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Sample records for gene nt4-ant-shepherdin induce

  1. Induced pluripotency with endogenous and inducible genes

    SciTech Connect

    Duinsbergen, Dirk; Eriksson, Malin; Hoen, Peter A.C. 't; Frisen, Jonas; Mikkers, Harald

    2008-10-15

    The recent discovery that two partly overlapping sets of four genes induce nuclear reprogramming of mouse and even human cells has opened up new possibilities for cell replacement therapies. Although the combination of genes that induce pluripotency differs to some extent, Oct4 and Sox2 appear to be a prerequisite. The introduction of four genes, several of which been linked with cancer, using retroviral approaches is however unlikely to be suitable for future clinical applications. Towards developing a safer reprogramming protocol, we investigated whether cell types that express one of the most critical reprogramming genes endogenously are predisposed to reprogramming. We show here that three of the original four pluripotency transcription factors (Oct4, Klf4 and c-Myc or MYCER{sup TAM}) induced reprogramming of mouse neural stem (NS) cells exploiting endogenous SoxB1 protein levels in these cells. The reprogrammed neural stem cells differentiated into cells of each germ layer in vitro and in vivo, and contributed to mouse development in vivo. Thus a combinatorial approach taking advantage of endogenously expressed genes and inducible transgenes may contribute to the development of improved reprogramming protocols.

  2. Radiation-induced gene responses

    SciTech Connect

    Woloschak, G.E.; Paunesku, T.; Shearin-Jones, P.; Oryhon, J.

    1996-12-31

    In the process of identifying genes that are differentially regulated in cells exposed to ultraviolet radiation (UV), we identified a transcript that was repressed following the exposure of cells to a combination of UV and salicylate, a known inhibitor of NF-kappaB. Sequencing this band determined that it has identify to lactate dehydrogenase, and Northern blots confirmed the initial expression pattern. Analysis of the sequence of the LDH 5` region established the presence of NF-kappaB, Sp1, and two Ap-2 elements; two partial AP- 1; one partial RE, and two halves of E-UV elements were also found. Electromobility shift assays were then performed for the AP-1, NF- kappaB, and E-UV elements. These experiments revealed that binding to NF-kappaB was induced by UV but repressed with salicylic acid; UV did not affect AP-1 binding, but salicylic acid inhibited it alone or following UV exposure; and E-UV binding was repressed by UV, and salicylic acid had little effect. Since the binding of no single element correlated with the expression pattern of LDH, it is likely that multiple elements govern UV/salicylate-mediated expression.

  3. Virus-induced gene complementation in tomato

    PubMed Central

    Kong, Jinhua; Chen, Weiwei; Shen, Jiajia; Qin, Cheng; Lai, Tongfei; Zhang, Pengcheng; Wang, Ying; Wu, Chaoqun; Yang, Xin; Hong, Yiguo

    2013-01-01

    Virus-induced gene complementation (VIGC), a plant virus technology based on Potato virus X for transient overexpression of endogenous genes complemented tomato mutants, resulting in non-ripening fruits to ripen. This efficient “gain-of-function” approach involves no stable transformation, and reveals a fruit-specific transcriptional network that may exist among key transcription factors in modulating tomato ripening. Thus, VIGC represents a novel and feasible strategy for gene functional analysis in plants. PMID:24305652

  4. Virus induced gene silencing of Arabidopsis gene homologues in wheat identify genes conferring improved drought tolerance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In a non-model staple crop like wheat, functional validation of potential drought stress responsive genes identified in Arabidopsis could provide gene targets for wheat breeding. Virus induced gene silencing (VIGS) of genes of interest can overcome the inherent problems of polyploidy and limited tra...

  5. Heat induces gene amplification in cancer cells

    SciTech Connect

    Yan, Bin; Ouyang, Ruoyun; Huang, Chenghui; Liu, Franklin; Neill, Daniel; Li, Chuanyuan; Dewhirst, Mark

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. Black-Right-Pointing-Pointer Hyperthermia induces DNA double strand breaks. Black-Right-Pointing-Pointer DNA double strand breaks are considered to be required for the initiation of gene amplification. Black-Right-Pointing-Pointer The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 Degree-Sign C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) {gamma}H2AX immunostaining to detect {gamma}H2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 Degree-Sign C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 Degree-Sign C for 30 min induces DNA double strand breaks in HCT116 cells as shown by {gamma}H2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and

  6. Tetracycline inducible gene manipulation in serotonergic neurons.

    PubMed

    Weber, Tillmann; Renzland, Insa; Baur, Max; Mönks, Simon; Herrmann, Elke; Huppert, Verena; Nürnberg, Frank; Schönig, Kai; Bartsch, Dusan

    2012-01-01

    The serotonergic (5-HT) neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA) mouse line (TPH2-tTA) that allows temporal and spatial control of tetracycline (Ptet) controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb) by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ). In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic β-galactosidase expression which could be completely suppressed with doxycycline (Dox). Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20) were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We generated a

  7. HIFU-induced gene activation in vitro

    NASA Astrophysics Data System (ADS)

    Liu, Yunbo; Zhong, Pei; Kon, Takashi; Li, Chuanyuan

    2001-05-01

    This work investigated the inducible gene activation in cancer cells that were sublethally injured during HIFU treatment. HeLa cells were transfected by an adenovirus vector that encodes GFP under the control of hsp70B promoter, leading to about 65% transfection efficiency. A volume of 10 μL transfected HeLa cells in suspension (5×107 cells/ml) were placed at the bottom of a PCR tube so that the cell suspension could be heated to a peak temperature of 50°C, 60°C, and 70°C for 120, 10, and 1 s, respectively, by a focused 1.1-MHz HIFU transducer operated at a peak negative pressure of -2.7 MPa at different duty cycles. One day after HIFU treatment, cell viability was determined to be 63%, 35%, and 18%, respectively, based on Trypan Blue exclusion test. Importantly, in all test groups, inducible GFP expression was detected in about 40%-50% of the surviving cells with GFP intensity increased by 25-fold based on flow cytometry analysis. These results demonstrate that even under the short exposure duration of HIFU treatment, inducible gene expression could be produced in sublethally injured cell population in vitro. Further studies are underway to explore the optimal HIFU condition for gene activation in vivo.

  8. Salt induced gene expression in Prosopis farcta

    SciTech Connect

    Heimer, I.M.; Golan, A.; Lips, H.

    1987-04-01

    The authors hypothesize that in facultative halophytes, the genes which impart salt tolerance are expressed when the plants are exposed to salt. As a first step towards possible identification of these genes, they examined salt induced changes of gene expression in the facultative halophyte Prosopis farcta at the protein level, by SDS-PAGE. Exposure to salt of aseptically grown, two-week old seedlings, was carried out in one of two ways: (1) a one step transfer of seedlings from medium without salt to that with the indicated concentrations followed by 5 hr or 24 hr incubation periods. During the last 2 hrs of each incubation period the seedlings were pulse-labelled with /sup 35/S Sulfate or L-Methionine; (2) a gradual increase of the salt concentration at 50 mM increments at 2-4 day intervals. Two days after reaching the desired salt concentration, the seedlings were pulse-labelled for 2 hrs with /sup 35/S sulfate or L-methionine. Protein from roots were extracted and analyzed. Polypeptides were visualized by staining with coomassie blue or by fluorography. Qualitative as well as quantitative changes of gene expression as induced by salt could be observed. Their significance regarding salt tolerance will be discussed.

  9. Gravity-Induced Gene Expression in Plants.

    NASA Astrophysics Data System (ADS)

    Sederoff, Heike; Heber, Steffen; Howard, Brian; Myburg-Nichols, Henrietta; Hammond, Rebecca; Salinas-Mondragon, Raul; Brown, Christopher S.

    Plants sense changes in their orientation towards the vector of gravity and respond with directional growth. Several metabolites in the signal transduction cascade have been identified. However, very little is known about the interaction between these sensing and signal transduction events and even less is known about their role in the differential growth response. Gravity induced changes in transcript abundance have been identified in Arabidopsis whole seedlings and root apices (Moseyko et al. 2002; Kimbrough et al. 2004). Gravity induced transcript abundance changes can be observed within less than 1 min after stimulation (Salinas-Mondragon et al. 2005). Gene expression however requires not only transcription but also translation of the mRNA. Translation can only occur when mRNA is associated with ribosomes, even though not all mRNA associated with ribosomes is actively translated. To approximate translational capacity we quantified whole genome transcript abundances in corn stem pulvini during the first hour after gravity stimulation in total and poly-ribosomal fractions. As in Arabidopsis root apices, transcript abundances of several clusters of genes responded to gravity stimulation. The vast majority of these transcripts were also found to associate with polyribosomes in the same temporal and quantitative pattern. These genes are transcriptionally regulated by gravity stimulation, but do not exhibit translational regulation. However, a small group of genes showed increased transcriptional regulation after gravity stimulation, but no association with polysomes. These transcripts likely are translationally repressed. The mechanism of translational repression for these transcripts is unknown. Based on the hypothesis that the genes essential for gravitropic responses should be expressed in most or all species, we compared the temporal gravity induced expression pattern of all orthologs identified between maize and Arabidopsis. A small group of genes showed high

  10. Targeted gene silencing to induce permanent sterility.

    PubMed

    Dissen, G A; Lomniczi, A; Boudreau, R L; Chen, Y H; Davidson, B L; Ojeda, S R

    2012-08-01

    A non-surgical method to induce sterility would be a useful tool to control feral populations of animals. Our laboratories have experience with approaches aimed at targeting brain cells in vivo with vehicles that deliver a payload of either inhibitory RNAs or genes intended to correct cellular dysfunction. A combination/modification of these methods may provide a useful framework for the design of approaches that can be used to sterilize cats and dogs. For this approach to succeed, it has to meet several conditions: it needs to target a gene essential for fertility. It must involve a method that can selectively silence the gene of interest. It also needs to deliver the silencing agent via a minimally invasive method. Finally, the silencing effect needs to be sustained for many years, so that expansion of the targeted population can be effectively prevented. In this article, we discuss this subject and provide a succinct account of our previous experience with: (i) molecular reagents able to disrupt reproductive cyclicity when delivered to regions of the brain involved in the control of reproduction and (ii) molecular reagents able to ameliorate neuronal disease when delivered systemically using a novel approach of gene therapy. PMID:22827375

  11. Virus-Induced Gene Silencing in Ornametal Plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-Induced Gene Silencing (VIGS) provides an attractive tool for high throughput analysis of the functional effects of gene knock-down. Virus genomes are engineered to include fragments of target host genes, and the infected plant recognizes and silences the target genes as part of its viral defe...

  12. Virus-Induced gene silencing in ornamental plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-Induced Gene Silencing (VIGS) provides an attractive tool for high throughput analysis of the functional effects of gene knock-down. Virus genomes are engineered to include fragments of target host genes, and the infected plant recognizes and silences the target genes as part of its viral defe...

  13. GENE METHYLATION CHANGES IN TUMOR SUPPRESSOR GENES INDUCED BY ARSENIC

    EPA Science Inventory

    The choice of a dose-response model used for extrapolation can be influenced by knowledge of mechanism of action. We have already showed that arsenic affects methylation of the human p53 gene promoter. Evidence that genes other than the p53 tumor suppressor gene are affected woul...

  14. Insect and wound induced GUS gene expression from a Beta vulgaris proteinase inhibitor gene promoter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inducible gene promoters that are specifically activated by pathogen invasion or insect pest attack are needed for effective expression of resistance genes to control plant diseases. In the present study, a promoter from a serine proteinase inhibitor gene (BvSTI) shown to be up-regulated in resist...

  15. Strong Magnetic Field Induced Changes of Gene Expression in Arabidopsis

    NASA Astrophysics Data System (ADS)

    Paul, A.-L.; Ferl, R. J.; Klingenberg, B.; Brooks, J. S.; Morgan, A. N.; Yowtak, J.; Meisel, M. W.

    2005-07-01

    We review our studies of the biological impact of magnetic field strengths of up to 30 T on transgenic arabidopsis plants engineered with a stress response gene consisting of the alcohol dehydrogenase (Adh) gene promoter driving the β-glucuronidase (GUS) gene reporter. Field strengths in excess of 15 T induce expression of the Adh/GUS transgene in the roots and leaves. Microarray analyses indicate that such field strengths have a far reaching effect on the genome. Wide spread induction of stress-related genes and transcription factors, and a depression of genes associated with cell wall metabolism are prominent examples.

  16. Inducible and combinatorial gene manipulation in mouse brain

    PubMed Central

    Dogbevia, Godwin K.; Marticorena-Alvarez, Ricardo; Bausen, Melanie; Sprengel, Rolf; Hasan, Mazahir T.

    2015-01-01

    We have deployed recombinant adeno-associated viruses equipped with tetracycline-controlled genetic switches to manipulate gene expression in mouse brain. Here, we show a combinatorial genetic approach for inducible, cell type-specific gene expression and Cre/loxP mediated gene recombination in different brain regions. Our chemical-genetic approach will help to investigate ‘when’, ‘where’, and ‘how’ gene(s) control neuronal circuit dynamics, and organize, for example, sensory signal processing, learning and memory, and behavior. PMID:25954155

  17. EVIDENCE FOR THE MACROPHAGE INDUCING GENE IN MYCOBACTERIUM INTRACELLULARE

    EPA Science Inventory

    Background: The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and possibly others. Organisms belonging to the MAC are phylogenetically closely related, opportunistic pathogens. The macrophage inducing gene (mig) is the only well-des...

  18. IL-10 induces gene expression in macrophages: partial overlap with IL-5 but not with IL-4 induced genes.

    PubMed

    Stumpo, Rita; Kauer, Manfred; Martin, Stephan; Kolb, Hubert

    2003-10-01

    The hypothesis that IL-10, in addition to down-regulating pro-inflammatory activities of macrophages, induces an alternative state of macrophage reactivity was tested. We therefore conducted a systematic search for genes induced by IL-10 using the method of suppression subtractive hybridisation. Of an initial 1,300 candidate clones obtained, several screening rounds led to the identification of 51 clones which were reproducibly at least twofold up-regulated in mouse J774 macrophages in response to treatment with IL-10. Of these, 41 genes were homologous to known genes involved in cell metabolism or immunoregulation, five contained novel sequences and another five were homologous to ESTs without known function. One major finding was that about 25% of the IL-10 genes were also found expressed in response to IFNgamma, and several of these also reappeared in IL-4 or IL-5 induced mRNA species. Hence, Th1 and Th2 type cytokines may elicit a common basal activation response in macrophages. The second major finding was that 57% of IL-10 induced genes reappeared in IL-5 induced mRNA but no more than 18% were also found in IL-4 induced mRNA of J774 cells. We conclude that the gene expression response to IL-10 in macrophages is partially different from the response to IL-5 and is substantially different from the response to IL-4, which suggests an unexpected diversity of biological phenotypes induced by different Th2 type cytokines. PMID:14561490

  19. Virus-induced gene silencing (VIGS) in barley seedling leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is one of the most potent reverse genetics technologies for gene functional characterization. This method exploits a dsRNA-mediated antiviral defense mechanism in plants. Using this method allows researchers to generate rapid phenotypic data in a relatively rapid ...

  20. Synthetic riboswitches that induce gene expression in diverse bacterial species.

    PubMed

    Topp, Shana; Reynoso, Colleen M K; Seeliger, Jessica C; Goldlust, Ian S; Desai, Shawn K; Murat, Dorothée; Shen, Aimee; Puri, Aaron W; Komeili, Arash; Bertozzi, Carolyn R; Scott, June R; Gallivan, Justin P

    2010-12-01

    We developed a series of ligand-inducible riboswitches that control gene expression in diverse species of Gram-negative and Gram-positive bacteria, including human pathogens that have few or no previously reported inducible expression systems. We anticipate that these riboswitches will be useful tools for genetic studies in a wide range of bacteria. PMID:20935124

  1. Mechanisms of radiation-induced gene responses

    SciTech Connect

    Woloschak, G.E.; Paunesku, T.

    1996-10-01

    In the process of identifying genes differentially expressed in cells exposed ultraviolet radiation, we have identified a transcript having a 26-bp region that is highly conserved in a variety of species including Bacillus circulans, yeast, pumpkin, Drosophila, mouse, and man. When the 5` region (flanking region or UTR) of a gene, the sequence is predominantly in +/+ orientation with respect to the coding DNA strand; while in the coding region and the 3` region (UTR), the sequence is most frequently in the +/-orientation with respect to the coding DNA strand. In two genes, the element is split into two parts; however, in most cases, it is found only once but with a minimum of 11 consecutive nucleotides precisely depicting the original sequence. The element is found in a large number of different genes with diverse functions (from human ras p21 to B. circulans chitonase). Gel shift assays demonstrated the presence of a protein in HeLa cell extracts that binds to the sense and antisense single-stranded consensus oligomers, as well as to the double- stranded oligonucleotide. When double-stranded oligomer was used, the size shift demonstrated as additional protein-oligomer complex larger than the one bound to either sense or antisense single-stranded consensus oligomers alone. It is speculated either that this element binds to protein(s) important in maintaining DNA is a single-stranded orientation for transcription or, alternatively that this element is important in the transcription-coupled DNA repair process.

  2. Antipsychotic Induced Gene Regulation in Multiple Brain Regions

    PubMed Central

    Girgenti, Matthew James; Nisenbaum, Laura K.; Bymaster, Franklin; Terwilliger, Rosemarie; Duman, Ronald S; Newton, Samuel Sathyanesan

    2010-01-01

    The molecular mechanism of action of antipsychotic drugs is not well understood. Their complex receptor affinity profiles indicate that their action could extend beyond dopamine receptor blockade. Single gene expression studies and high-throughput gene profiling have shown the induction of genes from several molecular classes and functional categories. Using a focused microarray approach we investigated gene regulation in rat striatum, frontal cortex and hippocampus after chronic administration of haloperidol or olanzapine. Regulated genes were validated by in-situ hybridization, realtime PCR and immunohistochemistry. Only limited overlap was observed in genes regulated by haloperidol and olanzapine. Both drugs elicited maximal gene regulation in the striatum and least in the hippocampus. Striatal gene induction by haloperidol was predominantly in neurotransmitter signaling, G-protein coupled receptors and transcription factors. Olanzapine prominently induced retinoic acid and trophic factor signaling genes in the frontal cortex. The data also revealed the induction of several genes that could be targeted in future drug development efforts. The study uncovered the induction of several novel genes, including somatostatin receptors and metabotropic glutamate receptors. The results demonstrating the regulation of multiple receptors and transcription factors suggests that both typical and atypical antipsychotics could possess a complex molecular mechanism of action. PMID:20070867

  3. Low doses of neutrons induce changes in gene expression

    SciTech Connect

    Woloschak, G.E.; Chang-Liu, C.M. ); Panozzo, J.; Libertin, C.R. )

    1993-01-01

    Studies were designed to identify genes induced following low-dose neutron but not following [gamma]-ray exposure in fibroblasts. Our past work had shown differences in the expression of [beta]-protein kinase C and c-fos genes, both being induced following [gamma]-ray but not neutron exposure. We have identified two genes that are induced following neutron, but not [gamma]-ray, exposure: Rp-8 (a gene induced by apoptosis) and the long terminal repeat (LTR) of the human immunodeficiency (HIV). Rp-8 mRNA induction was demonstrated in Syrian hamster embryo fibroblasts and was found to be induced in cells exposed to neutrons administered at low (0.5 cGy/min) and at high dose rate (12 cGy/min). The induction of transcription from the LTR of HIV was demonstrated in HeLa cells bearing a transfected construct of the chloramphenicol acetyl transferase (CAT) gene driven by the HIV-LTR promoter. Measures of CAT activity and CAT transcripts following irradiation demonstrated an unresponsiveness to [gamma] rays over a broad range of doses. Twofold induction of the HIV-LTR was detected following neutron exposure (48 cGy) administered at low (0.5 cGy/min) but not high (12 cGy/min) dose rates. Ultraviolet-mediated HIV-LTR induction was inhibited by low-dose-rate neutron exposure.

  4. Low doses of neutrons induce changes in gene expression

    SciTech Connect

    Woloschak, G.E.; Chang-Liu, C.M.; Panozzo, J.; Libertin, C.R.

    1993-06-01

    Studies were designed to identify genes induced following low-dose neutron but not following {gamma}-ray exposure in fibroblasts. Our past work had shown differences in the expression of {beta}-protein kinase C and c-fos genes, both being induced following {gamma}-ray but not neutron exposure. We have identified two genes that are induced following neutron, but not {gamma}-ray, exposure: Rp-8 (a gene induced by apoptosis) and the long terminal repeat (LTR) of the human immunodeficiency (HIV). Rp-8 mRNA induction was demonstrated in Syrian hamster embryo fibroblasts and was found to be induced in cells exposed to neutrons administered at low (0.5 cGy/min) and at high dose rate (12 cGy/min). The induction of transcription from the LTR of HIV was demonstrated in HeLa cells bearing a transfected construct of the chloramphenicol acetyl transferase (CAT) gene driven by the HIV-LTR promoter. Measures of CAT activity and CAT transcripts following irradiation demonstrated an unresponsiveness to {gamma} rays over a broad range of doses. Twofold induction of the HIV-LTR was detected following neutron exposure (48 cGy) administered at low (0.5 cGy/min) but not high (12 cGy/min) dose rates. Ultraviolet-mediated HIV-LTR induction was inhibited by low-dose-rate neutron exposure.

  5. Three light-inducible heat shock genes of Chlamydomonas reinhardtii.

    PubMed Central

    von Gromoff, E D; Treier, U; Beck, C F

    1989-01-01

    Genomic clones representing three Chlamydomonas reinhardtii genes homologous to the Drosophila hsp70 heat shock gene were isolated. The mRNAs of genes hsp68, hsp70, and hsp80 could be translated in vitro into proteins of Mr 68,000, 70,000, and 80,000, respectively. Transcription of these genes increased dramatically upon heat shock, and the corresponding mRNAs rapidly accumulated, reaching a peak at around 30 min after a shift to the elevated temperature. Light also induced the accumulation of the mRNAs encoded by these heat shock genes. A shift of dark-grown cells to light resulted in a drastic increase in mRNA levels, which reached a maximum at around 1 h after the shift. Thus, in Chlamydomonas, expression of hsp70-homologous heat shock genes appears to be regulated by thermal stress and light. Images PMID:2779571

  6. Salmonella induces prominent gene expression in the rat colon

    PubMed Central

    Rodenburg, Wendy; Keijer, Jaap; Kramer, Evelien; Roosing, Susanne; Vink, Carolien; Katan, Martijn B; van der Meer, Roelof; Bovee-Oudenhoven, Ingeborg MJ

    2007-01-01

    Background Salmonella enteritidis is suggested to translocate in the small intestine. In vivo it induces gene expression changes in the ileal mucosa and Peyer's patches. Stimulation of Salmonella translocation by dietary prebiotics fermented in colon suggests involvement of the colon as well. However, effects of Salmonella on colonic gene expression in vivo are largely unknown. We aimed to characterize time dependent Salmonella-induced changes of colonic mucosal gene expression in rats using whole genome microarrays. For this, rats were orally infected with Salmonella enteritidis to mimic a foodborne infection and colonic gene expression was determined at days 1, 3 and 6 post-infection (n = 8 rats per time-point). As fructo-oligosaccharides (FOS) affect colonic physiology, we analyzed colonic mucosal gene expression of FOS-fed versus cellulose-fed rats infected with Salmonella in a separate experiment. Colonic mucosal samples were isolated at day 2 post-infection. Results Salmonella affected transport (e.g. Chloride channel calcium activated 6, H+/K+ transporting Atp-ase), antimicrobial defense (e.g. Lipopolysaccharide binding protein, Defensin 5 and phospholipase A2), inflammation (e.g. calprotectin), oxidative stress related genes (e.g. Dual oxidase 2 and Glutathione peroxidase 2) and Proteolysis (e.g. Ubiquitin D and Proteosome subunit beta type 9). Furthermore, Salmonella translocation increased serum IFNγ and many interferon-related genes in colonic mucosa. The gene most strongly induced by Salmonella infection was Pancreatitis Associated Protein (Pap), showing >100-fold induction at day 6 after oral infection. Results were confirmed by Q-PCR in individual rats. Stimulation of Salmonella translocation by dietary FOS was accompanied by enhancement of the Salmonella-induced mucosal processes, not by induction of other processes. Conclusion We conclude that the colon is a target tissue for Salmonella, considering the abundant changes in mucosal gene expression

  7. Light-Inducible Gene Regulation with Engineered Zinc Finger Proteins

    PubMed Central

    Polstein, Lauren R.; Gersbach, Charles A.

    2014-01-01

    The coupling of light-inducible protein-protein interactions with gene regulation systems has enabled the control of gene expression with light. In particular, heterodimer protein pairs from plants can be used to engineer a gene regulation system in mammalian cells that is reversible, repeatable, tunable, controllable in a spatiotemporal manner, and targetable to any DNA sequence. This system, Light-Inducible Transcription using Engineered Zinc finger proteins (LITEZ), is based on the blue light-induced interaction of GIGANTEA and the LOV domain of FKF1 that drives the localization of a transcriptional activator to the DNA-binding site of a highly customizable engineered zinc finger protein. This chapter provides methods for modifying LITEZ to target new DNA sequences, engineering a programmable LED array to illuminate cell cultures, and using the modified LITEZ system to achieve spatiotemporal control of transgene expression in mammalian cells. PMID:24718797

  8. Down-Regulation of Gene Expression by RNA-Induced Gene Silencing

    NASA Astrophysics Data System (ADS)

    Travella, Silvia; Keller, Beat

    Down-regulation of endogenous genes via post-transcriptional gene silencing (PTGS) is a key to the characterization of gene function in plants. Many RNA-based silencing mechanisms such as post-transcriptional gene silencing, co-suppression, quelling, and RNA interference (RNAi) have been discovered among species of different kingdoms (plants, fungi, and animals). One of the most interesting discoveries was RNAi, a sequence-specific gene-silencing mechanism initiated by the introduction of double-stranded RNA (dsRNA), homologous in sequence to the silenced gene, which triggers degradation of mRNA. Infection of plants with modified viruses can also induce RNA silencing and is referred to as virus-induced gene silencing (VIGS). In contrast to insertional mutagenesis, these emerging new reverse genetic approaches represent a powerful tool for exploring gene function and for manipulating gene expression experimentally in cereal species such as barley and wheat. We examined how RNAi and VIGS have been used to assess gene function in barley and wheat, including molecular mechanisms involved in the process and available methodological elements, such as vectors, inoculation procedures, and analysis of silenced phenotypes.

  9. High Intensity Focused Ultrasound induced Gene Activation in Solid Tumors

    NASA Astrophysics Data System (ADS)

    Liu, Yunbo; Kon, Takashi; Li, Chuanyuan; Zhong, Pei

    2006-05-01

    In this work, the feasibility of using high intensity focused ultrasound (HIFU) to activate trans-gene expression in a mouse tumor model was investigated. 4T1 cancer cells were implanted subcutaneously in the hind limbs of Balb/C mice and adenovirus luciferase gene vectors under the control of heat shock protein 70B promoter (Adeno-hsp70B-Luc) were injected intratumoraly for gene transfection. One day following the virus injection, the transfected tumors were heated to a peak temperature of 55, 65, 75, and 85°C, respectively, in 10s at multiple sites around the center of the tumor using a HIFU transducer operated at either 1.1-MHz (fundamental) or 3.3-MHz (3rd harmonic) frequency. Inducible luciferase gene expression was found to vary from 15-fold to 120-fold of the control group following 1.1-MHz HIFU exposure. The maximum gene activation was produced at a peak temperature of 65˜75°C one day following HIFU exposure and decayed gradually to baseline level within 7 days. The inducible gene activation produced by 3.3-MHz HIFU exposure (75°C-10s) was found to be comparable to that produced by hyperthermia (42°C-30min). Altogether, these results demonstrate the feasibility of using HIFU as a simple and versatile physical means to regulate trans-gene expression in vivo. This unique feature may be explored in the future for a synergistic combination of HIFU-induced thermal ablation with heat-induced gene therapy for improved cancer therapy.

  10. Synapsins are late activity-induced genes regulated by birdsong

    PubMed Central

    Velho, Tarciso A. F.; Mello, Claudio V.

    2008-01-01

    The consolidation of long-lasting sensory memories requires the activation of gene expression programs in the brain. In spite of considerable knowledge about the early components of this response, little is known about late components (i.e. genes regulated 2-6 hr after stimulation) and the relationship between early and late genes. Birdsong represents one of the best natural behaviors to study sensory-induced gene expression in awake, freely behaving animals. Here we show that the expression of several isoforms of synapsins, a group of phosphoproteins thought to regulate the dynamics of synaptic vesicle storage and release, is induced by auditory stimulation with birdsong in the caudomedial nidopallium (NCM) of the zebra finch (Taeniopygia guttata) brain. This induction occurs mainly in excitatory (non-GABAergic) neurons and is modulated (suppressed) by early song-inducible proteins. We also show that ZENK, an early song-inducible transcription factor, interacts with the syn3 promoter in vivo, consistent with a direct regulatory effect and an emerging novel view of ZENK action. These results demonstrate that synapsins are a late component of the genomic response to neuronal activation and that their expression depends on a complex set of regulatory interactions between early and late regulated genes. PMID:19005052

  11. INDUCIBLE RNAi-MEDIATED GENE SILENCING USING NANOSTRUCTURED GENE DELIVERY ARRAYS

    SciTech Connect

    Mann, David George James; McKnight, Timothy E; Mcpherson, Jackson; Hoyt, Peter R; Melechko, Anatoli Vasilievich; Simpson, Michael L; Sayler, Gary Steven

    2008-01-01

    RNA interference has become a powerful biological tool over the last decade. In this study, a tetracycline-inducible shRNA vector system was designed for silencing CFP expression and introduced alongside the yfp marker gene into Chinese hamster ovary cells using spatially indexed vertically aligned carbon nanofiber arrays (VACNFs) in a gene delivery process termed impalefection. The VACNF architecture provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of interfaced cells, and repeated monitoring of single cells over time. 24 hours after nanofiber-mediated delivery, 53.1% 10.4% of the cells that expressed the yfp marker gene were also fully silenced by the inducible CFP-silencing shRNA vector. Additionally, efficient CFP-silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enables rapid analysis of gene silencing effects using RNAi in single cells and cell populations.

  12. High magnetic field induced changes of gene expression in arabidopsis

    PubMed Central

    Paul, Anna-Lisa; Ferl, Robert J; Meisel, Mark W

    2006-01-01

    Background High magnetic fields are becoming increasingly prevalent components of non-invasive, biomedical imaging tools (such as MRI), thus, an understanding of the molecular impacts associated with these field strengths in biological systems is of central importance. The biological impact of magnetic field strengths up to 30 Tesla were investigated in this study through the use of transgenic Arabidopsis plants engineered with a stress response gene consisting of the alcohol dehydrogenase (Adh) gene promoter driving the β-glucuronidase (GUS) gene reporter. Methods Magnetic field induced Adh/GUS activity was evaluated with histochemical staining to assess tissue specific expression and distribution, and with quantitative, spectrofluometric assays to measure degree of activation. The evaluation of global changes in the Arabidopsis genome in response to exposure to high magnetic fields was facilitated with Affymetrix Gene Chip microarrays. Quantitative analyses of gene expression were performed with quantitative real-time polymerase-chain-reaction (qRT-PCR). Results Field strengths in excess of about 15 Tesla induce expression of the Adh/GUS transgene in the roots and leaves. From the microarray analyses that surveyed 8000 genes, 114 genes were differentially expressed to a degree greater than 2.5 fold over the control. These results were quantitatively corroborated by qRT-PCR examination of 4 of the 114 genes. Conclusion The data suggest that magnetic fields in excess of 15 Tesla have far-reaching effect on the genome. The wide-spread induction of stress-related genes and transcription factors, and a depression of genes associated with cell wall metabolism, are prominent examples. The roles of magnetic field orientation of macromolecules and magnetophoretic effects are discussed as possible factors that contribute to the mounting of this response. PMID:17187667

  13. Roles of factorial noise in inducing bimodal gene expression

    NASA Astrophysics Data System (ADS)

    Liu, Peijiang; Yuan, Zhanjiang; Huang, Lifang; Zhou, Tianshou

    2015-06-01

    Some gene regulatory systems can exhibit bimodal distributions of mRNA or protein although the deterministic counterparts are monostable. This noise-induced bimodality is an interesting phenomenon and has important biological implications, but it is unclear how different sources of expression noise (each source creates so-called factorial noise that is defined as a component of the total noise) contribute separately to this stochastic bimodality. Here we consider a minimal model of gene regulation, which is monostable in the deterministic case. Although simple, this system contains factorial noise of two main kinds: promoter noise due to switching between gene states and transcriptional (or translational) noise due to synthesis and degradation of mRNA (or protein). To better trace the roles of factorial noise in inducing bimodality, we also analyze two limit models, continuous and adiabatic approximations, apart from the exact model. We show that in the case of slow gene switching, the continuous model where only promoter noise is considered can exhibit bimodality; in the case of fast switching, the adiabatic model where only transcriptional or translational noise is considered can also exhibit bimodality but the exact model cannot; and in other cases, both promoter noise and transcriptional or translational noise can cooperatively induce bimodality. Since slow gene switching and large protein copy numbers are characteristics of eukaryotic cells, whereas fast gene switching and small protein copy numbers are characteristics of prokaryotic cells, we infer that eukaryotic stochastic bimodality is induced mainly by promoter noise, whereas prokaryotic stochastic bimodality is induced primarily by transcriptional or translational noise.

  14. Roles of factorial noise in inducing bimodal gene expression.

    PubMed

    Liu, Peijiang; Yuan, Zhanjiang; Huang, Lifang; Zhou, Tianshou

    2015-06-01

    Some gene regulatory systems can exhibit bimodal distributions of mRNA or protein although the deterministic counterparts are monostable. This noise-induced bimodality is an interesting phenomenon and has important biological implications, but it is unclear how different sources of expression noise (each source creates so-called factorial noise that is defined as a component of the total noise) contribute separately to this stochastic bimodality. Here we consider a minimal model of gene regulation, which is monostable in the deterministic case. Although simple, this system contains factorial noise of two main kinds: promoter noise due to switching between gene states and transcriptional (or translational) noise due to synthesis and degradation of mRNA (or protein). To better trace the roles of factorial noise in inducing bimodality, we also analyze two limit models, continuous and adiabatic approximations, apart from the exact model. We show that in the case of slow gene switching, the continuous model where only promoter noise is considered can exhibit bimodality; in the case of fast switching, the adiabatic model where only transcriptional or translational noise is considered can also exhibit bimodality but the exact model cannot; and in other cases, both promoter noise and transcriptional or translational noise can cooperatively induce bimodality. Since slow gene switching and large protein copy numbers are characteristics of eukaryotic cells, whereas fast gene switching and small protein copy numbers are characteristics of prokaryotic cells, we infer that eukaryotic stochastic bimodality is induced mainly by promoter noise, whereas prokaryotic stochastic bimodality is induced primarily by transcriptional or translational noise. PMID:26172735

  15. Virus-Induced Gene Silencing in Hexaploid Wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Functional genomics analysis in hexaploid wheat is greatly impeded by the genetic redundancy of polyploidy and the difficulties in generating large numbers of transgenic plants required in insertional mutagenesis strategies. Virus-induced gene silencing (VIGS), however, is a strategy for creating g...

  16. A geminivirus-induced gene silencing system for gene function validation in cassava.

    PubMed

    Fofana, Ismael B F; Sangaré, Abdourahamane; Collier, Ray; Taylor, Christopher; Fauquet, Claude M

    2004-11-01

    We have constructed an African cassava mosaic virus (ACMV) based gene-silencing vector as a reverse genetics tool for gene function analysis in cassava. The vector carrying a fragment from the Nicotiana tabacum sulfur gene (su), encoding one unit of the chloroplast enzyme magnesium chelatase, was used to induce the silencing of the cassava orthologous gene resulting in yellow-white spots characteristic of the inhibition of su expression. This result suggests that well developed sequence databases from model plants like Arabidopsis thaliana, Nicotiana benthamiana, N. tabacum, Lycopersicon esculentum and others could be used as a major source of information and sequences for functional genomics in cassava. Furthermore, a fragment of the cassava CYP79D2 endogenous gene, sharing 89% homology with CYP79D1 endogenous gene was inserted into the ACMV vector. The resultant vector was inducing the down regulation of the expression of these two genes which catalyze the first-dedicated step in the synthesis of linamarin, the major cyanogenic glycoside in cassava. At 21 days post-inoculation (dpi), a 76% reduction of linamarin content was observed in silenced leaves. Using transgenic plants expressing antisense RNA of CYP79D1 and CYP79D2, Siritunga and Sayre (2003) obtained several lines with a reduction level varying from 60% to 94%. This result provides the first example of direct comparison of the efficiency of a virus-induced gene silencing (VIGS) system and the transgenic approach for suppression of a biosynthetic pathway. The ACMV VIGS system will certainly be a complement and in some cases an alternative to the transgenic approach, for gene discovery and gene function analysis in cassava. PMID:15630624

  17. Hypergravity-induced changes in gene expression in Arabidopsis hypocotyls

    NASA Astrophysics Data System (ADS)

    Yoshioka, R.; Soga, K.; Wakabayashi, K.; Takeba, G.; Hoson, T.

    2003-05-01

    Under hypergravity conditions, the cell wall of stem organs becomes mechanically rigid and elongation growth is suppressed, which can be recognized as the mechanism for plants to resist gravitational force. The changes in gene expression by hypergravity treatment were analyzed in Arabidopsis hypocotyls by the differential display method, for identifying genes involved in hypergravity-induced growth suppression. Sixty-two cDNA clones were expressed differentially between the control and 300 g conditions: the expression levels of 39 clones increased, whereas those of 23 clones decreased under hypergravity conditions. Sequence analysis and database searching revealed that 12 clones, 9 up-regulated and 3 down-regulated, have homology to known proteins. The expression of these genes was further analyzed using RT-PCR. Finally, six genes were confirmed to be up-regulated by hypergravity. One of such genes encoded 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGR), which catalyzes a reaction producing mevalonic acid, a key precursor ofterpenoids such as membrane sterols and several types of hormones. The expression of HMGR gene increased within several hours after hypergravity treatment. Also, compactin, an inhibitor of HMGR, prevented hypergravity-induced growth suppression, suggesting that HMGR is involved in suppression of Arabidopsis hypocotyl growth by hypergravity. In addition, hypergravity increased the expression levels of genes encoding CCR1 and ERD15, which were shown to take part in the signaling pathway of environmental stimuli such as temperature and water, and those of the α-tubulin gene. These genes may be involved in a series of cellular events leading to growth suppression of stem organs under hypergravity conditions.

  18. Benzoic Acid-Inducible Gene Expression in Mycobacteria

    PubMed Central

    Dragset, Marte S.; Barczak, Amy K.; Kannan, Nisha; Mærk, Mali; Flo, Trude H.; Valla, Svein; Rubin, Eric J.; Steigedal, Magnus

    2015-01-01

    Conditional expression is a powerful tool to investigate the role of bacterial genes. Here, we adapt the Pseudomonas putida-derived positively regulated XylS/Pm expression system to control inducible gene expression in Mycobacterium smegmatis and Mycobacterium tuberculosis, the causative agent of human tuberculosis. By making simple changes to a Gram-negative broad-host-range XylS/Pm-regulated gene expression vector, we prove that it is possible to adapt this well-studied expression system to non-Gram-negative species. With the benzoic acid-derived inducer m-toluate, we achieve a robust, time- and dose-dependent reversible induction of Pm-mediated expression in mycobacteria, with low background expression levels. XylS/Pm is thus an important addition to existing mycobacterial expression tools, especially when low basal expression is of particular importance. PMID:26348349

  19. Foxtail Mosaic Virus-Induced Gene Silencing in Monocot Plants.

    PubMed

    Liu, Na; Xie, Ke; Jia, Qi; Zhao, Jinping; Chen, Tianyuan; Li, Huangai; Wei, Xiang; Diao, Xianmin; Hong, Yiguo; Liu, Yule

    2016-07-01

    Virus-induced gene silencing (VIGS) is a powerful technique to study gene function in plants. However, very few VIGS vectors are available for monocot plants. Here we report that Foxtail mosaic virus (FoMV) can be engineered as an effective VIGS system to induce efficient silencing of endogenous genes in monocot plants including barley (Hordeum vulgare L.), wheat (Triticum aestivum) and foxtail millet (Setaria italica). This is evidenced by FoMV-based silencing of phytoene desaturase (PDS) and magnesium chelatase in barley, of PDS and Cloroplastos alterados1 in foxtail millet and wheat, and of an additional gene IspH in foxtail millet. Silencing of these genes resulted in photobleached or chlorosis phenotypes in barley, wheat, and foxtail millet. Furthermore, our FoMV-based gene silencing is the first VIGS system reported for foxtail millet, an important C4 model plant. It may provide an efficient toolbox for high-throughput functional genomics in economically important monocot crops. PMID:27225900

  20. Non-targeted effects of virus-induced gene silencing vectors on host endogenous gene expression.

    PubMed

    Oláh, Enikő; Pesti, Réka; Taller, Dénes; Havelda, Zoltán; Várallyay, Éva

    2016-09-01

    Virus-induced gene silencing (VIGS) uses recombinant viruses to study gene function; however, the effect of the virus vector itself on the gene expression of the host is not always considered. In our work, we investigated non-targeted gene expression changes of the host in order to see how often these changes appear. Effects of various VIGS vector infections were analysed by monitoring gene expression levels of housekeeping genes by Northern blot analysis in four different hosts. We found that non-targeted changes happens very often. More importantly, these non-targeted effects can cause drastic changes in the gene-expression pattern of host genes that are usually used as references in these studies. We have also found that in a tobacco rattle virus (TRV)-based VIGS, the presence of foreign sequences in the cloning site of the vector can also have a non-targeted effect, and even the use of an internal control can lead to unpredicted changes. Our results show that although VIGS is a very powerful technique, the VIGS vector, as a pathogen of the host, can cause unwanted changes in its gene-expression pattern, highlighting the importance of careful selection of both the genes to be tested and those to be used as references in the planned experiments. PMID:27283101

  1. Microarray studies of psychostimulant-induced changes in gene expression.

    PubMed

    Yuferov, Vadim; Nielsen, David; Butelman, Eduardo; Kreek, Mary Jeanne

    2005-03-01

    Alterations in the expression of multiple genes in many brain regions are likely to contribute to psychostimulant-induced behaviours. Microarray technology provides a powerful tool for the simultaneous interrogation of gene expression levels of a large number of genes. Several recent experimental studies, reviewed here, demonstrate the power, limitations and progress of microarray technology in the field of psychostimulant addiction. These studies vary in the paradigms of cocaine or amphetamine administration, drug doses, route and also mode of administration, duration of treatment, animal species, brain regions studied and time of tissue collection after final drug administration. The studies also utilize different microarray platforms and statistical techniques for analysis of differentially expressed genes. These variables influence substantially the results of these studies. It is clear that current microarray techniques cannot detect small changes reliably in gene expression of genes with low expression levels, including functionally significant changes in components of major neurotransmission systems such as glutamate, dopamine, opioid and GABA receptors, especially those that may occur after chronic drug administration or drug withdrawal. However, the microarray studies reviewed here showed cocaine- or amphetamine-induced alterations in the expression of numerous genes involved in the modulation of neuronal growth, cytoskeletal structures, synaptogenesis, signal transduction, apoptosis and cell metabolism. Application of laser capture microdissection and single-cell cDNA amplification may greatly enhance microarray studies of gene expression profiling. The combination of rapidly evolving microarray technology with established methods of neuroscience, molecular biology and genetics, as well as appropriate behavioural models of drug reinforcement, may provide a productive approach for delineating the neurobiological underpinnings of drug responses that lead to

  2. Identification of genes induced by neuregulin in cultured myotubes.

    PubMed

    Fu, A K; Cheung, W M; Ip, F C; Ip, N Y

    1999-09-01

    The formation of the neuromuscular junction (NMJ) involves a series of inductive interactions between motor neurons and muscle fibers. The neural signals proposed to induce the mRNA expression of acetylcholine receptors in muscle include neuregulin (NRG). In the present study, we have employed RNA fingerprinting by arbitrarily primed PCR analysis to identify the differentially expressed transcripts following NRG treatment in cultured myotubes. Nine partial cDNA fragments were isolated; the mRNA expression of eight of these genes was found to be up-regulated by NRG. The spatial and temporal expression profiles of these NRG-regulated genes in rat tissues during development suggest potential functional roles during the formation of NMJ in vivo. Our findings not only allowed the identification of novel genes, but also suggested possible functions for some known genes that are consistent with their potential roles at the NMJ. Furthermore, the identification of G-protein beta1 subunit and G-protein-coupled receptor as NRG-regulated genes has provided the first demonstration that activation of the NRG signaling pathway can induce the expression of components in the G-protein signaling cascade. PMID:10576892

  3. Evaluating the ability of the barley stripe mosaic virus-induced gene silencing system to simultaneously silence two wheat genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is an important tool for rapid assessment of gene function in plants. The ability of the Barley Stripe Mosaic Virus (BSMV) VIGS system to simultaneously silence two genes was assessed by comparing the extent of down-regulation of the wheat PDS and SGT1 genes afte...

  4. Evaluating the Ability of the Barley Stripe Mosaic Virus-Induced Gene Silencing System to Simultaneously Silence Two Wheat Genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is an important tool for rapid assessment of gene function in plants. The ability of the Barley stripe mosaic virus (BSMV) VIGS system to simultaneously silence two genes was assessed by comparing the extent of down-regulation of the wheat PDS and SGT1 genes afte...

  5. Gene expression profiling of replicative and induced senescence.

    PubMed

    Purcell, Maggie; Kruger, Adele; Tainsky, Michael A

    2014-01-01

    Cellular senescence is a cell cycle arrest accompanied by high expression of cyclin dependent kinase inhibitors which counteract overactive growth signals, which serves as a tumor suppressive mechanism. Senescence can be a result of telomere shortening (natural or replicative senescence) or DNA damage resulting from exogenous stressors (induced senescence). Here, we performed gene expression profiling through RNA-seq of replicative senescence, adriamycin-induced senescence, H2O2-induced senescence, and 5-aza-2-deoxycytidine-induced senescence in order to profile the pathways controlling various types of senescence. Overall, the pathways common to all 4 types of senescence were related to inflammation and the innate immune system. It was also evident that 5-aza-induced senescence mirrors natural replicative senescence due to telomere shortening. We also examined the prevalence of senescence-associated secretory phenotype (SASP) factors in the RNA-seq data, showing that it is a common characteristic of all 4 types of senescence. In addition, we could discriminate changes in gene expression due to quiescence during cellular senescence from those that were specific to senescence. PMID:25483067

  6. Applications and advantages of virus-induced gene silencing for gene function studies in plants.

    PubMed

    Burch-Smith, Tessa M; Anderson, Jeffrey C; Martin, Gregory B; Dinesh-Kumar, S P

    2004-09-01

    Virus-induced gene silencing (VIGS) is a recently developed gene transcript suppression technique for characterizing the function of plant genes. The approach involves cloning a short sequence of a targeted plant gene into a viral delivery vector. The vector is used to infect a young plant, and in a few weeks natural defense mechanisms of the plant directed at suppressing virus replication also result in specific degradation of mRNAs from the endogenous plant gene that is targeted for silencing. VIGS is rapid (3-4 weeks from infection to silencing), does not require development of stable transformants, allows characterization of phenotypes that might be lethal in stable lines, and offers the potential to silence either individual or multiple members of a gene family. Here we briefly review the discoveries that led to the development of VIGS and what is known about the experimental requirements for effective silencing. We describe the methodology of VIGS and how it can be optimized and used for both forward and reverse genetics studies. Advantages and disadvantages of VIGS compared with other loss-of-function approaches available for plants are discussed, along with how the limitations of VIGS might be overcome. Examples are reviewed where VIGS has been used to provide important new insights into the roles of specific genes in plant development and plant defense responses. Finally, we examine the future prospects for VIGS as a powerful tool for assessing and characterizing the function of plant genes. PMID:15315635

  7. INDUCIBLE RNAi-MEDIATED GENE SILENCING USING NANOSTRUCTURED GENE DELIVERY ARRAYS

    SciTech Connect

    Mann, David George James; McKnight, Timothy E; Mcpherson, Jackson; Hoyt, Peter R; Melechko, Anatoli Vasilievich; Simpson, Michael L; Sayler, Gary Steven

    2008-01-01

    RNA interference has become a powerful biological tool over the last decade. In this study, a tetracycline-inducible shRNA vector system was designed for silencing CFP expression and delivered alongside the yfp marker gene into Chinese hamster ovary cells using impalefection on spatially indexed vertically aligned carbon nanofiber arrays (VACNFs). The VACNF architecture provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of interfaced cells, and repeated monitoring of single cells over time. Following impalefection and tetracycline induction, 53.1% 10.4% of impalefected cells were fully silenced by the inducible CFP-silencing shRNA vector. Additionally, efficient CFP-silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enables rapid analysis of gene silencing effects using RNAi in single cells and cell populations.

  8. Screening Helicobacter pylori genes induced during infection of mouse stomachs

    PubMed Central

    Singh, Aparna; Hodgson, Nathaniel; Yan, Ming; Joo, Jungsoo; Gu, Lei; Sang, Hong; Gregory-Bryson, Emmalena; Wood, William G; Ni, Yisheng; Smith, Kimberly; Jackson, Sharon H; Coleman, William G

    2012-01-01

    AIM: To investigate the effect of in vivo environment on gene expression in Helicobacter pylori (H. pylori) as it relates to its survival in the host. METHODS: In vivo expression technology (IVET) systems are used to identify microbial virulence genes. We modified the IVET-transcriptional fusion vector, pIVET8, which uses antibiotic resistance as the basis for selection of candidate genes in host tissues to develop two unique IVET-promoter-screening vectors, pIVET11 and pIVET12. Our novel IVET systems were developed by the fusion of random Sau3A DNA fragments of H. pylori and a tandem-reporter system of chloramphenicol acetyltransferase and beta-galactosidase. Additionally, each vector contains a kanamycin resistance gene. We used a mouse macrophage cell line, RAW 264.7 and mice, as selective media to identify specific genes that H. pylori expresses in vivo. Gene expression studies were conducted by infecting RAW 264.7 cells with H. pylori. This was followed by real time polymerase chain reaction (PCR) analysis to determine the relative expression levels of in vivo induced genes. RESULTS: In this study, we have identified 31 in vivo induced (ivi) genes in the initial screens. These 31 genes belong to several functional gene families, including several well-known virulence factors that are expressed by the bacterium in infected mouse stomachs. Virulence factors, vacA and cagA, were found in this screen and are known to play important roles in H. pylori infection, colonization and pathogenesis. Their detection validates the efficacy of these screening systems. Some of the identified ivi genes have already been implicated to play an important role in the pathogenesis of H. pylori and other bacterial pathogens such as Escherichia coli and Vibrio cholerae. Transcription profiles of all ivi genes were confirmed by real time PCR analysis of H. pylori RNA isolated from H. pylori infected RAW 264.7 macrophages. We compared the expression profile of H. pylori and RAW 264

  9. Early antiviral response and virus-induced genes in fish.

    PubMed

    Verrier, Eloi R; Langevin, Christelle; Benmansour, Abdenour; Boudinot, Pierre

    2011-12-01

    In fish as in mammals, virus infections induce changes in the expression of many host genes. Studies conducted during the last fifteen years revealed a major contribution of the interferon system in fish antiviral response. This review describes the screening methods applied to compare the impact of virus infections on the transcriptome in different fish species. These approaches identified a "core" set of genes that are strongly induced in most viral infections. The "core" interferon-induced genes (ISGs) are generally conserved in vertebrates, some of them inhibiting a wide range of viruses in mammals. A selection of ISGs -PKR, vig-1/viperin, Mx, ISG15 and finTRIMs - is further analyzed here to illustrate the diversity and complexity of the mechanisms involved in establishing an antiviral state. Most of the ISG-based pathways remain to be directly determined in fish. Fish ISGs are often duplicated and the functional specialization of multigenic families will be of particular interest for future studies. PMID:21414349

  10. CXCR4 gene transfer prevents pressure overload induced heart failure

    PubMed Central

    LaRocca, Thomas J.; Jeong, Dongtak; Kohlbrenner, Erik; Lee, Ahyoung; Chen, JiQiu; Hajjar, Roger J.; Tarzami, Sima T.

    2012-01-01

    Stem cell and gene therapies are being pursued as strategies for repairing damaged cardiac tissue following myocardial infarction in an attempt to prevent heart failure. The chemokine receptor-4 (CXCR4) and its ligand, CXCL12, play a critical role in stem cell recruitment post-acute myocardial infarction. Whereas progenitor cell migration via the CXCL12/CXCR4 axis is well characterized, little is known about the molecular mechanisms of CXCR4 mediated modulation of cardiac hypertrophy and failure. We used gene therapy to test the effects of CXCR4 gene delivery on adverse ventricular remodeling due to pressure overload. We assessed the effect of cardiac overexpression of CXCR4 during trans-aortic constriction (TAC) using a cardiotropic adeno-associated viral vector (AAV9) carrying the CXCR4 gene. Cardiac overexpression of CXCR4 in mice with pressure overload prevented ventricular remodeling, preserved capillary density and maintained function as determined by echocardiography and in vivo hemodynamics. In isolated adult rat cardiac myocytes, CXCL12 treatment prevented isoproterenol induced hypertrophy and interrupted the calcineurin/NFAT pathway. Finally, a complex involving the L-type calcium channel, β2-adenoreceptor, and CXCR4 (Cav1.2/β2AR/CXCR4) was identified in healthy cardiac myocytes and was shown to dissociate as a consequence of heart failure. CXCR4 administered to the heart via gene transfer prevents pressure overload induced heart failure. The identification of CXCR4 participation in a Cav1.2-β2AR regulatory complex provides further insight into the mechanism by which CXCR4 modulates calcium homeostasis and chronic pressure overload responses in the cardiac myocyte. Together these results suggest AAV9.CXCR4 gene therapy is a potential therapeutic approach for congestive heart failure. PMID:22668785

  11. Two host-inducible genes of Rhizobium fredii and characterization of the inducing compound.

    PubMed Central

    Sadowsky, M J; Olson, E R; Foster, V E; Kosslak, R M; Verma, D P

    1988-01-01

    Random transcription fusions with Mu d1(Kan lac) generated three mutants in Rhizobium fredii (strain USDA 201) which showed induction of beta-galactosidase when grown in root exudate of the host plants Glycine max, Phaseolus vulgaris, and Vigna ungliculata. Two genes were isolated from a library of total plasmid DNA of one of the mutants, 3F1. These genes, present in tandem on a 4.2-kilobase HindIII fragment, appear in one copy each on the symbiotic plasmid and do not hybridize to the Rhizobium meliloti common nodulation region. They comprise two separate transcriptional units coding for about 450 and 950 nucleotides, both of which are transcribed in the same direction. The two open reading frames are separated by 586 base pairs, and the 5H regions of the two genes show a common sequence. No similarity was found with the promoter areas of Rhizobium trifolii, R. meliloti, or Bradyrhizobium japonicum nif genes and with any known nodulation genes. Regions homologous to both sequences were detected in EcoRI digests of genomic DNAs from B. japonicum USDA 110, USDA 122, and 61A76, but not in genomic DNA from R. trifolii, Rhizobium leguminosarum, or Rhizobium phaseoli. Mass spectrometry and nuclear magnetic resonance analysis indicated that the inducing compound has properties of 4',7-dihydroxyisoflavone, daidzein. These results suggest that, in addition to common nodulation genes, several other genes appear to be specifically induced by compounds in the root exudate of the host plants. Images PMID:2447061

  12. Efficient Virus-Induced Gene Silencing in Solanum rostratum

    PubMed Central

    Meng, Lan-Huan; Wang, Rui-Heng; Zhu, Ben-Zhong; Zhu, Hong-Liang; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Solanum rostratum is a “super weed” that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum. PMID:27258320

  13. Octylphenol induced gene expression in testes of Frog, Rana chensinensis.

    PubMed

    Li, Xinyi; Liu, Jia; Zhang, Yuhui

    2016-06-01

    Octylphenol (OP) is an endocrine-disrupting chemical (EDC), which can disrupt the reproductive system. To understand the effect of OP, a subtractive cDNA library was constructed using suppression subtractive hybridization (SSH) to identify alterations of gene transcription in the testes of the frog Rana chensinensis after OP exposure. Two hundred positive clones were selected and 134 sequences of gene fragments were produced from the subtractive library randomly. These genes were identified to be involved in metabolic process, cellular process, biological regulation, stimulus, immune system and female pregnancy process. In order to verify the efficiency of the subtractive cDNA library, PSG9 and PAPP-A were analyzed further as two representatives of differentially expressed transcription genes using semi-quantitative RT-PCR. Our result was the first successful construction of the subtractive cDNA library in frog testes after OP treatment. Based on this cDNA library, OP was shown to affect multiple physiological processes including inducing immune response, disrupting the steroid hormone synthesis and influencing spermatogenesis in the testis by up-regulation of specific genes. PMID:26896894

  14. Bitumen fume-induced gene expression profile in rat lung

    SciTech Connect

    Gate, Laurent . E-mail: laurent.gate@inrs.fr; Langlais, Cristina; Micillino, Jean-Claude; Nunge, Herve; Bottin, Marie-Claire; Wrobel, Richard; Binet, Stephane

    2006-08-15

    Exposure to bitumen fumes during paving and roofing activities may represent an occupational health risk. To date, most of the studies performed on the biological effect of asphalt fumes have been done with regard to their content in carcinogenic polycyclic aromatic hydrocarbons (PAH). In order to gain an additional insight into the mechanisms of action of bitumen fumes, we studied their pulmonary effects in rodents following inhalation using the microarray technology. Fisher 344 rats were exposed for 5 days, 6 h/day to bitumen fumes generated at road paving temperature (170 {sup o}C) using a nose-only exposition device. With the intention of studying the early transcriptional events induced by asphalt fumes, lung tissues were collected immediately following exposure and gene expression profiles in control and exposed rats were determined by using oligonucleotide microarrays. Data analysis revealed that genes involved in lung inflammatory response as well as genes associated with PAH metabolization and detoxification were highly expressed in bitumen-exposed animals. In addition, the expression of genes related to elastase activity and its inhibition which are associated with emphysema was also modulated. More interestingly genes coding for monoamine oxidases A and B involved in the metabolism of neurotransmitters and xenobiotics were downregulated in exposed rats. Altogether, these data give additional information concerning the bitumen fumes biological effects and would allow to better review the health effects of occupational asphalt fumes exposure.

  15. Virus-induced gene silencing in eggplant (Solanum melongena).

    PubMed

    Liu, Haiping; Fu, Daqi; Zhu, Benzhong; Yan, Huaxue; Shen, Xiaoying; Zuo, Jinhua; Zhu, Yi; Luo, Yunbo

    2012-06-01

    Eggplant (Solanum melongena) is an economically important vegetable requiring investigation into its various genomic functions. The current limitation in the investigation of genomic function in eggplant is the lack of effective tools available for conducting functional assays. Virus-induced gene silencing (VIGS) has played a critical role in the functional genetic analyses. In this paper, TRV-mediated VIGS was successfully elicited in eggplant. We first cloned the CDS sequence of PDS (PHYTOENE DESATURASE) in eggplant and then silenced the PDS gene. Photo-bleaching was shown on the newly-developed leaves four weeks after agroinoculation, indicating that VIGS can be used to silence genes in eggplant. To further illustrate the reliability of VIGS in eggplant, we selected Chl H, Su and CLA1 as reporters to elicit VIGS using the high-pressure spray method. Suppression of Chl H and Su led to yellow leaves, while the depletion of CLA1 resulted in albino. In conclusion, four genes, PDS, Chl H, Su (Sulfur), CLA1, were down-regulated significantly by VIGS, indicating that the VIGS system can be successfully applied in eggplant and is a reliable tool for the study of gene function. PMID:22268843

  16. Efficient Virus-Induced Gene Silencing in Solanum rostratum.

    PubMed

    Meng, Lan-Huan; Wang, Rui-Heng; Zhu, Ben-Zhong; Zhu, Hong-Liang; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Solanum rostratum is a "super weed" that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum. PMID:27258320

  17. Gas-inducible product gene expression in bioreactors.

    PubMed

    Weber, Wilfried; Rimann, Markus; de Glutz, François-Nicolas; Weber, Eric; Memmert, Klaus; Fussenegger, Martin

    2005-05-01

    Inducible transgene expression technologies are of unmatched potential for biopharmaceutical manufacturing of unstable, growth-impairing and cytotoxic proteins as well as conditional metabolic engineering to improve desired cell phenotypes. Currently available transgene dosing modalities which rely on physical parameters or small-molecule drugs for transgene fine-tuning compromise downstream processing and/or are difficult to implement technologically. The recently designed gas-inducible acetaldehyde-inducible regulation (AIR) technology takes advantage of gaseous acetaldehyde to modulate product gene expression levels. At regulation effective concentrations gaseous acetaldehyde is physiologically inert and approved as food additive by the Federal Drug Administration (FDA). During standard bioreactor operation, gaseous acetaldehyde could simply be administered using standard/existing gas supply tubing and eventually eliminated by stripping with inducer-free air. We have determined key parameters controlling acetaldehyde transfer in three types of bioreactors and designed a mass balance-based model for optimal product gene expression fine-tuning using gaseous acetaldehyde. Operating a standard stirred-tank bioreactor set-up at 10 L scale we have validated AIR technology using CHO-K1-derived serum-free suspension cultures transgenic for gas-inducible production of human interferon-beta (IFN-beta). Gaseous acetaldehyde-inducible IFN-beta production management was fully reversible while maintaining cell viability at over 95% during the entire process. Compatible with standard bioreactor design and downstream processing procedures AIR-based technology will foster novel opportunities for pilot and large-scale manufacturing of difficult-to-produce protein pharmaceuticals. PMID:15885616

  18. Inducible Gene Manipulations in Brain Serotonergic Neurons of Transgenic Rats

    PubMed Central

    Tews, Björn; Bartsch, Dusan

    2011-01-01

    The serotonergic (5-HT) system has been implicated in various physiological processes and neuropsychiatric disorders, but in many aspects its role in normal and pathologic brain function is still unclear. One reason for this might be the lack of appropriate animal models which can address the complexity of physiological and pathophysiological 5-HT functioning. In this respect, rats offer many advantages over mice as they have been the animal of choice for sophisticated neurophysiological and behavioral studies. However, only recently technologies for the targeted and tissue specific modification of rat genes - a prerequisite for a detailed study of the 5-HT system - have been successfully developed. Here, we describe a rat transgenic system for inducible gene manipulations in 5-HT neurons. We generated a Cre driver line consisting of a tamoxifen-inducible CreERT2 recombinase under the control of mouse Tph2 regulatory sequences. Tissue-specific serotonergic Cre recombinase expression was detected in four transgenic TPH2-CreERT2 rat founder lines. For functional analysis of Cre-mediated recombination, we used a rat Cre reporter line (CAG-loxP.EGFP), in which EGFP is expressed after Cre-mediated removal of a loxP-flanked lacZ STOP cassette. We show an in-depth characterisation of this rat Cre reporter line and demonstrate its applicability for monitoring Cre-mediated recombination in all major neuronal subpopulations of the rat brain. Upon tamoxifen induction, double transgenic TPH2-CreERT2/CAG-loxP.EGFP rats show selective and efficient EGFP expression in 5-HT neurons. Without tamoxifen administration, EGFP is only expressed in few 5-HT neurons which confirms minimal background recombination. This 5-HT neuron specific CreERT2 line allows Cre-mediated, inducible gene deletion or gene overexpression in transgenic rats which provides new opportunities to decipher the complex functions of the mammalian serotonergic system. PMID:22140568

  19. Virus-induced gene silencing of Arabidopsis thaliana gene homologues in wheat identifies genes conferring improved drought tolerance

    PubMed Central

    Lapitan, Nora

    2013-01-01

    In a non-model staple crop like wheat (Triticum aestivumI L.), functional validation of potential drought stress responsive genes identified in Arabidopsis could provide gene targets for breeding. Virus-induced gene silencing (VIGS) of genes of interest can overcome the inherent problems of polyploidy and limited transformation potential that hamper functional validation studies in wheat. In this study, three potential candidate genes shown to be involved in abiotic stress response pathways in Arabidopsis thaliana were selected for VIGS experiments in wheat. These include Era1 (enhanced response to abscisic acid), Cyp707a (ABA 8’-hydroxylase), and Sal1 (inositol polyphosphate 1-phosphatase). Gene homologues for these three genes were identified in wheat and cloned in the viral vector barley stripe mosaic virus (BSMV) in the antisense direction, followed by rub inoculation of BSMV viral RNA transcripts onto wheat plants. Quantitative real-time PCR showed that VIGS-treated wheat plants had significant reductions in target gene transcripts. When VIGS-treated plants generated for Era1 and Sal1 were subjected to limiting water conditions, they showed increased relative water content, improved water use efficiency, reduced gas exchange, and better vigour compared to water-stressed control plants inoculated with RNA from the empty viral vector (BSMV0). In comparison, the Cyp707a-silenced plants showed no improvement over BSMV0-inoculated plants under limited water condition. These results indicate that Era1 and Sal1 play important roles in conferring drought tolerance in wheat. Other traits affected by Era1 silencing were also studied. Delayed seed germination in Era1-silenced plants suggests this gene may be a useful target for developing resistance to pre-harvest sprouting. PMID:23364940

  20. Virus-induced gene silencing of fiber-related genes in cotton.

    PubMed

    Tuttle, John R; Haigler, Candace H; Robertson, Dominique Niki

    2015-01-01

    Virus-Induced Gene Silencing (VIGS) is a useful method for transient downregulation of gene expression in crop plants. The geminivirus Cotton leaf crumple virus (CLCrV) has been modified to serve as a VIGS vector for persistent gene silencing in cotton. Here the use of Green Fluorescent Protein (GFP) is described as a marker for identifying silenced tissues in reproductive tissues, a procedure that requires the use of transgenic plants. Suggestions are given for isolating and cloning combinations of target and marker sequences so that the total length of inserted foreign DNA is between 500 and 750 bp. Using this strategy, extensive silencing is achieved with only 200-400 bp of sequence homologous to an endogenous gene, reducing the possibility of off-target silencing. Cotyledons can be inoculated using either the gene gun or Agrobacterium and will continue to show silencing throughout fruit and fiber development. CLCrV is not transmitted through seed, and VIGS is limited to genes expressed in the maternally derived seed coat and fiber in the developing seed. This complicates the use of GFP as a marker for VIGS because cotton fibers must be separated from unsilenced tissue in the seed to determine if they are silenced. Nevertheless, fibers from a large number of seeds can be rapidly screened following placement into 96-well plates. Methods for quantifying the extent of silencing using semiquantitative RT-PCR are given. PMID:25740368

  1. Functional analyses of cellulose synthase genes in flax (Linum usitatissimum) by virus-induced gene silencing.

    PubMed

    Chantreau, Maxime; Chabbert, Brigitte; Billiard, Sylvain; Hawkins, Simon; Neutelings, Godfrey

    2015-12-01

    Flax (Linum usitatissimum) bast fibres are located in the stem cortex where they play an important role in mechanical support. They contain high amounts of cellulose and so are used for linen textiles and in the composite industry. In this study, we screened the annotated flax genome and identified 14 distinct cellulose synthase (CESA) genes using orthologous sequences previously identified. Transcriptomics of 'primary cell wall' and 'secondary cell wall' flax CESA genes showed that some were preferentially expressed in different organs and stem tissues providing clues as to their biological role(s) in planta. The development for the first time in flax of a virus-induced gene silencing (VIGS) approach was used to functionally evaluate the biological role of different CESA genes in stem tissues. Quantification of transcript accumulation showed that in many cases, silencing not only affected targeted CESA clades, but also had an impact on other CESA genes. Whatever the targeted clade, inactivation by VIGS affected plant growth. In contrast, only clade 1- and clade 6-targeted plants showed modifications in outer-stem tissue organization and secondary cell wall formation. In these plants, bast fibre number and structure were severely impacted, suggesting that the targeted genes may play an important role in the establishment of the fibre cell wall. Our results provide new fundamental information about cellulose biosynthesis in flax that should facilitate future plant improvement/engineering. PMID:25688574

  2. Cigarette smoke induces methylation of the tumor suppressor gene NISCH

    PubMed Central

    Ostrow, Kimberly Laskie; Michalidi, Christina; Guerrero-Preston, Rafael; Hoque, Mohammad O.; Greenberg, Alissa; Rom, William; Sidransky, David

    2013-01-01

    We have previously identified a putative tumor suppressor gene, NISCH, whose promoter is methylated in lung tumor tissue as well as in plasma obtained from lung cancer patients. NISCH was observed to be more frequently methylated in smoker lung cancer patients than in non-smoker lung cancer patients. Here, we investigated the effect of tobacco smoke exposure on methylation of the NISCH gene. We tested methylation of NISCH after oral keratinocytes were exposed to mainstream and side stream cigarette smoke extract in culture. Methylation of the promoter region of the NISCH gene was also evaluated in plasma obtained from lifetime non-smokers and light smokers (< 20 pack/year), with and without lung tumors, and heavy smokers (20+ pack/year) without disease. Promoter methylation of NISCH was tested by quantitative fluorogenic real-time PCR in all samples. Promoter methylation of NISCH occurred after exposure to mainstream tobacco smoke as well as to side stream tobacco smoke in normal oral keratinocyte cell lines. NISCH methylation was also detected in 68% of high-risk, heavy smokers without detectable tumors. Interestingly, in light smokers, NISCH methylation was present in 69% of patients with lung cancer and absent in those without disease. Our pilot study indicates that tobacco smoke induces methylation changes in the NISCH gene promoter before any detectable cancer. Methylation of the NISCH gene was also found in lung cancer patients’ plasma samples. After confirming these findings in longitudinally collected plasma samples from high-risk populations (such as heavy smokers), examining patients for hypermethylation of the NISCH gene may aid in identifying those who should undergo additional screening for lung cancer. PMID:23503203

  3. JC virus induces altered patterns of cellular gene expression: Interferon-inducible genes as major transcriptional targets

    SciTech Connect

    Verma, Saguna; Ziegler, Katja; Ananthula, Praveen; Co, Juliene K.G.; Frisque, Richard J.; Yanagihara, Richard; Nerurkar, Vivek R. . E-mail: nerurkar@pbrc.hawaii.edu

    2006-02-20

    Human polyomavirus JC (JCV) infects 80% of the population worldwide. Primary infection, typically occurring during childhood, is asymptomatic in immunocompetent individuals and results in lifelong latency and persistent infection. However, among the severely immunocompromised, JCV may cause a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Virus-host interactions influencing persistence and pathogenicity are not well understood, although significant regulation of JCV activity is thought to occur at the level of transcription. Regulation of the JCV early and late promoters during the lytic cycle is a complex event that requires participation of both viral and cellular factors. We have used cDNA microarray technology to analyze global alterations in gene expression in JCV-permissive primary human fetal glial cells (PHFG). Expression of more than 400 cellular genes was altered, including many that influence cell proliferation, cell communication and interferon (IFN)-mediated host defense responses. Genes in the latter category included signal transducer and activator of transcription 1 (STAT1), interferon stimulating gene 56 (ISG56), myxovirus resistance 1 (MxA), 2'5'-oligoadenylate synthetase (OAS), and cig5. The expression of these genes was further confirmed in JCV-infected PHFG cells and the human glioblastoma cell line U87MG to ensure the specificity of JCV in inducing this strong antiviral response. Results obtained by real-time RT-PCR and Western blot analyses supported the microarray data and provide temporal information related to virus-induced changes in the IFN response pathway. Our data indicate that the induction of an antiviral response may be one of the cellular factors regulating/controlling JCV replication in immunocompetent hosts and therefore constraining the development of PML.

  4. Virus-induced gene silencing and transient gene expression in soybean using Bean pod mottle virus infectious clones

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is a powerful and rapid approach for determining the functions of plant genes. The basis of VIGS is that a viral genome is engineered so that it can carry fragments of plant genes, typically in the 200-300 base pair size range. The recombinant viruses are used to ...

  5. Targeted genes and interacting proteins of hypoxia inducible factor-1

    PubMed Central

    Liu, Wei; Shen, Shao-Ming; Zhao, Xu-Yun; Chen, Guo-Qiang

    2012-01-01

    Heterodimeric transcription factor hypoxia inducible factor-1 (HIF-1) functions as a master regulator of oxygen homeostasis in almost all nucleated mammalian cells. The fundamental process adapted to cellular oxygen alteration largely depends on the refined regulation on its alpha subunit, HIF-1α. Recent studies have unraveled expanding and critical roles of HIF-1α, involving in a multitude of developmental, physiological, and pathophysiological processes. This review will focus on the current knowledge of HIF-1α-targeting genes and its interacting proteins, as well as the concomitant functional relationships between them. PMID:22773957

  6. Pneumococcal Hydrogen Peroxide–Induced Stress Signaling Regulates Inflammatory Genes

    PubMed Central

    Loose, Maria; Hudel, Martina; Zimmer, Klaus-Peter; Garcia, Ernesto; Hammerschmidt, Sven; Lucas, Rudolf; Chakraborty, Trinad; Pillich, Helena

    2015-01-01

    Microbial infections can induce aberrant responses in cellular stress pathways, leading to translational attenuation, metabolic restriction, and activation of oxidative stress, with detrimental effects on cell survival. Here we show that infection of human airway epithelial cells with Streptococcus pneumoniae leads to induction of endoplasmic reticulum (ER) and oxidative stress, activation of mitogen-associated protein kinase (MAPK) signaling pathways, and regulation of their respective target genes. We identify pneumococcal H2O2 as the causative agent for these responses, as both catalase-treated and pyruvate oxidase-deficient bacteria lacked these activities. Pneumococcal H2O2 induced nuclear NF-κB translocation and transcription of proinflammatory cytokines. Inhibition of translational arrest and ER stress by salubrinal or of MAPK signaling pathways attenuate cytokine transcription. These results provide strong evidence for the notion that inhibition of translation is an important host pathway in monitoring harmful pathogen-associated activities, thereby enabling differentiation between pathogenic and nonpathogenic bacteria. PMID:25183769

  7. Putting the diet back into diet-induced obesity: diet-induced hypothalamic gene expression.

    PubMed

    Mercer, Julian G; Archer, Zoë A

    2008-05-01

    A wealth of detailed mechanistic information relating to obesity and body weight regulation has emerged from study of single gene mutation models, and continues to be generated by engineered rodent models targeting specific genes. However, as an early step in translational research, many researchers are turning to models of diet-induced obesity. Interpretation of data generated from such models is not aided by the variety of diets and rodent strains employed in these studies and a strong case could be made for rationalisation. Differences in experimental protocol, which may deploy a single obligatory solid diet, a choice of solid diets, or liquid/solid combinations, and which may or may not allow a preferred macronutrient composition to be selected, mean that different models of diet-induced obesity achieve that obesity by different routes. The priority should be to mimic the palatability- and choice-driven over-consumption that probably underlies the majority of human obesity. Some of the hypothalamic energy balance genes apparently 'recognise' developing diet-induced obesity as indicated by counter-regulatory changes in expression levels. However, substantial changes in gene expression on long-term exposure to obesogenic diets are not able to prevent weight gain. Forebrain reward systems are widely assumed to be overriding hypothalamic homeostatic energy balance systems under these circumstances. More mechanism-based research at the homeostatic/reward/diet interface may enable diets to be manipulated with therapeutic benefit, or define the contribution of these interactions to susceptibility to diet-induced obesity. PMID:18342851

  8. Gene Dosage Imbalance Contributes to Chromosomal Instability-Induced Tumorigenesis.

    PubMed

    Clemente-Ruiz, Marta; Murillo-Maldonado, Juan M; Benhra, Najate; Barrio, Lara; Pérez, Lidia; Quiroga, Gonzalo; Nebreda, Angel R; Milán, Marco

    2016-02-01

    Chromosomal instability (CIN) is thought to be a source of mutability in cancer. However, CIN often results in aneuploidy, which compromises cell fitness. Here, we used the dosage compensation mechanism (DCM) of Drosophila to demonstrate that chromosome-wide gene dosage imbalance contributes to the deleterious effects of CIN-induced aneuploidy and its pro-tumorigenic action. We present evidence that resetting of the DCM counterbalances the damaging effects caused by CIN-induced changes in X chromosome number. Importantly, interfering with the DCM suffices to mimic the cellular effects of aneuploidy in terms of reactive oxygen species (ROS) production, JNK-dependent cell death, and tumorigenesis upon apoptosis inhibition. We unveil a role of ROS in JNK activation and a variety of cellular and tissue-wide mechanisms that buffer the deleterious effects of CIN, including DNA-damage repair, activation of the p38 pathway, and cytokine induction to promote compensatory proliferation. Our data reveal the existence of robust compensatory mechanisms that counteract CIN-induced cell death and tumorigenesis. PMID:26859353

  9. Functionalized nanoparticles for AMF-induced gene and drug delivery

    NASA Astrophysics Data System (ADS)

    Biswas, Souvik

    The properties and broad applications of nano-magnetic colloids have generated much interest in recent years. Specially, Fe3O4 nanoparticles have attracted a great deal of attention since their magnetic properties can be used for hyperthermia treatment or drug targeting. For example, enhanced levels of intracellular gene delivery can be achieved using Fe3O4 nano-vectors in the presence of an external magnetic field, a process known as 'magnetofection'. The low cytotoxicity, tunable particle size, ease of surface functionalization, and ability to generate thermal energy using an external alternating magnetic field (AMF) are properties have propelled Fe3O4 research to the forefront of nanoparticle research. The strategy of nanoparticle-mediated, AMF-induced heat generation has been used to effect intracellular hyperthermia. One application of this 'magnetic hyperthermia' is heat activated local delivery of a therapeutic effector (e.g.; drug or polynucleotide). This thesis describes the development of a magnetic nano-vector for AMF-induced, heat-activated pDNA and small molecule delivery. The use of heat-inducible vectors, such as heat shock protein ( hsp) genes, is a promising mode of gene therapy that would restrict gene expression to a local region by focusing a heat stimulus only at a target region. We thus aimed to design an Fe3O4 nanoparticle-mediated gene transfer vehicle for AMF-induced localized gene expression. We opted to use 'click' oximation techniques to assemble the magnetic gene transfer vector. Chapter 2 describes the synthesis, characterization, and transfection studies of the oxime ether lipid-based nano-magnetic vectors MLP and dMLP. The synthesis and characterization of a novel series of quaternary ammonium aminooxy reagents (2.1--2.4) is described. These cationic aminooxy compounds were loaded onto nanoparticles for ligation with carbonyl groups and also to impart a net positive charge on the nanoparticle surface. Our studies indicated that the

  10. Genomic Analysis Reveals Contrasting PIFq Contribution to Diurnal Rhythmic Gene Expression in PIF-Induced and -Repressed Genes.

    PubMed

    Martin, Guiomar; Soy, Judit; Monte, Elena

    2016-01-01

    Members of the PIF quartet (PIFq; PIF1, PIF3, PIF4, and PIF5) collectively contribute to induce growth in Arabidopsis seedlings under short day (SD) conditions, specifically promoting elongation at dawn. Their action involves the direct regulation of growth-related and hormone-associated genes. However, a comprehensive definition of the PIFq-regulated transcriptome under SD is still lacking. We have recently shown that SD and free-running (LL) conditions correspond to "growth" and "no growth" conditions, respectively, correlating with greater abundance of PIF protein in SD. Here, we present a genomic analysis whereby we first define SD-regulated genes at dawn compared to LL in the wild type, followed by identification of those SD-regulated genes whose expression depends on the presence of PIFq. By using this sequential strategy, we have identified 349 PIF/SD-regulated genes, approximately 55% induced and 42% repressed by both SD and PIFq. Comparison with available databases indicates that PIF/SD-induced and PIF/SD-repressed sets are differently phased at dawn and mid-morning, respectively. In addition, we found that whereas rhythmicity of the PIF/SD-induced gene set is lost in LL, most PIF/SD-repressed genes keep their rhythmicity in LL, suggesting differential regulation of both gene sets by the circadian clock. Moreover, we also uncovered distinct overrepresented functions in the induced and repressed gene sets, in accord with previous studies in other examined PIF-regulated processes. Interestingly, promoter analyses showed that, whereas PIF/SD-induced genes are enriched in direct PIF targets, PIF/SD-repressed genes are mostly indirectly regulated by the PIFs and might be more enriched in ABA-regulated genes. PMID:27458465

  11. Genomic Analysis Reveals Contrasting PIFq Contribution to Diurnal Rhythmic Gene Expression in PIF-Induced and -Repressed Genes

    PubMed Central

    Martin, Guiomar; Soy, Judit; Monte, Elena

    2016-01-01

    Members of the PIF quartet (PIFq; PIF1, PIF3, PIF4, and PIF5) collectively contribute to induce growth in Arabidopsis seedlings under short day (SD) conditions, specifically promoting elongation at dawn. Their action involves the direct regulation of growth-related and hormone-associated genes. However, a comprehensive definition of the PIFq-regulated transcriptome under SD is still lacking. We have recently shown that SD and free-running (LL) conditions correspond to “growth” and “no growth” conditions, respectively, correlating with greater abundance of PIF protein in SD. Here, we present a genomic analysis whereby we first define SD-regulated genes at dawn compared to LL in the wild type, followed by identification of those SD-regulated genes whose expression depends on the presence of PIFq. By using this sequential strategy, we have identified 349 PIF/SD-regulated genes, approximately 55% induced and 42% repressed by both SD and PIFq. Comparison with available databases indicates that PIF/SD-induced and PIF/SD-repressed sets are differently phased at dawn and mid-morning, respectively. In addition, we found that whereas rhythmicity of the PIF/SD-induced gene set is lost in LL, most PIF/SD-repressed genes keep their rhythmicity in LL, suggesting differential regulation of both gene sets by the circadian clock. Moreover, we also uncovered distinct overrepresented functions in the induced and repressed gene sets, in accord with previous studies in other examined PIF-regulated processes. Interestingly, promoter analyses showed that, whereas PIF/SD-induced genes are enriched in direct PIF targets, PIF/SD-repressed genes are mostly indirectly regulated by the PIFs and might be more enriched in ABA-regulated genes. PMID:27458465

  12. Moderate malnutrition in rats induces somatic gene mutations.

    PubMed

    Pacheco-Martínez, M Monserrat; Cortés-Barberena, Edith; Cervantes-Ríos, Elsa; Del Carmen García-Rodríguez, María; Rodríguez-Cruz, Leonor; Ortiz-Muñiz, Rocío

    2016-07-01

    The relationship between malnutrition and genetic damage has been widely studied in human and animal models, leading to the observation that interactions between genotoxic exposure and micronutrient status appear to affect genomic stability. A new assay has been developed that uses the phosphatidylinositol glycan class A gene (Pig-a) as a reporter for measuring in vivo gene mutation. The Pig-a assay can be employed to evaluate mutant frequencies (MFs) in peripheral blood reticulocytes (RETs) and erythrocytes (RBCs) using flow cytometry. In the present study, we assessed the effects of malnutrition on mutagenic susceptibility by exposing undernourished (UN) and well-nourished (WN) rats to N-ethyl-N-nitrosourea (ENU) and measuring Pig-a MFs. Two week-old UN and WN male Han-Wistar rats were treated daily with 0, 20, or 40mg/kg ENU for 3 consecutive days. Blood was collected from the tail vein one day before ENU treatment (Day-1) and after ENU administration on Days 7, 14, 21, 28, 35, 42, 49, 56 and 63. Pig-a MFs were measured in RETs and RBCs as the RET(CD59-) and RBC(CD59-) frequencies. In the vehicle control groups, the frequencies of mutant RETs and RBCs were significantly higher in UN rats compared with WN rats at all sampling times. The ENU treatments increased RET and RBC MFs starting at Day 7. Although ENU-induced Pig-a MFs were consistently lower in UN rats than in WN rats, these differences were not significant. To understand these responses, further studies should use other mutagens and nucleated surrogate cells and examine the types of mutations induced in UN and WN rats. PMID:26994962

  13. Rapamycin induces pluripotent genes associated with avoidance of replicative senescence

    PubMed Central

    Pospelova, Tatiana V; Bykova, Tatiana V; Zubova, Svetlana G; Katolikova, Natalia V; Yartzeva, Natalia M; Pospelov, Valery A

    2013-01-01

    Primary rodent cells undergo replicative senescence, independent from telomere shortening. We have recently shown that treatment with rapamycin during passages 3–7 suppressed replicative senescence in rat embryonic fibroblasts (REFs), which otherwise occurred by 10–14 passages. Here, we further investigated rapamycin-primed cells for an extended number of passages. Rapamycin-primed cells continued to proliferate without accumulation of senescent markers. Importantly, these cells retained the ability to undergo serum starvation- and etoposide-induced cell cycle arrest. The p53/p21 pathway was functional. This indicates that rapamycin did not cause either transformation or loss of cell cycle checkpoints. We found that rapamycin activated transcription of pluripotent genes, oct-4, sox-2, nanog, as well as further upregulated telomerase (tert) gene. The rapamycin-derived cells have mostly non-rearranged, near-normal karyotype. Still, when cultivated for a higher number of passages, these cells acquired a chromosomal marker within the chromosome 3. We conclude that suppression mTORC1 activity may prevent replicative senescence without transformation of rodent cells. PMID:24296616

  14. Fetal Globin Gene Inducers: Novel Agents & New Potential

    PubMed Central

    Perrine, Susan P.; Castaneda, Serguei A.; Chui, David H.; Faller, Douglas V.; Berenson, Ronald J.; Fucharoen, Suthat

    2013-01-01

    Inducing expression of endogenous fetal globin (γ-globin) gene expression to 60-70% of alpha globin synthesis produces β-thalassemia trait globin synthetic ratios and can reduce anemia to a mild level. Several classes of therapeutics have induced γ-globin expression in beta thalassemia patients and subsequently raised total hemoglobin levels, demonstrating proof-of-concept of the approach. Butyrate treatment eliminated transfusion requirements in formerly transfusion-dependent patients with treatment for as long as 7 years. However, prior generations were not readily applicable for widespread use. Currently, a novel oral dual-action therapeutic sodium 2,2-dimethylbutyrate is in clinical trials, an oral decitabine formulation is under development, and agents with complementary mechanisms of action can be applied in combined regimens. Identification of 3 major genetic trait loci which modulate clinical severity provides avenues for developing tailored regimens. These refinements offer renewed potential to apply fetal globin induction as a treatment approach in patient-friendly regimens that can be used world-wide. PMID:20712788

  15. Replication-induced transcription of an autorepressed gene: The replication initiator gene of plasmid P1

    PubMed Central

    Mukhopadhyay, Suman; Chattoraj, Dhruba K.

    2000-01-01

    The replication origin of plasmid P1 contains an array of five repeats (iterons) that bind the plasmid-encoded initiator RepA. Within the array lies the repA promoter, which becomes largely repressed on RepA binding (autorepression). One might expect that extra iterons produced on plasmid replication would titrate RepA and release the repression. The promoter, however, is induced poorly by extra iterons. The P1 copy number is reduced by extra iterons in the presence of the autorepressed repA gene but not when additional RepA is provided from constitutive sources. It has been proposed that the iteron-bound RepA couples with the promoter-bound RepA and thereby maintains repression. Although not the product of replication, we find that the act of replication itself can renew RepA synthesis. Replication apparently cleans the promoter of bound RepA and provides a window of opportunity for repA transcription. We propose that replication-induced transcription is required to ensure initiator availability in a system that is induced poorly when challenged with additional initiator binding sites. PMID:10840063

  16. An improved chemically inducible gene switch that functions in the monocotyledonous plant sugar cane.

    PubMed

    Kinkema, Mark; Geijskes, R Jason; Shand, Kylie; Coleman, Heather D; De Lucca, Paulo C; Palupe, Anthony; Harrison, Mark D; Jepson, Ian; Dale, James L; Sainz, Manuel B

    2014-03-01

    Chemically inducible gene switches can provide precise control over gene expression, enabling more specific analyses of gene function and expanding the plant biotechnology toolkit beyond traditional constitutive expression systems. The alc gene expression system is one of the most promising chemically inducible gene switches in plants because of its potential in both fundamental research and commercial biotechnology applications. However, there are no published reports demonstrating that this versatile gene switch is functional in transgenic monocotyledonous plants, which include some of the most important agricultural crops. We found that the original alc gene switch was ineffective in the monocotyledonous plant sugar cane, and describe a modified alc system that is functional in this globally significant crop. A promoter consisting of tandem copies of the ethanol receptor inverted repeat binding site, in combination with a minimal promoter sequence, was sufficient to give enhanced sensitivity and significantly higher levels of ethanol inducible gene expression. A longer CaMV 35S minimal promoter than was used in the original alc gene switch also substantially improved ethanol inducibility. Treating the roots with ethanol effectively induced the modified alc system in sugar cane leaves and stem, while an aerial spray was relatively ineffective. The extension of this chemically inducible gene expression system to sugar cane opens the door to new opportunities for basic research and crop biotechnology. PMID:24142380

  17. Methods for Virus-Induced Gene Silencing in Hexaploid Wheat using barley stripe mosaic virus vectors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is a useful functional genomics tool for rapidly creating gene knockout phenotypes that can be used to infer gene function. Until recently, VIGS has only been possible in dicotyledonous plants. However, the development of vectors based on barley stripe mosaic vi...

  18. Identification of promising host-induced silencing targets among genes preferentially transcribed in haustoria of Puccinia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Expression of dsRNA fragments of rust pathogen genes in wheat seedlings through the barley stripe mosaic virus (BSMV) based host-induced gene silencing (HIGS) system can reduce the expression of the corresponding genes in the rust fungus. The highest levels of suppression have generally been observe...

  19. A gold nanoparticle pentapeptide: gene fusion to induce therapeutic gene expression in mesenchymal stem cells.

    PubMed

    Muroski, Megan E; Morgan, Thomas J; Levenson, Cathy W; Strouse, Geoffrey F

    2014-10-22

    Mesenchymal stem cells (MSC) have been identified as having great potential as autologous cell therapeutics to treat traumatic brain injury and spinal injury as well as neuronal and cardiac ischemic events. All future clinical applications of MSC cell therapies must allow the MSC to be harvested, transfected, and induced to express a desired protein or selection of proteins to have medical benefit. For the full potential of MSC cell therapy to be realized, it is desirable to systematically alter the protein expression of therapeutically beneficial biomolecules in harvested MSC cells with high fidelity in a single transfection event. We have developed a delivery platform on the basis of the use of a solid gold nanoparticle that has been surface modified to produce a fusion containing a zwitterionic, pentapeptide designed from Bax inhibiting peptide (Ku70) to enhance cellular uptake and a linearized expression vector to induce enhanced expression of brain-derived neurotrophic factor (BDNF) in rat-derived MSCs. Ku70 is observed to effect >80% transfection following a single treatment of femur bone marrow isolated rat MSCs with efficiencies for the delivery of a 6.6 kbp gene on either a Au nanoparticle (NP) or CdSe/ZnS quantum dot (QD). Gene expression is observed within 4 d by optical measurements, and secretion is observed within 10 d by Western Blot analysis. The combination of being able to selectively engineer the NP, to colocalize biological agents, and to enhance the stability of those agents has provided the strong impetus to utilize this novel class of materials to engineer primary MSCs. PMID:25198921

  20. Regulation of gene expression during the vegetative incompatibility reaction in Podospora anserina. Characterization of three induced genes.

    PubMed Central

    Bourges, N; Groppi, A; Barreau, C; Clavé, C; Bégueret, J

    1998-01-01

    Vegetative incompatibility in fungi limits the formation of viable heterokaryons. It results from the coexpression of incompatible genes in the heterokaryotic cells and leads to a cell death reaction. In Podospora anserina, a modification of gene expression takes place during this reaction, including a strong decrease of total RNA synthesis and the appearance of a new set of proteins. Using in vitro translation of mRNA and separation of protein products by two-dimensional gel electrophoresis, we have shown that the mRNA content of cells is qualitatively modified during the progress of the incompatibility reaction. Thus, gene expression during vegetative incompatibility is regulated, at least in part, by variation of the mRNA content of specific genes. A subtractive cDNA library enriched in sequences preferentially expressed during incompatibility was constructed. This library was used to identify genomic loci corresponding to genes whose mRNA is induced during incompatibility. Three such genes were characterized and named idi genes for genes induced during incompatibility. Their expression profiles suggest that they may be involved in different steps of the incompatibility reaction. The putative IDI proteins encoded by these genes are small proteins with signal peptides. IDI-2 protein is a cysteine-rich protein. IDI-2 and IDI-3 proteins display some similarity in a tryptophan-rich region. PMID:9755195

  1. The bat gene of Halobacterium halobium encodes a trans-acting oxygen inducibility factor.

    PubMed Central

    Gropp, F; Betlach, M C

    1994-01-01

    Oxygen and light affect the expression of the bacterioopsin gene (bop), which encodes a light-driven proton pump in the purple membrane of Halobacterium halobium. This response is thought to be mediated by a set of genes located adjacent to the bop gene. DNA fragments containing either the bop gene or the entire bop gene cluster reversed the phenotype of purple membrane-deficient strains with mutations in the bop gene. Purple membrane synthesis was constitutive in one of these strains transformed with the bop gene alone. The same strain transformed with the bop gene cluster was inducible by low oxygen tension. Moreover, another strain that constitutively expresses purple membrane remained constitutive when transformed with the bop gene alone but the phenotype of the strain changed to inducible when transformed with the bop gene cluster. Additional experiments have confirmed that one of the genes of the bop gene cluster, the bat gene, encodes a trans-acting factor that is necessary and sufficient to confer inducibility of purple membrane synthesis by low oxygen tension. Images PMID:8202511

  2. The HIN-200 family: More than interferon-inducible genes?

    SciTech Connect

    Ludlow, Louise E.A.; Johnstone, Ricky W.; Clarke, Christopher J.P. . E-mail: chris.clarke@petermac.org

    2005-08-01

    The HIN-200 family was initially grouped together based on their hemopoietic expression, interferon-inducibility, nuclear localization, and characteristic 200 amino-acid domains. In this review, we performed a comprehensive search of genome databases and determined the location of previously characterized and predicted genes within the human, mouse, and rat HIN-200 loci. Several novel proteins were predicted in the mouse and rat. We also discuss recent advances in our understanding of this family of proteins and highlight the most important findings. In addition to a role in interferon biology, there is now good evidence supporting a role for these proteins as regulators of cell proliferation and differentiation. The activity of HIN-200 proteins is not restricted to the hemopoietic system as they are expressed and can function in a variety of other cells and tissues. The importance of HIN-200 proteins in disease now is beginning to be understood as they appear to be involved in autoimmunity and may act as tumor suppressor proteins.

  3. Tetranectin gene deletion induces Parkinson's disease by enhancing neuronal apoptosis.

    PubMed

    Chen, Zhifeng; Wang, Ersong; Hu, Rong; Sun, Yu; Zhang, Lei; Jiang, Jue; Zhang, Ying; Jiang, Hong

    Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). We previously identified tetranectin (TET) as a potential biomarker for PD whose expression is downregulated in the cerebrospinal fluid of PD patients. In the present study, we investigate the role of TET in neurodegeneration in vitro and in vivo. Our results showed that siRNA knockdown of TET decreased cell viability and the number of tyrosine hydroxylase (TH) positive cells, whereas it increased caspase-3 activity and the Bax/Bcl-2 ratio in cultured primary dopaminergic neurons. Overexpression of TET protected dopaminergic neurons against neuronal apoptosis in 1-methyl-4-phenylpyridinium cell culture model in vitro. In TET knockdown mouse model of PD, TET gene deletion decreased the number of TH positive cells in the SNpc, induced apoptosis via the p53/Bax pathway, and significantly impaired the motor behavior of transgenic mice. The findings suggest that TET plays a neuroprotective role via reducing neuron apoptosis and could be a valuable biomarker or potential therapeutic target for the treatment of patients with PD. PMID:26597345

  4. p53-induced Gene 3 Mediates Cell Death Induced by Glutathione Peroxidase 3*

    PubMed Central

    Wang, Hui; Luo, Katherine; Tan, Lang-Zhu; Ren, Bao-Guo; Gu, Li-Qun; Michalopoulos, George; Luo, Jian-Hua; Yu, Yan P.

    2012-01-01

    Expression of glutathione peroxidase 3 (GPx3) is down-regulated in a variety of human malignancies. Both methylation and deletion of GPx3 gene underlie the alterations of GPx3 expression in prostate cancer. A strong correlation between the down-regulation of GPx3 expression and progression of prostate cancer and the suppression of prostate cancer xenografts in SCID mice by forced expression of GPx3 suggests a tumor suppression role of GPx3 in prostate cancer. However, the mechanism of GPx3-mediated tumor suppression remains unclear. In this report, GPx3 was found to interact directly with p53-induced gene 3 (PIG3). Forced overexpression of GPx3 in prostate cancer cell lines DU145 and PC3 as well as immortalized prostate epithelial cells RWPE-1 increased apoptotic cell death. Expression of GPx3x73c, a peroxidase-negative OPAL codon mutant, in DU145 and PC3 cells also increased cell death. The induced expression of GPx3 in DU145 and PC3 cells resulted in an increase in reactive oxygen species and caspase-3 activity. These activities were abrogated by either knocking down PIG3 or mutating the PIG3 binding motif in GPx3 or binding interference from a peptide corresponding to PIG3 binding motif in GPx3. In addition, UV-treated RWPE-1 cells underwent apoptotic death, which was partially prevented by knocking down GPx3 or PIG3, suggesting that GPx3-PIG3 signaling is critical for UV-induced apoptosis. Taken together, these results reveal a novel signaling pathway of GPx3-PIG3 in the regulation of cell death in prostate cancer. PMID:22461624

  5. Gene Therapy Induces Antigen-Specific Tolerance in Experimental Collagen-Induced Arthritis

    PubMed Central

    Jirholt, Pernilla; Turesson, Olof; Wing, Kajsa; Holmdahl, Rikard; Kihlberg, Jan; Stern, Anna; Mårtensson, Inga-Lill; Henningsson, Louise; Gustafsson, Kenth; Gjertsson, Inger

    2016-01-01

    Here, we investigate induction of immunological tolerance by lentiviral based gene therapy in a mouse model of rheumatoid arthritis, collagen II-induced arthritis (CIA). Targeting the expression of the collagen type II (CII) to antigen presenting cells (APCs) induced antigen-specific tolerance, where only 5% of the mice developed arthritis as compared with 95% of the control mice. In the CII-tolerized mice, the proportion of Tregs as well as mRNA expression of SOCS1 (suppressors of cytokine signaling 1) increased at day 3 after CII immunization. Transfer of B cells or non-B cell APC, as well as T cells, from tolerized to naïve mice all mediated a certain degree of tolerance. Thus, sustainable tolerance is established very early during the course of arthritis and is mediated by both B and non-B cells as APCs. This novel approach for inducing tolerance to disease specific antigens can be used for studying tolerance mechanisms, not only in CIA but also in other autoimmune diseases. PMID:27159398

  6. Infrared laser-induced gene expression in targeted single cells of Caenorhabditis elegans.

    PubMed

    Suzuki, Motoshi; Toyoda, Naoya; Shimojou, Masaki; Takagi, Shin

    2013-05-01

    Since the dawn of transgenic technology some 40 years ago, biologists have sought ways to manipulate, at their discretion, the expression of particular genes of interest in living organisms. The infrared laser-evoked gene operator (IR-LEGO) is a recently developed system for inducing gene expression in living organisms in a targeted fashion. It exploits the highly efficient capacity of an infrared laser for heating cells, to provide a high level of gene expression driven by heat-inducible promoters. By irradiating living specimens with a laser under a microscope, heat shock responses can be induced in individual cells, thereby inducing a particular gene, under the control of a heat shock promoter, in specifically targeted cells. In this review we first summarize previous attempts to drive transgene expression in organisms by using heat shock promoters, and then introduce the basic principle of the IR-LEGO system, and its applications. PMID:23614811

  7. Functional Inducible Nitric Oxide Synthase Gene Variants Associate With Hypertension

    PubMed Central

    Nikkari, Seppo T.; Määttä, Kirsi M.; Kunnas, Tarja A.

    2015-01-01

    Abstract Increased inducible nitric oxide synthase (iNOS) activity and expression has been associated with hypertension, but less is known whether the 2 known functional polymorphic sites in the iNOS gene (g.–1026 C/A (rs2779249), g.2087 G/A (rs2297518)) affect susceptibility to hypertension. The objective of this study was to investigate the association between the genetic variants of iNOS and diagnosed hypertension in a Finnish cohort. This study included 320 hypertensive cases and 439 healthy controls. All participants were 50-year-old men and women and the data were collected from the Tampere adult population cardiovascular risk study (TAMRISK). DNA was extracted from buccal swabs and iNOS single nucleotide polymorphisms (SNPs) were analyzed using KASP genotyping PCR. Data analysis was done by logistic regression. At the age of 50 years, the SNP rs2779249 (C/A) associated significantly with hypertension (P = 0.009); specifically, subjects carrying the A-allele had higher risk of hypertension compared to those carrying the CC genotype (OR = 1.47; CI = 1.08–2.01; P = 0.015). In addition, a 15-year follow-up period (35, 40, and 45 years) of the same individuals showed that carriers of the A-allele had more often hypertension in all of the studied age-groups. The highest risk for developing hypertension was obtained among 35-year-old subjects (odds ratio [OR] 3.83; confidence interval [CI] = 1.20–12.27; P = 0.024). Those carrying variant A had also significantly higher readings of both systolic (P = 0.047) and diastolic (P = 0.048) blood pressure during the follow-up. No significant associations between rs2297518 (G/A) variants alone and hypertension were found. However, haplotype analysis of rs2779249 and rs2297518 revealed that individuals having haplotype H3 which combines both A alleles (CA–GA, 19.7% of individuals) was more commonly found in the hypertensive group than in the normotensive group (OR = 2.01; CI = 1

  8. Isoliquiritigenin, a strong nod gene- and glyceollin resistance-inducing flavonoid from soybean root exudate.

    PubMed Central

    Kape, R; Parniske, M; Brandt, S; Werner, D

    1992-01-01

    Isoflavonoid signal molecules from soybean (Glycine max (L.) Merr.) seed and root exudate induce the transcription of nodulation (nod) genes in Bradyrhizobium japonicum. In this study, a new compound with symbiotic activity was isolated from soybean root exudate. The isolated 2',4',4-trihydroxychalcone (isoliquiritigenin) is characterized by its strong inducing activity for the nod genes of B. japonicum. These genes are already induced at concentrations 1 order of magnitude below those required of the previously described isoflavonoid inducers genistein and daidzein. Isoliquiritigenin is also a potent inducer of glyceollin resistance in B. japonicum, which renders this bacterium insensitive to potentially bactericidal concentrations of glyceollin, the phytoalexin of G. max. No chemotactic effect of isoliquiritigenin was observed. The highly efficient induction of nod genes and glyceollin resistance by isoliquiritigenin suggests the ecological significance of this compound, although it is not a major flavonoid constituent of the soybean root exudate in quantitative terms. PMID:1622242

  9. Identification of Chromosomal Shigella flexneri Genes Induced by the Eukaryotic Intracellular Environment

    PubMed Central

    Runyen-Janecky, L. J.; Payne, S. M.

    2002-01-01

    Upon entry into the eukaryotic cytosol, the facultative intracellular bacterium Shigella flexneri is exposed to an environment that may necessitate the expression of particular genes for it to survive and grow intracellularly. To identify genes that are induced in response to the intracellular environment, we screened a library containing fragments of the S. flexneri chromosome fused to a promoterless green fluorescent protein gene (gfp). Bacteria containing promoter fusions that had a higher level of gfp expression when S. flexneri was intracellular (in Henle cells) than when S. flexneri was extracellular (in Luria-Bertani broth) were isolated by using fluorescence-activated cell sorting. Nine different genes with increased expression in Henle cells were identified. Several genes (uhpT, bioA, and lysA) were involved in metabolic processes. The uhpT gene, which encoded a sugar phosphate transporter, was the most frequently isolated gene and was induced by glucose-6-phosphate in vitro. Two of the intracellularly induced genes (pstS and phoA) encode proteins involved in phosphate acquisition and were induced by phosphate limitation in vitro. Additionally, three iron-regulated genes (sufA, sitA, and fhuA) were identified. The sufA promoter was derepressed in iron-limiting media and was also induced by oxidative stress. To determine whether intracellularly induced genes are required for survival or growth in the intracellular environment, we constructed mutations in the S. flexneri uhpT and pstS genes by allelic exchange. The uhpT mutant could not use glucose-6-phosphate as a sole carbon source in vitro but exhibited normal plaque formation on Henle cell monolayers. The pstS mutant had no apparent growth defect in low-phosphate media in vitro but formed smaller plaques on Henle cell monolayers than the parent strain. Both mutants were as effective as the parent strain in inducing apoptosis in a macrophage cell line. PMID:12117948

  10. Bifidobacterium bifidum actively changes the gene expression profile induced by Lactobacillus acidophilus in murine dendritic cells.

    PubMed

    Weiss, Gudrun; Rasmussen, Simon; Nielsen Fink, Lisbeth; Jarmer, Hanne; Nøhr Nielsen, Birgit; Frøkiaer, Hanne

    2010-01-01

    Dendritic cells (DC) play a pivotal regulatory role in activation of both the innate as well as the adaptive immune system by responding to environmental microorganisms. We have previously shown that Lactobacillus acidophilus induces a strong production of the pro-inflammatory and Th1 polarizing cytokine IL-12 in DC, whereas bifidobacteria do not induce IL-12 but inhibit the IL-12 production induced by lactobacilli. In the present study, genome-wide microarrays were used to investigate the gene expression pattern of murine DC stimulated with Lactobacillus acidophilus NCFM and Bifidobacterium bifidum Z9. L. acidophilus NCFM strongly induced expression of interferon (IFN)-beta, other virus defence genes, and cytokine and chemokine genes related to the innate and the adaptive immune response. By contrast, B. bifidum Z9 up-regulated genes encoding cytokines and chemokines related to the innate immune response. Moreover, B. bifidum Z9 inhibited the expression of the Th1-promoting genes induced by L. acidophilus NCFM and had an additive effect on genes of the innate immune response and Th2 skewing genes. The gene encoding Jun dimerization protein 2 (JDP2), a transcription factor regulating the activation of JNK, was one of the few genes only induced by B. bifidum Z9. Neutralization of IFN-beta abrogated L. acidophilus NCFM-induced expression of Th1-skewing genes, and blocking of the JNK pathway completely inhibited the expression of IFN-beta. Our results indicate that B. bifidum Z9 actively inhibits the expression of genes related to the adaptive immune system in murine dendritic cells and that JPD2 via blocking of IFN-beta plays a central role in this regulatory mechanism. PMID:20548777

  11. Mapping the genes for haloperidol-induced catalepsy.

    PubMed

    Kanes, S; Dains, K; Cipp, L; Gatley, J; Hitzemann, B; Rasmussen, E; Sanderson, S; Silverman, M; Hitzemann, R

    1996-05-01

    The strain means for haloperidol-induced catalepsy were determined in the 26 strain BXD recombinant inbred series. The ED50 values ranged from 0.55 mg/kg (strain 30) to 7.9 mg/kg (strain 2). Heritability for the catalepsy response was 0.78 and the number of effective loci was estimated to be four. The strain means were correlated with the strain distribution patterns for 1300 marker loci of known chromosomal location and polymorphic between the C57Bl/6J and DBA/2J strains. Six quantitative trait loci (QTLs) were identified at P < .01. Two of the six QTLs were confirmed in a sample of B6XD2 F2 animals (n = 144), phenotyped for haloperidol response and genotyped for microsatellites closely linked to the QTLs. The confirmed QTL on chromosome 4 is near the b locus. The confirmed QTL on chromosome 9 is closely linked to Drd2, the D2 dopamine receptor gene. One hundred of the F2 individuals were phenotyped for D2 dopamine receptor binding using the ligand [125I] epidepride as the ligand. Consistent with previous results, the nonresponsive F2 individuals showed modestly higher receptor binding in all brain regions examined: the nucleus accumbens core, the nucleus accumbens shell, the lateral caudate putamen, the dorsomedial caudate putamen, the substantia nigra zona compacta and the ventral tegmental area. The DBA/2J allele of the chromosome 9 QTL was associated with higher receptor binding in all brain areas except the ventral tegmental area. Overall, the data illustrate that either near or part of Drd2 is a QTL which has significant effects on both haloperidol response and D2 dopamine receptor binding. However, the data also illustrate that most of the genetic variance in either haloperidol response or D2 dopamine receptor binding is not associated with Drd2. PMID:8627512

  12. Glomerulonephritis-induced changes in kidney gene expression in rats

    PubMed Central

    Pavkovic, Mira; Riefke, Björn; Frisk, Anna-Lena; Gröticke, Ina; Ellinger-Ziegelbauer, Heidrun

    2015-01-01

    We investigated a glomerulonephritis (GN) model in rats induced by nephrotoxic serum (NTS) which contains antibodies against the glomerular basement membrane (GBM). The anti-GBM GN model in rats is widely used since its biochemical and histopathological characteristics are similar to crescentic nephritis and Goodpasture's disease in humans (Pusey, 2003[2]). Male Wistar Kyoto (WKY) and Sprague–Dawley (SD) rats were dosed once with 1, 2.5 and 5 ml/kg nephrotoxic serum (NTS) or 1.5 and 5 ml/kg NTS, respectively. GN and tubular damage were observed histopathologically in all treated rats after 14 days. To obtain insight into molecular processes during GN pathogenesis, mRNA expression was investigated in WKY and SD kidneys using Affymetrix's GeneChip Rat genome 230_2.0 arrays (GSE64265). The immunopathological processes during GN are still not fully understood and likely involve both innate and adaptive immunity. In the present study, several hundred mRNAs were found deregulated, which functionally were mostly associated with inflammation and regeneration. The β-chain of the major histocompatibility complex class II RT1.B (Rt1-Bb) and complement component 6 (C6) were identified as two mRNAs differentially expressed between WKY and SD rat strains which could be related to known different susceptibilities to NTS of different rat strains; both were increased in WKY and decreased in SD rats (Pavkovic et al., 2015 [1]). Increased Rt1-Bb expression in WKY rats could indicate a stronger and more persistent cellular reaction of the adaptive immune system in this strain, in line with findings indicating adaptive immune reactions during GN. The complement cascade is also known to be essential for GN development, especially terminal cascade products like C6. PMID:26697341

  13. Relationship between gene responses and symptoms induced by Rice grassy stunt virus

    PubMed Central

    Satoh, Kouji; Yoneyama, Kaori; Kondoh, Hiroaki; Shimizu, Takumi; Sasaya, Takahide; Choi, Il-Ryong; Yoneyama, Koichi; Omura, Toshihiro; Kikuchi, Shoshi

    2013-01-01

    Rice grassy stunt virus (RGSV) is a serious threat to rice production in Southeast Asia. RGSV is a member of the genus Tenuivirus, and it induces leaf yellowing, stunting, and excess tillering on rice plants. Here we examined gene responses of rice to RGSV infection to gain insight into the gene responses which might be associated with the disease symptoms. The results indicated that (1) many genes related to cell wall synthesis and chlorophyll synthesis were predominantly suppressed by RGSV infection; (2) RGSV infection induced genes associated with tillering process; (3) RGSV activated genes involved in inactivation of gibberellic acid and indole-3-acetic acid; and (4) the genes for strigolactone signaling were suppressed by RGSV. These results suggest that these gene responses to RGSV infection account for the excess tillering specific to RGSV infection as well as other symptoms by RGSV, such as stunting and leaf chlorosis. PMID:24151491

  14. A viral satellite DNA vector-induced transcriptional gene silencing via DNA methylation of gene promoter in Nicotiana benthamiana.

    PubMed

    Ju, Zheng; Wang, Lei; Cao, Dongyan; Zuo, Jinhua; Zhu, Hongliang; Fu, Daqi; Luo, Yunbo; Zhu, Benzhong

    2016-09-01

    Virus-induced gene silencing (VIGS) has been widely used for plant functional genomics study at the post-transcriptional level using various DNA or RNA viral vectors. However, while virus-induced transcriptional gene silencing (VITGS) via DNA methylation of gene promoter was achieved using several plant RNA viral vectors, it has not yet been done using a satellite DNA viral vector. In this study, a viral satellite DNA associated with tomato yellow leaf curl China virus (TYLCCNV), which has been modified as a VIGS vector in previous research, was developed as a VITGS vector. Firstly, the viral satellite DNA VIGS vector was further optimized to a more convenient p1.7A+2mβ vector with high silencing efficiency of the phytoene desaturase (PDS) gene in Nicotiana benthamiana plants. Secondly, the constructed VITGS vector (TYLCCNV:35S), which carried a portion of the cauliflower mosaic virus 35S promoter, could successfully induce heritable transcriptional gene silencing (TGS) of the green fluorescent protein (GFP) gene in the 35S-GFP transgenic N. benthamiana line 16c plants. Moreover, bisulfite sequencing results revealed higher methylated cytosine residues at CG, CHG and CHH sites of the 35S promoter sequence in TYLCCNV:35S-inoculated plants than in TYLCCNV-inoculated line 16c plants (control). Overall, these results demonstrated that the viral satellite DNA vector could be used as an effective VITGS vector to study DNA methylation in plant genomes. PMID:27422476

  15. Light-dependent expression of flg22-induced defense genes in Arabidopsis

    PubMed Central

    Sano, Satoshi; Aoyama, Mayu; Nakai, Kana; Shimotani, Koji; Yamasaki, Kanako; Sato, Masa H.; Tojo, Daisuke; Suwastika, I. Nengah; Nomura, Hironari; Shiina, Takashi

    2014-01-01

    Chloroplasts have been reported to generate retrograde immune signals that activate defense gene expression in the nucleus. However, the roles of light and photosynthesis in plant immunity remain largely elusive. In this study, we evaluated the effects of light on the expression of defense genes induced by flg22, a peptide derived from bacterial flagellins which acts as a potent elicitor in plants. Whole-transcriptome analysis of flg22-treated Arabidopsis thaliana seedlings under light and dark conditions for 30 min revealed that a number of (30%) genes strongly induced by flg22 (>4.0) require light for their rapid expression, whereas flg22-repressed genes include a significant number of genes that are down-regulated by light. Furthermore, light is responsible for the flg22-induced accumulation of salicylic acid (SA), indicating that light is indispensable for basal defense responses in plants. To elucidate the role of photosynthesis in defense, we further examined flg22-induced defense gene expression in the presence of specific inhibitors of photosynthetic electron transport: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB). Light-dependent expression of defense genes was largely suppressed by DBMIB, but only partially suppressed by DCMU. These findings suggest that photosynthetic electron flow plays a role in controlling the light-dependent expression of flg22-inducible defense genes. PMID:25346742

  16. Preferential effects of the chemotherapeutic DNA crosslinking agent mitomycin C on inducible gene expression in vivo.

    PubMed

    Caron, R M; Hamilton, J W

    1995-01-01

    The immediate effects of a single dose of the chemotherapeutic DNA crosslinking agent, mitomycin C (MMC), on the expression of several constitutive and drug-inducible genes were examined in a simple in vivo system, the 14 day chick embryo. We observed no effect of MMC on the steady-state mRNA expression of the constitutively expressed beta-actin, transferrin, or albumin genes. In contrast, MMC treatment significantly altered both the basal and drug-inducible mRNA expression of two glutethimide-inducible genes, 5-aminolevulinic acid (ALA) synthase and cytochrome P450 CYP2H1. The basal expression of these genes was transiently but significantly increased over a 24 hr period following a single dose of MMC. Conversely, MMC significantly suppressed the glutethimide-inducible expression of these genes when administered 1 to 24 hr prior to the inducing drug. The effects of MMC on both basal and drug-inducible ALA synthase and CYP2H1 mRNA expression were principally a result of changes in the transcription rates of these genes. In contrast, MMC treatment had little or no effect on glutethimide-induced expression of ALA synthase or CYP2H1 when administered 1 hr after the inducing drug, suggesting that a very early event in the induction process represents the target for these MMC effects. Covalent binding studies demonstrated that the effects of MMC on gene expression were closely correlated temporally with formation of [3H]-porfiromycin-DNA adducts. These results support the hypothesis that genotoxic chemicals specifically target their effects to inducible genes in vivo. PMID:7875125

  17. Shigella dysenteriae Modulates BMP Pathway to Induce Mucin Gene Expression In Vivo and In Vitro

    PubMed Central

    Gopal, Ashidha; Iyer, Soumya Chidambaram; Gopal, Udhayakumar; Devaraj, Niranjali; Halagowder, Devaraj

    2014-01-01

    Mucosal epithelial cells in the intestine act as the first line of host defense against pathogens by increasing mucin production for clearance. Despite this fact, the underlying molecular mechanisms by which Shigella dysenteriae transduce mucin gene expression remain poorly defined. The goal of this study was to determine the role of Bone morphogenetic protein (BMP) pathway in mucin gene expression during S. dysenteriae infection. In this study we demonstrate that S. dysenteriae activates BMP signaling to induce MUC2 and MUC5AC gene expression in rat ileal loop model and in vitro. We also observed that BMP pathway regulates CDX2 expression which plays a critical role in induction of MUC2 gene during S. dysenteriae infection. In SMAD4 silenced cells S. dysenteriae infection did not abrogate MUC2 and MUC5AC gene expression whereas in CDX2 silenced cells it induces differential expression of MUC5AC gene. These results suggest that SMAD4-CDX2 induces MUC2 gene expression whereas SMAD4 directly influences differential expression of MUC5AC gene. Altogether, our results show that during S. dysenteriae infection the BMP pathway modulates inflammatory transcription factors CDX2 and SMAD4 to induce MUC2 and MUC5AC gene expression which plays a key role in the regulation of host mucosal defense thereby paving a cue for therapeutic application. PMID:25365201

  18. Spatial Control of Gene Expression within a Scaffold by Localized Inducer Release

    PubMed Central

    Baraniak, Priya R.; Nelson, Devin M.; Leeson, Cory E.; Katakam, Anand K.; Friz, Jennifer L.; Cress, Dean E.; Hong, Yi; Guan, Jianjun; Wagner, William R.

    2011-01-01

    Gene expression can be controlled in genetically modified cells by employing an inducer/promoter system where presence of the inducer molecule regulates the timing and level of gene expression. By applying the principles of controlled release, it should be possible to control gene expression on a biomaterial surface by the presence or absence of inducer release from the underlying material matrix, thus avoiding alternative techniques that rely upon uptake of relatively labile DNA from material surfaces. To evaluate this concept, a modified ecdysone-responsive gene expression system was transfected into B16 murine cells and the ability of an inducer ligand, which was released from elastomeric poly(ester urethane) urea (PEUU), to initiate gene expression was studied. The synthetic inducer ligand was first loaded into PEUU to demonstrate extended release of the bioactive molecule at various loading densities over a one year period in vitro. Patterning films of PEUU variably-loaded with inducer resulted in spatially controlled cell expression of the gene product (green fluorescent protein, GFP). In porous scaffolds made from PEUU by salt leaching, where the central region was exclusively loaded with inducer, cells expressed GFP predominately in the loaded central regions whereas expression was minimal in outer regions where ligand was omitted. This scaffold system may ultimately provide a means to precisely control progenitor cell commitment in a spatially-defined manner in vivo for soft tissue repair and regeneration. PMID:21269687

  19. Estradiol-induced gene expression in largemouth bass (Micropterus salmoides)

    USGS Publications Warehouse

    Bowman, C.J.; Kroll, K.J.; Gross, T.G.; Denslow, N.D.

    2002-01-01

    Vitellogenin (Vtg) and estrogen receptor (ER) gene expression levels were measured in largemouth bass to evaluate the activation of the ER-mediated pathway by estradiol (E2). Single injections of E2 ranging from 0.0005 to 5 mg/kg up-regulated plasma Vtg in a dose-dependent manner. Vtg and ER mRNAs were measured using partial cDNA sequences corresponding to the C-terminal domain for Vtg and the ligand-binding domain of ER?? sequences. After acute E2-exposures (2 mg/kg), Vtg and ER mRNAs and plasma Vtg levels peaked after 2 days. The rate of ER mRNA accumulation peaked 36-42 h earlier than Vtg mRNA. The expression window for ER defines the primary response to E2 in largemouth bass and that for Vtg a delayed primary response. The specific effect of E2 on other estrogen-regulated genes was tested during these same time windows using differential display RT-PCR. Specific up-regulated genes that are expressed in the same time window as Vtg were ERp72 (a membrane-bound disulfide isomerase) and a gene with homology to an expressed gene identified in zebrafish. Genes that were expressed in a pattern that mimics the ER include the gene for zona radiata protein ZP2, and a gene with homology to an expressed gene found in winter flounder. One gene for fibrinogen ?? was down-regulated and an unidentified gene was transiently up-regulated after 12 h of exposure and returned to basal levels by 48 h. Taken together these studies indicate that the acute molecular response to E2 involves a complex network of responses over time. ?? 2002 Elsevier Science Ireland Ltd. All rights reserved.

  20. The gene coding for the mustard trypsin inhibitor-2 is discontinuous and wound-inducible.

    PubMed

    Ceci, L R; Spoto, N; de Virgilio, M; Gallerani, R

    1995-05-01

    The gene coding for the mustard trypsin inhibitor-2 has been isolated from a genomic library and characterized. Comparison of genomic and cDNA sequences indicates that the gene is interrupted by an intron of 193 bp. The eukaryotic peculiar regulatory sequences have been detected in the 5' flanking region of the gene. In addition, a decanucleotide has been detected that is highly similar to the proposed G-box and to the ABRE motifs required for the gene expression induced by methyl jasmonate and abscissic acid. Northern blot analysis demonstrates that the gene is expressed in immature seeds as well as in wounded leaves. PMID:7750566

  1. METHYL METHANESULFONATE-INDUCED GENE EXPRESSION CHANGES IN HUMAN SKIN FIBROBLASTS

    EPA Science Inventory

    METHYL METHANESULFONATE-INDUCED GENE EXPRESSION CHANGES IN HUMAN SKIN FIBROBLASTS. Geremy W. Knapp, Alan Tennant, and Russell D. Owen. Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, U. S. Environmental Protection Agency, Re...

  2. AGE-RELATED GENE EXPRESSION CHANGES IN HUMAN SKIN FIBROBLASTS INDUCED BY MMS

    EPA Science Inventory

    Age-Related Gene Expression Changes In Human Skin Fibroblasts Induced By methyl methanesulfonate. Geremy W. Knapp, Alan H. Tennant, and Russell D. Owen. Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, U. S. Environmental Prote...

  3. Identification of novel light-induced genes in the suprachiasmatic nucleus

    PubMed Central

    Porterfield, Veronica M; Piontkivska, Helen; Mintz, Eric M

    2007-01-01

    Background The transmission of information about the photic environment to the circadian clock involves a complex array of neurotransmitters, receptors, and second messenger systems. Exposure of an animal to light during the subjective night initiates rapid transcription of a number of immediate-early genes in the suprachiasmatic nucleus of the hypothalamus. Some of these genes have known roles in entraining the circadian clock, while others have unknown functions. Using laser capture microscopy, microarray analysis, and quantitative real-time PCR, we performed a comprehensive screen for changes in gene expression immediately following a 30 minute light pulse in suprachiasmatic nucleus of mice. Results The results of the microarray screen successfully identified previously known light-induced genes as well as several novel genes that may be important in the circadian clock. Newly identified light-induced genes include early growth response 2, proviral integration site 3, growth-arrest and DNA-damage-inducible 45 beta, and TCDD-inducible poly(ADP-ribose) polymerase. Comparative analysis of promoter sequences revealed the presence of evolutionarily conserved CRE and associated TATA box elements in most of the light-induced genes, while other core clock genes generally lack this combination of promoter elements. Conclusion The photic signalling cascade in the suprachiasmatic nucleus activates an array of immediate-early genes, most of which have unknown functions in the circadian clock. Detected evolutionary conservation of CRE and TATA box elements in promoters of light-induced genes suggest that the functional role of these elements has likely remained the same over evolutionary time across mammalian orders. PMID:18021443

  4. Transposon-induced nuclear mutations that alter chloroplast gene expression

    SciTech Connect

    Barkan, A.

    1992-01-01

    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.

  5. Inducible and Reversible Lentiviral and Recombination Mediated Cassette Exchange (RMCE) Systems for Controlling Gene Expression

    PubMed Central

    Bersten, David C.; Sullivan, Adrienne E.; Li, Dian; Bhakti, Veronica; Bent, Stephen J.; Whitelaw, Murray L.

    2015-01-01

    Manipulation of gene expression to invoke loss of function (LoF) or gain of function (GoF) phenotypes is important for interrogating complex biological questions both in vitro and in vivo. Doxycycline (Dox)-inducible gene expression systems are commonly used although success is often limited by high background and insufficient sensitivity to Dox. Here we develop broadly applicable platforms for reliable, tightly controlled and reversible Dox-inducible systems for lentiviral mediated generation of cell lines or FLP Recombination-Mediated Cassette Exchange (RMCE) into the Collagen 1a1 (Col1a1) locus (FLP-In Col1a1) in mouse embryonic stem cells. We significantly improve the flexibility, usefulness and robustness of the Dox-inducible system by using Tetracycline (Tet) activator (Tet-On) variants which are more sensitive to Dox, have no background activity and are expressed from single Gateway-compatible constructs. We demonstrate the usefulness of these platforms in ectopic gene expression or gene knockdown in multiple cell lines, primary neurons and in FLP-In Col1a1 mouse embryonic stem cells. We also improve the flexibility of RMCE Dox-inducible systems by generating constructs that allow for tissue or cell type-specific Dox-inducible expression and generate a shRNA selection algorithm that can effectively predict potent shRNA sequences able to knockdown gene expression from single integrant constructs. These platforms provide flexible, reliable and broadly applicable inducible expression systems for studying gene function. PMID:25768837

  6. MOLECULAR ANALYSIS OF MUTATIONS INDUCED BY MUTAGENS IN THE TK GENE OF MOUSE LYMPHOMA CELLS

    EPA Science Inventory

    MOLECULAR ANALYSIS OF MUTATIONS INDUCED BY BROMATE AND N- ETHYL-N-NITROSOUREA IN THE TK GENE OF MOUSE L YMPHOMA CELLS

    The mouse lymphoma assay is widely used to identify chemical mutagens The Tk +1- gene located on an autosome in mouse lymphoma cells may recover a wide ra...

  7. Wounding induces expression of genes involved in tuber closing layer and wound-periderm development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little is known about the coordinate induction of genes that may be involved in important wound-healing events. In this study, wound-healing events were determined together with wound-induced expression profiles of selected cell cycle, cell wall protein, and pectin methyl esterase genes using tuber...

  8. Probiotic bacteria change Escherichia coli-induced gene expression in cultured colonocytes: Implications in intestinal pathophysiology

    PubMed Central

    Panigrahi, Pinaki; Braileanu, Gheorghe T; Chen, Hegang; Stine, O Colin

    2007-01-01

    AIM: To investigate the change in eukaryotic gene expression profile in Caco-2 cells after infection with strains of Escherichia coli and commensal probiotic bacteria. METHODS: A 19200 gene/expressed sequence tag gene chip was used to examine expression of genes after infection of Caco-2 cells with strains of normal flora E. coli, Lactobacillus plantarum, and a combination of the two. RESULTS: The cDNA microarray revealed up-regulation of 155 and down-regulation of 177 genes by E. coli. L. plantarum up-regulated 45 and down-regulated 36 genes. During mixed infection, 27 genes were up-regulated and 59 were down-regulated, with nullification of stimulatory/inhibitory effects on most of the genes. Expression of several new genes was noted in this group. CONCLUSION: The commensal bacterial strains used in this study induced the expression of a large number of genes in colonocyte-like cultured cells and changed the expression of several genes involved in important cellular processes such as regulation of transcription, protein biosynthesis, metabolism, cell adhesion, ubiquitination, and apoptosis. Such changes induced by the presence of probiotic bacteria may shape the physiologic and pathologic responses they trigger in the host. PMID:18081226

  9. Virus induced gene silencing (VIGS) for functional analysis of wheat genes involved in Zymoseptoria tritici susceptibility and resistance

    PubMed Central

    Lee, Wing-Sham; Rudd, Jason J.; Kanyuka, Kostya

    2015-01-01

    Virus-induced gene silencing (VIGS) has emerged as a powerful reverse genetic technology in plants supplementary to stable transgenic RNAi and, in certain species, as a viable alternative approach for gene functional analysis. The RNA virus Barley stripe mosaic virus (BSMV) was developed as a VIGS vector in the early 2000s and since then it has been used to study the function of wheat genes. Several variants of BSMV vectors are available, with some requiring in vitro transcription of infectious viral RNA, while others rely on in planta production of viral RNA from DNA-based vectors delivered to plant cells either by particle bombardment or Agrobacterium tumefaciens. We adapted the latest generation of binary BSMV VIGS vectors for the identification and study of wheat genes of interest involved in interactions with Zymoseptoria tritici and here present detailed and the most up-to-date protocols. PMID:26092793

  10. FORMALDEHYDE-INDUCED GENE EXPRESSION IN F344 RAT NASAL RESPIRATORY EPITHELIUM.

    EPA Science Inventory

    Formaldehyde-induced gene expression in F344 rat nasal respiratory epithelium

    ABSTRACT

    Formaldehyde, an occupational and environmental toxicant used extensively in the manufacturing of many household and personal use products, is known to induce squamous cell carci...

  11. Bioinforrnatics of Gene Expression Profiling Data Provide Mechanistic Understanding of Acute Ozone-Induced Lung injury

    EPA Science Inventory

    Acute ozone-induced pulmonary injury and inflammation are well characterized. A few studies have used gene expression profiling to determine the types of changes induced by ozone; however the mechanisms or the pathways involved are less well understood. We presumed that robust bi...

  12. Protective effects of L-selenomethionine on space radiation induced changes in gene expression.

    PubMed

    Stewart, J; Ko, Y-H; Kennedy, A R

    2007-06-01

    Ionizing radiation can produce adverse biological effects in astronauts during space travel. Of particular concern are the types of radiation from highly energetic, heavy, charged particles known as HZE particles. The aims of our studies are to characterize HZE particle radiation induced biological effects and evaluate the effects of L-selenomethionine (SeM) on these adverse biological effects. In this study, microarray technology was used to measure HZE radiation induced changes in gene expression, as well as to evaluate modulation of these changes by SeM. Human thyroid epithelial cells (HTori-3) were irradiated (1 GeV/n iron ions) in the presence or in the absence of 5 microM SeM. At 6 h post-irradiation, all cells were harvested for RNA isolation. Gene Chip U133Av2 from Affymetrix was used for the analysis of gene expression, and ANOVA and EASE were used for a determination of the genes and biological processes whose differential expression is statistically significant. Results of this microarray study indicate that exposure to small doses of radiation from HZE particles, 10 and 20 cGy from iron ions, induces statistically significant differential expression of 196 and 610 genes, respectively. In the presence of SeM, differential expression of 77 out of 196 genes (exposure to 10 cGy) and 336 out of 610 genes (exposure to 20 cGy) is abolished. In the presence or in the absence of SeM, radiation from HZE particles induces differential expression of genes whose products have roles in the induction of G1/S arrest during the mitotic cell cycle, as well as heat shock proteins. Some of the genes, whose expressions were affected by radiation from HZE particles and were unchanged in irradiated cells treated with SeM, have been shown to have altered expression levels in cancer cells. The conclusions of this report are that radiation from HZE particles can induce differential expression of many genes, some of which are known to play roles in the same processes that have

  13. A specific library of randomly integrated reporter genes for the isolation of inducible functions by cell sorting

    SciTech Connect

    Lapeyre, J.N.; Marini, F.; Gratzner, H.G. AMC ImmunoDiagnostics, Houston, TX )

    1993-01-01

    A library of cells containing randomly integrated reporter genes has been constructed. The purpose of this library is to enable the isolation of genes of interest which are inducible by radiation, biological response modifiers, cytokines, or other agents. These genes are located near reporter genes which can be induced by the upstream promoter of the gene of interest. The reporter gene, Lac Z, was randomly inserted into the genome by retroviral transduction and subsequent selection of the neo[sup r] gene with gentamycin. Studies of radiation inducible genes were undertaken, whereby cells with the radiation sensitive function were isolated by sorting the cells fluorescent after staining with the beta gal substrate, fluorescein digalactoside (FDG). This gene-tagging approach is an improvement over the cDNA library subtraction protocol in that a single library of cells with random marker gene integration can be repeatedly and sequentially probed by sorting under different, selective conditions, dependent upon the genes to be characterized.

  14. Preventing High Fat Diet-induced Obesity and Improving Insulin Sensitivity through Neuregulin 4 Gene Transfer

    PubMed Central

    Ma, Yongjie; Gao, Mingming; Liu, Dexi

    2016-01-01

    Neuregulin 4 (NRG4), an epidermal growth factor-like signaling molecule, plays an important role in cell-to-cell communication during tissue development. Its function to regulate energy metabolism has recently been reported. This current study was designed to assess the preventive and therapeutic effects of NRG4 overexpression on high fat diet (HFD)-induced obesity. Using the hydrodynamic gene transfer method, we demonstrate that Nrg4 gene transfer in mice suppressed the development of diet-induced obesity, but did not affect pre-existing adiposity and body weight in obese mice. Nrg4 gene transfer curbed HFD-induced hepatic steatosis by inhibiting lipogenesis and PPARγ-mediated lipid storage. Concurrently, overexpression of NRG4 reduced chronic inflammation in both preventive and treatment studies, evidenced by lower mRNA levels of macrophage marker genes including F4/80, Cd68, Cd11b, Cd11c, and macrophage chemokine Mcp1, resulting in improved insulin sensitivity. Collectively, these results demonstrate that overexpression of the Nrg4 gene by hydrodynamic gene delivery prevents HFD-induced weight gain and fatty liver, alleviates obesity-induced chronic inflammation and insulin resistance, and supports the health benefits of NRG4 in managing obesity and obesity-associated metabolic disorders. PMID:27184920

  15. Sugar-inducible expression of a gene for beta-amylase in Arabidopsis thaliana.

    PubMed Central

    Mita, S; Suzuki-Fujii, K; Nakamura, K

    1995-01-01

    The levels of beta-amylase activity and of the mRNA for beta-amylase in rosette leaves of Arabidopsis thaliana (L.) Heynh. increased significantly, with the concomitant accumulation of starch, when whole plants or excised mature leaves were supplied with sucrose. A supply of glucose or fructose, but not of mannitol or sorbitol, to plants also induced the expression of the gene for beta-amylase, and the induction occurred not only in rosette leaves but also in roots, stems, and bracts. These results suggest that the gene for beta-amylase of Arabidopsis is subject to regulation by a carbohydrate metabolic signal, and expression of the gene in various tissues may be regulated by the carbon partitioning and sink-source interactions in the whole plant. The sugar-inducible expression of the gene in Arabidopsis was severely repressed in the absence of light. The sugar-inducible expression in the light was not inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea or by chloramphenicol, but it was inhibited by cycloheximide. These results suggest that a light-induced signal and de novo synthesis of proteins in the cytoplasm are involved in the regulation. A fusion gene composed of the 5' upstream region of the gene for beta-amylase from Arabidopsis and the coding sequence of beta-glucuronidase showed the sugar-inducible expression in a light-dependent manner in rosette leaves of transgenic Arabidopsis. PMID:7716246

  16. Hydroxycamptothecin-induced apoptotic gene expression profiling by PCR array in human Tenon's capsule fibroblasts.

    PubMed

    Tang, Wei; Zhang, Yinong; Zhang, Qing; Wang, Qinghua; Wu, Zhifeng

    2015-01-01

    Studies have indicated that hydroxycamptothecin (HCPT) induces apoptosis of fibroblasts. In this study, we investigated the apoptotic gene expression profiling in HCPT-treated -human Tenon's capsule fibroblasts (HTCFs) and identify the most implicated gene in apoptotic signaling of HTCFs by HCPT. Method HTCFs were incubated with HCPT at 0, 0.25 and 4 mg/L for 24, 48 and 72 h, respectively. Anti-proliferative effects were measured by MTT assay. Apoptosis was determined using the Annexin V-FITC/PI apoptosis detection kit and apoptotic cells were identified by flow cytometry. A PCR array was employed to analyze the most implicated apoptotic genes during HCPT-induced apoptosis in HTCFs. Results from our studies showed that HCPT induced apoptosis in HTCFs in a concentration-and time-dependent manner. The apoptotic HTCFs was increased by 9.38% with a multiplicity at 4 mg/L HCPT. PCR array demonstrated remarkable changes in 88 apoptotic genes, including 9 up-regulated genes and 36 down-regulated genes. HCPT treatment induced the upregulation of CHOP and downregulation of XIAP in HTCFs. To conclude, our results support HCPT induced the apoptosis of HTCFs, involving the activation of mitochondrial and endoplasmic reticulum stresses as well as the downregulation expression of XIAP. PMID:26045770

  17. Preventing High Fat Diet-induced Obesity and Improving Insulin Sensitivity through Neuregulin 4 Gene Transfer.

    PubMed

    Ma, Yongjie; Gao, Mingming; Liu, Dexi

    2016-01-01

    Neuregulin 4 (NRG4), an epidermal growth factor-like signaling molecule, plays an important role in cell-to-cell communication during tissue development. Its function to regulate energy metabolism has recently been reported. This current study was designed to assess the preventive and therapeutic effects of NRG4 overexpression on high fat diet (HFD)-induced obesity. Using the hydrodynamic gene transfer method, we demonstrate that Nrg4 gene transfer in mice suppressed the development of diet-induced obesity, but did not affect pre-existing adiposity and body weight in obese mice. Nrg4 gene transfer curbed HFD-induced hepatic steatosis by inhibiting lipogenesis and PPARγ-mediated lipid storage. Concurrently, overexpression of NRG4 reduced chronic inflammation in both preventive and treatment studies, evidenced by lower mRNA levels of macrophage marker genes including F4/80, Cd68, Cd11b, Cd11c, and macrophage chemokine Mcp1, resulting in improved insulin sensitivity. Collectively, these results demonstrate that overexpression of the Nrg4 gene by hydrodynamic gene delivery prevents HFD-induced weight gain and fatty liver, alleviates obesity-induced chronic inflammation and insulin resistance, and supports the health benefits of NRG4 in managing obesity and obesity-associated metabolic disorders. PMID:27184920

  18. Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives

    PubMed Central

    Gajbhiye, Virendra; Escalante, Leah; Chen, Guojun; Laperle, Alex; Zheng, Qifeng; Steyer, Benjamin; Gong, Shaoqin; Saha, Krishanu

    2014-01-01

    Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives. PMID:24232694

  19. Methanol inducible genes obtained from pichia and methods of use

    SciTech Connect

    Stroman, D.W.; Brust, P.F.; Ellis, S.B.; Gingeras, T.R.; Harpold, M.M.; Tschopp, J.F.

    1989-02-28

    A method is described for isolating the p76 gene from a methanol assimilating yeast, the method comprising: (a) digesting total DNA with at least one restriction enzyme to give digested DNA; (b) size fractionating the digested DNA by agarose electrophoresis; (c) denaturating and binding the size fractionated DNA from step (b) to nitrocellulose filter to give bound DNA; (d) hybridizing the bound DNA with labeled p76 gene from Pichia pastoris; (e) identifying the unique size fragment of bound DNA which cross-hybridizes with the labeled p76 gene from P. pastoris; (f) size fractionating additional DNA which has been digested in accordance with step (a) to recover for cloning the unique size fragment of DNA identified in step (e).

  20. Radiation-induced gene expression in the nematode Caenorhabditis elegans

    NASA Technical Reports Server (NTRS)

    Nelson, Gregory A.; Jones, Tamako A.; Chesnut, Aaron; Smith, Anna L.

    2002-01-01

    We used the nematode C. elegans to characterize the genotoxic and cytotoxic effects of ionizing radiation in a simple animal model emphasizing the unique effects of charged particle radiation. Here we demonstrate by RT-PCR differential display and whole genome microarray hybridization experiments that gamma rays, accelerated protons and iron ions at the same physical dose lead to unique transcription profiles. 599 of 17871 genes analyzed (3.4%) showed differential expression 3 hrs after exposure to 3 Gy of radiation. 193 were up-regulated, 406 were down-regulated and 90% were affected only by a single species of radiation. A novel statistical clustering technique identified the regulatory relationships between the radiation-modulated genes and showed that genes affected by each radiation species were associated with unique regulatory clusters. This suggests that independent homeostatic mechanisms are activated in response to radiation exposure as a function of track structure or ionization density.

  1. Fluoroquinolone-induced gene transfer in multidrug-resistant Salmonella

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fluoroquinolones are broad spectrum antibiotics that inhibit bacterial DNA gyrase and topoisomerase activity. Bacterial exposure to fluoroquinolones can cause DNA damage and induce a bacterial SOS response to stimulate repair of damaged DNA. Certain prophages (integrated in bacterial chromosomes) ...

  2. Isolation of the alkane inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis

    EPA Science Inventory

    The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a λgt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were i...

  3. ISOLATION OF THE ALKANE INDUCIBLE CYTOCHROME P450 (P450ALK) GENE FROM THE YEAST CANDIDA TROPICALIS

    EPA Science Inventory

    The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a gtll library. solation of the gene has been identified on the basis of its inducibility and partial DNA sequence. ranscripts of this gene were indu...

  4. Virus-induced gene silencing of soybean rust resistance genes in Glycine tomentella

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean rust, incited by the fungal pathogen Phakopsora pachyrhizi, is a serious foliar soybean disease capable of causing major economic yield loss. Specific resistance to P. pachyrhizi is known and single dominant genes have been identified in soybean (Rpp1-4), but these genes have been deemed ine...

  5. Rapid Determination of Gene Function by Virus-induced Gene Silencing in Wheat and Barley

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cereal crops are essential components to the human and animal food supply. Solutions to many of the problems challenging cereal production will require identification of genes responsible for particular traits. Unfortunately, the process of identifying gene function is very slow and complex in...

  6. Establishment of a system for conditional gene expression using an inducible tRNA suppressor gene.

    PubMed Central

    Dingermann, T; Werner, H; Schütz, A; Zündorf, I; Nerke, K; Knecht, D; Marschalek, R

    1992-01-01

    We investigated the use of the prokaryotic tetracycline operator-repressor system as a regulatory device to control the expression of Dictyostelium discoideum tRNA genes. The tetO1 operator fragment was inserted at three different positions in front of a tRNA(Glu) (Am) suppressor gene from D. discoideum, and the tetracycline repressor gene was expressed under the control of a constitutive actin 6 promoter. The effectiveness of this approach was determined by monitoring the expression of a beta-galactosidase gene engineered to contain a stop codon that could be suppressed by the tRNA. When these constructs were introduced into Dictyostelium cells, the repressor bound to the operator in front of the tRNA gene and prevented expression of the suppressor tRNA. Addition of tetracycline (30 micrograms/ml) to the growth medium prevented repressor binding, allowed expression of the suppressor tRNA, and resulted in beta-galactosidase synthesis. The operator-repressor complex interfered with tRNA gene transcription when the operator was inserted immediately upstream (position +1 or -7) of the mature tRNA coding region. Expression of a tRNA gene carrying the operator at position -46 did not respond to repressor binding. This system could be used to control the synthesis of any protein, provided the gene contained a translational stop signal. Images PMID:1508201

  7. Rapid Determination of Gene Function by Virus-Induced Gene Silencing in Wheat and Barley

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cereal crops are essential components to the human and animal food supply. Solutions to many of the problems challenging cereal production will require identification of genes responsible for particular traits. Unfortunately, the process of identifying gene function is very slow and complex in ...

  8. Permethrin induces overexpression of multiple genes in Aedes aegypti.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using the PCR-select subtractive cDNA hybridization technique, 18 different genes were isolated from a permethrin-treated vs acetone-treated Aedes aegypti subtractive library. QPCR results revealed that eight of the 18 gene’s transcriptional levels in permethrin-treated Ae. aegypti were at least 2- ...

  9. Gene expression profile analysis in astaxanthin-induced Haematococcus pluvialis using a cDNA microarray.

    PubMed

    Eom, Hyunsuk; Lee, Choul-Gyun; Jin, EonSeon

    2006-05-01

    The unicellular green alga Haematococcus pluvialis (Volvocales) is known for the ketocarotenoid astaxanthin (3, 3'-dihydroxy-beta, beta-carotene-4, 4'-dione) accumulation, which is induced under unfavorable culture conditions. In this work, we used cDNA microarray analysis to screen differentially expressed genes in H. pluvialis under astaxanthin-inductive culture conditions, such as combination of cell exposure to high irradiance and nutrient deprivation. Among the 965 genes in the cDNA array, there are 144 genes exhibiting differential expression (twofold changes) under these conditions. A significant decrease in the expression of photosynthesis-related genes was shown in astaxanthin-accumulating cells (red cells). Defense- or stress-related genes and signal transduction genes were also induced in the red cells. A comparison of microarray and real-time PCR analysis showed good correlation between the differentially expressed genes by the two methods. Our results indicate that the cDNA microarray approach, as employed in this work, can be relied upon and used to monitor gene expression profiles in H. pluvialis. In addition, the genes that were differentially expressed during astaxanthin induction are suitable candidates for further study and can be used as tools for dissecting the molecular mechanism of this unique pigment accumulation process in the green alga H. pluvialis. PMID:16320067

  10. Identification of genes in Xanthomonas campestris pv. vesicatoria induced during its interaction with tomato.

    PubMed

    Tamir-Ariel, Dafna; Navon, Naama; Burdman, Saul

    2007-09-01

    Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease of tomato and pepper. The disease process is interactive and very intricate and involves a plethora of genes in the pathogen and in the host. In the pathogen, different genes are activated in response to the changing environment to enable it to survive, adapt, evade host defenses, propagate, and damage the host. To understand the disease process, it is imperative to broaden our understanding of the gene machinery that participates in it, and the most reliable way is to identify these genes in vivo. Here, we have adapted a recombinase-based in vivo expression technology (RIVET) to study the genes activated in X. campestris pv. vesicatoria during its interaction with one of its hosts, tomato. This is the first study that demonstrates the feasibility of this approach for identifying in vivo induced genes in a plant pathogen. RIVET revealed 61 unique X. campestris pv. vesicatoria genes or operons that delineate a picture of the different processes involved in the pathogen-host interaction. To further explore the role of some of these genes, we generated knockout mutants for 13 genes and characterized their ability to grow in planta and to cause disease symptoms. This analysis revealed several genes that may be important for the interaction of the pathogen with its host, including a citH homologue gene, encoding a citrate transporter, which was shown to be required for wild-type levels of virulence. PMID:17573477

  11. Inducible Knockdown of Plasmodium Gene Expression Using the glmS Ribozyme

    PubMed Central

    Prommana, Parichat; Uthaipibull, Chairat; Wongsombat, Chayaphat; Kamchonwongpaisan, Sumalee; Yuthavong, Yongyuth; Knuepfer, Ellen; Holder, Anthony A.; Shaw, Philip J.

    2013-01-01

    Conventional reverse genetic approaches for study of Plasmodium malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control P. falciparum gene expression in which target genes bearing a glmS ribozyme in the 3′ untranslated region are efficiently knocked down in transgenic P. falciparum parasites in response to glucosamine inducer. Using reporter genes, we show that the glmS ribozyme cleaves reporter mRNA in vivo leading to reduction in mRNA expression following glucosamine treatment. Glucosamine-induced ribozyme activation led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The glmS ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). Glucosamine treatment of transgenic parasites led to rapid and efficient knockdown of PfDHFR-TS mRNA and protein. PfDHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The glmS ribozyme may thus be a tool for study of essential genes in P. falciparum and other parasite species amenable to transfection. PMID:24023691

  12. Cloning and characterization of a new multi-stress inducible metallothionein gene in Tetrahymena pyriformis.

    PubMed

    Fu, Chengjie; Miao, Wei

    2006-06-01

    A new multi-stress-inducible metallothionein (MT) gene isoform has been cloned and characterized from the ciliate Tetrahymena pyriformis. Both the 5'- and 3'-UT regions of the Tp-MT2 gene are very different from the previously reported Tp-MT1 isoform in this organism and from other described MT genes in Tetrahymena pigmentosa and Tetrahymena thermophila. The putative protein sequence of Tp-MT2 contains cysteine clusters with characteristics of the typical Tetrahymena Cd-inducible MT genes. However, the sequence has a special feature of four intragenic tandem repeats within its first half, with a conserved structural pattern x(5/8)CCCx(6)CCx(6)CxCxNCxCCK. To investigate the transcriptional activities of both Tp-MT2 and Tp-MT1 genes toward heavy metals (Cd, Hg, Cu, Zn) and H(2)O(2), the mRNA levels of these two isoforms were evaluated by means of real-time quantitative PCR. Results showed that Tp-MT2 had a higher basal expression level than Tp-MT1 and both genes were induced by Cd, Hg, Cu, and Zn ions after short exposure (1h), although to different extents. Cd was the most effective metal inducer of both two isoforms, but the relative expression level of Tp-MT2 was much lower than that of Tp-MT1. Different expression patterns were also shown between the two genes when treated with Cd over a period of 24h. We suggest that TpMT-1 plays the role of a multi-inducible stress gene, while TpMT-2 may have a more specific function in basal metal homeostasis although it may have undergone a functional differentiation process. The putative functional significance and evolutionary mode of the TpMT-2 isoform are discussed. PMID:16621695

  13. Thermally induced osteocyte damage initiates pro-osteoclastogenic gene expression in vivo.

    PubMed

    Dolan, Eimear B; Tallon, David; Cheung, Wing-Yee; Schaffler, Mitchell B; Kennedy, Oran D; McNamara, Laoise M

    2016-06-01

    Bone is often subject to harsh temperatures during orthopaedic procedures resulting in thermally induced bone damage, which may affect the healing response. Postsurgical healing of bone is essential to the success of surgery, therefore, an understanding of the thermally induced responses of bone cells to clinically relevant temperatures in vivo is required. Osteocytes have been shown to be integrally involved in the bone remodelling cascade, via apoptosis, in micro-damage systems. However, it is unknown whether this relationship is similar following thermal damage. Sprague-Dawley rat tibia were exposed to clinically relevant temperatures (47°C or 60°C) to investigate the role of osteocytes in modulating remodelling related factors. Immunohistochemistry was used to quantify osteocyte thermal damage (activated caspase-3). Thermally induced pro-osteoclastogenic genes (Rankl, Opg and M-csf), in addition to genes known to mediate osteoblast and osteoclast differentiation via prostaglandin production (Cox2), vascularization (Vegf) and inflammatory (Il1a) responses, were investigated using gene expression analysis. The results demonstrate that heat-treatment induced significant bone tissue and cellular damage. Pro-osteoclastogenic genes were upregulated depending on the amount of temperature elevation compared with the control. Taken together, the results of this study demonstrate the in vivo effect of thermally induced osteocyte damage on the gene expression profile. PMID:27335224

  14. Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.

    PubMed

    Mercer, Andrew C; Gaj, Thomas; Sirk, Shannon J; Lamb, Brian M; Barbas, Carlos F

    2014-10-17

    The construction of increasingly sophisticated synthetic biological circuits is dependent on the development of extensible tools capable of providing specific control of gene expression in eukaryotic cells. Here, we describe a new class of synthetic transcription factors that activate gene expression in response to extracellular chemical stimuli. These inducible activators consist of customizable transcription activator-like effector (TALE) proteins combined with steroid hormone receptor ligand-binding domains. We demonstrate that these ligand-responsive TALE transcription factors allow for tunable and conditional control of gene activation and can be used to regulate the expression of endogenous genes in human cells. Since TALEs can be designed to recognize any contiguous DNA sequence, the conditional gene regulatory system described herein will enable the design of advanced synthetic gene networks. PMID:24251925

  15. Virus-induced gene complementation reveals a transcription factor network in modulation of tomato fruit ripening

    PubMed Central

    Zhou, Tao; Zhang, Hang; Lai, Tongfei; Qin, Cheng; Shi, Nongnong; Wang, Huizhong; Jin, Mingfei; Zhong, Silin; Fan, Zaifeng; Liu, Yule; Wu, Zirong; Jackson, Stephen; Giovannoni, James J.; Rolin, Dominique; Gallusci, Philippe; Hong, Yiguo

    2012-01-01

    Plant virus technology, in particular virus-induced gene silencing, is a widely used reverse- and forward-genetics tool in plant functional genomics. However the potential of virus technology to express genes to induce phenotypes or to complement mutants in order to understand the function of plant genes is not well documented. Here we exploit Potato virus X as a tool for virus-induced gene complementation (VIGC). Using VIGC in tomato, we demonstrated that ectopic viral expression of LeMADS-RIN, which encodes a MADS-box transcription factor (TF), resulted in functional complementation of the non-ripening rin mutant phenotype and caused fruits to ripen. Comparative gene expression analysis indicated that LeMADS-RIN up-regulated expression of the SBP-box (SQUAMOSA promoter binding protein-like) gene LeSPL-CNR, but down-regulated the expression of LeHB-1, an HD-Zip homeobox TF gene. Our data support the hypothesis that a transcriptional network may exist among key TFs in the modulation of fruit ripening in tomato. PMID:23150786

  16. DNase I hypersensitive sites within the inducible qa gene cluster of Neurospora crassa.

    PubMed Central

    Baum, J A; Giles, N H

    1986-01-01

    DNase I hypersensitive regions were mapped within the 17.3-kilobase qa (quinic acid) gene cluster of Neurospora crassa. The 5'-flanking regions of the five qa structural genes and the two qa regulatory genes each contain DNase I hypersensitive sites under noninducing conditions and generally exhibit increases in DNase I cleavage upon induction of transcription with quinic acid. The two large intergenic regions of the qa gene cluster appear to be similarly organized with respect to the positions of constitutive and inducible DNase I hypersensitive sites. Inducible hypersensitive sites on the 5' side of one qa gene, qa-x, appear to be differentially regulated. Employing these and previously published data, we have identified a conserved sequence element that may mediate the activator function of the qa-1F regulatory gene. Variants of the 16-base-pair consensus sequence are consistently found within DNase I-protected regions adjacent to inducible DNase I hypersensitive sites within the gene cluster. Images PMID:2944110

  17. Gene profiling of the erythro- and megakaryoblastic leukaemias induced by the Graffi murine retrovirus

    PubMed Central

    2010-01-01

    Background Acute erythro- and megakaryoblastic leukaemias are associated with very poor prognoses and the mechanism of blastic transformation is insufficiently elucidated. The murine Graffi leukaemia retrovirus induces erythro- and megakaryoblastic leukaemias when inoculated into NFS mice and represents a good model to study these leukaemias. Methods To expand our understanding of genes specific to these leukaemias, we compared gene expression profiles, measured by microarray and RT-PCR, of all leukaemia types induced by this virus. Results The transcriptome level changes, present between the different leukaemias, led to the identification of specific cancerous signatures. We reported numerous genes that may be potential oncogenes, may have a function related to erythropoiesis or megakaryopoiesis or have a poorly elucidated physiological role. The expression pattern of these genes has been further tested by RT-PCR in different samples, in a Friend erythroleukaemic model and in human leukaemic cell lines. We also screened the megakaryoblastic leukaemias for viral integrations and identified genes targeted by these integrations and potentially implicated in the onset of the disease. Conclusions Taken as a whole, the data obtained from this global gene profiling experiment have provided a detailed characterization of Graffi virus induced erythro- and megakaryoblastic leukaemias with many genes reported specific to the transcriptome of these leukaemias for the first time. PMID:20102610

  18. Requirement for metabolic activation of acetylaminofluorene to induce multidrug gene expression.

    PubMed Central

    Gant, T W; Schrenk, D; Silverman, J A; Thorgeirsson, S S

    1994-01-01

    Previously we have demonstrated that several xenobiotics can induce multidrug (mdr) gene expression in cultures of primary isolated hepatocytes. One of the best of these xenobiotic inducers in rat hepatocytes is 2-acetylaminofluorene (2-AAF), which induces mdr expression by an enhancement of mdr gene transcription. In all species studied to date, AAF is extensively and variously metabolized. In this study we have sought to determine if AAF per se or a metabolite is responsible for mediating the increase in mdr gene transcription and expression. This study demonstrates that AAF per se is not active, but that the effect of AAF we have observed on mdr gene transcription and expression in the rat is due to the formation of a reactive metabolite(s). Our data indicate that this reactive metabolite is probably N-acetoxy-2-aminofluorene or the sulfate ester of N-hydroxy-AAF. The requirement for the formation of one of these metabolites may explain the differences in species response to AAF, in terms of mdr gene expression, that we have observed. We hypothesize that the mechanism by which mdr gene transcription is increased in response to AAF involves a covalent interaction between a reactive metabolite and an mdr gene regulatory protein. Our current work is concerned with the exploration of this hypothesis. PMID:7889850

  19. Cigarette Smoke Modulates Expression of Human Rhinovirus-Induced Airway Epithelial Host Defense Genes

    PubMed Central

    Proud, David; Hudy, Magdalena H.; Wiehler, Shahina; Zaheer, Raza S.; Amin, Minaa A.; Pelikan, Jonathan B.; Tacon, Claire E.; Tonsaker, Tabitha O.; Walker, Brandie L.; Kooi, Cora; Traves, Suzanne L.; Leigh, Richard

    2012-01-01

    Human rhinovirus (HRV) infections trigger acute exacerbations of chronic obstructive pulmonary disease (COPD) and asthma. The human airway epithelial cell is the primary site of HRV infection and responds to infection with altered expression of multiple genes, the products of which could regulate the outcome to infection. Cigarette smoking aggravates asthma symptoms, and is also the predominant risk factor for the development and progression of COPD. We, therefore, examined whether cigarette smoke extract (CSE) modulates viral responses by altering HRV-induced epithelial gene expression. Primary cultures of human bronchial epithelial cells were exposed to medium alone, CSE alone, purified HRV-16 alone or to HRV-16+ CSE. After 24 h, supernatants were collected and total cellular RNA was isolated. Gene array analysis was performed to examine mRNA expression. Additional experiments, using real-time RT-PCR, ELISA and/or western blotting, validated altered expression of selected gene products. CSE and HRV-16 each induced groups of genes that were largely independent of each other. When compared to gene expression in response to CSE alone, cells treated with HRV+CSE showed no obvious differences in CSE-induced gene expression. By contrast, compared to gene induction in response to HRV-16 alone, cells exposed to HRV+CSE showed marked suppression of expression of a number of HRV-induced genes associated with various functions, including antiviral defenses, inflammation, viral signaling and airway remodeling. These changes were not associated with altered expression of type I or type III interferons. Thus, CSE alters epithelial responses to HRV infection in a manner that may negatively impact antiviral and host defense outcomes. PMID:22808255

  20. Technical advances in trigger-induced RNA interference gene silencing in the parasite Entamoeba histolytica.

    PubMed

    Khalil, Mohamed I; Foda, Bardees M; Suresh, Susmitha; Singh, Upinder

    2016-03-01

    Entamoeba histolytica has a robust endogenous RNA interference (RNAi) pathway. There are abundant 27 nucleotide (nt) anti-sense small RNAs (AS sRNAs) that target genes for silencing and the genome encodes many genes involved in the RNAi pathway such as Argonaute proteins. Importantly, an E. histolytica gene with numerous AS sRNAs can function as a "trigger" to induce silencing of a gene that is fused to the trigger. Thus, the amebic RNAi pathway regulates gene expression relevant to amebic biology and has additionally been harnessed as a tool for genetic manipulation. In this study we have further improved the trigger-induced gene silencing method. We demonstrate that rather than using the full-length gene, a short portion of the coding region fused to a trigger is sufficient to induce silencing; the first 537 bp of the E. histolytica rhomboid gene (EhROM1) fused in-frame to the trigger was sufficient to silence EhROM1. We also demonstrated that the trigger method could silence two amebic genes concomitantly; fusion of the coding regions of EhROM1 and transcription factor, EhMyb, in-frame to a trigger gene resulted in both genes being silenced. Alternatively, two genes can be silenced sequentially: EhROM1-silenced parasites with no drug selection plasmid were transfected with trigger-EhMyb, resulting in parasites with both EhROM1 and EhMyb silenced. With all approaches tested, the trigger-mediated silencing was substantive and silencing was maintained despite loss of the G418 selectable marker. All gene silencing was associated with generation of AS sRNAs to the silenced gene. We tested the reversibility of the trigger system using inhibitors of histone modifications but found that the silencing was highly stable. This work represents a technical advance in the trigger gene silencing method in E. histolytica. Approaches that readily silence multiple genes add significantly to the genetic toolkit available to the ameba research community. PMID:26747561

  1. CHARACTERIZATION OF THE ALKANE-INDUCIBLE CYTOCHROME P450 (P450ALK) GENE FROM THE YEAST CANDIDA TROPICALIS: IDENTIFICATION OF A NEW P450 GENE FAMILY

    EPA Science Inventory

    The P450ALK gene, which is inducible by the assimilation of alkane in Candida tropicalis, was sequenced and characterized. tructural features described in promoter and terminator regions of Saccharomyces yeast genes are present in the P450alk gene and some particular structures a...

  2. Human CYP1A1 gene: cosegregation of the enzyme inducibility phenotype and an RFLP.

    PubMed Central

    Petersen, D D; McKinney, C E; Ikeya, K; Smith, H H; Bale, A E; McBride, O W; Nebert, D W

    1991-01-01

    The human CYP1A1 (cytochrome P1450) gene encodes an enzyme involved in the activation of procarcinogens, such as benzo[a]pyrene, to the ultimate reactive intermediate. Approximately 10% of the human population exhibit high CYP1A1 inducibility, and Kouri et al. reported that the high-inducibility phenotype might be at greater risk than low-inducibility individuals for cigarette smoke-induced bronchogenic carcinoma. In one 3-generation family of 15 individuals, we show here that the high-CYP1A1-inducibility phenotype segregates concordantly with an infrequent polymorphic site located 450 bases downstream from the CYP1A1 gene. Our findings are consistent with the study of Kawajiri et al., who demonstrated an association between this polymorphism and an increased incidence of squamous-cell lung cancer. Our data suggest that the CYP1A1 structural gene, or a region near this gene, might be correlated with the inducibility phenotype. Images Figure 3 PMID:1707592

  3. Myostatin gene mutated mice induced with tale nucleases.

    PubMed

    Zhou, Fangfang; Sun, Ruilin; Chen, Hongyan; Fei, Jian; Lu, Daru

    2015-01-01

    Myostain gene (MSTN) is expressed primarily in skeletal muscle, and negatively regulates skeletal muscle mass; it has been suggested that mice with MSTN inhibition have reduced adiposity and improved insulin sensitivity. Therefore, it is important to establish a fast and effective gene editing method. In this report, we established the myostatin mutated-mouse model by microinjection of Transcription Activator-Like Effector Nucleases (TALENs) mRNA within the mouse fertilized oocytes and achieved high rates of mutagenesis of the mouse MSTN in C57BL/6J. Six of 45 born mice carried target mutations and we appointed one as the parental mating with wild mouse to produce the F1 and backcross to produce the F2 generation. All the mutations of the mice were examined quickly and efficiently by high-resolution melting curve analysis (HRMA) and then verified by direct sequencing. We obtained the homozygous of the F2 generation which transmitted the mutant alleles to the progeny with 100% efficiency. Mutant mice exhibited increases in muscle mass comparable to those observed in wild-type mice. Therefore, combining TALEN-mediated gene targeting with HRMA technology is a superior method of constructing genetically modified mice through microinjection in the mouse fertilized oocytes with high efficiency and short time of selection. PMID:25695746

  4. Inducible long-term gene expression in brain with adeno-associated virus gene transfer.

    PubMed

    Haberman, R P; McCown, T J; Samulski, R J

    1998-12-01

    Recombinant adeno-associated virus (rAAV) vectors hold promise for treating a number of neurological disorders due to the ability to deliver long-term gene expression without toxicity or immune response. Critical to these endeavors will be controlled expression of the therapeutic gene in target cells. We have constructed and tested a dual cassette rAAV vector carrying a reporter gene under the control of the tetracycline-responsive system and the tetracycline transactivator. Transduction in vitro resulted in stable expression from the vector that can be suppressed 20-fold by tetracycline treatment. In vivo experiments, carried out to 6 weeks, demonstrated that vector-transduced expression is sustained until doxycycline administration upon which reporter gene expression is reduced. Moreover, the suppression of vector-driven expression can be reversed by removal of the drug. These studies demonstrate long-term regulated gene expression from rAAV vectors. This system will provide a valuable approach for controlling vector gene expression both in vitro and in vivo. PMID:10023439

  5. Interferon induced IFIT family genes in host antiviral defense

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Secretion of interferons (IFNs) from virus-infected cells is a hallmark of host antiviral immunity and in fact, IFNs exert their antiviral activities through the induction of antiviral proteins. The IFN-induced protein with tetratricopeptide repeats (IFITs) family is among hundreds of IF stimulated ...

  6. Tert-butylhydroquinone ameliorates doxorubicin-induced cardiotoxicity by activating Nrf2 and inducing the expression of its target genes

    PubMed Central

    Wang, Lin-Feng; Su, Su-Wen; Wang, Lei; Zhang, Guo-Qiang; Zhang, Rong; Niu, Yu-Jie; Guo, Yan-Su; Li, Chun-Yan; Jiang, Wen-Bo; Liu, Yi; Guo, Hui-Cai

    2015-01-01

    Oxidative stress plays an important role in doxorubicin (DOX)-induced cardiotoxicity. Nuclear factor E2-related factor-2 (Nrf2) is a transcription factor that orchestrates the antioxidant and cytoprotective responses to oxidative stress. In the present study, we tested whether tert-butylhydroquinone (tBHQ) could protect against DOX-induced cardiotoxicity in vivo and, if so, whether the protection was associated with the up-regulation of the Nrf2 pathway. The results showed that treatment with tBHQ significantly decreased the DOX-induced cardiac injury in wild-type mice. Moreover, tBHQ ameliorated the DOX-induced oxidative stress and apoptosis. Further studies suggested that tBHQ increased the nuclear accumulation of Nrf2 and the Nrf2-regulated gene expression, including heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxido-reductase-1 (NQO-1) expression. Knocking out Nrf2 in mice abolished the protective effect of tBHQ on the DOX-induced cardiotoxicity. These results indicate that tBHQ has a beneficial effect on DOX-induced cardiotoxicity, and this effect was associated with the enhanced expression of Nrf2 and its downstream antioxidant genes, HO-1 and NQO-1. PMID:26692920

  7. Tamoxifen Induces Expression of Immune Response-Related Genes in Cultured Normal Human Mammary Epithelial Cells

    PubMed Central

    Schild-Hay, Laura J.; Leil, Tarek A.; Divi, Rao L.; Olivero, Ofelia, A.; Weston, Ainsley; Poirier, Miriam C.

    2008-01-01

    Use of tamoxifen (TAM) is associated with a 50% reduction in breast cancer incidence and an increase in endometrial cancer incidence. Here, we documented TAM-induced gene expression changes in cultured normal human mammary epithelial cells (NHMEC strains numbered 5, 16 and 40), established from tissue taken at reduction mammoplasty from 3 individuals. Cells exposed to 0, 10 or 50 μM TAM for 48 hours were evaluated for (E)-α-(deoxyguanosin-N2-yl)-tamoxifen (dG-N2-TAM) adduct formation by TAM-DNA (DNA modified with dG-N2-TAM) chemiluminescence immunoassay (CIA), gene expression changes using NCI DNA-oligonucleotide microarray, and real time (RT)-PCR. At 48 hr, cells exposed to 10 μM and 50 μM TAM were 85.6% and 48.4% viable, respectively, and there were no measurable dG-N2-TAM adducts. For microarray, cells were exposed to 10 μM TAM and genes with expression changes of ≥ 3-fold were as follows: thirteen genes up-regulated and one down-related for strain 16; seventeen genes up-regulated for strain 5; and eleven genes up-regulated for strain 40. Interferon-inducible genes (IFITM1, IFIT1, IFNA1, MXI and GIP3), and a potassium ion channel (KCNJ1) were up-regulated in all 3 strains. No significant expression changes were found for genes related to estrogen or xenobiotic metabolism. RT-PCR revealed up-regulation of interferon α (IFNA1) and confirmed the TAM-induced up-regulation of the genes identified by microarray, with the exception of GIP3 and MX1, which were not up-regulated in strain 40. Induction of interferon-related genes in the three NHMEC strains suggests that, in addition to hormonal effects, TAM exposure may enhance immune response in normal breast tissue. PMID:19155303

  8. An Inducible Caspase-9 Suicide Gene to Improve the Safety of Therapy Using Human Induced Pluripotent Stem Cells.

    PubMed

    Yagyu, Shigeki; Hoyos, Valentina; Del Bufalo, Francesca; Brenner, Malcolm K

    2015-09-01

    Human induced pluripotent stem cells (hiPSC) hold promise for regenerative therapies, though there are several safety concerns including the risk of oncogenic transformation or unwanted adverse effects associated with hiPSC or their differentiated progeny. Introduction of the inducible caspase-9 (iC9) suicide gene, which is activated by a specific chemical inducer of dimerization (CID), is one of the most appealing safety strategies for cell therapies and is currently being tested in multicenter clinical trials. Here, we show that the iC9 suicide gene with a human EF1α promoter can be introduced into hiPSC by lentiviral transduction. The transduced hiPSC maintain their pluripotency, including their capacity for unlimited self-renewal and the potential to differentiate into three germ layer tissues. Transduced hiPSC are eliminated within 24 hours of exposure to pharmacological levels of CID in vitro, with induction of apoptosis in 94-99% of the cells. Importantly, the iC9 suicide gene can eradicate tumors derived from hiPSC in vivo. In conclusion, we have developed a direct and efficient hiPSC killing system that provides a necessary safety mechanism for therapies using hiPSC. We believe that our iC9 suicide gene will be of value in clinical applications of hiPSC-based therapy. PMID:26022733

  9. Interlaboratory evaluation of rat hepatic gene expression changes induced by methapyrilene.

    PubMed Central

    Waring, Jeffrey F; Ulrich, Roger G; Flint, Nick; Morfitt, David; Kalkuhl, Arno; Staedtler, Frank; Lawton, Michael; Beekman, Johanna M; Suter, Laura

    2004-01-01

    Several studies using microarrays have shown that changes in gene expression provide information about the mechanism of toxicity induced by xenobiotic agents. Nevertheless, the issue of whether gene expression profiles are reproducible across different laboratories remains to be determined. To address this question, several members of the Hepatotoxicity Working Group of the International Life Sciences Institute Health and Environmental Sciences Institute evaluated the liver gene expression profiles of rats treated with methapyrilene (MP). Animals were treated at one facility, and RNA was distributed to five different sites for gene expression analysis. A preliminary evaluation of the number of modulated genes uncovered striking differences between the five different sites. However, additional data analysis demonstrated that these differences had an effect on the absolute gene expression results but not on the outcome of the study. For all users, unsupervised algorithms showed that gene expression allows the distinction of the high dose of MP from controls and low dose. In addition, the use of a supervised analysis method (support vector machines) made it possible to correctly classify samples. In conclusion, the results show that, despite some variability, robust gene expression changes were consistent between sites. In addition, key expression changes related to the mechanism of MP-induced hepatotoxicity were identified. These results provide critical information regarding the consistency of microarray results across different laboratories and shed light on the strengths and limitations of expression profiling in drug safety analysis. PMID:15033593

  10. The Mycoplasma hyorhinis p37 Protein Rapidly Induces Genes in Fibroblasts Associated with Inflammation and Cancer

    PubMed Central

    Gomersall, Amber Cathie; Li, Song Feng; Parish, Roger W.

    2015-01-01

    The p37 protein at the surface of Mycoplasma hyorhinis cells forms part of a high-affinity transport system and has been found associated with animal and human cancers. Here we show in NIH3T3 fibroblasts, p37 rapidly induces the expression of genes implicated in inflammation and cancer progression. This gene activation was principally via the Tlr4 receptor. Activity was lost from p37 when the C-terminal 20 amino acids were removed or the four amino acids specific for the hydrogen bonding of thiamine pyrophosphate had been replaced by valine. Blocking the IL6 receptor or inhibiting STAT3 signalling resulted in increased p37-induced gene expression. Since cancer associated fibroblasts support growth, invasion and metastasis via their ability to regulate tumour-related inflammation, the rapid induction in fibroblasts of pro-inflammatory genes by p37 might be expected to influence cancer development. PMID:26512722

  11. Wnt signaling induces transcription, spatial proximity, and translocation of fusion gene partners in human hematopoietic cells.

    PubMed

    Ugarte, Giorgia D; Vargas, Macarena F; Medina, Matías A; León, Pablo; Necuñir, David; Elorza, Alvaro A; Gutiérrez, Soraya E; Moon, Randall T; Loyola, Alejandra; De Ferrari, Giancarlo V

    2015-10-01

    Chromosomal translocations are frequently associated with a wide variety of cancers, particularly hematologic malignancies. A recurrent chromosomal abnormality in acute myeloid leukemia is the reciprocal translocation t(8;21) that fuses RUNX1 and ETO genes. We report here that Wnt/β-catenin signaling increases the expression of ETO and RUNX1 genes in human hematopoietic progenitors. We found that β-catenin is rapidly recruited into RNA polymerase II transcription factories (RNAPII-Ser5) and that ETO and RUNX1 genes are brought into close spatial proximity upon Wnt3a induction. Notably, long-term treatment of cells with Wnt3a induces the generation a frequent RUNX1-ETO translocation event. Thus, Wnt/β-catenin signaling induces transcription and translocation of RUNX1 and ETO fusion gene partners, opening a novel window to understand the onset/development of leukemia. PMID:26333776

  12. In vivo footprinting of the estrogen-inducible vitellogenin II gene from chicken.

    PubMed Central

    Philipsen, J N; Hennis, B C; Ab, G

    1988-01-01

    Protein-DNA interactions in the promoter region of the chicken vitellogenin II gene were analyzed by in vivo dimethylsulphate footprinting with expressing and non-expressing tissues. The reactivity of G-residues is essentially the same in erythrocytes, oviduct and control liver, not expressing the gene. In the expressing estrogen-induced liver we find a number of G-residues with altered reactivities. These G's are located within distinct sequences: the estrogen responsive elements, a sequence resembling the NF-1 recognition motive, and several elements which are conserved between yolk protein genes. The expression-dependent binding of proteins to these sites was confirmed by DNaseI footprinting applied to nuclei isolated from estrogen-induced and control liver. Estradiol appears to establish a transcription complex comprising a number of distinct proteins bound to different sites in the 5' flanking region of the vitellogenin II gene. Images PMID:3186442

  13. Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas

    SciTech Connect

    Hahn, M.; Hayward, W.S.

    1988-06-01

    The authors determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myce genes contained missense mutations. This strongly supports the notion that the c-myc photo-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product.

  14. The agricultural antibiotic carbadox induces phage-mediated gene transfer in Salmonella

    PubMed Central

    Bearson, Bradley L.; Allen, Heather K.; Brunelle, Brian W.; Lee, In Soo; Casjens, Sherwood R.; Stanton, Thaddeus B.

    2013-01-01

    Antibiotics are used for disease therapeutic or preventative effects in humans and animals, as well as for enhanced feed conversion efficiency in livestock. Antibiotics can also cause undesirable effects in microbial populations, including selection for antibiotic resistance, enhanced pathogen invasion, and stimulation of horizontal gene transfer. Carbadox is a veterinary antibiotic used in the US during the starter phase of swine production for improved feed efficiency and control of swine dysentery and bacterial swine enteritis. Carbadox has been shown in vitro to induce phage-encoded Shiga toxin in Shiga toxin-producing Escherichia coli (STEC) and a phage-like element transferring antibiotic resistance genes in Brachyspira hyodysenteriae, but the effect of carbadox on prophages in other bacteria is unknown. This study examined carbadox exposure on prophage induction and genetic transfer in Salmonella enterica serovar Typhimurium, a human foodborne pathogen that frequently colonizes swine without causing disease. S. Typhimurium LT2 exposed to carbadox induced prophage production, resulting in bacterial cell lysis and release of virions that were visible by electron microscopy. Carbadox induction of phage-mediated gene transfer was confirmed by monitoring the transduction of a sodCIII::neo cassette in the Fels-1 prophage from LT2 to a recipient Salmonella strain. Furthermore, carbadox frequently induced generalized transducing phages in multidrug-resistant phage type DT104 and DT120 isolates, resulting in the transfer of chromosomal and plasmid DNA that included antibiotic resistance genes. Our research indicates that exposure of Salmonella to carbadox induces prophages that can transfer virulence and antibiotic resistance genes to susceptible bacterial hosts. Carbadox-induced, phage-mediated gene transfer could serve as a contributing factor in bacterial evolution during animal production, with prophages being a reservoir for bacterial fitness genes in the

  15. Attenuation of serum inducibility of immediate early genes by oncoproteins in tyrosine kinase signaling pathways.

    PubMed Central

    Yu, C L; Prochownik, E V; Imperiale, M J; Jove, R

    1993-01-01

    Immediate early genes involved in controlling cell proliferation are rapidly and transiently induced following stimulation of susceptible cells with serum. To study how oncoproteins regulate immediate early genes, we examined serum inducibility of these genes in cells transformed by various oncoproteins. We found that induction of the immediate early gene, c-fos, by serum stimulation was markedly attenuated in four independent cell lines stably transformed by the v-Src tyrosine kinase. Cells chronically transformed by other oncoproteins implicated in tyrosine kinase signaling pathways, including v-Sis, v-Ras, and v-Raf, showed the same pattern of attenuation. In contrast, serum inducibility of c-fos was not attenuated in cells transformed by simian virus 40, which is thought to transform cells through a different pathway. Cell cycle analyses showed that proliferation of these transformed cell lines could be arrested effectively in 0.1% serum, demonstrating that the attenuation was not simply due to continuous cycling of transformed cells after serum deprivation. Moreover, serum inducibility of other immediate early genes, including c-jun, junB, egr-1, and NGFI-B, also was strikingly attenuated by these same oncoproteins. Nuclear run-on transcription assays established that this attenuation of serum inducibility occurred at the transcriptional level. Finally, flow cytometric analysis demonstrated that serum-starved v-Src-transformed cells were viable and able to progress into S phase of the cell cycle after serum stimulation, even though the induction of immediate early genes was greatly attenuated in these cells. Our results suggest that activation of immediate early genes is repressed by chronic stimulation of tyrosine kinase signaling pathways in transformed cells. Images PMID:8384301

  16. Rhizobium meliloti Genes Encoding Catabolism of Trigonelline Are Induced under Symbiotic Conditions.

    PubMed

    Boivin, C.; Camut, S.; Malpica, C. A.; Truchet, G.; Rosenberg, C.

    1990-12-01

    Rhizobium meliloti trc genes controlling the catabolism of trigonelline, a plant secondary metabolite often abundant in legumes, are closely linked to nif-nod genes on the symbiotic megaplasmid pSym [Boivin, C., Malpica, C., Rosenberg, C., Denarie, J., Goldman, A., Fleury, V., Maille, M., Message, B., and Tepfer, D. (1989). In Molecular Signals in the Microbe-Plant Symbiotic and Pathogenic Systems. (Berlin: Springer-Verlag), pp. 401-407]. To investigate the role of trigonelline catabolism in the Rhizobium-legume interaction, we studied the regulation of trc gene expression in free-living and in endosymbiotic bacteria using Escherichia coli lacZ as a reporter gene. Experiments performed with free-living bacteria indicated that trc genes were organized in at least four transcription units and that the substrate trigonelline was a specific inducer for three of them. Noninducing trigonelline-related compounds such as betaines appeared to antagonize the inducing effect of trigonelline. None of the general or symbiotic regulatory genes ntrA, dctB/D, or nodD seemed to be involved in trigonelline catabolism. trc fusions exhibiting a low basal and a high induced [beta]-galactosidase activity when present on pSym were used to monitor trc gene expression in alfalfa tissue under symbiotic conditions. Results showed that trc genes are induced during all the symbiotic steps, i.e., in the rhizosphere, infection threads, and bacteroids of alfalfa, suggesting that trigonelline is a nutrient source throughout the Rhizobium-legume association. PMID:12354952

  17. Differential gene expression and lipid metabolism in fatty liver induced by acute ethanol treatment in mice

    SciTech Connect

    Yin Huquan; Kim, Mingoo; Kim, Ju-Han; Kong, Gu; Kang, Kyung-Sun; Kim, Hyung-Lae; Yoon, Byung-IL; Lee, Mi-Ock; Lee, Byung-Hoon

    2007-09-15

    Ethanol induces cumulative liver damage including steatosis, steatohepatitis and cirrhosis. The aim of this study is to investigate the global intrahepatic gene expression profile in the mouse liver treated with ethanol. A single oral dose of 0.5 or 5 g/kg ethanol was administered to male ICR mice, and liver samples were obtained after 6, 24 and 72 h. Histopathological evaluation showed typical fatty livers in the high-dose group at 24 h. Microarray analysis identified 28 genes as being ethanol responsive (two-way ANOVA; p < 0.05), after adjustment by the Benjamini-Hochberg multiple testing correction; these genes displayed {>=} 2-fold induction or repression. The expression of genes that are known to be involved in fatty acid synthesis was examined. The transcript for lipogenic transcription factor, sterol regulatory element (SRE)-binding factor 1 (Srebf1), was upregulated by acute ethanol exposure. Of the genes known to contain SRE or SRE-like sequences and to be regulated by SRE-binding protein 1 (SREBP1), those encoding malic enzyme (Mod1), ATP-citrate lyase (Acly), fatty acid synthase (Fasn) and stearyl-CoA desaturase (Scd1) were induced by ethanol. Quantitative real-time PCR confirmed the changes in the expression levels of the selected genes. The change in the Srebf1 mRNA level correlates well with that of the SREBP1 protein expression as well as its binding to the promoters of the target genes. The present study identifies differentially expressed genes that can be applied to the biomarkers for alcohol-binge-induced fatty liver. These results support the hypothesis by which ethanol-induced steatosis in mice is mediated by the fatty acid synthetic pathway regulated by SREBP1.

  18. Virus-induced gene silencing in the rapid cycling columbine Aquilegia coerulea "Origami".

    PubMed

    Sharma, Bharti; Kramer, Elena M

    2013-01-01

    Aquilegia Origami is an emerging model system for ecology and evolution, which has numerous genetic and genomic tools. Virus-induced gene silencing (VIGS) has been established as an effective approach to study gene function in Aquilegia. In the current protocol, we demonstrate VIGS using Agrobacterium strain GV3101 carrying tobacco rattle virus (TRV)-based constructs to infect Aquilegia coerulea "Origami" plants via vacuum infiltration. PMID:23386296

  19. IRF1 marks activated genes in SLE and can induce target gene expression

    PubMed Central

    Zhang, Zhe; Shi, Lihua; Song, Li; Ephrem, Elshaddai; Petri, Michelle; Sullivan, Kathleen E.

    2014-01-01

    Objective IRF1 both mediates responses to type I interferons and the induction of interferons. It has been implicated in murine lupus models as a critical mediator of inflammation. A previous study of chromatin modifications in SLE patient monocytes implicated IRF1 as associated with increased histone acetylation in SLE patients. This study directly investigated IRF1 binding sites on chromatin using ChIP-seq. Methods Nine female SLE patients and seven female controls were examined. Monocytes were purified from peripheral blood and subjected to library preparation using a validated antibody to IRF1. The effect of IRF1 on target gene expression was confirmed using an overexpression system in cell lines and co-immunoprecipitation was used to define protein interactions. Results IRF1 binding around transcribed regions was increased in SLE patient monocytes but histone modifications at potential IRF1 binding sites without detectable IRF1 binding were also increased. IRF1 overexpression was sufficient to drive transcription of target genes. IRF1 overexpression was also able to alter histone modifications at a focus set of target genes and the use of an IRF1 inhibitor decreased both expression and histone modifications at target genes. IRF1 was found to interact with a select set of histone modifying enzymes and other transcription factors. Conclusions IRF1 is an important signaling protein in the interferon pathway. IRF1 not only activates gene expression as a transcription factor but may perpetuate disease by leading to a dysregulated epigenome. PMID:25418955

  20. Sense Transgene-Induced Post-Transcriptional Gene Silencing in Tobacco Compromises the Splicing of Endogenous Counterpart Genes

    PubMed Central

    Shin, Mi-Rae; Natsuume, Masaya; Matsumoto, Takashi; Hanaoka, Mitsumasa; Imai, Misaki; Iijima, Ken; Oka, Shin-ichiro; Adachi, Eri; Kodama, Hiroaki

    2014-01-01

    Sense transgene-induced post-transcriptional gene silencing (S-PTGS) is thought to be a type of RNA silencing in which ARGONAUTE1 directs the small interfering RNA (siRNA)-mediated cleavage of a target mRNA in the cytoplasm. Here, we report that the altered splicing of endogenous counterpart genes is a main cause for the reduction of their mature mRNA levels. After the S-PTGS of a tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) gene, 3′-truncated, polyadenylated endo-NtFAD3 transcripts and 5′-truncated, intron-containing endo-NtFAD3 transcripts were detected in the total RNA fraction. Although transcription proceeded until the last exon of the endogenous NtFAD3 gene, intron-containing NtFAD3 transcripts accumulated in the nucleus of the S-PTGS plants. Several intron-containing NtFAD3 transcripts harboring most of the exon sequences were generated when an endogenous silencing suppressor gene, rgs-CaM, was overexpressed in the S-PTGS plants. These intron-containing NtFAD3 splice variants were generated in the presence of NtFAD3 siRNAs that are homologous to the nucleotide sequences of these splice variants. The results of this study indicate that the inhibition of endo-NtFAD3 gene expression is primarily directed via the alteration of splicing and not by cytoplasmic slicer activity. Our results suggest that the transgene and intron-containing endogenous counterpart genes are differentially suppressed in S-PTGS plants. PMID:24586294

  1. Regulation of sesquiterpene cyclase gene expression. Characterization of an elicitor- and pathogen-inducible promoter.

    PubMed Central

    Yin, S; Mei, L; Newman, J; Back, K; Chappell, J

    1997-01-01

    The promoter for a tobacco (Nicotiana tabacum) sesquiterpene cyclase gene, a key regulatory step in sesquiterpene phytoalexin biosynthesis, has been analyzed. The EAS4 promoter was fused to the beta-glucuronidase (GUS) reporter gene, and the temporal and spatial expression patterns of GUS activity were examined in stably transformed plants and in transient expression assays using electroporated protoplasts of tobacco. No GUS activity was observed in any tissues under normal growth conditions. A low level of GUS activity was detected in wounded leaf, root, and stem tissues, whereas a much higher level was observed when these tissues were challenged with elicitors or microbial pathogens. The GUS expression pattern directed by the EAS4 promoter was identical to the induction patterns observed for the endogenous sesquiterpene cyclase genes. Neither exogenous salicylic acid nor methyl jasmonate induced GUS expression; and H2O2 induced GUS expression to only a limited extent. Although the EAS4 promoter contains cis-sequences resembling previously identified transcriptional control motifs, other cis-sequences important for quantitative and qualitative gene expression were identified by deletion and gain-of-function analyses. The EAS4 promoter differs from previously described pathogen-/elicitor-inducible promoters because it only supports inducible gene expression and directs unique spatial expression patterns. PMID:9342864

  2. Activity analysis and preliminary inducer screening of the chicken DAZL gene promoter.

    PubMed

    Zhang, Lei; Zhu, Rui; Zuo, Qisheng; Li, Dong; Lian, Chao; Tang, Beibei; Xiao, Tianrong; Zhang, Yani; Li, Bichun

    2015-01-01

    This study was aimed at identifying the active control area of chicken DAZL gene core promoter, to screen optimum inducers of the DAZL gene, thus to enhance the differentiation of embryonic stem cells into spermatogonial stem cells. Fragments of chicken DAZL gene promoter were cloned into fluorescent reporter plasmids and transfected into DF-1 cells. Then Dual-Luciferase® Reporter Assay System was used to identify the activity of the DAZL gene under different inducers. Our studies showed that the DAZL core promoter region for the Suqin yellow chicken was -383 to -39 bp. The dual-luciferase® reporter showed that all-trans retinoic acid (ATRA), a retinoic acid receptor alpha agonist (tamibarotene/Am80), or estradiol (E2) could significantly enhance DAZL transcription. The in vitro inductive culture of chicken ESCs demonstrated that, with ATRA treatment, DAZL transcription peaked at 6 days and then decreased slowly; whereas, DAZL transcription was continuous and peaked at 10 days with Am80 treatment. E2 treatment significantly increased DAZL expression after 8 days. All three treatments were associated with the appearance of male germ cell (MGC)-like cells on day 10. These results provide the optimum inducer screening of the DAZL gene and lay the foundation for further screening of compounds that can induce the differentiation of ESCs into MGCs in vitro. PMID:25807265

  3. Activity Analysis and Preliminary Inducer Screening of the Chicken DAZL Gene Promoter

    PubMed Central

    Zhang, Lei; Zhu, Rui; Zuo, Qisheng; Li, Dong; Lian, Chao; Tang, Beibei; Xiao, Tianrong; Zhang, Yani; Li, Bichun

    2015-01-01

    This study was aimed at identifying the active control area of chicken DAZL gene core promoter, to screen optimum inducers of the DAZL gene, thus to enhance the differentiation of embryonic stem cells into spermatogonial stem cells. Fragments of chicken DAZL gene promoter were cloned into fluorescent reporter plasmids and transfected into DF-1 cells. Then Dual-Luciferase® Reporter Assay System was used to identify the activity of the DAZL gene under different inducers. Our studies showed that the DAZL core promoter region for the Suqin yellow chicken was −383 to −39 bp. The dual-luciferase® reporter showed that all-trans retinoic acid (ATRA), a retinoic acid receptor alpha agonist (tamibarotene/Am80), or estradiol (E2) could significantly enhance DAZL transcription. The in vitro inductive culture of chicken ESCs demonstrated that, with ATRA treatment, DAZL transcription peaked at 6 days and then decreased slowly; whereas, DAZL transcription was continuous and peaked at 10 days with Am80 treatment. E2 treatment significantly increased DAZL expression after 8 days. All three treatments were associated with the appearance of male germ cell (MGC)-like cells on day 10. These results provide the optimum inducer screening of the DAZL gene and lay the foundation for further screening of compounds that can induce the differentiation of ESCs into MGCs in vitro. PMID:25807265

  4. A light-inducible CRISPR/Cas9 system for control of endogenous gene activation

    PubMed Central

    Polstein, Lauren R.; Gersbach, Charles A.

    2015-01-01

    Optogenetic systems enable precise spatial and temporal control of cell behavior. We engineered a light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light. This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive dCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes. PMID:25664691

  5. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    SciTech Connect

    Sidjanin, D.; Grdina, D.; Woloschak, G.E.

    1994-11-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation induced by ultraviolet radiation. These experiments investigated the ability of 254 nm radiation on cell cycle progression and gene expression in rabbit lens epithelial cell line N/N1003A. No changes in expression of c-fos, c-jun, alpha- tubulin, or vimentin was observed following UV exposure. Using flow cytometry, an accumulation of cells in G1/S phase of the cell cycle 1 hr following exposure. The observed changes in gene expression, especially the decreased histone transcripts reported here may play a role in UV induced inhibition of cell cycle progression.

  6. Cord blood administration induces oligodendrocyte survival through alterations in gene expression

    PubMed Central

    Rowe, D.D.; Leonardo, C.C.; Hall, A.A.; Shahaduzzaman, M.D.; Collier, L.A.; Willing, A.E.; Pennypacker, K.R.

    2010-01-01

    Oligodendrocytes (OLs), the predominant cell type found in cerebral white matter, are essential for structural integrity and proper neural signaling. Very little is known concerning stroke-induced OL dysfunction. Our laboratory has shown that infusion of human umbilical cord blood (HUCB) cells protects striatal white matter tracts in vivo and directly protects mature primary OL cultures from oxygen glucose deprivation (OGD). Microarray studies of RNA prepared from OL cultures subjected to OGD and treated with HUCB cells showed an increase in the expression of 33 genes associated with OL proliferation, survival, and repair functions, such as myelination. The microarray results were verified using quantitative RT-PCR for the following eight genes: U2AF homology motif kinase 1 (Uhmk1), insulin induce gene 1 (Insig1), metallothionein ( Mt3), tetraspanin 2 (Tspan2), peroxiredoxin 4 (Prdx4), stathmin-like 2 (Stmn2), myelin oligodendrocyte glycoprotein (MOG), and versican (Vcan). Immunohistochemistry showed that MOG, Prdx4, Uhmk1, Insig1 and Mt3 protein expression were upregluated in the ipsilateral white matter tracts of rats infused with HUCB cells 48 hrs after middle cerebral artery occlusion (MCAO). Furthermore, promoter region analysis of these genes revealed common transcription factor binding sites, providing insight into the shared signal transduction pathways activated by HUCB cells to enhance transcription of these genes. These results show expression of genes induced by HUCB cell therapy that could confer oligoprotection from ischemia. PMID:20883670

  7. A CRISPR-based screen identifies genes essential for West Nile virus-induced cell death

    PubMed Central

    Ma, Hongming; Dang, Ying; Wu, Yonggan; Jia, Gengxiang; Anaya, Edgar; Zhang, Junli; Abraham, Sojan; Choi, Jang-Gi; Shi, Guojun; Qi, Ling; Manjunath, N.; Wu, Haoquan

    2015-01-01

    Summary West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen followed by a second screen with a sub-library. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J1, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the endoplasmic reticulum-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death. PMID:26190106

  8. Use of differential fluorescence induction and optical trapping to isolate environmentally induced genes.

    PubMed

    Allaway, D; Schofield, N A; Leonard, M E; Gilardoni, L; Finan, T M; Poole, P S

    2001-06-01

    The techniques of differential fluorescence induction (DFI) and optical trapping (OT) have been combined to allow the identification of environmentally induced genes in single bacterial cells. Designated DFI-OT, this technique allows the in situ isolation of genes driving the expression of green fluorescent protein (Gfp) using temporal and spatial criteria. A series of plasmid-based promoter probe vectors (pOT) was developed for the construction of random genomic libraries that are linked to gfpUV or egfp. Bacteria that do not express Gfp on laboratory medium (i.e. non-fluorescent) were inoculated into the environment, and induced genes were detected with a combined fluorescence/optical trapping microscope. Using this selection strategy, rhizosphere-induced genes with homology to thiamine pyrophosphorylase (thiE) and cyclic glucan synthase (ndvB) were isolated. Other genes were expressed late in the stationary phase or as a consequence of surface-dependent growth, including fixND and metX, and a putative ABC transporter of putrescine. This strategy provides a unique ability to combine spatial, temporal and physical information to identify environmental regulation of bacterial gene expression. PMID:11472504

  9. Analysis of Stress Responsive Genes Induced by Single-Walled Carbon Nanotubes in BJ Foreskin Cells

    PubMed Central

    Sarkar, Shubhashish; Sharma, Chidananda; Yog, Rajeshwari; Periakaruppan, Adaikkappan; Jejelowo, Olufisayo; Thomas, Renard; Barrera, Enrique V.; Rice-Ficht, Allison C.; Wilson, Bobby L.; Ramesh, Govindarajan T.

    2009-01-01

    Nanotechnology is finding its use as a potential technology in consumer products, defense, electronics, and medical applications by exploiting the properties of nanomaterials. Single-walled carbon nanotubes are novel forms of these nanomaterials with potential for large applications. However, the toxicity studies on this material are not explored in detail and therefore limiting its use. It has been earlier reported that single-walled carbon nanotubes induces oxidative stress and also dictates activation of specific signaling pathway in keratinocytes. The present study explores the effect of single-walled carbon nanotubes on stress genes in human BJ Foreskin cells. The results show induction of oxidative stress in BJ Foreskin cells by single-walled carbon nanotubes and increase in stress responsive genes. The genes included inducible genes like HMOX1, HMOX2, and Cyp1B1. In addition we validated increase for four genes by SWCNT, namely ATM, CCNC, DNAJB4, and GADD45A by RT-PCR. Moreover results of the altered stress related genes have been discussed and that partially explains some of the toxic responses induced by single-walled carbon nanotubes. PMID:17450800

  10. Potential of GRID2 receptor gene for preventing TNF-induced neurodegeneration in autism.

    PubMed

    Kalkan, Zeynep; Durasi, İlknur Melis; Sezerman, Ugur; Atasever-Arslan, Belkis

    2016-05-01

    Autism is one of the most common subtypes of autism spectrum disorder (ASD). Recent studies suggested a relationship between immune-dependent coding genes and ASD, indicating that long term neuroimmunological anomalies affect brain development and synaptic transmission among neural networks. Furthermore, various studies focused on biomarker potential of TNF-α in autism. Ionotropic receptors are also studied as potential marker for autism since altered gene expression levels are observed in autistic patients. GRID2 is a candidate ionotropic receptor which is involved glutamate transfer. In this study, to propose TNF-α dependent cellular processes involved in autism aetiology in relation to GRID2 we performed a bioinformatic network analysis and identified potential pathways and genes that are involved in TNF-α induced changes at GRID2 receptor levels. As a result, we ascertained the GRID2 receptor gene as a candidate gene and further studied the association between GRID2 expression levels and TNF-induced neurodegeneration. Our bioinformatic analyses and experimental results revealed that TNF-α regulates GRID2 gene expression by activating Cdc42 and GOPC genes. Moreover, increased TNF-α levels leads to increase of caspase-3 protein levels triggering neuronal apoptosis leading to neuronal deficiency, which is one of the major symptoms of autism. The study is the first to show the role of TNF-α in regulation of GRID2 gene expression and its signalling pathway. As a result, GRID2 gene can be a suppressor in TNF-induced neurodegeneration which may help to understand the main factors leading to autism. PMID:27019035

  11. Transcriptomic Sequencing Reveals a Set of Unique Genes Activated by Butyrate-Induced Histone Modification.

    PubMed

    Li, Cong-Jun; Li, Robert W; Baldwin, Ransom L; Blomberg, Le Ann; Wu, Sitao; Li, Weizhong

    2016-01-01

    Butyrate is a nutritional element with strong epigenetic regulatory activity as a histone deacetylase inhibitor. Based on the analysis of differentially expressed genes in the bovine epithelial cells using RNA sequencing technology, a set of unique genes that are activated only after butyrate treatment were revealed. A complementary bioinformatics analysis of the functional category, pathway, and integrated network, using Ingenuity Pathways Analysis, indicated that these genes activated by butyrate treatment are related to major cellular functions, including cell morphological changes, cell cycle arrest, and apoptosis. Our results offered insight into the butyrate-induced transcriptomic changes and will accelerate our discerning of the molecular fundamentals of epigenomic regulation. PMID:26819550

  12. Transcriptomic Sequencing Reveals a Set of Unique Genes Activated by Butyrate-Induced Histone Modification

    PubMed Central

    Li, Cong-Jun; Li, Robert W.; Baldwin, Ransom L.; Blomberg, Le Ann; Wu, Sitao; Li, Weizhong

    2016-01-01

    Butyrate is a nutritional element with strong epigenetic regulatory activity as a histone deacetylase inhibitor. Based on the analysis of differentially expressed genes in the bovine epithelial cells using RNA sequencing technology, a set of unique genes that are activated only after butyrate treatment were revealed. A complementary bioinformatics analysis of the functional category, pathway, and integrated network, using Ingenuity Pathways Analysis, indicated that these genes activated by butyrate treatment are related to major cellular functions, including cell morphological changes, cell cycle arrest, and apoptosis. Our results offered insight into the butyrate-induced transcriptomic changes and will accelerate our discerning of the molecular fundamentals of epigenomic regulation. PMID:26819550

  13. MR VIGS: microRNA-based virus-induced gene silencing in plants.

    PubMed

    Chen, Weiwei; Zhang, Qi; Kong, Junhua; Hu, Feng; Li, Bin; Wu, Chaoqun; Qin, Cheng; Zhang, Pengcheng; Shi, Nongnong; Hong, Yiguo

    2015-01-01

    In plants, microRNA (miRNA)-based virus-induced gene silencing, dubbed MR VIGS, is a powerful technique to delineate the biological functions of genes. By targeting to a specific sequence, miRNAs can knock down expression of genes with fewer off-target effects. Here, using a modified Cabbage leaf curling virus (CaLCuV) and Tobacco rattle virus (TRV) as vectors, we describe two virus-based miRNA expression systems to perform MR VIGS for plant functional genomics assays. PMID:25740363

  14. Identification of genes regulated during mechanical load-induced cardiac hypertrophy

    NASA Technical Reports Server (NTRS)

    Johnatty, S. E.; Dyck, J. R.; Michael, L. H.; Olson, E. N.; Abdellatif, M.; Schneider, M. (Principal Investigator)

    2000-01-01

    Cardiac hypertrophy is associated with both adaptive and adverse changes in gene expression. To identify genes regulated by pressure overload, we performed suppressive subtractive hybridization between cDNA from the hearts of aortic-banded (7-day) and sham-operated mice. In parallel, we performed a subtraction between an adult and a neonatal heart, for the purpose of comparing different forms of cardiac hypertrophy. Sequencing more than 100 clones led to the identification of an array of functionally known (70%) and unknown genes (30%) that are upregulated during cardiac growth. At least nine of those genes were preferentially expressed in both the neonatal and pressure over-load hearts alike. Using Northern blot analysis to investigate whether some of the identified genes were upregulated in the load-independent calcineurin-induced cardiac hypertrophy mouse model, revealed its incomplete similarity with the former models of cardiac growth. Copyright 2000 Academic Press.

  15. A Study on Effect of Electroacupuncture on Gene Expression in Hypothalamus of Rats with Stress-Induced Prehypertension Based on Gene Chip Technology

    PubMed Central

    Xie, Xiaojia; Guo, Yan; Liu, Qingguo; Wang, Zhaoyang; Guo, Changqing

    2015-01-01

    Objective. To explore the effect of electroacupuncture (EA) on gene expression in the hypothalamus of rats with stress-induced prehypertension and try to reveal its biological mechanism with gene chip technology. Methods. The stress-induced hypertensive rat model was prepared by combining electric foot-shocks with generated noise. Molding cycle lasted for 14 days and EA intervention was applied on model + EA group during model preparation. Rat Gene 2.0 Array technology was used for the determination of gene expression profiles and the screened key genes were verified by real-time fluorescence quantitative PCR method. Results. Compared with the blank group, 234 genes were upregulated and 73 were downregulated in the model group. Compared with the model group, 110 genes were upregulated and 273 genes were downregulated in model + EA group. The PCR results of the key genes including HSPB1, P2RX4, PPP1R14A, and TH are consistent with that of gene chip test. Conclusion. EA could significantly lower blood pressure of stress-induced prehypertension rats and affect its gene expression profile in hypothalamus. Genes and their signal transduction pathway that related to the contraction of vascular smooth muscle, concentration of Ca2+, and excitability of sympathetic nerve may be involved in EA's antihypertensive mechanism. PMID:26229544

  16. 20-hydroxyecdysone upregulates Atg genes to induce autophagy in the Bombyx fat body

    PubMed Central

    Tian, Ling; Ma, Li; Guo, Enen; Deng, Xiaojuan; Ma, Sanyuan; Xia, Qingyou; Cao, Yang; Li, Sheng

    2013-01-01

    Autophagy is finely regulated at multiple levels and plays crucial roles in development and disease. In the fat body of the silkworm, Bombyx mori, autophagy occurs and Atg gene expression peaks during the nonfeeding molting and pupation stages when the steroid hormone (20-hydroxyecdysone; 20E) is high. Injection of 20E into the feeding larvae upregulated Atg genes and reduced TORC1 activity resulting in autophagy induction in the fat body. Conversely, RNAi knockdown of the 20E receptor partner (USP) or targeted overexpression of a dominant negative mutant of the 20E receptor (EcRDN) in the larval fat body reduced autophagy and downregulated the Atg genes, confirming the importance of 20E-induction of Atg gene expression during pupation. Moreover, in vitro treatments of the larval fat body with 20E upregulated the Atg genes. Five Atg genes were potentially 20E primary-responsive, and a 20E response element was identified in the Atg1 (ortholog of human ULK1) promoter region. Furthermore, RNAi knockdown of 4 key genes (namely Br-C, E74, HR3 and βftz-F1) in the 20E-triggered transcriptional cascade reduced autophagy and downregulated Atg genes to different levels. Taken together, we conclude that in addition to blocking TORC1 activity for autophagosome initiation, 20E upregulates Atg genes to induce autophagy in the Bombyx fat body. PMID:23674061

  17. Gene expression during imidacloprid-induced hormesis in green peach aphid.

    PubMed

    Ayyanath, Murali-Mohan; Cutler, G Christopher; Scott-Dupree, Cynthia D; Prithiviraj, Balakrishnan; Kandasamy, Saveetha; Prithiviraj, Kalyani

    2014-07-01

    Imidacloprid-induced hormesis in the form of stimulated reproduction has previously been reported in green peach aphid, Myzus persicae. Changes in gene expression accompanying this hormetic response have not been previously investigated. In this study, expression of stress response (Hsp60), dispersal (OSD, TOL and ANT), and developmental (FPPS I) genes were examined for two generations during imidacloprid-induced reproductive stimulation in M. persicae. Global DNA methylation was also measured to test the hypothesis that changes in gene expression are heritable. At hormetic concentrations, down-regulation of Hsp60 was followed by up-regulation of this gene in the subsequent generation. Likewise, expression of dispersal-related genes and FPPS I varied with concentration, life stage, and generation. These results indicate that reproductive hormesis in M. persicae is accompanied by a complex transgenerational pattern of up- and down-regulation of genes that likely reflects trade-offs in gene expression and related physiological processes during the phenotypic dose-response. Moreover, DNA methylation in second generation M. persicae occurred at higher doses than in first-generation aphids, suggesting that heritable adaptability to low doses of the stressor might have occurred. PMID:25249837

  18. Gene expression profiling of breast cells induced by X-rays and heavy ions.

    PubMed

    Roy, D; Guida, P; Zhou, G; Echiburu-Chau, C; Calaf, G M

    2008-05-01

    Several genetic aberrations and gene expression changes have been shown to occur when cells are exposed to various types of radiation. The integrity of DNA depends upon several processes that include DNA damage recognition and repair, replication, transcription and cell cycle regulation. Ionizing radiation has many sources, including radon decay from the soil and X-rays from medical practice. Epidemiological evidence indicates a risk for cancer by inducing genetic alterations through DNA damage, and molecular alterations have been reported in epidemiological studies of the A-bomb survivors. A spontaneously immortalized human breast epithelial cell model, MCF-10F, was used to examine the gene expression profiling of breast cells induced by X-ray and heavy ion exposure, by a cDNA expression array of DNA damage and repair genes. This cell line was exposed to 10, 50, 100 and 200 cGy of either X-rays or heavy ions and gene expression profiles were studied. Results indicated that out of a total of 161 genes, 38 were differentially expressed by X-ray treatment and 24 by heavy ion (Fe(+2)) treatment. Eight genes were common to both treatments and were confirmed by Northern blot analysis: BRCA1, BIRC2/CIAP1, CENP-E, DDB1, MRE11A, RAD54/ATRX, Wip1 and XPF/ERCC4. A number of candidate genes reported here may be useful molecular biomarkers of radiation exposure in breast cells. PMID:18425356

  19. Targeted gene conversion induced by triplex-directed psoralen interstrand crosslinks in mammalian cells.

    PubMed

    Liu, Yaobin; Nairn, Rodney S; Vasquez, Karen M

    2009-10-01

    Correction of a defective gene is a promising approach for both basic research and clinical gene therapy. However, the absence of site-specific targeting and the low efficiency of homologous recombination in human cells present barriers to successful gene targeting. In an effort to overcome these barriers, we utilized triplex-forming oligonucleotides (TFOs) conjugated to a DNA interstrand crosslinking (ICL) agent, psoralen (pTFO-ICLs), to improve the gene targeting efficiency at a specific site in DNA. Gene targeting events were monitored by the correction of a deletion on a recipient plasmid with the homologous sequence from a donor plasmid in human cells. The mechanism underlying this event is stimulation of homologous recombination by the pTFO-ICL. We found that pTFO-ICLs are efficient in inducing targeted gene conversion (GC) events in human cells. The deletion size in the recipient plasmid influenced both the recombination frequency and spectrum of recombinants; i.e. plasmids with smaller deletions had a higher frequency and proportion of GC events. The polarity of the pTFO-ICL also had a prominent effect on recombination. Our results suggest that pTFO-ICL induced intermolecular recombination provides an efficient method for targeted gene correction in mammalian cells. PMID:19726585

  20. Among-lake reciprocal transplants induce convergent expression of immune genes in threespine stickleback.

    PubMed

    Stutz, William E; Schmerer, Matthew; Coates, Jessica L; Bolnick, Daniel I

    2015-09-01

    Geographic variation in parasite communities can drive evolutionary divergence in host immune genes. However, biotic and abiotic environmental variation can also induce plastic differences in immune function among populations. At present, there is little information concerning the relative magnitudes of heritable vs. induced immune divergence in natural populations. We examined immune gene expression profiles of threespine stickleback (Gasterosteus aculeatus) from six lakes on Vancouver Island, British Columbia. Parasite community composition differs between lake types (large or small, containing limnetic- or benthic-like stickleback) and between watersheds. We observed corresponding differences in immune gene expression profiles among wild-caught stickleback, using a set of seven immune genes representing distinct branches of the immune system. To evaluate the role of environmental effects on this differentiation, we experimentally transplanted wild-caught fish into cages in their native lake, or into a nearby foreign lake. Transplanted individuals' immune gene expression converged on patterns typical of their destination lake, deviating from their native expression profile. Transplant individuals' source population had a much smaller effect, suggesting relatively weak genetic underpinning of population differences in immunity, as viewed through gene expression. This strong environmental regulation of immune gene expression provides a counterpoint to the large emerging literature documenting microevolution and genetic diversification of immune function. Our findings illustrate the value of studying immunity in natural environmental settings where the immune system has evolved and actively functions. PMID:26118468

  1. Virus induced gene silencing of a gene repressing flowering in sugar beet.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Exposure to a prolonged cold period during winter is necessary for flowering in the next spring in many biennial plants - a process termed vernalization. We have described BvFL1, a vernalization gene in sugar beet (Beta vulgaris), which is a repressor of flowering that is downregulated in response ...

  2. Gene expression profiling in undifferentiated thyroid carcinoma induced by high-dose radiation.

    PubMed

    Bang, Hyun Soon; Choi, Moo Hyun; Kim, Cha Soon; Choi, Seung Jin

    2016-06-01

    Published gene expression studies for radiation-induced thyroid carcinogenesis have used various methodologies. In this study, we identified differential gene expression in a human thyroid epithelial cell line after exposure to high-dose γ-radiation. HTori-3 cells were exposed to 5 or 10 Gy of ionizing radiation using two dose rates (high-dose rate: 4.68 Gy/min, and low-dose rate: 40 mGy/h) and then implanted into the backs of BALB/c nude mice after 4 (10 Gy) or 5 weeks (5 Gy). Decreases in cell viability, increases in giant cell frequency, anchorage-independent growth in vitro, and tumorigenicity in vivo were observed. Particularly, the cells irradiated with 5 Gy at the high-dose rate or 10 Gy at the low-dose rate demonstrated more prominent tumorigenicity. Gene expression profiling was analyzed via microarray. Numerous genes that were significantly altered by a fold-change of >50% following irradiation were identified in each group. Gene expression analysis identified six commonly misregulated genes, including CRYAB, IL-18, ZNF845, CYP24A1, OR4N4 and VN1R4, at all doses. These genes involve apoptosis, the immune response, regulation of transcription, and receptor signaling pathways. Overall, the altered genes in high-dose rate (HDR) 5 Gy and low-dose rate (LDR) 10 Gy were more than those of LDR 5 Gy and HDR 10 Gy. Thus, we investigated genes associated with aggressive tumor development using the two dosage treatments. In this study, the identified gene expression profiles reflect the molecular response following high doses of external radiation exposure and may provide helpful information about radiation-induced thyroid tumors in the high-dose range. PMID:27006382

  3. Gene expression profiling in undifferentiated thyroid carcinoma induced by high-dose radiation

    PubMed Central

    Bang, Hyun Soon; Choi, Moo Hyun; Kim, Cha Soon; Choi, Seung Jin

    2016-01-01

    Published gene expression studies for radiation-induced thyroid carcinogenesis have used various methodologies. In this study, we identified differential gene expression in a human thyroid epithelial cell line after exposure to high-dose γ-radiation. HTori-3 cells were exposed to 5 or 10 Gy of ionizing radiation using two dose rates (high-dose rate: 4.68 Gy/min, and low-dose rate: 40 mGy/h) and then implanted into the backs of BALB/c nude mice after 4 (10 Gy) or 5 weeks (5 Gy). Decreases in cell viability, increases in giant cell frequency, anchorage-independent growth in vitro, and tumorigenicity in vivo were observed. Particularly, the cells irradiated with 5 Gy at the high-dose rate or 10 Gy at the low-dose rate demonstrated more prominent tumorigenicity. Gene expression profiling was analyzed via microarray. Numerous genes that were significantly altered by a fold-change of >50% following irradiation were identified in each group. Gene expression analysis identified six commonly misregulated genes, including CRYAB, IL-18, ZNF845, CYP24A1, OR4N4 and VN1R4, at all doses. These genes involve apoptosis, the immune response, regulation of transcription, and receptor signaling pathways. Overall, the altered genes in high-dose rate (HDR) 5 Gy and low-dose rate (LDR) 10 Gy were more than those of LDR 5 Gy and HDR 10 Gy. Thus, we investigated genes associated with aggressive tumor development using the two dosage treatments. In this study, the identified gene expression profiles reflect the molecular response following high doses of external radiation exposure and may provide helpful information about radiation-induced thyroid tumors in the high-dose range. PMID:27006382

  4. IKK{epsilon} modulates RSV-induced NF-{kappa}B-dependent gene transcription

    SciTech Connect

    Bao Xiaoyong; Indukuri, Hemalatha; Liu Tianshuang; Liao Suiling; Tian, Bing; Brasier, Allan R.; Garofalo, Roberto P.; Casola, Antonella

    2010-12-20

    Respiratory syncytial virus (RSV), a negative-strand RNA virus, is the most common cause of epidemic respiratory disease in infants and young children. RSV infection of airway epithelial cells induces the expression of immune/inflammatory genes through the activation of a subset of transcription factors, including Nuclear Factor-{kappa}B (NF-{kappa}B). In this study we have investigated the role of the non canonical I{kappa}B kinase (IKK){epsilon} in modulating RSV-induced NF-{kappa}B activation. Our results show that inhibition of IKK{epsilon} activation results in significant impairment of viral-induced NF-{kappa}B-dependent gene expression, through a reduction in NF-{kappa}B transcriptional activity, without changes in nuclear translocation or DNA-binding activity. Absence of IKK{epsilon} results in a significant decrease of RSV-induced NF-{kappa}B phosphorylation on serine 536, a post-translational modification important for RSV-induced NF-{kappa}B-dependent gene expression, known to regulate NF-{kappa}B transcriptional activity without affecting nuclear translocation. This study identifies a novel mechanism by which IKK{epsilon} regulates viral-induced cellular signaling.

  5. Identification of genes regulating TRAIL-induced apoptosis in rheumatoid arthritis fibroblasts-like synoviocytes.

    PubMed

    Audo, R; Hegglin, A; Severac, D; Dantec, C; Combe, B; Hahne, M; Morel, J

    2015-10-01

    We previously described that sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis varied in rheumatoid arthritis fibroblasts-like synoviocytes (RAFLS) from one patient to another and was correlated with disease severity. Therefore, we screened for genes differentially expressed in RAFLS sensitive and resistant to TRAIL-induced apoptosis. The sensitivity of RAFLS was defined based on the percentage of TRAIL-induced apoptosis: 0-10% for resistant cells and >25% for sensitive RAFLS. We performed transcriptomic comparison between RAFLS-S (n=6) and RAFLS-R (n=6) and then examined the implication of identified candidates in the regulation of apoptosis using small interference RNA (siRNA). Microarray analysis revealed 10 functional genes differentially expressed according to TRAIL sensitivity. These factors are implicated in different functions, such as the respiratory chain (ND3), the transport of lipids (OSBP2, PLTP), the regulation of signaling linked to extracellular factors (SULF2, GALNT1, SIAE) or the regulation of gene expression (TET2 and LARP6). We confirmed differential expression for GALNT1 and LARP6 by quantitative reverse transcriptase-PCR. Using siRNA extinction, we demonstrated the implication of GALNT1, SULF2 and LARP6 in the control of TRAIL-induced responses. These results are of particular interest as GALNT1 and LARP6 have been implicated in the regulation of cell death and may represent interesting targets to induce apoptosis of RAFLS. PMID:26247836

  6. IKKε MODULATES RSV-INDUCED NF-κB-DEPENDENT GENE TRANSCRIPTION

    PubMed Central

    Bao, Xiaoyong; Indukuri, Hemalatha; Liu, Tianshuang; Liao, Sui-Ling; Tian, Bing; Brasier, Allan R.; Garofalo, Roberto P.; Casola, Antonella

    2010-01-01

    Respiratory syncytial virus (RSV), a negative-strand RNA virus, is the most common cause of epidemic respiratory disease in infants and young children. RSV infection of airway epithelial cells induces the expression of immune/inflammatory genes through the activation of a subset of transcription factors, including Nuclear Factor-κB (NF-κB). In this study we have investigated the role of the non canonical IκB kinase (IKK)ε in modulating RSV-induced NF-κB activation. Our results show that inhibition of IKKε activation results in significant impairment of viral-induced NF-κB-dependent gene expression, through a reduction in NF-κB transcriptional activity, without changes in nuclear translocation or DNA-binding activity. Absence of IKKε results in a significant decrease of RSV-induced NF-κB phosphorylation on serine 536, a post-translational modification important for RSV-induced NF-κB-dependent gene expression, known to regulate NF-κB transcriptional activity without affecting nuclear translocation. This study identifies a novel mechanism by which IKKε regulates viral-induced cellular signaling. PMID:20961594

  7. Multiple copies of a bile acid-inducible gene in Eubacterium sp. strain VPI 12708.

    PubMed Central

    Gopal-Srivastava, R; Mallonee, D H; White, W B; Hylemon, P B

    1990-01-01

    Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium which possesses inducible bile acid 7-dehydroxylation activity. Several new polypeptides are produced in this strain following induction with cholic acid. Genes coding for two copies of a bile acid-inducible 27,000-dalton polypeptide (baiA1 and baiA2) have been previously cloned and sequenced. We now report on a gene coding for a third copy of this 27,000-dalton polypeptide (baiA3). The baiA3 gene has been cloned in lambda DASH on an 11.2-kilobase DNA fragment from a partial Sau3A digest of the Eubacterium DNA. DNA sequence analysis of the baiA3 gene revealed 100% homology with the baiA1 gene within the coding region of the 27,000-dalton polypeptides. The baiA2 gene shares 81% sequence identity with the other two genes at the nucleotide level. The flanking nucleotide sequences associated with the baiA1 and baiA3 genes are identical for 930 bases in the 5' direction from the initiation codon and for at least 325 bases in the 3' direction from the stop codon, including the putative promoter regions for the genes. An additional open reading frame (occupying from 621 to 648 bases, depending on the correct start codon) was found in the identical 5' regions associated with the baiA1 and baiA3 clones. The 5' sequence 930 bases upstream from the baiA1 and baiA3 genes was totally divergent. The baiA2 gene, which is part of a large bile acid-inducible operon, showed no homology with the other two genes either in the 5' or 3' direction from the polypeptide coding region, except for a 15-base-pair presumed ribosome-binding site in the 5' region. These studies strongly suggest that a gene duplication (baiA1 and baiA3) has occurred and is stably maintained in this bacterium. Images PMID:2376563

  8. Influence factors and gene expression patterns during MeJa-induced gummosis in peach.

    PubMed

    Li, Minji; Liu, Meiyan; Peng, Futian; Fang, Long

    2015-06-15

    Jasmonates (JAs) play important roles in gummosis in peach. Mechanical damage, methyl jasmonate (MeJa), and ethylene can induce gummosis on peach shoots in the field. In this study, we used MeJa (2%, w/w) to induce gummosis on current-year shoots in peach on high temperature (35°C). Based on the experimental model, we studied the influence of factors on the development of peach gummosis. Our experimental results showed that high temperature could promote gummosis development induced by MeJa. Exogenous CaCl2 treatment reduced the degree of gummosis by increasing the calcium content in shoots, which is conducive to the synthesis and maintenance of the cell wall. Using digital gene expression (DGE), 3831 differentially expressed genes were identified in the MeJa treatment versus the control. By analyzing changes in gene expression associated with cell wall degradation, genes encoding pectin methylesterase (PME) and endo-polygalacturonase (PG) were found to be significantly induced, suggesting that they are key enzymes in cell wall degradation that occurs during MeJa-induced gummosis. Genes for glycosyltransferase (GT) and cellulose synthase (CS) were also significantly upregulated by MeJa. This result suggests that MeJa treatment not only promotes the degradation of polysaccharides to destroy the cell wall, but also promotes the synthesis of new polysaccharides. We also analyzed changes in gene expression associated with sugar metabolism, senescence, and defense. MeJa treatment affected the expression of genes related to sugar metabolism and promoted plant senescence. Among the defense genes, the expression pattern of phenylalanine ammonium lyase (PAL) suggested that PAL may play an important role in protecting against the effects of MeJa treatment. Our experimental results showed that MeJa treatment can promote the biosynthesis and signal transduction of ethylene in peach shoots; they can induce gummosis on peach shoots respectively, and there are overlaps between

  9. Inducible Gene Expression in Tumors Colonized by Modified Oncolytic Vaccinia Virus Strains

    PubMed Central

    Huppertz, Sascha; Zhang, Qian; Geissinger, Ulrike; Härtl, Barbara; Gentschev, Ivaylo

    2014-01-01

    ABSTRACT Exogenous gene induction of therapeutic, diagnostic, and safety mechanisms could be a considerable improvement in oncolytic virotherapy. Here, we introduced a doxycycline-inducible promoter system (comprised of a tetracycline repressor, several promoter constructs, and a tet operator sequence) into oncolytic recombinant vaccinia viruses (rVACV), which were further characterized in detail. Experiments in cell cultures as well as in tumor-bearing mice were analyzed to determine the role of the inducible-system components. To accomplish this, we took advantage of the optical reporter construct, which resulted in the production of click-beetle luciferase as well as a red fluorescent protein. The results indicated that each of the system components could be used to optimize the induction rates and had an influence on the background expression levels. Depending on the given gene to be induced in rVACV-colonized tumors of patients, we discuss the doxycycline-inducible promoter system adjustment and further optimization. IMPORTANCE Oncolytic virotherapy of cancer can greatly benefit from the expression of heterologous genes. It is reasonable that some of those heterologous gene products could have detrimental effects either on the cancer patient or on the oncolytic virus itself if they are expressed at the wrong time or if the expression levels are too high. Therefore, exogenous control of gene expression levels by administration of a nontoxic inducer will have positive effects on the safety as well as the therapeutic outcome of oncolytic virotherapy. In addition, it paves the way for the introduction of new therapeutic genes into the genome of oncolytic viruses that could not have been tested otherwise. PMID:25056902

  10. Fine ambient particles induce oxidative stress and metal binding genes in human alveolar macrophages.

    PubMed

    Huang, Yuh-Chin T; Li, Zhuowei; Carter, Jacqueline D; Soukup, Joleen M; Schwartz, David A; Yang, Ivana V

    2009-11-01

    Exposure to pollutant particles increased respiratory morbidity and mortality. The alveolar macrophages (AMs) are one cell type in the lung directly exposed to particles. Upon contact with particles, AMs are activated and produce reactive oxygen species, but the scope of this oxidative stress response remains poorly defined. In this study, we determined the gene expression profile in human AMs exposed to particles, and sought to characterize the global response of pro- and antioxidant genes. We exposed AMs obtained by bronchoscopy from normal individuals to Chapel Hill particulate matter of 2.5-microm diameter or smaller (PM(2.5); 1 microg/ml) or vehicle for 4 hours (n = 6 independent samples). mRNAs were extracted, amplified, and hybridized to Agilent human 1A microarray. Significant genes were identified by significance analysis of microarrays (false discovery rate, 10%; P < or = 0.05) and mapped with Gene Ontology in the Database for Annotation, Visualization, and Integrated Discovery. We found 34 and 41 up- and down-regulated genes, respectively; 22 genes (approximately 30%) were involved in metal binding, and 11 were linked to oxidative stress, including up-regulation of five metallothionein (MT)-1 isoforms. Exogenous MT1 attenuated PM(2.5)-induced H2O2 release. PM(2.5) premixed with MT1 stimulated less H2O2 release. Knockdown of MT1F gene increased PM(2.5)-induced H2O2 release. PM(2.5) at 1 microg/ml did not increase H2O2 release. Mount St. Helens PM(2.5) and acid-extracted Chapel Hill PM(2.5), both poor in metals, did not induce MT1F or H2O2 release. Our results show that PM(2.5) induced a gene expression profile prevalent with genes related to metal binding and oxidative stress in human AMs, independent of oxidative stress. Metals associated with PM may play an important role in particle-induced gene changes. PMID:19251948

  11. Fine Ambient Particles Induce Oxidative Stress and Metal Binding Genes in Human Alveolar Macrophages

    PubMed Central

    Huang, Yuh-Chin T.; Li, Zhuowei; Carter, Jacqueline D.; Soukup, Joleen M.; Schwartz, David A.; Yang, Ivana V.

    2009-01-01

    Exposure to pollutant particles increased respiratory morbidity and mortality. The alveolar macrophages (AMs) are one cell type in the lung directly exposed to particles. Upon contact with particles, AMs are activated and produce reactive oxygen species, but the scope of this oxidative stress response remains poorly defined. In this study, we determined the gene expression profile in human AMs exposed to particles, and sought to characterize the global response of pro- and antioxidant genes. We exposed AMs obtained by bronchoscopy from normal individuals to Chapel Hill particulate matter of 2.5-μm diameter or smaller (PM2.5; 1 μg/ml) or vehicle for 4 hours (n = 6 independent samples). mRNAs were extracted, amplified, and hybridized to Agilent human 1A microarray. Significant genes were identified by significance analysis of microarrays (false discovery rate, 10%; P ≤ 0.05) and mapped with Gene Ontology in the Database for Annotation, Visualization, and Integrated Discovery. We found 34 and 41 up- and down-regulated genes, respectively; 22 genes (∼30%) were involved in metal binding, and 11 were linked to oxidative stress, including up-regulation of five metallothionein (MT)-1 isoforms. Exogenous MT1 attenuated PM2.5-induced H2O2 release. PM2.5 premixed with MT1 stimulated less H2O2 release. Knockdown of MT1F gene increased PM2.5-induced H2O2 release. PM2.5 at 1 μg/ml did not increase H2O2 release. Mount St. Helens PM2.5 and acid-extracted Chapel Hill PM2.5, both poor in metals, did not induce MT1F or H2O2 release. Our results show that PM2.5 induced a gene expression profile prevalent with genes related to metal binding and oxidative stress in human AMs, independent of oxidative stress. Metals associated with PM may play an important role in particle-induced gene changes. PMID:19251948

  12. Metal-dependent SV40 viruses containing inducible enhancers from the upstream region of metallothionein genes.

    PubMed Central

    Serfling, E; Lübbe, A; Dorsch-Häsler, K; Schaffner, W

    1985-01-01

    We have isolated SV40 recombinant viruses which are dependent on heavy metal ions for efficient propagation. They were obtained after-co-transfection of enhancerless SV40 DNA (the so-called enhancer trap) with sonicated DNA from the mouse metallothionein-I (mMT-I) or human metallothionein-IIA (hMT-IIA) upstream regions. To substitute for the SV40 enhancer, these viruses have incorporated a segment of the immediate upstream region of the metallothionein genes. Two recombinant viruses of the SVMT-I type carry segments of the mMT-I gene from positions -73 to -187 and -39 to -194 inverted with respect to their natural configuration. The overlapping segment contains two of the four metal-responsive elements involved in the induction of the mMT-I gene by heavy metal ions. The SVMT-II recombinant virus contains a segment of the hMT-IIA gene from position -39 to -366 which harbors the metal- and hormone-responsive elements of the hMT-IIA gene. Insertion of the mMT-I segment downstream of a rabbit beta-globin test gene enhances beta-globin transcription upon metal ion stimulation. This shows that the immediate upstream region of the mouse metalliothionein-I gene, when detached from its TATA box, can act as an inducible enhancer. It may be generally true that the enhancer/promoters of inducible genes are composed of several regulatory sequence elements which are interspersed with constitutive elements. The number and spatial arrangement of these elements probably determines the basal versus induced level of expression. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:2419129

  13. Delay-Induced Transient Increase and Heterogeneity in Gene Expression in Negatively Auto-Regulated Gene Circuits

    PubMed Central

    Maithreye, R.; Sarkar, Ram Rup; Parnaik, Veena K.; Sinha, Somdatta

    2008-01-01

    A generic feature in all intracellular biochemical processes is the time required to complete the whole sequence of reactions to yield any observable quantity-from gene expression to circadian rhythms. This widespread phenomenon points towards the importance of time delay in biological functions. Theoretically time delay is known to be the source of instability, and has been attributed to lead to oscillations or transient dynamics in several biological functions. Negative feedback loops, common in biochemical pathways, have been shown to provide stability and withstand considerable variations and random perturbations of biochemical parameters. The interaction of these two opposing factors-of instability and homeostasis-are features that are widespread in intracellular processes. To test the effect of these divergent forces in the dynamics of gene expression, we have designed and constructed simple negatively auto-regulated gene circuits consisting of a basic regulator and transcriptional repressor module, and compared it with one, which has delayed repression. We show, both theoretically and experimentally, that delayed repression induces transient increase and heterogeneity in gene expression before the gain of stability effected by the negative feedback. This design, therefore, seems to be suitable for conferring both stability and variability in cells required for adaptive response to a noisy environment. PMID:18698420

  14. CaMK4 Gene Deletion Induces Hypertension

    PubMed Central

    Santulli, Gaetano; Cipolletta, Ersilia; Sorriento, Daniela; Del Giudice, Carmine; Anastasio, Antonio; Monaco, Sara; Maione, Angela Serena; Condorelli, Gianluigi; Puca, Annibale; Trimarco, Bruno; Illario, Maddalena; Iaccarino, Guido

    2012-01-01

    Background The expression of calcium/calmodulin-dependent kinase IV (CaMKIV) was hitherto thought to be confined to the nervous system. However, a recent genome-wide analysis indicated an association between hypertension and a single-nucleotide polymorphism (rs10491334) of the human CaMKIV gene (CaMK4), which suggests a role for this kinase in the regulation of vascular tone. Methods and Results To directly assess the role of CaMKIV in hypertension, we characterized the cardiovascular phenotype of CaMK4−/− mice. They displayed a typical hypertensive phenotype, including high blood pressure levels, cardiac hypertrophy, vascular and kidney damage, and reduced tolerance to chronic ischemia and myocardial infarction compared with wild-type littermates. Interestingly, in vitro experiments showed the ability of this kinase to activate endothelial nitric oxide synthase. Eventually, in a population study, we found that the rs10491334 variant associates with a reduction in the expression levels of CaMKIV in lymphocytes from hypertensive patients. Conclusions Taken together, our results provide evidence that CaMKIV plays a pivotal role in blood pressure regulation through the control of endothelial nitric oxide synthase activity. (J Am Heart Assoc. 2012;1:e001081 doi: 10.1161/JAHA.112.001081.) PMID:23130158

  15. Sugar-Dependent Gibberellin-Induced Chalcone Synthase Gene Expression in Petunia Corollas.

    PubMed Central

    Moalem-Beno, D.; Tamari, G.; Leitner-Dagan, Y.; Borochov, A.; Weiss, D.

    1997-01-01

    The induction of anthocyanin synthesis and anthocyanin biosynthetic gene expression in detached petunia (Petunia hybrida) corollas by gibberellic acid (GA3) requires sucrose. Neither sucrose nor GA3 alone can induce these processes. We found that GA3 enhances sucrose uptake by 20 to 30%, and we tested whether this is the mechanism by which the hormone induces gene expression. Changing the intracellular level of sucrose with the inhibitors p-chloromercuribenzenesulfonic acid and vanadate did not inhibit the induction of chalcone synthase gene (chs) expression by GA3. Growing detached corollas in various sucrose concentrations did not affect the induction of the gene but did affect its level of expression and the level of anthocyanin accumulated. Only metabolic sugars promoted GA3-induced anthocyanin accumulation. Mannitol and sorbitol had no effect and 3-O-methylglucose only slightly promoted chs expression and anthocyanin accumulation. Our results do not support the suggestion that sugars act as specific signals in the activation of anthocyanin biosynthetic gene expression during petunia corolla development. We suggest that sugars are essential as general sources of carbohydrates for carbon metabolism, upon which the induction of pigmentation is dependent. PMID:12223616

  16. Rescue Effects and Underlying Mechanisms of Intragland Shh Gene Delivery on Irradiation-Induced Hyposalivation.

    PubMed

    Hai, Bo; Zhao, Qingguo; Qin, Lizheng; Rangaraj, Dharanipathy; Gutti, Veera R; Liu, Fei

    2016-05-01

    Irreversible hypofunction of salivary glands is common in head and neck cancer survivors treated with radiotherapy and can only be temporarily relieved with current treatments. We found in an inducible sonic hedgehog (Shh) transgenic mouse model that transient activation of the Hedgehog pathway after irradiation rescued salivary gland function in males by preserving salivary stem/progenitor cells and parasympathetic innervation. To translate these findings into feasible clinical application, we evaluated the effects of Shh gene transfer to salivary glands of wild-type mice on irradiation-induced hyposalivation. Shh or control GFP gene was delivered by noninvasive retrograde ductal instillation of corresponding adenoviral vectors. In both male and female mice, Shh gene delivery efficiently activated Hedgehog/Gli signaling, and significantly improved stimulated saliva secretion and preserved saliva-producing acinar cells after irradiation. In addition to preserving parasympathetic innervation through induction of neurotrophic factors, Shh gene delivery also alleviated the irradiation damage of the microvasculature, likely via inducing angiogenic factors, but did not expand the progeny of cells responsive to Hedgehog/Gli signaling. These data indicate that transient activation of the Hedgehog pathway by gene delivery is promising to rescue salivary function after irradiation in both sexes, and the Hedgehog/Gli pathway may function mainly in cell nonautonomous manners to achieve the rescue effect. PMID:27021743

  17. Liver lipid molecules induce PEPCK-C gene transcription and attenuate insulin action

    SciTech Connect

    Chen Guoxun

    2007-09-28

    Cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) plays key roles in gluconeogenesis, glyceroneogenesis, and cataplerosis. Experiments were designed to examine the effects of endogenous lipid molecules from rat livers on the expression of PEPCK-C gene in primary rat hepatocytes. The lipid extracts prepared from livers of Zucker fatty, lean, and Wistar rats induced the expression levels of PEPCK-C transcripts. Insulin-mediated reduction of PEPCK-C gene expression was attenuated by the same treatment. The lipid extracts induced the relative luciferase activity of reporter gene constructs that contain a 2.2-kb 5' promoter fragment of PEPCK-C gene, but not the construct that contains only the 3' untranslated region (UTR) of its mRNA. The estimated half life of PEPCK-C transcripts in the presence of the lipid extract is the same as that in the absence of it. My results demonstrate for the first time that endogenous lipid molecules induce PEPCK-C gene transcription and attenuate insulin action in liver.

  18. Isolation of nine gene sequences induced by silica in murine macrophages

    SciTech Connect

    Segade, F.; Claudio, E.; Wrobel, K.; Ramos, S.; Lazo, P.S.

    1995-03-01

    Macrophage activation by silica is the initial step in the development of silicosis. To identify genes that might be involved in silica-mediated activation, RAW 264.7 mouse macrophages were treated with silica for 48 h, and a subtracted cDNA library enriched for silica-induced genes (SIG) was constructed and differently screened. Nine cDNA clones (designated SIG-12, -14, -20, -41, -61, -81, -91, and -111) were partially sequenced and compared with sequences in GenBank/EMBL databases. SIG-12, -14, and -20 corresponded to the genes for ribosomal proteins L13A, L32, and L26, respectively. SIG-61 is the mouse homologue of p21 RhoC. SIG-91 is identical to the 67-kDa high-affinity laminin receptor. Four genes were not identified and are novel. All of the mRNAs corresponding to the nine cloned cDNAs were inducible by silica. Steady-state levels of mRNAs in RAW 264.7 cells treated with various macrophage activators and inducers of signal transduction pathways were determined. A complex pattern of induction and repression was found, indicating that upon phagocytosis of silica particles, many regulatory mechanisms of genes expression are simultaneously triggered. 55 refs., 4 figs., 1 tab.

  19. Microarray analysis of acaricide inducible gene expression in the southern cattle tick, Rhipicephalus (Boophilus) microplus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acaricide-inducible differential gene expression was studied in larvae of Rhipicephalus (Boophilus) microplus using a microarray-based approach. The acaricides used were: coumaphos, permethrin, ivermectin, and amitraz. The microarrays contained over 13,000 probes, having been derived from a previous...

  20. Transcriptomic sequencing reveals a set of unique genes activated by butyrate-induced histone modification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Butyrate is a nutritional element with strong epigenetic regulatory activity as an inhibitor of histone deacetylases (HDACs). Based on the analysis of differentially expressed genes induced by butyrate in the bovine epithelial cell using deep RNA-sequencing technology (RNA-seq), a set of unique gen...

  1. Hyperglycemia induces iNOS gene expression and consequent nitrosative stress via JNK activation

    PubMed Central

    YANG, Peixin; CAO, Yuanning; LI, Hua

    2010-01-01

    Objective Maternal diabetes adversely impacts embryonic development. We test the hypothesis that hyperglycemia-induced JNK1/2 activation mediates iNOS induction. Study Design Levels of iNOS mRNA and nitrosylated protein were determined in cultured C57BL/6J conceptuses exposed to hyperglycemia (300 mg/dl glucose) and C57BL/6J embryos exposed to streptozotocin-induced diabetes. iNOS-luciferase activity and endogenous reactive nitrogen species were determined in transfected PYS-2 (mouse teratocarcinoma) cells exposed to hyperglycemia (450 mg/dl glucose). Results Hyperglycemia increased iNOS mRNA and SP600125, a potent JNK1/2 inhibitor, abolished this effect. Hyperglycemia increased iNOS-luciferase activities and SP600125 blocked this effect. Diabetes increased iNOS mRNA and jnk2 gene deletion abrogated this effect. Correlated with iNOS gene induction, both hyperglycemia in vitro and diabetes in vivo enhanced the production of reactive nitrogen species and increased protein nitrosylation. jnk2 gene deletion blocked diabetes-induced protein nitrosylation. Conclusion JNK1/2 activation mediates hyperglycemia-induced iNOS gene expression and consequent nitrosative stress in diabetic embryopathy. PMID:20541731

  2. Age and vitamin E-induced changes in Gene Expression Profiles of T cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    T cell is vulnerable to age associated changes and vitamin E has been shown to improve T cell functions in the old. We studied the gene expression profile of T cells to better understand the underlying mechanisms of age and vitamin E-induced changes in T cell function. Young and old C57BL mice were ...

  3. The necrosis-inducing Phytophthora protein gene family of Phytophthora capsici is involved in pathogenicity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytophthora capsici is one of the most important pathogens limiting vegetable production worldwide. Necrosis-inducing Phytophthora protein (NPP), ocurring in phylogenetically distant organisms, is phytotoxic for dicotyledonous plants, but the mechanism of action has not been established. A gene fam...

  4. MICROARRAY ANALYSIS OF DICHLOROACETIC ACID-INDUCED CHANGES IN GENE EXPRESSION

    EPA Science Inventory


    MICROARRAY ANALYSIS OF DICHLOROACETIC ACID-INDUCED CHANGES IN GENE EXPRESSION

    Dichloroacetic acid (DCA) is a major by-product of water disinfection by chlorination. Several studies have demonstrated the hepatocarcinogenicity of DCA in rodents when administered in dri...

  5. Dual-responsive aggregation-induced emission-active supramolecular nanoparticles for gene delivery and bioimaging.

    PubMed

    Dong, Ruijiao; Ravinathan, Screenath P; Xue, Lizhe; Li, Nan; Zhang, Yingjian; Zhou, Linzhu; Cao, Chengxi; Zhu, Xinyuan

    2016-06-28

    Dual-responsive aggregation-induced emission-active supramolecular fluorescent nanoparticles are reported, which have the ability to undergo a unique morphological transition combining with a cooperative optical variation in response to pH and light stimuli. The dynamic supramolecular nanoparticles show excellent biocompatibility and effective plasmid DNA condensation capability, further achieving efficient in vitro gene delivery and bioimaging. PMID:27251637

  6. Co-silencing the mirabilis antiviral protein permits virus-induced gene silencing in Mirabilis jalapa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is an attractive and rapid technique for loss of function assay that can reveal the phenotype of embryo-lethal sequences and avoids the need for time consuming transformation and regeneration processes. Among various VIGS vectors that have been explored, the tobac...

  7. EFFECTS OF DIETARY FOLATE ON ARSENIC-INDUCED GENE EXPRESSION IN MICE

    EPA Science Inventory

    Effects of Dietary Folate on Arsenic-induced Gene Expression in Mice

    Arsenic, a drinking water contaminant, is a known carcinogen. Human exposure to inorganic arsenic has been linked to tumors of skin, bladder, lung, and to a lesser extent, kidney and liver. Dietary fola...

  8. Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives

    NASA Astrophysics Data System (ADS)

    Gajbhiye, Virendra; Escalante, Leah; Chen, Guojun; Laperle, Alex; Zheng, Qifeng; Steyer, Benjamin; Gong, Shaoqin; Saha, Krishanu

    2013-12-01

    Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives.Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained

  9. A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus

    PubMed Central

    Liscombe, David K.; O’Connor, Sarah E.

    2011-01-01

    The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by Madagascar periwinkle (Catharanthus roseus) plants. Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003–0.01% yields. Metabolic engineering efforts to improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. We have developed a VIGS method to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro. PMID:21802100

  10. Differential cellular gene expression induced by hepatitis B and C viruses.

    PubMed

    Otsuka, Motoyuki; Aizaki, Hideki; Kato, Naoya; Suzuki, Tetsuro; Miyamura, Tatsuo; Omata, Masao; Seki, Naohiko

    2003-01-10

    Hepatitis B virus (HBV) is a hepatotropic virus that causes acute and chronic hepatocellular injury and hepatocellular carcinoma. To clarify how HBV proteins regulate host cellular gene expression, we used our in-house cDNA microarray and HepG2.2.15 cells, which are derived from HepG2 cells and produce all HBV proteins. Of 2304 genes investigated, several genes were differentially expressed in HepG2.2.15 cells compared with HepG2 cells. These genes included insulin-like growth factor II and alpha-fetoprotein, consistent with previous reports. Furthermore, we previously performed similar microarray analyses to clarify the effects of hepatitis C virus (HCV) proteins on host cells, using a HepG2-derivative cell line, which produces all HCV proteins. Using these two microarray results, we compared the differences in cellular gene expression induced by HBV and HCV proteins. The expression of the majority of genes investigated differed only slightly between HBV and HCV protein-producing cells. However, HBV and HCV proteins clearly regulated several genes in a reciprocal manner. Combined, these microarray results shed new light on the effects of HBV proteins on cellular gene expression and on the differences in the pathogenic activities of these two hepatitis viruses. PMID:12504104

  11. Multi-walled carbon nanotube-induced gene expression in vitro: concordance with in vivo studies

    PubMed Central

    Snyder-Talkington, Brandi N.; Dong, Chunlin; Zhao, Xiangyi; Dymacek, Julian; Porter, Dale W.; Wolfarth, Michael G.; Castranova, Vincent; Qian, Yong; Guo, Nancy L.

    2014-01-01

    There is a current interest in reducing the in vivo toxicity testing of nanomaterials in animals by increasing toxicity testing using in vitro cellular assays; however, toxicological results are seldom concordant between in vivo and in vitro models. This study compared global multi-walled carbon nanotube (MWCNT)-induced gene expression from human lung epithelial and microvascular endothelial cells in monoculture and coculture with gene expression from mouse lungs exposed to MWCNT. Using a cutoff of 10% false discovery rate and 1.5 fold change, we determined that there were more concordant genes (gene expression both up- or downregulated in vivo and in vitro) expressed in both cell types in coculture than in monoculture. When reduced to only those genes involved in inflammation and fibrosis, known outcomes of in vivo MWCNT exposure, there were more disease-related concordant genes expressed in coculture than monoculture. Additionally, different cellular signaling pathways are activated in response to MWCNT dependent upon culturing conditions. As coculture gene expression better correlated with in vivo gene expression, we suggest that cellular cocultures may offer enhanced in vitro models for nanoparticle risk assessment and the reduction of in vivo toxicological testing. PMID:25511174

  12. Gene expression profiles of murine fatty liver induced by the administration of valproic acid

    SciTech Connect

    Lee, Min-Ho; Hong, Il; Kim, Mingoo; Lee, Byung Hoon; Kim, Ju-Han; Kang, Kyung-Sun; Kim, Hyung-Lae; Yoon, Byung-Il; Chung, Heekyoung; Kong, Gu; Lee, Mi-Ock . E-mail: molee@snu.ac.kr

    2007-04-01

    Valproic acid (VPA) has been used as anticonvulsants, however, it induces hepatotoxicity such as microvesicular steatosis and necrosis in the liver. To explore the mechanisms of VPA-induced steatosis, we profiled the gene expression patterns of the mouse liver that were altered by treatment with VPA using microarray analysis. VPA was orally administered as a single dose of 100 mg/kg (low-dose) or 1000 mg/kg (high-dose) to ICR mice and the animals were killed at 6, 24, or 72 h after treatment. Serum alanine aminotransferase and aspartate aminotransferase levels were not significantly altered in the experimental animals. However, symptoms of steatosis were observed at 72 h with low-dose and at 24 h and 72 h with high-dose. After microarray data analysis, 1910 genes were selected by two-way ANOVA (P < 0.05) as VPA-responsive genes. Hierarchical clustering revealed that gene expression changes depended on the time rather than the dose of VPA treatment. Gene profiling data showed striking changes in the expression of genes associated with lipid, fatty acid, and steroid metabolism, oncogenesis, signal transduction, and development. Functional categorization of 1156 characteristically up- and down-regulated genes (cutoff > 1.5-fold) revealed that 60 genes were involved in lipid metabolism that was interconnected with biological pathways for biosynthesis of triglyceride and cholesterol, catabolism of fatty acid, and lipid transport. This gene expression profile may be associated with the known steatogenic hepatotoxicity of VPA and it may provide useful information for prediction of hepatotoxicity of unknown chemicals or new drug candidates through pattern recognition.

  13. Fast and sensitive detection of indels induced by precise gene targeting.

    PubMed

    Yang, Zhang; Steentoft, Catharina; Hauge, Camilla; Hansen, Lars; Thomsen, Allan Lind; Niola, Francesco; Vester-Christensen, Malene B; Frödin, Morten; Clausen, Henrik; Wandall, Hans H; Bennett, Eric P

    2015-05-19

    The nuclease-based gene editing tools are rapidly transforming capabilities for altering the genome of cells and organisms with great precision and in high throughput studies. A major limitation in application of precise gene editing lies in lack of sensitive and fast methods to detect and characterize the induced DNA changes. Precise gene editing induces double-stranded DNA breaks that are repaired by error-prone non-homologous end joining leading to introduction of insertions and deletions (indels) at the target site. These indels are often small and difficult and laborious to detect by traditional methods. Here we present a method for fast, sensitive and simple indel detection that accurately defines indel sizes down to ±1 bp. The method coined IDAA for Indel Detection by Amplicon Analysis is based on tri-primer amplicon labelling and DNA capillary electrophoresis detection, and IDAA is amenable for high throughput analysis. PMID:25753669

  14. Functional analysis of an auxin-inducible DNA-binding protein gene.

    PubMed

    Bernstein, Any; Mangeon, Amanda; Almeida-Engler, Janice; Engler, Gilbert; Van Montagu, Marc; Sachetto-Martins, Gilberto; de Oliveira, Dulce Eleonora

    2015-01-01

    Over the past decades, several studies indicate a correlation between the phytohormone auxin and cell division. The molecular players of this signaling pathway are now being uncovered. DNA Binding Protein1 from Arabidopsis (AtDBP1) is an auxin-inducible gene able to bind DNA non-specifically. In this work the tissue-expression pattern of this gene was investigated. Promoter-GUS analysis demonstrated that the AtDBP1 promoter is active in regions exhibiting intense cell division such as meristems and nematode feeding sites. Also, the promoter expression was modulated upon incubation with cell cycle blockers, indicating a potential role in cell division for this gene. Lastly, AtDBP1 antisense plants presented a higher insensitivity to auxin, and interfered negatively with auxin-induced callus formation and reduced apical dominance. PMID:25482757

  15. Functional analysis of an auxin-inducible DNA-binding protein gene

    PubMed Central

    Bernstein, Any; Mangeon, Amanda; Almeida-Engler, Janice; Engler, Gilbert; Montagu, Marc Van; Sachetto-Martins, Gilberto; de Oliveira, Dulce Eleonora

    2015-01-01

    Over the past decades, several studies indicate a correlation between the phytohormone auxin and cell division. The molecular players of this signaling pathway are now being uncovered. DNA Binding Protein1 from Arabidopsis (AtDBP1) is an auxin-inducible gene able to bind DNA non-specifically. In this work the tissue-expression pattern of this gene was investigated. Promoter-GUS analysis demonstrated that the AtDBP1 promoter is active in regions exhibiting intense cell division such as meristems and nematode feeding sites. Also, the promoter expression was modulated upon incubation with cell cycle blockers, indicating a potential role in cell division for this gene. Lastly, AtDBP1 antisense plants presented a higher insensitivity to auxin, and interfered negatively with auxin–induced callus formation and reduced apical dominance. PMID:25482757

  16. Molecular characterization of a GA-inducible gene, Cvsus1, in developing watermelon seeds.

    PubMed

    Kim, Joonyul; Jun, Sung-Hoon; Kang, Hong-Gyu; Lee, Jinwon; An, Gynheung

    2002-10-31

    To understand the molecular mechanisms that control seed development, we isolated a seed-preferential gene from ESTs of developing watermelon seeds. The gene Cvsus1 encodes a protein that is 86% identical to the Vicia faba sucrose synthase expressed in developing seeds. RNA blot analysis showed that Cvsus1 was preferentially expressed in watermelon seeds. We also investigated gene expression levels both in pollinated seeds and in parthenocarpic seeds, which lack zygotic tissues. Whereas the transcript level of Cvsus1 was rapidly increased during normal seed development, the expression was not significantly increased in the parthenocarpic seeds. However, treating the parthenocarpic fruits with GA3 strongly induced Cvsus1 expression, up to the level accumulated in pollinated seeds. These results suggest that Cvsus1 is induced in maternal tissues via signals from the zygotic tissues, and that GA may be one of those signals. PMID:12442898

  17. A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize1[OPEN

    PubMed Central

    Mei, Yu; Kernodle, Bliss M.; Hill, John H.

    2016-01-01

    Plant viruses have been widely used as vectors for foreign gene expression and virus-induced gene silencing (VIGS). A limited number of viruses have been developed into viral vectors for the purposes of gene expression or VIGS in monocotyledonous plants, and among these, the tripartite viruses Brome mosaic virus and Cucumber mosaic virus have been shown to induce VIGS in maize (Zea mays). We describe here a new DNA-based VIGS system derived from Foxtail mosaic virus (FoMV), a monopartite virus that is able to establish systemic infection and silencing of endogenous maize genes homologous to gene fragments inserted into the FoMV genome. To demonstrate VIGS applications of this FoMV vector system, four genes, phytoene desaturase (functions in carotenoid biosynthesis), lesion mimic22 (encodes a key enzyme of the porphyrin pathway), iojap (functions in plastid development), and brown midrib3 (caffeic acid O-methyltransferase), were silenced and characterized in the sweet corn line Golden × Bantam. Furthermore, we demonstrate that the FoMV infectious clone establishes systemic infection in maize inbred lines, sorghum (Sorghum bicolor), and green foxtail (Setaria viridis), indicating the potential wide applications of this viral vector system for functional genomics studies in maize and other monocots. PMID:27208311

  18. Functional study of a salt-inducible TaSR gene in Triticum aestivum.

    PubMed

    Ma, Xiao-Li; Cui, Wei-Na; Zhao, Qian; Zhao, Jing; Hou, Xiao-Na; Li, Dong-Yan; Chen, Zhao-Liang; Shen, Yin-Zhu; Huang, Zhan-Jing

    2016-01-01

    The gene expression chip of a salt-tolerant wheat mutant under salt stress was used to clone a salt-induced gene with unknown functions. This gene was designated as TaSR (Triticum aestivum salt-response gene) and submitted to GenBank under accession number EF580107. Quantitative polymerase chain reaction (PCR) analysis showed that gene expression was induced by salt stress. Arabidopsis and rice (Oryza sativa) plants expressing TaSR presented higher salt tolerance than the controls, whereas AtSR mutant and RNA interference rice plants were more sensitive to salt. Under salt stress, TaSR reduced Na(+) concentration and improved cellular K(+) and Ca(2+) concentrations; this gene was also localized on the cell membrane. β-Glucuronidase (GUS) staining and GUS fluorescence quantitative determination were conducted through fragmentation cloning of the TaSR promoter. Salt stress-responsive elements were detected at 588-1074 bp upstream of the start codon. GUS quantitative tests of the full-length promoter in different tissues indicated that promoter activity was highest in the leaf under salt stress. Bimolecular fluorescence complementation and yeast two-hybrid screening further showed the correlation of TaSR with TaPRK and TaKPP. In vitro phosphorylation of TaSR and TaPRK2697 showed that TaPRK2697 did not phosphorylate TaSR. This study revealed that the novel TaSR may be used to improve plant tolerance to salt stress. PMID:25855206

  19. Heterogeneity of epigenetic changes at ischemia/reperfusion- and endotoxin-induced acute kidney injury genes

    PubMed Central

    Mar, Daniel; Gharib, Sina A.; Zager, Richard A.; Johnson, Ali; Denisenko, Oleg; Bomsztyk, Karol

    2015-01-01

    Aberrant gene expression is a molecular hallmark of acute kidney injury (AKI). Since epigenetic processes control gene expression in a cell- and environment-defined manner, understanding the epigenetic pathways that regulate genes altered by AKI may open vital new insights into the complexities of disease pathogenesis and identify possible therapeutic targets. Here we used matrix chromatin immunoprecipitation and integrative analysis to study twenty key permissive and repressive epigenetic histone marks at transcriptionally induced Tnf, Ngal, Kim-1 and Icam-1 genes in mouse models of AKI; unilateral renal ischemia/reperfusion, lipopolysaccharide (LPS) and their synergistically injurious combination. Results revealed unexpected heterogeneity of transcriptional and epigenetic responses. Tnf and Ngal were transcriptionally upregulated in response to both treatments individually, and to combination treatment. Kim-1 was induced by ischemia/reperfusion and Icam-1 by LPS only. Epigenetic alterations at these genes exhibited distinct time-dependent changes that shared some similarities, such as reduction in repressive histone modifications, but also had major ischemia/reperfusion vs. endotoxin differences. Thus, diversity of changes at AKI genes in response to different insults indicates involvement of several epigenetic pathways. This could be exploited pharmacologically through rational-drug design to alter the course and improve clinical outcomes of this syndrome. PMID:26061546

  20. Heterogeneity of epigenetic changes at ischemia/reperfusion- and endotoxin-induced acute kidney injury genes.

    PubMed

    Mar, Daniel; Gharib, Sina A; Zager, Richard A; Johnson, Ali; Denisenko, Oleg; Bomsztyk, Karol

    2015-10-01

    Aberrant gene expression is a molecular hallmark of acute kidney injury (AKI). As epigenetic processes control gene expression in a cell- and environment-defined manner, understanding the epigenetic pathways that regulate genes altered by AKI may open vital new insights into the complexities of disease pathogenesis and identify possible therapeutic targets. Here we used matrix chromatin immunoprecipitation and integrative analysis to study 20 key permissive and repressive epigenetic histone marks at transcriptionally induced Tnf, Ngal, Kim-1, and Icam-1 genes in mouse models of AKI; unilateral renal ischemia/reperfusion, lipopolysaccharide (LPS), and their synergistically injurious combination. Results revealed unexpected heterogeneity of transcriptional and epigenetic responses. Tnf and Ngal were transcriptionally upregulated in response to both treatments individually, and to combination treatment. Kim-1 was induced by ischemia/reperfusion and Icam-1 by LPS only. Epigenetic alterations at these genes exhibited distinct time-dependent changes that shared some similarities, such as reduction in repressive histone modifications, and also had major ischemia/reperfusion versus endotoxin differences. Thus, diversity of changes at AKI genes in response to different insults indicates involvement of several epigenetic pathways. This could be exploited pharmacologically through rational-drug design to alter the course and improve clinical outcomes of this syndrome. PMID:26061546

  1. UVB-induced gene expression in the skin of Xiphophorus maculatus Jp 163 B☆

    PubMed Central

    Yang, Kuan; Boswell, Mikki; Walter, Dylan J.; Downs, Kevin P.; Gaston-Pravia, Kimberly; Garcia, Tzintzuni; Shen, Yingjia; Mitchell, David L.; Walter, Ronald B.

    2014-01-01

    Xiphophorus fish and interspecies hybrids represent long-standing models to study the genetics underlying spontaneous and induced tumorigenesis. The recent release of the Xiphophorus maculatus genome sequence will allow global genetic regulation studies of genes involved in the inherited susceptibility to UVB-induced melanoma within select backcross hybrids. As a first step toward this goal, we report results of an RNA-Seq approach to identify genes and pathways showing modulated transcription within the skin of X. maculatus Jp 163 B upon UVB exposure. X. maculatus Jp 163 B were exposed to various doses of UVB followed by RNA-Seq analysis at each dose to investigate overall gene expression in each sample. A total of 357 genes with a minimum expression change of 4-fold (p-adj < 0.05) were identified as responsive to UVB. The molecular genetic response of Xiphophorus skin to UVB exposure permitted assessment of; (1) the basal expression level of each transcript for each skin sample, (2) the changes in expression levels for each gene in the transcriptome upon exposure to increasing doses of UVB, and (3) clusters of genes that exhibit similar patterns of change in expression upon UVB exposure. These data provide a foundation for understanding the molecular genetic response of fish skin to UVB exposure. PMID:24556253

  2. Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression

    NASA Astrophysics Data System (ADS)

    Makino, Yuichi; Cao, Renhai; Svensson, Kristian; Bertilsson, Göran; Asman, Mikael; Tanaka, Hirotoshi; Cao, Yihai; Berkenstam, Anders; Poellinger, Lorenz

    2001-11-01

    Alteration of gene expression is a crucial component of adaptive responses to hypoxia. These responses are mediated by hypoxia-inducible transcription factors (HIFs). Here we describe an inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, which is a basic helix-loop-helix (bHLH)/PAS protein structurally related to HIFs. IPAS contains no endogenous transactivation function but demonstrates dominant negative regulation of HIF-mediated control of gene expression. Ectopic expression of IPAS in hepatoma cells selectively impairs induction of genes involved in adaptation to a hypoxic environment, notably the vascular endothelial growth factor (VEGF) gene, and results in retarded tumour growth and tumour vascular density in vivo. In mice, IPAS was predominantly expressed in Purkinje cells of the cerebellum and in corneal epithelium of the eye. Expression of IPAS in the cornea correlates with low levels of expression of the VEGF gene under hypoxic conditions. Application of an IPAS antisense oligonucleotide to the mouse cornea induced angiogenesis under normal oxygen conditions, and demonstrated hypoxia-dependent induction of VEGF gene expression in hypoxic corneal cells. These results indicate a previously unknown mechanism for negative regulation of angiogenesis and maintenance of an avascular phenotype.

  3. Naked gene therapy of hepatocyte growth factor for dextran sulfate sodium-induced colitis in mice

    SciTech Connect

    Kanbe, Takamasa |; Murai, Rie; Mukoyama, Tomoyuki; Murawaki, Yoshiyuki |; Hashiguchi, Ko-ichi; Yoshida, Yoko; Tsuchiya, Hiroyuki; Kurimasa, Akihiro; Harada, Ken-ichi; Yashima, Kazuo; Nishimuki, Eiji; Shabana, Noriko; Kishimoto, Yukihiro; Kojyo, Haruhiko; Miura, Kunihiko; Kawasaki, Hironaka; Murawaki, Yoshikazu; Shiota, Goshi . E-mail: gshiota@grape.med.tottori-u.ac.jp

    2006-07-14

    Ulcerative colitis (UC) is progressive and relapsing disease. To explore the therapeutic effects of naked gene therapy of hepatocyte growth factor (HGF) on UC, the SR{alpha} promoter driving HGF gene was intrarectally administered to the mice in which colitis was induced by dextran sulfate sodium (DSS). Expression of the transgene was seen in surface epithelium, lamina propria, and muscularis mucosae. The HGF-treated mice showed reduced colonic mucosal damage and increased body weights, compared with control mice (P < 0.01 and P < 0.05, respectively). The HGF-treated mice displayed increased number of PCNA-positive cells and decreased number of apoptotic cells than in control mice (P < 0.01, each). Phosphorylated AKT was dramatically increased after HGF gene administration, however, phosphorylated ERK1/2 was not altered. Microarray analysis revealed that HGF induced expression of proliferation- and apoptosis-associated genes. These data suggest that naked HGF gene delivery causes therapeutic effects through regulation of many downstream genes.

  4. A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize.

    PubMed

    Mei, Yu; Zhang, Chunquan; Kernodle, Bliss M; Hill, John H; Whitham, Steven A

    2016-06-01

    Plant viruses have been widely used as vectors for foreign gene expression and virus-induced gene silencing (VIGS). A limited number of viruses have been developed into viral vectors for the purposes of gene expression or VIGS in monocotyledonous plants, and among these, the tripartite viruses Brome mosaic virus and Cucumber mosaic virus have been shown to induce VIGS in maize (Zea mays). We describe here a new DNA-based VIGS system derived from Foxtail mosaic virus (FoMV), a monopartite virus that is able to establish systemic infection and silencing of endogenous maize genes homologous to gene fragments inserted into the FoMV genome. To demonstrate VIGS applications of this FoMV vector system, four genes, phytoene desaturase (functions in carotenoid biosynthesis), lesion mimic22 (encodes a key enzyme of the porphyrin pathway), iojap (functions in plastid development), and brown midrib3 (caffeic acid O-methyltransferase), were silenced and characterized in the sweet corn line Golden × Bantam. Furthermore, we demonstrate that the FoMV infectious clone establishes systemic infection in maize inbred lines, sorghum (Sorghum bicolor), and green foxtail (Setaria viridis), indicating the potential wide applications of this viral vector system for functional genomics studies in maize and other monocots. PMID:27208311

  5. Foxtail Mosaic Virus-Induced Gene Silencing in Monocot Plants1[OPEN

    PubMed Central

    Liu, Na; Xie, Ke; Jia, Qi; Zhao, Jinping; Chen, Tianyuan; Li, Huangai; Wei, Xiang; Diao, Xianmin; Hong, Yiguo

    2016-01-01

    Virus-induced gene silencing (VIGS) is a powerful technique to study gene function in plants. However, very few VIGS vectors are available for monocot plants. Here we report that Foxtail mosaic virus (FoMV) can be engineered as an effective VIGS system to induce efficient silencing of endogenous genes in monocot plants including barley (Hordeum vulgare L.), wheat (Triticum aestivum) and foxtail millet (Setaria italica). This is evidenced by FoMV-based silencing of phytoene desaturase (PDS) and magnesium chelatase in barley, of PDS and Cloroplastos alterados1 in foxtail millet and wheat, and of an additional gene IspH in foxtail millet. Silencing of these genes resulted in photobleached or chlorosis phenotypes in barley, wheat, and foxtail millet. Furthermore, our FoMV-based gene silencing is the first VIGS system reported for foxtail millet, an important C4 model plant. It may provide an efficient toolbox for high-throughput functional genomics in economically important monocot crops. PMID:27225900

  6. Light-induced expression of fatty acid desaturase genes

    PubMed Central

    Kis, Mihály; Zsiros, Otto; Farkas, Tibor; Wada, Hajime; Nagy, Ferenc; Gombos, Zoltán

    1998-01-01

    In cyanobacterial cells, fatty acid desaturation is one of the crucial steps in the acclimation processes to low-temperature conditions. The expression of all the four acyl lipid desaturase genes of Synechocystis PCC 6803 was studied as a function of temperature and separately as a function of light. We used cells grown at 25°C in light-activated heterotrophic growth conditions. In these cells, the production of α-linolenic acid and 18:4 fatty acids was negligible and the synthesis of γ-linolenic acid was remarkably suppressed compared with those of the cells grown photoautotrophically. The cells grown in the light in the presence of glucose showed no difference in fatty acid composition compared with cells grown photoautotrophically. The level of desC mRNA for Δ9 desaturase was not affected by either the temperature or the light. It was constitutively expressed at 25°C with and without illumination. The level of desB transcripts was negligible in the dark-grown cells and was enhanced about 10-fold by exposure of the cells to light. The maximum level of expression occurred within 15 min. The level of desA and desD mRNAs was higher in dark-grown cells than that of desB mRNA for ω3 desaturase. However, the induction of both desA and desD mRNAs for Δ12 and Δ6 desaturases, respectively, was enhanced by light about 10-fold. Rifampicin, chloramphenicol, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea completely blocked the induction of the expression of desA, desB, and desD. Consequently, we suggest the regulatory role of light via photosynthetic processes in the induction of the expression of acyl lipid desaturases. PMID:9539715

  7. Compressive forces induce osteogenic gene expression in calvarial osteoblasts.

    PubMed

    Rath, Bjoern; Nam, Jin; Knobloch, Thomas J; Lannutti, John J; Agarwal, Sudha

    2008-01-01

    Bone cells and their precursors are sensitive to changes in their biomechanical environment. The importance of mechanical stimuli has been observed in bone homeostasis and osteogenesis, but the mechanisms responsible for osteogenic induction in response to mechanical signals are poorly understood. We hypothesized that compressive forces could exert an osteogenic effect on osteoblasts and act in a dose-dependent manner. To test our hypothesis, electrospun poly(epsilon-caprolactone) (PCL) scaffolds were used as a 3-D microenvironment for osteoblast culture. The scaffolds provided a substrate allowing cell exposure to levels of externally applied compressive force. Pre-osteoblasts adhered, proliferated and differentiated in the scaffolds and showed extensive matrix synthesis by scanning electron microscopy (SEM) and increased Young's modulus (136.45+/-9.15 kPa) compared with acellular scaffolds (24.55+/-8.5 kPa). Exposure of cells to 10% compressive strain (11.81+/-0.42 kPa) resulted in a rapid induction of bone morphogenic protein-2 (BMP-2), runt-related transcription factor 2 (Runx2), and MAD homolog 5 (Smad5). These effects further enhanced the expression of genes and proteins required for extracellular matrix (ECM) production, such as alkaline phosphatase (Akp2), collagen type I (Col1a1), osteocalcin/bone gamma carboxyglutamate protein (OC/Bglap), osteonectin/secreted acidic cysteine-rich glycoprotein (ON/Sparc) and osteopontin/secreted phosphoprotein 1 (OPN/Spp1). Exposure of cell-scaffold constructs to 20% compressive strain (30.96+/-2.82 kPa) demonstrated that these signals are not osteogenic. These findings provide the molecular basis for the experimental and clinical observations that appropriate physical activities or microscale compressive loading can enhance fracture healing due in part to the anabolic osteogenic effects. PMID:18191137

  8. Characterization of Chemically Induced Liver Injuries Using Gene Co-Expression Modules

    PubMed Central

    Tawa, Gregory J.; AbdulHameed, Mohamed Diwan M.; Yu, Xueping; Kumar, Kamal; Ippolito, Danielle L.; Lewis, John A.; Stallings, Jonathan D.; Wallqvist, Anders

    2014-01-01

    Liver injuries due to ingestion or exposure to chemicals and industrial toxicants pose a serious health risk that may be hard to assess due to a lack of non-invasive diagnostic tests. Mapping chemical injuries to organ-specific damage and clinical outcomes via biomarkers or biomarker panels will provide the foundation for highly specific and robust diagnostic tests. Here, we have used DrugMatrix, a toxicogenomics database containing organ-specific gene expression data matched to dose-dependent chemical exposures and adverse clinical pathology assessments in Sprague Dawley rats, to identify groups of co-expressed genes (modules) specific to injury endpoints in the liver. We identified 78 such gene co-expression modules associated with 25 diverse injury endpoints categorized from clinical pathology, organ weight changes, and histopathology. Using gene expression data associated with an injury condition, we showed that these modules exhibited different patterns of activation characteristic of each injury. We further showed that specific module genes mapped to 1) known biochemical pathways associated with liver injuries and 2) clinically used diagnostic tests for liver fibrosis. As such, the gene modules have characteristics of both generalized and specific toxic response pathways. Using these results, we proposed three gene signature sets characteristic of liver fibrosis, steatosis, and general liver injury based on genes from the co-expression modules. Out of all 92 identified genes, 18 (20%) genes have well-documented relationships with liver disease, whereas the rest are novel and have not previously been associated with liver disease. In conclusion, identifying gene co-expression modules associated with chemically induced liver injuries aids in generating testable hypotheses and has the potential to identify putative biomarkers of adverse health effects. PMID:25226513

  9. Novel drought-inducible genes in the highly drought-tolerant cowpea: cloning of cDNAs and analysis of the expression of the corresponding genes.

    PubMed

    Iuchi, S; Yamaguchi-Shinozaki, K; Urao, T; Terao, T; Shinozaki, K

    1996-12-01

    Ten cDNAs of genes that were induced by dehydration stress were cloned by differential screening from the highly drought-tolerant legume, cowpea (Vigna unguiculata), a major crop in West Africa. The clones were collectively named CPRD (cowpea clones responsive to dehydration). Northern blot analysis revealed that nine of the CPRD genes were induced by dehydration stress, but the timing of induction of mRNA synthesis varied among the CPRD genes. We analyzed the effects of other environmental stresses on the expression of the CPRD8, CPRD14 and CPRD22 genes, and we found that these genes were strongly induced by high-salinity stress but not by cold or heat stress. Drought-stressed cowpea plants accumulated abscisic acid (ABA) to a level that was 160 times higher than that in unstressed plants. The CPRD8 and CPRD22 genes were induced to a significant extent by the application of exogenous ABA but the CPRD14 gene was not. These results indicate the existence of at least two signal-transduction pathways between the detection of water stress and the expression of CPRD genes in cowpea. Sequence analysis of CPRD8 and CPRD22 cDNAs revealed that they encoded putative proteins that were related to old yellow enzyme and group 2 LEA proteins, respectively. The protein encoded by CPRD14 exhibited sequence homology to dihydroflavonol-4-reductase (DFR) and vestitone reductase (VR). Old yellow enzyme, DFR and VR have not been identified as drought-inducible proteins in other plants, whereas LEA genes have been well characterized as drought-inducible genes. The various gene products might function to protect cells from environmental stress. PMID:9032963

  10. Radiation-Inducible Caspase-8 Gene Therapy for Malignant Brain Tumors

    SciTech Connect

    Tsurushima, Hideo Yuan Xuan; Dillehay, Larry E.; Leong, Kam W.

    2008-06-01

    Purpose: Patients with malignant gliomas have a poor prognosis. To explore a novel and more effective approach for the treatment of patients with malignant gliomas, we designed a strategy that combines caspase-8 (CSP8) gene therapy and radiation treatment (RT). In addition, the specificity of the combined therapy was investigated to decrease the unpleasant effects experienced by the surrounding normal tissue. Methods and Materials: We constructed the plasmid pEGR-green fluorescence protein that included the radiation-inducible early growth response gene-1 (Egr-1) promoter and evaluated its characteristics. The pEGR-CSP8 was constructed and included the Egr-1 promoter and CSP8 complementary DNA. Assays that evaluated the apoptosis inducibility and cytotoxicity caused by CSP8 gene therapy combined with RT were performed using U251 and U87 glioma cells. The pEGR-CSP8 was transfected into the subcutaneous U251 glioma cells of nude mice by means of in vivo electroporation. The in vivo effects of CSP8 gene therapy combined with RT were evaluated. Results: The Egr-1 promoter yielded a better response with fractionated RT than with single-dose RT. In the assay of apoptosis inducibility and cytotoxicity, pEGR-CSP8 showed response for RT. The pEGR-CSP8 combined with RT is capable of inducing cell death effectively. In mice treated with pEGR-CSP8 and RT, apoptotic cells were detected in pathologic sections, and a significant difference was observed in tumor volumes. Conclusions: Our results indicate that radiation-inducible gene therapy may have great potential because this can be spatially or temporally controlled by exogenous RT and is safe and specific.

  11. β-Cryptoxanthin Alleviates Diet-Induced Nonalcoholic Steatohepatitis by Suppressing Inflammatory Gene Expression in Mice

    PubMed Central

    Kobori, Masuko; Ni, Yinhua; Takahashi, Yumiko; Watanabe, Natsumi; Sugiura, Minoru; Ogawa, Kazunori; Nagashimada, Mayumi; Kaneko, Shuichi; Naito, Shigehiro; Ota, Tsuguhito

    2014-01-01

    Recent nutritional epidemiological surveys showed that serum β-cryptoxanthin inversely associates with the risks for insulin resistance and liver dysfunction. Consumption of β-cryptoxanthin possibly prevents nonalcoholic steatohepatitis (NASH), which is suggested to be caused by insulin resistance and oxidative stress from nonalcoholic fatty liver disease. To evaluate the effect of β-cryptoxanthin on diet-induced NASH, we fed a high-cholesterol and high-fat diet (CL diet) with or without 0.003% β-cryptoxanthin to C56BL/6J mice for 12 weeks. After feeding, β-cryptoxanthin attenuated fat accumulation, increases in Kupffer and activated stellate cells, and fibrosis in CL diet-induced NASH in the mice. Comprehensive gene expression analysis showed that although β-cryptoxanthin histochemically reduced steatosis, it was more effective in inhibiting inflammatory gene expression change in NASH. β-Cryptoxanthin reduced the alteration of expression of genes associated with cell death, inflammatory responses, infiltration and activation of macrophages and other leukocytes, quantity of T cells, and free radical scavenging. However, it showed little effect on the expression of genes related to cholesterol and other lipid metabolism. The expression of markers of M1 and M2 macrophages, T helper cells, and cytotoxic T cells was significantly induced in NASH and reduced by β-cryptoxanthin. β-Cryptoxanthin suppressed the expression of lipopolysaccharide (LPS)-inducible and/or TNFα-inducible genes in NASH. Increased levels of the oxidative stress marker thiobarbituric acid reactive substances (TBARS) were reduced by β-cryptoxanthin in NASH. Thus, β-cryptoxanthin suppresses inflammation and the resulting fibrosis probably by primarily suppressing the increase and activation of macrophages and other immune cells. Reducing oxidative stress is likely to be a major mechanism of inflammation and injury suppression in the livers of mice with NASH. PMID:24858832

  12. Coordinated induction of Nrf2 target genes protects against iron nitrilotriacetate (FeNTA)-induced nephrotoxicity

    SciTech Connect

    Tanaka, Yuji; Aleksunes, Lauren M. |; Goedken, Michael J.; Chen, Chuan; Reisman, Scott A.; Manautou, Jose E.; Klaassen, Curtis D.

    2008-09-15

    The iron chelate, ferric nitrilotriacetate (FeNTA), induces acute proximal tubular necrosis as a consequence of lipid peroxidation and oxidative tissue damage. Chronic exposure of FeNTA leads to a high incidence of renal adenocarcinomas in rodents. NF-E2-related factor 2 (Nrf2) is a transcription factor that is activated by oxidative stress and electrophiles, and regulates the basal and inducible expression of numerous detoxifying and antioxidant genes. To determine the roles of Nrf2 in regulating renal gene expression and protecting against oxidative stress-induced kidney damage, wild-type and Nrf2-null mice were administered FeNTA. Renal Nrf2 protein translocated to the nucleus at 6h after FeNTA treatment. FeNTA increased mRNA levels of Nrf2 target genes, including NQO1, GCLC, GSTpi1/2, Mrp1, 2, and 4 in kidneys from wild-type mice, but not Nrf2-null mice. Protein expression of NQO1, a prototypical Nrf2 target gene, was increased in wild-type mice, with no change in Nrf2-null mice. FeNTA produced more nephrotoxicity in Nrf2-null mice than wild-type mice as indicated by higher serum urea nitrogen and creatinine levels, as more urinary NAG, stronger 4-hydroxynonenal protein adduct staining, and more extensive proximal tubule damage. Furthermore, pretreatment with CDDO-Im, a potent small molecule Nrf2 activator, protected mice against FeNTA-induced renal toxicity. Collectively, these results suggest that activation of Nrf2 protects mouse kidneys from FeNTA-induced oxidative stress damage by coordinately up-regulating the expression of cytoprotective genes.

  13. GATA2 Mediates Thyrotropin-Releasing Hormone-Induced Transcriptional Activation of the Thyrotropin β Gene

    PubMed Central

    Ohba, Kenji; Sasaki, Shigekazu; Matsushita, Akio; Iwaki, Hiroyuki; Matsunaga, Hideyuki; Suzuki, Shingo; Ishizuka, Keiko; Misawa, Hiroko; Oki, Yutaka; Nakamura, Hirotoshi

    2011-01-01

    Thyrotropin-releasing hormone (TRH) activates not only the secretion of thyrotropin (TSH) but also the transcription of TSHβ and α-glycoprotein (αGSU) subunit genes. TSHβ expression is maintained by two transcription factors, Pit1 and GATA2, and is negatively regulated by thyroid hormone (T3). Our prior studies suggest that the main activator of the TSHβ gene is GATA2, not Pit1 or unliganded T3 receptor (TR). In previous studies on the mechanism of TRH-induced activation of the TSHβ gene, the involvements of Pit1 and TR have been investigated, but the role of GATA2 has not been clarified. Using kidney-derived CV1 cells and pituitary-derived GH3 and TαT1 cells, we demonstrate here that TRH signaling enhances GATA2-dependent activation of the TSHβ promoter and that TRH-induced activity is abolished by amino acid substitution in the GATA2-Zn finger domain or mutation of GATA-responsive element in the TSHβ gene. In CV1 cells transfected with TRH receptor expression plasmid, GATA2-dependent transactivation of αGSU and endothelin-1 promoters was enhanced by TRH. In the gel shift assay, TRH signal potentiated the DNA-binding capacity of GATA2. While inhibition by T3 is dominant over TRH-induced activation, unliganded TR or the putative negative T3-responsive element are not required for TRH-induced stimulation. Studies using GH3 cells showed that TRH-induced activity of the TSHβ promoter depends on protein kinase C but not the mitogen-activated protein kinase, suggesting that the signaling pathway is different from that in the prolactin gene. These results indicate that GATA2 is the principal mediator of the TRH signaling pathway in TSHβ expression. PMID:21533184

  14. Functional analysis of the TMPRSS2:ERG fusion gene in cisplatin‑induced cell death.

    PubMed

    Wu, Junqi; Chi, Linfeng; Chen, Zhanghui; Lu, Xianghong; Xiao, Suping; Zhang, Guanglin; Luo, Jindan; Chen, Ge-Ming; Yang, Jun

    2016-04-01

    The TMPRSS2:E‑twenty‑six (ETS) gene fusion occurs frequently in a high proportion of patients with prostate cancer (PCa) in Western countries, and the aberrant expression of TMPRSS2: v‑ETS avian erythroblastosis virus E26 oncogene homolog (ERG), the most common form of the corresponding protein, can regulate cell migration and contribute to tumor invasion and metastasis. However, its association with other cellular events, and in particular, cell death, remain unknown. To examine the function of such fusion genes, an expression plasmid containing the TMPRSS2:ERG (T1/E5) sequence (ΔERG) from a patient sample was constructed and transiently transfected into DU145 cells, which do not express the fusion gene. It was found that the overexpression of ΔERG significantly inhibited the ability of cisplatin to induce apoptosis in DU145 cells. By contrast, VCaP cells, which do contain TMPRSS2:ERG, were sensitized to cisplatin‑induced apoptosis through siRNA inhibition of the fusion gene. To elucidate the underlying mechanism, a stable cell line expressing the ΔERG gene was constructed. Expression of ΔERG did not affect cell migration, but did protect cells from DNA damage and apoptosis induced by cisplatin. Furthermore, knockdown of ΔERG by short interfering RNA resulted in cells regaining their sensitivity to cisplatin. Finally, the gene coding for activating transcription factor 5, which is important for cell survival, may be upregulated by ΔERG. Taken together, these data point to a new function of the TMPRSS2:ERG fusion gene in regulating the apoptotic pathway. PMID:26935606

  15. Barcode Sequencing Screen Identifies SUB1 as a Regulator of Yeast Pheromone Inducible Genes.

    PubMed

    Sliva, Anna; Kuang, Zheng; Meluh, Pamela B; Boeke, Jef D

    2016-01-01

    The yeast pheromone response pathway serves as a valuable model of eukaryotic mitogen-activated protein kinase (MAPK) pathways, and transcription of their downstream targets. Here, we describe application of a screening method combining two technologies: fluorescence-activated cell sorting (FACS), and barcode analysis by sequencing (Bar-Seq). Using this screening method, and pFUS1-GFP as a reporter for MAPK pathway activation, we readily identified mutants in known mating pathway components. In this study, we also include a comprehensive analysis of the FUS1 induction properties of known mating pathway mutants by flow cytometry, featuring single cell analysis of each mutant population. We also characterized a new source of false positives resulting from the design of this screen. Additionally, we identified a deletion mutant, sub1Δ, with increased basal expression of pFUS1-GFP. Here, in the first ChIP-Seq of Sub1, our data shows that Sub1 binds to the promoters of about half the genes in the genome (tripling the 991 loci previously reported), including the promoters of several pheromone-inducible genes, some of which show an increase upon pheromone induction. Here, we also present the first RNA-Seq of a sub1Δ mutant; the majority of genes have no change in RNA, but, of the small subset that do, most show decreased expression, consistent with biochemical studies implicating Sub1 as a positive transcriptional regulator. The RNA-Seq data also show that certain pheromone-inducible genes are induced less in the sub1Δ mutant relative to the wild type, supporting a role for Sub1 in regulation of mating pathway genes. The sub1Δ mutant has increased basal levels of a small subset of other genes besides FUS1, including IMD2 and FIG1, a gene encoding an integral membrane protein necessary for efficient mating. PMID:26837954

  16. Barcode Sequencing Screen Identifies SUB1 as a Regulator of Yeast Pheromone Inducible Genes

    PubMed Central

    Sliva, Anna; Kuang, Zheng; Meluh, Pamela B.; Boeke, Jef D.

    2016-01-01

    The yeast pheromone response pathway serves as a valuable model of eukaryotic mitogen-activated protein kinase (MAPK) pathways, and transcription of their downstream targets. Here, we describe application of a screening method combining two technologies: fluorescence-activated cell sorting (FACS), and barcode analysis by sequencing (Bar-Seq). Using this screening method, and pFUS1-GFP as a reporter for MAPK pathway activation, we readily identified mutants in known mating pathway components. In this study, we also include a comprehensive analysis of the FUS1 induction properties of known mating pathway mutants by flow cytometry, featuring single cell analysis of each mutant population. We also characterized a new source of false positives resulting from the design of this screen. Additionally, we identified a deletion mutant, sub1Δ, with increased basal expression of pFUS1-GFP. Here, in the first ChIP-Seq of Sub1, our data shows that Sub1 binds to the promoters of about half the genes in the genome (tripling the 991 loci previously reported), including the promoters of several pheromone-inducible genes, some of which show an increase upon pheromone induction. Here, we also present the first RNA-Seq of a sub1Δ mutant; the majority of genes have no change in RNA, but, of the small subset that do, most show decreased expression, consistent with biochemical studies implicating Sub1 as a positive transcriptional regulator. The RNA-Seq data also show that certain pheromone-inducible genes are induced less in the sub1Δ mutant relative to the wild type, supporting a role for Sub1 in regulation of mating pathway genes. The sub1Δ mutant has increased basal levels of a small subset of other genes besides FUS1, including IMD2 and FIG1, a gene encoding an integral membrane protein necessary for efficient mating. PMID:26837954

  17. The Xenopus Emx genes identify presumptive dorsal telencephalon and are induced by head organizer signals.

    PubMed

    Pannese, M; Lupo, G; Kablar, B; Boncinelli, E; Barsacchi, G; Vignali, R

    1998-04-01

    We have isolated and studied the expression pattern of Xemx1 and Xemx2 genes in Xenopus laevis. Xemx genes are the homologues of mouse Emx genes, related to Drosophila empty spiracles. They are expressed in selected regions of the developing brain, particularly in the telencephalon, and, outside the brain, in the otic vesicles, olfactory placodes, visceral arches and the developing excretory system. We also report on experiments concerning the tissue and molecular signals responsible for their activation in competent ectoderm. Xemx genes are activated in ectoderm conjugated with head organizer tissue, but not with tail organizer tissue. Furthermore, they are not activated in animal cap either by noggin or by Xnr3, thus suggesting that a different inducer or the integration of several signals may be responsible for their activation. PMID:9545539

  18. Noise-induced multistability in the regulation of cancer by genes and pseudogenes

    NASA Astrophysics Data System (ADS)

    Petrosyan, K. G.; Hu, Chin-Kun

    2016-07-01

    We extend a previously introduced model of stochastic gene regulation of cancer to a nonlinear case having both gene and pseudogene messenger RNAs (mRNAs) self-regulated. The model consists of stochastic Boolean genetic elements and possesses noise-induced multistability (multimodality). We obtain analytical expressions for probabilities for the case of constant but finite number of microRNA molecules which act as a noise source for the competing gene and pseudogene mRNAs. The probability distribution functions display both the global bistability regime as well as even-odd number oscillations for a certain range of model parameters. Statistical characteristics of the mRNA's level fluctuations are evaluated. The obtained results of the extended model advance our understanding of the process of stochastic gene and pseudogene expressions that is crucial in regulation of cancer.

  19. Gene trapping identifies a putative tumor suppressor and a new inducer of cell migration

    SciTech Connect

    Guardiola-Serrano, Francisca; Haendeler, Judith; Lukosz, Margarete; Sturm, Karsten; Melchner, Harald von; Altschmied, Joachim

    2008-11-28

    Tumor necrosis factor alpha (TNF{alpha}) is a pleiotropic cytokine involved in apoptotic cell death, cellular proliferation, differentiation, inflammation, and tumorigenesis. In tumors it is secreted by tumor associated macrophages and can have both pro- and anti-tumorigenic effects. To identify genes regulated by TNF{alpha}, we performed a gene trap screen in the mammary carcinoma cell line MCF-7 and recovered 64 unique, TNF{alpha}-induced gene trap integration sites. Among these were the genes coding for the zinc finger protein ZC3H10 and for the transcription factor grainyhead-like 3 (GRHL3). In line with the dual effects of TNF{alpha} on tumorigenesis, we found that ZC3H10 inhibits anchorage independent growth in soft agar suggesting a tumor suppressor function, whereas GRHL3 strongly stimulated the migration of endothelial cells which is consistent with an angiogenic, pro-tumorigenic function.

  20. Inducible gene expression from the plastid genome by a synthetic riboswitch

    PubMed Central

    Verhounig, Andreas; Karcher, Daniel; Bock, Ralph

    2010-01-01

    Riboswitches are natural RNA sensors that regulate gene expression in response to ligand binding. Riboswitches have been identified in prokaryotes and eukaryotes but are unknown in organelles (mitochondria and plastids). Here we have tested the possibility to engineer riboswitches for plastids (chloroplasts), a genetic system that largely relies on translational control of gene expression. To this end, we have used bacterial riboswitches and modified them in silico to meet the requirements of translational regulation in plastids. These engineered switches were then tested for functionality in vivo by stable transformation of the tobacco chloroplast genome. We report the identification of a synthetic riboswitch that functions as an efficient translational regulator of gene expression in plastids in response to its exogenously applied ligand theophylline. This riboswitch provides a novel tool for plastid genome engineering that facilitates the tightly regulated inducible expression of chloroplast genes and transgenes and thus has wide applications in functional genomics and biotechnology. PMID:20308585

  1. NGF deprivation-induced gene expression: after ten years, where do we stand?

    PubMed

    Freeman, Robert S; Burch, Robert L; Crowder, Robert J; Lomb, David J; Schoell, Matthew C; Straub, Jennifer A; Xie, Liang

    2004-01-01

    Nerve growth factor (NGF) is required for the survival of developing sympathetic and sensory neurons. In the absence of NGF, these neurons undergo protein synthesis-dependent apoptosis. Ten years have gone by since the first reports of specific genes being upregulated during NGF deprivation-induced cell death. Over the last decade, a few additional genes (DP5, Bim, SM-20) have been added to a list that began with cyclin D1 and c-jun. In this chapter, we discuss the evidence that these genes act as regulators of neuronal cell death. We also suggest a hypothesis for how one gene, SM-20, may function to suppress a self-protection mechanism in NGF-deprived neurons. PMID:14699960

  2. Immunogenic response induced by wzm and wzt gene deletion mutants from Brucella abortus S19.

    PubMed

    Wang, Xiu-Ran; Yan, Guang-Mou; Zhang, Rui; Lang, Xu-Long; Yang, Yan-Ling; Li, Xiao-Yan; Chen, Si; Qian, Jing; Wang, Xing-Long

    2014-02-01

    Brucellosis is an infectious disease affecting humans and animals worldwide. Effective methods of control include inducing immunity in animals by vaccination and elimination. Brucella abortus S19 is one of the popular vaccines for control of cattle brucellosis, as it has low virulence. In this paper, allelic exchange plasmids of wzm and wzt genes were constructed and partially knocked out to evaluate the effects on the induction of immunity to Brucella abortus S19 mutants. Cytokine secretion in vitro, INF-γ induction in vivo and antibody dynamics were evaluated. These data suggested that the immunity-eliciting ability of the wzm and wzt gene deletion mutants was similar, although reduced compared with the S19 strain. The results demonstrated that the wzt gene may be more important in the regulation of the induction of immunity than the wzm gene. PMID:24247358

  3. Inducible gene expression from the plastid genome by a synthetic riboswitch.

    PubMed

    Verhounig, Andreas; Karcher, Daniel; Bock, Ralph

    2010-04-01

    Riboswitches are natural RNA sensors that regulate gene expression in response to ligand binding. Riboswitches have been identified in prokaryotes and eukaryotes but are unknown in organelles (mitochondria and plastids). Here we have tested the possibility to engineer riboswitches for plastids (chloroplasts), a genetic system that largely relies on translational control of gene expression. To this end, we have used bacterial riboswitches and modified them in silico to meet the requirements of translational regulation in plastids. These engineered switches were then tested for functionality in vivo by stable transformation of the tobacco chloroplast genome. We report the identification of a synthetic riboswitch that functions as an efficient translational regulator of gene expression in plastids in response to its exogenously applied ligand theophylline. This riboswitch provides a novel tool for plastid genome engineering that facilitates the tightly regulated inducible expression of chloroplast genes and transgenes and thus has wide applications in functional genomics and biotechnology. PMID:20308585

  4. Marek's disease virus challenge induced immune-related gene expression and chicken repeat 1 (CR1) methylation alterations in chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marek’s disease virus (MDV) challenge induces lymphoma in susceptible chickens. Host genes, especially immune related genes, are activated by the virus. DNA methylation is an epigenetic mechanism that governs gene transcription. In the present study, we found that expression of signal transducer and...

  5. Inducibility of the avidin gene by progesterone is suppressed during estrogen-induced cytodifferentiation.

    PubMed

    Joensuu, T; Niemelä, A; Kunnas, T; Salomaa, S; Alho, H; Vilja, P; Ylikomi, T; Kulomaa, M; Tuohimaa, P

    1992-12-01

    We have studied epithelial differentiation of the chick oviduct as induced by diethylstilbestrol (DES) and 17 beta-estradiol (E2). The proportion of goblet cells in the oviduct was slightly higher after E2 than after DES treatment. Also avidin induction by progesterone was stronger following DES than E2 priming. In the estrogen pretreated oviduct epithelium, avidin expression was induced by progesterone in the surface epithelial cells, protodifferentiated gland cells and tubular gland cells, but not in goblet cells. During prolonged estrogen treatment, however, the inducibility of avidin by progesterone ceased in tubular gland cells but not in surface epithelial cells. The estrogen action on the expression of avidin could be explained by estrogen-induced terminal differentiation of the epithelial gland cells or by a direct effect of estrogen on the progesterone action, for instance interaction of estrogen receptor and progesterone receptor in the regulation of transcription. PMID:1472452

  6. Atypical antipsychotics induce both proinflammatory and adipogenic gene expression in human adipocytes in vitro

    SciTech Connect

    Sárvári, Anitta K.; Veréb, Zoltán; Uray, Iván P.; Fésüs, László; Balajthy, Zoltán

    2014-08-08

    Highlights: • Antipsychotics modulate the expression of adipogenic genes in human adipocytes. • Secretion of proinflammatory cytokine IL8 and MCP-1 is induced by antipsychotics. • Adipocyte-dependent inflammatory abnormality could develop during chronic treatment. • Infiltrated macrophages would further enhance proinflammatory cytokine production. - Abstract: Schizophrenia requires lifelong treatment, potentially causing systemic changes in metabolic homeostasis. In the clinical setting, antipsychotic treatment may differentially lead to weight gain among individual patients, although the molecular determinants of such adverse effects are currently unknown. In this study, we investigated changes in the expression levels of critical regulatory genes of adipogenesis, lipid metabolism and proinflammatory genes during the differentiation of primary human adipose-derived stem cells (ADSCs). These cells were isolated from patients with body mass indices <25 and treated with the second-generation antipsychotics olanzapine, ziprasidone, clozapine, quetiapine, aripiprazole and risperidone and the first-generation antipsychotic haloperidol. We found that antipsychotics exhibited a marked effect on key genes involved in the regulation of cell cycle, signal transduction, transcription factors, nuclear receptors, differentiation markers and metabolic enzymes. In particular, we observed an induction of the transcription factor NF-KB1 and NF-KB1 target genes in adipocytes in response to these drugs, including the proinflammatory cytokines TNF-α, IL-1β, IL-8 and MCP-1. In addition, enhanced secretion of both IL8 and MCP-1 was observed in the supernatant of these cell cultures. In addition to their remarkable stimulatory effects on proinflammatory gene transcription, three of the most frequently prescribed antipsychotic drugs, clozapine, quetiapine and aripiprazole, also induced the expression of essential adipocyte differentiation genes and the adipocyte hormones leptin

  7. Sonoporation Increases Therapeutic Efficacy of Inducible and Constitutive BMP2/7 In Vivo Gene Delivery

    PubMed Central

    Hofmann, Anna T.; Slezak, Paul; Schuetzenberger, Sebastian; Kaipel, Martin; Schwartz, Ernst; Neef, Anne; Nomikou, Nikolitsa; Nau, Thomas; van Griensven, Martijn; McHale, Anthony P.; Redl, Heinz

    2014-01-01

    Abstract An ideal novel treatment for bone defects should provide regeneration without autologous or allogenous grafting, exogenous cells, growth factors, or biomaterials while ensuring spatial and temporal control as well as safety. Therefore, a novel osteoinductive nonviral in vivo gene therapy approach using sonoporation was investigated in ectopic and orthotopic models. Constitutive or regulated, doxycycline-inducible, bone morphogenetic protein 2 and 7 coexpression plasmids were repeatedly applied for 5 days. Ectopic and orthotopic gene transfer efficacy was monitored by coapplication of a luciferase plasmid and bioluminescence imaging. Orthotopic plasmid DNA distribution was investigated using a novel plasmid-labeling method. Luciferase imaging demonstrated an increased trend (61% vs. 100%) of gene transfer efficacy, and micro-computed tomography evaluation showed significantly enhanced frequency of ectopic bone formation for sonoporation compared with passive gene delivery (46% vs. 100%) dependent on applied ultrasound power. Bone formation by the inducible system (83%) was stringently controlled by doxycycline in vivo, and no ectopic bone formation was observed without induction or with passive gene transfer without sonoporation. Orthotopic evaluation in a rat femur segmental defect model demonstrated an increased trend of gene transfer efficacy using sonoporation. Investigation of DNA distribution demonstrated extensive binding of plasmid DNA to bone tissue. Sonoporated animals displayed a potentially increased union rate (33%) without extensive callus formation or heterotopic ossification. We conclude that sonoporation of BMP2/7 coexpression plasmids is a feasible, minimally invasive method for osteoinduction and that improvement of bone regeneration by sonoporative gene delivery is superior to passive gene delivery. PMID:24164605

  8. Developing tTA transgenic rats for inducible and reversible gene expression.

    PubMed

    Zhou, Hongxia; Huang, Cao; Yang, Min; Landel, Carlisle P; Xia, Pedro Yuxing; Liu, Yong-Jian; Xia, Xu Gang

    2009-01-01

    To develop transgenic lines for conditional expression of desired genes in rats, we generated several lines of the transgenic rats carrying the tetracycline-controlled transactivator (tTA) gene. Using a vigorous, ubiquitous promoter to drive the tTA transgene, we obtained widespread expression of tTA in various tissues. Expression of tTA was sufficient to strongly activate its reporter gene, but was below the toxicity threshold. We examined the dynamics of Doxycycline (Dox)-regulated gene expression in transgenic rats. In the two transmittable lines, tTA-mediated activation of the reporter gene was fully subject to regulation by Dox. Dox dose-dependently suppressed tTA-activated gene expression. The washout time for the effects of Dox was dose-dependent. We tested a complex regime of Dox administration to determine the optimal effectiveness and washout duration. Dox was administered at a high dose (500 microg/ml in drinking water) for two days to reach the effective concentration, and then was given at a low dose (20 microg/ml) to maintain effectiveness. This regimen of Dox administration can achieve a quick switch between ON and OFF statuses of tTA-activated gene expression. In addition, administration of Dox to pregnant rats fully suppressed postnatal tTA-activated gene expression in their offspring. Sufficient levels of Dox are present in mother's milk to produce maximal efficacy in nursing neonates. Administration of Dox to pregnant or nursing rats can provide a continual suppression of tTA-dependent gene expression during embryonic and postnatal development. The tTA transgenic rat allows for inducible and reversible gene expression in the rat; this important tool will be valuable in the development of genetic rat models of human diseases. PMID:19214245

  9. Effects of laser parameters on propagation characteristics of laser-induced stress wave for gene transfer

    NASA Astrophysics Data System (ADS)

    Ando, Takahiro; Sato, Shunichi; Terakawa, Mitsuhiro; Ashida, Hiroshi; Obara, Minoru

    2010-02-01

    Laser-based gene delivery is attractive as a new method for topical gene therapy because of the high spatial controllability of laser energy. Previously, we demonstrated that an exogenous gene can be transferred to cells both in vitro and in vivo by applying nanosecond pulsed laser-induced stress waves (LISWs) or photomechanical waves (PMWs). In this study, we investigated effects of laser parameters on the propagation characteristics of LISWs in soft tissue phantoms and depth-dependent properties of gene transfection. Temporal pressure profiles of LISWs were measured with a hydrophone, showing that with a larger laser spot diameter, LISWs can be propagated more efficiently in phantoms with keeping flat wavefront. Phantoms with various thicknesses were placed on the rat dorsal skin that had been injected with plasmid DNA coding for reporter gene, and LISWs were applied from the top of the phantom. Efficient gene expression was observed in the rat skin that had interacted with LISWs propagating through a 15-mm-thick phantom. These results would be useful to determine appropriate laser parameters for gene delivery to deep-located tissue by transcutaneous application of LISWs.

  10. Effects of L-theanine on posttraumatic stress disorder induced changes in rat brain gene expression.

    PubMed

    Ceremuga, Tomás Eduardo; Martinson, Stephanie; Washington, Jason; Revels, Robert; Wojcicki, Jessica; Crawford, Damali; Edwards, Robert; Kemper, Joshua Luke; Townsend, William Luke; Herron, Geno M; Ceremuga, George Allen; Padron, Gina; Bentley, Michael

    2014-01-01

    Posttraumatic stress disorder (PTSD) is characterized by the occurrence of a traumatic event that is beyond the normal range of human experience. The future of PTSD treatment may specifically target the molecular mechanisms of PTSD. In the US, approximately 20% of adults report taking herbal products to treat medical illnesses. L-theanine is the amino acid in green tea primarily responsible for relaxation effects. No studies have evaluated the potential therapeutic properties of herbal medications on gene expression in PTSD. We evaluated gene expression in PTSD-induced changes in the amygdala and hippocampus of Sprague-Dawley rats. The rats were assigned to PTSD-stressed and nonstressed groups that received either saline, midazolam, L-theanine, or L-theanine + midazolam. Amygdala and hippocampus tissue samples were analyzed for changes in gene expression. One-way ANOVA was used to detect significant difference between groups in the amygdala and hippocampus. Of 88 genes examined, 17 had a large effect size greater than 0.138. Of these, 3 genes in the hippocampus and 5 genes in the amygdala were considered significant (P < 0.05) between the groups. RT-PCR analysis revealed significant changes between groups in several genes implicated in a variety of disorders ranging from PTSD, anxiety, mood disorders, and substance dependence. PMID:25165739