kanngurn, Samornmas; Jaruratanasirikul, Somchit; Tubtawee, Teeravut; Chaiyapan, Walawee; Patrapinyokul, Sakda; Chiengkriwate, Piyawan

    2010-09-01

    While a germline activating mutation of the luteinizing hormone receptor (LHR) gene is known to cause autonomous production of testosterone from testicular Leydig cells in male-limited precocious puberty, only a few studies have addressed the role of somatic LHR mutation in testicular pathology. The authors report a case of a 6-year-old boy who developed secondary sex characteristics including facial acne, enlarging genitalia, and aggressive behavior, for which serial biochemical evaluation confirmed the status of peripheral precocious puberty. Examination revealed asymmetrical testicular volume, following which a left testicular tumor was detected through ultrasonography. A left orchiectomy was performed, and histopathology revealed a well-circumscribed Leydig cell tumor Molecular study of the exon 11 of the LHR gene revealed a missense mutation at the nucleotide position 1,732, leading to a substitution of histidine for aspartic acid at codon 578. Interestingly, the substitution was consistent with all previously reported LHR alteration in pediatric Leydig cell adenoma, but which had never before been reported in male-limited precocious puberty, suggesting that the mutation is a molecular signature of the adenoma. PMID:20873084

  9. Silencing of Reporter Gene Expression in Skin Using siRNAs and Expression of Plasmid DNA Delivered by a Soluble Protrusion Array Device (PAD)

    PubMed Central

    Gonzalez-Gonzalez, Emilio; Speaker, Tycho J; Hickerson, Robyn P; Spitler, Ryan; Flores, Manuel A; Leake, Devin; Contag, Christopher H; Kaspar, Roger L

    2010-01-01

    Despite rapid progress in the development of potent and selective small interfering RNA (siRNA) agents for skin disorders, translation to the clinic has been hampered by the lack of effective, patient-friendly delivery technologies. The stratum corneum poses a formidable barrier to efficient delivery of large and/or charged macromolecules including siRNAs. Intradermal siRNA injection results in effective knockdown of targeted gene expression but is painful and the effects are localized to the injection site. The use of microneedle arrays represents a less painful delivery method and may have utility for the delivery of nucleic acids, including siRNAs. For this purpose, we developed a loadable, dissolvable protrusion array device (PAD) that allows skin barrier penetration. The PAD tips dissolve upon insertion, forming a gel-like plug that releases functional cargo. PAD-mediated delivery of siRNA (modified for enhanced stability and cellular uptake) resulted in effective silencing of reporter gene expression in a transgenic reporter mouse model. PAD delivery of luciferase reporter plasmids resulted in expression in cells of the ear, back, and footpad skin as assayed by intravital bioluminescence imaging. These results support the use of PADs for delivery of functional nucleic acids to cells in the skin with an efficiency that may support clinical translation. PMID:20571543

  10. Oxygen-sensing reporter strain of Pseudomonas fluorescens for monitoring the distribution of low-oxygen habitats in soil.

    PubMed

    Højberg, O; Schnider, U; Winteler, H V; Sørensen, J; Haas, D

    1999-09-01

    The root-colonizing bacterium Pseudomonas fluorescens CHA0 was used to construct an oxygen-responsive biosensor. An anaerobically inducible promoter of Pseudomonas aeruginosa, which depends on the FNR (fumarate and nitrate reductase regulation)-like transcriptional regulator ANR (anaerobic regulation of arginine deiminase and nitrate reductase pathways), was fused to the structural lacZ gene of Escherichia coli. By inserting the reporter fusion into the chromosomal attTn7 site of P. fluorescens CHA0 by using a mini-Tn7 transposon, the reporter strain, CHA900, was obtained. Grown in glutamate-yeast extract medium in an oxystat at defined oxygen levels, the biosensor CHA900 responded to a decrease in oxygen concentration from 210 x 10(2) Pa to 2 x 10(2) Pa of O(2) by a nearly 100-fold increase in beta-galactosidase activity. Half-maximal induction of the reporter occurred at about 5 x 10(2) Pa. This dose response closely resembles that found for E. coli promoters which are activated by the FNR protein. In a carbon-free buffer or in bulk soil, the biosensor CHA900 still responded to a decrease in oxygen concentration, although here induction was about 10 times lower and the low oxygen response was gradually lost within 3 days. Introduced into a barley-soil microcosm, the biosensor could report decreasing oxygen concentrations in the rhizosphere for a 6-day period. When the water content in the microcosm was raised from 60% to 85% of field capacity, expression of the reporter gene was elevated about twofold above a basal level after 2 days of incubation, suggesting that a water content of 85% caused mild anoxia. Increased compaction of the soil was shown to have a faster and more dramatic effect on the expression of the oxygen reporter than soil water content alone, indicating that factors other than the water-filled pore space influenced the oxygen status of the soil. These experiments illustrate the utility of the biosensor for detecting low oxygen concentrations in the rhizosphere and other soil habitats. PMID:10473420

  11. Mutations in the Spt4, Spt5, and Spt6 Genes Alter Transcription of a Subset of Histone Genes in Saccharomyces Cerevisiae

    PubMed Central

    Compagnone-Post, P. A.; Osley, M. A.

    1996-01-01

    The SPT4, SPT5, and SPT6 gene products define a class of transcriptional repressors in Saccharomyces cerevisiae that are thought to function through their effects on chromatin assembly or stability. Mutations in these genes confer a similar range of phenotypes to mutations in HIR genes, which encode transcriptional repressors that regulate expression of many of the core histone genes. Here we show that mutations in the three SPT genes also affect transcription of the histone genes that reside at the HTA1-HTB1 locus. HTA1-lacZ transcription was reduced in each spt mutant background, an effect that required a negative site in the HTA1 promoter. The transcriptional effect could be reversed by the overproduction of histones H2A and H2B in an spt4 mutant and histones H3 and H4 in all three spt mutants. Suppression of the spt4 transcriptional defect was dependent on the overproduction of both histones H2A and H2B, and required the presence of N-terminal amino acids in both histones. The results are consistent with the idea that the effects of the spt mutations on nucleosome assembly and/or stability activate repressors of HTA1 transcription. PMID:8844144

  12. Development of a semi-quantitative plate-based alpha-galactosidase gene reporter for Schizosaccharomyces pombe and its use to isolate a constitutively active Mam2.

    PubMed

    Goddard, Alan; Ladds, Graham; Davey, John

    2005-01-15

    To extend the tools available for biochemical and genetical analysis in the fission yeast Schizosaccharomyces pombe we have investigated the development of gene reporter systems using the secreted alpha-galactosidase encoded by the Sz. pombe ORF SPAC869.07c (CAB60017), which we propose naming Mel1p to reflect its structural and functional similarity to MEL1p in Saccharomyces cerevisiae. The alpha-galactosidase activity can be monitored in liquid assays and converted the colourless substrate 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside (X-alpha-gal) into an insoluble blue product that was suitable for semi quantitative plate-based assays; colonies expressing the highest levels of alpha-galactosidase developed the most intense blue colour. Unlike assays based on beta-galactosidase, the Sz. pombe colonies develop the blue colouration under normal growth conditions, avoiding the need to replicate colonies to fresh plates for analysis. It is therefore suitable for screening large numbers of colonies. To illustrate the use of mel1 as a reporter we linked expression to the sxa2 gene promoter to provide a convenient readout for signalling through the pheromone response pathway. The sxa2 > mel1 strain identified constitutively active Mam2 pheromone receptors from a randomly mutagenised library. There was an approximate correlation between the intensity of the blue colour developed by each mutant colony and its level of constitutive activity and we identified a subset of mutants with low constitutive activity that could not have been isolated by a previous screen using nutritional selection. The mel1 alpha-galactosidase activity identified and characterised in this study can be easily adapted to provide a gene reporter for many biological processes and is a new addition to the research tools available in Sz. pombe. PMID:15580593

  13. Brief Report: The Dopamine-3-Receptor Gene ("DRD3") Is Associated with Specific Repetitive Behavior in Autism Spectrum Disorder (ASD)

    ERIC Educational Resources Information Center

    Staal, Wouter G.; de Krom, Mariken; de Jonge, Maretha V.

    2012-01-01

    Recently the "DRD3" gene has been associated with ASD in two independent samples. Follow up analysis of the risk allele of the SNP rs167771 in 91 subjects revealed a significant association with a specific type of repetitive behavior: the factor "insistence on sameness" (IS) derived from the Autism Diagnostic Interview. This risk allele was…

  14. Inducible clindamycin resistance due to expression of erm genes in Staphylococcus aureus: report from a tertiary care Hospital Karachi, Pakistan

    Microsoft Academic Search

    Naima Fasih; Seema Irfan; Afia Zafar; Erum Khan; Rumina Hasan

    2010-01-01

    OBJECTIVE: To assess the frequency of phenotypic expression of inducible resistance of clindamycin due to expression of erm genes, in clinical isolates of Staphylococcus aureus (S. aureus), by double disk diffusion test (D-test).METHOD: This was a cross sectional study conducted in the clinical laboratory of Aga Khan University Hospital, Karachi. A total of 2432, non duplicate clinical isolates of S.

  15. Replication-Deficient Adenovirus Vector Transfer of gfpReporter Gene into Supraoptic Nucleus and Subfornical Organ Neurons

    Microsoft Academic Search

    Elisardo C. Vasquez; Ralph F. Johnson; Terry G. Beltz; Ronald E. Haskell; Beverly L. Davidson; Alan Kim Johnson

    1998-01-01

    The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats

  16. (Analysis of cyanobacterial photosystem 2 genes by cloning and mutagenesis): Progress report, May 1986 to December 1986

    SciTech Connect

    Not Available

    1986-01-01

    We have isolated proteins that appear to be regulated by changes in environmental conditions, either iron or light. Iron-regulated proteins include: a 36 kDa protein that is involved in iron-acquisition, and a 34 kDa intrinsic thylakoid chlorophyll-binding protein associated with PS II. Utilizing a lambda gtll library we have cloned and sequenced the 36 kDa protein and are performing site-directed mutagenesis to determine the function of the protein. Insertion of a transposon into this gene and transformation of the mutated gene into a wild type host resulted in a strain which is unable to grow under low-iron conditions. We have obtained a 42 kDa carotenoid-binding protein that is induced upon growth in high light conditions, and cloned the gene for this protein. We have also isolated a carotenoid-binding protein that is localized exclusively in the plasmalemma. We have also cloned the 33 kDa extrinsic maganese-binding protein that is involved in oxygen-evolution. The gene has been cloned and sequenced and contains a 28 amino acid signal sequence. 4 figs.

  17. CELL DIVISION GENE CLUSTER IN SPIROPLASMA KUNKELII: FUNCTIONAL CHARACTERIZATION OF FTSZ AND THE FIRST REPORT OF FTSA IN MOLLICUTES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ftsZ gene, found in representatives of all bacterial groups, encodes an essential protein engaged in prokaryotic cell division. We cloned and characterized the ftsZ homologue (ftsZsk) from a pathogenic strain of the wall-less bacterium Spiroplasma kunkelii that causes corn stunt disease. The 1...

  18. LuSens: a keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification.

    PubMed

    Ramirez, Tzutzuy; Mehling, Annette; Kolle, Susanne N; Wruck, Christoph J; Teubner, Wera; Eltze, Tobias; Aumann, Alexandra; Urbisch, Daniel; van Ravenzwaay, Ben; Landsiedel, Robert

    2014-12-01

    Allergic contact dermatitis can develop following repeated exposure to allergenic substances. To date, hazard identification is still based on animal studies as non-animal alternatives have not yet gained global regulatory acceptance. Several non-animal methods addressing key-steps of the adverse outcome pathway (OECD, 2012) will most likely be needed to fully address this effect. Among the initial cellular events is the activation of keratinocytes and currently only one method, the KeratinoSens™, has been formally validated to address this event. In this study, a further method, the LuSens assay, that uses a human keratinocyte cell line harbouring a reporter gene construct composed of the antioxidant response element (ARE) of the rat NADPH:quinone oxidoreductase 1 gene and the luciferase gene. The assay was validated in house using a selection of 74 substances which included the LLNA performance standards. The predictivity of the LuSens assay for skin sensitization hazard identification was comparable to other non-animal methods, in particular to the KeratinoSens™. When used as part of a testing battery based on the OECD adverse outcome pathway for skin sensitization, a combination of the LuSens assay, the DPRA and a dendritic cell line activation test attained predictivities similar to that of the LLNA. PMID:25172300

  19. Use of a transposon with luciferase as a reporter to identify environmentally responsive genes in a cyanobacterium

    SciTech Connect

    Wolk, C.P.; Yuping Cai; Panoff, J.M. (Michigan State Univ., East Lansing (United States))

    1991-06-15

    Anabaena, a filamentous cyanobacterium, is of developmental interest because, when deprived of fixed nitrogen, it shows patterned differentiation of N{sub 2}-fixing cells called heterocysts. To help elucidate its early responses to a decrease in nitrogen, the authors used a derivative of transposon Tn5 to generate transcriptional fusions of promoterless bacterial luciferase genes, luxAB, to the Anabaena genome. Genes that responded to removal of fixed nitrogen or to other environmental shifts by increased or decreased transcription were identified by monitoring the luminescence of colonies from transposon-generated libraries. The Tn5 derivative transposed in Anabaena at ca. 1-4 {times} 10{sup {minus}5} per cell and permitted high-resolution mapping of its position and orientation in the genome and facile cloning of contiguous genomic DNA.

  20. [Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene]. Progress report, [March 15, 1989--April 14, 1990

    SciTech Connect

    Okita, T.W.

    1990-12-31

    The long term aim of this project is to assess the feasibility of increasing the conversion of photosynthate into starch via manipulation of the gene that encodes for ADPglucose pyrophosphorylase, a key regulatory enzyme of starch biosynthesis. In developing storage tissues such as cereal seeds and tubers, starch biosynthesis is regulated by the gene activation and expression of ADPglucose pyrophosphorylase, starch synthase, branching enzyme and other ancillary starch modifying enzymes, as well as the allosteric-controlled behavior of ADPglucose pyrophosphorylase activity. During the last two years we have obtained information on the structure of this enzyme from both potato tuber and rice endosperm, using a combination of biochemical and molecular biological approaches. Moreover, we present evidence that this enzyme may be localized at discrete regions of the starch grain within the amyloplast, and plays a role in controlling overall starch biosynthesis in potato tubers.

  1. A visual reporter system for virus-induced gene silencing in tomato fruit based on anthocyanin accumulation.

    PubMed

    Orzaez, Diego; Medina, Aurora; Torre, Sara; Fernández-Moreno, Josefina Patricia; Rambla, José Luis; Fernández-Del-Carmen, Asun; Butelli, Eugenio; Martin, Cathie; Granell, Antonio

    2009-07-01

    Virus-induced gene silencing (VIGS) is a powerful tool for reverse genetics in tomato (Solanum lycopersicum). However, the irregular distribution of the effects of VIGS hampers the identification and quantification of nonvisual phenotypes. To overcome this limitation, a visually traceable VIGS system was developed for fruit, comprising two elements: (1) a transgenic tomato line (Del/Ros1) expressing Antirrhinum majus Delila and Rosea1 transcription factors under the control of the fruit-specific E8 promoter, showing a purple-fruited, anthocyanin-rich phenotype; and (2) a modified tobacco rattle virus VIGS vector incorporating partial Rosea1 and Delila sequences, which was shown to restore the red-fruited phenotype upon agroinjection in Del/Ros1 plants. Dissection of silenced areas for subsequent chemometric analysis successfully identified the relevant metabolites underlying gene function for three tomato genes, phytoene desaturase, TomloxC, and SlODO1, used for proof of concept. The C-6 aldehydes derived from lipid 13-hydroperoxidation were found to be the volatile compounds most severely affected by TomloxC silencing, whereas geranial and 6-methyl-5-hepten-2-one were identified as the volatiles most severely reduced by phytoene desaturase silencing in ripening fruit. In a third example, silencing of SlODO1, a tomato homolog of the ODORANT1 gene encoding a myb transcription factor, which regulates benzenoid metabolism in petunia (Petunia hybrida) flowers, resulted in a sharp accumulation of benzaldehyde in tomato fruit. Together, these results indicate that fruit VIGS, enhanced by anthocyanin monitoring, can be a powerful tool for reverse genetics in the study of the metabolic networks operating during fruit ripening. PMID:19429602

  2. A 796 bp PsPR10 gene promoter fragment increased root-specific expression of the GUS reporter gene under the abiotic stresses and signal molecules in tobacco.

    PubMed

    Xu, Xiangbin; Guo, Sai; Chen, Kai; Song, Hongmiao; Liu, Junjun; Guo, Longbiao; Qian, Qian; Wang, Huizhong

    2010-10-01

    A 1681 bp PsPR10 promoter was isolated from Pinus strobus and a series of 5'-deletions were fused to the ?-glucuronidase (GUS) reporter gene and introduced into tobacco. GUS activity in P796 (-796 to +69) construct transgenic plant roots was similar with that of P1681 and higher than those of the P513 (-513 to +69) and P323 (-323 to +69) transgenic plants. Moreover, the abiotic stresses of NaCl, PEG 6000 and mannitol, and salicylic acid (SA), abscisic acid (ABA) and jasmonic acid (JA) induced higher GUS activity in the roots of P796 transgenic tobacco. This study provides a potential inducible root-specific promoter for transgenic plants. PMID:20495947

  3. Excess congenital non-synonymous variation in leukemia-associated genes in MLL? infant leukemia: a Children's Oncology Group report

    PubMed Central

    Valentine, M C; Linabery, A M; Chasnoff, S; Hughes, A E O; Mallaney, C; Sanchez, N; Giacalone, J; Heerema, N A; Hilden, J M; Spector, L G; Ross, J A; Druley, T E

    2014-01-01

    Infant leukemia (IL) is a rare sporadic cancer with a grim prognosis. Although most cases are accompanied by MLL rearrangements and harbor very few somatic mutations, less is known about the genetics of the cases without MLL translocations. We performed the largest exome-sequencing study to date on matched non-cancer DNA from pairs of mothers and IL patients to characterize congenital variation that may contribute to early leukemogenesis. Using the COSMIC database to define acute leukemia-associated candidate genes, we find a significant enrichment of rare, potentially functional congenital variation in IL patients compared with randomly selected genes within the same patients and unaffected pediatric controls. IL acute myeloid leukemia (AML) patients had more overall variation than IL acute lymphocytic leukemia (ALL) patients, but less of that variation was inherited from mothers. Of our candidate genes, we found that MLL3 was a compound heterozygote in every infant who developed AML and 50% of infants who developed ALL. These data suggest a model by which known genetic mechanisms for leukemogenesis could be disrupted without an abundance of somatic mutation or chromosomal rearrangements. This model would be consistent with existing models for the establishment of leukemia clones in utero and the high rate of IL concordance in monozygotic twins. PMID:24301523

  4. Functional polymorphisms in the serotonin 1B receptor gene (HTR1B) predict self-reported anger and hostility among young men

    PubMed Central

    Conner, Tamlin S.; Jensen, Kevin P.; Tennen, Howard; Furneaux, Henry M.; Kranzler, Henry R.; Covault, Jonathan

    2011-01-01

    Objective To examine associations between haplotypes of the serotonin 1B receptor gene and individual differences in anger and hostility. Methods Data were analyzed from a study of 361 university students (47% male). Participants were genotyped at 5 polymorphisms in the HTR1B gene (rs11568817, rs130058, rs6296, rs6297, rs13212041), including promoter and 3?UTR polymorphisms with opposite functional effects on gene expression. Participants reported their emotional states across 30 consecutive days for up to four years. Haplotype pairs were constructed statistically and assigned to a level of HTR1B expression based on the presence of the functional polymorphisms. Results Six haplotypes accounted for >97% of chromosomes. Three low expression haplotypes contained the 3?UTR variant (rs13212041 A-allele) that enables a microRNA-mediated reduction in expression. One intermediate expression haplotype contained the 3?UTR A-allele paired with the high-activity promoter. Two high expression haplotypes contained the 3?UTR variant (rs13212041 G-allele) that attenuates microRNA-mediated reduction in expression. Men with low expression haplotypes reported greater anger and hostility than men with one or two high expression haplotypes. Diplotype classification accounted for 8.4% of the variance in men’s anger and hostility, primarily due to the 3?UTR polymorphism (rs13212041), but with some contribution of the functional promoter combination (rs11568817, rs130058). Associations with anger and hostility were not found in women. Conclusions These findings extend our understanding of the genetic basis of anger and hostility by showing that newly characterized HTR1B haplotypes, particularly those with rs13212041, which modulates microRNA-mediated regulation of HTR1B expression, may have important implications for aggression-related phenotypes among young men. PMID:19350534

  5. A novel ER?-mediated reporter gene assay for screening estrogenic/antiestrogenic chemicals based on LLC-MK2 cells.

    PubMed

    Huang, Xiaoming; Huang, Jie; Zhang, Lishi; Zhu, Yanfeng; Li, Yun

    2014-12-01

    Low concentration of endocrine-disrupting chemicals (EDCs) may lead to serious consequences in animals and human, so it is essential to develop an effective assay for EDCs detection. In this study, we developed a novel ER?-mediated reporter gene assay based on the LLC-MK2 cells by co-transfecting pERE-sv40-Luc, hER?-pcDNA3.1, and pRL-tk. Then we determined 17?-estradiol (E2) and some estrogenic/antiestrogenic chemicals to verify the validity of this assay. Data showed that the assay possesses a concentration-dependent responses to E2 and diethylstilbestrol (DES) from 10(-12?)M to 10(-8?)M with EC(50) 3.4?×?10(-10?)M and 5.9?×?10(-10?)M, and ICI 182,780 completely blocks the luciferase activity induced by 10(-9?)M E2. Bisphenol A (BPA), nonylphenol (NP), genistein (GS), and tamoxifen (TAM) also showed corresponding estrogenic or antiestrogenic activity at test concentrations. All evidences proved that the LLC-MK2 reporter gene assay was specific and sensitive to estrogen receptor (ER) agonistic and antagonistic chemicals. PMID:25045971

  6. Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering.

    PubMed

    Kimura, Yukiko; Hisano, Yu; Kawahara, Atsuo; Higashijima, Shin-ichi

    2014-01-01

    The type II bacterial CRISPR/Cas9 system is rapidly becoming popular for genome-engineering due to its simplicity, flexibility, and high efficiency. Recently, targeted knock-in of a long DNA fragment via homology-independent DNA repair has been achieved in zebrafish using CRISPR/Cas9 system. This raised the possibility that knock-in transgenic zebrafish could be efficiently generated using CRISPR/Cas9. However, how widely this method can be applied for the targeting integration of foreign genes into endogenous genomic loci is unclear. Here, we report efficient generation of knock-in transgenic zebrafish that have cell-type specific Gal4 or reporter gene expression. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, a sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. We have succeeded in establishing stable knock-in transgenic fish with several different constructs for 4 genetic loci at a frequency being exceeding 25%. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic zebrafish. PMID:25293390

  7. Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknuellt protein

    SciTech Connect

    Yamaguchi, Masamitsu; Hirose, Fumiko; Nishida, Yasuyoshi; Matsukage, Akio (Aichi Cancer Center Research Inst., Nagoya (Japan))

    1991-10-01

    A 631-bp fragment containing the 5{prime}-flanking region of the Drosophila melanogaster proliferating-cell nuclear antigen (PCNA) gene was placed upstream of the chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in Drosophila Kc cells transfected with this plasmid and a set of 5{prime}-deletion derivatives revealed that the promoter function resided within a 192-bp region. Cotransfection with a zerknuellt (zen)-expressing plasmid specifically repressed CAT expression. However, cotransfection with expression plasmids for a nonfunctional zen mutation, even skipped, or bicoid showed no significant effect on CAT expression. RNase protection analysis revealed that the repression by zen was at the transcription step. The target sequence of zen was mapped within the 34-bp region of the PCNA gene promoter, even though it lacked zen protein-binding sites. Transgenic flies carrying the PCNA gene regulatory region fused with lacZ were established. These results indicate that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter.

  8. Replication-deficient adenovirus vector transfer of gfp reporter gene into supraoptic nucleus and subfornical organ neurons

    NASA Technical Reports Server (NTRS)

    Vasquez, E. C.; Johnson, R. F.; Beltz, T. G.; Haskell, R. E.; Davidson, B. L.; Johnson, A. K.

    1998-01-01

    The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats were injected with Ad (2 x 10(6) pfu) and sacrificed 1-7 days later for cell culture of the SON and of the SFO. In the SON, GFP fluorescence was visualized in both neuronal and nonneuronal cells while only neurons in the SFO expressed GFP. Successful in vitro transfection of cultured cells from the SON and SFO was also achieved with Ad (2 x 10(6) to 2 x 10(8) pfu). The expression of GFP in in vitro transfected cells was higher in nonneuronal (approximately 28% in SON and SFO) than neuronal (approximately 4% in SON and 10% in SFO) cells. The expression of GFP was time and viral concentration related. No apparent alterations in cellular morphology of transfected cells were detected and electrophysiological characterization of transfected cells was similar between GFP-expressing and nonexpressing neurons. We conclude that (1) GFP is an effective marker for gene transfer in living SON and SFO cells, (2) Ad infects both neuronal and nonneuronal cells, (3) Ad is taken up by axonal projections from the SON and retrogradely transported to the SFO where it is expressed at detectable levels, and (4) Ad does not adversely affect neuronal viability. These results demonstrate the feasibility of using adenoviral vectors to deliver genes to the SFO-SON axis. Copyright 1998 Academic Press.

  9. Human SP-A1 (SFTPA1) variant-specific 3? UTRs and poly(A) tail differentially affect the in vitro translation of a reporter gene

    PubMed Central

    Silveyra, Patricia; Wang, Guirong

    2010-01-01

    Human surfactant protein A (SP-A) is encoded by two functional genes (SFTPA1, SFTPA2) with a high degree of sequence identity. Sequence differences among these genes and their genetic variants have been observed at the 5? and 3? untranslated regions (UTRs). In this work, we studied the impact on translation of the SFTPA1 (hSP-A1) and SFTPA2 (hSP-A2) gene 5? UTR splice variants and 3? UTR sequence variants, in the presence or absence of poly(A) tail. We generated constructs containing the luciferase reporter gene flanked upstream by one of the hSP-A 5? UTR splice variants and/or downstream by one hSP-A 3? UTR sequence variant. mRNA transcripts were prepared by in vitro transcription and used for either in vitro translation with a rabbit reticulocyte lysate or transient transfection of the lung adenocarcinoma cell line NCI-H441. The luciferase activity results indicate that hSP-A 5? UTR and 3? UTR together have an additive effect on translation. In this context, the hSP-A1 6A3 and 6A4 3? UTR variants exhibited higher translation efficiency than the 6A2 variant (P <0.05), whereas no significant difference was observed between the two hSP-A2 3? UTRs studied (1A0, 1A3). Further sequence analysis revealed that a deletion of an 11-nucleotide (nt) element in both the 6A3 and 6A4 3? UTR variants changes the predicted secondary structure stability and the number of putative miRNA binding sites. Removal of this 11-nt element in the 6A2 3? UTR resulted in increased translation, and the opposite effect was observed when the 11-nt element was cloned in a guest 3? UTR (6A3, 6A4). These results indicate that sequence differences among hSP-A gene variants may account for differential regulation at the translational level. PMID:20693318

  10. Characterization of the regulatory regions in the human desmoglein genes encoding the pemphigus foliaceous and pemphigus vulgaris antigens.

    PubMed Central

    Adams, M J; Reichel, M B; King, I A; Marsden, M D; Greenwood, M D; Thirlwell, H; Arnemann, J; Buxton, R S; Ali, R R

    1998-01-01

    The adhesive proteins in the desmosome type of cell junction consist of two members of the cadherin superfamily, the desmogleins and desmocollins. Both desmogleins and desmocollins occur as at least three different isoforms with various patterns of expression. The molecular mechanisms controlling the differential expression of the desmosomal cadherin isoforms are not yet known. We have begun an investigation of desmoglein gene expression by cloning and analysing the promoters of the human genes coding for the type 1 and type 3 desmogleins (DSG1 and DSG3). The type 1 isoform is restricted to the suprabasal layers of the epidermis and is the autoantigen in the autoimmune blistering skin disease pemphigus foliaceous. The type 3 desmoglein isoform is also expressed in the epidermis, but in lower layers than the type 1 isoform, and is the autoantigen in pemphigus vulgaris. Phage lambda genomic clones were obtained containing 4.2 kb upstream of the translation start site of DSG1 and 517 bp upstream of the DSG3 start site. Sequencing of 660 bp upstream of DSG1 and 517 bp upstream of DSG3 revealed that there was no obvious TATA box, but a possible CAAT box was present at -238 in DSG1 and at -193 in DSG3 relative to the translation start site. Primer extension analysis and RNase protection experiments revealed four putative transcription initiation sites for DSG1 at positions -163, -151, -148 and -141, and seven closely linked sites for DSG3, the longest being at -140 relative to the translation start site. The sequences at these possible sites at -166 to -159 in DSG1 (TTCAGTCC) and at -124 to -117 in DSG3 (CTTAGACT) have some similarity to the initiator sequence (CTCANTCT) described for a TATA-less promoter often from -3 to +5, and the true transcription initiator site might therefore be the A residue in these sequences. There were two regions of similarity between the DSG1 and DSG3 promoters just upstream of the transcription initiation sites, of 20 and 13 bp, separated by 41 bp in DSG1 and 36 bp in DSG3. The significance of these regions of similarity remains to be elucidated, but the results suggest that they represent a point at which these two desmoglein genes are co-ordinately regulated. Analysis of the upstream sequences revealed GC-rich regions and consensus binding sites for transcription factors including AP-1 and AP-2. Exon boundaries were conserved compared with the classical cadherin E-cadherin, but the equivalent of the second cadherin intron was lacking. A 4.2 kb region of the human DSG1 promoter sequence was linked to the lacZ gene reporter gene in such a way that there was only one translation start site, and this construct was used to generate transgenic mice. We present the first transgenic analysis of a promoter region taken from a desmosomal cadherin gene. Our results suggest that the 4.2 kb upstream region of DSG1 does not contain all the regulatory elements necessary for correct expression of this gene but might have elements that regulate activity during hair growth. PMID:9405290

  11. Cytokine gene associations with self-report ratings of morning and evening fatigue in oncology patients and their family caregivers.

    PubMed

    Dhruva, Anand; Aouizerat, Bradley E; Cooper, Bruce; Paul, Steven M; Dodd, Marylin; West, Claudia; Wara, William; Lee, Kathryn; Dunn, Laura B; Langford, Dale J; Merriman, John D; Baggott, Christina; Cataldo, Janine; Ritchie, Christine; Kober, Kord M; Leutwyler, Heather; Miaskowski, Christine

    2015-03-01

    The purpose of this study was to evaluate for differences in variations in pro- and anti-inflammatory cytokine genes between participants who were classified as having low and high levels of morning and evening fatigue and to evaluate for differences in phenotypic characteristics between these two groups. In a sample of 167 oncology outpatients with breast, prostate, lung, or brain cancer and 85 of their family caregivers, growth mixture modeling was used to identify latent classes of individuals based on ratings of morning and evening fatigue obtained prior to, during, and for 4 months following completion of radiation therapy. Differences in single nucleotide polymorphisms and haplotypes in 15 cytokine genes were evaluated between the latent classes. Multiple logistic regression was used to assess the effect of phenotypic and genotypic characteristics on morning and evening fatigue class membership. Associations were found between morning fatigue and number of comorbidities as well as variations in tumor necrosis factor alpha (TNFA) rs1800629 and rs3093662. Evening fatigue was associated with caring for children at home and variations in interleukin 4 (IL4) rs2243248 and TNFA rs2229094. Younger age and lower performance status were associated with both morning and evening fatigue. These findings suggest that inflammatory mediators are associated with the development of morning and evening fatigue. However, because different phenotypic characteristics and genomic markers are associated with diurnal variations in fatigue, morning and evening fatigue may be distinct but related symptoms. PMID:24872120

  12. HSD17B1 expression enhances estrogen signaling stimulated by the low active estrone, evidenced by an estrogen responsive element-driven reporter gene in vivo.

    PubMed

    Järvensivu, Päivi; Saloniemi-Heinonen, Taija; Awosanya, Michael; Koskimies, Pasi; Saarinen, Niina; Poutanen, Matti

    2015-06-01

    Hydroxysteroid (17beta) dehydrogenase 1 (HSD17B1) belongs to a family of short-chain-dehydrogenases. The enzyme utilizes NAD(P) and NAD(P)H as cofactors, and catalyzes the reversible reaction between estrone (E1) and estradiol (E2) in vitro. Of these steroids, E1 presents with lower estrogenic activity, but is converted to highly active E2 by HSD17B1. HSD17B1 is expressed especially in tissues with a high E2-producing capacity such as human ovaries and placenta, but also in several peripheral estrogen target tissues in humans, and inhibiting the enzyme activity is, thus, considered a promising approach to treat estrogen-dependent diseases. By analyzing transgenic mice universally expressing human HSD17B1 and carrying estrogen-response element (ERE)-driven luciferase reporter gene (Bi-transgenic ERELuc-HSD17B1TG mice) we showed a markedly higher reporter gene activity in various peripheral tissues of these mice as compared with ERELuc mice, indicating enhanced estrogen response generated by human HSD17B1 expression. An increased response after E1 administration was also evident in the Bi-TG mice, indicated by the increased uterus growth response and by the higher ERELuc reporter gene activity in the uterus. Moreover, a HSD17B1 inhibitor significantly reduced E1-induced increase in the uterus weight and uterine epithelial proliferation in the Bi-TG mice. Also the E1-induced ERELuc activity in the inhibitor-treated uterus was reduced by the HSD17B1 inhibitor in immature mice ex vivo, as well as in the liver of adult mice. The data, thus, demonstrate the potential use of the Bi-TG mice as a preclinical in vivo model for screening the efficacy of HSD17B1 inhibitors. As compared with the existing models, the Bi-TG mice present with luciferase activity as an additional, easily quantitative endpoint for the estrogen action. PMID:25617485

  13. Analysis of extracellular alginate lyase (alyA) expression and its regulatory region in a marine bacterial strain, Pseudoalteromonas atlantica AR06, using a gfp gene reporter system.

    PubMed

    Matsushima, Ryoji; Watanabe, Ryuichi; Tsuda, Masataka; Suzuki, Toshiyuki

    2013-06-01

    Extracellular alginate lyase (alyA) has an important role in the use of alginate in the marine bacterial strain Pseudoalteromonas atlantica AR06. Green fluorescent protein (GFP) is a convenient, useful tool for visualization of gene expression, and here we introduced the gfp gene at the end of alyA by homologous recombination in AR06. The gfp gene was expressed as a polycistronic transcript in reporter strain ARAG (AR06 alyA-gfp). The analysis of GFP fluorescence of ARAG in minimal medium containing a carbon source (sucrose, mannitol, glucose, or glucuronate) revealed that the alyA was induced by alginate and regulated by carbon catabolite repression by the coexistence of each carbon substrate (sucrose, mannitol, and glucose). The transcription start site of alyA was identified 192 bp upstream from translation start site of alyA and the 10-bp sequence was a palindrome in which the transcription start site [G] was at the end. Thirteen kinds of plasmids were constructed for promoter analysis of alyA, which contains between 215 and 618 bp of upstream sequence from the translation start and gfp. Longer sequences than 339 bp were capable to increase the GFP fluorescence responding to alginate in the homologous recombination alyA deletion mutant strain ARA? (AR06 ?alyA). The 339-bp upstream sequence was proved sufficiency for reproduction of the catabolite repression of alyA. These results indicate that (a) transcription of alyA is probably regulated by substrate recognition systems driven by multiple factors and (b) regulation of alyA requires a cis element on the upstream region of alyA. PMID:23179456

  14. FACS identifies unique cocaine-induced gene regulation in selectively activated adult striatal neurons

    PubMed Central

    Guez-Barber, Danielle; Fanous, Sanya; Golden, Sam A.; Schrama, Regina; Koya, Eisuke; Stern, Anna L.; Bossert, Jennifer M.; Harvey, Brandon K.; Picciotto, Marina R.; Hope, Bruce T.

    2011-01-01

    Numerous studies with the neural activity marker Fos indicate that cocaine activates only a small proportion of sparsely distributed striatal neurons. Until now, efficient methods were not available to assess neuroadaptations induced specifically within these activated neurons. We used fluorescence-activated cell sorting (FACS) to purify striatal neurons activated during cocaine-induced locomotion in naïve and cocaine-sensitized cfos-lacZ transgenic rats. Activated neurons were labeled with an antibody against ?-galactosidase, the protein product of the lacZ gene. Cocaine induced a unique gene expression profile selectively in the small proportion of activated neurons that was not observed in the non-activated majority of neurons. These genes included altered levels of the immediate early genes arc, fosB, and nr4a3, as well as genes involved in p38 MAPK signaling and cell-type specificity. We propose that this FACS method can be used to study molecular neuroadaptations in specific neurons encoding the behavioral effects of abused drugs and other learned behaviors. PMID:21411666

  15. Cloning and nucleotide sequence analysis of psaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologous to previously reported Streptococcus sp. adhesins.

    PubMed Central

    Sampson, J S; O'Connor, S P; Stinson, A R; Tharpe, J A; Russell, H

    1994-01-01

    Gene psaA, which encodes the Streptococcus pneumoniae 37-kDa protein, was cloned in Escherichia coli, and its complete nucleotide sequence was determined. Analysis of the sequence of the 2.4-kb cloned fragment revealed three open reading frames (ORFs). ORF2, which is 933 bp long, was identified as psaA. The two other ORFs identified flank psaA. ORF1, located upstream of psaA, is 836 nucleotides long and encodes a protein with a calculated molecular mass of 29,843 Da. The sequence for ORF3, located downstream of psaA, was only partially determined. Northern (RNA) blot analysis of pneumococcal RNA suggests that psaA is transcribed as part of a polycistronic message. Analysis of the primary structure of the protein encoded by this gene indicated significant similarity to two previously reported streptococcal proteins, SsaB (80% similarity) and FimA (92.3% similarity), from S. sanguis and S. parasanguis, respectively. These two homologous proteins have been shown to be associated with bacterial adhesion, and the possibility of a similar role for PsaA is hypothesized. Images PMID:7505262

  16. Development of a transgenic hairy root system in jute (Corchorus capsularis L.) with gusA reporter gene through Agrobacterium rhizogenes mediated co-transformation.

    PubMed

    Chattopadhyay, Tirthartha; Roy, Sheuli; Mitra, Adinpunya; Maiti, Mrinal K

    2011-04-01

    Transgenic hairy root system is important in several recalcitrant plants, where Agrobacterium tumefaciens-mediated plant transformation and generation of transgenic plants are problematic. Jute (Corchorus spp.), the major fibre crop in Indian subcontinent, is one of those recalcitrant plants where in vitro tissue culture has provided a little success, and hence, Agrobacterium-mediated genetic transformation remains to be a challenging proposition in this crop. In the present work, a system of transgenic hairy roots in Corchorus capsularis L. has been developed through genetic transformation by Agrobacterium rhizogenes harbouring two plasmids, i.e. the natural Ri plasmid and a recombinant binary vector derived from the disarmed Ti plasmid of A. tumefaciens. Our findings indicate that the system is relatively easy to establish and reproducible. Molecular analysis of the independent lines of transgenic hairy roots revealed the transfer of relevant transgenes from both the T-DNA parts into the plant genome, indicating the co-transformation nature of the event. High level expression and activity of the gusA reporter gene advocate that the transgenic hairy root system, thus developed, could be applicable as gene expression system in general and for root functional genomics in particular. Furthermore, these transgenic hairy roots can be used in future as explants for plantlet regeneration to obtain stable transgenic jute plants. PMID:21153028

  17. RECOMBINANT AVIAN ADENO-ASSOCIATED VIRUS: TRANSGENE EXPRESSION IN VIVO AND ENHANCEMENT OF EXPRESSION IN VITRO

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant avian adeno-associated virus coding for the LacZ gene were used to inoculate embryonating chicken eggs, to assess the usefulness of the system for the expression of a transgene in vivo. The results obtained indicate significantly higher levels of expression of the reporter gene at variou...

  18. A fatal infection caused by sequence type 398 methicillin-resistant Staphylococcus aureus carrying the Panton-Valentine leukocidin gene: A case report in Japan.

    PubMed

    Koyama, Hiroshi; Sanui, Masamitsu; Saga, Tomoo; Harada, Sohei; Ishii, Yoshikazu; Tateda, Kazuhiro; Lefor, Alan Kawarai

    2015-07-01

    Methicillin-resistant Staphylococcus aureus (MRSA) has now been recognized as a common pathogen in the community. Sequence type (ST) 398 MRSA is generally considered as an emerging zoonotic agent spreading among livestock and personnel who have direct contact with animals, mainly in Europe. A 37-year-old Chinese woman receiving steroid therapy for systemic lupus erythematosus with general fatigue and myalgia was brought to the emergency department in critical condition. Her condition deteriorated despite aggressive management and she died on day 7. Her blood culture revealed ST398 MRSA-SCCmec V with Panton-Valentine Leukocidin (PVL) gene. This is the first case report of a fatal infection caused by this lineage. According to the results of molecular analyses, the isolate from this particular patient's blood was genetically close to a lineage detected in China, and is less likely to be related to an animal-associated lineage. PMID:25922086

  19. Flow-type electroporation chips for gene transfection

    NASA Astrophysics Data System (ADS)

    Lin, Yu-Cheng; Jen, Chung-Min; Huang, Ming-Yuan; Lin, Xi-Zhang

    2000-08-01

    Electroporation is a technique with which DNA molecules can be delivered into cells in a chamber using high electric field pulses. The limited amount of target cells and the potential risk from the high voltage are the two drawbacks in this technique. This study aimed to fabricate an electroporation chip to manage large amount of cells continuously with a lower applied voltage. The electroporation chip, consisting of a micro-channel with thin film electrodes made of gold or platinum wire electrodes on both sides, was fabricated on PMMA material using evaporation, photolithography, wet- etching, lift-off, and fusion-bonding methods. The suspension fluid of Huh-7 cell lines (1 x 106 cells/ml) mixed with 10 micrometers plasmids equipped with lacZ genes in a volume of 500 (mu) l flowed through the channel with a variety of flow rates under a series of square pulses. The transfection rate was evaluated with blue-staining cells under X-Gal stain 24 hours later. The dimensions of the channel were 5 mm wide, 0.2 mm high, and 25 mm long. Two types of electrodes, parallel-plate type and parallel-line type electrodes, were fabricated and tested in these experiments. The fabricated microchip can deliver genes into the flowing of cells. The electric pulse frequency that determines the shock number for each cell for a fixed flow rate can be optimized for better transfection and survival rates.

  20. Similarities in the endocrine-disrupting potencies of indoor dust and flame retardants by using human osteosarcoma (U2OS) cell-based reporter gene assays.

    PubMed

    Suzuki, Go; Tue, Nguyen Minh; Malarvannan, Govindan; Sudaryanto, Agus; Takahashi, Shin; Tanabe, Shinsuke; Sakai, Shin-ichi; Brouwer, Abraham; Uramaru, Naoto; Kitamura, Shigeyuki; Takigami, Hidetaka

    2013-03-19

    Indoor dust is a sink for many kinds of pollutants, including flame retardants (FRs), plasticizers, and their contaminants and degradation products. These pollutants can be migrated to indoor dust from household items such as televisions and computers. To reveal high-priority end points of and contaminant candidates in indoor dust, using CALUX reporter gene assays based on human osteosarcoma (U2OS) cell lines, we evaluated and characterized the endocrine-disrupting potencies of crude extracts of indoor dust collected from Japan (n = 8), the United States (n = 21), Vietnam (n = 10), the Philippines (n = 17), and Indonesia (n = 10) and for 23 selected FRs. The CALUX reporter gene assays used were specific for compounds interacting with the human androgen receptor (AR), estrogen receptor ? (ER?), progesterone receptor (PR), glucocorticoid receptor (GR), and peroxisome proliferator-activated receptor ?2 (PPAR?2). Indoor dust extracts were agonistic to ER?, GR, and PPAR?2 and antagonistic against AR, PR, GR, and PPAR?2. In comparison, a majority of FRs was agonistic to ER? and PPAR?2 only, and some FRs demonstrated receptor-specific antagonism against all tested nuclear receptors. Hierarchical clustering clearly indicated that agonism of ER? and antagonism of AR and PR were common, frequently detected end points for indoor dust and tested FRs. Given our previous results regarding the concentrations of FRs in indoor dust and in light of our current results, candidate contributors to these effects include not only internationally controlled brominated FRs but also alternatives such as some phosphorus-containing FRs. In the context of indoor pollution, high-frequency effects of FRs such as agonism of ER? and antagonism of AR and PR are candidate high-priority end points for further investigation. PMID:23398518

  1. A Putatively Functional Polymorphism in the HTR2C Gene is Associated with Depressive Symptoms in White Females Reporting Significant Life Stress

    PubMed Central

    Brummett, Beverly H.; Babyak, Michael A.; Williams, Redford B.; Harris, Kathleen Mullan; Jiang, Rong; Kraus, William E.; Singh, Abanish; Costa, Paul T.; Georgiades, Anastasia; Siegler, Ilene C.

    2014-01-01

    Psychosocial stress is well known to be positively associated with subsequent depressive symptoms. Cortisol response to stress may be one of a number of biological mechanisms that links psychological stress to depressive symptoms, although the precise causal pathway remains unclear. Activity of the x-linked serotonin 5-HTR2C receptor has also been shown to be associated with depression and with clinical response to antidepressant medications. We recently demonstrated that variation in a single nucleotide polymorphism on the HTR2C gene, rs6318 (Ser23Cys), is associated with different cortisol release and short-term changes in affect in response to a series of stress tasks in the laboratory. Based on this observation, we decided to examine whether rs6318 might moderate the association between psychosocial stress and subsequent depressive symptoms. In the present study we use cross-sectional data from a large population-based sample of young adult White men (N?=?2,366) and White women (N?=?2,712) in the United States to test this moderation hypothesis. Specifically, we hypothesized that the association between self-reported stressful life events and depressive symptoms would be stronger among homozygous Ser23 C females and hemizygous Ser23 C males than among Cys23 G carriers. In separate within-sex analyses a genotype-by-life stress interaction was observed for women (p?=?.022) but not for men (p?=?.471). Homozygous Ser23 C women who reported high levels of life stress had depressive symptom scores that were about 0.3 standard deviations higher than female Cys23 G carriers with similarly high stress levels. In contrast, no appreciable difference in depressive symptoms was observed between genotypes at lower levels of stress. Our findings support prior work that suggests a functional SNP on the HTR2C gene may confer an increased risk for depressive symptoms in White women with a history of significant life stress. PMID:25514629

  2. Testing of chemicals for genetic activity with Saccharomyces cerevisiae: a report of the U.S. Environmental Protection Agency Gene-Tox Program.

    PubMed

    Zimmermann, F K; von Borstel, R C; von Halle, E S; Parry, J M; Siebert, D; Zetterberg, G; Barale, R; Loprieno, N

    1984-05-01

    The yeast Saccharomyces cerevisiae is a unicellular fungus that can be cultured as a stable haploid or a stable diploid . Diploid cultures can be induced to undergo meiosis in a synchronous fashion under well-defined conditions. Consequently, yeasts can be used to study genetic effects both in mitotic and in meiotic cells. Haploid strains have been used to study the induction of point mutations. In addition to point mutation induction, diploid strains have been used for studying mitotic recombination, which is the expression of the cellular repair activities induced by inflicted damage. Chromosomal malsegregation in mitotic and meiotic cells can also be studied in appropriately marked strains. Yeast has a considerable potential for endogenous activation, provided the tests are performed with appropriate cells. Exogenous activation has been achieved with S9 rodent liver in test tubes as well as in the host-mediated assay, where cells are injected into rodents. Yeast cells can be recovered from various organs and tested for induced genetic effects. The most commonly used genetic end point has been mitotic recombination either as mitotic crossing-over or mitotic gene conversion. A number of different strains are used by different authors. This also applies to haploid strains used for monitoring induction of point mutations. Mitotic chromosome malsegregation has been studied mainly with strain D6 and meiotic malsegregation with strain DIS13 . Data were available on tests with 492 chemicals, of which 249 were positive, as reported in 173 articles or reports. The genetic test/carcinogenicity accuracy was 0.74, based on the carcinogen listing established in the Gene-Tox Program. The yeast tests supplement the bacterial tests for detecting agents that act via radical formation, antibacterial drugs, and other chemicals interfering with chromosome segregation and recombination processes. PMID:6374444

  3. Gene targeting in Arabidopsis thaliana

    Microsoft Academic Search

    Ursula Halfter; Peter-Christian Morris; Lothar Willmitzer

    1992-01-01

    Gene targeting of a chromosomally integrated transgene in Arabidopsis thaliana is reported. A chimeric gene consisting of the promoter of the 35S RNA of CaMV, the polyadenylation signal of the octopine synthase gene and the coding region of the bacterial hygromycin phosphotransferase gene (hpt), which was rendered non-functional by deletion of 19 bp, was introduced into the genome of A.

  4. A new mutation of the PCNT gene in a Colombian patient with microcephalic osteodysplastic primordial dwarfism type II: a case report

    PubMed Central

    2014-01-01

    Introduction Microcephalic osteodysplastic primordial dwarfism is a syndrome characterized by the presence of intrauterine growth restriction, post-natal growth deficiency and microcephaly. Microcephalic osteodysplastic primordial dwarfism type II is the most distinctive syndrome in this group of entities. Individuals affected by this disease present at an adult height of less than 100cm, a post-pubertal head circumference of 40cm or less, mild mental retardation, an outgoing personality and bone dysplasia. Case presentation We report the first case of a five-year-old Colombian boy of mixed race ancestry (mestizo), with clinical features of microcephaly, prominent and narrow nose, arched palate, amelogenesis imperfecta, short stature, tall and narrow pelvis, disproportionate shortening of fore-arms and legs, and mild coxa vara. Analysis of the PCNT gene by sequencing showed the presence of a nucleotide change in exon 10, c. 1468C>T, evidencing a new mutation not reported in the literature for microcephalic osteodysplastic primordial dwarfism. Conclusion The new mutation identified in this case could be associated with the severity of the phenotypic expression of the disease, resulting in the extreme short stature of the patient. Further studies are required to reach an explanation that can justify such findings, and it is vital to emphasize the importance of detection and follow-up by the epidemiological surveillance groups in birth defects and rare diseases. PMID:24928221

  5. Association between chemerin rs17173608 and vaspin rs2236242 gene polymorphisms and the metabolic syndrome, a preliminary report.

    PubMed

    Hashemi, Mohammad; Rezaei, Hamzeh; Eskandari-Nasab, Ebrahim; Kaykhaei, Mahmoud Ali; Zakeri, Zahra; Taheri, Mohsen

    2012-12-01

    Metabolic syndrome (MeS) is associated with increased risk for type 2 diabetes mellitus (T2DM) and cardiovascular disease. There is some evidence indicating that adipokines play a role in the development of MeS. The present study was aimed to investigate the impact of chemerin rs17173608 and vaspin rs2236242 gene polymorphisms with the risk of MeS in a sample of Iranian population. This case-control study was done on 151 subjects with MeS and 149 without MeS, as defined by NCEP-ATPIII. Tetra amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) was designed to detect the polymorphisms. Our finding showed that there are significant differences in genotype frequencies between the groups regarding vaspin rs2236242 polymorphism (?(2)=18.74, p<0.0001). A significant protection against MeS was found for vaspin rs2236242 in allele and genotypes (Odd Ratio [OR]=0.52; 95% confidence interval [CI]=0.37-0.72; p=0.0001, T vs A; OR=0.49; 95%CI=0.29-0.82; p=0.007, TT vs TA and OR=0.17; 95%CI=0.07-0.40; p<0.0001, TT vs AA). Our finding showed positive association between chemerin rs17173608 polymorphism and risk of MeS (?(2)=7.70, p=0.021). The G allele increased the risk of MeS (OR=1.78; 95% CI=1.14-2.78; p=0.012) as compared to the T allele. In conclusion, our data suggest for the first time a significant association between vaspin rs2236242 and chemerin rs17173608 polymorphisms and the MeS in Zahedan, southeast Iran. Further studies with large sample size and different ethnicities are required to confirm our findings. PMID:22982016

  6. nodSU, two new nod genes of the broad host range Rhizobium strain NGR234 encode host-specific nodulation of the tropical tree Leucaena leucocephala.

    PubMed

    Lewin, A; Cervantes, E; Chee-Hoong, W; Broughton, W J

    1990-01-01

    Rhizobium species strain NGR234 nodulates at least 35 diverse genera of legumes as well as the nonlegume Parasponia andersonii. Most nodulation genes are located on the 500-kilobase pair symbiotic plasmid, pNGR234a. Previously, three plasmid-borne host range determinants (HsnI, HsnII, and HsnIII) were identified by their ability to extend the nodulation capacity of heterologous rhizobia to include Vigna unguiculata. In this study, we show that HsnII contains two new nod-box linked hsn genes, nodS and nodU.nodS controls nodulation of the tropical tree Leucaena leucocephala, while the nodSU genes regulate nodulation of the pasture legume Desmodium intortum and the grain legume V. unguiculata. Regulation of the nod-box upstream of nodSU by the flavonoid naringenin was shown using a fusion with a promoterless lacZ gene. Determination of the nucleotide sequence of the nodS gene did not reveal homology with any gene in the EMBL library, although Bradyrhizobium japonicum USDA110 contains both nodS and nodU (M. Göttfert, S. Hitz, and H. Hennecke, Molecular Plant-Microbe Interactions 3:308-316, 1990). We suggest that broad host range in NGR234 is controlled in part by a nodD gene which interacts with a wide range of flavonoids, and in part by host-specific nod genes such as nodS. PMID:2134856

  7. cis-acting sequences required for expression of the divergently transcribed Drosophila melanogaster Sgs-7 and Sgs-8 glue protein genes.

    PubMed Central

    Hofmann, A; Garfinkel, M D; Meyerowitz, E M

    1991-01-01

    The Sgs-7 and Sgs-8 glue genes at 68C are divergently transcribed and are separated by 475 bp. Fusion genes with Adh or lacZ coding sequences were constructed, and the expression of these genes, with different amounts of upstream sequences present, was tested by a transient expression procedure and by germ line transformation. A cis-acting element for both genes is located asymmetrically in the intergenic region between -211 and -43 bp relative to Sgs-7. It is required for correct expression of both genes. This element can confer the stage- and tissue-specific expression pattern of glue genes on a heterologous promoter. An 86-bp portion of the element, from -133 to -48 bp relative to Sgs-7, is shown to be capable of enhancing the expression of a truncated and therefore weakly expressed Sgs-3 fusion gene. Recently described common sequence motifs of glue gene regulatory elements (T. Todo, M. Roark, K. Vijay Raghavan, C. A. Mayeda, and E.M. Meyerowitz, Mol. Cell. Biol. 10:5991-6002, 1990) are located within this 86-bp region. PMID:1903838

  8. Crosstalk between Desmoglein 2 and Patched 1 accelerates chemical-induced skin tumorigenesis

    PubMed Central

    Brennan-Crispi, Donna M.; Hossain, Claudia; Sahu, Joya; Brady, Mary; Riobo, Natalia A.; Mahoney, M? G.

    2015-01-01

    Aberrant activation of Hedgehog (Hh) signaling is causative of BCCs and has been associated with a fraction of SCCs. Desmoglein 2 (Dsg2) is an adhesion protein that is upregulated in many cancers and overexpression of Dsg2 in the epidermis renders mice more susceptible to squamous-derived neoplasia. Here we examined a potential crosstalk between Dsg2 and Hh signaling in skin tumorigenesis. Our findings show that Dsg2 modulates Gli1 expression, in vitro and in vivo. Ectopic expression of Dsg2 on Ptc1+/lacZ background enhanced epidermal proliferation and interfollicular activation of the Hh pathway. Furthermore, in response to DMBA/TPA, the Dsg2/Ptc1+/lacZ mice developed squamous lessons earlier than the WT, Ptc1+/lacZ, and Inv-Dsg2 littermates. Additionally, DMBA/TPA induced BCC formation in all mice harboring the Ptc1+/lacZ gene and the presence of Dsg2 in Dsg2/Ptc1+/lacZ mice doubled the BCC tumor burden. Reporter analysis revealed activation of the Hh pathway in the BCC tumors. However, in the SCCs we observed Hh activity only in the underlying dermis of the tumors. Furthermore, Dsg2/Ptc1+/lacZ mice demonstrated enhanced MEK/Erk1/2 activation within the tumors and expression of Shh in the dermis. In summary, our results demonstrate that Dsg2 modulates Hh signaling, and this synergy may accelerate skin tumor development by different mechanisms. PMID:25871385

  9. Crosstalk between Desmoglein 2 and Patched 1 accelerates chemical-induced skin tumorigenesis.

    PubMed

    Brennan-Crispi, Donna M; Hossain, Claudia; Sahu, Joya; Brady, Mary; Riobo, Natalia A; Mahoney, M? G

    2015-04-20

    Aberrant activation of Hedgehog (Hh) signaling is causative of BCCs and has been associated with a fraction of SCCs. Desmoglein 2 (Dsg2) is an adhesion protein that is upregulated in many cancers and overexpression of Dsg2 in the epidermis renders mice more susceptible to squamous-derived neoplasia. Here we examined a potential crosstalk between Dsg2 and Hh signaling in skin tumorigenesis. Our findings show that Dsg2 modulates Gli1 expression, in vitro and in vivo. Ectopic expression of Dsg2 on Ptc1(+/lacZ) background enhanced epidermal proliferation and interfollicular activation of the Hh pathway. Furthermore, in response to DMBA/TPA, the Dsg2/Ptc1+/lacZ mice developed squamous lessons earlier than the WT, Ptc1(+/lacZ), and Inv-Dsg2 littermates. Additionally, DMBA/TPA induced BCC formation in all mice harboring the Ptc1(+/lacZ) gene and the presence of Dsg2 in Dsg2/Ptc1(+/lacZ) mice doubled the BCC tumor burden. Reporter analysis revealed activation of the Hh pathway in the BCC tumors. However, in the SCCs we observed Hh activity only in the underlying dermis of the tumors. Furthermore, Dsg2/Ptc1(+/lacZ) mice demonstrated enhanced MEK/Erk1/2 activation within the tumors and expression of Shh in the dermis. In summary, our results demonstrate that Dsg2 modulates Hh signaling, and this synergy may accelerate skin tumor development by different mechanisms. PMID:25871385

  10. Induction of Rhizobium meliloti nodC gene by Alnus incana compounds.

    PubMed

    Ma?ek, W

    1996-01-01

    An expression of nodC promoter of R. meliloti 1021, cloned in front of the Escherichia coli lacZ gene, was used to study the presence of R. meliloti nodC gene-inducing compound(s) in extracts and exudates of A. incana seeds and roots. The regulatory gene nodD was expressed at comparable level in bacterial culture of R. meliloti with and without the studied plant extracts and exudates, whereas the nodC-lacZ fusion expression was increased 4 times by seed exudates of grey alder and 2 times by seed extracts and root ingredients in comparison to the nodC-lacZ fusion expression in R. meliloti grown in a medium free of plant compounds. Induction of R. meliloti nodC gene expression by A. incana substances was also supported in plant test. Sterile filtrate of coculture of R. meliloti with seed exudates of A. incana induced root hair deformations and nodule-like structures on Medicago sativa. PMID:8914264

  11. Evaluation of Jeffamine®-cored PAMAM dendrimers as an efficient in vitro gene delivery system.

    PubMed

    Aydin, Zeynep; Akbas, Fahri; Senel, Mehmet; Koc, S Naci

    2012-10-01

    In this study, we investigated gene delivery properties of Jeffamine-cored polyamidoamine (PAMAM) dendrimers (JCPDs). The effects of dendrimer concentration, generation, and core size on the gene delivery have been analyzed. The experimental results showed that the JCPD effectively delivered plasmid DNA inside the HeLa cells, and the transfection efficiency improved considerably as the number of generation increased. The cytotoxicity of JCPD in different concentration was tested for HeLa cell line. JCPD was complexed with a lacZ gene carrying plasmid and tested for transfection efficiency using quantitative ?-galactosidase expression assay. Additionally, confocal microscopy results revealed that JCPD effectively delivered green fluorescent protein-expressing plasmid into HeLa cells and produced fluorescent signal with satisfactory efficiency. The highest transfection efficiency was obtained from JCPDs G4 and G5, which mixed with expression plasmid vectors at a 10/1 weight ratio. These results indicated that under optimized conditions, JCPD can be considered as an efficient transfection reagent and can be effectively used for gene delivery applications. PMID:22610890

  12. High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns

    Microsoft Academic Search

    Yong-Li Xiao; Julia C Redman; Erin L Monaghan; Jun Zhuang; Beverly A Underwood; William A Moskal; Wei Wang; Hank C Wu; Christopher D Town

    2010-01-01

    BACKGROUND: Although the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in

  13. Expression of Runx2 transcription factor in non-skeletal tissues, sperm and brain.

    PubMed

    Jeong, Jae-Hwan; Jin, Jung-Sook; Kim, Hyun-Nam; Kang, Sang-Min; Liu, Julie C; Lengner, Christopher J; Otto, Florian; Mundlos, Stefan; Stein, Janet L; van Wijnen, Andre J; Lian, Jane B; Stein, Gary S; Choi, Je-Yong

    2008-11-01

    Runx2 is a master transcription factor for chondrocyte and osteoblast differentiation and bone formation. However expression of Runx2 (by RT-PCR), has been reported in non-skeletal tissues such as breast, T cells and testis. To better define Runx2 activity in non-skeletal tissues, we examined transgenic (Tg) mice expressing LacZ gene under control of 3.0 kb (3 kb Tg) or 1.0 kb (1 kb Tg) of the Runx2 distal (P1) promoter, Runx2 LacZ knock-in (Runx2(+/LacZ)) and Runx2/P1 LacZ knock-in (Runx2/P1(+/LacZ)). In the Runx2 3 kb Tg mouse, beta-galactosidase (beta-gal) expression appeared in various non-skeletal tissues including testis, skin, adrenal gland and brain. beta-gal expression from both 3 kb and 1 kb Tg, reflecting activity of the Runx2 promoter, was readily detectable in seminiferous tubules of the testis and the epididymis. At the single cell level, beta-gal was detected in spermatids and mature sperms not in sertoli or Leydig cells. We also detected a positive signal from the Runx2(+/LacZ) and Runx2/P1(+/LacZ) mice. Indeed, Runx2 expression was observed in isolated mature sperms, which was confirmed by RT-PCR and Western blot analysis. Runx2, however, was not related to sex determination and sperm motility. Runx2 mediated beta-gal activity is also found robustly in the hippocampus and frontal lobe of the brain in Runx2(+/LacZ). Collectively, these results indicate that Runx2 is expressed in several non-skeletal tissues particularly sperms of testis and hippocampus of brain. It suggests that Runx2 may play an important role in male reproductive organ testis and brain. PMID:18636555

  14. RB1 gene mutation up-date, a meta-analysis based on 932 reported mutations available in a searchable database

    PubMed Central

    Valverde, José R; Alonso, Javier; Palacios, Itziar; Pestaña, Ángel

    2005-01-01

    Background Retinoblastoma, a prototype of hereditary cancer, is the most common intraocular tumour in children and potential cause of blindness from therapeutic eye ablation, second tumours in germ line carrier's survivors, and even death when left untreated. The molecular scanning of RB1 in search of germ line mutations lead to the publication of more than 900 mutations whose knowledge is important for genetic counselling and the characterization of phenotypic-genotypic relationships. Results A searchable database (RBGMdb) has been constructed with 932 published RB1 mutations. The spectrum of these mutations has been analyzed with the following results: 1) the retinoblastoma protein is frequently inactivated by deletions and nonsense mutations while missense mutations are the main inactivating event in most genetic diseases. 2) Near 40% of RB1 gene mutations are recurrent and gather in sixteen hot points, including twelve nonsense, two missense and three splicing mutations. The remainder mutations are scattered along RB1, being most frequent in exons 9, 10, 14, 17, 18, 20, and 23. 3) The analysis of RB1 mutations by country of origin of the patients identifies two groups in which the incidence of nonsense and splicing mutations show differences extremely significant, and suggest the involvement of predisposing ethnic backgrounds. 4) A significant association between late age at diagnosis and splicing mutations in bilateral retinoblastoma patients suggests the occurrence of a delayed-onset genotype. 5) Most of the reported mutations in low-penetrance families fall in three groups: a) Mutations in regulatory sequences at the promoter resulting in low expression of a normal Rb; b) Missense and in-frame deletions affecting non-essential sequence motifs which result in a partial inactivation of Rb functions; c) Splicing mutations leading to the reduction of normal mRNA splicing or to alternative splicing involving either true oncogenic or defective (weak) alleles. Conclusion The analysis of RB1 gene mutations logged in the RBGMdb has shown relevant phenotype-genotype relationships and provided working hypothesis to ascertain mechanisms linking certain mutations to ethnicity, delayed onset of the disease and low-penetrance. Gene profiling of tumors will help to clarify the genetic background linked to ethnicity and variable expressivity or delayed onset phenotypes. PMID:16269091

  15. kdgREcc negatively regulates genes for pectinases, cellulase, protease, HarpinEcc, and a global RNA regulator in Erwinia carotovora subsp. carotovora.

    PubMed

    Liu, Y; Jiang, G; Cui, Y; Mukherjee, A; Ma, W L; Chatterjee, A K

    1999-04-01

    Erwinia carotovora subsp. carotovora produces extracellular pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt). The concerted actions of these enzymes largely determine the virulence of this plant-pathogenic bacterium. E. carotovora subsp. carotovora also produces HarpinEcc, the elicitor of the hypersensitive reaction. We document here that KdgREcc (Kdg, 2-keto-3-deoxygluconate; KdgR, general repressor of genes involved in pectin and galacturonate catabolism), a homolog of the E. chrysanthemi repressor, KdgREch and the Escherichia coli repressor, KdgREco, negatively controls not only the pectinases, Pel and Peh, but also Cel, Prt, and HarpinEcc production in E. carotovora subsp. carotovora. The levels of pel-1, peh-1, celV, and hrpNEcc transcripts are markedly affected by KdgREcc. The KdgREcc- mutant is more virulent than the KdgREcc+ parent. Thus, our data for the first time establish a global regulatory role for KdgREcc in E. carotovora subsp. carotovora. Another novel observation is the negative effect of KdgREcc on the transcription of rsmB (previously aepH), which specifies an RNA regulator controlling exoenzyme and HarpinEcc production. The levels of rsmB RNA are higher in the KdgREcc- mutant than in the KdgREcc+ parent. Moreover, by DNase I protection assays we determined that purified KdgREcc protected three 25-bp regions within the transcriptional unit of rsmB. Alignment of the protected sequences revealed the 21-mer consensus sequence of the KdgREcc-binding site as 5'-G/AA/TA/TGAAA[N6]TTTCAG/TG/TA-3'. Two such KdgREcc-binding sites occur in rsmB DNA in a close proximity to each other within nucleotides +79 and +139 and the third KdgREcc-binding site within nucleotides +207 and +231. Analysis of lacZ transcriptional fusions shows that the KdgR-binding sites negatively affect the expression of rsmB. KdgREcc also binds the operator DNAs of pel-1 and peh-1 genes and represses expression of a pel1-lacZ and a peh1-lacZ transcriptional fusions. We conclude that KdgREcc affects extracellular enzyme production by two ways: (i) directly, by inhibiting the transcription of exoenzyme genes; and (ii) indirectly, by preventing the production of a global RNA regulator. Our findings support the idea that KdgREcc affects transcription by promoter occlusion, i.e., preventing the initiation of transcription, and by a roadblock mechanism, i.e., by affecting the elongation of transcription. PMID:10198003

  16. Cytochrome P4501A induction in avian hepatocyte cultures exposed to polychlorinated biphenyls: Comparisons with AHR1-mediated reporter gene activity and in ovo toxicity

    SciTech Connect

    Manning, Gillian E., E-mail: gmann017@uottawa.ca [Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5 (Canada); Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Mundy, Lukas J., E-mail: lukas.mundy@ec.gc.ca [Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5 (Canada); Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Crump, Doug, E-mail: doug.crump@ec.gc.ca [Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada)] [Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Jones, Stephanie P., E-mail: stephanie.jones@ec.gc.ca [Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Chiu, Suzanne, E-mail: suzanne.chiu@ec.gc.ca [Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada)] [Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Klein, Jeff, E-mail: jeffery@well-labs.com [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3chsM5 (Canada)] [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3chsM5 (Canada); Konstantinov, Alex, E-mail: alex@well-labs.com [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3chsM5 (Canada)] [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3chsM5 (Canada); Potter, Dave, E-mail: dpotter@well-labs.com [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3chsM5 (Canada)] [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3chsM5 (Canada); Kennedy, Sean W., E-mail: sean.kennedy@ec.gc.ca [Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5 (Canada); Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada)

    2013-01-01

    Avian-specific toxic equivalency factors (TEFs) were developed by the World Health Organization to simplify environmental risk assessments of dioxin-like compounds (DLCs), but TEFs do not account for differences in the toxic and biochemical potencies of DLCs among species of birds. Such variability may be due to differences in species sensitivity to individual DLCs. The sensitivity of avian species to DLCs was recently associated with the identity of amino acids 324 and 380 in the aryl hydrocarbon receptor 1 (AHR1) ligand binding domain. A luciferase reporter gene (LRG) assay, measuring AHR1-mediated induction of a cytochrome P450 1A5 (CYP1A5) reporter gene, in combination with a species' AHR1 ligand binding domain sequence, were also shown to predict avian species sensitivity to polychlorinated biphenyls (PCBs) and PCB relative potency in a given species. The goals of the present study were to (1) characterize the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and PCBs 126, 77, 105 and 118 on induction of ethoxyresorufin O-deethylase (EROD) activity and CYP1A4/5 mRNA in chicken, ring-necked pheasant and Japanese quail embryo hepatocytes and (2) compare these in vitro results to those previously generated by the LRG assay and in ovo toxicity studies. EROD activity and CYP1A4/5 mRNA expression data support and complement the findings of the LRG assay. CYP1A enzyme activity and mRNA expression were significantly correlated both with luciferase activity and in ovo toxicity induced by PCBs. Relative potency values were generally similar between the LRG and EROD assays and indicate that the relative potency of some PCBs may differ among species. -- Highlights: ? The chicken isn't the most sensitive species to CYP1A induction by PCB 105 and 118. ? The relative potency of PCBs differs between avian species. ? EROD activity was correlated with luciferase activity from the LRG assay. ? EROD activity was a better predictor of toxicity than CYP1A4/5 mRNA expression.

  17. Expression of the human cystic fibrosis transmembrane conductance regulator gene in the mouse lung after in vivo intratracheal plasmid-mediated gene transfer.

    PubMed Central

    Yoshimura, K; Rosenfeld, M A; Nakamura, H; Scherer, E M; Pavirani, A; Lecocq, J P; Crystal, R G

    1992-01-01

    As an approach to gene therapy for the respiratory manifestations of cystic fibrosis (CF), in vivo plasmid-mediated direct transfer of the normal CF transmembrane conductance regulator (CFTR) gene to the airway epithelium was investigated in mice. To evaluate the feasibility of this strategy, pRSVL, a plasmid composed of a firefly luciferase gene driven by the Rous sarcoma virus long terminal repeat (RSV-LTR), along with cationic liposomes was instilled into the trachea of C57BI/6NCR mice. With administration of 200-400 micrograms plasmid DNA, luciferase expression could be detected in the mouse lung homogenates for at least 4 wk. With this background, a CFTR expression plasmid vector (pRSVCFTR) constructed by replacing the luciferase cDNA from pRSVL with the normal human CFTR cDNA was evaluated in vivo in mice. Intratracheal instillation of pRSVCFTR with cationic liposomes followed by analysis of mouse lung RNA by polymerase chain reaction amplification (after conversion of mRNA to cDNA) using a RSV-LTR specific sense primer and a human CFTR-specific antisense primer demonstrated human CFTR mRNA transcripts from one day to 4 wk after instillation. Further, in vivo evaluation of beta-galactosidase activity after intratracheal administration of an E. coli lacZ gene expression plasmid vector directed by the cytomegalovirus promoter (pCMV beta) demonstrated that the airway epithelium was the major target of transfer and expression of the exogenous gene. These observations demonstrate successful plasmid-mediated gene transfer to the airway epithelium in vivo. This strategy may be feasible as a form of gene therapy to prevent the pulmonary manifestations of CF. Images PMID:1377820

  18. Establishment of mesenchymal stem cells derived from bone marrow and synovium of transgenic rats expressing dual reporter genes

    NASA Astrophysics Data System (ADS)

    Horie, Masafumi; Sekiya, Ichiro; Muneta, Takeshi; Murakami, Takashi; Kobayashi, Eiji

    2008-02-01

    Mesenchymal stem cells (MSCs) are an attractive cell source for regenerative medicine because they can be harvested in a relatively less invasive manner, easily isolated, and expanded with multipotentiality. Bone marrow seems to be the most commonly used tissue as a source for MSCs at present. However, there are emerging reports to describe that MSCs exist in most mesenchymal tissues. We have recently compared in vivo chondrogenic potential in MSCs derived from various mesenchymal tissues and demonstrated that synovium-MSCs and bone marrow-MSCs possessed greater chondrogenic ability than other mesenchymal tissue-derived MSCs. This indicates that those MSCs are promising cellular sources for cartilage regeneration. As the fate of synovium-MSCs is unclear after transplantation, we herein established MSCs using double transgenic rats expressing either Luciferase/GFP or Luciferase/LacZ. The cellular fate of MSCs could be traced by an in vivo luciferase-based luminescent imaging system, and also followed histologically by green fluorescence and by X-gal staining, respectively. Thus, both synovium-MSCs and bone marrow-MSCs expressing Luciferase/GFP or Luciferase/LacZ provide powerful tools not only for cell tracking in vivo but also for histological analysis, leading to a compelling experimental model of cartilage regeneration with cell therapy.

  19. Unscheduled DNA synthesis tests. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

    PubMed

    Mitchell, A D; Casciano, D A; Meltz, M L; Robinson, D E; San, R H; Williams, G M; Von Halle, E S

    1983-12-01

    The utility of unscheduled DNA synthesis (UDS) testing for screening potentially hazardous chemicals was evaluated using the published papers and technical reports available to the UDS Work Group. A total of 244 documents were reviewed. Based on criteria defined in advance for evaluation of the results, 169 were rejected. From the 75 documents accepted, results were reviewed for 136 chemicals tested using autoradiographic approaches and for 147 chemicals tested using liquid scintillation counting (LSC) procedures; 38 chemicals were tested by both approaches to measure UDS. Since there were no documents available that provided detailed recommendations of UDS screening protocols or criteria for evaluating the results, the UDS Work Group presents suggested protocols and evaluation criteria suitable for measuring and evaluating UDS by autoradiography in primary rat hepatocytes and diploid human fibroblasts and by the LSC approach in diploid human fibroblasts. UDS detection is an appropriate system for inclusion in carcinogenicity and mutagenicity testing programs, because it measures the repair of DNA damage induced by many classes of chemicals over the entire mammalian genome. However, for this system to be utilized effectively, appropriate metabolic activation systems for autoradiographic measurements of UDS in human diploid fibroblasts must be developed, the nature of hepatocyte-to-hepatocyte variability in UDS responses must be determined, and the three suggested protocols must be thoroughly evaluated by using them to test a large number of coded chemicals of known in vivo mutagenicity and carcinogenicity. PMID:6358881

  20. Mutagenicity of Ochratoxin A and Its Hydroquinone Metabolite in the SupF Gene of the Mutation Reporter Plasmid Ps189

    PubMed Central

    Akman, Steven A.; Adams, Marissa; Case, Doug; Park, Gyungse; Manderville, Richard A.

    2012-01-01

    Ochratoxin A (OTA) is a mycotoxin that enhances renal tumor formation in the outer medulla of male rat kidney. Direct DNA damage and subsequent mutagenicity may contribute to these processes. In this study we have determined whether OTA in the absence or presence of activated rat liver microsomes (RLM) or redox-active transition metals (Fe(III) or Cu(II)) causes promutagenic DNA damage in the supF gene of the mutation reporter plasmid pS189 replicating in human Ad293 cells. In addition, we have assessed the mutagenicity of the hydroquinone metabolite (OTHQ) of OTA in the absence or presence of cysteine without added cofactors. Our results show that oxidation of OTA, either by RLM or by transition metal ions, activates OTA to a directly genotoxic mutagen(s). The Fe(III)/OTA system was the most potent mutagen in our experimental system, causing a 32-fold increase in mutant fraction (MF) above the spontaneous control MF. The Cu(II)/OTA system caused a 9-fold increase in MF, while a 6–10-fold increase in MF was observed for OTA in the presence of RLM. The OTHQ metabolite is also mutagenic, especially in the presence of cysteine, in which a 6-fold increase in MF was observed. Our data provide further insight into OTA bioactivation that may account for its in vivo mutagenicity in male rat kidney. PMID:22606376

  1. Construction of a new minicircle DNA carrying an enhanced green florescent protein reporter gene for efficient expression into mammalian cell lines.

    PubMed

    Sanei Ata-Abadi, Nafiseh; Dormiani, Kianoush; Khazaie, Yahya; Ghaedi, Kamran; Forouzanfar, Mahboobeh; Lachinani, Liana; Rezaei, Naeimeh; Kiani-Esfahani, Abbas; Nasr-Esfahani, Mohammad Hossein

    2015-07-01

    The presence of a bacterial backbone in conventional eukaryotic expression plasmids may cause undesirable effects by triggering the immune responses in mammals and repression of episomal transgene expression. To avoid these problems, researchers have proposed the use of minicircle DNAs which are episomal vectors that have lost their bacterial backbone using a site-specific recombinase mediated recombination. In the present study, we have constructed a new minicircle DNA vector that carries an enhanced green florescent protein (EGFP) reporter gene using phage ?C31 integrase-mediated recombination and homing endonuclease ISceI-mediated purification in E. coli. ?C31 integrase expression was under the control of the araBAD promoter, whereas ISceI endonuclease was controlled by the tac promoter. This vector was transfected into CHO-K1 cells, which showed transient expression of EGFP up to 14 generations. Similar results were obtained upon transient transfection into HEK cells. In addition, PCR results on genomic DNA, demonstrated the EGFP-minicircle was episomal and did not integrate into the host genome. Our constructed parental plasmid expresses EGFP and could be used for the generation of episomal minicircle DNA with intent to carry out transient transfection of interested DNA fragments into the eukaryotic cells for various purposes. PMID:25736052

  2. Rapid activity-directed screening of estrogens by parallel coupling of liquid chromatography with a functional gene reporter assay and mass spectrometry.

    PubMed

    Jonker, Willem; Lamoree, Marja H; Houtman, Corine J; Hamers, Timo; Somsen, Govert W; Kool, Jeroen

    2015-08-01

    In this study we developed a new LC nanofractionation platform that combines a human cell (BG1.Luc) gene reporter assay with a high resolution mass spectrometer for the detection and identification of estrogenic and anti-estrogenic compounds in environmental waters. The selection of this assay was based on its high sensitivity and selectivity, which is required for environmental trace level detection. We modified an autosampler and controlled it with in-house developed software to collect fractions in the low second range in microtiter plates. This ensured that chromatographic separation was maintained and allowed straightforward hyphenation with the bioassay. After bioassay testing, bioassay chromatograms were reconstructed and directly correlated with MS chromatograms that were obtained in parallel. This enabled to pinpoint bioactives in the MS chromatogram within a single fractionation cycle and results in a significant increase in throughput compared to traditional EDA studies. The sensitivity of the platform was low enough for environmental waters (80nM for bisphenol A and 320pM and 3.2nM for estradiol and estriol, respectively). In addition, the ability of the platform to detect anti-estrogens was successfully demonstrated as well. Finally, real samples were analysed. PMID:26116188

  3. Transient expression of a GUS reporter gene from cauliflower mosaic virus replacement vectors in the presence and absence of helper virus

    Microsoft Academic Search

    Rita Viaplana; David S. Turner; Simon N. Covey

    Vectors based upon the genome of cauliflower mosaic virus (CaMV) have only a limited capacity for replicating foreign DNA in plants. A helper virus system has been developed to complement CaMV constructs capable of carrying a large foreign gene (glucuronidase; GUS). GUS replaced part or all of the non-essential CaMV gene II and the essential genes III, IV and V.

  4. Depletion of the xynB2 gene upregulates ?-xylosidase expression in C. crescentus.

    PubMed

    Corrêa, Juliana Moço; Mingori, Moara Rodrigues; Gandra, Rinaldo Ferreira; Loth, Eduardo Alexandre; Seixas, Flávio Augusto Vicente; Simão, Rita de Cássia Garcia

    2014-01-01

    Caulobacter crescentus is able to express several enzymes involved in the utilization of lignocellulosic biomasses. Five genes, xynB1-5, that encode ?-xylosidases are present in the genome of this bacterium. In this study, the xynB2 gene, which encodes ?-xylosidase II (CCNA_02442), was cloned under the control of the PxylX promoter to generate the O-xynB2 strain, which overexpresses the enzyme in the presence of xylose. In addition, a null mutant strain, ?-xynB2, was created by two homologous recombination events where the chromosomal xynB2 gene was replaced by a copy that was disrupted by the spectinomycin-resistant cassette. We demonstrated that C. crescentus cells lacking ?-xylosidase II upregulates the xynB genes inducing ?-xylosidase activity. Transcriptional analysis revealed that xynB1 (RT-PCR analysis) and xynB2 (lacZ transcription fusion) gene expression was induced in the ?-xynB2 cells, and high ?-xylosidase activity was observed in the presence of different agro-industrial residues in the null mutant strain, a characteristic that can be explored and applied in biotechnological processes. In contrast, overexpression of the xynB2 gene caused downregulation of the expression and activity of the ?-xylosidase. For example, the ?-xylosidase activity that was obtained in the presence of sugarcane bagasse was 7-fold and 16-fold higher than the activity measured in the C. crescentus parental and O-xynB2 cells, respectively. Our results suggest that ?-xylosidase II may have a role in controlling the expression of the xynB1 and xynB2 genes in C. crescentus. PMID:24142353

  5. The cps gene cluster of Salmonella strain LT2 includes a second mannose pathway: sequence of two genes and relationship to genes in the rfb gene cluster

    Microsoft Academic Search

    Gordon Stevenson; Sang Jun Lee; Lajwant K. Romana; Peter R. Reeves

    1991-01-01

    We report the presence in Salmonella enterica strain LT2 (serovar thyphimurium) of duplicate genes for two steps in the synthesis of GDP-mannose. The previously known genes, rfbK (phosphomannomutase) and rfbM (mannose-l-phosphate guanyltransferase), are part of the gene cluster for the O antigen. The two new genes, cpsB and cpsG, respectively, are thought to be part of the gene cluster for

  6. Control of yeast gene expression by transposable elements: maximum expression requires a functional Ty activator sequence and a defective Ty promoter.

    PubMed Central

    Coney, L R; Roeder, G S

    1988-01-01

    Integration of a transposable element adjacent to a gene frequently results in an alteration in expression of the nearby gene. The purpose of our experiments was to identify cis-acting sequences within a yeast transposon (Ty) that are important for expression of the adjacent gene. The role of these sequences in Ty transcription was also analyzed in order to examine the relationship between Ty and adjacent gene expression. Three naturally occurring Ty elements located at the HIS4 locus were examined. These Ty elements differed by multiple sequence changes and had different effects on HIS4 expression. To determine which sequences were important to Ty and HIS4 expression, Ty::lacZ and Ty::HIS4::lacZ fusion genes were constructed and analyzed. Results of these experiments indicated that a sequence element is present in the Ty epsilon region that is necessary for HIS4 expression but which has only a modest effect on Ty transcription. Additionally, a mutation in the Ty promoter region decreased Ty transcription and increased HIS4 expression. The opposite effects of this mutation on Ty and adjacent gene expression were probably caused by promoter competition. PMID:2847026

  7. A therapeutic window for intravenous administration of autologous bone marrow after cerebral ischemia in adult rats

    Microsoft Academic Search

    Satoshi Iihoshi; Osamu Honmou; Kiyohiro Houkin; Kazuo Hashi; Jeffery D. Kocsis

    2004-01-01

    The primary objective of this study was to test the hypothesis that intravenous administration of autologous bone marrow cells could improve functional recovery after middle cerebral artery occlusion (MCAO) for 45 min in the rat and to determine specific time windows for efficacy. Mononuclear cells from autologous bone marrow were transfected with the LacZ reporter gene, and injected intravenously into

  8. brief communications nature methods | VOL.7 NO.11 | NOVEMBER2010 | 893

    E-print Network

    Cai, Long

    by individual researchers. The floxed Smad4 (Smad4f) knockout-first allele generated by the IKMC containsP sites flanking a critical Smad4 coding exon (Fig. 1b). This results in expression of a lacZ reporter and the neomycin resistance (neo) genes under control of the endogenous Smad4 promoter, which is active

  9. The NOT proteins are part of the CCR4 transcriptional complex and affect gene expression both positively and negatively.

    PubMed Central

    Liu, H Y; Badarinarayana, V; Audino, D C; Rappsilber, J; Mann, M; Denis, C L

    1998-01-01

    The CCR4 transcriptional regulatory complex consisting of CCR4, CAF1, DBF2 and other unidentified factors is one of several groups of proteins that affect gene expression. Using mass spectrometry, we have identified the 195, 185 and 116 kDa species which are part of the CCR4 complex. The 195 and 185 kDa proteins were found to be NOT1 and the 116 kDa species was identical to NOT3. NOT1, 2, 3 and 4 proteins are part of a regulatory complex that negatively affects transcription. All four NOT proteins were found to co-immunoprecipitate with CCR4 and CAF1, and NOT1 co-purified with CCR4 and CAF1 through three chromatographic steps in a complex estimated to be 1.2x10(6) Da in size. Mutations in the NOT genes affected many of the same genes and processes that are affected by defects in the CCR4 complex components, including reduction in ADH2 derepression, defective cell wall integrity and increased sensitivity to monoand divalent ions. Similarly, ccr4, caf1 and dbf2 alleles negatively regulated FUS1-lacZ expression, as do defects in the NOT genes. These results indicate that the NOT proteins are physically and functionally part of the CCR4 complex which forms a unique and novel complex that affects transcription both positively and negatively. PMID:9463387

  10. A model system for testing gene vectors using murine tumor cells on the chorioallantoic membrane of the chick embryo.

    PubMed

    Dani, Sergio U; Espindola, Rachel

    2002-01-01

    We developed a model system for testing gene vectors, based on the growth of murine tumors on the chorioallantoic membrane (CAM) of embryonic chickens. The ability of selected murine cells to grow on the CAM was rated according to the following criteria: i) formation of tumor masses; ii) metastasis formation; iii) reproducibility; iv) yield, indicated as the number of embryos surviving to assessment time with visible tumors on the CAM; v) maintainability of the cell, both in the original host and the embryonic chick, or 'shuttle maintainability'; vi) detection by the naked eye, and vii) cost/benefit relation. The murine melanoma cell lineage, B16F10, which efficiently forms distinct, pigmented tumor masses and metastases on the CAM, performed better in this model than the murine B61 cell line. In vitro transduction of B16F10 cells with a recombinant adenovirus carrying a construct of the E. coli LacZ gene followed by inoculation onto the CAM resulted in beta-galactosidase expression in the tumor mass growing on the CAM. This model is potentially applicable to preclinical evaluation of gene vectors, especially for gene therapy of cancer. PMID:14963844

  11. The effect of the rpoSam allele on gene expression and stress resistance in Escherichia coli.

    PubMed

    Galbiati, Heloisa F; Taschner, Natalia P; Spira, Beny

    2014-08-01

    The RNA polymerase associated with RpoS transcribes many genes related to stationary phase and stress survival in Escherichia coli. The DNA sequence of rpoS exhibits a high degree of polymorphism. A C to T transition at position 99 of the rpoS ORF, which results in a premature amber stop codon often found in E. coli strains. The rpoSam mutant expresses a truncated and partially functional RpoS protein. Here, we present new evidence regarding rpoS polymorphism in common laboratory E. coli strains. One out of the six tested strains carries the rpoSam allele, but expressed a full-length RpoS protein owing to the presence of an amber supressor mutation. The rpoSam allele was transferred to a non-suppressor background and tested for RpoS level, stress resistance and for the expression of RpoS and sigma70-dependent genes. Overall, the rpoSam strain displayed an intermediate phenotype regarding stress resistance and the expression of ?(S)-dependent genes when compared to the wild-type rpoS(+) strain and to the rpoS null mutant. Surprisingly, overexpression of rpoSam had a differential effect on the expression of the ?(70)-dependent genes phoA and lacZ that, respectively, encode the enzymes alkaline phosphatase and ?-galactosidase. The former was enhanced while the latter was inhibited by high levels of RpoSam. PMID:24862098

  12. Gene Therapy

    PubMed Central

    Scheller, E.L.; Krebsbach, P.H.

    2009-01-01

    Gene therapy is defined as the treatment of disease by transfer of genetic material into cells. This review will explore methods available for gene transfer as well as current and potential applications for craniofacial regeneration, with emphasis on future development and design. Though non-viral gene delivery methods are limited by low gene transfer efficiency, they benefit from relative safety, low immunogenicity, ease of manufacture, and lack of DNA insert size limitation. In contrast, viral vectors are nature’s gene delivery machines that can be optimized to allow for tissue-specific targeting, site-specific chromosomal integration, and efficient long-term infection of dividing and non-dividing cells. In contrast to traditional replacement gene therapy, craniofacial regeneration seeks to use genetic vectors as supplemental building blocks for tissue growth and repair. Synergistic combination of viral gene therapy with craniofacial tissue engineering will significantly enhance our ability to repair and replace tissues in vivo. PMID:19641145

  13. Comparative analysis of mouse hepcidin 1 and 2 genes: evidence for different patterns of expression and co-inducibility during iron overload 1 1 The nucleotide sequence reported in this paper has been submitted to the GenBank Data Bank with accession number AY232841

    Microsoft Academic Search

    Gennady Ilyin; Brice Courselaud; Marie-Bérengère Troadec; Christelle Pigeon; Mehdi Alizadeh; Patricia Leroyer; Pierre Brissot; Olivier Loréal

    2003-01-01

    In contrast to the human genome, the mouse genome contains two HEPC genes encoding hepcidin, a key regulator of iron homeostasis. Here we report a comparative analysis of sequence, genomic structure, expression and iron regulation of mouse HEPC genes. The predicted processed 25 amino acid hepcidin 2 peptide share 68% identity with hepcidin 1 with perfect conservation of eight cysteine

  14. Chromosomal localisation of a pseudoautosomal growth gene(s).

    PubMed Central

    Ogata, T; Petit, C; Rappold, G; Matsuo, N; Matsumoto, T; Goodfellow, P

    1992-01-01

    Although recent molecular studies in patients with sex chromosome aberrations are consistent with a growth gene(s) being present in the pseudoautosomal region (PAR), the precise location has not been determined. In this report, we describe a Japanese boy and his mother with an interstitial deletion in Xp22.3 and review the correlation between genotype and stature in six cases of partial monosomy of the PAR. The results indicate that the region from DXYS20 to DXYS15 is the critical region for the putative growth gene(s). Images PMID:1404292

  15. Gene Concepts, Gene Talk, and Gene Patents

    E-print Network

    Torrance, Andrew W.

    2010-01-01

    Since the existence of a discrete unit of heredity was first proposed by Gregor Mendel, scientific concepts of the “gene” have undergone rapid evolution. Beyond obvious epistemic and operational importance to the scientific ...

  16. Structure of the DNA damage-inducible gene DDR48 and evidence for its role in mutagenesis in Saccharomyces cerevisiae.

    PubMed Central

    Treger, J M; McEntee, K

    1990-01-01

    The DDR48 gene of Saccharomyces cerevisiae is a member of a set of genes that displays increased transcription in response to treatments that produce DNA lesions or to heat-shock stress. Other members of this group include the DDRA2 and UBI4 genes. DNA sequence analysis of the DDR48 gene demonstrates the presence of two overlapping open reading frames, each of which has the capacity to encode a protein with a molecular mass of approximately 45 kilodaltons. Fusions of the DDR48 coding sequences to lacZ demonstrates that only one of these frames is expressed in yeast cells. The protein predicted from this sequence is extremely hydrophilic and contains multiple repeats of the peptide sequence Ser-Asn-Asn-X-Asp-Ser-Tyr-Gly where X is either Asn or Asp. Additionally, closely related sequences are found throughout the primary sequence. Primer extension data indicate that, after 4-nitroquinoline-1-oxide and heat-shock treatments, there are three major and two minor transcriptional start sites which are utilized. The function of the DDR48 gene was investigated by disrupting this gene in diploid cells. Viable haploid cells containing the DDR48 gene disruption were isolated after tetrad analysis. Although the ddr48 mutant showed a slightly altered sensitivity to killing by 4-nitroquinoline-1-oxide and to heat shock compared with the DDR48 haploid, the spontaneous mutation rate of reversion of a his4 mutation was reduced 6- to 14-fold in the ddr48 strain. These results implicate the DDR48 gene in the production or recovery of mutations in S. cerevisiae. Images PMID:2111448

  17. Hemato-endothelial differentiation from lentiviral-transduced human embryonic stem cells retains durable reporter gene expression under the control of ubiquitin promoter

    Microsoft Academic Search

    Hua Jiang; Xiaolong Lin; Youji Feng; Yi Xie; Jinlan Han; Yueping Zhang; Zack Z. Wang; Tong Chen

    2010-01-01

    Human embryonic stem (hES) cells are able to give rise to a variety of cell lineages under specific culture condition. An\\u000a effective strategy for stable genetic modification in hES cells may provide a powerful tool for study of human embryogenesis\\u000a and cell-based therapies. However, gene silences are documented in hES cells. In current study, we investigated whether genes\\u000a controlled under

  18. Iron regulation of gene expression in the Bradyrhizobium japonicum/soybean symbiosis. Final technical report, June 1, 1991--May 31, 1995

    SciTech Connect

    Guerinot, M.L.

    1996-02-08

    B.japonicum produces ALA in a reaction catalyzed by the product of the hemA gene. Expression of the gene is affected by iron availability. To address the question of how the 5 prime untranslated region of the hemA transcript is involved in iron regulation, evenly spaced 10bp deletions within the hemA leader region was constructed and effects on hemA-lacZ expression were determined.

  19. Transient gene expression of foreign genes in preheated protoplasts: stimulation of expression of transfected genes lacking heat shock elements

    Microsoft Academic Search

    Nehama Zakai; Nurit Ballas; Maty Hershkovitz; Shimshon Broido; Raquel Ram; Abraham Loyter

    1993-01-01

    Transfection of preheated petunia protoplasts with several biologically active DNA constructs resulted in a significantly higher gene expression than that observed in transfected unheated protoplasts. It was observed with supercoiled, linearized and single-stranded DNA structures that stimulation of transient gene expression in preheated protoplasts was neither dependent on the reporter gene nor on the regulatory elements used. Heat treatment at

  20. Involvement of the 5'-untranslated region in cold-regulated expression of the rbpA1 gene in the cyanobacterium Anabaena variabilis M3.

    PubMed Central

    Sato, N; Nakamura, A

    1998-01-01

    Transcript of the rbpA1 gene in Anabaena variabilis accumulates significantly at low growth temperatures below 28 degreesC. This accumulation was maximal at 16 degreesC. Accumulation of the rbpA1 transcript was completely abolished by rifampicin, but not by chloramphenicol. Photosynthesis was not required for this cold-induced accumulation. This accumulation of transcript was partly accounted for by increased stability of the rbpA1 transcript at low temperature. Expression of chimeric genes containing 3'-deleted rbpA1 sequences fused to the lacZ gene was regulated by low temperature when almost the entire 5'-untranslated region (5'-UTR) remained undeleted. Further deletion resulted in constitutive expression of the chimeric gene. The 5'-UTR sequence formed two types of complexes in vitro with protein extract from cells grown at 38 degreesC, but not with extract from the 22 degreesC grown cells. Affinity purification identified polypeptides of 75 and 32 kDa in Complex 1 and a 72 kDa polypeptide in Complex 2. These results are compatible with a model in which expression of the rbpA1 gene is regulated by transcriptional derepression at low temperature, although additional mechanisms, such as regulation of mRNA stability, might also contribute to temperature-dependent regulation. PMID:9547280

  1. LfrR Is a Repressor That Regulates Expression of the Efflux Pump LfrA in Mycobacterium smegmatis

    Microsoft Academic Search

    Silvia Buroni; Giulia Manina; Paola Guglierame; Maria Rosalia Pasca; Giovanna Riccardi; Edda De Rossi

    2006-01-01

    The lfrA gene of Mycobacterium smegmatis encodes an efflux pump which mediates resistance to different fluoroquinolones, cationic dyes, and anthracyclines. The deletion of the lfrR gene, coding for a putative repressor and localized upstream of lfrA, increased the lfrA expression. In this study, reverse transcription- PCR experiments showed that the two genes are organized as an operon, and lacZ reporter

  2. Gene-environment dependence creates spurious gene-environment interaction.

    PubMed

    Dudbridge, Frank; Fletcher, Olivia

    2014-09-01

    Gene-environment interactions have the potential to shed light on biological processes leading to disease and to improve the accuracy of epidemiological risk models. However, relatively few such interactions have yet been confirmed. In part this is because genetic markers such as tag SNPs are usually studied, rather than the causal variants themselves. Previous work has shown that this leads to substantial loss of power and increased sample size when gene and environment are independent. However, dependence between gene and environment can arise in several ways including mediation, pleiotropy, and confounding, and several examples of gene-environment interaction under gene-environment dependence have recently been published. Here we show that under gene-environment dependence, a statistical interaction can be present between a marker and environment even if there is no interaction between the causal variant and the environment. We give simple conditions under which there is no marker-environment interaction and note that they do not hold in general when there is gene-environment dependence. Furthermore, the gene-environment dependence applies to the causal variant and cannot be assessed from marker data. Gene-gene interactions are susceptible to the same problem if two causal variants are in linkage disequilibrium. In addition to existing concerns about mechanistic interpretations, we suggest further caution in reporting interactions for genetic markers. PMID:25152454

  3. Adenovirus-mediated gene delivery to hypothalamic magnocellular neurons in mice

    NASA Technical Reports Server (NTRS)

    Vasquez, E. C.; Beltz, T. G.; Meyrelles, S. S.; Johnson, A. K.

    1999-01-01

    Vasopressin is synthesized by magnocellular neurons in supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei and released by their axon terminals in the neurohypophysis (NH). With its actions as an antidiuretic hormone and vasoactive agent, vasopressin plays a pivotal role in the control of body fluids and cardiovascular homeostasis. Because of its well-defined neurobiology and functional importance, the SON/PVN-NH system is ideal to establish methods for gene transfer of genetic material into specific pathways in the mouse central nervous system. In these studies, we compared the efficiency of transferring the gene lacZ, encoding for beta-galactosidase (beta-gal), versus a gene encoding for green fluorescent protein by using replication-deficient adenovirus (Ad) vectors in adult mice. Transfection with viral concentrations up to 2 x 10(7) plaque-forming units per coverslip of NH, PVN, and SON in dissociated, cultured cells caused efficient transfection without cytotoxicity. However, over an extended period of time, higher levels (50% to 75% of the cells) of beta-gal expression were detected in comparison with green fluorescent protein (5% to 50% of the cells). With the use of a stereotaxic approach, the pituitary glands of mice were injected with Ad (4 x 10(6) plaque-forming units). In material from these animals, we were able to visualize the expression of the beta-gal gene in the NH and in magnocellular neurons of both the PVN and SON. The results of these experiments indicate that Ad-Rous sarcoma virus promoter-beta-gal is taken up by nerve terminals at the injection site (NH) and retrogradely transported to the soma of the neurons projecting to the NH. We conclude that the application of these experimental approaches will provide powerful tools for physiological studies and potential approaches to deliver therapeutic genes to treat diseases.

  4. Gene Therapy

    PubMed Central

    Baum, Bruce J

    2014-01-01

    Applications of gene therapy have been evaluated in virtually every oral tissue, and many of these have proved successful at least in animal models. While gene therapy will not be used routinely in the next decade, practitioners of oral medicine should be aware of the potential of this novel type of treatment that doubtless will benefit many patients with oral diseases. PMID:24372817

  5. Trichoderma genes

    DOEpatents

    Foreman, Pamela (Los Altos, CA); Goedegebuur, Frits (Vlaardingen, NL); Van Solingen, Pieter (Naaldwijk, NL); Ward, Michael (San Francisco, CA)

    2012-06-19

    Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

  6. Gene Identification

    NSDL National Science Digital Library

    Chuck Daniels

    This module will examine the "Language" of genes and illustrate how basic statistical methods can be applied to the problem of gene prediction. The merger of computational sciences with biology, and the challenges facing BioinFormatics, will also be explored through the use of analysis tools available at the National Center for Biotechnology InFormation (NCBI).

  7. Gene Control

    NSDL National Science Digital Library

    WGBH Educational Foundation

    2003-09-26

    The development of creatures that appear to have nothing in common is directed by a surprisingly small number of genes. In this video segment, learn about the power of master control genes. Footage from The Secret of Life: Birth, Sex & Death.

  8. Cloning and expression of two homologous genes of Bacillus thuringiensis subsp. israelensis which encode 130-kilodalton mosquitocidal proteins.

    PubMed Central

    Ward, E S; Ellar, D J

    1988-01-01

    Two homologous genes encoding 130-kilodalton (kDa) mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis have been cloned and expressed in Escherichia coli or Bacillus subtilis or both. One of these genes, pPC130, was expressed as a lacZ transcriptional fusion in E. coli at a level sufficient to produce phase-bright inclusions, which were purified and shown to be toxic to Aedes aegypti larvae. The second gene, pCH130, was expressed at a low level in recombinant E. coli cells and was therefore cloned in B. subtilis as a transcriptional fusion of the promoter sequences corresponding to a B. thuringiensis subsp. israelensis 27-kDa delta-endotoxin (E. S. Ward, A. R. Ridley, D. J. Ellar, and J. A. Todd, J. Mol. Biol. 191:13-22, 1986) and the structural gene. Recombinant B. subtilis cells produced phase-bright inclusions during late sporulation; these were partially purified and shown to be toxic to A. aegypti larvae at an LC50 (concentration required to cause 50% mortality of larvae after 24 h of assay) which is significantly lower than that of the pPC130 protein. Neither 130-kDa protein was hemolytic under the assay conditions. Comparison of the nucleotide sequences of these two genes indicates that they share a high degree of homology in the C-terminal portions, but relatively little similarity in the N termini. In addition, significant homologies were found between the pCH130 gene and the HD-1 Dipel gene of B. thuringiensis subsp. kurstaki (H. E. Schnepf, H. C. Wong, and H. R. Whiteley, J. Biol. Chem. 260:6264-6272, 1985). Images PMID:2828321

  9. The HXT1 gene product of Saccharomyces cerevisiae is a new member of the family of hexose transporters.

    PubMed Central

    Lewis, D A; Bisson, L F

    1991-01-01

    Two novel genes affecting hexose transport in the yeast Saccharomyces cerevisiae have been identified. The gene HXT1 (hexose transport), isolated from plasmid pSC7, was sequenced and found to encode a hydrophobic protein which is highly homologous to the large family of sugar transporter proteins from eucaryotes and procaryotes. Multicopy expression of the HXT1 gene restored high-affinity glucose transport to the snf3 mutant, which is deficient in a significant proportion of high-affinity glucose transport. HXT1 was unable to complement the snf3 growth defect in low copy number. The HXT1 protein was found to contain 12 putative membrane-spanning domains with a central hydrophilic domain and hydrophilic N- and C-terminal domains. The HXT1 protein is 69% identical to GAL2 and 66% identical to HXT2, and all three proteins were found to have a putative leucine zipper motif at a consensus location in membrane-spanning domain 2. Disruption of the HXT1 gene resulted in loss of a portion of high-affinity glucose and mannose transport, and wild-type levels of transport required both the HXT1 and SNF3 genes. Unexpectedly, expression of beta-galactosidase activity by using a fusion of the lacZ gene to the HXT1 promoter in a multicopy plasmid was maximal during lag and early exponential phases of growth, decreasing approximately 100-fold upon further entry into exponential growth. Deletion analysis of pSC7 revealed the presence of another gene (called ORF2) capable of suppressing the snf3 null mutant phenotype by restoring high-affinity glucose transport and increased low-affinity transport. PMID:2046678

  10. Novel frame shift mutations ('A' deletion) observed in exon 9 of Wilms' tumor (WT1) gene in a patient reported with glomerulosclerosis.

    PubMed

    Pasupuleti, Santhosh Kumar; Katari, Venkatesh; Lokanathan, Srikanth; Uppu, Venkateswara Prasad; Thummaginjala, Syama Sundar; Akkamgari, Ram Prasad Reddy; Ayapati, Tyagi; Kottu, Radhika; Potukuchi, Venkata Gurunadha Krishna Sarma

    2014-08-01

    Wilms' tumor-suppressor gene-1 (WT1) is a transcription factor that contains four zinc-finger motifs at the C-terminus and plays a crucial role in kidney and gonad development. We have identified primitive glomeruloid formation using immunohistochemistry in a patient who was clinically diagnosed with a Wilms' tumor. In order to understand the involvement of mutations in the WT1 gene, the genomic DNA was isolated from peripheral blood of the patient (18/F). Exon 9 of the WT1 gene was amplified and sequenced. The obtained sequence was BLAST searched against the transcript variants (TV) of the WT1 gene. An amplified exon 9 sequence of the WT1 gene showing similarity with exon 9 of TV-A, F and exon 10 of TV-B, D and E with a deletion of single nucleotide 'A' causing frame shift in the 4th zinc finger domain of the WT1 protein resulted in Wilms' tumor condition. The deletion position is variable with different transcript variants and they are present at: for TV-A c.1592delA, p.468, for TV-F c.1053delA, p.259, for TV-B c.1643delA, p.485, for TV-D c.1652 delA, p.488, and for TV-E c.1095delA, p.273; all these variations resulted in frame shift mutation. In order to substantiate these results in silico analysis was carried out; the structural superimposition of wild type and mutant WT1 structures showed that the mutated region exhibited a different confirmation with RMSD of 1.759Å. Therefore, these results conclusively explain the mutation in the WT1 gene that leads to structural changes contributing to glomerulosclerosis. PMID:24853201

  11. [Genetic engineering with a gene encoding a soybean storage protein to identify DNA sequences to control its expression]: Annual report, 1993

    SciTech Connect

    Beachy, R.N.

    1993-12-31

    The {beta}-conglycinins are soybean storage proteins encoded by genes that are tightly regulated both spatially and temporally. The author has studied the Soybean Embryo Factors that bind to the cis elements that are presumably involved in regulating the expression of these gene promoters using both in vitro binding assays and in vivo expression assays in transgenic plants. The results obtained to date have made it evident that there are no clear correlations between the in vivo and the in vitro results, i.e., changes in single nucleotides that alter protein:DNA interactions can have little or no impact on expression of the promoters in vivo. In contrast, the CATGCAT (RY element) sequence, for which no binding proteins have been identified, appear to be very important for controlling gene expression. Although the author has been attempting to isolate and characterize the SEF 3 and SEF 4 proteins he has to date not been successful using protein expression libraries derived from embryo cDNAs. He is continuing experiments of this type, as well as more standard protein purification procedures. He has constructed a number of chimeric promoters with different upstream and downstream regulatory sequences in an attempt to identify, by expression assays in transgenic plants, those sequences that are uniquely responsible for the temporal and spatially regulated expression of the {beta}-conglycinin genes. Because of the results of previously published work from this laboratory, the author concluded that the core promoters themselves may be responsible for the regulation. Therefore, he has focused his efforts on these sequences, the RY element, and the SEF 3 binding sequences. In a follow-up to the studies to modify the expression of genes in seeds, he will express several types of human and other animal genes in seeds of transgenic plants.

  12. Expression and fate of CAT reporter gene microinjected into fertilized medaka ( Oryzias latipes ) eggs in the form of plasmid DNA, recombinant phage particles and its DNA

    Microsoft Academic Search

    S. S. C. Chong; J. R. Vielkind

    1989-01-01

    Fertilized medaka (Oryzias latipes) eggs were cytoplasmically injected with the chloramphenicol acetyltransferase (CAT) gene encompassed in supercoiled and linear plasmid DNA, as well as in intact recombinant phage particles and DNA isolated from the phage. Expression for the CAT plasmid DNA was highest at the gastrula\\/neurula stage, while for the DNA of the phage, it peaked in the 1-week old

  13. Sequential strategy to identify a susceptibility gene for schizophrenia: Report of potential linkage on chromosome 22q12-q13.1: Part 1

    Microsoft Academic Search

    Ann E. Pulver; P. S. Wolyniec; V. K. Lasseter; Laura Kasch; Gerald Nestadt; Stylianos Antonarakis; David Housman; Haig H. Kazazian; Deborah Meyers; Jurg Ott; Malgorzata Lamacz; Kung-Yee Liang; John Hanfelt; Gail Ullrich; Nicola DeMarchi; Elango Ramu; Paul R. McHugh; Lawrence Adler; Marion Thomas; William T. Carpenter; Theo Manschreck; C. T. Gordon; Michelle Kimberland; Robert Babb; Jennifer Puck; Barton Childs

    1994-01-01

    To identify genes responsible for the susceptibility for schizophrenia, and to test the hypothesis that schizophrenia is etiologically heterogeneous, we have studied 39 multiplex families from a systematic sample of schizophrenic patients. Using a complex autosomal dominant model, which considers only those with a diagnosis of schizophrenia or schizoaffective disorder as affected, a random search of the genome for detection

  14. Identification of a novel de novo mutation in the NIPBL gene in an Iranian patient with Cornelia de Lange syndrome: A case report

    PubMed Central

    2011-01-01

    Background Cornelia de Lange syndrome is characterized by dysmorphic facial features, hirsutism, severe growth and developmental delay. Germline mutations in the NIPBL gene with an autosomal dominant pattern and in the SMC1A gene with an X-linked pattern have been identified in Cornelia de Lange syndrome. Case presentation A two-month-old Iranian boy who showed multiple congenital anomalies was referred to the genetic center of a welfare organization in southwest Iran. He was the second child of a non-consanguineous marriage, born after full term with normal delivery. His birth weight was 3110 g, his length was 46 cm and his head circumference was 30 cm. Both parents were clinically asymptomatic, with no positive history of any deformity in their respective families. Conclusions Sequencing of the NIPBL gene from our patient revealed a single-base deletion of thymidine in exon 10 (c.516delT). This mutation presumably results in premature termination at codon 526. We did not observe this mutation in the parents of our patient with Cornelia de Lange syndrome. The results presented here enlarge the spectrum of NIPBL gene mutations associated with Cornelia de Lange syndrome by identifying a novel de novo mutation in an Iranian patient with Cornelia de Lange syndrome and further support the hypothesis that NIPBL mutations are disease-causing mutations leading to Cornelia de Lange syndrome. PMID:21707975

  15. Gastrointestinal Mucins of Fut2-Null Mice Lack Terminal Fucosylation without Affecting Colonization by Candida albicans

    PubMed Central

    Hurd, Elizabeth A.; Holmén, Jessica M.; Hansson, Gunnar C.; Domino, Steven E.

    2006-01-01

    Post-translational modification of apomucins by the sequential action of glycosyltransferases is required to produce mature mucins. The “Secretor” gene (FUT2) encodes an ?(1,2)fucosyltransferase (E.C. 2.4.1.69) that catalyzes addition of terminal ?(1,2)fucose residues on mucins and other molecules in mucosal epithelium. Mutant mice containing targeted replacement of Fut2 with the bacterial reporter gene lacZ were studied to determine the affect of the loss of Fut2 on glycosylation of mucins in the gastrointestinal tract. By whole organ X-gal staining, lacZ activity is prominently expressed in the foveolar pit and chief cells of the glandular stomach, Brunner's glands of the duodenum, and goblet cells in the large intestine of Fut2-LacZ null mice. Staining with Aleuria aurantia agglutinin demonstrates loss of l-fucosylated epithelial glycans throughout the gastrointestinal tract of Fut2-LacZ null mice, however, histologic appearance of the tissues appears normal. Analysis of oligosaccharides released from insoluble colonic mucins, largely Muc2, by mass spectrometry shows complete lack of terminal fucosylation of O-linked oligosaccharides in Fut2-LacZ null mice. Precursor glycans accumulate with no evidence of compensation by other fucosyltransferases or sialyltransferases on mucin glycosylation. Since Candida albicans has been reported to adhere to intestinal mucins creating a potential reservoir associated with vaginitis, Fut2-LacZ null and wild type mice were inoculated by gastric lavage with C. albicans. We observe no difference in colonization between genotypes suggesting mucin terminal fucosylation does not significantly influence C. albicans-host interaction in the intestine, highlighting that infections caused by the same organism at different mucosal surfaces are not equal. PMID:15958416

  16. Sequencing, chromosomal inactivation, and functional expression in Escherichia coli of ppsR, a gene which represses carotenoid and bacteriochlorophyll synthesis in Rhodobacter sphaeroides.

    PubMed Central

    Penfold, R J; Pemberton, J M

    1994-01-01

    Sequencing of a DNA fragment that causes trans suppression of bacteriochlorophyll and carotenoid levels in Rhodobacter sphaeroides revealed two genes: orf-192 and ppsR. The ppsR gene alone is sufficient for photopigment suppression. Inactivation of the R. sphaeroides chromosomal copy of ppsR results in overproduction of both bacteriochlorophyll and carotenoid pigments. The deduced 464-amino-acid protein product of ppsR is homologous to the CrtJ protein of Rhodobacter capsulatus and contains a helix-turn-helix domain that is found in various DNA-binding proteins. Removal of the helix-turn-helix domain renders PpsR nonfunctional. The promoter of ppsR is located within the coding region of the upstream orf-192 gene. When this promoter is replaced by a lacZ promoter, ppsR is expressed in Escherichia coli. An R. sphaeroides DNA fragment carrying crtD', -E, and -F and bchC, -X, -Y, and -Z' exhibited putative promoter activity in E. coli. This putative promoter activity could be suppressed by PpsR in both E. coli and R. sphaeroides. These results suggest that PpsR is a transcriptional repressor. It could potentially act by binding to a putative regulatory palindrome found in the 5' flanking regions of a number of R. sphaeroides and R. capsulatus photosynthesis genes. PMID:8188588

  17. Gene Cloning

    NSDL National Science Digital Library

    WGBH Educational Foundation

    2009-12-07

    This interactive activity adapted from the University of Nebraska's Library of Crop Technologies details the steps involved in producing clones of genes that can then be used to transform the characteristics of an organism.

  18. On sports and genes.

    PubMed

    Zilberman-Schapira, Gili; Chen, Jieming; Gerstein, Mark

    2012-12-01

    Our genes influence our athletic ability. However, the causal genetic factors and mechanisms, and the extent of their effects, remain largely elusive. Many studies investigate this association between specific genes and athletic performance. Such studies have increased in number over the past few years, as recent developments and patents in DNA sequencing have made large amounts of sequencing data available for such analysis. In this paper, we consider four of the most intensively studied genes in relation to athletic ability: angiotensin I-converting enzyme, alpha-actinin 3, peroxismose proliferator-activator receptor alpha and nitric oxide synthase 3. We investigate the connection between genotype and athletic phenotype in the context of these four genes in various sport fields and across different ethnicities and genders. We do an extensive literature survey on these genes and the polymorphisms (single nucleotide polymorphisms or indels) found to be associated with athletic performance. We also present, for each of these polymorphisms, the allele frequencies in the different ethnicities reported in the pilot phase of the 1000 Genomes Project - arguably the largest human genome-sequencing endeavor to date. We discuss the considerable success, and significant drawbacks, of past research along these lines, and propose interesting directions for future research. PMID:22762737

  19. Gene therapy for sarcoma.

    PubMed

    Fruehauf, S; Veldwijk, M R; Berlinghoff, S; Basara, N; Baum, C; Flasshove, M; Hegewisch-Becker, S; Kröger, N; Licht, T; Moritz, T; Hengge, U R; Zeller, W J; Laufs, S

    2002-01-01

    Soft tissue sarcomas are mesenchymal tumors which respond poorly to systemic therapy. Recent studies suggest a higher response rate with an increased doxorubicin dosage. However, this was parallel with a profound hematotoxicity in 75% of patients. Transfer of the human multidrug resistance 1 (MDR1) gene to normal hematopoietic stem cells and transplantation may significantly reduce the hematotoxicity of anthracyclin-based chemotherapy. To test this concept of supportive gene therapy in advance of a clinical study, we transduced mobilized peripheral blood progenitor cells (PBPC) with the retroviral vector SF91m3 containing the human MDR1 gene, transplanted these cells to immune-deficient mice, allowed 6 weeks for engraftment to occur and treated the animals with MDR1-based chemotherapy. In the MDR1-transduced group the human leukocytes were significantly protected from the toxicity of chemotherapy (p < 0.05). While the gene transfer rate was in the range of 10% and thus comparable to recent clinical trials, the gene expression was 59% of transduced cells and thus significantly higher than previously reported for less-advanced vectors. On the other hand, ifosfamide, a drug which has been used successfully for stem cell mobilization, is active in soft tissue sarcoma. Due to these favorable characteristics sarcoma is an attractive target to test the efficacy of MDR1 gene therapy in a clinical setting. Gene therapeutic strategies may also be used to directly target sarcoma cells, e.g. by transfer of suicide genes. We found that adenoassociated virus 2 (AAV-2) vectors efficiently transduce human HS-1 and HT1080 sarcoma cells (>90%) while other tumor cell lines and primary human PBPC were less susceptible. The thymidine kinase (TK) suicide gene was cloned into an AAV-2 vector and a complete kill of TK-transduced HS-1 and HT1080 cells was observed following exposure to aciclovir or ganciclovir (GCV), while >90% of mock-transduced HS-1 cells survived at these dosages. Transplantation of those sarcoma cells to nonobese diabetic (NOD)/LtSz-severe-combined immunodeficient (scid)/scid (NOD/SCID) mice resulted in a survival of >5 months in the AAV-TK-transduced/GCV-treated group, while the mice in the mock-transduced/GCV-treated group had died after 3 weeks. These data show that soft tissue sarcomas are a particularly suitable model system for the development and clinical testing of new gene therapeutic concepts. PMID:12426490

  20. Two genes required for cell fusion during yeast conjugation: evidence for a pheromone-induced surface protein.

    PubMed Central

    Trueheart, J; Boeke, J D; Fink, G R

    1987-01-01

    We characterized two genes, FUS1 and FUS2, which are required for fusion of Saccharomyces cerevisiae cells during conjugation. Mutations in these genes lead to an interruption of the mating process at a point just before cytoplasmic fusion; the partition dividing the mating pair remains undissolved several hours after the cells have initially formed a stable "prezygote." Fusion is only moderately impaired when the two parents together harbor one or two mutant fus genes, and it is severely compromised only when three or all four fus genes are inactivated. Cloning of FUS1 and FUS2 revealed that they share some functional homology; FUS1 on a high-copy number plasmid can partially suppress a fus2 mutant, and vice versa. FUS1 remains essentially unexpressed in vegetative cells, but is strongly induced by incubation of haploid cells with the appropriate mating pheromone. Immunofluorescence microscopy of alpha factor-induced a cells harboring a fus1-LACZ fusion showed the fusion protein to be localized at the cell surface, concentrated at one end of the cell (the shmoo tip). FUS1 maps near HIS4, and the intervening region (including BIK1, a gene required for nuclear fusion) was sequenced along with FUS1. The sequence of FUS1 revealed the presence of three copies of a hexamer (TGAAAC) conserved in the 5' noncoding regions of other pheromone-inducible genes. The deduced FUS1 protein sequence exhibits a striking concentration of serines and threonines at the amino terminus (46%; 33 of 71), followed by a 25-amino acid hydrophobic stretch and a predominantly hydrophilic carboxy terminus, which contains several potential N-glycosylation sites (Asn-X-Ser/Thr). This sequence suggests that FUS1 encodes a membrane-anchored glycoprotein with both N- and O-linked sugars. Images PMID:3302672

  1. Cytotoxicity and gene induction by some essential oils in the yeast Saccharomyces cerevisiae.

    PubMed

    Bakkali, F; Averbeck, S; Averbeck, D; Zhiri, A; Idaomar, M

    2005-08-01

    In order to get an insight into the possible genotoxicity of essential oils (EOs) used in traditional pharmacological applications we tested five different oils extracted from the medicinal plants Origanum compactum, Coriandrum sativum, Artemisia herba alba, Cinnamomum camphora (Ravintsara aromatica) and Helichrysum italicum (Calendula officinalis) for genotoxic effects using the yeast Saccharomyces cerevisiae. Clear cytotoxic effects were observed in the diploid yeast strain D7, with the cells being more sensitive to EOs in exponential than in stationary growth phase. The cytotoxicity decreased in the following order: Origanum compactum>Coriandrum sativum>Artemisia herba alba>Cinnamomum camphora>Helichrysum italicum. In the same order, all EOs, except that derived from Helichrysum italicum, clearly induced cytoplasmic petite mutations indicating damage to mitochondrial DNA. However, no nuclear genetic events such as point mutations or mitotic intragenic or intergenic recombination were induced. The capacity of EOs to induce nuclear DNA damage-responsive genes was tested using suitable Lac-Z fusion strains for RNR3 and RAD51, which are genes involved in DNA metabolism and DNA repair, respectively. At equitoxic doses, all EOs demonstrated significant gene induction, approximately the same as that caused by hydrogen peroxide, but much lower than that caused by methyl methanesulfonate (MMS). EOs affect mitochondrial structure and function and can stimulate the transcriptional expression of DNA damage-responsive genes. The induction of mitochondrial damage by EOs appears to be closely linked to overall cellular cytotoxicity and appears to mask the occurrence of nuclear genetic events. EO-induced cytotoxicity involves oxidative stress, as is evident from the protection observed in the presence of ROS inhibitors such as glutathione, catalase or the iron-chelating agent deferoxamine. PMID:15975845

  2. Genetic analysis of cryIIIA gene expression in Bacillus thuringiensis.

    PubMed

    Salamitou, S; Agaisse, H; Bravo, A; Lereclus, D

    1996-08-01

    The Bacillus thuringiensis (Bt) cryIIIA gene is regulated by a different mechanism from that of most of the other cry genes. Its expression begins during late-exponential growth and not during sporulation as for the other classes of cry genes. Moreover, in Bacillus subtilis, cryIIIA expression is independent of the major sporulation-specific sigma factors and is increased in a spoOA genetic background. We used lacZ fusions and primer-extension analysis to follow the time-course of cryIIIA transcription in Bt wild-type and in various Spo- genetic backgrounds (spoOA, sigE and sigK). cryIIIA was activated from the end of vegetative growth to stage II of sporulation (t3) in the wild-type strain. Thereafter, transcription from the same promoter continued, at a decreasing rate, until the end of stage III. In the spoOA mutant strain, the same promoter was activated for at least 15 h during the stationary phase. cryIIIA activation in the sigK genetic background was similar to that in the wild-type but was extended in a sigma E mutant strain. Thus cryIIIA expression in Bt is not directly dependent on the major sporulation-specific sigma factors. Furthermore, an event linked with the thE-dependent period of sporulation ends cryIIIA activation, although transcription of this gene does not switch off before the end of stage III. PMID:8760917

  3. Reconstruction of a Functional Human Gene Network, with an Application for Prioritizing Positional Candidate Genes

    PubMed Central

    Franke, Lude; Bakel, Harm van; Fokkens, Like; de Jong, Edwin D.; Egmont-Petersen, Michael; Wijmenga, Cisca

    2006-01-01

    Most common genetic disorders have a complex inheritance and may result from variants in many genes, each contributing only weak effects to the disease. Pinpointing these disease genes within the myriad of susceptibility loci identified in linkage studies is difficult because these loci may contain hundreds of genes. However, in any disorder, most of the disease genes will be involved in only a few different molecular pathways. If we know something about the relationships between the genes, we can assess whether some genes (which may reside in different loci) functionally interact with each other, indicating a joint basis for the disease etiology. There are various repositories of information on pathway relationships. To consolidate this information, we developed a functional human gene network that integrates information on genes and the functional relationships between genes, based on data from the Kyoto Encyclopedia of Genes and Genomes, the Biomolecular Interaction Network Database, Reactome, the Human Protein Reference Database, the Gene Ontology database, predicted protein-protein interactions, human yeast two-hybrid interactions, and microarray coexpressions. We applied this network to interrelate positional candidate genes from different disease loci and then tested 96 heritable disorders for which the Online Mendelian Inheritance in Man database reported at least three disease genes. Artificial susceptibility loci, each containing 100 genes, were constructed around each disease gene, and we used the network to rank these genes on the basis of their functional interactions. By following up the top five genes per artificial locus, we were able to detect at least one known disease gene in 54% of the loci studied, representing a 2.8-fold increase over random selection. This suggests that our method can significantly reduce the cost and effort of pinpointing true disease genes in analyses of disorders for which numerous loci have been reported but for which most of the genes are unknown. PMID:16685651

  4. Genes, brain, and behavior: development gone awry in autism? A report on the 23rd Annual International Symposium of the Center for the Study of Gene Structure and Function.

    PubMed

    Lewis, Michael J; Dictenberg, Jason B

    2010-09-01

    Autism and its highly variable symptomology were the themes of the 23rd Annual International Symposium of the Center for the Study of Gene Structure and Function at Hunter College in New York City, held 15 January 2010. The meeting explored the extensive research on autism from several perspectives-integrating research on genetics, neuroscience, and behavior-from researchers presenting new and innovative approaches to understanding the autism spectrum. Early diagnosis, intervention, and genetics were major themes because they are seen as essential areas in which progress is needed before the rise in numbers of cases of autism throughout the world, which some describe as approaching an epidemic, can be stemmed. Several genetic, neurobiological, and behavioral markers of autism have been identified that may ultimately provide the basis for early identification, and that presently define the key areas requiring intensive intervention. PMID:20860674

  5. Gene 209 (1998) 157165 Characterization of the P25 silk gene and associated insertion elements in

    E-print Network

    ?urovec, Michal

    1998-01-01

    sequences reported for the regulatory regions of Bombyx silk genes are not obvious in Galleria P25 of such silk glands secretes a core of the silk fibre, whereas the fibroin, and P25 (Tanaka et al., 1993; Z of expression were Abbreviations: Fib-H, gene for heavy-chain fibroin; Fib-L, gene for studied in Bombyx silk

  6. Gene Trap Lines Define Domains of Gene Regulation in Arabidopsis Petals and Stamens

    Microsoft Academic Search

    Naomi Nakayama; Juana M. Arroyo; Joseph Simorowski; Bruce May; Robert Martienssen; Vivian F. Irisha

    2005-01-01

    marking epidermis and vasculature, as well as lines demarcating the proximodistal or abaxial\\/adaxial axes of the organs. Interestingly, reporter gene expression was typically restricted along the proximodistal axis of petals and stamens, indicating the importance of this developmental axis in patterning of gene expression domains in these organs. We identified novel domains of gene expression along the axis marking the

  7. Gene Trap Lines Define Domains of Gene Regulation in Arabidopsis Petals and StamensW?

    PubMed Central

    Nakayama, Naomi; Arroyo, Juana M.; Simorowski, Joseph; May, Bruce; Martienssen, Robert; Irish, Vivian F.

    2005-01-01

    To identify genes involved in Arabidopsis thaliana petal and stamen organogenesis, we used a gene trap approach to examine the patterns of reporter expression at each stage of flower development of 1765 gene trap lines. In 80 lines, the reporter gene showed petal- and/or stamen-specific expression or lack of expression, or expression in distinct patterns within the petals and/or the stamens, including distinct suborgan domains of expression, such as tissue-specific lines marking epidermis and vasculature, as well as lines demarcating the proximodistal or abaxial/adaxial axes of the organs. Interestingly, reporter gene expression was typically restricted along the proximodistal axis of petals and stamens, indicating the importance of this developmental axis in patterning of gene expression domains in these organs. We identified novel domains of gene expression along the axis marking the midregion of the petals and apical and basal parts of the anthers. Most of the genes tagged in these 80 lines were identified, and their possible functions in petal and/or stamen differentiation are discussed. We also scored the floral phenotypes of the 1765 gene trap lines and recovered two mutants affecting previously uncharacterized genes. In addition to revealing common domains of gene expression, the gene trap lines reported here provide both useful markers and valuable starting points for reverse genetic analyses of the differentiation pathways in petal and stamen development. PMID:16055634

  8. Gene Clustering Based on RNAi Phenotypes of Ovary-Enriched Genes in C. elegans

    Microsoft Academic Search

    Fabio Piano; Aaron J. Schetter; Diane G. Morton; Kristin C. Gunsalus; Valerie Reinke; Stuart K. Kim; Kenneth J. Kemphues

    2002-01-01

    Recently, a set of 766 genes that are enriched in the ovary as compared to the soma was identified by microarray analysis [1]. Here, we report a functional analysis of 98% of these genes by RNA interference (RNAi). Over half the genes tested showed at least one detectable phenotype, most commonly embryonic lethality, consistent with the expectation that ovary transcripts

  9. Final Scientific/Technical Report for DOE Award No. DE-FG02-03ER15426: Role of Arabidopsis PINHEAD gene in meristem function

    SciTech Connect

    Dr. M. Kathryn Barton

    2011-11-29

    The shoot apical meristems of land plants are small mounds of hundreds of cells located at the tips of branches. It is from these small clusters of cells that essentially all above ground plant biomass and therefore much of our energy supply originates. Several key genes have been discovered that are necessary for cells in the shoot apical meristem to take on stem cell properties. The goal of this project is to understand how the synthesis and accumulation of the mRNAs and proteins encoded by these genes is controlled. A thorough understanding of the molecules that control the growth of shoot apical meristems in plants will help us to manipulate food, fiber and biofuel crops to better feed, clothe and provide energy for humans.

  10. [Congenital adrenal hyperplasia due to lack of 17?-hydroxylase: a report of a new mutation in the gene CYP17A1].

    PubMed

    Perales Martínez, J I; Pina Marqués, B; de Arriba Muñoz, A; Mayayo Dehesa, E; Labarta Aizpún, J I; Loidi Fernández, L

    2015-01-01

    P450c17 enzyme catalyses two different reactions: the 17?-hydroxylation of progesterone and pregnenolone, and segmenting the carbon 17-20 binding from the 17,20lyase producing adrenal androgens. This enzyme is coded by the CYP17A1 gene. The case is presented of a 14 year old patient with delayed pubertal development and a high blood pressure for height and age. 46,XX karyotype. Hormonal studies highlighted hypergonadotropic hypogonadism, adrenal insufficiency and mineralocorticoid excess. Subsequent genetic studies showed a homozygous mutation in the CYP17A1 gene (c.753+G>A), not previously described, which is responsible for the pathophysiology of 17?-hydroxylase deficiency. This entity is a rare form of congenital adrenal hyperplasia. The disease often goes unnoticed until adolescence or early adult life, and should be suspected in 46,XY individuals with ambiguous genitalia or 46,XX with delayed puberty associated with hypertension and/or hypokalaemia. PMID:24593890

  11. Progress reports on immune gene therapy for stage IV renal cell cancer using lethally irradiated granulocyte-macrophage colony-stimulating factor-transduced autologous renal cancer cells

    Microsoft Academic Search

    Kenzaburo Tani; Yukoh Nakazaki; Hidenori Hase; Keisuke Takahashi; Miyuki Azuma; Junko Ohata; Reiko Kitamura; Fumihiko Komine; Maki Oiwa; Atsuko Masunaga; Taira Maekawa; Noriharu Satoh; Daiki Adachi; Yasushi Soda; Utako Machida; Muneomi Endo; Tomoko Yamazaki; Kiyoshi Watari; Arinobu Tojo; Naohide Yamashita; Shinji Tomikawa; Masazumi Eriguchi; Hirofumi Hamada; Yoshiaki Wakumoto; Kisaburo Hanazawa; Koh Okumura; Makoto Fujime; Taro Shuin; Kouji Kawai; Hideyuki Akaza; Shirley Clift; Dale Ando; Stephan Sherwin; Richard Mulligan; Shigetaka Asano

    2000-01-01

    There is no effective treatment for patients with stage IV renal cell cancer (RCC), although the introduction of new therapy is imminent. Cancer gene therapy is currently considered to be one of the most promising therapeutic modalities in the field of cancer treatment. Based on the results of animal studies, vaccination using autologous granulocyte-macrophage colony-stimulating factor-transduced renal cancer cells appears

  12. The phn Genes of Burkholderia sp. Strain RP007 Constitute a Divergent Gene Cluster for Polycyclic Aromatic Hydrocarbon Catabolism

    Microsoft Academic Search

    ANDREW D. LAURIE; GARETH LLOYD-JONES

    1999-01-01

    Cloning and molecular ecological studies have underestimated the diversity of polycyclic aromatic hydro- carbon (PAH) catabolic genes by emphasizing classical nah-like (nah, ndo, pah, and dox) sequences. Here we report the description of a divergent set of PAH catabolic genes, the phn genes, which although isofunctional to the classical nah-like genes, show very low homology. This phn locus, which contains

  13. Temsirolimus in the treatment of renal cell carcinoma associated with Xp11.2 translocation/TFE gene fusion proteins: a case report and review of literature

    PubMed Central

    Parikh, Jigarkumar; Coleman, Teresa; Messias, Nidia; Brown, James

    2009-01-01

    Xp11.2 translocation renal cell carcinomas (TRCCs) are a rare family of tumors newly recognized by the World Health Organization (WHO) in 2004. These tumors result in the fusion of partner genes to the TFE3 gene located on Xp11.2. They are most common in the pediatric population, but have been recently implicated in adult renal cell carcinoma (RCC) presenting at an early age. TFE3-mediated direct transcriptional upregulation of the Met tyrosine kinase receptor triggers dramatic activation of downstream signaling pathways including the protein kinase B (Akt)/phosphatidylinositol-3 kinase (PI3K) and mammalian target of rapamycin (mTOR) pathways. Temsirolimus is an inhibitor of mammalian target of rapamycin (mTOR) kinase, a component of intracellular signaling pathways involved in the growth and proliferation of malignant cells. Here we present a case of a 22-year old female who has been treated with temsirolimus for her Xp11.2/TFE3 gene fusion RCC. PMID:21139932

  14. Fusion genes as putative microbial drug targets in H. pylori

    Microsoft Academic Search

    Meena K. Sakharkar; Kishore R. Sakharkar; Vincent T. K. Chow

    2006-01-01

    Fusion genes have been reported as a means of enabling the development of novel or enhanced functions. In this report, we analyzed fusion genes in the genomes of two Helicobacter pylori strains (26695 and J99) and identified 32 fusion genes that are present as neighbours in one strain (components) and are fused in the second (composite), and vice-versa. The mechanism

  15. Origin of New Genes: Evidence from Experimental and Computational Analyses

    Microsoft Academic Search

    Manyuan Long; Michael Deutsch; Wen Wang; Esther Betrán; Frédéric G. Brunet; Jianming Zhang

    2003-01-01

    Exon shuffling is an essential molecular mechanism for the formation of new genes. Many cases of exon shuffling have been reported in vertebrate genes. These discoveries revealed the importance of exon shuffling in the origin of new genes. However, only a few cases of exon shuffling were reported from plants and invertebrates, which gave rise to the assertion that the

  16. Origin of new genes: evidence from experimental and computational analyses

    Microsoft Academic Search

    Manyuan Long; Michael Deutsch; Wen Wang; Jianming Zhang

    2003-01-01

    Exon shuffling is an essential molecular mechanism for the formation of new genes. Many cases of exon shuffling have been reported in vertebrate genes. These discoveries revealed the importance of exon shuffling in the origin of new genes. However, only a few cases of exon shuffling were reported from plants and invertebrates, which gave rise to the assertion that the

  17. Gene length and expression level shape genomic novelties

    PubMed Central

    Grishkevich, Vladislav

    2014-01-01

    Gene duplication and alternative splicing are important mechanisms in the production of genomic novelties. Previous work has shown that a gene’s family size and the number of splice variants it produces are inversely related, although the underlying reason is not well understood. Here, we report that gene length and expression level together explain this relationship. We found that gene lengths correlate with both gene duplication and alternative splicing: Longer genes are less likely to produce duplicates and more likely to exhibit alternative splicing. We show that gene length is a dynamic property, increasing with evolutionary time—due in part to the insertions of transposable elements—and decreasing following partial gene duplications. However, gene length alone does not account for the relationship between alternative splicing and gene duplication. A gene’s expression level appears both to impose a strong constraint on its length and to restrict gene duplications. Furthermore, high gene expression promotes alternative splicing, in particular for long genes, and alternatively, short genes with low expression levels have large gene families. Our analysis of the human and mouse genomes shows that gene length and expression level are primary genic properties that together account for the relationship between gene duplication and alternative splicing and bias the origin of novelties in the genome. PMID:25015383

  18. Transgenic Arabidopsis Gene Expression System

    NASA Technical Reports Server (NTRS)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  19. Foreign gene transfer into Chinese shrimps ( Penaeus chinensis ) with gene gun

    Microsoft Academic Search

    Zhiyi Liu; Jianhai Xiang; Guoying Zhou; Zuxun Gong

    2001-01-01

    Plasmids pG DNA-RZ1 with aGFP (green fluorescent protein) reporter gene and a ribozyme gene incising penaeid white spot baculovirus (WSBV) were first introduced\\u000a into the fertilized eggs of Chinese shrimps by gene gun. The treated and control samples of different development stages were\\u000a observed with a fluorescent microscope. The transient expression ofGFP gene was high in nauplius and zoea larvae.

  20. A new hyperrecombination mutation identifies a novel yeast gene, THP1, connecting transcription elongation with mitotic recombination.

    PubMed Central

    Gallardo, M; Aguilera, A

    2001-01-01

    Given the importance of the incidence of recombination in genomic instability, it is of great interest to know the elements or processes controlling recombination in mitosis. One such process is transcription, which has been shown to induce recombination in bacteria, yeast, and mammals. To further investigate the genetic control of the incidence of recombination and genetic instability and, in particular, its connection with transcription, we have undertaken a search for hyperrecombination mutants among a large number of strains deleted in genes of unknown function. We have identified a new gene, THP1 (YOL072w), whose deletion mutation strongly stimulates recombination between repeats. In addition, thp1 Delta impairs transcription, a defect that is particularly strong at the level of elongation through particular DNA sequences such as lacZ. The hyperrecombination phenotype of thp1 Delta cells is fully dependent on transcription elongation of the repeat construct. When transcription is impeded either by shutting off the promoter or by using a premature transcription terminator, hyperrecombination between repeats is abolished, providing new evidence that transcription-elongation impairment may be a source of recombinogenic substrates in mitosis. We show that Thp1p and two other proteins previously shown to control transcription-associated recombination, Hpr1p and Tho2p, act in the same "pathway" connecting transcription elongation with the incidence of mitotic recombination. PMID:11139493

  1. A gene-specific requirement for FACT during transcription is related to the chromatin organization of the transcribed region.

    PubMed

    Jimeno-González, Silvia; Gómez-Herreros, Fernando; Alepuz, Paula M; Chávez, Sebastián

    2006-12-01

    The FACT complex stimulates transcription elongation on nucleosomal templates. In vivo experiments also involve FACT in the reassembly of nucleosomes traversed by RNA polymerase II. Since several features of chromatin organization vary throughout the genome, we wondered whether FACT is equally required for all genes. We show in this study that the in vivo depletion of Spt16, one of the subunits of Saccharomyces cerevisiae FACT, strongly affects transcription of three genes, GAL1, PHO5, and Kluyveromyces lactis LAC4, which exhibit positioned nucleosomes at their transcribed regions. In contrast, showing a random nucleosome structure, YAT1 and Escherichia coli lacZ are only mildly influenced by Spt16 depletion. We also show that the effect of Spt16 depletion on GAL1 expression is suppressed by a histone mutation and that the insertion of a GAL1 fragment, which allows the positioning of two nucleosomes, at the 5' end of YAT1 makes the resulting transcription unit sensitive to Spt16 depletion. These results indicate that FACT requirement for transcription depends on the chromatin organization of the 5' end of the transcribed region. PMID:17000768

  2. High-Density Microarray-Mediated Gene Expression Profiling of Escherichia coli

    PubMed Central

    Wei, Yan; Lee, Jian-Ming; Richmond, Craig; Blattner, Frederick R.; Rafalski, J. Antoni; LaRossa, Robert A.

    2001-01-01

    A nearly complete collection of 4,290 Escherichia coli open reading frames was amplified and arrayed in high density on glass slides. To exploit this reagent, conditions for RNA isolation from E. coli cells, cDNA production with attendant fluorescent dye incorporation, DNA-DNA hybridization, and hybrid quantitation have been established. A brief isopropyl-?-d-thiogalactopyranoside (IPTG) treatment elevated lacZ, lacY, and lacA transcript content about 30-fold; in contrast, most other transcript titers remained unchanged. Distinct RNA expression patterns between E. coli cultures in the exponential and transitional phases of growth were catalogued, as were differences associated with culturing in minimal and rich media. The relative abundance of each transcript was estimated by using hybridization of a genomic DNA-derived, fluorescently labeled probe as a correction factor. This inventory provided a quantitative view of the steady-state level of each mRNA species. Genes the expression of which was detected by this method were enumerated, and results were compared with the current understanding of E. coli physiology. PMID:11133948

  3. Human gene therapy: Methods and materials. December 1985-April 1990 (A Bibliography from the Biobusiness data base). Report for December 1985-April 1990

    SciTech Connect

    Not Available

    1990-04-01

    This bibliography contains citations concerning the rapid evolution of technologies geared toward the genetic identification and treatment of diseases. Emphasis is placed upon development and application of genetic engineering techniques for the production of biopharmaceuticals. Other topics include the use of DNA (deoxyribonucleic acid) probes for gene isolation and disease marker identification, methods for replacing missing or defective genetic material, and mapping of the human genome. Governmental regulation, and moral and ethical implications are briefly reviewed. (Contains 299 citations fully indexed and including a title list.)

  4. Identification of a DNA segment that is necessary and sufficient for alpha-specific gene control in Saccharomyces cerevisiae: implications for regulation of alpha-specific and a-specific genes.

    PubMed Central

    Jarvis, E E; Hagen, D C; Sprague, G F

    1988-01-01

    STE3 mRNA is present only in Saccharomyces cerevisiae alpha cells, not in a or a/alpha cells, and the transcript level increases about fivefold when cells are treated with a-factor mating pheromone. Deletions in the 5' noncoding region of STE3 defined a 43-base-pair (bp) upstream activation sequence (UAS) that can impart both modes of regulation to a CYC1-lacZ fusion when substituted for the native CYC1 UAS. UAS activity required the alpha 1 product of MAT alpha, which is known to be required for transcription of alpha-specific genes. A chromosomal deletion that removed only 14 bp of the STE3 UAS reduced STE3 transcript levels 50- to 100-fold, indicating that the UAS is essential for expression. The STE3 UAS shares a 26-bp homology with the 5' noncoding sequences of the only other known alpha-specific genes, MF alpha 1 and MF alpha 2. We view the homology as having two components--a nearly palindromic 16-bp "P box" and an adjacent 10-bp "Q box." A synthetic STE3 P box was inactive as a UAS; a perfect palindrome P box was active in all three cell types. We propose that the P box is the binding site for a transcription activator, but that alpha 1 acting via the Q box is required for this activator to bind to the imperfect P boxes of alpha-specific genes. Versions of the P box are also found upstream of a-specific genes, within the binding sites of the repressor alpha 2 encoded by MAT alpha. Thus, the products of MAT alpha may render gene expression alpha or a-specific by controlling access of the same transcription activator to its binding site, the P box. Images PMID:3275872

  5. Gene therapy on the move

    PubMed Central

    Kaufmann, Kerstin B; Büning, Hildegard; Galy, Anne; Schambach, Axel; Grez, Manuel

    2013-01-01

    The first gene therapy clinical trials were initiated more than two decades ago. In the early days, gene therapy shared the fate of many experimental medicine approaches and was impeded by the occurrence of severe side effects in a few treated patients. The understanding of the molecular and cellular mechanisms leading to treatment- and/or vector-associated setbacks has resulted in the development of highly sophisticated gene transfer tools with improved safety and therapeutic efficacy. Employing these advanced tools, a series of Phase I/II trials were started in the past few years with excellent clinical results and no side effects reported so far. Moreover, highly efficient gene targeting strategies and site-directed gene editing technologies have been developed and applied clinically. With more than 1900 clinical trials to date, gene therapy has moved from a vision to clinical reality. This review focuses on the application of gene therapy for the correction of inherited diseases, the limitations and drawbacks encountered in some of the early clinical trials and the revival of gene therapy as a powerful treatment option for the correction of monogenic disorders. PMID:24106209

  6. Evidence of efficient stop codon readthrough in four mammalian genes

    E-print Network

    Loughran, Gary

    Stop codon readthrough is used extensively by viruses to expand their gene expression. Until recent discoveries in Drosophila, only a very limited number of readthrough cases in chromosomal genes had been reported. Analysis ...

  7. Gene family matters: expanding the HGNC resource.

    PubMed

    Daugherty, Louise C; Seal, Ruth L; Wright, Mathew W; Bruford, Elspeth A

    2012-01-01

    The HUGO Gene Nomenclature Committee (HGNC) assigns approved gene symbols to human loci. There are currently over 33,000 approved gene symbols, the majority of which represent protein-coding genes, but we also name other locus types such as non-coding RNAs, pseudogenes and phenotypic loci. Where relevant, the HGNC organise these genes into gene families and groups. The HGNC website http://www.genenames.org/ is an online repository of HGNC-approved gene nomenclature and associated resources for human genes, and includes links to genomic, proteomic and phenotypic information. In addition to this, we also have dedicated gene family web pages and are currently expanding and generating more of these pages using data curated by the HGNC and from information derived from external resources that focus on particular gene families. Here, we review our current online resources with a particular focus on our gene family data, using it to highlight our new Gene Symbol Report and gene family data downloads. PMID:23245209

  8. The Human Blue Opsin Promoter Directs Transgene Expression in Short-Wave Cones and Bipolar Cells in the Mouse Retina

    Microsoft Academic Search

    J. Chen; C. L. Tucker; B. Woodford; A. Szel; J. Lem; A. Gianella-Borradori; M. I. Simon; E. Bogenmann

    1994-01-01

    Transgenic mouse lines were generated using either 3.8 or 1.1 kb of 5' upstream flanking sequence from the human blue opsin gene fused to the lacZ or human growth hormone reporter gene. Mice were analyzed for appropriate cell-specific and developmental expression patterns. In 13 independently derived lines of animals, transgene expression was limited to photoreceptor and inner nuclear layer cells.

  9. Estrogenic activity of triterpene glycosides in yeast two-hybrid assay

    Microsoft Academic Search

    S. N. Kovalchuk; V. B. Kozhemyako; L. N. Atopkina; A. S. Silchenko; S. A. Avilov; V. I. Kalinin; V. A. Rasskazov; D. L. Aminin

    2006-01-01

    Estrogenic potency of six triterpene glycosides, Holothurin A, Holotoxin A1, Frondoside A, Cucumarioside A2-2 and Cauloside C, that are natural products and semi-synthesized Ginsenoside-Rh2, were examined with yeast two-hybrid system, including expressed genes of human estrogen receptor, hER?, the co-activator TIF2 and lacZ as a reporter gene. Only Ginsenoside-Rh2 exhibited significant moderate estrogenic activity in the concentration range of 10?7

  10. Effect of Subinhibitory Antibiotic Concentrations on Polysaccharide Intercellular Adhesin Expression in Biofilm-Forming Staphylococcus epidermidis

    Microsoft Academic Search

    SHWAN RACHID; KNUT OHLSEN; WOLFGANG WITTE; JORG HACKER; WILMA ZIEBUHR

    2000-01-01

    Biofilm production is an important step in the pathogenesis of Staphylococcus epidermidis polymer-associated infections and depends on the expression of the icaADBC operon leading to the synthesis of a polysaccharide intercellular adhesin. A chromosomally encoded reporter gene fusion between the ica promoter and the beta-galactosidase gene lacZ from Escherichia coli was constructed and used to investigate the influence of both

  11. Granulocytic sarcoma of the small intestine with CBF?\\/ MYH11 fusion gene: report of an aleukaemic case and review of the literature

    Microsoft Academic Search

    Sandra G Xavier; Evandro M Fagundes; Roc??o Hassan; Carlos Bacchi; Mônika Conchon; Daniel G Tabak; Nelson Spector; Ilana R Zalcberg

    2003-01-01

    Granulocytic sarcomas (GS) are rare extramedullary tumours composed of immature myeloid cells. Inversion of chromosome 16 [inv(16)] is a cytogenetic marker for M4Eo subtype of acute myeloid leukaemia (AML). The possibility of an association between the development of granulocytic sarcoma of the small intestine (GSSI) and the M4Eo subtype of AML was suggested in nine previous case reports.Here we report

  12. Final report: FASEB Summer Research Conference on ''Post-transcriptional control of gene expression: Effectors of mRNA decay'' [agenda and attendees list

    SciTech Connect

    Maquat, Lynne

    2002-12-01

    The goal of this meeting was to provide an interactive forum for scientists working on prokaryotic and eukaryotic mRNA decay. A special seminar presented by a leader in the field of mRNA decay in S. cerevisiae focused on what is known and what needs to be determined, not only for yeast but for other organisms. The large attendance (110 participants) reflects the awareness that mRNA decay is a key player in gene regulation in a way that is affected by the many steps that precede mRNA formation. Sessions were held on the following topics: mRNA transport and mRNP; multicomponent eukaryotic nucleases; nonsense-mediated mRNA decay and nonsense-associated altered splicing; Cis-acting sequences/Trans-acting factors of mRNA decay; translational accuracy; multicomponent bacterial nucleases; interplay between mRNA polyadenylation, translation and decay in prokaryotes and prokaryotic organelles; and RNA interference and other RNA mediators of gene expression. In addition to the talks and two poster sessions, there were three round tables: (1) Does translation occur in the nucleus? (2) Differences and similarities in the mechanisms of mRNA decay in different eukaryotes, and (3) RNA surveillance in bacteria?

  13. The Rox1 repressor of the Saccharomyces cerevisiae hypoxic genes is a specific DNA-binding protein with a high-mobility-group motif.

    PubMed Central

    Balasubramanian, B; Lowry, C V; Zitomer, R S

    1993-01-01

    The ROX1 gene encodes a repressor of the hypoxic functions of the yeast Saccharomyces cerevisiae. The DNA sequence of the gene was determined and found to encode a protein of 368 amino acids. The amino-terminal third of the protein contains a high-mobility-group motif characteristic of DNA-binding proteins. To determine whether the Rox1 repressor bound DNA, the gene was expressed in Escherichia coli cells as a fusion to the maltose-binding protein and this fusion was partially purified by amylose affinity chromatography. By using a gel retardation assay, both the fusion protein and Rox1 itself were found to bind specifically to a synthetic 32-bp DNA containing the hypoxic consensus sequence. We assessed the role of the general repressor Ssn6 in ANB1 repression. An ANB1-lacZ fusion was expressed constitutively in an ssn6 deletion strain, and deletion of the Rox1 binding sites in the ANB1 upstream region did not increase the level of derepression, suggesting that Ssn6 exerts its effect through Rox1. Finally, ROX1 was mapped to yeast chromosome XVI, near the ARO7-OSM2 locus. Images PMID:8413209

  14. Vulnerability genes or plasticity genes?

    PubMed Central

    Belsky, J; Jonassaint, C; Pluess, M; Stanton, M; Brummett, B; Williams, R

    2009-01-01

    The classic diathesis–stress framework, which views some individuals as particularly vulnerable to adversity, informs virtually all psychiatric research on behavior–gene–environment (G × E) interaction. An alternative framework of ‘differential susceptibility' is proposed, one which regards those most susceptible to adversity because of their genetic make up as simultaneously most likely to benefit from supportive or enriching experiences—or even just the absence of adversity. Recent G × E findings consistent with this perspective and involving monoamine oxidase-A, 5-HTTLPR (5-hydroxytryptamine-linked polymorphic region polymorphism) and dopamine receptor D4 (DRD4) are reviewed for illustrative purposes. Results considered suggest that putative ‘vulnerability genes' or ‘risk alleles' might, at times, be more appropriately conceptualized as ‘plasticity genes', because they seem to make individuals more susceptible to environmental influences—for better and for worse. PMID:19455150

  15. Klotho gene delivery suppresses Nox2 expression and attenuates oxidative stress in rat aortic smooth muscle cells via the cAMP-PKA pathway.

    PubMed

    Wang, Yuhong; Kuro-o, Makoto; Sun, Zhongjie

    2012-06-01

    Klotho is a recently discovered anti-aging gene. The purpose of this study was to investigate whether klotho gene transfer attenuates superoxide production and oxidative stress in rat aorta smooth muscle (RASM) cells. RASM cells were transfected with AAV plasmids carrying mouse klotho full-length cDNA (mKL) or LacZ as a control. Klotho gene transfer increased klotho expression in RASM cells. Notably, klotho gene expression decreased Nox2 NADPH oxidase protein expression but did not affect Nox2 mRNA expression, suggesting that the inhibition may occur at the posttranscriptional level. Klotho gene transfer decreased intracellular superoxide production and oxidative stress in RASM cells. Klotho gene expression also significantly attenuated the angiotensin II (AngII)-induced superoxide production, oxidative damage, and apoptosis. Interestingly, klotho gene delivery dose dependently increased the intracellular cAMP level and PKA activity in RASM cells. Rp-cAMP, a competitive inhibitor of cAMP, abolished the klotho-induced increase in PKA activity, indicating that klotho activated PKA via cAMP. Notably, inhibition of cAMP-dependent PKA activity by RP-cAMP abolished klotho-induced inhibition of Nox2 protein expression, suggesting an important role of cAMP-dependent PKA in this process. This finding revealed a previously unidentified role of klotho in regulating Nox2 protein expression in RASM cells. Klotho not only downregulated Nox2 protein expression and intracellular superoxide production but also attenuated AngII-induced superoxide production, oxidative damage, and apoptosis. The klotho-induced suppression of Nox2 protein expression may be mediated by the cAMP-PKA pathway. PMID:22260450

  16. A systematic RNAi screen for longevity genes in C. elegans

    Microsoft Academic Search

    Benjamin Hamilton; Yuqing Dong; Mami Shindo; Wenyu Liu; Ian Odell; Gary Ruvkun; Siu Sylvia Lee

    2005-01-01

    We report here the first genome-wide functional genomic screen for longevity genes. We systematically surveyed Caenorhabditis elegans genes using large-scale RNA interference (RNAi), and found that RNAi inactivation of 89 genes extend C. elegans lifespan. Components of the daf-2\\/insulin-like signaling pathway are recovered, as well as genes that regulate metabolism, signal transduction, protein turnover, and gene expression. Many of these

  17. Oxygen, nitrate, and molybdenum regulation of dmsABC gene expression in Escherichia coli.

    PubMed Central

    Cotter, P A; Gunsalus, R P

    1989-01-01

    Escherichia coli can respire anaerobically using either trimethylamine-N-oxide (TMAO) or dimethyl sulfoxide (DMSO) as the terminal electron acceptor for oxidative phosphorylation. To determine whether the regulation of the dmsABC genes, which encode a membrane-associated TMAO/DMSO reductase, are transcriptionally controlled in response to the availability of alternate electron acceptors, we constructed an operon fusion between the dmsA gene, along with its associated regulatory region, and lacZ+. Expression of dmsA'-lacZ was stimulated 65-fold by anaerobiosis versus aerobiosis, while nitrate caused a 12-fold repression. Its expression, however, was unaffected by the presence of the alternate electron acceptors DMSO, TMAO, and fumarate. Anaerobic induction of dmsA'-lacZ was defective in an fnr mutant, thus establishing that Fnr is responsible for anaerobic activation of dmsABC. Repression of dmsA'-lacZ expression by nitrate was independent of oxygen and was shown to be mediated by the products of two genes, narL (frdR2) and narX. dmsA'-lacZ expression was also altered in chlD strains that are defective in molybdenum transport but not in chlA and chlE strains that are defective in molybdopterin cofactor biosynthesis, thus establishing that the molybdenum ion but not the ability to form a functional cofactor is required for regulation. Molybdenum was required both for complete induction of dmsA'-lacZ expression during anaerobic growth and for complete repression of dmsA'-lacZ by nitrate. Additionally, expression of dmsABC varied depending on the carbon source. Expression was highest when cells were grown on sorbitol. PMID:2544558

  18. A chloride-inducible gene expression cassette and its use in induced lysis of Lactococcus lactis.

    PubMed

    Sanders, J W; Venema, G; Kok, J

    1997-12-01

    A chloride-inducible promoter previously isolated from the chromosome of Lactococcus lactis (J. W. Sanders, G. Venema, J. Kok, and K. Leenhouts, Mol. Gen. Genet., in press) was exploited for the inducible expression of homologous and heterologous genes. An expression cassette consisting of the positive-regulator gene gadR, the chloride-inducible promoter Pgad, and the translation initiation signals of gadC was amplified by PCR. The cassette was cloned upstream of Escherichia coli lacZ, the holin-lysin cassette (lytPR) of the lactococcal bacteriophage r1t, and the autolysin gene of L. lactis, acmA. Basal activity of Pgad resulted in a low level of expression of all three proteins. Growth in the presence of 0.5 M NaCl of a strain containing the gadC::lacZ fusion resulted in a 1,500-fold increase of beta-galactosidase activity. The background activity levels of LytPR and AcmA had no deleterious effects on cell growth, but induction of lysin expression by addition of 0.5 M NaCl resulted in inhibition of growth. Lysis was monitored by following the release of the cytoplasmic marker enzyme PepX. Released PepX activity was maximal at 1 day after induction of lytPR expression with 0.1 M NaCl. Induction of acmA expression resulted in slower release of PepX from the cells. The presence of the inducing agent NaCl resulted in the stabilization of osmotically fragile cells. PMID:9406408

  19. Down syndrome and the genes of human chromosome 21: current knowledge and future potentials. Report on the Expert workshop on the biology of chromosome 21 genes: towards gene-phenotype correlations in Down syndrome. Washington D.C., September 28-October 1, 2007.

    PubMed

    Pritchard, M; Reeves, R H; Dierssen, M; Patterson, D; Gardiner, K J

    2008-01-01

    Down syndrome (DS), trisomy of human chromosome 21, is the most common genetic cause of intellectual disability. With an incidence in some countries as high as one in approximately 700 live births, and a complex, extensive and variably severe phenotype, Down syndrome is a significant medical and social challenge. In recent years, there has been a rapid increase in information on the functions of the genes of human chromosome 21, as well as in techniques and resources for their analysis. A recent workshop brought together experts on the molecular biology of Down syndrome and chromosome 21 with interested researchers in other fields to discuss advances and potentials for generating gene-phenotype correlations. An additional goal of the workshop was to work towards identification of targets for therapeutics that will correct features of DS. A knowledge-based approach to therapeutics also requires the correlation of chromosome 21 gene function with phenotypic features. PMID:18544929

  20. Brains, genes, and primates.

    PubMed

    Izpisua Belmonte, Juan Carlos; Callaway, Edward M; Caddick, Sarah J; Churchland, Patricia; Feng, Guoping; Homanics, Gregg E; Lee, Kuo-Fen; Leopold, David A; Miller, Cory T; Mitchell, Jude F; Mitalipov, Shoukhrat; Moutri, Alysson R; Movshon, J Anthony; Okano, Hideyuki; Reynolds, John H; Ringach, Dario; Sejnowski, Terrence J; Silva, Afonso C; Strick, Peter L; Wu, Jun; Zhang, Feng

    2015-05-01

    One of the great strengths of the mouse model is the wide array of genetic tools that have been developed. Striking examples include methods for directed modification of the genome, and for regulated expression or inactivation of genes. Within neuroscience, it is now routine to express reporter genes, neuronal activity indicators, and opsins in specific neuronal types in the mouse. However, there are considerable anatomical, physiological, cognitive, and behavioral differences between the mouse and the human that, in some areas of inquiry, limit the degree to which insights derived from the mouse can be applied to understanding human neurobiology. Several recent advances have now brought into reach the goal of applying these tools to understanding the primate brain. Here we describe these advances, consider their potential to advance our understanding of the human brain and brain disorders, discuss bioethical considerations, and describe what will be needed to move forward. PMID:25950631

  1. Gene-gene, gene-environment, gene-nutrient interactions and single nucleotide polymorphisms of inflammatory cytokines.

    PubMed

    Nadeem, Amina; Mumtaz, Sadaf; Naveed, Abdul Khaliq; Aslam, Muhammad; Siddiqui, Arif; Lodhi, Ghulam Mustafa; Ahmad, Tausif

    2015-05-15

    Inflammation plays a significant role in the etiology of type 2 diabetes mellitus (T2DM). The rise in the pro-inflammatory cytokines is the essential step in glucotoxicity and lipotoxicity induced mitochondrial injury, oxidative stress and beta cell apoptosis in T2DM. Among the recognized markers are interleukin (IL)-6, IL-1, IL-10, IL-18, tissue necrosis factor-alpha (TNF-?), C-reactive protein, resistin, adiponectin, tissue plasminogen activator, fibrinogen and heptoglobins. Diabetes mellitus has firm genetic and very strong environmental influence; exhibiting a polygenic mode of inheritance. Many single nucleotide polymorphisms (SNPs) in various genes including those of pro and anti-inflammatory cytokines have been reported as a risk for T2DM. Not all the SNPs have been confirmed by unifying results in different studies and wide variations have been reported in various ethnic groups. The inter-ethnic variations can be explained by the fact that gene expression may be regulated by gene-gene, gene-environment and gene-nutrient interactions. This review highlights the impact of these interactions on determining the role of single nucleotide polymorphism of IL-6, TNF-?, resistin and adiponectin in pathogenesis of T2DM. PMID:25987962

  2. Repetitive sequence environment distinguishes housekeeping genes

    PubMed Central

    Eller, C. Daniel; Regelson, Moira; Merriman, Barry; Nelson, Stan; Horvath, Steve; Marahrens, York

    2007-01-01

    Housekeeping genes are expressed across a wide variety of tissues. Since repetitive sequences have been reported to influence the expression of individual genes, we employed a novel approach to determine whether housekeeping genes can be distinguished from tissue-specific genes their repetitive sequence context. We show that Alu elements are more highly concentrated around housekeeping genes while various longer (>400-bp) repetitive sequences ("repeats"), including Long Interspersed Nuclear Element 1 (LINE-1) elements, are excluded from these regions. We further show that isochore membership does not distinguish housekeeping genes from tissue-specific genes and that repetitive sequence environment distinguishes housekeeping genes from tissue-specific genes in every isochore. The distinct repetitive sequence environment, in combination with other previously published sequence properties of housekeeping genes, were used to develop a method of predicting housekeeping genes on the basis of DNA sequence alone. Using expression across tissue types as a measure of success, we demonstrate that repetitive sequence environment is by far the most important sequence feature identified to date for distinguishing housekeeping genes. PMID:17141428

  3. Gene fusion in Helicobacter pylori: making the ends meet.

    PubMed

    Sakharkar, Kishore R; Sakharkar, Meena K; Chow, Vincent T K

    2006-01-01

    Fusion genes have been reported as a means of enabling the development of novel or enhanced functions. In this report, we analyzed fusion genes in the genomes of two Helicobacter pylori strains (26695 and J99) and identified 32 fusion genes that are present as neighbours in one strain (components) and are fused in the second (composite), and vice-versa. The mechanism for each case of gene fusion is explored. 28 out of 32 genes identified as fusion products in this analysis were reported as essential genes in the previously documented transposon mutagenesis of H. pylori strain G27. This observation suggests the potential of the products of fusion genes as putative microbial drug targets. These results underscore the utility of bacterial genomic sequence comparisons for understanding gene evolution and for in silico drug target identification in the post-genomic era. PMID:16541196

  4. Evolution of the Aflatoxin Gene Cluster

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Why Aspergillus species produce aflatoxin remains an unsolved question. In this report, we suggest that evolution of the aflatoxin biosynthesis gene cluster has been a multistep process. More than 300 million years ago, a primordial cluster of genes allowed production of anthraquinones that may ha...

  5. Molecular analysis of the recA gene and SOS box of the purple non-sulfur bacterium Rhodopseudomonas palustris no. 7.

    PubMed

    Dumay, V; Inui, M; Yukawa, H

    1999-05-01

    The recA gene of the purple non-sulfur bacterium Rhodopseudomonas palustris no. 7 was isolated by a PCR-based method and sequenced. The complete nucleotide sequence consists of 1089 bp encoding a polypeptide of 363 amino acids which is most closely related to the RecA proteins from species of Rhizobiaceae and Rhodospirillaceae. A recA-deficient strain of R. palustris no. 7 was obtained by gene replacement. As expected, this strain exhibited increased sensitivity to DNA-damaging agents. Transcriptional fusions of the recA promoter region to lacZ confirmed that the R. palustris no. 7 recA gene is inducible by DNA damage. Primer extension analysis of recA mRNA located the recA gene transcriptional start. A sequential deletion of the fusion plasmid was used to delimit the promoter region of the recA gene. A gel mobility shift assay demonstrated that a DNA-protein complex is formed at this promoter region. This DNA-protein complex was not formed when protein extracts from cells treated with DNA-damaging agents were used, indicating that the binding protein is a repressor. Comparison of the minimal R. palustris no. 7 recA promoter region with the recA promoter sequences from other alpha-Proteobacteria revealed the presence of the conserved sequence GAACA-N6-G(A/T)AC. Site-directed mutations that changed this consensus sequence abolished the DNA-damage-mediated expression of the R. palustris recA gene, confirming that this sequence is the SOS box of R. palustris and probably plays the same role in other alpha-Proteobacteria. PMID:10376844

  6. Facile high-throughput forward chemical genetic screening by in situ monitoring of glucuronidase-based reporter gene expression in Arabidopsis thaliana

    PubMed Central

    Halder, Vivek; Kombrink, Erich

    2015-01-01

    The use of biologically active small molecules to perturb biological functions holds enormous potential for investigating complex signaling networks. However, in contrast to animal systems, the search for and application of chemical tools for basic discovery in the plant sciences, generally referred to as “chemical genetics,” has only recently gained momentum. In addition to cultured cells, the well-characterized, small-sized model plant Arabidopsis thaliana is suitable for cultivation in microplates, which allows employing diverse cell- or phenotype-based chemical screens. In such screens, a chemical's bioactivity is typically assessed either through scoring its impact on morphological traits or quantifying molecular attributes such as enzyme or reporter activities. Here, we describe a facile forward chemical screening methodology for intact Arabidopsis seedlings harboring the ?-glucuronidase (GUS) reporter by directly quantifying GUS activity in situ with 4-methylumbelliferyl-?-D-glucuronide (4-MUG) as substrate. The quantitative nature of this screening assay has an obvious advantage over the also convenient histochemical GUS staining method, as it allows application of statistical procedures and unbiased hit selection based on threshold values as well as distinction between compounds with strong or weak bioactivity. At the same time, the in situ bioassay is very convenient requiring less effort and time for sample handling in comparison to the conventional quantitative in vitro GUS assay using 4-MUG, as validated with several Arabidopsis lines harboring different GUS reporter constructs. To demonstrate that the developed assays is particularly suitable for large-scale screening projects, we performed a pilot screen for chemical activators or inhibitors of salicylic acid-mediated defense signaling using the Arabidopsis PR1p::GUS line. Importantly, the screening methodology provided here can be adopted for any inducible GUS reporter line. PMID:25688251

  7. Overgrowth and trisomy 15q26.1-qter including the IGF1 receptor gene: report of two families and review of the literature

    Microsoft Academic Search

    Laurence Faivre; Philippe Gosset; Valérie Cormier-Daire; Sylvie Odent; Jeanne Amiel; Irina Giurgea; Marie-Cécile Nassogne; Laurent Pasquier; Arnold Munnich; Serge Romana; Marguerite Prieur; Michel Vekemans; Marie-Christine de Blois; Catherine Turleau

    2002-01-01

    Overgrowth is rarely associated with chromosomal imbalances. Here we report on four children from two unrelated families presenting with overgrowth and a terminal duplication of the long arm of chromosome 15 diagnosed using cytogenetic and FISH studies. In both cases, chromosome analysis of the parents showed a balanced translocation involving 15q26.1-qter. Molecular and cytogenetic studies showed three copies of the

  8. A novel mutation of the HNF1B gene associated with hypoplastic glomerulocystic kidney disease and neonatal renal failure: a case report and mutation update.

    PubMed

    Alvelos, Maria Inês; Rodrigues, Magda; Lobo, Luísa; Medeira, Ana; Sousa, Ana Berta; Simão, Carla; Lemos, Manuel Carlos

    2015-02-01

    Hepatocyte nuclear factor 1 beta (HNF1B) plays an important role in embryonic development, namely in the kidney, pancreas, liver, genital tract, and gut. Heterozygous germline mutations of HNF1B are associated with the renal cysts and diabetes syndrome (RCAD). Affected individuals may present a variety of renal developmental abnormalities and/or maturity-onset diabetes of the young (MODY). A Portuguese 19-month-old male infant was evaluated due to hypoplastic glomerulocystic kidney disease and renal dysfunction diagnosed in the neonatal period that progressed to stage 5 chronic renal disease during the first year of life. His mother was diagnosed with a solitary hypoplastic microcystic left kidney at age 20, with stage 2 chronic renal disease established at age 35, and presented bicornuate uterus, pancreatic atrophy, and gestational diabetes. DNA sequence analysis of HNF1B revealed a novel germline frameshift insertion (c.110_111insC or c.110dupC) in both the child and the mother. A review of the literature revealed a total of 106 different HNF1B mutations, in 236 mutation-positive families, comprising gross deletions (34%), missense mutations (31%), frameshift deletions or insertions (15%), nonsense mutations (11%), and splice-site mutations (8%). The study of this family with an unusual presentation of hypoplastic glomerulocystic kidney disease with neonatal renal dysfunction identified a previously unreported mutation of the HNF1B gene, thereby expanding the spectrum of known mutations associated with renal developmental disorders. PMID:25700310

  9. Gene Ontology Driven Classification of Gene

    E-print Network

    Spang, Rainer

    expression patterns Gene Ontology · Structure knowledge about genes · Directed acyclic graph · Represents · No biological knowledge #12;Introduction 23-Jul-02 5 / 17Claudio Lottaz: GO driven classification of gene knowledge on · Molecular function · Bilogical process · Cellular component · Genes are annotated to nodes

  10. Gene Expression Mural: Visualizing Gene Expression Databases

    E-print Network

    Gene Expression Mural: Visualizing Gene Expression Databases Mathew Clement, Margaret Ellis, Josh@cs.vt.edu Abstract The Gene Expression Mural is a tool designed for managing the vast amount of information produced quantifying the expression level of gene sequences in a number of conditions [1]. Software tools integrating

  11. Evidence That the Stilbene-Derived Optical Brightener M2R Enhances Autographa californicaM Nucleopolyhedrovirus Infection of Trichoplusia niand Heliothis virescensby Preventing Sloughing of Infected Midgut Epithelial Cells

    Microsoft Academic Search

    Jan O. Washburn; Bruce A. Kirkpatrick; Eric Haas-Stapleton; Loy E. Volkman

    1998-01-01

    Previous reports have shown that the stilbene-derived optical brightener M2R increases mortality and hastens death of larval lepidopterans orally challenged with baculoviruses. The mechanism of viral enhancement, however, has not been elucidated. We used a reporter gene recombinant ofAutographa californicaMNPV (AcMNPV-hsp70\\/lacZ) to investigate the enhancing effects of M2R on pathogenesis, mortality and time to death in larvae ofTrichoplusia ni,andHeliothis virescensinoculated

  12. Sperm-mediated gene transfer

    Microsoft Academic Search

    Anthony W. S. Chan; C. Marc Luetjens; Gerald P. Schatten

    2000-01-01

    Since 1989, a new method for the production of transgenic animals has been available, namely sperm- mediated gene transfer (SMGT), based on the intrinsic ability of sperm cells to bind and internalise exogenous DNA molecules and to transfer them into the oocyte at fertilisation. We first described the SMGT procedure in a small animal model, with high efficiency reported in

  13. Random forests-based differential analysis of gene sets for gene expression data.

    PubMed

    Hsueh, Huey-Miin; Zhou, Da-Wei; Tsai, Chen-An

    2013-04-10

    In DNA microarray studies, gene-set analysis (GSA) has become the focus of gene expression data analysis. GSA utilizes the gene expression profiles of functionally related gene sets in Gene Ontology (GO) categories or priori-defined biological classes to assess the significance of gene sets associated with clinical outcomes or phenotypes. Many statistical approaches have been proposed to determine whether such functionally related gene sets express differentially (enrichment and/or deletion) in variations of phenotypes. However, little attention has been given to the discriminatory power of gene sets and classification of patients. In this study, we propose a method of gene set analysis, in which gene sets are used to develop classifications of patients based on the Random Forest (RF) algorithm. The corresponding empirical p-value of an observed out-of-bag (OOB) error rate of the classifier is introduced to identify differentially expressed gene sets using an adequate resampling method. In addition, we discuss the impacts and correlations of genes within each gene set based on the measures of variable importance in the RF algorithm. Significant classifications are reported and visualized together with the underlying gene sets and their contribution to the phenotypes of interest. Numerical studies using both synthesized data and a series of publicly available gene expression data sets are conducted to evaluate the performance of the proposed methods. Compared with other hypothesis testing approaches, our proposed methods are reliable and successful in identifying enriched gene sets and in discovering the contributions of genes within a gene set. The classification results of identified gene sets can provide an valuable alternative to gene set testing to reveal the unknown, biologically relevant classes of samples or patients. In summary, our proposed method allows one to simultaneously assess the discriminatory ability of gene sets and the importance of genes for interpretation of data in complex biological systems. The classifications of biologically defined gene sets can reveal the underlying interactions of gene sets associated with the phenotypes, and provide an insightful complement to conventional gene set analyses. PMID:23219997

  14. Gorgeous mosaic of mitochondrial genes created by horizontal transfer and gene conversion

    PubMed Central

    Hao, Weilong; Richardson, Aaron O.; Zheng, Yihong; Palmer, Jeffrey D.

    2010-01-01

    The best known outcome of horizontal gene transfer (HGT) is the introduction of novel genes, but other outcomes have been described. When a transferred gene has a homolog in the recipient genome, the native gene may be functionally replaced (and subsequently lost) or partially overwritten by gene conversion with transiently present foreign DNA. Here we report the discovery, in two lineages of plant mitochondrial genes, of novel gene combinations that arose by conversion between coresident native and foreign homologs. These lineages have undergone intricate conversion between native and foreign copies, with conversion occurring repeatedly and differentially over the course of speciation, leading to radiations of mosaic genes involved in respiration and intron splicing. Based on these findings, we develop a model—the duplicative HGT and differential gene conversion model—that integrates HGT and ongoing gene conversion in the context of speciation. Finally, we show that one of these HGT-driven gene-conversional radiations followed two additional types of conversional chimerism, namely, intramitochondrial retroprocessing and interorganellar gene conversion across the 2 billion year divide between mitochondria and chloroplasts. These findings expand our appreciation of HGT and gene conversion as creative evolutionary forces, establish plant mitochondria as a premiere system for studying the evolutionary dynamics of HGT and its genetic reverberations, and recommend careful examination of bacterial and other genomes for similar, likely overlooked phenomena. PMID:21115831

  15. Identification of Development and Pathogenicity Related Gene in Botrytis cinerea via Digital Gene Expression Profile

    PubMed Central

    Zhao, Bin; Si, He Long; Sun, Zhi Ying; Xu, Zheng; Chen, Zhan; Zhang, Jin lin; Xing, Ji Hong; Dong, Jin Gao

    2015-01-01

    Background: Botrytis cinerea, a haploid Euascomycete fungus that infects numerous crops, has been used as a model system for studying molecular phytopathology. Botrytis cinerea adopts various modes of infection, which are mediated by a number of pathogenicity and virulence-related genes. Many of these genes have not been reported previously. Objectives: This study aimed to investigate development and pathogenicity-related genes between a novel nonpathogenic mutant and the Wild Type (WT) in B. cinerea. Materials and Methods: Digital Gene Expression (DGE) tag profiling can reveal novel genes that may be involved in development and pathogenicity of plant pathogen. A large volume of B. cinerea tag-seq was generated to identify differential expressed genes by the Illumina DGE tag pro?ling technology. Results: A total of 4,182,944 and 4,182,021 clean tags were obtained from the WT and a nonpathogenic mutant stain (BCt89), respectively, and 10,410 differentially expressed genes were identified. In addition, 84 genes were expressed in the WT only while 34 genes were expressed in the mutant only. A total of 664 differentially expressed genes were involved in 91 Kyoto Encyclopedia of Genes and Genome pathways, including signaling and metabolic pathways. Conclusions: Expression levels of 1,426 genes were significantly up-regulated in the mutant compared to WT. Furthermore, 301 genes were down-regulated with False Discovery Rates (FDR) of < 0.001 and absolute value of log2 Ratio of ? 1. PMID:26034553

  16. Origin and Ascendancy of a Chimeric Fusion Gene: The ?/?-Globin Gene of Paenungulate Mammals

    PubMed Central

    Opazo, Juan C.; Sloan, Angela M.; Campbell, Kevin L.

    2009-01-01

    The ?-globin gene (HBD) of eutherian mammals exhibits a propensity for recombinational exchange with the closely linked ?-globin gene (HBB) and has been independently converted by the HBB gene in multiple lineages. Here we report the presence of a chimeric ?/? fusion gene in the African elephant (Loxodonta africana) that was created by unequal crossing-over between misaligned HBD and HBB paralogs. The recombinant chromosome that harbors the ?/? fusion gene in elephants is structurally similar to the “anti-Lepore” duplication mutant of humans (the reciprocal exchange product of the hemoglobin Lepore deletion mutant). However, the situation in the African elephant is unique in that the chimeric ?/? fusion gene supplanted the parental HBB gene and is therefore solely responsible for synthesizing the ?-chain subunits of adult hemoglobin. A phylogenetic survey of ?-like globin genes in afrotherian and xenarthran mammals revealed that the origin of the chimeric ?/? fusion gene and the concomitant inactivation of the HBB gene predated the radiation of “Paenungulata,” a clade of afrotherian mammals that includes three orders: Proboscidea (elephants), Sirenia (dugongs and manatees), and Hyracoidea (hyraxes). The reduced fitness of the human Hb Lepore deletion mutant helps to explain why independently derived ?/? fusion genes (which occur on an anti-Lepore chromosome) have been fixed in a number of mammalian lineages, whereas the reciprocal ?/? fusion gene (which occurs on a Lepore chromosome) has yet to be documented in any nonhuman mammal. This illustrates how the evolutionary fates of chimeric fusion genes can be strongly influenced by their recombinational mode of origin. PMID:19332641

  17. Identification of RpoS (?S)-Regulated Genes in Salmonella enterica Serovar Typhimurium

    PubMed Central

    Ibanez-Ruiz, Magdalena; Robbe-Saule, Véronique; Hermant, Daniel; Labrude, Séverine; Norel, Françoise

    2000-01-01

    The rpoS gene encodes the alternative sigma factor ?S (RpoS) and is required for survival of bacteria under starvation and stress conditions. It is also essential for Salmonella virulence in mice. Most work on the RpoS regulon has been in the closely related enterobacterial species Escherichia coli. To characterize the RpoS regulon in Salmonella, we isolated 38 unique RpoS-activated lacZ gene fusions from a bank of Salmonella enterica serovar Typhimurium mutants harboring random Tn5B21 mutations. Dependence on RpoS varied from 3-fold to over 95-fold, and all gene fusions isolated were regulated by growth phase. The identities of 21 RpoS-dependent fusions were determined by DNA sequence analysis. Seven of the fusions mapped to DNA regions in Salmonella serovar Typhimurium that do not match any known E. coli sequence, suggesting that the composition of the RpoS regulon differs markedly in the two species. The other 14 fusions mapped to 13 DNA regions very similar to E. coli sequences. None of the insertion mutations in DNA regions common to both species appeared to affect Salmonella virulence in BALB/c mice. Of these, only three (otsA, katE, and poxB) are located in known members of the RpoS regulon. Ten insertions mapped in nine open reading frames of unknown function (yciF, yehY, yhjY, yncC, yjgB, yahO, ygaU, ycgB, and yeaG) appear to be novel members of the RpoS regulon. One insertion, that in mutant C52::H87, was in the noncoding region upstream from ogt, encoding a O6-methylguanine DNA methyltransferase involved in repairing alkylation damage in DNA. The ogt coding sequence is very similar to the E. coli homolog, but the ogt 5? flanking regions were found to be markedly different in the two species, suggesting genetic rearrangements. Using primer extension assays, a specific ogt mRNA start site was detected in RNAs of the Salmonella serovar Typhimurium wild-type strains C52 and SL1344 but not in RNAs of the mutant strains C52K (rpoS), SL1344K (rpoS), and C52::H87. In mutant C52::H87, Tn5B21 is inserted at the ogt mRNA start site, with lacZ presumably transcribed from the identified RpoS-regulated promoter. These results indicate that ogt gene expression in Salmonella is regulated by RpoS in stationary phase of growth in rich medium, a finding that suggests a novel role for RpoS in DNA repair functions. PMID:11004173

  18. Progress in Gene Therapy for Prostate Cancer

    PubMed Central

    Ahmed, Kamran A.; Davis, Brian J.; Wilson, Torrence M.; Wiseman, Gregory A.; Federspiel, Mark J.; Morris, John C.

    2012-01-01

    Gene therapy has held promise to correct various disease processes. Prostate cancer represents the second leading cause of cancer death in American men. A number of clinical trials involving gene therapy for the treatment of prostate cancer have been reported. The ability to efficiently transduce tumors with effective levels of therapeutic genes has been identified as a fundamental barrier to effective cancer gene therapy. The approach utilizing gene therapy in prostate cancer patients at our institution attempts to address this deficiency. The sodium-iodide symporter (NIS) is responsible for the ability of the thyroid gland to transport and concentrate iodide. The characteristics of the NIS gene suggest that it could represent an ideal therapeutic gene for cancer therapy. Published results from Mayo Clinic researchers have indicated several important successes with the use of the NIS gene and prostate gene therapy. Studies have demonstrated that transfer of the human NIS gene into prostate cancer using adenovirus vectors in vitro and in vivo results in efficient uptake of radioactive iodine and significant tumor growth delay with prolongation of survival. Preclinical successes have culminated in the opening of a phase I trial for patients with advanced prostate disease which is currently accruing patients. Further study will reveal the clinical promise of NIS gene therapy in the treatment of prostate as well as other malignancies. PMID:23181221

  19. Gene Switches

    NSDL National Science Digital Library

    2013-07-30

    In this activity, learners explore how genetic switches function and the role of genetic switches in the process of evolution. To make these concepts less abstract and more understandable, learners first view a series of video clips and animations from the HHMI DVD (or online) "Evolution: Constant Change and Common Threads." Then, learners construct a model of a gene switch using craft materials or FridgiGears (magnetic gears). This activity can be done as a demonstration, a student inquiry activity, or a combination of the two.

  20. Initial report of a genome search for the affective disorder predisposition gene in the Old Order Amish pedigrees: Chromosomes 1 and 11

    SciTech Connect

    Gerhard, D.S.; Bland, S.D. [Washington Univ. School of Medicine, St. Louis, MO (United States); LaBuda, M.C. [NIDA Addiction Research Center, Baltimore, MD (United States)] [and others

    1994-12-15

    Family data have suggested that some forms of major affective disorder are genetic. Certain of the Old Order Amish pedigrees have a familial form of the disease. In this report we present the results of genetic analyses under autosomal dominant mode of transmission with reduce penetrance and three different disease hierarchies. The pedigrees were genotyped with 28 markers from chromosome 1 and 23 markers from chromosomes 11. None of the markers result in a significantly positive lod score. 49 refs., 1 fig., 4 tabs.

  1. Fever, genes, and epilepsy.

    PubMed

    Baulac, Stéphanie; Gourfinkel-An, Isabelle; Nabbout, Rima; Huberfeld, Gilles; Serratosa, Jose; Leguern, Eric; Baulac, Michel

    2004-07-01

    About 13% of patients with epilepsy have a history of febrile seizures (FS). Studies of familial forms suggest a genetic component to the epidemiological link. Indeed, in certain monogenic forms of FS, for which several loci have been reported, some patients develop epilepsy with a higher risk than in the general population. Patients with generalised epilepsy with febrile seizures plus (GEFS+) can have typical and isolated FS, FS lasting more beyond age 6 years, and subsequent afebrile (typically generalised) seizures. Mutations associated with GEFS+ were identified in genes for subunits of the voltage-gated sodium channel and the gamma2 subunit of the ligand-gated GABAA receptor. Screening for these genes in patients with severe myoclonic epilepsy in infancy showed de novo mutations of the alpha1 subunit of the voltage-gated sodium channel. Antecedent FS are commonly observed in temporal-lobe epilepsy (TLE). In sporadic mesial TLE-characterised by the sequence of complex FS in childhood, hippocampal sclerosis, and refractory temporal-lobe seizures-association studies suggested the role of several susceptibility genes. Work on some large pedigrees also suggests that FS and temporal-lobe seizures may have a common genetic basis, whether hippocampus sclerosis is present or not. The molecular defects identified in the genetic associations of FS and epileptic seizures are very attractive models to aid our understanding of epileptogenesis and susceptibility to seizure-provoking factors, especially fever. PMID:15207799

  2. Gene transfer mediated by alpha2-macroglobulin.

    PubMed Central

    Schneider, H; Huse, K; Birkenmeier, G; Otto, A; Scholz, G H

    1996-01-01

    alpha2-Macroglobulin covalently linked to poly(L)-lysine can be used as a vehicle for receptor-mediated gene transfer. This modified alpha2-macroglobulin maintains its ability to bind to the alpha2-macroglobulin receptor, and was shown to introduce a luciferase reporter gene plasmid into HepG2 human hepatoma cells in vitro. The alpha2-macroglobulin receptor is a very large and multifunctional cell surface receptor, whose rapid and efficient internalization rate makes it attractive for gene therapy, e.g. for hepatic gene targeting via injection into the portal vein. PMID:8871570

  3. Cytological Features of a Variant NUT Midline Carcinoma of the Lung Harboring the NSD3-NUT Fusion Gene: A Case Report and Literature Review

    PubMed Central

    Kuroda, Shiho; Kurita, Akira; Muraki, Mari; Aoshima, Yoichiro; Tanioka, Fumihiko

    2015-01-01

    Background. Nuclear protein in testis (NUT) midline carcinoma (NMC) is a very rare and aggressive malignancy. In more than two-thirds of these NMC cases, a fusion between NUT and BRD4 or BRD3 has been documented; other variants are rare. The cytology of NMC itself has been sparsely documented and that of variant NMC has never been reported. Case Presentation. A 36-year-old woman was admitted because of a rapidly progressing lung tumor with metastases to the breast and bone. We recently reported this patient as the first case of a variant NMC of the lung harboring an NSD3-NUT fusion, based on immunohistochemical and genetic analyses. Cytological material was available for the present review. A highly cellular smear contained a predominantly noncohesive pattern of monomorphic cells with diameters 2–2.5 times greater than those of small lymphocytes, with a round-to-oval nucleus, slightly irregular nuclear contours, variably prominent nucleoli, scant cytoplasm, and identifiable mitotic figures. Foci of stratification and overt pearl formation, including a dyskeratocyte, were occasionally observed. The necrotic background contained naked nuclei, karyorrhectic debris, apoptotic cells, and macrophages phagocytizing karyorrhectic debris; nuclear crushing was noted. Conclusion. The cytological features of a variant NMC of the lung are described for the first time. PMID:25685583

  4. Cytological Features of a Variant NUT Midline Carcinoma of the Lung Harboring the NSD3-NUT Fusion Gene: A Case Report and Literature Review.

    PubMed

    Kuroda, Shiho; Suzuki, Shioto; Kurita, Akira; Muraki, Mari; Aoshima, Yoichiro; Tanioka, Fumihiko; Sugimura, Haruhiko

    2015-01-01

    Background. Nuclear protein in testis (NUT) midline carcinoma (NMC) is a very rare and aggressive malignancy. In more than two-thirds of these NMC cases, a fusion between NUT and BRD4 or BRD3 has been documented; other variants are rare. The cytology of NMC itself has been sparsely documented and that of variant NMC has never been reported. Case Presentation. A 36-year-old woman was admitted because of a rapidly progressing lung tumor with metastases to the breast and bone. We recently reported this patient as the first case of a variant NMC of the lung harboring an NSD3-NUT fusion, based on immunohistochemical and genetic analyses. Cytological material was available for the present review. A highly cellular smear contained a predominantly noncohesive pattern of monomorphic cells with diameters 2-2.5 times greater than those of small lymphocytes, with a round-to-oval nucleus, slightly irregular nuclear contours, variably prominent nucleoli, scant cytoplasm, and identifiable mitotic figures. Foci of stratification and overt pearl formation, including a dyskeratocyte, were occasionally observed. The necrotic background contained naked nuclei, karyorrhectic debris, apoptotic cells, and macrophages phagocytizing karyorrhectic debris; nuclear crushing was noted. Conclusion. The cytological features of a variant NMC of the lung are described for the first time. PMID:25685583

  5. Inactivation of the Carney complex gene 1 (PRKAR1A) alters spatiotemporal regulation of cAMP and cAMP-dependent protein kinase: a study using genetically encoded FRET-based reporters

    PubMed Central

    Cazabat, Laure; Ragazzon, Bruno; Varin, Audrey; Potier-Cartereau, Marie; Vandier, Christophe; Vezzosi, Delphine; Risk-Rabin, Marthe; Guellich, Aziz; Schittl, Julia; Lechêne, Patrick; Richter, Wito; Nikolaev, Viacheslav O.; Zhang, Jin; Bertherat, Jérôme; Vandecasteele, Grégoire

    2014-01-01

    Carney complex (CNC) is a hereditary disease associating cardiac myxoma, spotty skin pigmentation and endocrine overactivity. CNC is caused by inactivating mutations in the PRKAR1A gene encoding PKA type I alpha regulatory subunit (RI?). Although PKA activity is enhanced in CNC, the mechanisms linking PKA dysregulation to endocrine tumorigenesis are poorly understood. In this study, we used Förster resonance energy transfer (FRET)-based sensors for cAMP and PKA activity to define the role of RI? in the spatiotemporal organization of the cAMP/PKA pathway. RI? knockdown in HEK293 cells increased basal as well as forskolin or prostaglandin E1 (PGE1)-stimulated total cellular PKA activity as reported by western blots of endogenous PKA targets and the FRET-based global PKA activity reporter, AKAR3. Using variants of AKAR3 targeted to subcellular compartments, we identified similar increases in the response to PGE1 in the cytoplasm and at the outer mitochondrial membrane. In contrast, at the plasma membrane, the response to PGE1 was decreased along with an increase in basal FRET ratio. These results were confirmed by western blot analysis of basal and PGE1-induced phosphorylation of membrane-associated vasodilator-stimulated phosphoprotein. Similar differences were observed between the cytoplasm and the plasma membrane in human adrenal cells carrying a RI? inactivating mutation. RI? inactivation also increased cAMP in the cytoplasm, at the outer mitochondrial membrane and at the plasma membrane, as reported by targeted versions of the cAMP indicator Epac1-camps. These results show that RI? inactivation leads to multiple, compartment-specific alterations of the cAMP/PKA pathway revealing new aspects of signaling dysregulation in tumorigenesis. PMID:24122441

  6. Effects of G-gene Deletion and Replacement on Rabies Virus Vector Gene Expression

    PubMed Central

    Sato, Sho; Ohara, Shinya; Tsutsui, Ken-Ichiro; Iijima, Toshio

    2015-01-01

    The glycoprotein-gene (G gene) -deleted rabies virus (RV) vector is a powerful tool to examine the function and structure of neural circuits. We previously reported that the deletion of the G gene enhances the transgene expression level of the RV vector. However, the mechanism of this enhancement remains to be clarified. We presume that there are two possible factors for this enhancement. The first factor is the glycoprotein of RV, which shows cytotoxicity; thus, may cause a dysfunction in the translation process of infected cells. The second possible factor is the enhanced expression of the L gene, which encodes viral RNA polymerase. In the RV, it is known that the gene expression level is altered depending on the position of the gene. Since G-gene deletion displaces the L gene in the genome, the expression of the L gene and viral transcription may be enhanced. In this study, we compared the transgene expression level and viral transcription of three recombinant RV vectors. The effect of glycoprotein was examined by comparing the viral gene expression of G-gene-intact RV and G-gene-replaced RV. Despite the fact that the L-gene transcription level of these two RV vectors was similar, the G-gene-replaced RV vector showed higher viral transcription and transgene expression level than the G-gene-intact RV vector. To examine the effect of the position of the L gene, we compared the viral gene expression of the G-gene-deleted RV and G-gene-replaced RV. The G-gene-deleted RV vector showed higher L-gene transcription, viral transcription, and transgene expression level than the G-gene-replaced RV vector. These results indicate that G-gene deletion enhances the transgene expression level through at least two factors, the absence of glycoprotein and enhancement of L-gene expression. These findings enable investigators to design a useful viral vector that shows a controlled desirable transgene expression level in applications. PMID:26023771

  7. Comparing the presence of different genes in Salmonella subspecies I-IV and development of a diagnostic multiplex PCR method for identification of Salmonella subspecies.

    PubMed

    Münch, Sebastian; Wernery, Ulrich; Kinne, Jörg; Joseph, Marina; Braun, Peggy; Pees, Michael; Flieger, Antje; Fruth, Angelika; Rabsch, Wolfgang

    2013-01-01

    Reptile-associated salmonellosis in humans has become a growing problem worldwide. Reptiles are frequently asymptomatic carriers of Salmonella and therefore, they are considered as an important reservoir for these bacteria. The classical biochemical method for Salmonella subspecies detection is time consuming, especially in samples from reptiles since they frequently carry more than one Salmonella subspecies. The aim of this study was therefore to develop a multiplex PCR assay for a rapid and accurate differentiation of Salmonella subspecies I, II, IIa, IIIb and IV. In the present study, the occurrence of the genes invA, ttrCA, iroB, STM4075, sciA, STM3690, sadA, gatD, foxA, pagN, fljB, iucD, spvB, lacZ, iutA, mdcA and irp2 was examined in 41 Salmonella strains from Middle Eastern animals (mainly reptiles) by monoplex PCR. According to the results a multiplex PCR assay was developed based on the genes ttrCA, sciA, foxA, iutA. Compared to biochemical analysis this method allowed a fast identification of the subspecies from all the Middle Eastern Salmonella strains (n = 41), as well as 79 strains from German children (n = 18) with reptile associated salmonellosis and other humans and animals (n = 61) with salmonellosis.These results revealed the multiplex PCR as a fast assay for a specific identification of Salmonella subspecies I, II, IIIa, and IIIb. PMID:23367664

  8. Positional Cloning of a Novel Fanconi Anemia Gene, FANCD2

    Microsoft Academic Search

    Cynthia Timmers; Toshiyasu Taniguchi; James Hejna; Carol Reifsteck; Lora Lucas; Donald Bruun; Matthew Thayer; Barbara Cox; Susan Olson; Alan D. D'Andrea; Robb Moses; Markus Grompe

    2001-01-01

    Fanconi anemia (FA) is a genetic disease with birth defects, bone marrow failure, and cancer susceptibility. To date, genes for five of the seven known complementation groups have been cloned. Complementation group D is heterogeneous, consisting of two distinct genes, FANCD1 and FANCD2. Here we report the positional cloning of FANCD2. The gene consists of 44 exons, encodes a novel

  9. Molecular Diagnosis of Deafness: Impact of Gene Identification

    Microsoft Academic Search

    Shin-ichi Usami; Eiko Koda; Koji Tsukamoto; Akihiro Otsuka; Isamu Yuge; Kenji Asamura; Satoko Abe; Jiro Akita; Atsushi Namba

    2002-01-01

    Recent progress in identifying genes responsible for hearing loss enables the ENT clinician to apply molecular diagnosis by genetic testing. This article focuses on three genes, which are prevalent and therefore commonly encountered in the clinic. GJB2 (connexin 26) is currently recognized as the most prevalent gene responsible for congenital hearing loss in many countries. A series of reports revealed

  10. Expression and secretion of the Candida wickerhamii extracellular beta-glucosidase gene, bglB, in Saccharomyces cerevisiae.

    PubMed

    Skory, C D; Freer, S N; Bothast, R J

    1996-11-01

    The yeast Candida wickerhamii exports a cell-associated beta-glucosidase that is active against cellobiose and all soluble cellodextrins. Because of its unique ability to tolerate end-product inhibition by glucose, the bglB gene that encodes this enzyme was previously cloned and sequenced in this laboratory. Using several different promoters and constructs, bglB was expressed in the hosts Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae. Expression was initially performed in E. coli using either the lacZ or tac promoter. This resulted in intracellular expression of the BglB protein with the protein being rapidly fragmented. Secretion and glycosylation of active beta-glucosidase was achieved with several different S. cerevisiae constructs utilizing either the adh1 or the gal1 promoter on 2-micro replicating plasmids. When either the invertase (Suc2) or the BglB secretion signal was used, BglB protein remained associated with the cell wall and appeared to be hyperglycosylated. Expression in P. pastoris was also examined to determine if higher activity and expression could be achieved in a yeast host that usually does not hyperglycosylate. Using the alcohol oxidase promoter in conjunction with either the pho1 or the alpha-factor secretion signal, the recombinant enzyme was successfully secreted and glycosylated in P. pastoris. However, levels of protein expression from the chromosomally integrated vector were insufficient to detect activity. PMID:8929394

  11. Molecular biological enhancement of coal desulfurization: Cloning and expression of the sulfoxide/sulfone/sulfonate/sulfate genes in pseudomonads and Thiobacillae. Third quarterly report, January 1990--March 1990

    SciTech Connect

    Krawiec, S.

    1990-04-20

    This contract was established on July 20, 1989. This first quarterly report pertains to the isolation, identification, and acquisition of organisms, perceived refinements to the fluorescent spray plate technique, optimization of mating protocols, production of autotrophs, refinement of procedures for the isolation of plasmids, and definition of procedures to be used in the identification and use of R primes. A mating regime has been developed which effectively selects against soil transconjugants serving as donors and for recipients derived from P. aeruginosa 278583. Three methionine auxotrophs and a histidine auxotroph have been generated in P. aeruginosa 27853 by transposon mutagenesis. A new lysis by alkali method has been successfully used to prepare plasmids R68.45, pLA2917, and pRK2013.

  12. Gene Polymorphisms in Chronic Periodontitis

    PubMed Central

    Laine, Marja L.; Loos, Bruno G.; Crielaard, W.

    2010-01-01

    We aimed to conduct a review of the literature for gene polymorphisms associated with chronic periodontitis (CP) susceptibility. A comprehensive search of the literature in English was performed using the keywords: periodontitis, periodontal disease, combined with the words genes, mutation, or polymorphism. Candidate gene polymorphism studies with a case-control design and reported genotype frequencies in CP patients were searched and reviewed. There is growing evidence that polymorphisms in the IL1, IL6, IL10, vitamin D receptor, and CD14 genes may be associated with CP in certain populations. However, carriage rates of the rare (R)-allele of any polymorphism varied considerably among studies and most of the studies appeared under-powered and did not correct for other risk factors. Larger cohorts, well-defined phenotypes, control for other risk factors, and analysis of multiple genes and polymorphisms within the same pathway are needed to get a more comprehensive insight into the contribution of gene polymorphisms in CP. PMID:20339487

  13. Immunoglobulin genes of the turtles.

    PubMed

    Magadán-Mompó, Susana; Sánchez-Espinel, Christian; Gambón-Deza, Francisco

    2013-03-01

    The availability of reptile genomes for the use of the scientific community is an exceptional opportunity to study the evolution of immunoglobulin genes. The genome of Chrysemys picta bellii and Pelodiscus sinensis is the first one that has been reported for turtles. The scanning for immunoglobulin genes resulted in the presence of a complex locus for the immunoglobulin heavy chain (IGH). This IGH locus in both turtles contains genes for 13 isotypes in C. picta bellii and 17 in P. sinensis. These correspond with one immunoglobulin M, one immunoglobulin D, several immunoglobulins Y (six in C. picta bellii and eight in P. sinensis), and several immunoglobulins that are similar to immunoglobulin D2 (five in C. picta belli and seven in P. sinensis) that was previously described in Eublepharis macularius. It is worthy to note that IGHD2 are placed in an inverted transcriptional orientation and present sequences for two immunoglobulin domains that are similar to bird IgA domains. Furthermore, its phylogenetic analysis allows us to consider about the presence of IGHA gene in a primitive reptile, so we would be dealing with the memory of the gene that originated from the bird IGHA. In summary, we provide a clear picture of the immunoglobulins present in a turtle, whose analysis supports the idea that turtles emerged from the evolutionary line from the differentiation of birds and the presence of the IGHA gene present in a common ancestor. PMID:23208582

  14. Essential Bacillus subtilis genes

    Microsoft Academic Search

    K. Kobayashi; S. D. Ehrlichb; A. Albertini; G. Amati; K. Asaig Arnaudf; M. Arnaud; K. Asai; S. Ashikaga; S. Aymerich; P. Bessieres; F. Boland; S. C. Brignell; S. Bron; K. Bunai; J. Chapuis; L. C. Christiansen; A. Danchin; M. Débarbouillé; E. Dervyn; E. Deuerling; K. Devine; S. K. Devine; O. Dreesen; J. Errington; S. Fillinger; S. J. Foster; Y. Fujita; A. Galizzi; R. Gardan; C. Eschevins; T. Fukushima; K. Haga; C. R. Harwood; M. Hecker; D. Hosoya; M. F. Hullo; H. Kakeshita; D. Karamata; Y. Kasahara; F. Kawamura; K. Koga; P. Koski; R. Kuwana; D. Imamura; M. Ishimaru; S. Ishikawa; I. Ishio; D. Le Coq; A. Masson; C. Mauël; R. Meima; R. P. Mellado; A. Moir; S. Moriya; E. Nagakawa; H. Nanamiya; S. Nakai; P. Nygaard; M. Ogura; T. Ohanan; M. O'Reilly; M. O'Rourke; Z. Pragai; H. M. Pooley; G. Rapoport; J. P. Rawlins; L. A. Rivas; C. Rivolta; A. Sadaie; Y. Sadaie; M. Sarvas; T. Sato; H. H. Saxild; E. Scanlan; W. Schumann; J. F. Seegers; J. Sekiguchi; A. Sekowska; S. J. Seror; M. Simon; P. Stragier; R. Studer; H. Takamatsu; T. Tanaka; M. Takeuchi; H. B. Thomaides; V. Vagner; J. M. van Dijl; K. Watabe; A. Wipat; H. Yamamoto; M. Yamamoto; Y. Yamamoto; K. Yamane; K. Yata; K. Yoshida; H. Yoshikawa; U. Zuber; N. Ogasawara

    2003-01-01

    To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively

  15. Nucleotide sequence of the 5' region of the Bacillus licheniformis alpha-amylase gene: comparison with the B. amyloliquefaciens gene.

    PubMed Central

    Stephens, M A; Ortlepp, S A; Ollington, J F; McConnell, D J

    1984-01-01

    The DNA sequence of the 5' region of the Bacillus licheniformis alpha-amylase gene is reported. Comparison of the inferred amino acid sequence of the B. licheniformis alpha-amylase gene with that of the Bacillus amyloliquefaciens gene shows that whereas the amino acid sequences of the mature proteins have considerable homology, the sequences for the signal peptides are distinct. PMID:6609154

  16. Evolutionary Signatures amongst Disease Genes Permit Novel Methods for Gene Prioritization and Construction of Informative Gene-Based Networks

    PubMed Central

    Priedigkeit, Nolan; Wolfe, Nicholas; Clark, Nathan L.

    2015-01-01

    Genes involved in the same function tend to have similar evolutionary histories, in that their rates of evolution covary over time. This coevolutionary signature, termed Evolutionary Rate Covariation (ERC), is calculated using only gene sequences from a set of closely related species and has demonstrated potential as a computational tool for inferring functional relationships between genes. To further define applications of ERC, we first established that roughly 55% of genetic diseases posses an ERC signature between their contributing genes. At a false discovery rate of 5% we report 40 such diseases including cancers, developmental disorders and mitochondrial diseases. Given these coevolutionary signatures between disease genes, we then assessed ERC's ability to prioritize known disease genes out of a list of unrelated candidates. We found that in the presence of an ERC signature, the true disease gene is effectively prioritized to the top 6% of candidates on average. We then apply this strategy to a melanoma-associated region on chromosome 1 and identify MCL1 as a potential causative gene. Furthermore, to gain global insight into disease mechanisms, we used ERC to predict molecular connections between 310 nominally distinct diseases. The resulting “disease map” network associates several diseases with related pathogenic mechanisms and unveils many novel relationships between clinically distinct diseases, such as between Hirschsprung's disease and melanoma. Taken together, these results demonstrate the utility of molecular evolution as a gene discovery platform and show that evolutionary signatures can be used to build informative gene-based networks. PMID:25679399

  17. Homeobox genes expressed during echinoderm arm regeneration.

    PubMed

    Ben Khadra, Yousra; Said, Khaled; Thorndyke, Michael; Martinez, Pedro

    2014-04-01

    Regeneration in echinoderms has proved to be more amenable to study in the laboratory than the more classical vertebrate models, since the smaller genome size and the absence of multiple orthologs for different genes in echinoderms simplify the analysis of gene function during regeneration. In order to understand the role of homeobox-containing genes during arm regeneration in echinoderms, we isolated the complement of genes belonging to the Hox class that are expressed during this process in two major echinoderm groups: asteroids (Echinaster sepositus and Asterias rubens) and ophiuroids (Amphiura filiformis), both of which show an extraordinary capacity for regeneration. By exploiting the sequence conservation of the homeobox, putative orthologs of several Hox genes belonging to the anterior, medial, and posterior groups were isolated. We also report the isolation of a few Hox-like genes expressed in the same systems. PMID:24309817

  18. HLA Immune Function Genes in Autism

    PubMed Central

    Torres, Anthony R.; Westover, Jonna B.; Rosenspire, Allen J.

    2012-01-01

    The human leukocyte antigen (HLA) genes on chromosome 6 are instrumental in many innate and adaptive immune responses. The HLA genes/haplotypes can also be involved in immune dysfunction and autoimmune diseases. It is now becoming apparent that many of the non-antigen-presenting HLA genes make significant contributions to autoimmune diseases. Interestingly, it has been reported that autism subjects often have associations with HLA genes/haplotypes, suggesting an underlying dysregulation of the immune system mediated by HLA genes. Genetic studies have only succeeded in identifying autism-causing genes in a small number of subjects suggesting that the genome has not been adequately interrogated. Close examination of the HLA region in autism has been relatively ignored, largely due to e