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Sample records for plants constitutively expressing

  1. Enhanced salt tolerance in tomato plants constitutively expressing heat-shock protein in the endoplasmic reticulum.

    PubMed

    Fu, C; Liu, X X; Yang, W W; Zhao, C M; Liu, J

    2016-01-01

    The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates the unfolded protein response (UPR) signaling pathway. The UPR signaling pathway is associated with plant responses to adverse environmental conditions. Thus, changes in the UPR signaling pathway might affect plant abiotic tolerance. Here, the role of ER small heat-shock protein (ER-sHSP) in improving plant resistance to salt stress was explored. Under salt stress conditions, ER-sHSP transgenic plants were found to have more vigorous roots, maintain a higher relative water content, absorb less Na(+), accumulate more osmolytes and Ca(2+), and sustain less damage to the photosystem, compared to wild-type non-transgenic plants. Furthermore, we found that the constitutive expression of ER-sHSP under salt stress depressed the expression of other ER molecular chaperones. These results indicate that the constitutive expression of ER-sHSP enhanced salinity tolerance of tomato plants significantly, and alleviated the ER stress caused by the salt stress in plant cells. PMID:27421016

  2. Evolutionary change from induced to constitutive expression of an indirect plant resistance.

    PubMed

    Heil, Martin; Greiner, Sabine; Meimberg, Harald; Krüger, Ralf; Noyer, Jean-Louis; Heubl, Günther; Linsenmair, K Eduard; Boland, Wilhelm

    2004-07-01

    Induced plant resistance traits are expressed in response to attack and occur throughout the plant kingdom. Despite their general occurrence, the evolution of such resistances has rarely been investigated. Here we report that extrafloral nectar, a usually inducible trait, is constitutively secreted by Central American Acacia species that are obligately inhabited by ants. Extrafloral nectar is secreted as an indirect resistance, attracting ants that defend plants against herbivores. Leaf damage induces extrafloral nectar secretion in several plant species; among these are various Acacia species and other Fabaceae investigated here. In contrast, Acacia species obligately inhabited by symbiotic ants nourish these ants by secreting extrafloral nectar constitutively at high rates that are not affected by leaf damage. The phylogeny of the genus Acacia and closely related genera indicate that the inducibility of extrafloral nectar is the plesiomorphic or 'original' state, whereas the constitutive extrafloral nectar flow is derived within Acacia. A constitutive resistance trait has evolved from an inducible one, obviously in response to particular functional demands.

  3. Constitutive Expression of Mammalian Nitric Oxide Synthase in Tobacco Plants Triggers Disease Resistance to Pathogens

    PubMed Central

    Chun, Hyun Jin; Park, Hyeong Cheol; Koo, Sung Cheol; Lee, Ju Huck; Park, Chan Young; Choi, Man Soo; Kang, Chang Ho; Baek, Dongwon; Cheong, Yong Hwa; Yun, Dae-Jin; Chung, Woo Sik; Cho, Moo Je; Kim, Min Chul

    2012-01-01

    Nitric oxide (NO) is known for its role in the activation of plant defense responses. To examine the involvement and mode of action of NO in plant defense responses, we introduced calmodulin-dependent mammalian neuronal nitric oxide synthase (nNOS), which controls the CaMV35S promoter, into wild-type and NahG tobacco plants. Constitutive expression of nNOS led to NO production and triggered spontaneous induction of leaf lesions. Transgenic plants accumulated high amounts of H2O2, with catalase activity lower than that in the wild type. nNOS transgenic plants contained high levels of salicylic acid (SA), and they induced an array of SA-, jasmonic acid (JA)-, and/or ethylene (ET)-related genes. Consequently, NahG co-expression blocked the induction of systemic acquired resistance (SAR)-associated genes in transgenic plants, implying SA is involved in NO-mediated induction of SAR genes. The transgenic plants exhibited enhanced resistance to a spectrum of pathogens, including bacteria, fungi, and viruses. Our results suggest a highly ranked regulatory role for NO in SA-, JA-, and/or ET-dependent pathways that lead to disease resistance. PMID:23124383

  4. Constitutive expression of CaPLA1 conferred enhanced growth and grain yield in transgenic rice plants.

    PubMed

    Park, Ki Youl; Kim, Eun Yu; Seo, Young Sam; Kim, Woo Taek

    2016-03-01

    Phospholipids are not only important components of cell membranes, but participate in diverse processes in higher plants. In this study, we generated Capsicum annuum phospholipiase A1 (CaPLA1) overexpressing transgenic rice (Oryza sativa L.) plants under the control of the maize ubiquitin promoter. The T4 CaPLA1-overexpressing rice plants (Ubi:CaPLA1) had a higher root:shoot mass ratio than the wild-type plants in the vegetative stage. Leaf epidermal cells from transgenic plants had more cells than wild-type plants. Genes that code for cyclin and lipid metabolic enzymes were up-regulated in the transgenic lines. When grown under typical paddy field conditions, the transgenic plants produced more tillers, longer panicles and more branches per panicle than the wild-type plants, all of which resulted in greater grain yield. Microarray analysis suggests that gene expressions that are related with cell proliferation, lipid metabolism, and redox state were widely altered in CaPLA1-overexpressing transgenic rice plants. Ubi:CaPLA1 plants had a reduced membrane peroxidation state, as determined by malondialdehyde and conjugated diene levels and higher peroxidase activity than wild-type rice plants. Furthermore, three isoprenoid synthetic genes encoding terpenoid synthase, hydroxysteroid dehydrogenase and 3-hydroxy-3-methyl-glutaryl-CoA reductase were up-regulated in CaPLA1-overexpressing plants. We suggest that constitutive expression of CaPLA1 conferred increased grain yield with enhanced growth in transgenic rice plants by alteration of gene activities related with cell proliferation, lipid metabolism, membrane peroxidation state and isoprenoid biosynthesis.

  5. Constitutive expression of CaPLA1 conferred enhanced growth and grain yield in transgenic rice plants.

    PubMed

    Park, Ki Youl; Kim, Eun Yu; Seo, Young Sam; Kim, Woo Taek

    2016-03-01

    Phospholipids are not only important components of cell membranes, but participate in diverse processes in higher plants. In this study, we generated Capsicum annuum phospholipiase A1 (CaPLA1) overexpressing transgenic rice (Oryza sativa L.) plants under the control of the maize ubiquitin promoter. The T4 CaPLA1-overexpressing rice plants (Ubi:CaPLA1) had a higher root:shoot mass ratio than the wild-type plants in the vegetative stage. Leaf epidermal cells from transgenic plants had more cells than wild-type plants. Genes that code for cyclin and lipid metabolic enzymes were up-regulated in the transgenic lines. When grown under typical paddy field conditions, the transgenic plants produced more tillers, longer panicles and more branches per panicle than the wild-type plants, all of which resulted in greater grain yield. Microarray analysis suggests that gene expressions that are related with cell proliferation, lipid metabolism, and redox state were widely altered in CaPLA1-overexpressing transgenic rice plants. Ubi:CaPLA1 plants had a reduced membrane peroxidation state, as determined by malondialdehyde and conjugated diene levels and higher peroxidase activity than wild-type rice plants. Furthermore, three isoprenoid synthetic genes encoding terpenoid synthase, hydroxysteroid dehydrogenase and 3-hydroxy-3-methyl-glutaryl-CoA reductase were up-regulated in CaPLA1-overexpressing plants. We suggest that constitutive expression of CaPLA1 conferred increased grain yield with enhanced growth in transgenic rice plants by alteration of gene activities related with cell proliferation, lipid metabolism, membrane peroxidation state and isoprenoid biosynthesis. PMID:26803502

  6. Plant density and nutrient availability constrain constitutive and wound-induced expression of trypsin inhibitors in Brassica napus.

    PubMed

    Cipollini, D F; Bergelson, J

    2001-03-01

    We investigated the effects of plant density on plant size, leaf total soluble protein content, and constitutive and wound-induced levels of proteinaceous trypsin inhibitors in pot-grown Brassica napus seedlings in two greenhouse studies. We manipulated plant density by varying the number of intraspecific neighbors surrounding a target plant in the center of each pot. In general, constitutive and induced levels of trypsin inhibitors were significantly reduced by competition in a density-dependent manner, to the extent that induction was greatly reduced or abolished in target plants surrounded by six neighbors. To investigate whether the effects of plant density on inhibitor production were mediated by nutrient availability, we manipulated the concentration of a complete fertilizer applied to target plants surrounded by six neighbors in two greenhouse studies. In general, constitutive and wound-induced levels of inhibitors in plants surrounded by six neighbors were increased by nutrient addition in a dose-dependent manner, such that wound-induction was completely restored in competing plants under conditions of high nutrient availability. Leaf total soluble protein content, measured only in the second trial of each experiment, was not affected by any of the treatments. The effects of plant density, nutrient addition, and wounding on inhibitor levels in all experiments were independent of their effects on above-ground plant size at the time of wounding. Overall, our results suggest that decreasing nutrient availability mediates the density-dependent reductions in inhibitor levels in B. napus seedings.

  7. Constitutive expression of fluorescent protein by Aspergillus var. niger and Aspergillus carbonarius to monitor fungal colonization in maize plants.

    PubMed

    Palencia, Edwin Rene; Glenn, Anthony Elbie; Hinton, Dorothy Mae; Bacon, Charles Wilson

    2013-09-01

    Aspergillus niger and Aspergillus carbonarius are two species in the Aspergillus section Nigri (black-spored aspergilli) frequently associated with peanut (Arachis hypogea), maize (Zea mays), and other plants as pathogens. These infections are symptomless and as such are major concerns since some black aspergilli produce important mycotoxins, ochratoxins A, and the fumonisins. To facilitate the study of the black aspergilli-maize interactions with maize during the early stages of infections, we developed a method that used the enhanced yellow fluorescent protein (eYFP) and the monomeric red fluorescent protein (mRFP1) to transform A. niger and A. carbonarius, respectively. The results were constitutive expressions of the fluorescent genes that were stable in the cytoplasms of hyphae and conidia under natural environmental conditions. The hyphal in planta distribution in 21-day-old seedlings of maize were similar wild type and transformants of A. niger and A. carbonarius. The in planta studies indicated that both wild type and transformants internally colonized leaf, stem and root tissues of maize seedlings, without any visible disease symptoms. Yellow and red fluorescent strains were capable of invading epidermal cells of maize roots intercellularly within the first 3 days after inoculation, but intracellular hyphal growth was more evident after 7 days of inoculation. We also tested the capacity of fluorescent transformants to produce ochratoxin A and the results with A. carbonarius showed that this transgenic strain produced similar concentrations of this secondary metabolite. This is the first report on the in planta expression of fluorescent proteins that should be useful to study the internal plant colonization patterns of two ochratoxigenic species in the Aspergillus section Nigri.

  8. Constitutive expression of the xylanase inhibitor TAXI-III delays Fusarium head blight symptoms in durum wheat transgenic plants.

    PubMed

    Moscetti, Ilaria; Tundo, Silvio; Janni, Michela; Sella, Luca; Gazzetti, Katia; Tauzin, Alexandra; Giardina, Thierry; Masci, Stefania; Favaron, Francesco; D'Ovidio, Renato

    2013-12-01

    Cereals contain xylanase inhibitor (XI) proteins which inhibit microbial xylanases and are considered part of the defense mechanisms to counteract microbial pathogens. Nevertheless, in planta evidence for this role has not been reported yet. Therefore, we produced a number of transgenic plants constitutively overexpressing TAXI-III, a member of the TAXI type XI that is induced by pathogen infection. Results showed that TAXI-III endows the transgenic wheat with new inhibition capacities. We also showed that TAXI-III is correctly secreted into the apoplast and possesses the expected inhibition parameters against microbial xylanases. The new inhibition properties of the transgenic plants correlate with a significant delay of Fusarium head blight disease symptoms caused by Fusarium graminearum but do not significantly influence leaf spot symptoms caused by Bipolaris sorokiniana. We showed that this contrasting result can be due to the different capacity of TAXI-III to inhibit the xylanase activity of these two fungal pathogens. These results provide, for the first time, clear evidence in planta that XI are involved in plant defense against fungal pathogens and show the potential to manipulate TAXI-III accumulation to improve wheat resistance against F. graminearum.

  9. Biochemical and molecular characterization of transgenic Lotus japonicus plants constitutively over-expressing a cytosolic glutamine synthetase gene

    PubMed Central

    Ortega, Jose Luis; Temple, Stephen J.; Bagga, Suman; Ghoshroy, Soumitra; Sengupta-Gopalan, Champa

    2013-01-01

    Higher plants assimilate nitrogen in the form of ammonia through the concerted activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS enzyme is either located in the cytoplasm (GS1) or in the chloroplast (GS2). To understand how modulation of GS activity affects plant performance, Lotus japonicus L. plants were transformed with an alfalfa GS1 gene driven by the CaMV 35S promoter. The transformants showed increased GS activity and an increase in GS1 polypeptide level in all the organs tested. GS was analyzed by non-denaturing gel electrophoresis and ion-exchange chromatography. The results showed the presence of multiple GS isoenzymes in the different organs and the presence of a novel isoform in the transgenic plants. The distribution of GS in the different organs was analyzed by immunohistochemical localization. GS was localized in the mesophyll cells of the leaves and in the vasculature of the stem and roots of the transformants. Our results consistently showed higher soluble protein concentration, higher chlorophyll content and a higher biomass accumulation in the transgenic plants. The total amino acid content in the leaves and stems of the transgenic plants was 22–24% more than in the tissues of the non-transformed plants. The relative abundance of individual amino acid was similar except for aspartate/asparagine and proline, which were higher in the transformants. PMID:15197594

  10. Constitutive expression of fluorescent protein by Aspergillus var. niger and Aspergillus carbonarius to monitor fungal colonization in maize plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspergillus niger and A. carbonarius are two species in the Aspergillus section Nigri (black-spored aspergilli) frequently associated with peanut (Arachis hypogea), maize (Zea mays), and other plants as pathogens. These infections are symptomless and as such are major concerns since some black aspe...

  11. Constitutive expression of the tzs gene from Agrobacterium tumefaciens virG mutant strains is responsible for improved transgenic plant regeneration in cotton meristem transformation.

    PubMed

    Ye, Xudong; Chen, Yurong; Wan, Yuechun; Hong, Yun-Jeong; Ruebelt, Martin C; Gilbertson, Larry A

    2016-03-01

    KEY MESSAGE : virG mutant strains of a nopaline type of Agrobacterium tumefaciens increase the transformation frequency in cotton meristem transformation. Constitutive cytokinin expression from the tzs gene in the virG mutant strains is responsible for the improvement. Strains of Agrobacterium tumefaciens were tested for their ability to improve cotton meristem transformation frequency. Two disarmed A. tumefaciens nopaline strains with either a virGN54D constitutively active mutation or virGI77V hypersensitive induction mutation significantly increased the transformation frequency in a cotton meristem transformation system. The virG mutant strains resulted in greener explants after three days of co-culture in the presence of light, which could be attributed to a cytokinin effect of the mutants. A tzs knockout strain of virGI77V mutant showed more elongated, less green explants and decreased cotton transformation frequency, as compared to a wild type parental strain, suggesting that expression of the tzs gene is required for transformation frequency improvement in cotton meristem transformation. In vitro cytokinin levels in culture media were tenfold higher in the virGN54D strain, and approximately 30-fold higher in the virGI77V strain, in the absence of acetosyringone induction, compared to the wild type strain. The cytokinin level in the virGN54D strain is further increased upon acetosyringone induction, while the cytokinin level in the virGI77V mutant is decreased by induction, suggesting that different tzs gene expression regulation mechanisms are present in the two virG mutant strains. Based on these data, we suggest that the increased cytokinin levels play a major role in increasing Agrobacterium attachment and stimulating localized division of the attached plant cells. PMID:26650837

  12. Photoreceptor cells constitutively express functional TLR4

    PubMed Central

    Tu, Zhidan; Portillo, Jose-Andres; Howell, Scott; Bu, Hong; Subauste, Carlos S.; Al-Ubaidi, Muayyad R; Pearlman, Eric; Lin, Feng

    2010-01-01

    Toll-like receptor 4 (TLR4) is expressed on a number of cells including neurons in the brain. However, it has yet to be determined if TLR4 is expressed on photoreceptor cells in the retina. In this report, we examined primary photoreceptor cells and an established photoreceptor cell line (661W). We found that functional TLR4 is constitutively expressed on photoreceptor cells, and can be activated by LPS. We conclude that TLR4 on photoreceptor cells could directly contribute to retinal inflammatory diseases and photoreceptor cell survival. PMID:20801528

  13. Constitutive expression of DaCBF7, an Antarctic vascular plant Deschampsia antarctica CBF homolog, resulted in improved cold tolerance in transgenic rice plants.

    PubMed

    Byun, Mi Young; Lee, Jungeun; Cui, Li Hua; Kang, Yoonjee; Oh, Tae Kyung; Park, Hyun; Lee, Hyoungseok; Kim, Woo Taek

    2015-07-01

    Deschampsia antarctica is an Antarctic hairgrass that grows on the west coast of the Antarctic peninsula. In this report, we have identified and characterized a transcription factor, D. antarctica C-repeat binding factor 7 (DaCBF7), that is a member of the monocot group V CBF homologs. The protein contains a single AP2 domain, a putative nuclear localization signal, and the typical CBF signature. DaCBF7, like other monocot group V homologs, contains a distinct polypeptide stretch composed of 43 amino acids in front of the AP2 motif. DaCBF7 was predominantly localized to nuclei and interacted with the C-repeat/dehydration responsive element (CRT/DRE) core sequence (ACCGAC) in vitro. DaCBF7 was induced by abiotic stresses, including drought, cold, and salinity. To investigate its possible cellular role in cold tolerance, a transgenic rice system was employed. DaCBF7-overexpressing transgenic rice plants (Ubi:DaCBF7) exhibited markedly increased tolerance to cold stress compared to wild-type plants without growth defects; however, overexpression of DaCBF7 exerted little effect on tolerance to drought or salt stress. Transcriptome analysis of a Ubi:DaCBF7 transgenic line revealed 13 genes that were up-regulated in DaCBF7-overexpressing plants compared to wild-type plants in the absence of cold stress and in short- or long-term cold stress. Five of these genes, dehydrin, remorin, Os03g63870, Os11g34790, and Os10g22630, contained putative CRT/DRE or low-temperature responsive elements in their promoter regions. These results suggest that overexpression of DaCBF7 directly and indirectly induces diverse genes in transgenic rice plants and confers enhanced tolerance to cold stress.

  14. Constitutive over-expression of rice chymotrypsin protease inhibitor gene OCPI2 results in enhanced growth, salinity and osmotic stress tolerance of the transgenic Arabidopsis plants.

    PubMed

    Tiwari, Lalit Dev; Mittal, Dheeraj; Chandra Mishra, Ratnesh; Grover, Anil

    2015-07-01

    Protease inhibitors are involved primarily in defense against pathogens. In recent years, these proteins have also been widely implicated in response of plants to diverse abiotic stresses. Rice chymotrypsin protease inhibitor gene OCPI2 is highly induced under salt and osmotic stresses. The construct containing the complete coding sequence of OCPI2 cloned downstream to CaMV35S promoter was transformed in Arabidopsis and single copy, homozygous transgenic lines were produced. The transgenic plants exhibited significantly enhanced tolerance to NaCl, PEG and mannitol stress as compared to wild type plants. Importantly, the vegetative and reproductive growth of transgenic plants under unstressed, control conditions was also enhanced: transgenic plants were more vigorous than wild type, resulting into higher yield in terms of silique number. The RWC values and membrane stability index of transgenic in comparison to wild type plants was higher. Higher proline content was observed in the AtOCPI2 lines, which was associated with higher transcript expression of pyrroline-5-carboxylate synthase and lowered levels of proline dehydrogenase genes. The chymotrypsin protease activities were lower in the transgenic as against wild type plants, under both unstressed, control as well as stressed conditions. It thus appears that rice chymotrypsin protease inhibitor gene OCPI2 is a useful candidate gene for genetic improvement of plants against salt and osmotic stress. PMID:25910649

  15. Constitutive over-expression of rice chymotrypsin protease inhibitor gene OCPI2 results in enhanced growth, salinity and osmotic stress tolerance of the transgenic Arabidopsis plants.

    PubMed

    Tiwari, Lalit Dev; Mittal, Dheeraj; Chandra Mishra, Ratnesh; Grover, Anil

    2015-07-01

    Protease inhibitors are involved primarily in defense against pathogens. In recent years, these proteins have also been widely implicated in response of plants to diverse abiotic stresses. Rice chymotrypsin protease inhibitor gene OCPI2 is highly induced under salt and osmotic stresses. The construct containing the complete coding sequence of OCPI2 cloned downstream to CaMV35S promoter was transformed in Arabidopsis and single copy, homozygous transgenic lines were produced. The transgenic plants exhibited significantly enhanced tolerance to NaCl, PEG and mannitol stress as compared to wild type plants. Importantly, the vegetative and reproductive growth of transgenic plants under unstressed, control conditions was also enhanced: transgenic plants were more vigorous than wild type, resulting into higher yield in terms of silique number. The RWC values and membrane stability index of transgenic in comparison to wild type plants was higher. Higher proline content was observed in the AtOCPI2 lines, which was associated with higher transcript expression of pyrroline-5-carboxylate synthase and lowered levels of proline dehydrogenase genes. The chymotrypsin protease activities were lower in the transgenic as against wild type plants, under both unstressed, control as well as stressed conditions. It thus appears that rice chymotrypsin protease inhibitor gene OCPI2 is a useful candidate gene for genetic improvement of plants against salt and osmotic stress.

  16. Constitutive over-expression of rice ClpD1 protein enhances tolerance to salt and desiccation stresses in transgenic Arabidopsis plants.

    PubMed

    Mishra, Ratnesh Chandra; Richa; Grover, Anil

    2016-09-01

    Caseinolytic proteases (Clps) perform the important role of removing protein aggregates from cells, which can otherwise prove to be highly toxic. ClpD system is a two-component protease complex composed of a regulatory ATPase module ClpD and a proteolytic component ClpP. Under desiccation stress condition, rice ClpD1 (OsClpD1) gene encoding for the regulatory subunit, was represented by four variant transcripts differing mainly in the expanse of their N-terminal amino acids. These transcripts were expressed in a differential manner in response to salt, mannitol and polyethylene glycol stresses in rice. Purified OsClpD1.3 protein exhibited intrinsic chaperone activity, shown using citrate synthase as substrate. Arabidopsis (Col-0) plants over-expressing OsClpD1.3 open reading frame downstream to CaMV35S promoter (ClpD1.3 plants) showed higher tolerance to salt and desiccation stresses as compared to wild type plants. ClpD1.3 seedlings also showed enhanced growth during the early stages of seed germination under unstressed, control conditions. The free proline levels and starch breakdown activities were higher in the ClpD1.3 seedlings as compared to the wild type Arabidopsis seedlings. It thus emerges that increasing the potential of ClpD1 chaperoning activity may be of advantage in protection against abiotic stresses. PMID:27457985

  17. Constitutive nitrate reductase expression and inhibition in winged bean

    SciTech Connect

    Wu, Shenchuan; Harper, J.E. )

    1990-05-01

    It was found that NO{sub 3}{sup {minus}} had no effect on winged bean nitrate reductase activity (NRA). Similar NRA was expressed in plants grown on NO{sub 3}{sup {minus}}, urea, NH{sub 4}{sup +}, and nil N. This indicated that the primary NR expressed in winged bean was constitutive, rather than substrate-inducible. Maximum NRA in winged bean was obtained in the light. KClO{sub 3} was capable of inhibiting NRA of leaves if added to the root growth medium or to the NR assay medium, indicating possible competition with NO{sub 3}{sup {minus}} at the reduction site. While it has previously been shown that either cycloheximide alone, or both cycloheximide and chloramphenicol impair the synthesis of NR protein, our data unexpectedly demonstrated that cycloheximide had little effect on NRA, whereas chloramphenicol greatly inhibited the expression of NRA in winged bean. One interpretation is that chloroplasts may influence the activity and/or synthesis of constitutive NR proteins.

  18. Constitutive Expression of Rice MicroRNA528 Alters Plant Development and Enhances Tolerance to Salinity Stress and Nitrogen Starvation in Creeping Bentgrass.

    PubMed

    Yuan, Shuangrong; Li, Zhigang; Li, Dayong; Yuan, Ning; Hu, Qian; Luo, Hong

    2015-09-01

    MicroRNA528 (miR528) is a conserved monocot-specific small RNA that has the potential of mediating multiple stress responses. So far, however, experimental functional studies of miR528 are lacking. Here, we report that overexpression of a rice (Oryza sativa) miR528 (Osa-miR528) in transgenic creeping bentgrass (Agrostis stolonifera) alters plant development and improves plant salt stress and nitrogen (N) deficiency tolerance. Morphologically, miR528-overexpressing transgenic plants display shortened internodes, increased tiller number, and upright growth. Improved salt stress resistance is associated with increased water retention, cell membrane integrity, chlorophyll content, capacity for maintaining potassium homeostasis, CATALASE activity, and reduced ASCORBIC ACID OXIDASE (AAO) activity; while enhanced tolerance to N deficiency is associated with increased biomass, total N accumulation and chlorophyll synthesis, nitrite reductase activity, and reduced AAO activity. In addition, AsAAO and COPPER ION BINDING PROTEIN1 are identified as two putative targets of miR528 in creeping bentgrass. Both of them respond to salinity and N starvation and are significantly down-regulated in miR528-overexpressing transgenics. Our data establish a key role that miR528 plays in modulating plant growth and development and in the plant response to salinity and N deficiency and indicate the potential of manipulating miR528 in improving plant abiotic stress resistance.

  19. Constitutive Expression of Rice MicroRNA528 Alters Plant Development and Enhances Tolerance to Salinity Stress and Nitrogen Starvation in Creeping Bentgrass.

    PubMed

    Yuan, Shuangrong; Li, Zhigang; Li, Dayong; Yuan, Ning; Hu, Qian; Luo, Hong

    2015-09-01

    MicroRNA528 (miR528) is a conserved monocot-specific small RNA that has the potential of mediating multiple stress responses. So far, however, experimental functional studies of miR528 are lacking. Here, we report that overexpression of a rice (Oryza sativa) miR528 (Osa-miR528) in transgenic creeping bentgrass (Agrostis stolonifera) alters plant development and improves plant salt stress and nitrogen (N) deficiency tolerance. Morphologically, miR528-overexpressing transgenic plants display shortened internodes, increased tiller number, and upright growth. Improved salt stress resistance is associated with increased water retention, cell membrane integrity, chlorophyll content, capacity for maintaining potassium homeostasis, CATALASE activity, and reduced ASCORBIC ACID OXIDASE (AAO) activity; while enhanced tolerance to N deficiency is associated with increased biomass, total N accumulation and chlorophyll synthesis, nitrite reductase activity, and reduced AAO activity. In addition, AsAAO and COPPER ION BINDING PROTEIN1 are identified as two putative targets of miR528 in creeping bentgrass. Both of them respond to salinity and N starvation and are significantly down-regulated in miR528-overexpressing transgenics. Our data establish a key role that miR528 plays in modulating plant growth and development and in the plant response to salinity and N deficiency and indicate the potential of manipulating miR528 in improving plant abiotic stress resistance. PMID:26224802

  20. Light-inducible and constitutively expressed DNA-binding proteins recognizing a plant promoter element with functional relevance in light responsiveness.

    PubMed Central

    Weisshaar, B; Armstrong, G A; Block, A; da Costa e Silva, O; Hahlbrock, K

    1991-01-01

    Four cis-acting elements, designated as Boxes I, II, III and IV, have previously been identified as functionally relevant components of the light-responsive chalcone synthase (CHS) promoter in parsley (Petroselinum crispum). This paper describes the isolation of three cDNAs encoding proteins which bind specifically to Box II, one of two cis-acting elements found within a 52 bp CHS promoter region shown here to be sufficient for light responsiveness in parsley. The deduced amino acid sequences of all three proteins reveal conserved basic and leucine zipper domains characteristic of transcription factors of the bZIP class. Nucleotide sequences recognized by these factors contain an ACGT motif common to many cis-acting elements. Therefore, we have termed the proteins CPRF-1, -2 and -3 (Common Plant Regulatory Factor). The characteristics of CPRF-1 binding to Box II and the timing of transient CPRF-1 mRNA accumulation during light exposure of previously dark-grown parsley cells are consistent with the hypothesis that this factor participates in the light-mediated activation of the CHS gene in parsley. Images PMID:2050115

  1. Freedom of Expression in the Original Constitution: A Bicentennial Review.

    ERIC Educational Resources Information Center

    Kane, Peter E.

    The original United States Constitution of 1787, specifically in the communication related provisions of Article 1, deals extensively with communication issues, and provides an illustration of the tensions that existed between freedom and suppression. Philosophically, there was support for freedom of expression while in real world events there was…

  2. A comparison of constitutive promoters for expression of transgenes in alfalfa (Medicago sativa).

    PubMed

    Samac, Deborah A; Tesfaye, Mesfin; Dornbusch, Melinda; Saruul, Purev; Temple, Stephen J

    2004-08-01

    The activity of constitutive promoters was compared in transgenic alfalfa plants using two marker genes. Three promoters, the 35S promoter from cauliflower mosaic virus (CaMV), the cassava vein mosaic virus (CsVMV) promoter, and the sugarcane bacilliform badnavirus (ScBV) promoter were each fused to the beta-glucuronidase (gusA) gene. The highest GUS enzyme activity was obtained using the CsVMV promoter and all alfalfa cells assayed by in situ staining had high levels of enzyme activity. The 35S promoter was expressed in leaves, roots, and stems at moderate levels, but the promoter was not active in stem pith cells, root cortical cells, or in the symbiotic zones of nodules. The ScBV promoter was active primarily in vascular tissues throughout the plant. In leaves, GUS activity driven by the CsVMV promoter was approximately 24-fold greater than the activity from the 35S promoter and 38-fold greater than the activity from the ScBV promoter. Five promoters, the double 35S promoter, figwort mosaic virus (FMV) promoter, CsVMV promoter, ScBV promoter, and alfalfa small subunit Rubisco (RbcS) promoter were used to control expression of a cDNA from Trichoderma atroviride encoding an endochitinase (ech42). Highest chitinase activity in leaves, roots, and root nodules was obtained in plants containing the CsVMV:ech42 transgene. Plants expressing the endochitinase were challenged with Phoma medicaginis var. medicaginis, the causal agent of spring black stem and leaf spot of alfalfa. Although endochitinase activity in leaves of transgenic plants was 50- to 2650-fold greater than activity in control plants, none of the transgenic plants showed a consistent increase in disease resistance compared to controls. The high constitutive levels of both GUS and endochitinase activity obtained demonstrate that the CsVMV promoter is useful for high-level transgene expression in alfalfa.

  3. Constitutive knox1 gene expression in dandelion (Taraxacum officinale, Web.) changes leaf morphology from simple to compound.

    PubMed

    Müller, Kai J; He, Xinqiang; Fischer, Rainer; Prüfer, Dirk

    2006-10-01

    Seed plants with compound leaves constitute a polyphyletic group, but studies of diverse taxa show that genes of the class 1 KNOTTED-LIKE HOMEOBOX (KNOX1) family are often involved in compound leaf development. This suggests that knox1 genes have been recruited on multiple occasions during angiosperm evolution (Bharathan et al. in Science 296:1858-1860, 2002). In agreement with this, we demonstrate that the simple leaf of dandelion (Taraxacum officinale Web.) can be converted into a compound leaf by the constitutive expression of heterologous knox1 genes. Dandelion is a rosette plant of the family Asteraceae, characterised by simple leaves with deeply lobed margins and endogenous knox1 gene expression. Transgenic dandelion plants constitutively expressing the barley (Hordeum vulgare L.) hooded gene (bkn3, barley knox3) or the related bkn1 gene, developed compound leaves featuring epiphyllous rosettes. We discuss these results in the context of two current models of compound leaf formation.

  4. Constitutive and functional expression of YB-1 in microglial cells.

    PubMed

    Keilhoff, G; Titze, M; Esser, T; Langnaese, K; Ebmeyer, U

    2015-08-20

    Y-box-binding protein (YB-1) is a member of the cold-shock protein family and participates in a wide variety of DNA/RNA-dependent cellular processes including DNA repair, transcription, mRNA splicing, packaging, and translation. At the cellular level, YB-1 is involved in cell proliferation and differentiation, stress responses, and malignant cell transformation. A general role for YB-1 during inflammation has also been well described; however, there are minimal data concerning YB-1 expression in microglia, which are the immune cells of the brain. Therefore, we studied the expression of YB-1 in a clinically relevant global ischemia model for neurological injury following cardiac arrest. This model is characterized by massive neurodegeneration of the hippocampal CA1 region and the subsequent long-lasting activation of microglia. In addition, we studied YB-1 expression in BV-2 cells, which are an accepted microglia culture model. BV-2 cells were stressed by oxygen/glucose deprivation (OGD), OGD-relevant mediators, lipopolysaccharide (LPS), and phagocytosis-inducing cell debris and nanoparticles. Using quantitative polymerase chain reaction (PCR), we show constitutive expression of YB-1 transcripts in unstressed BV-2 cells. The functional upregulation of the YB-1 protein was demonstrated in microglia in vivo and in BV-2 cells in vitro. All stressors except for LPS were potent enhancers of the level of YB-1 protein, which appears to be regulated primarily by proteasomal degradation and, to a lesser extent, by the activation (phosphorylation) of the translation initiation factor eIF4E. The proteasome of BV-2 cells is impaired by OGD, which results in decreased protein degradation and therefore increased levels of YB-1 protein. LPS induces proteasome activity, which enables the level of YB-1 protein to remain at control levels despite enhanced protein ubiquitination. The proteasome inhibitor MG-132 was able to increase YB-1 protein levels in control and LPS

  5. Constitutive heterochromatin: a surprising variety of expressed sequences.

    PubMed

    Dimitri, Patrizio; Caizzi, Ruggiero; Giordano, Ennio; Carmela Accardo, Maria; Lattanzi, Giovanna; Biamonti, Giuseppe

    2009-08-01

    The organization of chromosomes into euchromatin and heterochromatin is amongst the most important and enigmatic aspects of genome evolution. Constitutive heterochromatin is a basic yet still poorly understood component of eukaryotic chromosomes, and its molecular characterization by means of standard genomic approaches is intrinsically difficult. Although recent evidence indicates that the presence of transcribed genes in constitutive heterochromatin is a conserved trait that accompanies the evolution of eukaryotic genomes, the term heterochromatin is still considered by many as synonymous of gene silencing. In this paper, we comprehensively review data that provide a clearer picture of transcribed sequences within constitutive heterochromatin, with a special emphasis on Drosophila and humans.

  6. Trade-off between constitutive and inducible resistance against herbivores is only partially explained by gene expression and glucosinolate production

    PubMed Central

    Rasmann, Sergio; Chassin, Estelle; Bilat, Julia; Glauser, Gaétan; Reymond, Philippe

    2015-01-01

    The hypothesis that constitutive and inducible plant resistance against herbivores should trade-off because they use the same resources and impose costs to plant fitness has been postulated for a long time. Negative correlations between modes of deployment of resistance and defences have been observed across and within species in common garden experiments. It was therefore tested whether that pattern of resistance across genotypes follows a similar variation in patterns of gene expression and chemical defence production. Using the genetically tractable model Arabidopsis thaliana and different modes of induction, including the generalist herbivore Spodoptera littoralis, the specialist herbivore Pieris brassicae, and jasmonate application, constitutive and inducibility of resistance was measured across seven A. thaliana accessions that were previously selected based on constitutive levels of defence gene expression. According to theory, it was found that modes of resistance traded-off among accessions, particularly against S. littoralis, in which accessions investing in high constitutive resistance did not increase it substantially after attack and vice-versa. Accordingly, the average expression of eight genes involved in glucosinolate production negatively predicted larval growth across the seven accessions. Glucosinolate production and genes related to defence induction on healthy and herbivore-damaged plants were measured next. Surprisingly, only a partial correlation between glucosinolate production, gene expression, and the herbivore resistance results was found. These results suggest that the defence outcome of plants against herbivores goes beyond individual molecules or genes but stands on a complex network of interactions. PMID:25716695

  7. Trade-off between constitutive and inducible resistance against herbivores is only partially explained by gene expression and glucosinolate production.

    PubMed

    Rasmann, Sergio; Chassin, Estelle; Bilat, Julia; Glauser, Gaétan; Reymond, Philippe

    2015-05-01

    The hypothesis that constitutive and inducible plant resistance against herbivores should trade-off because they use the same resources and impose costs to plant fitness has been postulated for a long time. Negative correlations between modes of deployment of resistance and defences have been observed across and within species in common garden experiments. It was therefore tested whether that pattern of resistance across genotypes follows a similar variation in patterns of gene expression and chemical defence production. Using the genetically tractable model Arabidopsis thaliana and different modes of induction, including the generalist herbivore Spodoptera littoralis, the specialist herbivore Pieris brassicae, and jasmonate application, constitutive and inducibility of resistance was measured across seven A. thaliana accessions that were previously selected based on constitutive levels of defence gene expression. According to theory, it was found that modes of resistance traded-off among accessions, particularly against S. littoralis, in which accessions investing in high constitutive resistance did not increase it substantially after attack and vice-versa. Accordingly, the average expression of eight genes involved in glucosinolate production negatively predicted larval growth across the seven accessions. Glucosinolate production and genes related to defence induction on healthy and herbivore-damaged plants were measured next. Surprisingly, only a partial correlation between glucosinolate production, gene expression, and the herbivore resistance results was found. These results suggest that the defence outcome of plants against herbivores goes beyond individual molecules or genes but stands on a complex network of interactions.

  8. Constitutively expressed DHAR and MDHAR influence fruit, but not foliar ascorbate levels in tomato

    PubMed Central

    Haroldsen, Victor M.; Chi-Ham, Cecilia L.; Kulkarni, Shashank; Lorence, Argelia; Bennett, Alan B.

    2012-01-01

    Vitamin C (l-ascorbate, AsA) is an essential nutrient required in key metabolic functions in humans and must be obtained from the diet, mainly from fruits and vegetables. Given its importance in human health and plant physiology we sought to examine the role of the ascorbate recycling enzymes monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) in tomato (Solanum lycopersicum), an economically important fruit crop. Cytosolic-targeted tomato genes Mdhar and Dhar were cloned and over-expressed under a constitutive promoter in tomato var. Micro-Tom. Lines with increased protein levels and enzymatic activity were identified and examined. Mature green and red ripe fruit from DHAR over-expressing lines had a 1.6 fold increase in AsA content in plants grown under relatively low light conditions (150 µmol m−2 s−1). Conversely, MDHAR over-expressers had significantly reduced AsA levels in mature green fruits by 0.7 fold. Neither over-expressing line had altered levels of AsA in foliar tissues. These results underscore a complex regulation of the AsA pool size in tomato. PMID:21875809

  9. Field tolerance to fungal pathogens of Brassica napus constitutively expressing a chimeric chitinase gene

    SciTech Connect

    Grison, R.; Grezes-Besset, B.; Lucante, N.

    1996-05-01

    Constitutive overexpression of a protein involved in plant defense mechanisms to disease is one of the strategies proposed to increase plant tolerance to fungal pathogens. A hybrid endochitinase gene under a constitutive promoter was introduced by Agrobacterium-mediated transformation into a winter-type oilseed rape (Brassica napus var. oleifera) inbred line. Progeny from transformed plants was challenged using three different fungal pathogens (Cylindrosporium concentricum, Phoma lingam, Sclerotinia sclerotiorum) in field trials at two different geographical locations. These plants exhibited an increased tolerance to disease as compared with the nontransgenic parental plants. 31 refs., 1 fig., 2 tabs.

  10. Carbon Costs of Constitutive and Expressed Resistance to a Non-Native Pathogen in Limber Pine

    PubMed Central

    2016-01-01

    Increasing the frequency of resistance to the non-native fungus Cronartium ribicola (causative agent of white pine blister rust, WPBR) in limber pine populations is a primary management objective to sustain high-elevation forest communities. However, it is not known to what extent genetic disease resistance is costly to plant growth or carbon economy. In this study, we measured growth and leaf-level physiology in (1) seedling families from seed trees that have previously been inferred to carry or not carry Cr4, the dominant R gene allele conferring complete, gene-for-gene resistance to WPBR in limber pine, and (2) populations that were and were not infected with C. ribicola. We found that, in the absence of C. ribicola exposure, there was no significant difference in carbon relations between families born from seed trees that harbor the resistance allele compared to those that lack it, either to plant growth and phenology or leaf-level photosynthetic traits. However, post-infection with C. ribicola, growth was significantly reduced in inoculation survivors expressing complete resistance compared to uninoculated seedlings. Furthermore, inoculation survivors exhibited significant increases in a suite of traits including photosynthetic rate, respiration rate, leaf N, and stomatal conductance and a decrease in photosynthetic water-use efficiency. The lack of constitutive carbon costs associated with Cr4 resistance in non-stressed limber pine is consistent with a previous report that the R gene allele is not under selection in the absence of C. ribicola and suggests that host resistance may not bear a constitutive cost in pathosystems that have not coevolved. However, under challenge by C. ribicola, complete resistance to WPBR in limber pine has a significant cost to plant growth, though enhanced carbon acquisition post-infection may offset this somewhat. These costs and effects on performance further complicate predictions of this species’ response in warmer future

  11. Constitutive expression of high-affinity sulfate transporter (HAST) gene in Indian mustard showed enhanced sulfur uptake and assimilation.

    PubMed

    Abdin, M Z; Akmal, M; Ram, M; Nafis, T; Alam, P; Nadeem, M; Khan, M A; Ahmad, A

    2011-07-01

    Lycopersicon esculantum sulfate transporter gene (LeST 1.1) encodes a high-affinity sulfate transporter (HAST) located in root epidermis. In this study, the LeST 1.1 gene was constitutively expressed in Indian mustard (Brassica juncea cv. Pusa Jai Kisan). Transgenic as well as untransformed plants were grown in sulfur-insufficient (25 and 50 μM) and sulfur-sufficient (1,000 μM) conditions for 30 days. Two-fold increase was noticed in the sulfate uptake rate of transgenic plants grown in both sulfur-insufficient and -sufficient conditions as compared to untransformed plants. The transgenic B. juncea plants were able to accumulate higher biomass and showed improved sulfur status even in sulfur-insufficient conditions when compared with untransformed plants. Chlorophyll content, ATP sulfurylase activity and protein content were also higher in transgenic plants than untranformed plants under sulfur-insufficient conditions. Our results, thus, clearly indicate that constitutive expression of LeST 1.1 gene in B. juncea had led to enhanced capacity of sulfur uptake and assimilation even in sulfur-insufficient conditions. This approach can also be used in other crops to enhance their sulfate uptake and assimilation potential under S-insufficient conditions. PMID:20938698

  12. Constitutive high-level SOS1 expression and absence of HKT1;1 expression in the salt-accumulating halophyte Salicornia dolichostachya.

    PubMed

    Katschnig, D; Bliek, T; Rozema, J; Schat, H

    2015-05-01

    We investigated the effects of salinity on ion accumulation and expression of candidate salt tolerance genes in the highly tolerant salt accumulating halophyte Salicornia dolichostachya and the taxonomically related glycophytic Spinacia oleracea. S. dolichostachya, in comparison with S. oleracea, constitutively expressed SOS1 at a high level, but did not detectably express HKT1;1. These findings suggest that the constitutive high level of shoot salt accumulation in S. dolichostachya is accomplished through enhancement of SOS1-mediated Na(+) xylem loading, in combination with complete suppression of HKT1;1-mediated Na(+) retrieval from the xylem. Our findings demonstrate the importance of gene expression comparisons between highly tolerant halophytes and taxonomically related glycophytes to improve the understanding of mechanisms of Na(+) movement and salt tolerance in plants. PMID:25804817

  13. Functional Characterization of a Strong Bi-directional Constitutive Plant Promoter Isolated from Cotton Leaf Curl Burewala Virus

    PubMed Central

    Khan, Zainul A.; Abdin, Malik Z.; Khan, Jawaid A.

    2015-01-01

    Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells. PMID:25799504

  14. Functional characterization of a strong bi-directional constitutive plant promoter isolated from cotton leaf curl Burewala virus.

    PubMed

    Khan, Zainul A; Abdin, Malik Z; Khan, Jawaid A

    2015-01-01

    Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.

  15. Constitutive and Inducible Expression of the rRNA Methylase Gene erm(B) in Campylobacter

    PubMed Central

    Deng, Fengru; Shen, Jianzhong; Zhang, Maojun; Wu, Congming

    2015-01-01

    Macrolides are the antimicrobials of choice for treating human campylobacteriosis. The recent emergence of erm(B) in Campylobacter bacteria threatens the utility of this class of antibiotics. Here we report the constitutive and inducible expression of erm(B) in Campylobacter isolates derived from diarrheal patients and food-producing animals. Constitutive expression of erm(B) was associated with insertion and deletion in the regulatory region of the gene, providing the first documentation of the differential expression of erm(B) in Campylobacter bacteria. PMID:26259800

  16. Pineapple translation factor SUI1 and ribosomal protein L36 promoters drive constitutive transgene expression patterns in Arabidopsis thaliana.

    PubMed

    Koia, Jonni; Moyle, Richard; Hendry, Caroline; Lim, Lionel; Botella, José Ramón

    2013-03-01

    The availability of a variety of promoter sequences is necessary for the genetic engineering of plants, in basic research studies and for the development of transgenic crops. In this study, the promoter and 5' untranslated regions of the evolutionally conserved protein translation factor SUI1 gene and ribosomal protein L36 gene were isolated from pineapple and sequenced. Each promoter was translationally fused to the GUS reporter gene and transformed into the heterologous plant system Arabidopsis thaliana. Both the pineapple SUI1 and L36 promoters drove GUS expression in all tissues of Arabidopsis at levels comparable to the CaMV35S promoter. Transient assays determined that the pineapple SUI1 promoter also drove GUS expression in a variety of climacteric and non-climacteric fruit species. Thus the pineapple SUI1 and L36 promoters demonstrate the potential for using translation factor and ribosomal protein genes as a source of promoter sequences that can drive constitutive transgene expression patterns.

  17. Constitutive expression of pathogenesis-related proteins PR-1, GRP, and PR-S in tobacco has no effect on virus infection.

    PubMed

    Linthorst, H J; Meuwissen, R L; Kauffmann, S; Bol, J F

    1989-03-01

    Samsun NN tobacco cells were transformed with chimeric genes for pathogenesis-related (PR) proteins derived from genomic (PR-1a, GRP) or cDNA (PR-S) clones under the transcriptional control of the cauliflower mosaic virus 35S promoter. Regenerated plants were assayed by RNA and protein gel blotting, and plants showing high specific expression of the inserted genes were selected for self-pollination and seed formation. Inspection of second generation transformants showed that constitutive expression of PR-1a, GRP, and PR-S in tobacco in general does not have an effect on the phenotypic appearance of the plants or the expression of other endogenous PR genes. Furthermore, constitutive expression of the above genes does not affect the susceptibility of the plants to infection with tobacco mosaic virus or alfalfa mosaic virus.

  18. Inducible and constitutive expression of an elicitor gene Hrip1 from Alternaria tenuissima enhances stress tolerance in Arabidopsis.

    PubMed

    Peng, Xue-Cong; Qiu, De-Wen; Zeng, Hong-Mei; Guo, Li-Hua; Yang, Xiu-Fen; Liu, Zheng

    2015-02-01

    Hrip1 is a novel hypersensitive response-inducing protein secreted by Alternaria tenuissima that activates defense responses and systemic acquired resistance in tobacco. This study investigates the role that Hrip1 plays in responses to abiotic and biotic stress using transgenic Arabidopsis thaliana expressing the Hrip1 gene under the control of the stress-inducible rd29A promoter or constitutive cauliflower mosaic virus 35S promoter. Bioassays showed that inducible Hrip1 expression in rd29A∷Hrip1 transgenic lines had a significantly higher effect on plant height, silique length, plant dry weight, seed germination and root length under salt and drought stress compared to expression in 35S∷Hrip1 lines and wild type plants. The level of enhancement of resistance to Botrytis cinerea by the 35S∷Hrip1 lines was higher than in the rd29A∷Hrip1 lines. Moreover, stress-related gene expression in the transgenic Arabidopsis lines was significantly increased by 200 mM NaCl and 200 mM mannitol treatments, and defense genes in the jasmonic acid and ethylene signaling pathway were significantly up-regulated after Botrytis inoculation in the Hrip1 transgenic plants. Furthermore, the activity of some antioxidant enzymes, such as peroxidase and catalase increased after salt and drought stress and Botrytis infection. These results suggested that the Hrip1 protein contributes to abiotic and biotic resistance in transgenic Arabidopsis and may be used as a useful gene for resistance breeding in crops. Although the constitutive expression of Hrip1 is suitable for biotic resistance, inducible Hrip1 expression is more responsive for abiotic resistance.

  19. Constitutive S-adenosylmethionine decarboxylase gene expression increases drought tolerance through inhibition of reactive oxygen species accumulation in Arabidopsis.

    PubMed

    Wi, Soo Jin; Kim, Soo Jin; Kim, Woo Taek; Park, Ky Young

    2014-05-01

    Using subtractive hybridization analysis, the S-adenosylmethionine decarboxylase (SAMDC) gene from Capsicum annuum was isolated and renamed CaSAMDC. We generated independent transgenic Arabidopsis (Arabidopsis thaliana) lines constitutively expressing a 35S::CaSAMDC construct. Drought tolerance was significantly enhanced in Arabidopsis T4 transgenic homozygous lines as compared to wild-type (WT) plants. The levels of main polyamines (PAs) were more significantly increased in CaSAMDC-overexpressing transgenic plants after 6 h of drought stress as compared to stressed WT plants. Basal transcription of polyamine oxidase (PAO) showed at a much higher level in unstressed-transgenic plants as compared to unstressed WT plants. However, the difference in PAO transcription level between WT and transgenic plants was reduced after drought stress. Cellular accumulation of reactive oxygen species (ROS) was significantly reduced following drought stress in transgenic Arabidopsis plants as compared to WT plants. These results were in agreement with additional observations that stress-induced ROS generation, as determined by qRT-PCR analysis of NADPH oxidase (RbohD and RbohF), was significantly suppressed while transcription of ROS-detoxifying enzymes was notably elevated in transgenic lines in response to drought stress. Further, ROS-induced transcription of the metacaspase II gene was remarkably inhibited in transgenic plants. Collectively, these results suggest that drought stress tolerance due to reduction of ROS production and enhancement of ROS detoxification can be attributed to elevation of PAs.

  20. Constitutive Expresser of Pathogenesis Related Genes 1 Is Required for Pavement Cell Morphogenesis in Arabidopsis.

    PubMed

    Han, Bing; Chen, Liang; Wang, Jing; Wu, Zhongliang; Yan, Longfeng; Hou, Suiwen

    2015-01-01

    For over 50 years, researchers have focused on the mechanisms underlying the important roles of the cytoskeleton in controlling the cell growth direction and cell expansion. In our study, we performed ethyl methane sulfonate mutagenesis on Col-0 background and identified two new CONSTITUTIVE EXPRESSER OF PATHOGENESIS RELATED GENES 1 (CPR1) alleles with pavement cell (PC) morphogenetic defects. Morphological characterizations showed that polar growth initiation and expansion of PCs are seriously suppressed in cpr1. Closer cytoskeleton investigation showed that the directional arrangement of microtubules (MTs) during PC development is defective and the cortical fine actin filaments cannot be aggregated effectively to form actin cable networks in cpr1 mutants. These results suggest that the abnormal PC morphogenesis in cpr1 is accompanying with the aberrant arrangement of cytoskeleton. Site-directed mutagenesis and knockout within the F-box-associated (FBA) domain, which is reported to be a motif for recognizing particular substrates of CPR1, proved that the FBA domain is indispensable for normal CPR1 regulation of the PC morphogenesis. Further genetic analysis indicated that the defects on PC morphogenesis of cpr1 depend on two lipase-like proteins, ENHANCED DISEASE SUSCEPTIBILITY 1 and PHYTOALEXIN DEFICIENT 4. Our results provide further insights into the relationship between the cytoskeleton and PC morphogenesis, and suggest that the cytoskeleton-mediated PC morphogenesis control might be tightly linked to plant defense responses. PMID:26193674

  1. MiniTn7-transposon delivery vectors for inducible or constitutive fluorescent protein expression in Enterobacteriaceae

    PubMed Central

    Remus-Emsermann, Mitja N. P.; Gisler, Pascal; Drissner, David

    2016-01-01

    Here we present the generation and function of two sets of bacterial plasmids that harbor fluorescent genes encoding either blue, cyan, yellow or red fluorescent proteins. In the first set, protein expression is controlled by the strong and constitutive nptII promoter whereas in the second set, the strong tac promoter was chosen that underlies LacIq regulation. Furthermore, the plasmids are mobilizable, contain Tn7 transposons and a temperature-sensitive origin of replication. Using Escherichia coli S17-1 as donor strain, the plasmids allow fast and convenient Tn7-transposon delivery into many enterobacterial hosts, such as the here-used E. coli O157:H7. This procedure omits the need of preparing competent recipient cells and antibiotic resistances are only transiently conferred to the recipients. As the fluorescence proteins show little to no overlap in fluorescence emission, the constructs are well suited for the study of multicolored synthetic bacterial communities during biofilm production or in host colonization studies, e.g. of plant surfaces. Furthermore, tac promoter-reporter constructs allow the generation of so-called reproductive success reporters, which allow to estimate past doublings of bacterial individuals after introduction into environments, emphasizing the role of individual cells during colonization. PMID:27445318

  2. Constitutive Expresser of Pathogenesis Related Genes 1 Is Required for Pavement Cell Morphogenesis in Arabidopsis.

    PubMed

    Han, Bing; Chen, Liang; Wang, Jing; Wu, Zhongliang; Yan, Longfeng; Hou, Suiwen

    2015-01-01

    For over 50 years, researchers have focused on the mechanisms underlying the important roles of the cytoskeleton in controlling the cell growth direction and cell expansion. In our study, we performed ethyl methane sulfonate mutagenesis on Col-0 background and identified two new CONSTITUTIVE EXPRESSER OF PATHOGENESIS RELATED GENES 1 (CPR1) alleles with pavement cell (PC) morphogenetic defects. Morphological characterizations showed that polar growth initiation and expansion of PCs are seriously suppressed in cpr1. Closer cytoskeleton investigation showed that the directional arrangement of microtubules (MTs) during PC development is defective and the cortical fine actin filaments cannot be aggregated effectively to form actin cable networks in cpr1 mutants. These results suggest that the abnormal PC morphogenesis in cpr1 is accompanying with the aberrant arrangement of cytoskeleton. Site-directed mutagenesis and knockout within the F-box-associated (FBA) domain, which is reported to be a motif for recognizing particular substrates of CPR1, proved that the FBA domain is indispensable for normal CPR1 regulation of the PC morphogenesis. Further genetic analysis indicated that the defects on PC morphogenesis of cpr1 depend on two lipase-like proteins, ENHANCED DISEASE SUSCEPTIBILITY 1 and PHYTOALEXIN DEFICIENT 4. Our results provide further insights into the relationship between the cytoskeleton and PC morphogenesis, and suggest that the cytoskeleton-mediated PC morphogenesis control might be tightly linked to plant defense responses.

  3. α(1D)-Adrenergic receptors constitutive activity and reduced expression at the plasma membrane.

    PubMed

    García-Sáinz, J Adolfo; Romero-Ávila, M Teresa; Medina, Luz Del Carmen

    2010-01-01

    Adrenergic receptors are a heterogeneous family of the G protein-coupled receptors that mediate the actions of adrenaline and noradrenaline. Adrenergic receptors comprise three subfamilies (α(1), α(2), and β, with three members each) and the α(1D)-adrenergic receptor is one of the members of the α(1) subfamily with some interesting traits. The α(1D)-adrenergic receptor is difficult to express, seems predominantly located intracellularly, and exhibits constitutive activity. In this chapter, we will describe in detail the conditions and procedures used to determine changes in intracellular free calcium concentration which has been instrumental to define the constitutive activity of these receptors. Taking advantage of the fact that truncation of the first 79 amino acids of α(1D)-adrenergic receptors markedly increased their membrane expression, we were able to show that constitutive activity is present in receptors truncated at the amino and carboxyl termini, which indicates that such domains are dispensable for this action. Constitutive activity could be observed in cells expressing either the rat or human α(1D)-adrenergic receptor orthologs. Such constitutive activity has been observed in native rat arteries and we will discuss the possible functional implications that it might have in the regulation of blood pressure.

  4. Fundamental role for HIF-1α in constitutive expression of human β defensin-1.

    PubMed

    Kelly, C J; Glover, L E; Campbell, E L; Kominsky, D J; Ehrentraut, S F; Bowers, B E; Bayless, A J; Saeedi, B J; Colgan, S P

    2013-11-01

    Antimicrobial peptides are secreted by the intestinal epithelium to defend from microbial threats. The role of human β defensin-1 (hBD-1) is notable because its gene (beta-defensin 1 (DEFB1)) is constitutively expressed and its antimicrobial activity is potentiated in the low-oxygen environment that characterizes the intestinal mucosa. Hypoxia-inducible factor (HIF) is stabilized even in healthy intestinal mucosa, and we identified that epithelial HIF-1α maintains expression of murine defensins. Extension to a human model revealed that basal HIF-1α is critical for the constitutive expression of hBD-1. Chromatin immunoprecipitation identified HIF-1α binding to a hypoxia response element in the DEFB1 promoter whose importance was confirmed by site-directed mutagenesis. We used 94 human intestinal samples to identify a strong expression correlation between DEFB1 and the canonical HIF-1α target GLUT1. These findings indicate that basal HIF-1α is critical for constitutive expression of enteric DEFB1 and support targeting epithelial HIF for restoration and maintenance of intestinal integrity.

  5. Construction of vectors for inducible and constitutive gene expression in Lactobacillus.

    PubMed

    Duong, Tri; Miller, Michael J; Barrangou, Rodolphe; Azcarate-Peril, M Andrea; Klaenhammer, Todd R

    2011-05-01

    Microarray analysis of the genome of Lactobacillus acidophilus identified a number of operons that were differentially expressed in response to carbohydrate source or constitutively expressed regardless of carbohydrate source. These included operons implicated in the transport and catabolism of fructooligosaccharides (FOS), lactose (lac), trehalose (tre) and genes directing glycolysis. Analysis of these operons identified a number of putative promoter and repressor elements, which were used to construct a series of expression vectors for use in lactobacilli, based on the broad host range pWV01 replicon. A β-glucuronidase (GusA3) reporter gene was cloned into each vector to characterize expression from each promoter. GUS reporter assays showed FOS, lac and tre based vectors to be highly inducible by their specific carbohydrate and repressed by glucose. Additionally, a construct based on the phosphoglycerate mutase (pgm) promoter was constitutively highly expressed. To demonstrate the potential utility of these vectors, we constructed a plasmid for the overexpression of the oxalate degradation pathway (Frc and Oxc) of L. acidophilus NCFM. This construct was able to improve oxalate degradation by L. gasseri ATCC 33323 and compliment a L. acidophilus oxalate-deficient mutant. Development of these expression vectors could support several novel applications, including the expression of enzymes, proteins, vaccines and biotherapeutics by intestinal lactobacilli. PMID:21375708

  6. Construction of vectors for inducible and constitutive gene expression in Lactobacillus

    PubMed Central

    Duong, Tri; Miller, Michael J.; Barrangou, Rodolphe; Azcarate‐Peril, M. Andrea; Klaenhammer, Todd R.

    2011-01-01

    Summary Microarray analysis of the genome of Lactobacillus acidophilus identified a number of operons that were differentially expressed in response to carbohydrate source or constitutively expressed regardless of carbohydrate source. These included operons implicated in the transport and catabolism of fructooligosaccharides (FOS), lactose (lac), trehalose (tre) and genes directing glycolysis. Analysis of these operons identified a number of putative promoter and repressor elements, which were used to construct a series of expression vectors for use in lactobacilli, based on the broad host range pWV01 replicon. A β‐glucuronidase (GusA3) reporter gene was cloned into each vector to characterize expression from each promoter. GUS reporter assays showed FOS, lac and tre based vectors to be highly inducible by their specific carbohydrate and repressed by glucose. Additionally, a construct based on the phosphoglycerate mutase (pgm) promoter was constitutively highly expressed. To demonstrate the potential utility of these vectors, we constructed a plasmid for the overexpression of the oxalate degradation pathway (Frc and Oxc) of L. acidophilus NCFM. This construct was able to improve oxalate degradation by L. gasseri ATCC 33323 and compliment a L. acidophilus oxalate‐deficient mutant. Development of these expression vectors could support several novel applications, including the expression of enzymes, proteins, vaccines and biotherapeutics by intestinal lactobacilli. PMID:21375708

  7. Assessment of Heat Shock Protein 70 Induction by Heat in Alfalfa Varieties and Constitutive Overexpression in Transgenic Plants

    PubMed Central

    Ferradini, Nicoletta; Iannacone, Rina; Capomaccio, Stefano; Metelli, Alessandra; Armentano, Nadia; Semeraro, Lucia; Cellini, Francesco; Veronesi, Fabio; Rosellini, Daniele

    2015-01-01

    Heat shock proteins (HSPs) are molecular chaperones involved in many cellular functions. It has been shown that mammalian cytosolic HSP70 binds antigenic peptides mediating the activation of the immune system, and that it plays a determining role in tumour immunogenicity. This suggests that HSP70 may be used for the production of conjugated vaccines. Human and plant HSPs share high sequence similarity and some important biological functions in vitro. In addition, plant HSPs have no endotoxic side effects. Extraction of HSP70 from plants for use as vaccine adjuvant requires enhancing its concentration in plant tissues. In this work, we explored the possibility to produce HSP70 in both transgenic and non-transgenic plants, using alfalfa as a model species. First, a transcriptional analysis of a constitutive and an inducible HSP70 genes was conducted in Arabidopsis thaliana. Then the coding sequence of the inducible form was cloned and introduced into alfalfa by Agrobacterium-mediated transformation, and the accumulation of the protein in leaf tissue of transgenic plants was demonstrated. We also tested diverse alfalfa varieties for heat-inducible expression of endogenous HSP70, revealing variety-specific responses to heat shock. PMID:25951604

  8. Assessment of heat shock protein 70 induction by heat in alfalfa varieties and constitutive overexpression in transgenic plants.

    PubMed

    Ferradini, Nicoletta; Iannacone, Rina; Capomaccio, Stefano; Metelli, Alessandra; Armentano, Nadia; Semeraro, Lucia; Cellini, Francesco; Veronesi, Fabio; Rosellini, Daniele

    2015-01-01

    Heat shock proteins (HSPs) are molecular chaperones involved in many cellular functions. It has been shown that mammalian cytosolic HSP70 binds antigenic peptides mediating the activation of the immune system, and that it plays a determining role in tumour immunogenicity. This suggests that HSP70 may be used for the production of conjugated vaccines. Human and plant HSPs share high sequence similarity and some important biological functions in vitro. In addition, plant HSPs have no endotoxic side effects. Extraction of HSP70 from plants for use as vaccine adjuvant requires enhancing its concentration in plant tissues. In this work, we explored the possibility to produce HSP70 in both transgenic and non-transgenic plants, using alfalfa as a model species. First, a transcriptional analysis of a constitutive and an inducible HSP70 genes was conducted in Arabidopsis thaliana. Then the coding sequence of the inducible form was cloned and introduced into alfalfa by Agrobacterium-mediated transformation, and the accumulation of the protein in leaf tissue of transgenic plants was demonstrated. We also tested diverse alfalfa varieties for heat-inducible expression of endogenous HSP70, revealing variety-specific responses to heat shock. PMID:25951604

  9. Assessment of heat shock protein 70 induction by heat in alfalfa varieties and constitutive overexpression in transgenic plants.

    PubMed

    Ferradini, Nicoletta; Iannacone, Rina; Capomaccio, Stefano; Metelli, Alessandra; Armentano, Nadia; Semeraro, Lucia; Cellini, Francesco; Veronesi, Fabio; Rosellini, Daniele

    2015-01-01

    Heat shock proteins (HSPs) are molecular chaperones involved in many cellular functions. It has been shown that mammalian cytosolic HSP70 binds antigenic peptides mediating the activation of the immune system, and that it plays a determining role in tumour immunogenicity. This suggests that HSP70 may be used for the production of conjugated vaccines. Human and plant HSPs share high sequence similarity and some important biological functions in vitro. In addition, plant HSPs have no endotoxic side effects. Extraction of HSP70 from plants for use as vaccine adjuvant requires enhancing its concentration in plant tissues. In this work, we explored the possibility to produce HSP70 in both transgenic and non-transgenic plants, using alfalfa as a model species. First, a transcriptional analysis of a constitutive and an inducible HSP70 genes was conducted in Arabidopsis thaliana. Then the coding sequence of the inducible form was cloned and introduced into alfalfa by Agrobacterium-mediated transformation, and the accumulation of the protein in leaf tissue of transgenic plants was demonstrated. We also tested diverse alfalfa varieties for heat-inducible expression of endogenous HSP70, revealing variety-specific responses to heat shock.

  10. A constitutive promoter directs expression of the nerve growth factor receptor gene

    SciTech Connect

    Sehgal, A.; Patil, N.; Chao, M.

    1988-08-01

    Expression of nerve growth factor receptor is normally restricted to cells derived from the neural crest in a developmentally regulated manner. The authors analyzed promoter sequences for the human nerve growth factor receptor gene and found that the receptor promoter resembles others which are associated with constitutively expressed genes that have housekeeping and growth-related functions. Unlike these other genes, the initiation of transcription occurred at one major site rather than at multiple sites. The constitutive nature of the nerve growth factor receptor promoter may account for the ability of this gene to be transcribed in a diverse number of heterologous cells after gene transfer. The intron-exon structure of the receptor gene indicated that structural features are precisely divided into discrete domains.

  11. Spatial and developmental profiling of miraculin accumulation in transgenic tomato fruits expressing the miraculin gene constitutively.

    PubMed

    Kim, You-Wang; Kato, Kazuhisa; Hirai, Tadayoshi; Hiwasa-Tanase, Kyoko; Ezura, Hiroshi

    2010-01-13

    We previously developed a transgenic tomato that expresses the miraculin gene using a constitutive promoter. In this study, we profiled the developmental and spatial accumulation of the miraculin protein and mRNA in transgenic tomato fruits. Miraculin mRNA expression was almost constant up to orange stage, and then the expression increased at red stage. The miraculin protein accumulated gradually during fruit development and reached its highest level at the overripe stage. At the red stage of fruit, miraculin protein was accumulated at the highest level in the exocarp, and similar in other fruit tissues: mesocarp, dissepiment, upper placenta, lower placenta and jelly. Moreover, the pattern of miraculin accumulation in fruit tissues was the same regardless of genetic background and position at which the miraculin gene was inserted in the genome. We also discuss suitable tomato types expressing miraculin for their commercial use. PMID:20014854

  12. Enhancing Antitumor Efficacy of Chimeric Antigen Receptor T Cells Through Constitutive CD40L Expression

    PubMed Central

    Curran, Kevin J; Seinstra, Beatrijs A; Nikhamin, Yan; Yeh, Raymond; Usachenko, Yelena; van Leeuwen, Dayenne G; Purdon, Terence; Pegram, Hollie J; Brentjens, Renier J

    2015-01-01

    Adoptive cell therapy with genetically modified T cells expressing a chimeric antigen receptor (CAR) is a promising therapy for patients with B-cell acute lymphoblastic leukemia. However, CAR-modified T cells (CAR T cells) have mostly failed in patients with solid tumors or low-grade B-cell malignancies including chronic lymphocytic leukemia with bulky lymph node involvement. Herein, we enhance the antitumor efficacy of CAR T cells through the constitutive expression of CD40 ligand (CD40L, CD154). T cells genetically modified to constitutively express CD40L (CD40L-modified T cells) demonstrated increased proliferation and secretion of proinflammatory TH1 cytokines. Further, CD40L-modified T cells augmented the immunogenicity of CD40+ tumor cells by the upregulated surface expression of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58, and CD70), human leukocyte antigen (HLA) molecules (Class I and HLA-DR), and the Fas-death receptor (CD95). Additionally, CD40L-modified T cells induced maturation and secretion of the proinflammatory cytokine interleukin-12 by monocyte-derived dendritic cells. Finally, tumor-targeted CD19-specific CAR/CD40L T cells exhibited increased cytotoxicity against CD40+ tumors and extended the survival of tumor-bearing mice in a xenotransplant model of CD19+ systemic lymphoma. This preclinical data supports the clinical application of CAR T cells additionally modified to constitutively express CD40L with anticipated enhanced antitumor efficacy. PMID:25582824

  13. Constitutive type VI secretion system expression gives Vibrio cholerae intra- and interspecific competitive advantages.

    PubMed

    Unterweger, Daniel; Kitaoka, Maya; Miyata, Sarah T; Bachmann, Verena; Brooks, Teresa M; Moloney, Jessica; Sosa, Oscar; Silva, David; Duran-Gonzalez, Jorge; Provenzano, Daniele; Pukatzki, Stefan

    2012-01-01

    The type VI secretion system (T6SS) mediates protein translocation across the cell membrane of Gram-negative bacteria, including Vibrio cholerae - the causative agent of cholera. All V. cholerae strains examined to date harbor gene clusters encoding a T6SS. Structural similarity and sequence homology between components of the T6SS and the T4 bacteriophage cell-puncturing device suggest that the T6SS functions as a contractile molecular syringe to inject effector molecules into prokaryotic and eukaryotic target cells. Regulation of the T6SS is critical. A subset of V. cholerae strains, including the clinical O37 serogroup strain V52, express T6SS constitutively. In contrast, pandemic strains impose tight control that can be genetically disrupted: mutations in the quorum sensing gene luxO and the newly described regulator gene tsrA lead to constitutive T6SS expression in the El Tor strain C6706. In this report, we examined environmental V. cholerae isolates from the Rio Grande with regard to T6SS regulation. Rough V. cholerae lacking O-antigen carried a nonsense mutation in the gene encoding the global T6SS regulator VasH and did not display virulent behavior towards Escherichia coli and other environmental bacteria. In contrast, smooth V. cholerae strains engaged constitutively in type VI-mediated secretion and displayed virulence towards prokaryotes (E. coli and other environmental bacteria) and a eukaryote (the social amoeba Dictyostelium discoideum). Furthermore, smooth V. cholerae strains were able to outcompete each other in a T6SS-dependent manner. The work presented here suggests that constitutive T6SS expression provides V. cholerae with an advantage in intraspecific and interspecific competition.

  14. CT-2576, an inhibitor of phospholipid signaling, suppresses constitutive and induced expression of human immunodeficiency virus.

    PubMed Central

    Leung, D W; Peterson, P K; Weeks, R; Gekker, G; Chao, C C; Kaplan, A H; Balantac, N; Tompkins, C; Underiner, G E; Bursten, S

    1995-01-01

    Viruses such as human immunodeficiency virus (HIV) require cellular activation for expression. Cellular activation in lymphoid cells is associated with augmented accumulation of certain phosphatidic acid (PA) species derived from the hydrolysis of glycan phosphatidylinositol (GPI). This suggests that activation of a phospholipid pathway may play a role in initiation of viral replication. To test this hypothesis, we examined the effect of tat gene expression on the production of cellular PA species, as the Tat protein is essential for HIV expression and has been implicated in activating the expression of multiple host cellular genes. Expression of tat increased the expression of PA. We then tested whether synthetic inhibitors of PA metabolism would inhibit activation of the HIV long terminal repeat by Tat and tumor necrosis factor alpha (TNF-alpha). CT-2576 suppressed both PA generation induced by Tat and HIV long terminal repeat-directed gene expression in response to Tat or TNF-alpha at a posttranscriptional step. CT-2576 also inhibited constitutive as well as TNF-alpha- and interleukin 6-induced expression of HIV p24 antigen in chronically infected U1 cells and in peripheral blood lymphocytes acutely infected with a clinical isolate of HIV. Pharmacological inhibition of synthesis of selected PA species may therefore provide a therapeutic approach to suppression of HIV replication. Images Fig. 1 Fig. 3 Fig. 5 PMID:7761405

  15. Constitutive GABA expression via a recombinant adeno-associated virus consistently attenuates neuropathic pain.

    PubMed

    Lee, Boyoung; Kim, Jaehyung; Kim, Sung Jin; Lee, Heuiran; Chang, Jin Woo

    2007-06-15

    Peripheral neuropathic pain is a common clinical problem with few existing treatments. Previously, we constructed rAAV bearing GAD65 and demonstrated that GAD65 and GABA can be constitutively produced in the CNS. To investigate the beneficial effects of GAD65 produced by rAAV and resulting GABA release in peripheral neuropathic pain, we established a neuropathic pain rat model. The direct administration of rAAV-GAD65 to dorsal root ganglion induced constitutive GAD65 expression, which was readily detected by immunohistochemistry. Both allodynic and hyperalgeic behavior tests suggested that neuropathic pain was noticeably reduced, along with the transgenic GAD65 expression. Moreover, the magnitude of pain relief was maintained during the entire experimental period. Concomitantly, the significant enhancement in GABA release following transgenic GAD65 expression was identified in vivo. Taken all together, these results provide evidence that persistent GAD65 and subsequent GABA expression in DRGs via rAAV effectively attenuates peripheral neuropathic pain for long period of time.

  16. T Cells Expressing Constitutively Active Akt Resist Multiple Tumor-associated Inhibitory Mechanisms

    PubMed Central

    Sun, Jiali; Dotti, Gianpietro; Huye, Leslie E; Foster, Aaron E; Savoldo, Barbara; Gramatges, Maria M; Spencer, David M; Rooney, Cliona M

    2010-01-01

    Adoptive transfer of antigen-specific cytotoxic T lymphocytes has shown promise for the therapy of cancer. However, tumor-specific T cells are susceptible to diverse inhibitory signals from the tumor microenvironment. The Akt/protein kinase B plays a central role in T-cell proliferation, function, and survival and we hypothesized that expression of constitutively active Akt (caAkt) in T cells could provide resistance to many of these tumor-associated inhibitory mechanisms. caAkt expression in activated human T cells increased proliferation and cytokine production, a likely result of their sustained expression of nuclear factor-κB (NF-κB) and provided resistance to apoptosis by upregulating antiapoptotic molecules. caAkt expressing T cells (caAkt-T-cells) were also relatively resistant to suppression by and conversion into regulatory T cells (Tregs). These characteristics provided a survival advantage to T cells cocultured with tumor cells in vitro; CD3/28-stimulated T cells expressing a chimeric antigen receptor (CAR) specific for disialoganglioside (GD2) that redirected their activity to the immunosuppressive, GD2-expressing neuroblastoma cell line, LAN-1, resisted tumor-induced apoptosis when co-expressing transgenic caAkt. In conclusion, caAkt-transduced T cells showed resistance to several evasion strategies employed by tumors and may therefore enhance the antitumor activity of adoptively transferred T lymphocytes. PMID:20842106

  17. Efficient production and evaluation of lignocellulolytic enzymes using a constitutive protein expression system in Penicillium oxalicum.

    PubMed

    Hu, Yibo; Xue, Haizhao; Liu, Guodong; Song, Xin; Qu, Yinbo

    2015-06-01

    Native lignocellulolytic enzyme systems secreted by filamentous fungi can be further optimized by protein engineering or supplementation of exogenous enzyme components. We developed a protein production and evaluation system in cellulase-producing fungus Penicillium oxalicum. First, by deleting the major amylase gene amy15A, a strain Δ15A producing few extracellular proteins on starch was constructed. Then, three lignocellulolytic enzymes (BGL4, Xyn10B, and Cel12A) with originally low expression levels were successfully expressed with selected constitutive promoters in strain Δ15A. BGL4 and Cel12A overexpression resulted in increased specific filter paper activity (FPA), while the overexpression of Xyn10B improved volumetric FPA but not specific FPA. By switching the culture medium, this platform is convenient to produce originally low-expressed lignocellulolytic enzymes in relatively high purities on starch and to evaluate the effect of their supplementation on the performance of a complex cellulase system on cellulose.

  18. Human CD4+ CD25+ Foxp3+ regulatory T cells do not constitutively express IL-35.

    PubMed

    Bardel, Emilie; Larousserie, Frédérique; Charlot-Rabiega, Pascaline; Coulomb-L'Herminé, Aurore; Devergne, Odile

    2008-11-15

    EBV-induced gene 3 (EBI3) can associate with p28 to form the heterodimeric cytokine IL-27, or with the p35 subunit of IL-12 to form the EBI3/p35 heterodimer, recently named IL-35. In mice, IL-35 has been shown to be constitutively expressed by CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg cells) and suggested to contribute to their suppressive activity. In this study, we investigated whether human Treg cells express IL-35. Double-staining analysis of human thymuses showed that neither Foxp3(+) nor CD25(+) cells coexpressed EBI3. Similarly, Foxp3(+) cells present in human lymph nodes, tonsils, spleens, and intestines did not express EBI3. Consistent with these in situ observations, Treg cells purified from blood or tonsils were negative for EBI3 by immunoblotting. Other human T cell subsets, including effector T cells, naive and memory CD4(+) T cells, CD8(+) and gammadelta T cells also did not constitutively express EBI3, which contrasts with IL-35 expression observed in murine CD8(+) and gammadelta T cells. Furthermore, although CD3/CD28 stimulation consistently induced low levels of EBI3 in various CD4(+) T cell subsets, no EBI3 could be detected in CD3/CD28-stimulated Treg cells. RT-PCR analysis showed that, whereas p35 transcripts were detected in both Teff and Treg cells, EBI3 transcripts were detected only in activated Teff cells, but not in resting or activated Treg cells. Thus, in contrast to their murine counterpart, human Treg cells do not express detectable amounts of IL-35.

  19. Cloning and constitutive expression of Deschampsia antarctica Cu/Zn superoxide dismutase in Pichia pastoris

    PubMed Central

    Sánchez-Venegas, Jaime R; Navarrete, Alejandro; Dinamarca, Jorge; Bravo Ramírez, León A; Moraga, Ana Gutiérrez; Gidekel, Manuel

    2009-01-01

    Background Deschampsia antarctica shows tolerance to extreme environmental factors such as low temperature, high light intensity and an increasing UV radiation as result of the Antarctic ozone layer thinning. It is very likely that the survival of this species is due to the expression of genes that enable it to tolerate high levels of oxidative stress. On that account, we planned to clone the D. antarctica Cu/ZnSOD gene into Pichia pastoris and to characterize the heterologous protein. Findings The Copper/Zinc superoxide dismutase (Cu/ZnSOD) gene, SOD gene, was isolated from a D. antarctica by cDNA library screening. This SOD gene was cloned in the expression vector pGAPZαA and successfully integrated into the genome of the yeast P. pastoris SMD1168H. A constitutive expression system for the expression of the recombinant SOD protein was used. The recombinant protein was secreted into the YPD culture medium as a glycosylated protein with a 32 mg/l expression yield. The purified recombinant protein possesses a specific activity of 440 U/mg. Conclusion D. antarctica Cu/ZnSOD recombinant protein was expressed in a constitutive system, and purified in a single step by means of an affinity column. The recombinant SOD was secreted to the culture medium as a glycoprotein, corresponding to approximately 13% of the total secreted protein. The recombinant protein Cu/ZnSOD maintains 60% of its activity after incubation at 40°C for 30 minutes and it is stable (80% of activity) between -20°C and 20°C. The recombinant SOD described in this study can be used in various biotechnological applications. PMID:19821975

  20. Conditional expression of constitutively active estrogen receptor {alpha} in chondrocytes impairs longitudinal bone growth in mice

    SciTech Connect

    Ikeda, Kazuhiro; Tsukui, Tohru; Imazawa, Yukiko; Horie-Inoue, Kuniko; Inoue, Satoshi

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Conditional transgenic mice expressing constitutively active estrogen receptor {alpha} (caER{alpha}) in chondrocytes were developed. Black-Right-Pointing-Pointer Expression of caER{alpha} in chondrocytes impaired longitudinal bone growth in mice. Black-Right-Pointing-Pointer caER{alpha} affects chondrocyte proliferation and differentiation. Black-Right-Pointing-Pointer This mouse model is useful for understanding the physiological role of ER{alpha}in vivo. -- Abstract: Estrogen plays important roles in the regulation of chondrocyte proliferation and differentiation, which are essential steps for longitudinal bone growth; however, the mechanisms of estrogen action on chondrocytes have not been fully elucidated. In the present study, we generated conditional transgenic mice, designated as caER{alpha}{sup ColII}, expressing constitutively active mutant estrogen receptor (ER) {alpha} in chondrocytes, using the chondrocyte-specific type II collagen promoter-driven Cre transgenic mice. caER{alpha}{sup ColII} mice showed retardation in longitudinal growth, with short bone lengths. BrdU labeling showed reduced proliferation of hypertrophic chondrocytes in the proliferating layer of the growth plate of tibia in caER{alpha}{sup ColII} mice. In situ hybridization analysis of type X collagen revealed that the maturation of hypertrophic chondrocytes was impaired in caER{alpha}{sup ColII} mice. These results suggest that ER{alpha} is a critical regulator of chondrocyte proliferation and maturation during skeletal development, mediating longitudinal bone growth in vivo.

  1. Constitutive expression and structural diversity of inducible isoform of nitric oxide synthase in human tissues.

    PubMed

    Park, C S; Park, R; Krishna, G

    1996-01-01

    Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) plays a major role in immune responses to bacteria and tumors, and the gene is induced by endotoxin and cytokines. However, we have detected iNOS cDNA sequences expressed constitutively at low level from human retinal, cerebellar and skeletal muscle tissues using northern-blot and RT-PCR analyses. In northern-blot analysis, two types (4.5 kb and 4.2 kb) of iNOS mRNA have been observed in retinal tissue, whereas only one type of mRNA was observed in cerebellum (4.5 kb) and skeletal muscle (4.2 kb). This result indicates that the presence of differential expression and/or structural diversity of the iNOS gene in various tissues, and some cells can express iNOS gene constitutively. We have also demonstrated a structural diversity formed by alternative splicing in the open reading frame sequence of the iNOS cDNA cloned from retinal tissue, which may reflect functional differences of iNOS gene.

  2. A minimal regulatory region maintains constitutive expression of the max gene.

    PubMed Central

    Peters, M A; Sollenberger, K G; Kao, T L; Taparowsky, E J

    1997-01-01

    Max is a basic helix-loop-helix/leucine zipper protein that forms heterodimers with the Myc family of proteins to promote cell growth and with the Mad/Mxi1 family of proteins to inhibit cell growth. The role of Max as the obligate binding partner for these two protein families necessitates the observed constitutive expression and relatively long half-life of the max mRNA under a variety of growth conditions. In this study, we have used the chicken max gene to map DNA elements maintaining max gene expression in vertebrate cells. We have identified a minimal regulatory region (MRR) that resides within 115 bp of the max translation initiation site and that possesses an overall structure typical of TATA-less promoters. Within the MRR are two consensus binding sites for Sp1, a ubiquitously expressed transcription factor that plays a role in the expression of many constitutive genes. Interestingly, we show that direct binding by Sp1 to these sites is not required for MRR-mediated transcription. Instead, the integrity of a 20-bp DNA element in the MRR is required for transcriptional activity, as is the interaction of this DNA element with a 90-kDa cellular protein. Our data suggest that it is the persistence of this 90-kDa protein in vertebrate cells which drives max gene expression, insulates the max promoter from the dramatic changes in transcription that accompany cell growth and development, and ensures that adequate levels of Max will be available to facilitate the function of the Myc, Mad, and Mxi1 families of proteins. PMID:9032230

  3. PLEXdb: Gene expression resources for plants and plant pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PLEXdb (Plant Expression Database), in partnership with community databases, supports comparisons of gene expression across multiple plant and pathogen species, promoting individuals and/or consortia to upload genome-scale data sets to contrast them to previously archived data. These analyses facili...

  4. Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems.

    PubMed

    Gottschamel, Johanna; Lössl, Andreas; Ruf, Stephanie; Wang, Yanliang; Skaugen, Morten; Bock, Ralph; Clarke, Jihong Liu

    2016-07-01

    Dengue fever is a disease in many parts of the tropics and subtropics and about half the world's population is at risk of infection according to the World Health Organization. Dengue is caused by any of the four related dengue virus serotypes DEN-1, -2, -3 and -4, which are transmitted to people by Aedes aegypti mosquitoes. Currently there is only one vaccine (Dengvaxia(®)) available (limited to a few countries) on the market since 2015 after half a century's intensive efforts. Affordable and accessible vaccines against dengue are hence still urgently needed. The dengue envelop protein domain III (EDIII), which is capable of eliciting serotype-specific neutralizing antibodies, has become the focus for subunit vaccine development. To contribute to the development of an accessible and affordable dengue vaccine, in the current study we have used plant-based vaccine production systems to generate a dengue subunit vaccine candidate in tobacco. Chloroplast genome engineering was applied to express serotype-specific recombinant EDIII proteins in tobacco chloroplasts using both constitutive and ethanol-inducible expression systems. Expression of a tetravalent antigen fusion construct combining EDIII polypeptides from all four serotypes was also attempted. Transplastomic EDIII-expressing tobacco lines were obtained and homoplasmy was verified by Southern blot analysis. Northern blot analyses showed expression of EDIII antigen-encoding genes. EDIII protein accumulation levels varied for the different recombinant EDIII proteins and the different expression systems, and reached between 0.8 and 1.6 % of total cellular protein. Our study demonstrates the suitability of the chloroplast compartment as a production site for an EDIII-based vaccine candidate against dengue fever and presents a Gateway(®) plastid transformation vector for inducible transgene expression. PMID:27116001

  5. Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack.

    PubMed

    Tavladoraki, P; Benvenuto, E; Trinca, S; De Martinis, D; Cattaneo, A; Galeffi, P

    1993-12-01

    Expression of viral genes in transgenic plants is a very effective tool for attenuating plant viral infection. Nevertheless, the lack of generality and risk issues related to the expression of viral genes in plants might limit the exploitation of this strategy. Expression in plants of antibodies against essential viral proteins could provide an alternative approach to engineer viral resistance. Recently, expression of complete or engineered antibodies has been successfully achieved in plants. The engineered single-chain Fv antibody scFv (refs 10, 11) is particularly suitable for expression in plants because of its small size and the lack of assembly requirements. Here we present evidence that constitutive expression in transgenic plants of a scFv antibody, directed against the plant icosahedral tombusvirus artichoke mottled crinkle virus, causes reduction of infection incidence and delay in symptom development. PMID:8247156

  6. A recursive network approach can identify constitutive regulatory circuits in gene expression data

    NASA Astrophysics Data System (ADS)

    Blasi, Monica Francesca; Casorelli, Ida; Colosimo, Alfredo; Blasi, Francesco Simone; Bignami, Margherita; Giuliani, Alessandro

    2005-03-01

    The activity of the cell is often coordinated by the organisation of proteins into regulatory circuits that share a common function. Genome-wide expression profiles might contain important information on these circuits. Current approaches for the analysis of gene expression data include clustering the individual expression measurements and relating them to biological functions as well as modelling and simulation of gene regulation processes by additional computer tools. The identification of the regulative programmes from microarray experiments is limited, however, by the intrinsic difficulty of linear methods to detect low-variance signals and by the sensitivity of the different approaches. Here we face the problem of recognising invariant patterns of correlations among gene expression reminiscent of regulation circuits. We demonstrate that a recursive neural network approach can identify genetic regulation circuits from expression data for ribosomal and genome stability genes. The proposed method, by greatly enhancing the sensitivity of microarray studies, allows the identification of important aspects of genetic regulation networks and might be useful for the discrimination of the different players involved in regulation circuits. Our results suggest that the constitutive regulatory networks involved in the generic organisation of the cell display a high degree of clustering depending on a modular architecture.

  7. Effect of chromosome constitution variations on the expression of Turner phenotype.

    PubMed

    Bispo, A V S; Dos Santos, L O; Burégio-Frota, P; Galdino, M B; Duarte, A R; Leal, G F; Araújo, J; Gomes, B; Soares-Ventura, E M; Muniz, M T C; Santos, N

    2013-01-01

    Turner syndrome (TS) is a chronic disease related to haploinsufficiency of genes that are normally expressed in both X chromosomes in patients with female phenotype that is associated with a wide range of somatic malformations. We made detailed cytogenetic and clinical analysis of 65 patients with TS from the region of Recife, Brazil, to determine the effects of different chromosome constitutions on expression of the TS phenotype. Overall, patients with X-monosomy exhibited a tendency to have more severe phenotypes with higher morbidity, showing its importance in TS prognosis. Additionally, we found rare genetic and phenotypic abnormalities associated with this syndrome. To the best of our knowledge, this is the first case of 45,X,t(11;12)(q22;q22) described as a TS karyotype. Turner patients usually have normal intelligence; however, moderate to severe levels of mental retardation were found in 5 TS cases, which is considerate a very uncommon feature in this syndrome. PMID:23546984

  8. Constitutive expression of a grapevine stilbene synthase gene in transgenic hop (Humulus lupulus L.) yields resveratrol and its derivatives in substantial quantities.

    PubMed

    Schwekendiek, Axel; Spring, Otmar; Heyerick, Arne; Pickel, Benjamin; Pitsch, Nicola T; Peschke, Florian; de Keukeleire, Denis; Weber, Gerd

    2007-08-22

    Resveratrol, a well-known phytoalexin and antioxidant, is produced by the action of stilbene synthase (STS) in some plant species. Hop (Humulus lupulus L.) plants of the Tettnang variety were transformed with a gene encoding for STS from grapevine. Under the control of the constitutive 35S cauliflower mosaic virus promoter, expression of the transgene resulted in accumulation of resveratrol and high levels of its glycosylated derivatives in leaves and inflorescences. Piceid, the predominant derivative, reached a concentration of up to 560 microg/g of fresh weight (f.w.) in hop cones, whereas no stilbenes were detected in nontransformed controls (wild-type). In transgenic plants the amounts of alpha- and beta-acids, naringenin chalcone, and prenylated flavonoids did not change significantly when compared with nontransformed plants. Transgenic plants showed normal morphology and flower development as did the nontransformed controls. The results clearly show that in hop constitutive expression of sts interferes neither with plant development nor with the biosynthesis of secondary metabolites relevant for the brewing industry. Since resveratrol is a well-known phytoalexin and antioxidant, sts transgenic hop plants could display enhanced pathogen resistance against microbial pathogens, exhibit new beneficial properties for health, and open new venues for metabolic engineering. PMID:17655245

  9. A constitutive unregulated expression of β-galactosidase in Lactobacillus fermentum M1.

    PubMed

    Arukha, Ananta Prasad; Mukhopadhyay, Bidhan Chandra; Mitra, Suranjita; Biswas, Swadesh Ranjan

    2015-02-01

    A constitutively β-galactosidase (LacL)-producing Lactobacillus fermentum M1 isolated from fermented milk was found to produce β-galactosidase in the presence of glucose. β-galactosidase activity produced in glucose (30 mM) medium was 2.17 U/mL as compared to 2.27 and 2.19 U/mL with galactose and lactose, respectively. When a combination of glucose (30 or 60 mM) with galactose (30 mM) was used as carbon source, β-galactosidase activity was not repressed rather was found increased when compared to carbon sources used individually. In real-time PCR analysis of mRNA synthesized on individual and combined carbon sources, repression of the lacL gene expression was not observed. This observation suggests that the strain M1 lacked normal carbon catabolite repression. Examination of nucleotide sequence of lacL identified two catabolite responsive elements (cre): cre1 located downstream near the promoter region and cre2 within the coding sequence. Each of which differed from the 14-bp consensus by a single nucleotide. In cre1, it is C in place of highly conserved T at position 1 in the consensus. In cre 2, it is G in place of C, a residue completely conserved at position 13. Since catabolite genes in Gram-positive bacteria are regulated by carbon catabolite protein A (CcpA) through interaction with DNA at a specific cis-acting cre, it is assumed that base changes at conserved position in the cre elements disrupt CcpA binding and thereby leading to constitutive expression of lacL gene. The study noted to be the first report about the constitutive production of β-galactosidase in L. fermentum.

  10. Expression of multiple proteins in transgenic plants

    DOEpatents

    Vierstra, Richard D.; Walker, Joseph M.

    2002-01-01

    A method is disclosed for the production of multiple proteins in transgenic plants. A DNA construct for introduction into plants includes a provision to express a fusion protein of two proteins of interest joined by a linking domain including plant ubiquitin. When the fusion protein is produced in the cells of a transgenic plant transformed with the DNA construction, native enzymes present in plant cells cleave the fusion protein to release both proteins of interest into the cells of the transgenic plant. Since the proteins are produced from the same fusion protein, the initial quantities of the proteins in the cells of the plant are approximately equal.

  11. Rho GTPase signaling promotes constitutive expression and release of TGF-β2 by human trabecular meshwork cells.

    PubMed

    Pervan, Cynthia L; Lautz, Jonathan D; Blitzer, Andrea L; Langert, Kelly A; Stubbs, Evan B

    2016-05-01

    Elevated intraocular pressure (IOP) is causally implicated in the pathophysiology of primary open-angle glaucoma (POAG). The molecular mechanisms responsible for elevated IOP remain elusive, but may involve aberrant expression and signaling of transforming growth factor (TGF)-β2 within the trabecular meshwork (TM). Consistent with previously published studies, we show here that exogenous addition of TGF-β2 to cultured porcine anterior segments significantly attenuates outflow facility in a time-dependent manner. By comparison, perfusing segments with a TGFβRI/ALK-5 antagonist (SB-431542) unexpectedly elicited a significant and sustained increase in outflow facility, implicating a role for TM-localized constitutive expression and release of TGF-β2. Consistent with this thesis, cultured primary or transformed (GTM3) quiescent human TM cells were found to constitutively express and secrete measurable amounts of biologically-active TGF-β2. Disrupting monomeric GTPase post-translational prenylation and activation with lovastatin or GGTI-298 markedly reduced constitutive TGF-β2 expression and release. Specifically, inhibiting the Rho subfamily of GTPases with C3 exoenzyme similarly reduced constitutive expression and secretion of TGF-β2. These findings suggest that Rho GTPase signaling, in part, regulates constitutive expression and release of biologically-active TGF-β2 from human TM cells. Localized constitutive expression and release of TGF-β2 by TM cells may promote or exacerbate elevation of IOP in POAG.

  12. Constitutive renal Rel/nuclear factor-κB expression in Lewis polycystic kidney disease rats

    PubMed Central

    Ta, Michelle H T; Schwensen, Kristina G; Liuwantara, David; Huso, David L; Watnick, Terry; Rangan, Gopala K

    2016-01-01

    AIM: To determine the temporal expression and pattern of Rel/nuclear factor (NF)-κB proteins in renal tissue in polycystic kidney disease (PKD). METHODS: The renal expression of Rel/NF-κB proteins was determined by immunohistochemistry, immunofluorescence and immunoblot analysis in Lewis polycystic kidney rats (LPK, a genetic ortholog of human nephronopthsis-9) from postnatal weeks 3 to 20. At each timepoint, renal disease progression and the mRNA expression of NF-κB-dependent genes (TNFα and CCL2) were determined. NF-κB was also histologically assessed in human PKD tissue. RESULTS: Progressive kidney enlargement in LPK rats was accompanied by increased renal cell proliferation and interstitial monocyte accumulation (peaking at weeks 3 and 10 respectively), and progressive interstitial fibrosis (with α smooth muscle actin and Sirius Red deposition significantly increased compared to Lewis kidneys from weeks 3 to 6 onwards). Rel/NF-κB proteins (phosphorylated-p105, p65, p50, c-Rel and RelB) were expressed in cystic epithelial cells (CECs) of LPK kidneys as early as postnatal week 3 and sustained until late-stage disease at week 20. From weeks 10 to 20, nuclear p65, p50, RelB and cytoplasmic IκBα protein levels, and TNFα and CCL2 expression, were upregulated in LPK compared to Lewis kidneys. NF-κB proteins were consistently expressed in CECs of human PKD. The DNA damage marker γ-H2AX was also identified in the CECs of LPK and human polycystic kidneys. CONCLUSION: Several NF-κB proteins are consistently expressed in CECs in human and experimental PKD. These data suggest that the upregulation of both the canonical and non-canonical pathways of NF-κB signaling may be a constitutive and early pathological feature of cystic renal diseases. PMID:27458563

  13. Integrating constitutive gene expression and chemoactivity: mining the NCI60 anticancer screen.

    PubMed

    Covell, David G

    2012-01-01

    Studies into the genetic origins of tumor cell chemoactivity pose significant challenges to bioinformatic mining efforts. Connections between measures of gene expression and chemoactivity have the potential to identify clinical biomarkers of compound response, cellular pathways important to efficacy and potential toxicities; all vital to anticancer drug development. An investigation has been conducted that jointly explores tumor-cell constitutive NCI60 gene expression profiles and small-molecule NCI60 growth inhibition chemoactivity profiles, viewed from novel applications of self-organizing maps (SOMs) and pathway-centric analyses of gene expressions, to identify subsets of over- and under-expressed pathway genes that discriminate chemo-sensitive and chemo-insensitive tumor cell types. Linear Discriminant Analysis (LDA) is used to quantify the accuracy of discriminating genes to predict tumor cell chemoactivity. LDA results find 15% higher prediction accuracies, using ∼30% fewer genes, for pathway-derived discriminating genes when compared to genes derived using conventional gene expression-chemoactivity correlations. The proposed pathway-centric data mining procedure was used to derive discriminating genes for ten well-known compounds. Discriminating genes were further evaluated using gene set enrichment analysis (GSEA) to reveal a cellular genetic landscape, comprised of small numbers of key over and under expressed on- and off-target pathway genes, as important for a compound's tumor cell chemoactivity. Literature-based validations are provided as support for chemo-important pathways derived from this procedure. Qualitatively similar results are found when using gene expression measurements derived from different microarray platforms. The data used in this analysis is available at http://pubchem.ncbi.nlm.nih.gov/andhttp://www.ncbi.nlm.nih.gov/projects/geo (GPL96, GSE32474).

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

    PubMed

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

    2014-06-01

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

  15. Selective lignin downregulation leads to constitutive defense response expression in alfalfa (Medicago sativa L.).

    PubMed

    Gallego-Giraldo, Lina; Jikumaru, Yusuke; Kamiya, Yuji; Tang, Yuhong; Dixon, Richard A

    2011-05-01

    • Downregulation of hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) in alfalfa (Medicago sativa) reduces lignin levels and improves forage quality and saccharification efficiency for bioethanol production. However, the plants have reduced stature. It was previously reported that HCT-down-regulated Arabidopsis have impaired auxin transport, but this has recently been disproved. • To address the basis for the phenotypes of lignin-modified alfalfa, we measured auxin transport, profiled a range of metabolites including flavonoids and hormones, and performed in depth transcriptome analyses. • Auxin transport is unaffected in HCT antisense alfalfa despite increased flavonoid biosynthesis. The plants show increased cytokinin and reduced auxin levels, and gibberellin levels and sensitivity are both reduced. Levels of salicylic, jasmonic and abscisic acids are elevated, associated with massive upregulation of pathogenesis and abiotic stress-related genes and enhanced tolerance to fungal infection and drought. • We suggest that HCT downregulated alfalfa plants exhibit constitutive activation of defense responses, triggered by release of bioactive cell wall fragments and production of hydrogen peroxide as a result of impaired secondary cell wall integrity.

  16. Constitutive and allergen-induced expression of eotaxin mRNA in the guinea pig lung

    PubMed Central

    1995-01-01

    Eotaxin is a member of the C-C family of chemokines and is related during antigen challenge in a guinea pig model of allergic airway inflammation (asthma). Consistent with its putative role in eosinophilic inflammation, eotaxin induces the selective infiltration of eosinophils when injected into the lung and skin. Using a guinea pig lung cDNA library, we have cloned full-length eotaxin cDNA. The cDNA encodes a protein of 96 amino acids, including a putative 23-amino acid hydrophobic leader sequence, followed by 73 amino acids composing the mature active eotaxin protein. The protein-coding region of this cDNA is 73, 71, 50, and 48% identical in nucleic acid sequence to those of human macrophage chemoattractant protein (MCP) 3, MCP-1, macrophage inflammatory protein (MIP) 1 alpha, and RANTES, respectively. Analysis of genomic DNA suggested that there is a single eotaxin gene in guinea pig which is apparently conserved in mice. High constitutive levels of eotaxin mRNA expression were observed in the lung, while the intestines, stomach, spleen, liver, heart, thymus, testes, and kidney expressed lower levels. To determine if eotaxin mRNA levels are elevated during allergen-induced eosinophilic airway inflammation, ovalbumin (OVA)-sensitized guinea pigs were challenged with aerosolized antigen. Compared with the lungs from saline-challenged animals, eotaxin mRNA levels increased sixfold within 3 h and returned to baseline by 6 h. Thus, eotaxin mRNA levels are increased in response to allergen challenge during the late phase response. The identification of constitutive eotaxin mRNA expression in multiple tissues suggests that in addition to regulating airway eosinophilia, eotaxin is likely to be involved in eosinophil recruitment into other tissues as well as in baseline tissue homing. PMID:7869037

  17. A constitutive expression system for cellulase secretion in Escherichia coli and its use in bioethanol production.

    PubMed

    Munjal, Neha; Jawed, Kamran; Wajid, Saima; Yazdani, Syed Shams

    2015-01-01

    The production of biofuels from lignocellulosic biomass appears to be attractive and viable due to the abundance and availability of this biomass. The hydrolysis of this biomass, however, is challenging because of the complex lignocellulosic structure. The ability to produce hydrolytic cellulase enzymes in a cost-effective manner will certainly accelerate the process of making lignocellulosic ethanol production a commercial reality. These cellulases may need to be produced aerobically to generate large amounts of protein in a short time or anaerobically to produce biofuels from cellulose via consolidated bioprocessing. Therefore, it is important to identify a promoter that can constitutively drive the expression of cellulases under both aerobic and anaerobic conditions without the need for an inducer. Using lacZ as reporter gene, we analyzed the strength of the promoters of four genes, namely lacZ, gapA, ldhA and pflB, and found that the gapA promoter yielded the maximum expression of the β-galactosidase enzyme under both aerobic and anaerobic conditions. We further cloned the genes for two cellulolytic enzymes, β-1,4-endoglucanase and β-1,4-glucosidase, under the control of the gapA promoter, and we expressed these genes in Escherichia coli, which secreted the products into the extracellular medium. An ethanologenic E. colistrain transformed with the secretory β-glucosidase gene construct fermented cellobiose in both defined and complex medium. This recombinant strain also fermented wheat straw hydrolysate containing glucose, xylose and cellobiose into ethanol with an 85% efficiency of biotransformation. An ethanologenic strain that constitutively secretes a cellulolytic enzyme is a promising platform for producing lignocellulosic ethanol.

  18. Constitutive expression of MKS1 confers susceptibility to Botrytis cinerea infection independent of PAD3 expression.

    PubMed

    Fiil, Berthe Katrine; Petersen, Morten

    2011-10-01

    Signal transduction through MAPK cascades is essential for eukaryotic cell response to various extracellular stimuli, such as the induction of innate immune responses. Arabidopsis thaliana relies in particular on three of its 20 MAPKs, MPK3,-4,-6, for a proper immune response. Recently we showed that one MPK4-substrate, MKS1, is required for basal resistance against the virulent Pseudomonas syringae and the oomycete Hyaloperonospora arabidopsidis. Overexpression of MKS1 (35S-MKS1) led to increased resistance to the same pathogens but also to an increased susceptibility towards the fungi Botrytis cinerea. MKS1 interacts with the transcription factor WRKY33, which in turn controls the regulation of PAD3 and CYP71A13, two genes, required for proper resistance to B. cinerea. Therefore, we tested if the increased susceptibility towards B. cinerea from 35S-MKS1 was due to deregulation of WRKY33 targets. PAD3 and CYP71A13 expression is similar in 35S-MKS1 and WT after B. cinerea treatment suggesting another mechanism controls 35S-MKS1 susceptibility.

  19. A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina

    DOE PAGES

    Linger, Jeffrey G.; Taylor, II, Larry E.; Baker, John O.; Vander Wall, Todd; Hobdey, Sarah E.; Podkaminer, Kara; Himmel, Michael E.; Decker, Stephen R.

    2015-03-18

    One of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is cellobiohydrolase I (cbh1or Cel7A) due to its enzymatic proficiency in cellulose depolymerization. However, production of Cel7A with native-like properties from heterologous expression systems has proven difficult. In this study, we develop a protein expression system in H. jecorina (Trichoderma reesei) useful for production and secretion of heterologous cellobiohydrolases from glycosyl hydrolase family 7. Building upon previous work in heterologous protein expression in filamentous fungi, we have integrated amore » native constitutive enolase promoter with the native cbh1 signal sequence. The results are the following: The constitutive eno promoter driving the expression of Cel7A allows growth on glucose and results in repression of the native cellulase system, severely reducing background endo- and other cellulase activity and greatly simplifying purification of the recombinant protein. Coupling this system to a Δcbh1 strain of H. jecorina ensures that only the recombinant Cel7A protein is produced. Two distinct transformant colony morphologies were observed and correlated with high and null protein production. Production levels in ‘fast’ transformants are roughly equivalent to those in the native QM6a strain of H. jecorina, typically in the range of 10 to 30 mg/L when grown in continuous stirred-tank fermenters. ‘Slow’ transformants showed no evidence of Cel7A production. Specific activity of the purified recombinant Cel7A protein is equivalent to that of native protein when assayed on pretreated corn stover, as is the thermal stability and glycosylation level. Purified Cel7A produced from growth on glucose demonstrated remarkably consistent specific activity. Purified Cel7A from the same strain grown on lactose

  20. S-like ribonuclease gene expression in carnivorous plants.

    PubMed

    Nishimura, Emi; Kawahara, Minako; Kodaira, Reina; Kume, Marina; Arai, Naoki; Nishikawa, Jun-ichi; Ohyama, Takashi

    2013-11-01

    Functions of S-like ribonucleases (RNases) differ considerably from those of S-RNases that function in self-incompatibility. Expression of S-like RNases is usually induced by low nutrition, vermin damage or senescence. However, interestingly, an Australian carnivorous plant Drosera adelae (a sundew), which traps prey with a sticky digestive liquid, abundantly secretes an S-like RNase DA-I in the digestive liquid even in ordinary states. Here, using D. adelae, Dionaea muscipula (Venus flytrap) and Cephalotus follicularis (Australian pitcher plant), we show that carnivorous plants use S-like RNases for carnivory: the gene da-I encoding DA-I and its ortholog cf-I of C. follicularis are highly expressed and constitutively active in each trap/digestion organ, while the ortholog dm-I of D. muscipula becomes highly active after trapping insects. The da-I promoter is unmethylated only in its trap/digestion organ, glandular tentacles (which comprise a small percentage of the weight of the whole plant), but methylated in other organs, which explains the glandular tentacles-specific expression of the gene and indicates a very rare gene regulation system. In contrast, the promoters of dm-I, which shows induced expression, and cf-I, which has constitutive expression, were not methylated in any organs examined. Thus, it seems that the regulatory mechanisms of the da-I, dm-I and cf-I genes differ from each other and do not correlate with the phylogenetic relationship. The current study suggests that under environmental pressure in specific habitats carnivorous plants have managed to evolve their S-like RNase genes to function in carnivory. PMID:23959189

  1. S-like ribonuclease gene expression in carnivorous plants.

    PubMed

    Nishimura, Emi; Kawahara, Minako; Kodaira, Reina; Kume, Marina; Arai, Naoki; Nishikawa, Jun-ichi; Ohyama, Takashi

    2013-11-01

    Functions of S-like ribonucleases (RNases) differ considerably from those of S-RNases that function in self-incompatibility. Expression of S-like RNases is usually induced by low nutrition, vermin damage or senescence. However, interestingly, an Australian carnivorous plant Drosera adelae (a sundew), which traps prey with a sticky digestive liquid, abundantly secretes an S-like RNase DA-I in the digestive liquid even in ordinary states. Here, using D. adelae, Dionaea muscipula (Venus flytrap) and Cephalotus follicularis (Australian pitcher plant), we show that carnivorous plants use S-like RNases for carnivory: the gene da-I encoding DA-I and its ortholog cf-I of C. follicularis are highly expressed and constitutively active in each trap/digestion organ, while the ortholog dm-I of D. muscipula becomes highly active after trapping insects. The da-I promoter is unmethylated only in its trap/digestion organ, glandular tentacles (which comprise a small percentage of the weight of the whole plant), but methylated in other organs, which explains the glandular tentacles-specific expression of the gene and indicates a very rare gene regulation system. In contrast, the promoters of dm-I, which shows induced expression, and cf-I, which has constitutive expression, were not methylated in any organs examined. Thus, it seems that the regulatory mechanisms of the da-I, dm-I and cf-I genes differ from each other and do not correlate with the phylogenetic relationship. The current study suggests that under environmental pressure in specific habitats carnivorous plants have managed to evolve their S-like RNase genes to function in carnivory.

  2. Constitutive expression of a grapevine polygalacturonase-inhibiting protein affects gene expression and cell wall properties in uninfected tobacco

    PubMed Central

    2011-01-01

    Background Polygalacturonase-inhibiting proteins (PGIPs) directly limit the effective ingress of fungal pathogens by inhibiting cell wall-degrading endopolygalacturonases (ePGs). Transgenic tobacco plants over-expressing grapevine (Vitis vinifera) Vvpgip1 have previously been shown to be resistant to Botrytis infection. In this study we characterized two of these PGIP over-expressing lines with known resistance phenotypes by gene expression and hormone profiling in the absence of pathogen infection. Results Global gene expression was performed by a cross-species microarray approach using a potato cDNA microarray. The degree of potential cross-hybridization between probes was modeled by a novel computational workflow designed in-house. Probe annotations were updated by predicting probe-to-transcript hybridizations and combining information derived from other plant species. Comparing uninfected Vvpgip1-overexpressing lines to wild-type (WT), 318 probes showed significant change in expression. Functional groups of genes involved in metabolism and associated to the cell wall were identified and consequent cell wall analysis revealed increased lignin-levels in the transgenic lines, but no major differences in cell wall-derived polysaccharides. GO enrichment analysis also identified genes responsive to auxin, which was supported by elevated indole-acetic acid (IAA) levels in the transgenic lines. Finally, a down-regulation of xyloglucan endotransglycosylase/hydrolases (XTHs), which are important in cell wall remodeling, was linked to a decrease in total XTH activity. Conclusions This evaluation of PGIP over-expressing plants performed under pathogen-free conditions to exclude the classical PGIP-ePG inhibition interaction indicates additional roles for PGIPs beyond the inhibition of ePGs. PMID:22078230

  3. Constitutive overexpression of the sucrose transporter SoSUT1 in potato plants increases arbuscular mycorrhiza fungal root colonization under high, but not under low, soil phosphorus availability.

    PubMed

    Gabriel-Neumann, Elke; Neumann, Günter; Leggewie, Georg; George, Eckhard

    2011-06-15

    The sucrose transporter SUT1 functions in phloem loading of photoassimilates in solanaceous plant species. In the present study, wildtype and transgenic potato plants with either constitutive overexpression or antisense inhibition of SUT1 were grown under high or low phosphorus (P) fertilization levels in the presence or absence of the arbuscular mycorrhizal (AM) fungus Glomus intraradices. At a low soil P fertilization level, the extent of AM fungal root colonization was not different among the genotypes. In all plants, the AM symbiosis contributed significantly to P uptake under these conditions. In response to a high soil P fertilization level, all genotypes showed a decrease in AM fungal root colonization, indicating that the expression level of SUT1 does not constitute a major mechanism of control over AM development in response to the soil P availability. However, plants with overexpression of SUT1 showed a higher extent of AM fungal root colonization compared with the other genotypes when the soil P availability was high. Whether an increased symbiotic C supply, alterations in the phytohormonal balance, or a decreased synthesis of antimicrobial compounds was the major cause for this effect requires further investigation. In plants with impaired phloem loading, a low C status of plant sink tissues did apparently not negatively affect plant C supply to the AM symbiosis. It is possible that, at least during vegetative and early generative growth, source rather than sink tissues exert control over amounts of C supplied to AM fungi.

  4. Constitutive overexpression of the sucrose transporter SoSUT1 in potato plants increases arbuscular mycorrhiza fungal root colonization under high, but not under low, soil phosphorus availability.

    PubMed

    Gabriel-Neumann, Elke; Neumann, Günter; Leggewie, Georg; George, Eckhard

    2011-06-15

    The sucrose transporter SUT1 functions in phloem loading of photoassimilates in solanaceous plant species. In the present study, wildtype and transgenic potato plants with either constitutive overexpression or antisense inhibition of SUT1 were grown under high or low phosphorus (P) fertilization levels in the presence or absence of the arbuscular mycorrhizal (AM) fungus Glomus intraradices. At a low soil P fertilization level, the extent of AM fungal root colonization was not different among the genotypes. In all plants, the AM symbiosis contributed significantly to P uptake under these conditions. In response to a high soil P fertilization level, all genotypes showed a decrease in AM fungal root colonization, indicating that the expression level of SUT1 does not constitute a major mechanism of control over AM development in response to the soil P availability. However, plants with overexpression of SUT1 showed a higher extent of AM fungal root colonization compared with the other genotypes when the soil P availability was high. Whether an increased symbiotic C supply, alterations in the phytohormonal balance, or a decreased synthesis of antimicrobial compounds was the major cause for this effect requires further investigation. In plants with impaired phloem loading, a low C status of plant sink tissues did apparently not negatively affect plant C supply to the AM symbiosis. It is possible that, at least during vegetative and early generative growth, source rather than sink tissues exert control over amounts of C supplied to AM fungi. PMID:21382646

  5. Enhancement of endothelial cell migration by constitutively active LPA{sub 1}-expressing tumor cells

    SciTech Connect

    Kitayoshi, Misaho; Kato, Kohei; Tanabe, Eriko; Yoshikawa, Kyohei; Fukui, Rie; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer Mutated LPA{sub 1} stimulates cell migration of endothelial cells. Black-Right-Pointing-Pointer VEGF expressions are increased by mutated LPA{sub 1}. Black-Right-Pointing-Pointer LPA signaling via mutated LPA{sub 1} is involved in angiogenesis. Black-Right-Pointing-Pointer Mutated LPA{sub 1} promotes cancer cell progression. -- Abstract: Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors (LPA receptors; LPA{sub 1} to LPA{sub 6}). They indicate a variety of cellular response by the interaction with LPA, including cell proliferation, migration and differentiation. Recently, we have reported that constitutive active mutated LPA{sub 1} induced the strong biological effects of rat neuroblastoma B103 cells. In the present study, we examined the effects of mutated LPA{sub 1} on the interaction between B103 cells and endothelial F-2 cells. Each LPA receptor expressing B103 cells were maintained in serum-free DMEM and cell motility assay was performed with a Cell Culture Insert. When F-2 cells were cultured with conditioned medium from Lpar1 and Lpar3-expressing cells, the cell motility of F-2 cells was significantly higher than control cells. Interestingly, the motile activity of F-2 cells was strongly induced by mutated LPA{sub 1} than other cells, correlating with the expression levels of vascular endothelial growth factor (Vegf)-A and Vegf-C. Pretreatment of LPA signaling inhibitors inhibited F-2 cell motility stimulated by mutated LPA{sub 1}. These results suggest that activation of LPA signaling via mutated LPA{sub 1} may play an important role in the promotion of angiogenesis in rat neuroblastoma cells.

  6. Inducible gene expression systems for plants.

    PubMed

    Borghi, Lorenzo

    2010-01-01

    Several systems for induction of transgene expression in plants have been described recently. Inducible systems were used mainly in tobacco, rice, Arabidopsis, tomato, and maize. Inducible systems offer researchers the possibility to deregulate gene expression levels at particular stages of plant development and in particular tissues of interest. The more precise temporal and spatial control, obtained by providing the transgenic plant with the appropriate chemical compound or treatment, permits to analyze also the function of those genes required for plant viability. In addition, inducible systems allow promoting local changes in gene expression levels without causing gross alterations to the whole plant development. Here, protocols will be presented to work with five different inducible systems: AlcR/AlcA (ethanol inducible); GR fusions, GVG, and pOp/LhGR (dexamethasone inducible); XVE/OlexA (beta-estradiol inducible); and heat shock induction. PMID:20734254

  7. Pantophysin is a ubiquitously expressed synaptophysin homologue and defines constitutive transport vesicles

    PubMed Central

    1996-01-01

    Certain properties of the highly specialized synaptic transmitter vesicles are shared by constitutively occurring vesicles. We and others have thus identified a cDNA in various nonneuroendocrine cell types of rat and human that is related to synaptophysin, one of the major synaptic vesicle membrane proteins, which we termed pantophysin. Here we characterize the gene structure, mRNA and protein expression, and intracellular distribution of pantophysin. Its mRNA is detected in murine cell types of nonneuroendocrine as well as of neuroendocrine origin. The intron/exon structure of the murine pantophysin gene is identical to that of synaptophysin except for the last intron that is absent in pantophysin. The encoded polypeptide of calculated mol wt 28,926 shares many sequence features with synaptophysin, most notably the four hydrophobic putative transmembrane domains, although the cytoplasmic end domains are completely different. Using antibodies against the unique carboxy terminus pantophysin can be detected by immunofluorescence microscopy in both exocrine and endocrine cells of human pancreas, and in cultured cells, colocalizing with constitutive secretory and endocytotic vesicle markers in nonneuroendocrine cells and with synaptophysin in cDNA-transfected epithelial cells. By immunoelectron microscopy, the majority of pantophysin reactivity is detected at vesicles with a diameter of < 100 nm that have a smooth surface and an electron-translucent interior. Using cell fractionation in combination with immunoisolation, these vesicles are enriched in a light fraction and shown to contain the cellular vSNARE cellubrevin and the ubiquitous SCAMPs in epithelial cells and synaptophysin in neuroendocrine or cDNA-transfected nonneuroendocrine cells and neuroendocrine tissues. Pantophysin is therefore a broadly distributed marker of small cytoplasmic transport vesicles independent of their content. PMID:8707851

  8. Ethanol inducible expression of a mesophilic cellulase avoids adverse effects on plant development

    PubMed Central

    2013-01-01

    Background Plant-produced biomass-degrading enzymes are promising tools for the processing of lignocellulose to fermentable sugars. A major limitation of in planta production is that high-level expression of such enzymes could potentially affect the structure and integrity of the plant cell wall and negatively influence plant growth and development. Results Here, we evaluate the impact on tobacco plant development of constitutive versus alcohol-inducible expression of the endoglucanase TrCel5A from the mesophilic fungus Trichoderma reesei. Using this system, we are able to demonstrate that constitutive expression of the enzyme, controlled by the doubled Cauliflower Mosaic Virus promoter, leads to lower cellulose content of the plant combined with severe effects on plant growth. However, using an alcohol-inducible expression of the endoglucanase in the plant leaves, we achieved similar enzymatic expression levels with no changes in the crystalline cellulose content. Conclusion We were able to produce significant amounts of cellulase in the plant leaves without detrimental effects to plant development. These results demonstrate the potential feasibility of an inducible expression system for producing biomass degrading enzymes in plants. PMID:23587418

  9. Peptide inhibition of constitutively activated epithelial Na(+) channels expressed in Xenopus oocytes.

    PubMed

    Ji, H L; Fuller, C M; Benos, D J

    1999-12-31

    The hypothesis that 30-amino acid peptides corresponding to the C-terminal portion of the beta- and/or gamma-rat epithelial sodium channel (rENaC) subunits block constitutively activated ENaC was tested by examining the effects of these peptides on wild-type (wt) rENaC (alphabetagamma-rENaC), truncated Liddle's mutants (alphabeta(T)gamma-, alphabetagamma(T)-, and alphabeta(T)gamma(T)-rENaC), and point mutants (alphabeta(Y)gamma-, alphabetagamma(Y)-rENaC) expressed in Xenopus oocytes. The chord conductances of alphabeta(T)gamma-, alphabetagamma(T)-, and alphabeta(T)gamma(T)-rENaC were 2- or 3-fold greater than for wt alphabetagamma-rENaC. Introduction of peptides into oocytes expressing alphabeta(T)gamma-, alphabetagamma(T)-, and alphabeta(T)gamma(T)-rENaC produced a concentration-dependent inhibition of the amiloride-sensitive Na(+) conductances, with apparent dissociation constants (K(d)) ranging from 1700 to 160 microM, depending upon whether individual peptides or their combination was used. Injection of peptides alone or in combination into oocytes expressing wt alphabetagamma-rENaC or single-point mutants did not affect the amiloride-sensitive whole-cell currents. The single channel conductances of all the mutant ENaCs were the same as that of wild type (alphabetagamma-). The single channel activities (N.P(o)) of the mutants were approximately 2.2-2.6-fold greater than wt alphabetagamma-rENaC (1.08 +/- 0.24, n = 7) and were reduced to 1.09 +/- 0.17 by 100 microM peptide mixture (n = 9). The peptides were without effect on the single channel properties of either wt or single-point mutants of rENaC. Our data demonstrate that the C-terminal peptides blocked the Liddle's truncation mutant (alphabeta(T)gamma(T)) expressed in Xenopus oocytes but not the single-point mutants (alphabeta(Y)gamma or alphabetagamma(Y)). Moreover, the blocking effect of both peptides in combination on alphabeta(T)gamma(T)-rENaC was synergistic. PMID:10608827

  10. Identification of chemical modulators of the constitutive activated receptor (CAR) in a gene expression compendium

    PubMed Central

    Oshida, Keiyu; Vasani, Naresh; Jones, Carlton; Moore, Tanya; Hester, Susan; Nesnow, Stephen; Auerbach, Scott; Geter, David R.; Aleksunes, Lauren M.; Thomas, Russell S.; Applegate, Dawn; Klaassen, Curtis D.; Corton, J. Christopher

    2015-01-01

    The nuclear receptor family member constitutive activated receptor (CAR) is activated by structurally diverse drugs and environmentally-relevant chemicals leading to transcriptional regulation of genes involved in xenobiotic metabolism and transport. Chronic activation of CAR increases liver cancer incidence in rodents, whereas suppression of CAR can lead to steatosis and insulin insensitivity. Here, analytical methods were developed to screen for chemical treatments in a gene expression compendium that lead to alteration of CAR activity. A gene expression biomarker signature of 83 CAR-dependent genes was identified using microarray profiles from the livers of wild-type and CAR-null mice after exposure to three structurally-diverse CAR activators (CITCO, phenobarbital, TCPOBOP). A rank-based algorithm (Running Fisher’s algorithm (p-value ≤ 10-4)) was used to evaluate the similarity between the CAR biomarker signature and a test set of 28 and 32 comparisons positive or negative, respectively, for CAR activation; the test resulted in a balanced accuracy of 97%. The biomarker signature was used to identify chemicals that activate or suppress CAR in an annotated mouse liver/primary hepatocyte gene expression database of ~1850 comparisons. CAR was activated by 1) activators of the aryl hydrocarbon receptor (AhR) in wild-type but not AhR-null mice, 2) pregnane X receptor (PXR) activators in wild-type and to lesser extents in PXR-null mice, and 3) activators of PPARα in wild-type and PPARα-null mice. CAR was consistently activated by five conazole fungicides and four perfluorinated compounds. Comparison of effects in wild-type and CAR-null mice showed that the fungicide propiconazole increased liver weight and hepatocyte proliferation in a CAR-dependent manner, whereas the perfluorinated compound perfluorooctanoic acid (PFOA) increased these endpoints in a CAR-independent manner. A number of compounds suppressed CAR coincident with increases in markers of

  11. Biologic and immunohistochemical analysis of interleukin-6 expression in vivo. Constitutive and induced expression in murine polymorphonuclear and mononuclear phagocytes.

    PubMed Central

    Terebuh, P. D.; Otterness, I. G.; Strieter, R. M.; Lincoln, P. M.; Danforth, J. M.; Kunkel, S. L.; Chensue, S. W.

    1992-01-01

    Interleukin-6 (IL-6) is considered an important multifunctional cytokine involved in the regulation of a variety of cellular responses, including the induction of acute-phase protein synthesis, lymphocyte activation, and hematopoiesis. In vitro studies have identified many cells that can produce IL-6, but the cellular sources under physiologic conditions have yet to be identified. Using immunoaffinity purified goat anti-murine IL-6, the authors performed immunohistochemical studies to localize cells expressing IL-6 in selected organs of normal and endotoxin challenged NIH-Swiss outbred mice. In the blood, findings were correlated with cell-associated bioactivity using the standard B9 cell proliferation assay. In normal mice, constitutive expression was seen in granulocytes, monocytes and their precursors as well as in bone marrow and splenic stromal macrophages. Hepatic macrophages were negative, as were lymphocytes, megakaryocytes, erythroid precursors, and endothelial cells. In the absence of significant serum levels of IL-6, cell-associated IL-6 bioactivity was detected in circulating polymorphonuclear leukocytes (PMNs), but not lymphocytes. After endotoxin challenge, there was a threefold increase in PMN IL-6 content from 1 to 3 hours followed by almost complete depletion at 6 hours. This correlated well with a threefold increase of IL-6 mRNA in the bone marrow followed by a decrease at 6 hours. This pattern also correlated with serum levels of IL-6, which peaked at 3 hours and dropped significantly by 6 hours. By 24 hours, cell-associated IL-6 showed recovery with no increase in serum levels. In vivo findings showing IL-6 expression in bone marrow macrophages support in vitro studies suggesting a role for IL-6 in hematopoiesis. Furthermore, PMNs as well as macrophages are likely important sources of IL-6 during inflammatory and septic states. Images Figure 1 Figure 5 PMID:1372159

  12. Ectopic expression of CsCTR1, a cucumber CTR-like gene, attenuates constitutive ethylene signaling in an Arabidopsis ctr1-1 mutant and expression pattern analysis of CsCTR1 in cucumber (Cucumis sativus).

    PubMed

    Bie, Beibei; Sun, Jin; Pan, Junsong; He, Huanle; Cai, Run

    2014-09-15

    The gaseous plant hormone ethylene regulates many aspects of plant growth, development and responses to the environment. Constitutive triple response 1 (CTR1) is a central regulator involved in the ethylene signal transduction pathway. To obtain a better understanding of this particular pathway in cucumber, the cDNA-encoding CTR1 (designated CsCTR1) was isolated from cucumber. A sequence alignment and phylogenetic analyses revealed that CsCTR1 has a high degree of homology with other plant CTR1 proteins. The ectopic expression of CsCTR1 in the Arabidopsis ctr1-1 mutant attenuates constitutive ethylene signaling of this mutant, suggesting that CsCTR1 indeed performs its function as negative regulator of the ethylene signaling pathway. CsCTR1 is constitutively expressed in all of the examined cucumber organs, including roots, stems, leaves, shoot apices, mature male and female flowers, as well as young fruits. CsCTR1 expression gradually declined during male flower development and increased during female flower development. Additionally, our results indicate that CsCTR1 can be induced in the roots, leaves and shoot apices by external ethylene. In conclusion, this study provides a basis for further studies on the role of CTR1 in the biological processes of cucumber and on the molecular mechanism of the cucumber ethylene signaling pathway.

  13. Photosynthetic gene expression in higher plants.

    PubMed

    Berry, James O; Yerramsetty, Pradeep; Zielinski, Amy M; Mure, Christopher M

    2013-11-01

    Within the chloroplasts of higher plants and algae, photosynthesis converts light into biological energy, fueling the assimilation of atmospheric carbon dioxide into biologically useful molecules. Two major steps, photosynthetic electron transport and the Calvin-Benson cycle, require many gene products encoded from chloroplast as well as nuclear genomes. The expression of genes in both cellular compartments is highly dynamic and influenced by a diverse range of factors. Light is the primary environmental determinant of photosynthetic gene expression. Working through photoreceptors such as phytochrome, light regulates photosynthetic genes at transcriptional and posttranscriptional levels. Other processes that affect photosynthetic gene expression include photosynthetic activity, development, and biotic and abiotic stress. Anterograde (from nucleus to chloroplast) and retrograde (from chloroplast to nucleus) signaling insures the highly coordinated expression of the many photosynthetic genes between these different compartments. Anterograde signaling incorporates nuclear-encoded transcriptional and posttranscriptional regulators, such as sigma factors and RNA-binding proteins, respectively. Retrograde signaling utilizes photosynthetic processes such as photosynthetic electron transport and redox signaling to influence the expression of photosynthetic genes in the nucleus. The basic C3 photosynthetic pathway serves as the default form used by most of the plant species on earth. High temperature and water stress associated with arid environments have led to the development of specialized C4 and CAM photosynthesis, which evolved as modifications of the basic default expression program. The goal of this article is to explain and summarize the many gene expression and regulatory processes that work together to support photosynthetic function in plants.

  14. Constitutive expression of nitrogen fixation (nif) genes of Klebsiella pneumoniae due to a DNA duplication.

    PubMed

    Sibold, L; Elmerich, C

    1982-01-01

    A spontaneous mutant of Klebsiella pneumoniae exhibiting nitrogen fixing activity in the presence of ammonia was isolated from a nifL ::Mu mutant. The main features of the nif constitutive mutation, designated nif-8388, were as follows: (i) neither ammonia nor bases repressed, but amino acids partially repressed, nitrogen fixation; (ii) the mutation caused an escape from the regulatory effect of glnA and glnG mutations of K. pneumoniae but not that of a glnF mutation; (iii) it enabled the activation of the nifH -lac fusion in the presence of oxygen with or without ammonia and a nifL -lac fusion in the presence of ammonia without oxygen; (iv) the mutation allowed nitrogen fixation at 37 degrees C when plasmid-borne. Restriction analysis and Southern hybridization using Mu DNA and the 8.1-kb nifQBALF EcoRI fragment as probes demonstrated that the nif-8388 mutation was a tandem duplication of 10 kb in the nifL region in which no Mu DNA was present. This duplication led to an operon fusion between nifLA and his since Nifc expression was shown to be increased with a specific inducer of the his operon. These results provide further evidence that the nifA product is a nif-specific activator, and that the nifL product is involved in oxygen repression and temperature control. In addition, they suggest that there is an autoactivation of nifLA transcription by the nifA product and that glnF could act in nif regulation by a mechanism other than the glnG-mediated control of nifLA transcription. PMID:6327278

  15. Gene transfer and expression in plants.

    PubMed

    Lorence, Argelia; Verpoorte, Robert

    2004-01-01

    Until recently, agriculture and plant breeding relied solely on the accumulated experience of generations of farmers and breeders that is, on sexual transfer of genes between plant species. However, recent developments in plant molecular biology and genomics now give us access to knowledge and understanding of plant genomes and the possibility of modifying them. This chapter presents an updated overview of the two most powerful technologies for transferring genetic material (DNA) into plants: Agrobacterium-mediated transformation and microparticle bombardment (biolistics). Some of the topics that are discussed in detail are the main variables controlling the transformation efficiency that can be achieved using each one of these approaches; the advantages and limitations of each methodology; transient versus stable transformation approaches; the potential of some in planta transformation systems; alternatives to developing transgenic plants without selection markers; the availability of diverse genetic tools generated as part of the genome sequencing of different plant species; transgene expression, gene silencing, and their association with regulatory elements; and prospects and ways to possibly overcome some transgene expression difficulties, in particular the use of matrix-attachment regions (MARs).

  16. The capability to synthesize phytochelatins and the presence of constitutive and functional phytochelatin synthases are ancestral (plesiomorphic) characters for basal land plants.

    PubMed

    Petraglia, Alessandro; De Benedictis, Maria; Degola, Francesca; Pastore, Giovanni; Calcagno, Margherita; Ruotolo, Roberta; Mengoni, Alessio; Sanità di Toppi, Luigi

    2014-03-01

    Bryophytes, a paraphyletic group which includes liverworts, mosses, and hornworts, have been stated as land plants that under metal stress (particularly cadmium) do not synthesize metal-binding peptides such as phytochelatins. Moreover, very little information is available to date regarding phytochelatin synthesis in charophytes, postulated to be the direct ancestors of land plants, or in lycophytes, namely very basal tracheophytes. In this study, it was hypothesized that basal land plants and charophytes have the capability to produce phytochelatins and possess constitutive and functional phytochelatin synthases. To verify this hypothesis, twelve bryophyte species (six liverworts, four mosses, and two hornworts), three charophytes, and two lycophyte species were exposed to 0-36 μM cadmium for 72 h, and then assayed for: (i) glutathione and phytochelatin quali-quantitative content by HPLC and mass spectrometry; (ii) the presence of putative phytochelatin synthases by western blotting; and (iii) in vitro activity of phytochelatin synthases. Of all the species tested, ten produced phytochelatins in vivo, while the other seven did not. The presence of a constitutively expressed and functional phytochelatin synthase was demonstrated in all the bryophyte lineages and in the lycophyte Selaginella denticulata, but not in the charophytes. Hence, current knowledge according to phytochelatins have been stated as being absent in bryophytes was therefore confuted by this work. It is argued that the capability to synthesize phytochelatins, as well as the presence of active phytochelatin synthases, are ancestral (plesiomorphic) characters for basal land plants. PMID:24449382

  17. The capability to synthesize phytochelatins and the presence of constitutive and functional phytochelatin synthases are ancestral (plesiomorphic) characters for basal land plants.

    PubMed

    Petraglia, Alessandro; De Benedictis, Maria; Degola, Francesca; Pastore, Giovanni; Calcagno, Margherita; Ruotolo, Roberta; Mengoni, Alessio; Sanità di Toppi, Luigi

    2014-03-01

    Bryophytes, a paraphyletic group which includes liverworts, mosses, and hornworts, have been stated as land plants that under metal stress (particularly cadmium) do not synthesize metal-binding peptides such as phytochelatins. Moreover, very little information is available to date regarding phytochelatin synthesis in charophytes, postulated to be the direct ancestors of land plants, or in lycophytes, namely very basal tracheophytes. In this study, it was hypothesized that basal land plants and charophytes have the capability to produce phytochelatins and possess constitutive and functional phytochelatin synthases. To verify this hypothesis, twelve bryophyte species (six liverworts, four mosses, and two hornworts), three charophytes, and two lycophyte species were exposed to 0-36 μM cadmium for 72 h, and then assayed for: (i) glutathione and phytochelatin quali-quantitative content by HPLC and mass spectrometry; (ii) the presence of putative phytochelatin synthases by western blotting; and (iii) in vitro activity of phytochelatin synthases. Of all the species tested, ten produced phytochelatins in vivo, while the other seven did not. The presence of a constitutively expressed and functional phytochelatin synthase was demonstrated in all the bryophyte lineages and in the lycophyte Selaginella denticulata, but not in the charophytes. Hence, current knowledge according to phytochelatins have been stated as being absent in bryophytes was therefore confuted by this work. It is argued that the capability to synthesize phytochelatins, as well as the presence of active phytochelatin synthases, are ancestral (plesiomorphic) characters for basal land plants.

  18. PLANEX: the plant co-expression database

    PubMed Central

    2013-01-01

    Background The PLAnt co-EXpression database (PLANEX) is a new internet-based database for plant gene analysis. PLANEX (http://planex.plantbioinformatics.org) contains publicly available GeneChip data obtained from the Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information (NCBI). PLANEX is a genome-wide co-expression database, which allows for the functional identification of genes from a wide variety of experimental designs. It can be used for the characterization of genes for functional identification and analysis of a gene’s dependency among other genes. Gene co-expression databases have been developed for other species, but gene co-expression information for plants is currently limited. Description We constructed PLANEX as a list of co-expressed genes and functional annotations for Arabidopsis thaliana, Glycine max, Hordeum vulgare, Oryza sativa, Solanum lycopersicum, Triticum aestivum, Vitis vinifera and Zea mays. PLANEX reports Pearson’s correlation coefficients (PCCs; r-values) that distribute from a gene of interest for a given microarray platform set corresponding to a particular organism. To support PCCs, PLANEX performs an enrichment test of Gene Ontology terms and Cohen’s Kappa value to compare functional similarity for all genes in the co-expression database. PLANEX draws a cluster network with co-expressed genes, which is estimated using the k-mean method. To construct PLANEX, a variety of datasets were interpreted by the IBM supercomputer Advanced Interactive eXecutive (AIX) in a supercomputing center. Conclusion PLANEX provides a correlation database, a cluster network and an interpretation of enrichment test results for eight plant species. A typical co-expressed gene generates lists of co-expression data that contain hundreds of genes of interest for enrichment analysis. Also, co-expressed genes can be identified and cataloged in terms of comparative genomics by using the ‘Co-expression gene compare’ feature

  19. Slower skeletal muscle phenotypes are critical for constitutive expression of Hsp70 in overloaded rat plantaris muscle.

    PubMed

    O'Neill, David E T; Aubrey, F Kris; Zeldin, David A; Michel, Robin N; Noble, Earl G

    2006-03-01

    Heat shock protein 72 (Hsp70) is constitutively expressed in rat hindlimb muscles, reportedly in proportion to their content of type I myosin heavy chain. This distribution pattern has been suggested to result from the higher recruitment and activity of such muscles and/or a specific relationship between myosin phenotype and Hsp70 content. To differentiate between these possibilities, the fiber-specific distribution of Hsp70 was examined in male Sprague-Dawley rat plantaris under control conditions, following a fast-to-slow phenotypic shift in response to surgically induced overload (O) and in response to O when the phenotypic shift was prevented by 3,5,3'-triiodo-dl-thyronine administration. Constitutive expression of Hsp70 was restricted to type I and IIa fibers in plantaris from control rats, and this fiber-specific pattern of expression was maintained following O of up to 28 days, although Hsp70 content in the O muscle doubled. When O (for 40 days) of the plantaris was combined with 3,5,3'-triiodo-dl-thyronine administration, despite typical hypertrophy in the overloaded plantaris, prevention of the normal phenotypic transformation also blocked the increased expression of Hsp70 observed in euthyroid controls. Collectively, these data suggest that chronic changes in constitutive expression of Hsp70 with altered contractile activity appear critically dependent on fast-to-slow phenotypic remodeling. PMID:16293703

  20. New Vectors for Chromosomal Integration Enable High-Level Constitutive or Inducible Magnetosome Expression of Fusion Proteins in Magnetospirillum gryphiswaldense

    PubMed Central

    Borg, Sarah; Hofmann, Julia; Pollithy, Anna; Lang, Claus

    2014-01-01

    The alphaproteobacterium Magnetospirillum gryphiswaldense biomineralizes magnetosomes, which consist of monocrystalline magnetite cores enveloped by a phospholipid bilayer containing specific proteins. Magnetosomes represent magnetic nanoparticles with unprecedented magnetic and physicochemical characteristics. These make them potentially useful in a number of biotechnological and biomedical applications. Further functionalization can be achieved by expression of foreign proteins via genetic fusion to magnetosome anchor peptides. However, the available genetic tool set for strong and controlled protein expression in magnetotactic bacteria is very limited. Here, we describe versatile vectors for either inducible or high-level constitutive expression of proteins in M. gryphiswaldense. The combination of an engineered native PmamDC promoter with a codon-optimized egfp gene (Mag-egfp) resulted in an 8-fold increase in constitutive expression and in brighter fluorescence. We further demonstrate that the widely used Ptet promoter is functional and tunable in M. gryphiswaldense. Stable and uniform expression of the EGFP and β-glucuronidase (GusA) reporters was achieved by single-copy chromosomal insertion via Tn5-mediated transposition. In addition, gene duplication by Mag-EGFP–EGFP fusions to MamC resulted in further increased magnetosome expression and fluorescence. Between 80 and 210 (for single MamC–Mag-EGFP) and 200 and 520 (for MamC–Mag-EGFP–EGFP) GFP copies were estimated to be expressed per individual magnetosome particle. PMID:24532068

  1. Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase

    NASA Technical Reports Server (NTRS)

    Perera, Imara Y.; Love, John; Heilmann, Ingo; Thompson, William F.; Boss, Wendy F.; Brown, C. S. (Principal Investigator)

    2002-01-01

    To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP(3)) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP(3). The transgenic cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP(3) compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP(3) in both wild-type and transgenic cells. However, even with stimulation, InsP(3) levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP(3) signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)), the lipid precursor of InsP(3), was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP(2) metabolism showed that the activity of the PtdInsP(2)-hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of (32)P into PtdInsP(2) in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP(2) synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP(2) synthesis as a regulatory step in this system.

  2. Hypoxia reduces constitutive and TNF-{alpha}-induced expression of monocyte chemoattractant protein-1 in human proximal renal tubular cells

    SciTech Connect

    Li Xuan; Kimura, Hideki . E-mail: hkimura@fmsrsa.fukui-med.ac.jp; Hirota, Kiichi; Sugimoto, Hidehiro; Yoshida, Haruyoshi

    2005-10-07

    Chronic hypoxia has been reported to be associated with macrophage infiltration in progressive forms of kidney disease. Here, we investigated the regulatory effects of hypoxia on constitutive and TNF-{alpha}-stimulated expression of monocyte chemoattractant protein-1 (MCP-1) in cultured human proximal renal tubular cells (HPTECs). Hypoxia reduced constitutive MCP-1 expression at the mRNA and protein levels in a time-dependent fashion for up to 48 h. Hypoxia also inhibited MCP-1 up-regulation by TNF-{alpha}. Treatment with actinomycin D showed that hypoxic down-regulation of MCP-1 expression resulted mainly from a decrease in the transcription but not the mRNA stability. Immunoblot and immunofluorescence analyses revealed that treatment with hypoxia or an iron chelator, desferrioxamine, induced nuclear accumulation of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) in HPTECs. Desferrioxamine mimicked hypoxia in the reduction of MCP-1 expression. However, overexpression of a dominant negative form of HIF-1{alpha} did not abolish the hypoxia-induced reduction of MCP-1 expression in HPTECs. These results suggest that hypoxia is an important negative regulator of monocyte chemotaxis to the renal inflamed interstitium, by reducing MCP-1 expression partly via hypoxia-activated signals other than the HIF-1 pathway.

  3. Virus-Derived Vectors for the Expression of Multiple Proteins in Plants.

    PubMed

    Saxena, Pooja; Thuenemann, Eva C; Sainsbury, Frank; Lomonossoff, George P

    2016-01-01

    This chapter constitutes a practical guide to using the "pEAQ" vector series for transient or stable expression of one or more protein(s) in Nicotiana benthamiana plants. The pEAQ vectors are a series of small binary vectors designed for controlled expression of multiple proteins in plants. To achieve high levels of expression, an expression system based on translational enhancement by the untranslated regions of RNA-2 from cowpea mosaic virus (CPMV), named CPMV-HT, is used. The expression vector pEAQ-HT combines the user-friendly pEAQ plasmid with CPMV-HT to provide a system for high-level expression of proteins in plants. PMID:26614280

  4. Constitutive expression of the maize genes B1 and C1 in transgenic Hi II maize results in differential tissue pigmentation and generates resistance to Helicoverpa zea.

    PubMed

    Johnson, Eric T; Berhow, Mark A; Dowd, Patrick F

    2010-02-24

    Anthocyanin biosynthesis in maize protects tissues from biotic and abiotic stresses. Constitutive expression of the maize B1 and C1 genes, which induces anthocyanin biosynthesis, resulted in transgenic plants with varied phenotypes. Some colored leaves were substantially resistant to thrips damage, while only leaves with the highest levels of cyanidin, the predominant anthocyanidin detected in all colored transgenic tissues, were resistant to corn earworm (CEW) larvae. Colored anthers were resistant to CEW feeding, and reductions in CEW growth were significantly correlated to levels of cyanidin in the anthers. Cyanidin chloride and cyanidin-3-glucoside chloride added to insect diet slowed the growth of CEW larvae. Attempts to produce 3'5'-hydroxylated anthocyanins in colored maize with the expression of a petunia F3'5'H hydroxylase gene were unsuccessful. PMID:20108901

  5. Expression of a constitutively active nitrate reductase variant in tobacco reduces tobacco-specific nitrosamine accumulation in cured leaves and cigarette smoke.

    PubMed

    Lu, Jianli; Zhang, Leichen; Lewis, Ramsey S; Bovet, Lucien; Goepfert, Simon; Jack, Anne M; Crutchfield, James D; Ji, Huihua; Dewey, Ralph E

    2016-07-01

    Burley tobaccos (Nicotiana tabacum) display a nitrogen-use-deficiency phenotype that is associated with the accumulation of high levels of nitrate within the leaf, a trait correlated with production of a class of compounds referred to as tobacco-specific nitrosamines (TSNAs). Two TSNA species, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), have been shown to be strong carcinogens in numerous animal studies. We investigated the potential of molecular genetic strategies to lower nitrate levels in burley tobaccos by overexpressing genes encoding key enzymes of the nitrogen-assimilation pathway. Of the various constructs tested, only the expression of a constitutively active nitrate reductase (NR) dramatically decreased free nitrate levels in the leaves. Field-grown tobacco plants expressing this NR variant exhibited greatly reduced levels of TSNAs in both cured leaves and mainstream smoke of cigarettes made from these materials. Decreasing leaf nitrate levels via expression of a constitutively active NR enzyme represents an exceptionally promising means for reducing the production of NNN and NNK, two of the most well-documented animal carcinogens found in tobacco products. PMID:26800860

  6. d-alpha-tocopherol inhibits collagen alpha 1(I) gene expression in cultured human fibroblasts. Modulation of constitutive collagen gene expression by lipid peroxidation.

    PubMed Central

    Houglum, K; Brenner, D A; Chojkier, M

    1991-01-01

    Ascorbic acid stimulates collagen gene transcription in cultured fibroblasts, and this effect is mediated through the induction of lipid peroxidation by ascorbic acid. Quiescent cultured fibroblasts in the absence of ascorbic acid have a high constitutive level of collagen production, but the mechanisms of collagen gene regulation in this unstimulated state are not known. Because lipid peroxidation also occurs in normal cells, we wondered if lipid peroxidation plays a role in the regulation of basal collagen gene expression. Inhibition of lipid peroxidation in cultured human fibroblasts with d-alpha-tocopherol or methylene blue decreased the synthesis of collagen, the steady-state levels of procollagen alpha 1(I) mRNA and the transcription of the procollagen alpha 1(I) gene. This effect on collagen gene expression was selective and not associated with cellular toxicity. Thus, these experiments suggest a role for lipid peroxidation in the modulation of constitutive collagen gene expression. Images PMID:2040703

  7. Constitutive activation of gene expression by thyroid hormone receptor results from reversal of p53-mediated repression.

    PubMed Central

    Qi, J S; Desai-Yajnik, V; Yuan, Y; Samuels, H H

    1997-01-01

    Thyroid hormone receptor (T3R) is a member of the steroid hormone receptor gene family of nuclear hormone receptors. In most cells T3R activates gene expression only in the presence of its ligand, L-triiodothyronine (T3). However, in certain cell types (e.g., GH4C1 cells) expression of T3R leads to hormone-independent constitutive activation. This activation by unliganded T3R occurs with a variety of gene promoters and appears to be independent of the binding of T3R to specific thyroid hormone response elements (TREs). Previous studies indicate that this constitutive activation results from the titration of an inhibitor of transcription. Since the tumor suppresser p53 is capable of repressing a wide variety of gene promoters, we considered the possibility that the inhibitor is p53. Evidence to support this comes from studies indicating that expression of p53 blocks T3R-mediated constitutive activation in GH4C1 cells. In contrast with hormone-independent activation by T3R, p53 had little or no effect on T3-dependent stimulation which requires TREs. In addition, p53 mutants which oligomerize with wild-type p53 and interfere with its function also increase promoter activity. This enhancement is of similar magnitude to but is not additive with the stimulation mediated by unliganded T3R, suggesting that they target the same factor. Since p53 mutants are known to target wild-type p53 in the cell, this suggests that T3R also interacts with p53 in vivo and that endogenous levels of p53 act to suppress promoter activity. Evidence supporting both functional and physical interactions of T3R and p53 in the cell is presented. The DNA binding domain (DBD) of T3R is important in mediating constitutive activation, and the receptor DBD appears to functionally interact with the N terminus of p53 in the cell. In vitro binding studies indicate that the T3R DBD is important for interaction of T3R with p53 and that this interaction is reduced by T3. These findings are consistent with

  8. IFN-α Is Constitutively Expressed in the Human Thymus, but Not in Peripheral Lymphoid Organs

    PubMed Central

    Colantonio, Arnaud D.; Epeldegui, Marta; Jesiak, Maria; Jachimowski, Loes; Blom, Bianca; Uittenbogaart, Christel H.

    2011-01-01

    Type I interferons have been typically studied for their effects in the context of bacterial or viral infections. However in this report, we provide evidence that Interferon-alpha (IFN-α) expressing cells are present in the thymus in the absence of infection. We show that pDC express the highest level of IFN-α and that MxA, which is exclusively expressed after engagement of the type I IFN receptor by IFN-α/β, is expressed in normal fetal and post-natal thymus, but not in the periphery. The highest level of MxA is expressed in mature thymocytes and pDC located in the medulla and at the cortico-medullary junction. The anti-microbial peptide LL-37, which is expressed in the thymus, when complexed with eukaryotic nucleic acids, induces the secretion of IFN-α by thymic pDC. This results in the upregulation of MxA expression in responsive thymocytes. We propose that the secretion of IFN-α in the thymus may function to regulate the rate of T cell development and modulate the requirements for the selection of developing T cells. PMID:21904619

  9. Plant trait expression responds to establishment timing.

    PubMed

    Brandt, Angela J; Leahy, S Conor; Zimmerman, Nicole M; Burns, Jean H

    2015-06-01

    Trait divergence between co-occurring individuals could decrease the strength of competition between these individuals, thus promoting their coexistence. To test this hypothesis, we manipulated establishment timing for four congeneric pairs of perennial plants and assessed trait plasticity. Because soil conditions can affect trait expression and competition, we grew the plants in field-collected soil from each congener. Competition was generally weak across species, but the order of establishment affected divergence in biomass between potmates for three congeneric pairs. The type of plastic response differed among genera, with trait means of early-establishing individuals of Rumex and Solanum spp. differing from late-establishing individuals, and trait divergence between potmates of Plantago and Trifolium spp. depending on which species established first. Consistent with adaptive trait plasticity, higher specific leaf area (SLA) and root-shoot ratio in Rumex spp. established later suggest that these individuals were maximizing their ability to capture light and soil resources. Greater divergence in SLA correlated with increased summed biomass of competitors, which is consistent with trait divergence moderating the strength of competition for some species. Species did not consistently perform better in conspecific or congener soil, but soil type influenced the effect of establishment order. For example, biomass divergence between Rumex potmates was greater in R. obtusifolius soil regardless of which species established first. These results suggest that plant responses to establishment timing act in a species-specific fashion, potentially enhancing coexistence in plant communities.

  10. Plant trait expression responds to establishment timing.

    PubMed

    Brandt, Angela J; Leahy, S Conor; Zimmerman, Nicole M; Burns, Jean H

    2015-06-01

    Trait divergence between co-occurring individuals could decrease the strength of competition between these individuals, thus promoting their coexistence. To test this hypothesis, we manipulated establishment timing for four congeneric pairs of perennial plants and assessed trait plasticity. Because soil conditions can affect trait expression and competition, we grew the plants in field-collected soil from each congener. Competition was generally weak across species, but the order of establishment affected divergence in biomass between potmates for three congeneric pairs. The type of plastic response differed among genera, with trait means of early-establishing individuals of Rumex and Solanum spp. differing from late-establishing individuals, and trait divergence between potmates of Plantago and Trifolium spp. depending on which species established first. Consistent with adaptive trait plasticity, higher specific leaf area (SLA) and root-shoot ratio in Rumex spp. established later suggest that these individuals were maximizing their ability to capture light and soil resources. Greater divergence in SLA correlated with increased summed biomass of competitors, which is consistent with trait divergence moderating the strength of competition for some species. Species did not consistently perform better in conspecific or congener soil, but soil type influenced the effect of establishment order. For example, biomass divergence between Rumex potmates was greater in R. obtusifolius soil regardless of which species established first. These results suggest that plant responses to establishment timing act in a species-specific fashion, potentially enhancing coexistence in plant communities. PMID:25616649

  11. Expression of bacterial genes in plant cells.

    PubMed Central

    Fraley, R T; Rogers, S G; Horsch, R B; Sanders, P R; Flick, J S; Adams, S P; Bittner, M L; Brand, L A; Fink, C L; Fry, J S; Galluppi, G R; Goldberg, S B; Hoffmann, N L; Woo, S C

    1983-01-01

    Chimeric bacterial genes conferring resistance to aminoglycoside antibiotics have been inserted into the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid and introduced into plant cells by in vitro transformation techniques. The chimeric genes contain the nopaline synthase 5' and 3' regulatory regions joined to the genes for neomycin phosphotransferase type I or type II. The chimeric genes were cloned into an intermediate vector, pMON120, and inserted into pTiB6S3 by recombination and then introduced into petunia and tobacco cells by cocultivating A. tumefaciens cells with protoplast-derived cells. Southern hybridization was used to confirm the presence of the chimeric genes in the transformed plant tissues. Expression of the chimeric genes was determined by the ability of the transformed cells to proliferate on medium containing normally inhibitory levels of kanamycin (50 micrograms/ml) or other aminoglycoside antibiotics. Plant cells transformed by wild-type pTiB6S3 or derivatives carrying the bacterial neomycin phosphotransferase genes with their own promoters failed to grow under these conditions. The significance of these results for plant genetic engineering is discussed. Images PMID:6308651

  12. A new series of vectors for constitutive, inducible or repressible gene expression in Candida guilliermondii.

    PubMed

    Defosse, Tatiana A; Melin, Céline; Obando Montoya, Erika J; Lanoue, Arnaud; Foureau, Emilien; Glévarec, Gaëlle; Oudin, Audrey; Simkin, Andrew J; Crèche, Joël; Atehortùa, Lucia; Giglioli-Guivarc'h, Nathalie; Clastre, Marc; Courdavault, Vincent; Papon, Nicolas

    2014-06-20

    The biotechnological potential of C. guilliermondii is now well established. This yeast species currently benefits from the availability of a convenient molecular toolbox including recipient strains, selectable markers and optimized transformation protocols. However, the number of expression systems for biotechnological applications in C. guilliermondii remains limited. We have therefore developed and characterized a new series of versatile controllable expression vectors for this yeast. While previous studies firmly demonstrated that knock-out systems represent efficient genetic strategies to interrupt yeast biochemical pathways at a specific step in C. guilliermondii, the set of expression plasmids described in this study will provide new powerful opportunities to boost homologous or heterologous biosynthetic routes by fine controlled over-expression approaches. PMID:24709398

  13. High-level expression, purification and characterization of a constitutively active thromboxane A2 receptor polymorphic variant.

    PubMed

    Xu, Bing; Chakraborty, Raja; Eilers, Markus; Dakshinamurti, Shyamala; O'Neil, Joe D; Smith, Steven O; Bhullar, Rajinder P; Chelikani, Prashen

    2013-01-01

    G protein-coupled receptors (GPCRs) exhibit some level of basal signaling even in the absence of a bound agonist. This basal or constitutive signaling can have important pathophysiological roles. In the past few years, a number of high resolution crystal structures of GPCRs have been reported, including two crystal structures of constitutively active mutants (CAM) of the dim-light receptor, rhodopsin. The structural characterizations of CAMs are impeded by the lack of proper expression systems. The thromboxane A2 receptor (TP) is a GPCR that mediates vasoconstriction and promotes thrombosis in response to the binding of thromboxane. Here, we report on the expression and purification of a genetic variant and CAM in TP, namely A160T, using tetracycline-inducible HEK293S-TetR and HEK293S (GnTI¯)-TetR cell lines. Expression of the TP and the A160T genes in these mammalian cell lines resulted in a 4-fold increase in expression to a level of 15.8 ±0.3 pmol of receptor/mg of membrane protein. The receptors expressed in the HEK293S (GnTI(-))-TetR cell line showed homogeneous glycosylation. The functional yield of the receptors using a single step affinity purification was 45 µg/10⁶ cells. Temperature- dependent secondary structure changes of the purified TP and A160T receptors were characterized using circular dichroism (CD) spectropolarimetry. The CD spectra shows that the loss of activity or thermal sensitivity that was previously observed for the A160T mutant, is not owing to large unfolding of the protein but rather to a more subtle effect. This is the first study to report on the successful high-level expression, purification, and biophysical characterization of a naturally occurring, diffusible ligand activated GPCR CAM. PMID:24086743

  14. PmiRExAt: plant miRNA expression atlas database and web applications

    PubMed Central

    Gurjar, Anoop Kishor Singh; Panwar, Abhijeet Singh; Gupta, Rajinder; Mantri, Shrikant S.

    2016-01-01

    High-throughput small RNA (sRNA) sequencing technology enables an entirely new perspective for plant microRNA (miRNA) research and has immense potential to unravel regulatory networks. Novel insights gained through data mining in publically available rich resource of sRNA data will help in designing biotechnology-based approaches for crop improvement to enhance plant yield and nutritional value. Bioinformatics resources enabling meta-analysis of miRNA expression across multiple plant species are still evolving. Here, we report PmiRExAt, a new online database resource that caters plant miRNA expression atlas. The web-based repository comprises of miRNA expression profile and query tool for 1859 wheat, 2330 rice and 283 maize miRNA. The database interface offers open and easy access to miRNA expression profile and helps in identifying tissue preferential, differential and constitutively expressing miRNAs. A feature enabling expression study of conserved miRNA across multiple species is also implemented. Custom expression analysis feature enables expression analysis of novel miRNA in total 117 datasets. New sRNA dataset can also be uploaded for analysing miRNA expression profiles for 73 plant species. PmiRExAt application program interface, a simple object access protocol web service allows other programmers to remotely invoke the methods written for doing programmatic search operations on PmiRExAt database. Database URL: http://pmirexat.nabi.res.in. PMID:27081157

  15. PmiRExAt: plant miRNA expression atlas database and web applications.

    PubMed

    Gurjar, Anoop Kishor Singh; Panwar, Abhijeet Singh; Gupta, Rajinder; Mantri, Shrikant S

    2016-01-01

    High-throughput small RNA (sRNA) sequencing technology enables an entirely new perspective for plant microRNA (miRNA) research and has immense potential to unravel regulatory networks. Novel insights gained through data mining in publically available rich resource of sRNA data will help in designing biotechnology-based approaches for crop improvement to enhance plant yield and nutritional value. Bioinformatics resources enabling meta-analysis of miRNA expression across multiple plant species are still evolving. Here, we report PmiRExAt, a new online database resource that caters plant miRNA expression atlas. The web-based repository comprises of miRNA expression profile and query tool for 1859 wheat, 2330 rice and 283 maize miRNA. The database interface offers open and easy access to miRNA expression profile and helps in identifying tissue preferential, differential and constitutively expressing miRNAs. A feature enabling expression study of conserved miRNA across multiple species is also implemented. Custom expression analysis feature enables expression analysis of novel miRNA in total 117 datasets. New sRNA dataset can also be uploaded for analysing miRNA expression profiles for 73 plant species. PmiRExAt application program interface, a simple object access protocol web service allows other programmers to remotely invoke the methods written for doing programmatic search operations on PmiRExAt database.Database URL:http://pmirexat.nabi.res.in. PMID:27081157

  16. Contraction of the type I IFN locus and unusual constitutive expression of IFN-α in bats.

    PubMed

    Zhou, Peng; Tachedjian, Mary; Wynne, James W; Boyd, Victoria; Cui, Jie; Smith, Ina; Cowled, Christopher; Ng, Justin H J; Mok, Lawrence; Michalski, Wojtek P; Mendenhall, Ian H; Tachedjian, Gilda; Wang, Lin-Fa; Baker, Michelle L

    2016-03-01

    Bats harbor many emerging and reemerging viruses, several of which are highly pathogenic in other mammals but cause no clinical signs of disease in bats. To determine the role of interferons (IFNs) in the ability of bats to coexist with viruses, we sequenced the type I IFN locus of the Australian black flying fox, Pteropus alecto, providing what is, to our knowledge, the first gene map of the IFN region of any bat species. Our results reveal a highly contracted type I IFN family consisting of only 10 IFNs, including three functional IFN-α loci. Furthermore, the three IFN-α genes are constitutively expressed in unstimulated bat tissues and cells and their expression is unaffected by viral infection. Constitutively expressed IFN-α results in the induction of a subset of IFN-stimulated genes associated with antiviral activity and resistance to DNA damage, providing evidence for a unique IFN system that may be linked to the ability of bats to coexist with viruses.

  17. Contraction of the type I IFN locus and unusual constitutive expression of IFN-α in bats.

    PubMed

    Zhou, Peng; Tachedjian, Mary; Wynne, James W; Boyd, Victoria; Cui, Jie; Smith, Ina; Cowled, Christopher; Ng, Justin H J; Mok, Lawrence; Michalski, Wojtek P; Mendenhall, Ian H; Tachedjian, Gilda; Wang, Lin-Fa; Baker, Michelle L

    2016-03-01

    Bats harbor many emerging and reemerging viruses, several of which are highly pathogenic in other mammals but cause no clinical signs of disease in bats. To determine the role of interferons (IFNs) in the ability of bats to coexist with viruses, we sequenced the type I IFN locus of the Australian black flying fox, Pteropus alecto, providing what is, to our knowledge, the first gene map of the IFN region of any bat species. Our results reveal a highly contracted type I IFN family consisting of only 10 IFNs, including three functional IFN-α loci. Furthermore, the three IFN-α genes are constitutively expressed in unstimulated bat tissues and cells and their expression is unaffected by viral infection. Constitutively expressed IFN-α results in the induction of a subset of IFN-stimulated genes associated with antiviral activity and resistance to DNA damage, providing evidence for a unique IFN system that may be linked to the ability of bats to coexist with viruses. PMID:26903655

  18. Contraction of the type I IFN locus and unusual constitutive expression of IFN-α in bats

    PubMed Central

    Zhou, Peng; Tachedjian, Mary; Wynne, James W.; Boyd, Victoria; Smith, Ina; Cowled, Christopher; Ng, Justin H. J.; Mok, Lawrence; Michalski, Wojtek P.; Mendenhall, Ian H.; Tachedjian, Gilda; Baker, Michelle L.

    2016-01-01

    Bats harbor many emerging and reemerging viruses, several of which are highly pathogenic in other mammals but cause no clinical signs of disease in bats. To determine the role of interferons (IFNs) in the ability of bats to coexist with viruses, we sequenced the type I IFN locus of the Australian black flying fox, Pteropus alecto, providing what is, to our knowledge, the first gene map of the IFN region of any bat species. Our results reveal a highly contracted type I IFN family consisting of only 10 IFNs, including three functional IFN-α loci. Furthermore, the three IFN-α genes are constitutively expressed in unstimulated bat tissues and cells and their expression is unaffected by viral infection. Constitutively expressed IFN-α results in the induction of a subset of IFN-stimulated genes associated with antiviral activity and resistance to DNA damage, providing evidence for a unique IFN system that may be linked to the ability of bats to coexist with viruses. PMID:26903655

  19. CACCC and GATA-1 sequences make the constitutively expressed alpha-globin gene erythroid-responsive in mouse erythroleukemia cells.

    PubMed Central

    Ren, S; Li, J; Atweh, G F

    1996-01-01

    Although the human alpha-globin and beta-globin genes are co-regulated in adult life, they achieve the same end by very different mechanisms. For example, a transfected beta-globin gene is expressed in an inducible manner in mouse erythroleukemia (MEL) cells while a transfected alpha-globin gene is constitutively expressed at a high level in induced and uninduced MEL cells. Interestingly, when the alpha-globin gene is transferred into MEL cells as part of human chromosome 16, it is appropriately expressed in an inducible manner. We explored the basis for the lack of erythroid-responsiveness of the proximal regulatory elements of the human alpha-globin gene. Since the alpha-globin gene is the only functional human globin gene that lacks CACCC and GATA-1 motifs, we asked whether their addition to the alpha-globin promoter would make the gene erythroid-responsive in MEL cells. The addition of each of these binding sites to the alpha-globin promoter separately did not result in inducibility in MEL cells. However, when both sites were added together, the alpha-globin gene became inducible in MEL cells. This suggests that erythroid non-responsiveness of the alpha-globin gene results from the lack of erythroid binding sites and is not necessarily a function of the constitutively active, GC rich promoter. PMID:8628660

  20. Enhancement of ganoderic acid production by constitutively expressing Vitreoscilla hemoglobin gene in Ganoderma lucidum.

    PubMed

    Li, Huan-Jun; He, Yi-Long; Zhang, De-Huai; Yue, Tong-Hui; Jiang, Lu-Xi; Li, Na; Xu, Jun-Wei

    2016-06-10

    The Vitreoscilla hemoglobin (VHb) gene was expressed in Ganoderma lucidum to enhance antitumor ganoderic acid (GA) production. The effects of VHb expression on the accumulation of GAs and lanosterol (intermediate) and the transcription of GA biosynthesis genes were also investigated. In VHb-expressing G. lucidum, the maximum concentrations of four individual GAs (GA-S, GA-T, GA-Mk and GA-Me) were 19.1±1.8, 34.6±2.1, 191.5±13.1 and 45.2±2.8μg/100mg dry weight, respectively, which were 1.4-, 2.2, 1.9- and 2.0-fold higher than those obtained in the wild-type strain. Moreover, the maximum lanosterol concentration in the strain expressing VHb was 1.28-fold lower than that in the wild-type strain. The transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, and lanosterol synthase genes were up-regulated by 1.6-, 1.5-, and 1.6-fold, respectively, in the strain expressing VHb. This work is beneficial in developing an efficient fermentation process for the hyperproduction of GAs. PMID:27080449

  1. Sex steroid hormones regulate constitutive expression of Cyp2e1 in female mouse liver

    PubMed Central

    Cheng, Jie; Gonzalez, Frank J.

    2013-01-01

    CYP2E1 is of paramount toxicological significance because it metabolically activates a large number of low-molecular-weight toxicants and carcinogens. In this context, factors that interfere with Cyp2e1 regulation may critically affect xenobiotic toxicity and carcinogenicity. The aim of this study was to investigate the role of female steroid hormones in the regulation of CYP2E1, as estrogens and progesterone are the bases of contraceptives and hormonal replacement therapy in menopausal women. Interestingly, a fluctuation in the hepatic expression pattern of Cyp2e1 was revealed in the different phases of the estrous cycle of female mice, with higher Cyp2e1 expression at estrus (E) and lower at methestrus (ME), highly correlated with that in plasma gonadal hormone levels. Depletion of sex steroids by ovariectomy repressed Cyp2e1 expression to levels similar to those detected in males and cyclic females at ME. Hormonal supplementation brought Cyp2e1 expression back to levels detected at E. The role of progesterone appeared to be more prominent than that of 17β-estradiol. Progesterone-induced Cyp2e1 upregulation could be attributed to inactivation of the insulin/PI3K/Akt/FOXO1 signaling pathway. Tamoxifen, an anti-estrogen, repressed Cyp2e1 expression potentially via activation of the PI3K/Akt/FOXO1 and GH/STAT5b-linked pathways. The sex steroid hormone-related changes in hepatic Cyp2e1 expression were highly correlated with those observed in Hnf-1α, β-catenin, and Srebp-1c. In conclusion, female steroid hormones are clearly involved in the regulation of CYP2E1, thus affecting the metabolism of a plethora of toxicants and carcinogenic agents, conditions that may trigger several pathologies or exacerbate the outcomes of various pathophysiological states. PMID:23548611

  2. Human papilloma virus transformed CaSki cells constitutively express high levels of functional SerpinB2.

    PubMed

    Major, Lee; Schroder, Wayne A; Gardner, Joy; Fish, Richard J; Suhrbier, Andreas

    2011-02-01

    Many malignant tissues, including human papilloma virus (HPV)-associated cancers, express SerpinB2, also known as plasminogen activator inhibitor type-2 (PAI-2). Whether SerpinB2 is expressed by the HPV-transformed cancer cells, and if so, whether SerpinB2 is mutated or behaves aberrantly remains unclear. Here we show that HPV-transformed CaSki cells express high levels of constitutive wild-type SerpinB2, with cellular distribution, glycosylation, secretion, cleavage, induction and urokinase binding similar to that reported for primary cells. Neutralization of secreted SerpinB2 failed to affect CaSki cell migration or growth. Lentivirus-based over-expression of SerpinB2 also had no effect on growth, and we were unable to confirm a role for SerpinB2 in binding or regulating expression of the retinoblastoma protein. CaSki cells thus emerge as a useful tool for studying SerpinB2, with the physiological function of SerpinB2 expression by tumor cells remaining controversial. Using CaSki cells as a source of endogenous SerpinB2, we confirmed that SerpinB2 efficiently binds the proteasomal subunit member β1. PMID:20974129

  3. Constitutive and inducible expression of HLA class II determinants by human osteoblast-like cells in vitro.

    PubMed Central

    Skjødt, H; Hughes, D E; Dobson, P R; Russell, R G

    1990-01-01

    Activated immune cells release cytokines which modulate the activity of bone cells in vitro. Expression of major histocompatibility complex (HLA in humans) class II determinants on bone surface cells may be important in local immune cell activation. In this study, expression of HLA-DR and DQ by cultured human bone cells (HBC) derived from normal trabecular bone surfaces was assessed by fluorescence-activated cell sorter (FACS) analysis and immunoperoxidase techniques using monoclonal antibodies. A subset of HBC (10-30%) expressed DR constitutively while 5-15% displayed DQ during long-term culture. HBC lacked a number of monocyte and lymphocyte markers. In addition, both DR+ and DR- HBC (FACS separated) produced osteocalcin stimulated by 1,25-dihydroxyvitamin D2 (1,25(OH)2D3). This suggests that both phenotypes belong to the osteoblast lineage. The number of DR+ HBC was increased by interferon-gamma (IFN gamma; 40-95% DR+ cells) whereas DQ+ HBC remained unchanged or was slightly increased (5-20% DQ+ cells). Moreover, 1,25(OH)2D3 enhanced IFN gamma-induced DR expression and at high concentration (10(-7) M) augmented DR expression by itself. Other major osteotropic factors, parathyroid hormone, interleukin 1, and calcitonin, did not affect HBC DR expression. The findings suggest that HBC may participate in activation of the immune system and that some osteotropic factors may regulate this function. PMID:2110190

  4. Regulatory elements involved in constitutive and phorbol ester-inducible expression of the plasminogen activator inhibitor type 2 gene promoter.

    PubMed Central

    Cousin, E; Medcalf, R L; Bergonzelli, G E; Kruithof, E K

    1991-01-01

    Gene transcription rates and mRNA levels of plasminogen activator inhibitor type 2 (PAI-2) are markedly induced by the tumor promoting agent phorbol 12-myristate 13-acetate (PMA) in human HT1080 fibrosarcoma cells. To identify promoter elements required for basal-, and phorbol ester-inducible expression, deletion mutants of the PAI-1 promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene, were transiently expressed in HT1080 cells. Constitutive CAT activity was expressed from constructs containing more than 215 bp of promoter sequence, whereas deletion to position -91 bp abolished CAT gene expression. Treatment of transfected cells with PMA resulted in a three- to ten-fold increase in CAT expression from all constructs except from the construct shortened to position -91. DNAse1 protection analysis of the promoter region between -215 and the transcription initiation site revealed numerous protected regions, including two AP1-like binding sites (AP1a and AP1b) and one CRE-like element. Site-directed mutagenesis of the AP1a site or of the CRE-like site resulted in the loss of basal CAT activity and abolished the PMA effect, whereas mutagenesis of AP1b only partially inhibited basal and PMA-mediated expression. Our results suggest that the PAI-2 promoter contains at least two elements required for basal gene transcription and PMA-mediated induction. Images PMID:1650454

  5. Inducible and constitutive expression of pMOL28-encoded nickel resistance in Alcaligenes eutrophus N9A.

    PubMed Central

    Siddiqui, R A; Schlegel, H G; Meyer, M

    1988-01-01

    The nickel and cobalt resistance plasmid pMOL28 was transferred by conjugation from its natural host Alcaligenes eutrophus CH34 to the susceptible A. eutrophus N9A. Strain N9A and its pMOL28-containing transconjugant M220 were studied in detail. At a concentration of 3.0 mM NiCl2, the wild-type N9A did not grow, while M220 started to grow at its maximum exponential growth rate after a lag of 12 to 24 h. When grown in the presence of subinhibitory concentrations (0.5 mM) of nickel salt, M220 grew actively at 3 mM NiCl2 without a lag, indicating that nickel resistance is an inducible property. Expression of nickel resistance required active growth in the presence of nickel salts at a concentration higher than 0.05 mM. Two mutants of M220 were isolated which expressed nickel resistance constitutively. When the plasmids, pMOL28.1 and pMOL28.2, carried by the mutants were transferred to strains H16 and CH34, the transconjugants expressed constitutive nickel resistance. This indicates that the mutation is plasmid located. Both mutants expressed constitutive resistance to nickel and cobalt. Physiological studies revealed the following differences between strain N9A and its pMOL28.1-harboring mutant derivatives. (i) The uptake of 63NiCl2 occurred more rapidly in the susceptible strain and reached a 30- to 60-fold-higher amount that in the pMOL28.1-harboring mutant; (ii) in intact cells of the susceptible strain N9A, the cytoplasmic hydrogenase was inhibited by 1 to 5 nM NiCl2, whereas 10 mM Ni2+ was needed to inhibit the hydrogenase of mutant cells; (iii) the minimal concentration of nickel chloride for the derepressed synthesis of cytoplasmic hydrogenase was lower in strain N9A (1 to 3 microM) than in the constitutive mutant (8 to 10 microM). PMID:3410828

  6. 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

  7. FABP4-Cre mediated expression of constitutively active ChREBP protects against obesity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Carbohydrate response element binding protein (ChREBP) regulates cellular glucose and lipid homeostasis. Although ChREBP is highly expressed in many key metabolic tissues, the role of ChREBP in most of those tissues and consequent effects on whole-body glucose and lipid metabolism are not well under...

  8. Constitutive Expression of Pluripotency-Associated Genes in Mesodermal Progenitor Cells (MPCs)

    PubMed Central

    Pacini, Simone; Carnicelli, Vittoria; Trombi, Luisa; Montali, Marina; Fazzi, Rita; Lazzarini, Edoardo; Giannotti, Stefano; Petrini, Mario

    2010-01-01

    Background We recently characterized a progenitor of mesodermal lineage (MPCs) from the human bone marrow of adults or umbilical cord blood. These cells are progenitors able to differentiate toward mesenchymal, endothelial and cardiomyogenic lineages. Here we present an extensive molecular characterization of MPCs, from bone marrow samples, including 39 genes involved in stem cell machinery, differentiation and cell cycle regulation. Methodology/Principal Findings MPCs are cytofluorimetrically characterized and quantitative RT-PCR was performed to evaluate the gene expression profile, comparing it with MSCs and hESCs lines. Immunofluorescence and dot-blot analysis confirm qRT-PCR data. MPCs exhibit an increased expression of OCT4, NANOG, SALL4, FBX15, SPP1 and to a lesser extent c-MYC and KLF4, but lack LIN28 and SOX2. MPCs highly express SOX15. Conclusions/Significance MPCs express many pluripotency-associated genes and show a peculiar Oct-4 molecular circuit. Understanding this unique molecular mechanism could lead to identifying MPCs as feasible, long telomeres, target cells for reprogramming with no up-regulation of the p53 pathway. Furthermore MPCs are easily and inexpensively harvested from human bone marrow. PMID:20360837

  9. Constitutive IFNα/β signaling maintains expression of signaling intermediaries for efficient cytokine responses.

    PubMed

    Messina, Nicole L; Clarke, Christopher J P; Johnstone, Ricky W

    2016-01-01

    Interferons (IFNs) are a family of immunoregulatory cytokines with important roles in anti-viral and anti-tumor responses. Type I and II IFNs bind distinct receptors and are associated with different stages of the immune response. There is however, considerable crosstalk between these two cytokines with enhancement of IFNγ responses following IFNα/β priming and loss of IFNα/β receptor (IFNAR) resulting in diminished IFNγ responses. In this study, we sought to define the mechanism of crosstalk between the type I and II IFNs. Our previous reports demonstrated reduced expression of the canonically activated transcription factor signal transducer and activator of transcription (STAT)1, in cells lacking the IFNAR α chain (IFNAR1). Therefore, we used microarray analysis to determine whether reconstitution of STAT1 in IFNAR1-deficient cells was sufficient to restore IFNγ responses. We identified several biological pathways, including the MHC class I antigen presentation pathway, in which STAT1 reconstitution was able to significantly rescue IFNγ-mediated gene regulation in Ifnar1 (-/-) cells. Notably, we also found that in addition to low basal expression of STAT1, cells lacking the IFNAR1 also had aberrant expression of multiple other transcription factors and signaling intermediaries. The studies described herein demonstrate that basal and regulated expression of signaling intermediaries is a mechanism for crosstalk between cytokines including type I and II IFNs. PMID:27512617

  10. Constitutive patterns of gene expression regulated by RNA-binding proteins

    PubMed Central

    2014-01-01

    Background RNA-binding proteins regulate a number of cellular processes, including synthesis, folding, translocation, assembly and clearance of RNAs. Recent studies have reported that an unexpectedly large number of proteins are able to interact with RNA, but the partners of many RNA-binding proteins are still uncharacterized. Results We combined prediction of ribonucleoprotein interactions, based on catRAPID calculations, with analysis of protein and RNA expression profiles from human tissues. We found strong interaction propensities for both positively and negatively correlated expression patterns. Our integration of in silico and ex vivo data unraveled two major types of protein–RNA interactions, with positively correlated patterns related to cell cycle control and negatively correlated patterns related to survival, growth and differentiation. To facilitate the investigation of protein–RNA interactions and expression networks, we developed the catRAPID express web server. Conclusions Our analysis sheds light on the role of RNA-binding proteins in regulating proliferation and differentiation processes, and we provide a data exploration tool to aid future experimental studies. PMID:24401680

  11. Acclimation of the crucifer Eutrema salsugineum to phosphate limitation is associated with constitutively high expression of phosphate-starvation genes.

    PubMed

    Velasco, Vera Marjorie Elauria; Mansbridge, John; Bremner, Samantha; Carruthers, Kimberley; Summers, Peter S; Sung, Wilson W L; Champigny, Marc J; Weretilnyk, Elizabeth A

    2016-08-01

    Eutrema salsugineum, a halophytic relative of Arabidopsis thaliana, was subjected to varying phosphate (Pi) treatments. Arabidopsis seedlings grown on 0.05 mm Pi displayed shortened primary roots, higher lateral root density and reduced shoot biomass allocation relative to those on 0.5 mm Pi, whereas Eutrema seedlings showed no difference in lateral root density and shoot biomass allocation. While a low Fe concentration mitigated the Pi deficiency response for Arabidopsis, Eutrema root architecture was unaltered, but adding NaCl increased Eutrema lateral root density almost 2-fold. Eutrema and Arabidopsis plants grown on soil without added Pi for 4 weeks had low shoot and root Pi content. Pi-deprived, soil-grown Arabidopsis plants were stunted with senescing older leaves, whereas Eutrema plants were visually indistinguishable from 2.5 mm Pi-supplemented plants. Genes associated with Pi starvation were analysed by RT-qPCR. EsIPS2, EsPHT1;4 and EsPAP17 showed up-regulated expression in Pi-deprived Eutrema plants, while EsPHR1, EsWRKY75 and EsRNS1 showed no induction. Absolute quantification of transcripts indicated that PHR1, WRKY75 and RNS1 were expressed at higher levels in Eutrema plants relative to those in Arabidopsis regardless of external Pi. The low phenotypic plasticity Eutrema displays to Pi supply is consistent with adaptation to chronic Pi deprivation in its extreme natural habitat. PMID:27038434

  12. Constitutive and xenobiotics-induced expression of a novel CYP3A gene from zebrafish larva

    SciTech Connect

    Tseng, H.-P.; Hseu, Tzong-Hsiung; Buhler, Donald R.; Wang, W.-D.; Hu, C.-H. . E-mail: chhu@mail.ntou.edu.tw

    2005-06-15

    In mammals, CYP3A isozymes collectively comprise the largest portion of the liver and small intestinal CYP protein. They are involved in the metabolism of an extensive range of endogenous substrates and xenobiotics and make a significant contribution to the termination of the action of steroid hormones. A full-length cDNA of CYP3A gene, named CYP3A65, was cloned from zebrafish by RT-PCR. The CYP3A65 mRNA was initially transcribed only in the liver and intestine upon hatching of the zebrafish embryos. Like the human CYP3A genes, CYP3A65 transcription in the foregut region was enhanced by treatment of the zebrafish larvae with the steroid dexamethasone and the macrocyclic antibiotic rifampicin. Differing from mammalian CYP3A genes, CYP3A65 transcription was also elicited by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) during early larval stages. Repression of AHR2 translation by antisense morpholino oligonucleotides abrogated both of constitutive and TCDD-stimulated CYP3A65 transcription in larval intestine. These findings suggested that the AHR2 signaling pathway plays an essential role in CYP3A65 transcription.

  13. Expression Atlas update—an integrated database of gene and protein expression in humans, animals and plants

    PubMed Central

    Petryszak, Robert; Keays, Maria; Tang, Y. Amy; Fonseca, Nuno A.; Barrera, Elisabet; Burdett, Tony; Füllgrabe, Anja; Fuentes, Alfonso Muñoz-Pomer; Jupp, Simon; Koskinen, Satu; Mannion, Oliver; Huerta, Laura; Megy, Karine; Snow, Catherine; Williams, Eleanor; Barzine, Mitra; Hastings, Emma; Weisser, Hendrik; Wright, James; Jaiswal, Pankaj; Huber, Wolfgang; Choudhary, Jyoti; Parkinson, Helen E.; Brazma, Alvis

    2016-01-01

    Expression Atlas (http://www.ebi.ac.uk/gxa) provides information about gene and protein expression in animal and plant samples of different cell types, organism parts, developmental stages, diseases and other conditions. It consists of selected microarray and RNA-sequencing studies from ArrayExpress, which have been manually curated, annotated with ontology terms, checked for high quality and processed using standardised analysis methods. Since the last update, Atlas has grown seven-fold (1572 studies as of August 2015), and incorporates baseline expression profiles of tissues from Human Protein Atlas, GTEx and FANTOM5, and of cancer cell lines from ENCODE, CCLE and Genentech projects. Plant studies constitute a quarter of Atlas data. For genes of interest, the user can view baseline expression in tissues, and differential expression for biologically meaningful pairwise comparisons—estimated using consistent methodology across all of Atlas. Our first proteomics study in human tissues is now displayed alongside transcriptomics data in the same tissues. Novel analyses and visualisations include: ‘enrichment’ in each differential comparison of GO terms, Reactome, Plant Reactome pathways and InterPro domains; hierarchical clustering (by baseline expression) of most variable genes and experimental conditions; and, for a given gene-condition, distribution of baseline expression across biological replicates. PMID:26481351

  14. Expression of human milk proteins in plants.

    PubMed

    Lönnerdal, Bo

    2002-06-01

    Human milk proteins are believed to have a multitude of biological activities benefiting the newborn infant. Such functions include antibacterial and antiviral activities, enhancement of the immune system and increased nutrient absorption. To date, only breast-fed infants have been exposed to these proteins. However, by using genetic engineering it is now possible to express these proteins in plants, such as rice, at very high levels. Recombinant human milk proteins can subsequently be added to infant formula and baby foods. Prior to such addition, safety tests and efficacy trials need to be conducted. The safety tests will initially be done in rats and then in humans. The efficacy trials should also evaluate stability against heat treatment (processing), pH (stomach conditions) and proteolytic enzymes (digestion). To date, we have expressed recombinant human lactoferrin, lysozyme and alpha1-antitrypsin in rice at very high expression levels. These recombinant proteins showed a stability and activities similar to those of the native milk proteins, suggesting that they may be able to exert biological activities in infants when added to formula or baby foods.

  15. Conserved but Attenuated Parental Gene Expression in Allopolyploids: Constitutive Zinc Hyperaccumulation in the Allotetraploid Arabidopsis kamchatica

    PubMed Central

    Paape, Timothy; Hatakeyama, Masaomi; Shimizu-Inatsugi, Rie; Cereghetti, Teo; Onda, Yoshihiko; Kenta, Tanaka; Sese, Jun; Shimizu, Kentaro K.

    2016-01-01

    Allopolyploidization combines parental genomes and often confers broader species distribution. However, little is known about parentally transmitted gene expression underlying quantitative traits following allopolyploidization because of the complexity of polyploid genomes. The allopolyploid species Arabidopsis kamchatica is a natural hybrid of the zinc hyperaccumulator Arabidopsis halleri and of the nonaccumulator Arabidopsis lyrata. We found that A. kamchatica retained the ability to hyperaccumulate zinc from A. halleri and grows in soils with both low and high metal content. Hyperaccumulation of zinc by A. kamchatica was reduced to about half of A. halleri, but is 10-fold greater than A. lyrata. Homeologs derived from A. halleri had significantly higher levels of expression of genes such as HEAVY METAL ATPASE4 (HMA4), METAL TRANSPORTER PROTEIN1 and other metal ion transporters than those derived from A. lyrata, which suggests cis-regulatory differences. A. kamchatica has on average about half the expression of these genes compared with A. halleri due to fixed heterozygosity inherent in allopolyploids. Zinc treatment significantly changed the ratios of expression of 1% of homeologous pairs, including genes putatively involved in metal homeostasis. Resequencing data showed a significant reduction in genetic diversity over a large genomic region (290 kb) surrounding the HMA4 locus derived from the A. halleri parent compared with the syntenic A. lyrata-derived region, which suggests different evolutionary histories. We also estimated that three A. halleri-derived HMA4 copies are present in A. kamchatica. Our findings support a transcriptomic model in which environment-related transcriptional patterns of both parents are conserved but attenuated in the allopolyploids. PMID:27413047

  16. Constitutive Nuclear Expression of Dentin Matrix Protein 1 Fails to Rescue the Dmp1-null Phenotype*

    PubMed Central

    Lin, Shuxian; Zhang, Qi; Cao, Zhengguo; Lu, Yongbo; Zhang, Hua; Yan, Kevin; Liu, Ying; McKee, Marc D.; Qin, Chunlin; Chen, Zhi; Feng, Jian Q.

    2014-01-01

    Dentin matrix protein 1 (DMP1) plays multiple roles in bone, tooth, phosphate homeostasis, kidney, salivary gland, reproductive cycles, and the development of cancer. In vitro studies have indicated two different biological mechanisms: 1) as a matrix protein, DMP1 interacts with αvβ3 integrin and activates MAP kinase signaling; and 2) DMP1 serves as a transcription co-factor. In vivo studies have demonstrated its key role in osteocytes. This study attempted to determine whether DMP1 functions as a transcription co-factor and regulates osteoblast functions. For gene expression comparisons using adenovirus constructs, we targeted the expression of DMP1 either to the nucleus only by replacing the endogenous signal peptide with a nuclear localization signal (NLS) sequence (referred to as NLSDMP1) or to the extracellular matrix as the WT type (referred to as SPDMP1) in MC3T3 osteoblasts. High levels of DMP1 in either form greatly increased osteogenic gene expression in an identical manner. However, the targeted NLSDMP1 transgene driven by a 3.6-kb rat Col 1α1 promoter in the nucleus of osteoblasts and osteocytes failed to rescue the phenotyope of Dmp1-null mice, whereas the SPDMP1 transgene rescued the rickets defect. These studies support the notion that DMP1 functions as an extracellular matrix protein, rather than as a transcription co-factor in vivo. We also show that DMP1 continues its expression in osteoblasts during postnatal development and that the deletion of Dmp1 leads to an increase in osteoblast proliferation. However, poor mineralization in the metaphysis indicates a critical role for DMP1 in both osteoblasts and osteocytes. PMID:24917674

  17. Constitutive Expression and Enzymatic Cleavage of ICAM-1 in the Spontaneously Hypertensive Rat

    PubMed Central

    Tong, Sheng; Neboori, Hanmanth J.; Tran, Edward D.; Schmid-Schönbein, Geert W.

    2011-01-01

    Background/Aims: Leukocyte adhesion to the endothelium is abnormal in hypertension. We have recently shown that spontaneously hypertensive rats (SHRs) have circulating leukocytes with enhanced CD18 receptor cleavage. In the current study, we investigate expression levels of its counter receptor, intercellular adhesion molecule (ICAM-1), and its possible proteolytic cleavage in the SHR and control Wistar rat. Methods ICAM-1 was labeled on tissue sections with two antibodies targeting its extracellular and intracellular domains and evaluated by light absorption measurements. The in situ cleavage of ICAM-1 was assessed by treating vessel sections with matrix metalloproteinase (MMP)-7, MMP-9 and elastase. Results SHRs showed a significant increase in ICAM-1 expression in liver and kidney compared with Wistar rats. The liver and kidney glomeruli exhibit a discrepancy in label density between intra- and extracellular antibodies, which suggests that enzymatic cleavage may be a factor determining ICAM-1 distribution. MMP-7 and MMP-9, which are elevated in SHR plasma, and elastase, which has elevated activity in SHR neutrophils, cleave the extracellular domain of ICAM-1 when applied to the tissue. Conclusion ICAM-1 expression in SHRs is upregulated in a tissue-specific manner. Proteolytic cleavage of the extracellular domain of ICAM-1 and accumulation in kidney glomeruli may play a role in the renal involvement of inflammation. PMID:21464573

  18. Constitutive Expression of GATA4 Dramatically Increases the Cardiogenic Potential of D3 Mouse Embryonic Stem Cells

    PubMed Central

    Laemmle, Lillian L.; Cohen, Justus B.; Glorioso, Joseph C.

    2016-01-01

    The transcription factor GATA binding protein 4 (GATA4) is a vital regulator of cardiac programming that acts by inducing the expression of many different genes involved in cardiomyogenesis. Here we generated a D3 mouse embryonic stem cell line that constitutively expresses high levels of GATA4 and show that these cells have dramatically increased cardiogenic potential compared to an eGFP-expressing control cell line. Embryoid bodies (EB) derived from the D3-GATA4 line displayed increased levels of cardiac gene expression and showed more abundant cardiomyocyte differentiation than control eGFP EB. These cells and two additional lines expressing lower levels of GATA4 provide a platform to screen previously untested cardiac genes and gene combinations for their ability to further increase the efficiency of cardiomyocyte differentiation beyond that achieved by transgenic GATA4 alone. Non-integrative delivery of identified gene combinations will aid in the production of differentiated cells for the treatment of ischemic cardiomyopathy. PMID:27441042

  19. Constitutive expression of Yes-associated protein (Yap) in adult skeletal muscle fibres induces muscle atrophy and myopathy.

    PubMed

    Judson, Robert N; Gray, Stuart R; Walker, Claire; Carroll, Andrew M; Itzstein, Cecile; Lionikas, Arimantas; Zammit, Peter S; De Bari, Cosimo; Wackerhage, Henning

    2013-01-01

    The aim of this study was to investigate the function of the Hippo pathway member Yes-associated protein (Yap, gene name Yap1) in skeletal muscle fibres in vivo. Specifically we bred an inducible, skeletal muscle fibre-specific knock-in mouse model (MCK-tTA-hYAP1 S127A) to test whether the over expression of constitutively active Yap (hYAP1 S127A) is sufficient to drive muscle hypertrophy or stimulate changes in fibre type composition. Unexpectedly, after 5-7 weeks of constitutive hYAP1 S127A over expression, mice suddenly and rapidly lost 20-25% body weight and suffered from gait impairments and kyphosis. Skeletal muscles atrophied by 34-40% and the muscle fibre cross sectional area decreased by ≈40% when compared to control mice. Histological analysis revealed evidence of skeletal muscle degeneration and regeneration, necrotic fibres and a NADH-TR staining resembling centronuclear myopathy. In agreement with the histology, mRNA expression of markers of regenerative myogenesis (embryonic myosin heavy chain, Myf5, myogenin, Pax7) and muscle protein degradation (atrogin-1, MuRF1) were significantly elevated in muscles from transgenic mice versus control. No significant changes in fibre type composition were detected using ATPase staining. The phenotype was largely reversible, as a cessation of hYAP1 S127A expression rescued body and muscle weight, restored muscle morphology and prevented further pathological progression. To conclude, high Yap activity in muscle fibres does not induce fibre hypertrophy nor fibre type changes but instead results in a reversible atrophy and deterioration. PMID:23544078

  20. Constitutive Expression of Yes-Associated Protein (Yap) in Adult Skeletal Muscle Fibres Induces Muscle Atrophy and Myopathy

    PubMed Central

    Judson, Robert N.; Gray, Stuart R.; Walker, Claire; Carroll, Andrew M.; Itzstein, Cecile; Lionikas, Arimantas; Zammit, Peter S.; De Bari, Cosimo; Wackerhage, Henning

    2013-01-01

    The aim of this study was to investigate the function of the Hippo pathway member Yes-associated protein (Yap, gene name Yap1) in skeletal muscle fibres in vivo. Specifically we bred an inducible, skeletal muscle fibre-specific knock-in mouse model (MCK-tTA-hYAP1 S127A) to test whether the over expression of constitutively active Yap (hYAP1 S127A) is sufficient to drive muscle hypertrophy or stimulate changes in fibre type composition. Unexpectedly, after 5–7 weeks of constitutive hYAP1 S127A over expression, mice suddenly and rapidly lost 20–25% body weight and suffered from gait impairments and kyphosis. Skeletal muscles atrophied by 34–40% and the muscle fibre cross sectional area decreased by ≈40% when compared to control mice. Histological analysis revealed evidence of skeletal muscle degeneration and regeneration, necrotic fibres and a NADH-TR staining resembling centronuclear myopathy. In agreement with the histology, mRNA expression of markers of regenerative myogenesis (embryonic myosin heavy chain, Myf5, myogenin, Pax7) and muscle protein degradation (atrogin-1, MuRF1) were significantly elevated in muscles from transgenic mice versus control. No significant changes in fibre type composition were detected using ATPase staining. The phenotype was largely reversible, as a cessation of hYAP1 S127A expression rescued body and muscle weight, restored muscle morphology and prevented further pathological progression. To conclude, high Yap activity in muscle fibres does not induce fibre hypertrophy nor fibre type changes but instead results in a reversible atrophy and deterioration. PMID:23544078

  1. Constitutive Expression of Human Telomerase Enhances the Proliferation Potential of Human Mesenchymal Stem Cells

    PubMed Central

    Bischoff, David S.; Makhijani, Nalini S.

    2012-01-01

    Abstract Human mesenchymal stem cells (hMSCs) are highly desirable cells for bone engineering due to the inherent multipotent nature of the cells. Unfortunately, there is a high degree of variability, as primary hMSC cultures quickly undergo replicative senescence with loss of proliferative potential as they are continually propagated in cell culture. We sought to reduce the variability of these cells by insertion and expression of human telomerase reverse transcriptase (TERT) to immortalize the cell line. hMSCs were transduced with a lentivirus containing the human TERT gene. The resulting cell line has been propagated through more than 70 population-doubling level (PDL) to date and continues to grow exhibiting the characteristic fibroblastic hMSC phenotype. Expression of TERT mRNA and protein activity was confirmed in the TERT-transduced cells. Mock-transduced hMSCs had almost undetectable levels of TERT mRNA and protein activity and lost proliferation potential at PDL 14. The enhanced growth capacity of the hMSC TERT cells was due to increased cell proliferation and reduced cellular senescence rather than due to inhibition of apoptosis. The multipotent nature of the TERT cells was confirmed by differentiation toward the osteoblastic and adipogenic lineages in vitro. Osteoblastic differentiation was confirmed by both expression of alkaline phosphate and mineral deposition visualized by Alizarin Red staining. Adipogenic differentiation was confirmed by production of lipid droplets, which were detected by Oil Red-O staining. In summary, we have generated a stable hMSC line that can be continually propagated and retains both osteoblastic and adipogenic differentiation potential. PMID:23515239

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

    PubMed

    Muranaka, Tomoaki; Kubota, Saya; Oyama, Tokitaka

    2013-12-01

    Gene expression is a fundamental cellular process and expression dynamics are of great interest in life science. We succeeded in monitoring cellular gene expression in a duckweed plant, Lemna gibba, using bioluminescent reporters. Using particle bombardment, epidermal and mesophyll cells were transfected with the luciferase gene (luc+) under the control of a constitutive [Cauliflower mosaic virus 35S (CaMV35S)] and a rhythmic [Arabidopsis thaliana CIRCADIAN CLOCK ASSOCIATED 1 (AtCCA1)] promoter. Bioluminescence images were captured using an EM-CCD (electron multiply charged couple device) camera. Luminescent spots of the transfected cells in the plant body were quantitatively measured at the single-cell level. Luminescence intensities varied over a 1,000-fold range among CaMV35S::luc+-transfected cells in the same plant body and showed a log-normal-like frequency distribution. We monitored cellular gene expression under light-dark conditions by capturing bioluminescence images every hour. Luminescence traces of ≥50 individual cells in a frond were successfully obtained in each monitoring procedure. Rhythmic and constitutive luminescence behaviors were observed in cells transfected with AtCCA1::luc+ and CaMV35S::luc+, respectively. Diurnal rhythms were observed in every AtCCA1::luc+-introduced cell with traceable luminescence, and slight differences were detected in their rhythmic waveforms. Thus the single-cell bioluminescence monitoring system was useful for the characterization of cellular gene expression in a plant body. PMID:24058151

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

    PubMed

    Muranaka, Tomoaki; Kubota, Saya; Oyama, Tokitaka

    2013-12-01

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

  4. Proton-Coupled Electron Transfer Constitutes the Photoactivation Mechanism of the Plant Photoreceptor UVR8.

    PubMed

    Mathes, Tilo; Heilmann, Monika; Pandit, Anjali; Zhu, Jingyi; Ravensbergen, Janneke; Kloz, Miroslav; Fu, Yinan; Smith, Brian O; Christie, John M; Jenkins, Gareth I; Kennis, John T M

    2015-07-01

    UVR8 is a novel UV-B photoreceptor that regulates a range of plant responses and is already used as a versatile optogenetic tool. Instead of an exogenous chromophore, UVR8 uniquely employs tryptophan side chains to accomplish UV-B photoreception. UV-B absorption by homodimeric UVR8 induces monomerization and hence signaling, but the underlying photodynamic mechanisms are not known. Here, by using a combination of time-resolved fluorescence and absorption spectroscopy from femto- to microseconds, we provide the first experimental evidence for the UVR8 molecular signaling mechanism. The results indicate that tryptophan residues at the dimer interface engage in photoinduced proton coupled electron transfer reactions that induce monomerization.

  5. Constitutive arsenite oxidase expression detected in arsenic-hypertolerant Pseudomonas xanthomarina S11.

    PubMed

    Koechler, Sandrine; Arsène-Ploetze, Florence; Brochier-Armanet, Céline; Goulhen-Chollet, Florence; Heinrich-Salmeron, Audrey; Jost, Bernard; Lièvremont, Didier; Philipps, Muriel; Plewniak, Frédéric; Bertin, Philippe N; Lett, Marie-Claire

    2015-04-01

    Pseudomonas xanthomarina S11 is an arsenite-oxidizing bacterium isolated from an arsenic-contaminated former gold mine in Salsigne, France. This bacterium showed high resistance to arsenite and was able to oxidize arsenite to arsenate at concentrations up to 42.72 mM As[III]. The genome of this strain was sequenced and revealed the presence of three ars clusters. One of them is located on a plasmid and is organized as an "arsenic island" harbouring an aio operon and genes involved in phosphorous metabolism, in addition to the ars genes. Neither the aioXRS genes nor a specific sigma-54-dependent promoter located upstream of aioBA genes, both involved in regulation of arsenite oxidase expression in other arsenite-oxidizing bacteria, could be identified in the genome. This observation is in accordance with the fact that no difference was observed in expression of arsenite oxidase in P. xanthomarina S11, whether or not the strain was grown in the presence of As[III].

  6. Identification and Characterization of a Stress-Inducible and a Constitutive Small Heat-Shock Protein Targeted to the Matrix of Plant Peroxisomes1[W

    PubMed Central

    Ma, Changle; Haslbeck, Martin; Babujee, Lavanya; Jahn, Olaf; Reumann, Sigrun

    2006-01-01

    Small heat-shock proteins (sHsps) are widespread molecular chaperones for which a peroxisomal localization has not yet been reported. The Arabidopsis (Arabidopsis thaliana) genome encodes two sHsps with putative peroxisomal targeting signals type 1 or 2 (PTS1 or PTS2). As demonstrated by double-labeling experiments using full-length fusion proteins with enhanced yellow fluorescent protein and deletion constructs lacking the putative targeting domains, AtHsp15.7 (At5g37670) and AtAcd31.2 (At1g06460) are targeted to the peroxisome matrix by a functional PTS1 (SKL>) and a functional PTS2 (RLx5HF), respectively. The peroxisomal localization of AtAcd31.2 was further confirmed by isolation of leaf peroxisomes from Arabidopsis by two successive sucrose density gradients, protein separation by one- and two-dimensional gel electrophoresis, and mass spectrometric protein identification. When AtHsp15.7 and AtAcd31.2 were heterologously expressed in yeast (Saccharomyces cerevisiae) and directed to the cytosol by deletion of the PTSs, both sHsps were able to complement the morphological phenotype of yeast mutants deficient in the cytosolic homologs ScHsp42 or ScHsp26. According to expression studies by reverse transcription-PCR, AtAcd31.2 is constitutively expressed, whereas AtHsp15.7 is hardly expressed under normal conditions but strongly induced by heat and oxidative stress, the latter of which was triggered by the catalase inhibitor 3-aminotriazole or the herbicide methyl viologen applied by watering of whole plants or infiltration of rosette leaves. Thus, plants are exceptional among eukaryotes in employing sHsps in the peroxisome matrix to prevent unspecific aggregation of partially denatured proteins under both physiological and stress conditions. PMID:16531488

  7. Waking up Streptomyces secondary metabolism by constitutive expression of activators or genetic disruption of repressors.

    PubMed

    Aigle, Bertrand; Corre, Christophe

    2012-01-01

    Streptomycete bacteria are renowned as a prolific source of natural products with diverse biological activities. Production of these metabolites is often subject to transcriptional regulation: the biosynthetic genes remain silent until the required environmental and/or physiological signals occur. Consequently, in the laboratory environment, many gene clusters that direct the biosynthesis of natural products with clinical potential are not expressed or at very low level preventing the production/detection of the associated metabolite. Genetic engineering of streptomycetes can unleash the production of many new natural products. This chapter describes the overexpression of pathway-specific activators, the genetic disruption of pathway-specific repressors, and the main strategy used to identify and characterize new natural products from these engineered Streptomyces strains.

  8. Expression profiling of constitutive mast cells reveals a unique identity within the immune system

    PubMed Central

    Dwyer, Daniel F.; Barrett, Nora A.; Austen, K. Frank

    2016-01-01

    Mast cells are evolutionarily ancient sentinel cells. Like basophils, mast cells express the high-affinity IgE receptor and are implicated in host defense and diverse immune-mediated diseases. To better characterize the function of these cells, we assessed the transcriptional profiles of mast cells isolated from peripheral connective tissues and basophils isolated from spleen and blood. We found that mast cells were transcriptionally distinct, clustering independently from all other profiled cells, and that mast cells demonstrated considerably greater heterogeneity across tissues than previously appreciated. We observed minimal homology between mast cells and basophils, which share more overlap with other circulating granulocytes than with mast cells. Derivation of mast cell and basophil transcriptional signatures underscores their differential capacity to detect environmental signals and influence the inflammatory milieu. PMID:27135604

  9. Plenary perspective: the complexity of constitutive and inducible gene expression in mononuclear phagocytes.

    PubMed

    Hume, David A

    2012-09-01

    Monocytes and macrophages differentiate from progenitor cells under the influence of colony-stimulating factors. Genome-scale data have enabled the identification of the set of genes that distinguishes macrophages from other cell types and the ways in which thousands of genes are regulated in response to pathogen challenge. Although there has been a focus on a small subset of lineage-enriched transcription factors, such as PU.1, more than one-half of the transcription factors in the genome can be expressed in macrophage lineage cells under some state of activation, and they interact in a complex network. The network architecture is conserved across species, but many of the target genes evolve rapidly and differ between mouse and human. The data and publication deluge related to macrophage biology require the development of new analytical tools and ways of presenting information in an accessible form.

  10. Transgenic plants expressing GLK1 and CCA1 having increased nitrogen assimilation capacity

    SciTech Connect

    Coruzzi, Gloria; Gutierrez, Rodrigo A.; Nero, Damion C.

    2012-04-10

    Provided herein are compositions and methods for producing transgenic plants. In specific embodiments, transgenic plants comprise a construct comprising a polynucleotide encoding CCA1, GLK1 or bZIP1, operably linked to a plant-specific promote, wherein the CCA1, GLK1 or bZIP1 is ectopically overexpressed in the transgenic plants, and wherein the promoter is optionally a constitutive or inducible promoter. In other embodiments, transgenic plants in which express a lower level of CCA1, GLK1 or bZIP1 are provided. Also provided herein are commercial products (e.g., pulp, paper, paper products, or lumber) derived from the transgenic plants (e.g., transgenic trees) produced using the methods provided herein.

  11. Chronic lung injury by constitutive expression of activation-induced cytidine deaminase leads to focal mucous cell metaplasia and cancer.

    PubMed

    Kitamura, Jiro; Uemura, Munehiro; Kurozumi, Mafumi; Sonobe, Makoto; Manabe, Toshiaki; Hiai, Hiroshi; Date, Hiroshi; Kinoshita, Kazuo

    2015-01-01

    Activation-induced cytidine deaminase (AID) is an enzyme required for antibody diversification, and it causes DNA mutations and strand breaks. Constitutive AID expression in mice invariably caused lung lesions morphologically similar to human atypical adenomatous hyperplasia (AAH), which can be a precursor of bronchioloalveolar carcinoma. Similar to AAH, mouse AAH-like lesion (MALL) exhibited signs of alveolar differentiation, judging from the expression of alveolar type II (AT2) cell marker surfactant protein C (SP-C). However, electron microscopy indicated that MALL, which possessed certain features of a mucous cell, is distinct from an AAH or AT2 cell. Although MALL developed in all individuals within 30 weeks after birth, lung tumors occurred in only 10%; this suggests that the vast majority of MALLs fail to grow into visible tumors. MALL expressed several recently described markers of lung alveolar regeneration such as p63, keratin 5, keratin 14, leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), and Lgr6. Increased cell death was observed in the lungs of AID transgenic mice compared with wild-type mice. Based on these observations, we speculate that MALL is a regenerating tissue compensating for cellular loss caused by AID cytotoxicity. AID expression in such regenerating tissue should predispose cells to malignant transformation via its mutagenic activity. PMID:25659078

  12. Seasonal photosynthetic gas exchange and water-use efficiency in a constitutive CAM plant, the giant saguaro cactus (Carnegiea gigantea).

    PubMed

    Bronson, Dustin R; English, Nathan B; Dettman, David L; Williams, David G

    2011-11-01

    Crassulacean acid metabolism (CAM) and the capacity to store large quantities of water are thought to confer high water use efficiency (WUE) and survival of succulent plants in warm desert environments. Yet the highly variable precipitation, temperature and humidity conditions in these environments likely have unique impacts on underlying processes regulating photosynthetic gas exchange and WUE, limiting our ability to predict growth and survival responses of desert CAM plants to climate change. We monitored net CO(2) assimilation (A(net)), stomatal conductance (g(s)), and transpiration (E) rates periodically over 2 years in a natural population of the giant columnar cactus Carnegiea gigantea (saguaro) near Tucson, Arizona USA to investigate environmental and physiological controls over carbon gain and water loss in this ecologically important plant. We hypothesized that seasonal changes in daily integrated water use efficiency (WUE(day)) in this constitutive CAM species would be driven largely by stomatal regulation of nighttime transpiration and CO(2) uptake responding to shifts in nighttime air temperature and humidity. The lowest WUE(day) occurred during time periods with extreme high and low air vapor pressure deficit (D(a)). The diurnal with the highest D(a) had low WUE(day) due to minimal net carbon gain across the 24 h period. Low WUE(day) was also observed under conditions of low D(a); however, it was due to significant transpiration losses. Gas exchange measurements on potted saguaro plants exposed to experimental changes in D(a) confirmed the relationship between D(a) and g(s). Our results suggest that climatic changes involving shifts in air temperature and humidity will have large impacts on the water and carbon economy of the giant saguaro and potentially other succulent CAM plants of warm desert environments. PMID:21822726

  13. Seasonal photosynthetic gas exchange and water-use efficiency in a constitutive CAM plant, the giant saguaro cactus (Carnegiea gigantea).

    PubMed

    Bronson, Dustin R; English, Nathan B; Dettman, David L; Williams, David G

    2011-11-01

    Crassulacean acid metabolism (CAM) and the capacity to store large quantities of water are thought to confer high water use efficiency (WUE) and survival of succulent plants in warm desert environments. Yet the highly variable precipitation, temperature and humidity conditions in these environments likely have unique impacts on underlying processes regulating photosynthetic gas exchange and WUE, limiting our ability to predict growth and survival responses of desert CAM plants to climate change. We monitored net CO(2) assimilation (A(net)), stomatal conductance (g(s)), and transpiration (E) rates periodically over 2 years in a natural population of the giant columnar cactus Carnegiea gigantea (saguaro) near Tucson, Arizona USA to investigate environmental and physiological controls over carbon gain and water loss in this ecologically important plant. We hypothesized that seasonal changes in daily integrated water use efficiency (WUE(day)) in this constitutive CAM species would be driven largely by stomatal regulation of nighttime transpiration and CO(2) uptake responding to shifts in nighttime air temperature and humidity. The lowest WUE(day) occurred during time periods with extreme high and low air vapor pressure deficit (D(a)). The diurnal with the highest D(a) had low WUE(day) due to minimal net carbon gain across the 24 h period. Low WUE(day) was also observed under conditions of low D(a); however, it was due to significant transpiration losses. Gas exchange measurements on potted saguaro plants exposed to experimental changes in D(a) confirmed the relationship between D(a) and g(s). Our results suggest that climatic changes involving shifts in air temperature and humidity will have large impacts on the water and carbon economy of the giant saguaro and potentially other succulent CAM plants of warm desert environments.

  14. The Dioxin Receptor Regulates the Constitutive Expression of the Vav3 Proto-Oncogene and Modulates Cell Shape and Adhesion

    PubMed Central

    Carvajal-Gonzalez, Jose M.; Mulero-Navarro, Sonia; Roman, Angel Carlos; Sauzeau, Vincent; Merino, Jaime M.; Bustelo, Xose R.

    2009-01-01

    The dioxin receptor (AhR) modulates cell plasticity and migration, although the signaling involved remains unknown. Here, we report a mechanism that integrates AhR into these cytoskeleton-related functions. Immortalized and mouse embryonic fibroblasts lacking AhR (AhR−/−) had increased cell area due to spread cytoplasms that reverted to wild-type morphology upon AhR re-expression. The AhR-null phenotype included increased F-actin stress fibers, depolarized focal adhesions, and enhanced spreading and adhesion. The cytoskeleton alterations of AhR−/− cells were due to down-regulation of constitutive Vav3 expression, a guanosine diphosphate/guanosine triphosphate exchange factor for Rho/Rac GTPases and a novel transcriptional target of AhR. AhR was recruited to the vav3 promoter and maintained constitutive mRNA expression in a ligand-independent manner. Consistently, AhR−/− fibroblasts had reduced Rac1 activity and increased activation of the RhoA/Rho kinase (Rock) pathway. Pharmacological inhibition of Rac1 shifted AhR+/+ fibroblasts to the null phenotype, whereas Rock inhibition changed AhR-null cells to the AhR+/+ morphology. Knockdown of vav3 transcripts by small interfering RNA induced cytoskeleton defects and changes in adhesion and spreading mimicking those of AhR-null cells. Moreover, vav3−/− MEFs, as AhR−/− mouse embryonic fibroblasts, had increased cell area and enhanced stress fibers. By modulating Vav3-dependent signaling, AhR could regulate cell shape, adhesion, and migration under physiological conditions and, perhaps, in certain pathological states. PMID:19158396

  15. Chemical constitution and effect of extracts of tomato plants byproducts on the enteric viral surrogates.

    PubMed

    Silva-Beltrán, Norma Patricia; Ruiz-Cruz, Saul; Chaidez, Cristobal; Ornelas-Paz, José de Jesús; López-Mata, Marco A; Márquez-Ríos, Enrique; Estrada, Maria Isabel

    2015-01-01

    Byproducts of tomato are known to include phenolic compounds but have not been studied in depth. In this study, the phenolic compositions of (stem, leaf, root, and whole plant) of two tomato cultivars, Pitenza and Floradade, were analyzed by HPLC-DAD. In parallel, the antiviral effects of crude extracts on viral surrogates, the bacteriophages MS2 and Av-05 were evaluated. The leaf extracts from the two varieties showed the highest concentration of phenolic compounds. The compounds identified were gallic acid, chlorogenic acid, ferulic acid, cafeic acid, rutin, and quercetin, and they represented 3174.3 and 1057.9 mg/100 g dried weight of the Pitenza and Floradade cultivars, respectively. MS2 and Av-05 titers at 5 mg/mL were reduced by 3.47 and 5.78 log10 PFU/mL and 3.78 and 4.93 log10 PFU/mL by Pitenza and Floradade cultivar leaf extract, respectively. These results show that tomato extracts are natural sources of bioactive substances with antiviral activity.

  16. Chemical constitution and effect of extracts of tomato plants byproducts on the enteric viral surrogates.

    PubMed

    Silva-Beltrán, Norma Patricia; Ruiz-Cruz, Saul; Chaidez, Cristobal; Ornelas-Paz, José de Jesús; López-Mata, Marco A; Márquez-Ríos, Enrique; Estrada, Maria Isabel

    2015-01-01

    Byproducts of tomato are known to include phenolic compounds but have not been studied in depth. In this study, the phenolic compositions of (stem, leaf, root, and whole plant) of two tomato cultivars, Pitenza and Floradade, were analyzed by HPLC-DAD. In parallel, the antiviral effects of crude extracts on viral surrogates, the bacteriophages MS2 and Av-05 were evaluated. The leaf extracts from the two varieties showed the highest concentration of phenolic compounds. The compounds identified were gallic acid, chlorogenic acid, ferulic acid, cafeic acid, rutin, and quercetin, and they represented 3174.3 and 1057.9 mg/100 g dried weight of the Pitenza and Floradade cultivars, respectively. MS2 and Av-05 titers at 5 mg/mL were reduced by 3.47 and 5.78 log10 PFU/mL and 3.78 and 4.93 log10 PFU/mL by Pitenza and Floradade cultivar leaf extract, respectively. These results show that tomato extracts are natural sources of bioactive substances with antiviral activity. PMID:25059828

  17. A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina

    SciTech Connect

    Linger, Jeffrey G.; Taylor, II, Larry E.; Baker, John O.; Vander Wall, Todd; Hobdey, Sarah E.; Podkaminer, Kara; Himmel, Michael E.; Decker, Stephen R.

    2015-03-18

    One of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is cellobiohydrolase I (cbh1or Cel7A) due to its enzymatic proficiency in cellulose depolymerization. However, production of Cel7A with native-like properties from heterologous expression systems has proven difficult. In this study, we develop a protein expression system in H. jecorina (Trichoderma reesei) useful for production and secretion of heterologous cellobiohydrolases from glycosyl hydrolase family 7. Building upon previous work in heterologous protein expression in filamentous fungi, we have integrated a native constitutive enolase promoter with the native cbh1 signal sequence. The results are the following: The constitutive eno promoter driving the expression of Cel7A allows growth on glucose and results in repression of the native cellulase system, severely reducing background endo- and other cellulase activity and greatly simplifying purification of the recombinant protein. Coupling this system to a Δcbh1 strain of H. jecorina ensures that only the recombinant Cel7A protein is produced. Two distinct transformant colony morphologies were observed and correlated with high and null protein production. Production levels in ‘fast’ transformants are roughly equivalent to those in the native QM6a strain of H. jecorina, typically in the range of 10 to 30 mg/L when grown in continuous stirred-tank fermenters. ‘Slow’ transformants showed no evidence of Cel7A production. Specific activity of the purified recombinant Cel7A protein is equivalent to that of native protein when assayed on pretreated corn stover, as is the thermal stability and glycosylation level. Purified Cel7A produced from growth on glucose demonstrated remarkably consistent specific activity. Purified Cel7A from the same strain grown on lactose

  18. Host Plant Effects on Nodulation and Competitiveness of the Bradyrhizobium japonicum Serotype Strains Constituting Serocluster 123

    PubMed Central

    Cregan, P. B.; Keyser, H. H.; Sadowsky, M. J.

    1989-01-01

    Strains in Bradyrhizobium japonicum serocluster 123 are the major indigenous competitors for nodulation in a large portion of the soybean production area of the United States. Serocluster 123 is defined by the serotype strains USDA 123, USDA 127, and USDA 129. The objective of the work reported here was to evaluate the ability of two soybean genotypes, PI 377578 and PI 417566, to restrict the nodulation and reduce the competitiveness of serotype strains USDA 123, USDA 127, and USDA 129 in favor of the highly effective strain CB1809 and to determine how these soybean genotypes alter the competitive relationships among the three serotype strains in the serocluster. The soybean genotypes PI 377578 and PI 417566 along with the commonly grown cultivar Williams were planted in soil essentially free of soybean rhizobia and inoculated with single-strain treatments of USDA 123, USDA 127, USDA 129, or CB1809 and six dual-strain competition treatments of USDA 123, USDA 127, or USDA 129 versus CB1809, USDA 123 versus USDA 127, USDA 123 versus USDA 129, and USDA 127 versus USDA 129. PI 377578 severely reduced the nodulation and competitiveness of USDA 123 and USDA 127, while PI 417566 similarly affected the nodulation and competitiveness of USDA 129. Thus, the two soybean genotypes can reduce the nodulation and competitiveness of each of the three serocluster 123 serotype strains. Our results indicate that host control of restricted nodulation and reduced competitiveness is quite specific and effectively discriminates between B. japonicum strains which are serologically related. PMID:16348029

  19. Distinctive features of plant organs characterized by global analysis of gene expression in Arabidopsis.

    PubMed

    Obayashi, Takeshi; Okegawa, Takashi; Sasaki-Sekimoto, Yuko; Shimada, Hiroshi; Masuda, Tatsuru; Asamizu, Erika; Nakamura, Yasukazu; Shibata, Daisuke; Tabata, Satoshi; Takamiya, Ken-ichiro; Ohta, Hiroyuki

    2004-02-29

    The distinctive features of plant organs are primarily determined by organ-specific gene expression. We analyzed the expression specificity of 8809 genes in 7 organs of Arabidopsis using a cDNA macroarray system. Using relative expression (RE) values between organs, many known and unknown genes specifically expressed in each organ were identified. We also analyzed the organ specificity of various gene groups using the GRE (group relative expression) value, the average of the REs of all genes in a group. Consequently, we found that many gene groups even ribosomal protein genes, have strong organ-specific expression. Clustering of the expression profiles revealed that the 8809 genes were classified into 9 major categories. Although 3451 genes were clustered into the largest category, which showed constitutive gene expression, 266 and 1005 genes were found to be root- and silique-specific genes, respectively. By this clustering, particular gene groups which showed multi-organ-specific expression profiles, such as bud-flower-specific, stem-silique-specific or bud-flower-root-specific profiles, could be effectively identified. From these results, major features of plant organs could be characterized by their distinct profiles of global gene expression. These data of organ-specific gene expression are available at our web site: Arabidopsis thaliana Tissue-Specific Expression Database, ATTED (http://www.atted.bio.titech.ac.jp/).

  20. Identification and characterization of a constitutively expressed Ctenopharyngodon idella ADAR1 splicing isoform (CiADAR1a).

    PubMed

    Liu, Xiancheng; Huang, Keyi; Hou, Qunhao; Sun, Zhicheng; Wang, Binhua; Lin, Gang; Li, Dongming; Liu, Yong; Xu, Xiaowen; Hu, Chengyu

    2016-10-01

    As one member of ADAR family, ADAR1 (adenosine deaminase acting on RNA 1) can convert adenosine to inosine within dsRNA. There are many ADAR1 splicing isoforms in mammals, including an interferon (IFN) inducible ∼150 kD protein (ADAR1-p150) and a constitutively expressed ∼110 kD protein (ADAR1-p110). The structural diversity of ADAR1 splicing isoforms may reflect their multiple functions. ADAR1 splicing isoforms were also found in fish. In our previous study, we have cloned and identified two different grass carp ADAR1 splicing isoforms, i.e. CiADAR1 and CiADAR1-like, both of them are IFN-inducible proteins. In this paper, we identified a novel CiADAR1 splicing isoform gene (named CiADAR1a). CiADAR1a gene contains 15 exons and 14 introns. Its full-length cDNA is comprised of a 5' UTR (359 bp), a 3' UTR (229 bp) and a 2952 bp ORF encoding a polypeptide of 983 amino acids with one Z-DNA binding domain, three dsRNA binding motifs and a highly conserved hydrolytic deamination domain. CiADAR1a was constitutively expressed in Ctenopharyngodon idella kidney (CIK) cells regardless of Poly I:C stimulation by Western blot assay. In normal condition, CiADAR1a was found to be present mainly in the nucleus. After treatment with Poly I:C, it gradually shifted to cytoplasm. To further investigate the mechanism of transcriptional regulation of CiADAR1a, we cloned and identified its promoter sequence. The transcriptional start site of CiADAR1a is mapped within the truncated exon 2. CiADAR1a promoter is 1303 bp in length containing 4 IRF-Es. In the present study, we constructed pcDNA3.1 eukaryotic expression vectors with IRF1 and IRF3 and co-transfected them with pGL3-CiADAR1a promoter into CIK cells. The results showed that neither the over-expression of IRF1 or IRF3 nor Poly I:C stimulation significantly impacted CiADAR1a promoter activity in CIK cells. Together, according to the molecular and expression characteristics, subcellular localization and transcriptional

  1. Cyclic AMP-dependent constitutive expression of gal operon: use of repressor titration to isolate operator mutations.

    PubMed Central

    Irani, M; Orosz, L; Busby, S; Taniguchi, T; Adhya, S

    1983-01-01

    When the gal operator region is present in a multicopy plasmid it binds to all ("titrates") the gal repressor and "induces" the chromosomal gal operon. To make operator mutations (Oa) with reduced affinity toward the repressor, plasmid DNA was irradiated with UV light and mutant derivatives were isolated that were unable to release the chromosomal gal genes from repression. Then with such an Oa plasmid operator revertants were isolated that had reacquired the ability to release repression. Both sets of mutations have been localized by DNA sequence analysis. When the Oa mutations were transferred from the plasmid to the chromosome by recombination these mutant operators were found to make gal expression constitutive (independent of repressor) but still dependent on cAMP, whereas the previously reported gal operator mutants (Oc) are constitutive both in the presence and in the absence of cAMP. The titration method of isolating mutants enables the isolation of strains with operator mutations that also affect normal promoter activity, and it provides an easy way to isolate revertants of operator mutations. Images PMID:6308647

  2. Antisense Suppression of a (+)-δ-Cadinene Synthase Gene in Cotton Prevents the Induction of This Defense Response Gene during Bacterial Blight Infection But Not Its Constitutive Expression1[w

    PubMed Central

    Townsend, Belinda J.; Poole, Andrew; Blake, Christopher J.; Llewellyn, Danny J.

    2005-01-01

    In cotton (Gossypium hirsutum) the enzyme (+)-δ-cadinene synthase (CDNS) catalyzes the first committed step in the biosynthesis of cadinane-type sesquiterpenes, such as gossypol, that provide constitutive and inducible protection against pests and diseases. A cotton cDNA clone encoding CDNS (cdn1-C4) was isolated from developing embryos and functionally characterized. Southern analysis showed that CDNS genes belong to a large multigene family, of which five genomic clones were studied, including three pseudogenes and one gene that may represent another subfamily of CDNS. CDNS expression was shown to be induced in cotton infected with either the bacterial blight or verticillium wilt pathogens. Constructs for the constitutive or seed-specific antisense suppression of cdn1-C4 were introduced into cotton by Agrobacterium-mediated transformation. Gossypol levels were not reduced in the seeds of transformants with either construct, nor was the induction of CDNS expression affected in stems of the constitutive antisense plants infected with Verticillium dahliae Kleb. However, the induction of CDNS mRNA and protein in response to bacterial blight infection of cotyledons was completely blocked in the constitutive antisense plants. These results suggest that cdn1-C4 may be involved specifically in the bacterial blight response and that the CDNS multigene family comprises a complex set of genes differing in their temporal and spatial regulation and responsible for different branches of the cotton sesquiterpene pathway. PMID:15849309

  3. Constitutive expression of CCR7 directs effector CD8 T cells into the splenic white pulp and impairs functional activity.

    PubMed

    Unsoeld, Heike; Voehringer, David; Krautwald, Stefan; Pircher, Hanspeter

    2004-09-01

    Antigenic stimulation down-regulates CCR7 on effector T cells. To analyze the importance of CCR7 down-regulation, transgenic (tg) mice constitutively expressing CCR7 were generated. CD8 T cells with defined Ag specificity were obtained by breeding CCR7-tg mice with P14 TCR-tg mice specific for lymphocytic choriomeningitis virus. Transgenic CCR7 expression did not impair proliferation of P14.CCR7 T cells induced by lymphocytic choriomeningitis virus infection, but prevented CCR7 down-regulation. Compared with wild-type P14 effector cells, P14.CCR7 effector cells, expressing the CCR7 transgene, were increased in the spleen, but decreased in blood and peripheral tissues. Moreover, P14.CCR7 effector cells localized almost exclusively in the splenic white pulp, whereas P14 effector cells were excluded from splenic white pulp cords and were found preferentially in the red pulp. Functional experiments further revealed that P14.CCR7 effector cells were impaired in rapid viral clearance and in inducing Ag-specific delayed-type hypersensitivity reactions. Thus, the present study demonstrates that down-regulation of CCR7 during CD8 T cell activation is important to release effector cells from the white pulp of the spleen, and highlights the importance of effector cell localization in providing rapid immunity. PMID:15322160

  4. Expression of constitutively active erythropoietin receptor in pyramidal neurons of cortex and hippocampus boosts higher cognitive functions in mice

    PubMed Central

    2011-01-01

    Background Erythropoietin (EPO) and its receptor (EPOR) are expressed in the developing brain and their transcription is upregulated in adult neurons and glia upon injury or neurodegeneration. We have shown neuroprotective effects and improved cognition in patients with neuropsychiatric diseases treated with EPO. However, the critical EPO targets in brain are unknown, and separation of direct and indirect effects has remained difficult, given the role of EPO in hematopoiesis and brain oxygen supply. Results Here we demonstrate that mice with transgenic expression of a constitutively active EPOR isoform (cEPOR) in pyramidal neurons of cortex and hippocampus exhibit enhancement of spatial learning, cognitive flexibility, social memory, and attentional capacities, accompanied by increased impulsivity. Superior cognitive performance is associated with augmented long-term potentiation of cEPOR expressing neurons in hippocampal slices. Conclusions Active EPOR stimulates neuronal plasticity independent of any hematopoietic effects and in addition to its neuroprotective actions. This property of EPOR signaling should be exploited for defining novel strategies to therapeutically enhance cognitive performance in disease conditions. PMID:21527022

  5. Loss-of-Function of Constitutive Expresser of Pathogenesis Related Genes5 Affects Potassium Homeostasis in Arabidopsis thaliana

    PubMed Central

    Borghi, Monica; Rus, Ana; Salt, David E.

    2011-01-01

    Here, we demonstrate that the reduction in leaf K+ observed in a mutant previously identified in an ionomic screen of fast neutron mutagenized Arabidopsis thaliana is caused by a loss-of-function allele of CPR5, which we name cpr5-3. This observation establishes low leaf K+ as a new phenotype for loss-of-function alleles of CPR5. We investigate the factors affecting this low leaf K+ in cpr5 using double mutants defective in salicylic acid (SA) and jasmonic acid (JA) signalling, and by gene expression analysis of various channels and transporters. Reciprocal grafting between cpr5 and Col-0 was used to determine the relative importance of the shoot and root in causing the low leaf K+ phenotype of cpr5. Our data show that loss-of-function of CPR5 in shoots primarily determines the low leaf K+ phenotype of cpr5, though the roots also contribute to a lesser degree. The low leaf K+ phenotype of cpr5 is independent of the elevated SA and JA known to occur in cpr5. In cpr5 expression of genes encoding various Cyclic Nucleotide Gated Channels (CNGCs) are uniquely elevated in leaves. Further, expression of HAK5, encoding the high affinity K+ uptake transporter, is reduced in roots of cpr5 grown with high or low K+ supply. We suggest a model in which low leaf K+ in cpr5 is driven primarily by enhanced shoot-to-root K+ export caused by a constitutive activation of the expression of various CNGCs. This activation may enhance K+ efflux, either indirectly via enhanced cytosolic Ca2+ and/or directly by increased K+ transport activity. Enhanced shoot-to-root K+ export may also cause the reduced expression of HAK5 observed in roots of cpr5, leading to a reduction in uptake of K+. All ionomic data presented is publically available at www.ionomicshub.org. PMID:22046278

  6. Constitutive Expression of OsIAA9 Affects Starch Granules Accumulation and Root Gravitropic Response in Arabidopsis

    PubMed Central

    Luo, Sha; Li, Qianqian; Liu, Shanda; Pinas, Nicholaas M.; Tian, Hainan; Wang, Shucai

    2015-01-01

    Auxin/Indole-3-Acetic Acid (Aux/IAA) genes are early auxin response genes ecoding short-lived transcriptional repressors, which regulate auxin signaling in plants by interplay with Auxin Response Factors (ARFs). Most of the Aux/IAA proteins contain four different domains, namely Domain I, Domain II, Domain III, and Domain IV. So far all Aux/IAA mutants with auxin-related phenotypes identified in both Arabidopsis and rice (Oryza sativa) are dominant gain-of-function mutants with mutations in Domain II of the corresponding Aux/IAA proteins, suggest that Aux/IAA proteins in both Arabidopsis and rice are largely functional redundantly, and they may have conserved functions. We report here the functional characterization of a rice Aux/IAA gene, OsIAA9. RT-PCR results showed that expression of OsIAA9 was induced by exogenously applied auxin, suggesting that OsIAA9 is an auxin response gene. Bioinformatic analysis showed that OsIAA9 has a repressor motif in Domain I, a degron in Domain II, and the conserved amino acid signatures for protein-protein interactions in Domain III and Domain IV. By generating transgenic plants expressing GFP-OsIAA9 and examining florescence in the transgenic plants, we found that OsIAA9 is localized in the nucleus. When transfected into protoplasts isolated from rosette leaves of Arabidopsis, OsIAA9 repressed reporter gene expression, and the repression was partially released by exogenously IAA. These results suggest that OsIAA9 is a canonical Aux/IAA protein. Protoplast transfection assays showed that OsIAA9 interacted ARF5, but not ARF6, 7, 8 and 19. Transgenic Arabidopsis plants expressing OsIAA9 have increased number of lateral roots, and reduced gravitropic response. Further analysis showed that OsIAA9 transgenic Arabidopsis plants accumulated fewer granules in their root tips and the distribution of granules was also affected. Taken together, our study showed that OsIAA9 is a transcriptional repressor, and it regulates gravitropic

  7. Constitutive expression of tdTomato protein as a cytotoxicity and proliferation marker for space radiation biology.

    PubMed

    Chishti, Arif A; Hellweg, Christine E; Berger, Thomas; Baumstark-Khan, Christa; Feles, Sebastian; Kätzel, Thorben; Reitz, Günther

    2015-01-01

    The radiation risk assessment for long-term space missions requires knowledge on the biological effectiveness of different space radiation components, e.g. heavy ions, on the interaction of radiation and other space environmental factors such as microgravity, and on the physical and biological dose distribution in the human body. Space experiments and ground-based experiments at heavy ion accelerators require fast and reliable test systems with an easy readout for different endpoints. In order to determine the effect of different radiation qualities on cellular proliferation and the biological depth dose distribution after heavy ion exposure, a stable human cell line expressing a novel fluorescent protein was established and characterized. tdTomato, a red fluorescent protein of the new generation with fast maturation and high fluorescence intensity, was selected as reporter of cell proliferation. Human embryonic kidney (HEK/293) cells were stably transfected with a plasmid encoding tdTomato under the control of the constitutively active cytomegalovirus (CMV) promoter (ptdTomato-N1). The stably transfected cell line was named HEK-ptdTomato-N1 8. This cytotoxicity biosensor was tested by ionizing radiation (X-rays and accelerated heavy ions) exposure. As biological endpoints, the proliferation kinetics and the cell density reached 100 h after irradiation reflected by constitutive expression of the tdTomato were investigated. Both were reduced dose-dependently after radiation exposure. Finally, the cell line was used for biological weighting of heavy ions of different linear energy transfer (LET) as space-relevant radiation quality. The relative biological effectiveness of accelerated heavy ions in reducing cellular proliferation peaked at an LET of 91 keV/μm. The results of this study demonstrate that the HEK-ptdTomato-N1 reporter cell line can be used as a fast and reliable biosensor system for detection of cytotoxic damage caused by ionizing radiation.

  8. Constitutive expression of tdTomato protein as a cytotoxicity and proliferation marker for space radiation biology

    NASA Astrophysics Data System (ADS)

    Chishti, Arif A.; Hellweg, Christine E.; Berger, Thomas; Baumstark-Khan, Christa; Feles, Sebastian; Kätzel, Thorben; Reitz, Günther

    2015-01-01

    The radiation risk assessment for long-term space missions requires knowledge on the biological effectiveness of different space radiation components, e.g. heavy ions, on the interaction of radiation and other space environmental factors such as microgravity, and on the physical and biological dose distribution in the human body. Space experiments and ground-based experiments at heavy ion accelerators require fast and reliable test systems with an easy readout for different endpoints. In order to determine the effect of different radiation qualities on cellular proliferation and the biological depth dose distribution after heavy ion exposure, a stable human cell line expressing a novel fluorescent protein was established and characterized. tdTomato, a red fluorescent protein of the new generation with fast maturation and high fluorescence intensity, was selected as reporter of cell proliferation. Human embryonic kidney (HEK/293) cells were stably transfected with a plasmid encoding tdTomato under the control of the constitutively active cytomegalovirus (CMV) promoter (ptdTomato-N1). The stably transfected cell line was named HEK-ptdTomato-N1 8. This cytotoxicity biosensor was tested by ionizing radiation (X-rays and accelerated heavy ions) exposure. As biological endpoints, the proliferation kinetics and the cell density reached 100 h after irradiation reflected by constitutive expression of the tdTomato were investigated. Both were reduced dose-dependently after radiation exposure. Finally, the cell line was used for biological weighting of heavy ions of different linear energy transfer (LET) as space-relevant radiation quality. The relative biological effectiveness of accelerated heavy ions in reducing cellular proliferation peaked at an LET of 91 keV/μm. The results of this study demonstrate that the HEK-ptdTomato-N1 reporter cell line can be used as a fast and reliable biosensor system for detection of cytotoxic damage caused by ionizing radiation.

  9. Repressor-mediated tissue-specific gene expression in plants

    DOEpatents

    Meagher, Richard B.; Balish, Rebecca S.; Tehryung, Kim; McKinney, Elizabeth C.

    2009-02-17

    Plant tissue specific gene expression by way of repressor-operator complexes, has enabled outcomes including, without limitation, male sterility and engineered plants having root-specific gene expression of relevant proteins to clean environmental pollutants from soil and water. A mercury hyperaccumulation strategy requires that mercuric ion reductase coding sequence is strongly expressed. The actin promoter vector, A2pot, engineered to contain bacterial lac operator sequences, directed strong expression in all plant vegetative organs and tissues. In contrast, the expression from the A2pot construct was restricted primarily to root tissues when a modified bacterial repressor (LacIn) was coexpressed from the light-regulated rubisco small subunit promoter in above-ground tissues. Also provided are analogous repressor operator complexes for selective expression in other plant tissues, for example, to produce male sterile plants.

  10. Constitutive expression of the pea ABA-responsive 17 (ABR17) cDNA confers multiple stress tolerance in Arabidopsis thaliana.

    PubMed

    Srivastava, Sanjeeva; Rahman, Muhammad H; Shah, Salehuzzaman; Kav, Nat N V

    2006-09-01

    The constitutive expression of the pea ABR17 (abscisic acid-responsive 17) cDNA, which is a member of the group 10 family of pathogenesis-related proteins (PR 10), in Arabidopsis thaliana is reported. The presence of ABR17 transcripts and the protein in the three transgenic lines is demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) and two-dimensional electrophoresis followed by tandem mass spectrometry, respectively. Three independently derived transgenic lines containing ABR17 germinated better in the presence of salt, cold temperature or both. Furthermore, the transgenic plants also exhibited enhanced tolerance to freezing temperature, suggesting the potential utility of the ABR17 gene to engineer multiple stress tolerance. In order to obtain insights into the mechanism underlying ABR17-mediated stress tolerance, we have compared the proteome of a transgenic line with that of its wild-type counterpart. Several proteins were observed to be significantly altered in the transgenic line, including some with a role(s) in photosynthesis, stress tolerance and the regulation of gene expression. Our findings are discussed within the context of available genes to engineer multiple stress tolerance as well as the biological activities of the ABR17 protein.

  11. Identification of centrarchid hepcidins and evidence that 17beta-estradiol disrupts constitutive expression of hepcidin-1 and inducible expression of hepcidin-2 in largemouth bass (Micropterus salmoides).

    PubMed

    Robertson, Laura S; Iwanowicz, Luke R; Marranca, Jamie Marie

    2009-06-01

    Hepcidin is a highly conserved antimicrobial peptide and iron-regulatory hormone. Here, we identify two hepcidin genes (hep-1 and hep-2) in largemouth bass (Micropterus salmoides) and smallmouth bass (Micropterus dolomieu). Hepcidin-1 contains a putative ATCUN metal-binding site in the amino-terminus that is missing in hepcidin-2, suggesting that hepcidin-1 may function as an iron-regulatory hormone. Both hepcidins are predominately expressed in the liver of largemouth bass, similar to other fish and mammals. Experimental exposure of pond-raised largemouth bass to 17beta-estradiol and/or the bacteria Edwardsiella ictaluri led to distinct changes in expression of hep-1 and hep-2. Estradiol reduced the constitutive expression of hep-1 in the liver. Bacterial exposure induced expression of hep-2, suggesting that hepcidin-2 may have an antimicrobial function, and this induction was abolished by estradiol. To our knowledge, this is the first report of the regulation of hepcidin expression by estradiol in either fish or mammals.

  12. Identification of centrarchid hepcidins and evidence that 17β-estradiol disrupts constitutive expression of hepcidin-1 and inducible expression of hepcidin-2 in largemouth bass (Micropterus salmoides)

    USGS Publications Warehouse

    Robertson, L.S.; Iwanowicz, L.R.; Marranca, J.M.

    2009-01-01

    Hepcidin is a highly conserved antimicrobial peptide and iron-regulatory hormone. Here, we identify two hepcidin genes (hep-1 and hep-2) in largemouth bass (Micropterus salmoides) and smallmouth bass (Micropterus dolomieu). Hepcidin-1 contains a putative ATCUN metal-binding site in the amino-terminus that is missing in hepcidin-2, suggesting that hepcidin-1 may function as an iron-regulatory hormone. Both hepcidins are predominately expressed in the liver of largemouth bass, similar to other fish and mammals. Experimental exposure of pond-raised largemouth bass to 17β-estradiol and/or the bacteria Edwardsiella ictaluri led to distinct changes in expression of hep-1 and hep-2. Estradiol reduced the constitutive expression of hep-1 in the liver. Bacterial exposure induced expression of hep-2, suggesting that hepcidin-2 may have an antimicrobial function, and this induction was abolished by estradiol. To our knowledge, this is the first report of the regulation of hepcidin expression by estradiol in either fish or mammals.

  13. Transient Expression of Viral Proteins in Plants Using Agrobacterium tumefaciens.

    PubMed

    Hitzeroth, Inga I; van Zyl, Albertha R

    2016-01-01

    Transient expression of viral proteins in plants is a novel alternative to other expression platforms. The viral proteins can be used as potential vaccines or in diagnostics. Nicotiana benthamiana leaves or whole plants are infiltrated with recombinant Agrobacterium that harbor the gene of interest. Protein expression in the plants is rapid and results are obtained within 2-7 days. Here we describe how to make electrocompetent Agrobacterium, how to transform Agrobacterium, how to infiltrate leaves or plants with the recombinant Agrobacterium, and lastly how to extract the protein for analysis by gel electrophoresis. PMID:27076324

  14. Yeast Fex1p Is a Constitutively Expressed Fluoride Channel with Functional Asymmetry of Its Two Homologous Domains*

    PubMed Central

    Smith, Kathryn D.; Gordon, Patricia B.; Rivetta, Alberto; Allen, Kenneth E.; Berbasova, Tetyana; Slayman, Clifford; Strobel, Scott A.

    2015-01-01

    Fluoride is a ubiquitous environmental toxin with which all biological species must cope. A recently discovered family of fluoride export (FEX) proteins protects organisms from fluoride toxicity by removing it from the cell. We show here that FEX proteins in Saccharomyces cerevisiae function as ion channels that are selective for fluoride over chloride and that these proteins are constitutively expressed at the yeast plasma membrane. Continuous expression is in contrast to many other toxin exporters in yeast, and this, along with the fact that two nearly duplicate proteins are encoded in the yeast genome, suggests that the threat posed by fluoride ions is frequent and detrimental. Structurally, eukaryotic FEX proteins consist of two homologous four-transmembrane helix domains folded into an antiparallel dimer, where the orientation of the two domains is fixed by a single transmembrane linker helix. Using phylogenetic sequence conservation as a guide, we have identified several functionally important residues. There is substantial functional asymmetry in the effect of mutation at corresponding sites in the two domains. Specifically, mutations to residues in the C-terminal domain proved significantly more detrimental to function than did similar mutations in the N-terminal domain. Our data suggest particular residues that may be important to anion specificity, most notably the necessity of a positive charge near the end of TMH1 in the C-terminal domain. It is possible that a cationic charge at this location may create an electrostatic well for fluoride ions entering the channel from the cytoplasm. PMID:26055717

  15. Constitutive expression of the cloned phenol hydroxylase gene(s) from Alcaligenes eutrophus JMP134 and concomitant trichloroethylene oxidation.

    PubMed Central

    Kim, Y; Ayoubi, P; Harker, A R

    1996-01-01

    Given the demonstrated phenol-dependent trichloroethylene (TCE) degradation in Alcaligenes eutrophus JMP134 (A. R. Harker and Y. Kim, Appl. Environ. Microbiol. 56:1179-1181, 1990), this work represents a purposeful effort to create a constitutive degrader of TCE. Genes responsible for phenol hydroxylase activity were identified by Tn5 transposon mutagenesis. Mutants lacked both phenol hydroxylase and catechol 2,3-dioxygenase activities. Southern blot analysis of total DNA showed that all mutants contained a single copy of Tn5 inserted in the same 11.5-kb EcoRI fragment. Complementation with a cosmid-based gene bank constructed from A. eutrophus AEK101 allowed the isolation of three recombinant cosmids carrying a common 16.8-kb HindIII fragment. Deletion and subcloning analysis localized the genes involved in phenol hydroxylase and catechol 2,3-dioxygenase activities. Partial sequence analysis of regions within the cloned phenol hydroxylase-expressing fragment shows significant homology to the oxygenase and oxidoreductase subunits of toluene-3-monooxygenase from Pseudomonas pickettii. The Tn5-induced phl mutant, carrying a recombinant plasmid expressing the phenol hydroxylase activity, degrades TCE in the absence of induction. Complete removal of TCE (50 microM) within 24 h was observed in minimal medium containing only 0.05% ethanol as a carbon source. The bacterium removed 200 microM TCE to below detectable levels within 2 days under noninducing and nonselective conditions. PMID:8795212

  16. Constitutive expression of the cloned phenol hydroxylase gene(s) from Alcaligenes eutrophus JMP134 and concomitant trichloroethylene oxidation.

    PubMed

    Kim, Y; Ayoubi, P; Harker, A R

    1996-09-01

    Given the demonstrated phenol-dependent trichloroethylene (TCE) degradation in Alcaligenes eutrophus JMP134 (A. R. Harker and Y. Kim, Appl. Environ. Microbiol. 56:1179-1181, 1990), this work represents a purposeful effort to create a constitutive degrader of TCE. Genes responsible for phenol hydroxylase activity were identified by Tn5 transposon mutagenesis. Mutants lacked both phenol hydroxylase and catechol 2,3-dioxygenase activities. Southern blot analysis of total DNA showed that all mutants contained a single copy of Tn5 inserted in the same 11.5-kb EcoRI fragment. Complementation with a cosmid-based gene bank constructed from A. eutrophus AEK101 allowed the isolation of three recombinant cosmids carrying a common 16.8-kb HindIII fragment. Deletion and subcloning analysis localized the genes involved in phenol hydroxylase and catechol 2,3-dioxygenase activities. Partial sequence analysis of regions within the cloned phenol hydroxylase-expressing fragment shows significant homology to the oxygenase and oxidoreductase subunits of toluene-3-monooxygenase from Pseudomonas pickettii. The Tn5-induced phl mutant, carrying a recombinant plasmid expressing the phenol hydroxylase activity, degrades TCE in the absence of induction. Complete removal of TCE (50 microM) within 24 h was observed in minimal medium containing only 0.05% ethanol as a carbon source. The bacterium removed 200 microM TCE to below detectable levels within 2 days under noninducing and nonselective conditions. PMID:8795212

  17. Expression of constitutively activated Akt in the mammary gland leads to excess lipid synthesis during pregnancy and lactation.

    PubMed

    Schwertfeger, Kathryn L; McManaman, James L; Palmer, Carol A; Neville, Margaret C; Anderson, Steven M

    2003-06-01

    Expression of constitutively activated Akt in the mammary glands of transgenic mice results in a delay in post-lactational involution. We now report precocious lipid accumulation in the alveolar epithelium of mouse mammary tumor virus-myr-Akt transgenic mice accompanied by a lactation defect that results in a 50% decrease in litter weight over the first 9 days of lactation. Although ductal structures and alveolar units develop normally during pregnancy, cytoplasmic lipid droplets appeared precociously in mammary epithelial cells in early pregnancy and were accompanied by increased expression of adipophilin, which is associated with lipid droplets. By late pregnancy the lipid droplets had become significantly larger than in nontransgenic mice, and they persisted into lactation. The fat content of milk from lactating myr-Akt transgenic mice was 65-70% by volume compared to 25-30% in wild-type mice. The diminished growth of pups nursed by transgenic mothers could result from the high viscosity of the milk and the inability of the pups to remove sufficient quantities of milk by suckling. Transduction of the CIT3 mammary epithelial cell line with a recombinant human adenovirus encoding myr-Akt resulted in an increase in glucose transport and lipid biosynthesis, suggesting that Akt plays an important role in regulation of lipid metabolism. PMID:12700340

  18. Expression of snowdrop lectin (GNA) in transgenic rice plants confers resistance to rice brown planthopper.

    PubMed

    Rao, K V; Rathore, K S; Hodges, T K; Fu, X; Stoger, E; Sudhakar, D; Williams, S; Christou, P; Bharathi, M; Bown, D P; Powell, K S; Spence, J; Gatehouse, A M; Gatehouse, J A

    1998-08-01

    Snowdrop lectin (Galanthus nivalis agglutinin; GNA) has been shown previously to be toxic towards rice brown planthopper (Nilaparvata lugens; BPH) when administered in artificial diet. BPH feeds by phloem abstraction, and causes 'hopper burn', as well as being an important virus vector. To evaluate the potential of the gna gene to confer resistance towards BPH, transgenic rice (Oryza sativa L.) plants were produced, containing the gna gene in constructs where its expression was driven by a phloem-specific promoter (from the rice sucrose synthase RSs1 gene) and by a constitutive promoter (from the maize ubiquitin ubi1 gene). PCR and Southern analyses on DNA from these plants confirmed their transgenic status, and that the transgenes were transmitted to progeny after self-fertilization. Western blot analyses revealed expression of GNA at levels of up to 2.0% of total protein in some of the transgenic plants. GNA expression driven by the RSs1 promoter was tissue-specific, as shown by immunohistochemical localization of the protein in the non-lignified vascular tissue of transgenic plants. Insect bioassays and feeding studies showed that GNA expressed in the transgenic rice plants decreased survival and overall fecundity (production of offspring) of the insects, retarded insect development, and had a deterrent effect on BPH feeding. gna is the first transgene to exhibit insecticidal activity towards sap-sucking insects in an important cereal crop plant.

  19. Methods and compositions for regulating gene expression in plant cells

    NASA Technical Reports Server (NTRS)

    Beachy, Roger N. (Inventor); Luis, Maria Isabel Ordiz (Inventor); Dai, Shunhong (Inventor)

    2010-01-01

    Novel chimeric plant promoter sequences are provided, together with plant gene expression cassettes comprising such sequences. In certain preferred embodiments, the chimeric plant promoters comprise the BoxII cis element and/or derivatives thereof. In addition, novel transcription factors are provided, together with nucleic acid sequences encoding such transcription factors and plant gene expression cassettes comprising such nucleic acid sequences. In certain preferred embodiments, the novel transcription factors comprise the acidic domain, or fragments thereof, of the RF2a transcription factor. Methods for using the chimeric plant promoter sequences and novel transcription factors in regulating the expression of at least one gene of interest are provided, together with transgenic plants comprising such chimeric plant promoter sequences and novel transcription factors.

  20. Ortho-aminoazotoluene activates mouse constitutive androstane receptor (mCAR) and increases expression of mCAR target genes.

    PubMed

    Smetanina, Mariya A; Pakharukova, Mariya Y; Kurinna, Svitlana M; Dong, Bingning; Hernandez, Juan P; Moore, David D; Merkulova, Tatyana I

    2011-08-15

    2'-3-dimethyl-4-aminoazobenzene (ortho-aminoazotoluene, OAT) is an azo dye and a rodent carcinogen that has been evaluated by the International Agency for Research on Cancer (IARC) as a possible (class 2B) human carcinogen. Its mechanism of action remains unclear. We examined the role of the xenobiotic receptor Constitutive Androstane Receptor (CAR, NR1I3) as a mediator of the effects of OAT. We found that OAT increases mouse CAR (mCAR) transactivation in a dose-dependent manner. This effect is specific because another closely related azo dye, 3'-methyl-4-dimethyl-aminoazobenzene (3'MeDAB), did not activate mCAR. Real-time Q-PCR analysis in wild-type C57BL/6 mice revealed that OAT induces the hepatic mRNA expression of the following CAR target genes: Cyp2b10, Cyp2c29, Cyp3a11, Ugt1a1, Mrp4, Mrp2 and c-Myc. CAR-null (Car(-/-)) mice showed no increased expression of these genes following OAT treatment, demonstrating that CAR is required for their OAT dependent induction. The OAT-induced CAR-dependent increase of Cyp2b10 and c-Myc expression was confirmed by Western blotting. Immunohistochemistry analysis of wild-type and Car(-/-) livers showed that OAT did not acutely induce hepatocyte proliferation, but at much later time points showed an unexpected CAR-dependent proliferative response. These studies demonstrate that mCAR is an OAT xenosensor, and indicate that at least some of the biological effects of this compound are mediated by this nuclear receptor. PMID:21672546

  1. Ortho-Aminoazotoluene activates mouse Constitutive Androstane Receptor (mCAR) and increases expression of mCAR target genes

    PubMed Central

    Smetanina, Mariya A.; Pakharukova, Mariya Y.; Kurinna, Svitlana M.; Dong, Bingning; Hernandez, Juan P.; Moore, David D.; Merkulova, Tatyana I.

    2011-01-01

    2'-3-dimethyl-4-aminoazobenzene (ortho-aminoazotoluene, OAT) is an azo dye and a rodent carcinogen that has been evaluated by the International Agency for Research on Cancer (IARC) as a possible (class 2B) human carcinogen. Its mechanism of action remains unclear. We examined the role of the xenobiotic receptor Constitutive Androstane Receptor (CAR, NR1I3) as a mediator of the effects of OAT. We found that OAT increases mouse CAR (mCAR) transactivation in a dose-dependent manner. This effect is specific because another closely related azo dye, 3'-methyl-4-dimethyl-aminoazobenzene (3'MeDAB), did not activate mCAR. Real-time Q-PCR analysis in wild-type C57BL/6 mice revealed that OAT induces the hepatic mRNA expression of the following CAR target genes: Cyp2b10, Cyp2c29, Cyp3a11, Ugt1a1, Mrp4, Mrp2 and c-Myc. CAR-null (Car−/−) mice showed no increased expression of these genes following OAT treatment, demonstrating that CAR is required for their OAT dependent induction. The OAT-induced CAR-dependent increase of Cyp2b10 and c-Myc expression was confirmed by Western blotting. Immunohistochemistry analysis of wild-type and Car−/− livers showed that OAT did not acutely induce hepatocyte proliferation, but at much later time points showed an unexpected CAR-dependent proliferative response. These studies demonstrate that mCAR is an OAT xenosensor, and indicate that at least some of the biological effects of this compound are mediated by this nuclear receptor. PMID:21672546

  2. Constitutive expression of genes encoding notch receptors and ligands in developing lymphocytes, nTreg cells and dendritic cells in the human thymus.

    PubMed

    Bento-de-Souza, Luciana; Victor, Jefferson R; Bento-de-Souza, Luiz C; Arrais-Santos, Magaly; Rangel-Santos, Andréia C; Pereira-Costa, Érica; Raniero-Fernandes, Elaine; Seixas-Duarte, Maria I; Oliveira-Filho, João B; Silva Duarte, Alberto J

    2016-01-01

    The thymus is the site of T cell maturation. Notch receptors (Notch1-4) and ligands (DLL1-3 and Jagged1-2) constitute one of several pathways involved in this process. Our data revealed differential constitutive expression of Notch genes and ligands in T lymphocytes and thymic dendritic cells (tDCs), suggesting their participation in human thymocyte maturation. nTreg analyses indicated that the Notch components function in parallel to promote maturation in the thymus. PMID:27504259

  3. Functional granulocyte/macrophage colony stimulating factor receptor is constitutively expressed on neoplastic plasma cells and mediates tumour cell longevity.

    PubMed

    Villunger, A; Egle, A; Kos, M; Egle, D; Tinhofer, I; Henn, T; Uberall, F; Maly, K; Greil, R

    1998-09-01

    It has been shown that granulocyte/macrophage colony stimulating factor (GM-CSF) is able to support myeloma cell propagation in cooperation with interleukin (IL)-6, the major growth factor for malignant plasma cells, although the biological mechanisms involved remain unknown. Therefore we investigated (i) the expression levels of the GM-CSF receptor (GM-CSFR) constituents in three malignant plasma cell lines and in native malignant plasma cells, (ii) the ability of the receptor to mediate common signalling pathways regulating proliferation and cell survival in malignant plasma cell lines, and (iii) the effects of GM-CSF on tumour cell biology. The GM-CSFRalpha subunit was detected in the malignant plasma cell lines RPMI-8226, MC/CAR, IM-9 as well as 6/6 native myeloma cell samples derived from the bone marrow of patients with overt disease. Furthermore, GM-CSFR expression was also detected in the CD19+ fraction from 2/3 bone marrow samples and 5/8 peripheral blood samples derived from patients with malignant plasma cell disorders, but not in the CD19+ fraction of peripheral blood from healthy donors. The expressed cytokine receptor alpha-subunit was able to constitute a functional signalling complex with the ubiquitously expressed GM-CSFRbeta subunit, as demonstrated by the fact that GM-CSF induced the p21-ras/mitogen-activated protein kinase (MAPK) signalling cascade in malignant plasma cell lines. Since this signalling cascade plays an essential role in the mediation of both proliferation and cell survival, we investigated the impact of GM-CSF on these two events. Application of GM-CSF led to an increase of DNA-synthesis in MC/CAR, IM-9 and RPMI-8226 cells. Furthermore, it increased longevity of these malignant plasma cell lines by reducing the rates of spontaneous apoptosis. We conclude that (i) the functional GM-CSFR is commonly expressed on malignant plasma cells and that (ii) GM-CSF promotes the clonal expansion of myeloma cells by inhibiting spontaneous

  4. Transgenic salt-tolerant sugar beet (Beta vulgaris L.) constitutively expressing an Arabidopsis thaliana vacuolar Na/H antiporter gene, AtNHX3, accumulates more soluble sugar but less salt in storage roots.

    PubMed

    Liu, Hua; Wang, Qiuqing; Yu, Mengmeng; Zhang, Yanyan; Wu, Yingbao; Zhang, Hongxia

    2008-09-01

    In Arabidopsis thaliana, six vacuolar Na(+)/H(+) antiporters (AtNHX1-6) were identified. Among them, AtNHX1, 2 and 5 are functional Na(+)/H(+) antiporters with the most abundant expression levels in seedling shoots and roots. However, the expression of AtNHX3 in Arabidopsis can only be detected by RT-PCR, and its physiological function still remains unclear. In this work, we demonstrate that constitutive expression of AtNHX3 in sugar beet (Beta vulgaris L.) conferred augmented resistance to high salinity on transgenic plants. In the presence of 300 or 500 mm NaCl, transgenic plants showed very high potassium accumulation in the roots and storage roots. Furthermore, the transcripts of sucrose phosphate synthase (SPS), sucrose synthase (SS) and cell wall sucrose invertase (SI) genes were maintained in transgenic plants. The accumulation of soluble sugar in the storage roots of transgenic plants grown under high salt stress condition was also higher. Our results implicate that AtNHX3 is also a functional antiporter responsible for salt tolerance by mediating K(+)/H(+) exchange in higher plants. The salt accumulation in leaves but not in the storage roots, and the increased yield of storage roots with enhanced constituent soluble sugar contents under salt stress condition demonstrate a great potential use of this gene in improving the quality and yield of crop plants.

  5. Transgenic maize plants expressing the Totivirus antifungal protein, KP4, are highly resistant to corn smut.

    PubMed

    Allen, Aron; Islamovic, Emir; Kaur, Jagdeep; Gold, Scott; Shah, Dilip; Smith, Thomas J

    2011-10-01

    The corn smut fungus, Ustilago maydis, is a global pathogen responsible for extensive agricultural losses. Control of corn smut using traditional breeding has met with limited success because natural resistance to U. maydis is organ specific and involves numerous maize genes. Here, we present a transgenic approach by constitutively expressing the Totivirus antifungal protein KP4, in maize. Transgenic maize plants expressed high levels of KP4 with no apparent negative impact on plant development and displayed robust resistance to U. maydis challenges to both the stem and ear tissues in the greenhouse. More broadly, these results demonstrate that a high level of organ independent fungal resistance can be afforded by transgenic expression of this family of antifungal proteins.

  6. Plant peroxisomes are degraded by starvation-induced and constitutive autophagy in tobacco BY-2 suspension-cultured cells

    PubMed Central

    Voitsekhovskaja, Olga V.; Schiermeyer, Andreas; Reumann, Sigrun

    2014-01-01

    Very recently, autophagy has been recognized as an important degradation pathway for quality control of peroxisomes in Arabidopsis plants. To further characterize the role of autophagy in plant peroxisome degradation, we generated stable transgenic suspension-cultured cell lines of heterotrophic Nicotiana tabacum L. cv. Bright Yellow 2 expressing a peroxisome-targeted version of enhanced yellow fluorescent protein. Indeed, this cell line model system proved advantageous for detailed cytological analyses of autophagy stages and for quantification of cellular peroxisome pools under different culturing conditions and upon inhibitor applications. Complementary biochemical, cytological, and pharmacological analyses provided convincing evidence for peroxisome degradation by bulk autophagy during carbohydrate starvation. This degradation was slowed down by the inhibitor of autophagy, 3-methyladenine (3-MA), but the 3-MA effect ceased at advanced stages of starvation, indicating that another degradation mechanism for peroxisomes might have taken over. 3-MA also caused an increase particularly in peroxisomal proteins and cellular peroxisome numbers when applied under nutrient-rich conditions in the logarithmic growth phase, suggesting a high turnover rate for peroxisomes by basal autophagy under non-stress conditions. Together, our data demonstrate that a great fraction of the peroxisome pool is subject to extensive autophagy-mediated turnover under both nutrient starvation and optimal growth conditions. Our analyses of the cellular pool size of peroxisomes provide a new tool for quantitative investigations of the role of plant peroxisomes in reactive oxygen species metabolism. PMID:25477890

  7. Ortho-aminoazotoluene activates mouse constitutive androstane receptor (mCAR) and increases expression of mCAR target genes

    SciTech Connect

    Smetanina, Mariya A.; Pakharukova, Mariya Y.; Kurinna, Svitlana M.; Dong, Bingning; Hernandez, Juan P.; Moore, David D.; Merkulova, Tatyana I.

    2011-08-15

    2'-3-dimethyl-4-aminoazobenzene (ortho-aminoazotoluene, OAT) is an azo dye and a rodent carcinogen that has been evaluated by the International Agency for Research on Cancer (IARC) as a possible (class 2B) human carcinogen. Its mechanism of action remains unclear. We examined the role of the xenobiotic receptor Constitutive Androstane Receptor (CAR, NR1I3) as a mediator of the effects of OAT. We found that OAT increases mouse CAR (mCAR) transactivation in a dose-dependent manner. This effect is specific because another closely related azo dye, 3'-methyl-4-dimethyl-aminoazobenzene (3'MeDAB), did not activate mCAR. Real-time Q-PCR analysis in wild-type C57BL/6 mice revealed that OAT induces the hepatic mRNA expression of the following CAR target genes: Cyp2b10, Cyp2c29, Cyp3a11, Ugt1a1, Mrp4, Mrp2 and c-Myc. CAR-null (Car{sup -/-}) mice showed no increased expression of these genes following OAT treatment, demonstrating that CAR is required for their OAT dependent induction. The OAT-induced CAR-dependent increase of Cyp2b10 and c-Myc expression was confirmed by Western blotting. Immunohistochemistry analysis of wild-type and Car{sup -/-} livers showed that OAT did not acutely induce hepatocyte proliferation, but at much later time points showed an unexpected CAR-dependent proliferative response. These studies demonstrate that mCAR is an OAT xenosensor, and indicate that at least some of the biological effects of this compound are mediated by this nuclear receptor. - Highlights: > The azo dye and mouse carcinogen OAT is a very effective mCAR activator. > OAT increases mCAR transactivation in a dose-dependent manner. > OAT CAR-dependently increases the expression of a specific subset of CAR target genes. > OAT induces an unexpectedly deferred, but CAR-dependent hepatocyte proliferation.

  8. Sex-related resistance to myocardial ischemia-reperfusion injury is associated with high constitutive ARC expression.

    PubMed

    Bouma, Wobbe; Noma, Mio; Kanemoto, Shinya; Matsubara, Muneaki; Leshnower, Bradley G; Hinmon, Robin; Gorman, Joseph H; Gorman, Robert C

    2010-05-01

    The female sex has been associated with improved myocardial salvage after ischemia and reperfusion (I/R). Estrogen, specifically 17beta-estradiol, has been demonstrated to mediate this phenomenon by limiting cardiomyocyte apoptosis. We sought to quantitatively assess the effect of sex, ovarian hormone loss, and I/R on myocardial Bax, Bcl-2, and apoptosis repressor with caspase recruitment domain (ARC) expression. Male (n = 48), female (n = 26), and oophorectomized female (n = 20) rabbits underwent 30 min of regional ischemia and 3 h of reperfusion. The myocardial area at risk and infarct size were determined using a double-staining technique and planimetry. In situ oligo ligation was used to assess apoptotic cell death. Western blot analysis was used to determine proapoptotic (Bax) and antiapoptotic (Bcl-2 and ARC) protein levels in all three ischemic groups and, additionally, in three nonischemic groups. Infarct size (43.7 +/- 3.2%) and apoptotic cell death (0.51 +/- 0.10%) were significantly attenuated in females compared with males (56.4 +/- 1.6%, P < 0.01, and 4.29 +/- 0.95%, P < 0.01) and oophorectomized females (55.7 +/- 3.4%, P < 0.05, and 4.36 +/- 0.51%, P < 0.01). Females expressed significantly higher baseline ARC levels (3.62 +/- 0.29) compared with males (1.78 +/- 0.18, P < 0.01) and oophorectomized females (1.08 +/- 0.26, P < 0.01). Males expressed a significantly higher baseline Bax-to-Bcl-2 ratio (4.32 +/- 0.99) compared with females (0.65 +/- 0.13, P < 0.01) and oophorectomized females (0.42 +/- 0.10, P < 0.01). I/R significantly reduced Bax-to-Bcl-2 ratios in males. In all other groups, ARC levels and Bax-to-Bcl-2 ratios did not significantly change. These results support the conclusion that in females, endogenous estrogen limits I/R-induced cardiomyocyte apoptosis by producing a baseline antiapoptotic profile, which is associated with estrogen-dependent high constitutive myocardial ARC expression.

  9. Constitutive androstane receptor activation by 2,4,6-triphenyldioxane-1,3 suppresses the expression of the gluconeogenic genes.

    PubMed

    Kachaylo, Ekaterina M; Yarushkin, Andrei A; Pustylnyak, Vladimir O

    2012-03-15

    The constitutive androstane receptor (CAR, NR1I3) has a central role in detoxification processes, regulating the expression of a set of genes involved in metabolism. The dual role of NR1I3 as both a xenosensor and as a regulator of endogenous energy metabolism has recently been accepted. Here, we investigated the mechanism of transcriptional regulation of the glucose metabolising genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) by the cis isomer of 2,4,6-triphenyldioxane-1,3 (cisTPD), a highly effective NR1I3 activator in rat liver. It was shown that expression of the gluconeogenic genes PEPCK and G6Pase was repressed by cisTPD treatment under fasting conditions. Western-blot analysis demonstrated a clear reduction in the intensity of PEPCK and G6Pase immunobands from the livers of cisTPD-treated animals relative to bands from the livers of control animals. Chromatin immunoprecipitation assays demonstrated that cisTPD prevents the binding of FOXO1 to the insulin response sequences in the PEPCK and G6Pase gene promoters in rat liver. Moreover, cisTPD-activated NR1I3 inhibited NR2A1 (HNF-4) transactivation by competing with NR2A1 for binding to the NR2A1-binding element (DR1-site) in the gluconeogenic gene promoters. Thus, our results are consistent with the hypothesis that the cisTPD-activated NR1I3 participates in the regulation of the gluconeogenic genes PEPCK and G6Pase. PMID:22296760

  10. A novel meningococcal outer membrane vesicle vaccine with constitutive expression of FetA: A phase I clinical trial

    PubMed Central

    Marsay, L.; Dold, C.; Green, C.A.; Rollier, C.S.; Norheim, G.; Sadarangani, M.; Shanyinde, M.; Brehony, C.; Thompson, A.J.; Sanders, H.; Chan, H.; Haworth, K.; Derrick, J.P.; Feavers, I.M.; Maiden, M.C.; Pollard, A.J.

    2015-01-01

    Summary Objectives Outer membrane vesicle (OMV) vaccines are used against outbreaks of capsular group B Neisseria meningitidis (MenB) caused by strains expressing particular PorA outer membrane proteins (OMPs). Ferric enterobactin receptor (FetA) is another variable OMP that induces type-specific bactericidal antibodies, and the combination of judiciously chosen PorA and FetA variants in vaccine formulations is a potential approach to broaden protection of such vaccines. Methods The OMV vaccine MenPF-1 was generated by genetically modifying N. meningitidis strain 44/76 to constitutively express FetA. Three doses of 25 μg or 50 μg of MenPF-1 were delivered intra-muscularly to 52 healthy adults. Results MenPF-1 was safe and well tolerated. Immunogenicity was measured by serum bactericidal assay (SBA) against wild-type and isogenic mutant strains. After 3 doses, the proportion of volunteers with SBA titres ≥1:4 (the putative protective titre) was 98% for the wild-type strain, and 77% for the strain 44/76 FetAonPorAoff compared to 51% in the strain 44/76 FetAoffPorAoff, demonstrating that vaccination with MenPF-1 simultaneously induced FetA and PorA bactericidal antibodies. Conclusion This study provides a proof-of-concept for generating bactericidal antibodies against FetA after OMV vaccination in humans. Prevalence-based choice of PorA and FetA types can be used to formulate a vaccine for broad protection against MenB disease. PMID:25982025

  11. Characterization of a novel type of MLSB resistance plasmid from Staphylococcus saprophyticus carrying a constitutively expressed erm(C) gene.

    PubMed

    Hauschild, Tomasz; Lüthje, Petra; Schwarz, Stefan

    2006-06-15

    An erm(C)-carrying plasmid of unusual size and restriction map, designated pSES22, was identified in a Staphylococcus saprophyticus strain and sequenced completely. Constitutive expression of the erm(C) gene from pSES22 is based on a novel 22-bp tandem duplication in the erm(C) translational attenuator. Comparative analysis of the deduced Erm(C) amino acid sequence revealed that Erm(C) from pSES22 - together with an Erm(C) methylase from S. hyicus - represented a separate branch in the homology tree of Erm(C) methylases. Structural comparisons showed that plasmid pSES22 differed distinctly from all other completely sequenced erm(C)-carrying resistance plasmids. However, pSES22 was similar to several members of a diverse group of small plasmids, all of which carried closely related plasmid backbones consisting of the genes repU and pre/mob, but differed in their resistance genes.

  12. MDCK cells expressing constitutively active Yes-associated protein (YAP) undergo apical extrusion depending on neighboring cell status.

    PubMed

    Chiba, Takanori; Ishihara, Erika; Miyamura, Norio; Narumi, Rika; Kajita, Mihoko; Fujita, Yasuyuki; Suzuki, Akira; Ogawa, Yoshihiro; Nishina, Hiroshi

    2016-01-01

    Cell competition is a cell-cell interaction by which a cell compares its fitness to that of neighboring cells. The cell with the relatively lower fitness level is the "loser" and actively eliminated, while the cell with the relatively higher fitness level is the "winner" and survives. Recent studies have shown that cells with high Yes-associated protein (YAP) activity win cell competitions but the mechanism is unknown. Here, we report the unexpected finding that cells overexpressing constitutively active YAP undergo apical extrusion and are losers, rather than winners, in competitions with normal mammalian epithelial cells. Inhibitors of metabolism-related proteins such as phosphoinositide-3-kinase (PI3K), mammalian target of rapamycin (mTOR), or p70S6 kinase (p70S6K) suppressed this apical extrusion, as did knockdown of vimentin or filamin in neighboring cells. Interestingly, YAP-overexpressing cells switched from losers to winners when co-cultured with cells expressing K-Ras (G12V) or v-Src. Thus, the role of YAP in deciding cell competitions depends on metabolic factors and the status of neighboring cells. PMID:27324860

  13. MDCK cells expressing constitutively active Yes-associated protein (YAP) undergo apical extrusion depending on neighboring cell status

    PubMed Central

    Chiba, Takanori; Ishihara, Erika; Miyamura, Norio; Narumi, Rika; Kajita, Mihoko; Fujita, Yasuyuki; Suzuki, Akira; Ogawa, Yoshihiro; Nishina, Hiroshi

    2016-01-01

    Cell competition is a cell-cell interaction by which a cell compares its fitness to that of neighboring cells. The cell with the relatively lower fitness level is the “loser” and actively eliminated, while the cell with the relatively higher fitness level is the “winner” and survives. Recent studies have shown that cells with high Yes-associated protein (YAP) activity win cell competitions but the mechanism is unknown. Here, we report the unexpected finding that cells overexpressing constitutively active YAP undergo apical extrusion and are losers, rather than winners, in competitions with normal mammalian epithelial cells. Inhibitors of metabolism-related proteins such as phosphoinositide-3-kinase (PI3K), mammalian target of rapamycin (mTOR), or p70S6 kinase (p70S6K) suppressed this apical extrusion, as did knockdown of vimentin or filamin in neighboring cells. Interestingly, YAP-overexpressing cells switched from losers to winners when co-cultured with cells expressing K-Ras (G12V) or v-Src. Thus, the role of YAP in deciding cell competitions depends on metabolic factors and the status of neighboring cells. PMID:27324860

  14. Expression of Recombinant Vaccines and Antibodies in Plants

    PubMed Central

    2014-01-01

    Plants are able to perform post-translational maturations of therapeutic proteins required for their functional biological activity and suitable in vivo pharmacokinetics. Plants can be a low-cost, large-scale production platform of recombinant biopharmaceutical proteins such as vaccines and antibodies. Plants, however, lack mechanisms of processing authentic human N-glycosylation, which imposes a major limitation in their use as an expression system for therapeutic glycoproducts. Efforts have been made to circumvent plant-specific N-glycosylation, as well as to supplement the plant's endogenous system with human glycosyltransferases for non-immunogenic and humanized N-glycan production. Herein we review studies on the potential of plants to serve as production systems for therapeutic and prophylactic biopharmaceuticals. We have especially focused on recombinant vaccines and antibodies and new expression strategies to overcome the existing problems associated with their production in plants. PMID:24937251

  15. Expression of recombinant vaccines and antibodies in plants.

    PubMed

    Ko, Kisung

    2014-06-01

    Plants are able to perform post-translational maturations of therapeutic proteins required for their functional biological activity and suitable in vivo pharmacokinetics. Plants can be a low-cost, large-scale production platform of recombinant biopharmaceutical proteins such as vaccines and antibodies. Plants, however, lack mechanisms of processing authentic human N-glycosylation, which imposes a major limitation in their use as an expression system for therapeutic glycoproducts. Efforts have been made to circumvent plant-specific N-glycosylation, as well as to supplement the plant's endogenous system with human glycosyltransferases for non-immunogenic and humanized N-glycan production. Herein we review studies on the potential of plants to serve as production systems for therapeutic and prophylactic biopharmaceuticals. We have especially focused on recombinant vaccines and antibodies and new expression strategies to overcome the existing problems associated with their production in plants.

  16. Enhanced water stress tolerance of transgenic maize plants over-expressing LEA Rab28 gene.

    PubMed

    Amara, Imen; Capellades, Montserrat; Ludevid, M Dolors; Pagès, Montserrat; Goday, Adela

    2013-06-15

    Late Embryogenesis Abundant (LEA) proteins participate in plant stress responses and contribute to the acquisition of desiccation tolerance. In this report Rab28 LEA gene has been over-expressed in maize plants under a constitutive maize promoter. The expression of Rab28 transcripts led to the accumulation and stability of Rab28 protein in the transgenic plants. Native Rab28 protein is localized to nucleoli in wild type maize embryo cells; here we find by whole-mount immunocytochemistry that in root cells of Rab28 transgenic and wild-type plants the protein is also associated to nucleolar structures. Transgenic plants were tested for stress tolerance and resulted in sustained growth under polyethyleneglycol (PEG)-mediated dehydration compared to wild-type controls. Under osmotic stress transgenic seedlings showed increased leaf and root areas, higher relative water content (RWC), reduced chlorophyll loss and lower Malondialdehyde (MDA) production in relation to wild-type plants. Moreover, transgenic seeds exhibited higher germination rates than wild-type seeds under water deficit. Overall, our results highlight the presence of transgenic Rab28 protein in nucleolar structures and point to the potential of group 5 LEA Rab28 gene as candidate to enhance stress tolerance in maize plants.

  17. Constitutive expression of a salinity-induced wheat WRKY transcription factor enhances salinity and ionic stress tolerance in transgenic Arabidopsis thaliana

    SciTech Connect

    Qin, Yuxiang; Tian, Yanchen; Han, Lu; Yang, Xinchao

    2013-11-15

    Highlights: •A class II WRKY transcription factor, TaWRKY79 was isolated and characterized. •TaWRKY79 was induced by NaCl or abscisic acid. •843 bp regulatory segment was sufficient to respond to ABA or NaCl treatment. •TaWRKY79 enhanced salinity and ionic tolerance while reduced sensitivity to ABA. •TaWRKY79 increased salinity and ionic tolerance in an ABA-dependent pathway. -- Abstract: The isolation and characterization of TaWRKY79, a wheat class II WRKY transcription factor, is described. Its 1297 bp coding region includes a 987 bp long open reading frame. TaWRKY79 was induced by stressing seedlings with either NaCl or abscisic acid (ABA). When a fusion between an 843 bp segment upstream of the TaWRKY79 coding sequence and GUS was introduced into Arabidopsis thaliana, GUS staining indicated that this upstream segment captured the sequence(s) required to respond to ABA or NaCl treatment. When TaWRKY79 was constitutively expressed as a transgene in A. thaliana, the transgenic plants showed an improved capacity to extend their primary root in the presence of either 100 mM NaCl, 10 mM LiCl or 2 μM ABA. The inference was that TaWRKY79 enhanced the level of tolerance to both salinity and ionic stress, while reducing the level of sensitivity to ABA. The ABA-related genes ABA1, ABA2 ABI1 and ABI5 were all up-regulated in the TaWRKY79 transgenic plants, suggesting that the transcription factor operates in an ABA-dependent pathway.

  18. A universal expression/silencing vector in plants.

    PubMed

    Peretz, Yuval; Mozes-Koch, Rita; Akad, Fuad; Tanne, Edna; Czosnek, Henryk; Sela, Ilan

    2007-12-01

    A universal vector (IL-60 and auxiliary constructs), expressing or silencing genes in every plant tested to date, is described. Plants that have been successfully manipulated by the IL-60 system include hard-to-manipulate species such as wheat (Triticum duram), pepper (Capsicum annuum), grapevine (Vitis vinifera), citrus, and olive (Olea europaea). Expression or silencing develops within a few days in tomato (Solanum lycopersicum), wheat, and most herbaceous plants and in up to 3 weeks in woody trees. Expression, as tested in tomato, is durable and persists throughout the life span of the plant. The vector is, in fact, a disarmed form of Tomato yellow leaf curl virus, which is applied as a double-stranded DNA and replicates as such. However, the disarmed virus does not support rolling-circle replication, and therefore viral progeny single-stranded DNA is not produced. IL-60 does not integrate into the plant's genome, and the construct, including the expressed gene, is not heritable. IL-60 is not transmitted by the Tomato yellow leaf curl virus's natural insect vector. In addition, artificial satellites were constructed that require a helper virus for replication, movement, and expression. With IL-60 as the disarmed helper "virus," transactivation occurs, resulting in an inducible expressing/silencing system. The system's potential is demonstrated by IL-60-derived suppression of a viral-silencing suppressor of Grapevine virus A, resulting in Grapevine virus A-resistant/tolerant plants.

  19. A Bacterial Biosensor for Oxidative Stress Using the Constitutively Expressed Redox-Sensitive Protein roGFP2

    PubMed Central

    Arias-Barreiro, Carlos R.; Okazaki, Keisuke; Koutsaftis, Apostolos; Inayat-Hussain, Salmaan H.; Tani, Akio; Katsuhara, Maki; Kimbara, Kazuhide; Mori, Izumi C.

    2010-01-01

    A highly specific, high throughput-amenable bacterial biosensor for chemically induced cellular oxidation was developed using constitutively expressed redox-sensitive green fluorescent protein roGFP2 in E. coli (E. coli-roGFP2). Disulfide formation between two key cysteine residues of roGFP2 was assessed using a double-wavelength ratiometric approach. This study demonstrates that only a few minutes were required to detect oxidation using E. coli-roGFP2, in contrast to conventional bacterial oxidative stress sensors. Cellular oxidation induced by hydrogen peroxide, menadione, sodium selenite, zinc pyrithione, triphenyltin and naphthalene became detectable after 10 seconds and reached the maxima between 80 to 210 seconds, contrary to Cd2+, Cu2+, Pb2+, Zn2+ and sodium arsenite, which induced the oxidation maximum immediately. The lowest observable effect concentrations (in ppm) were determined as 1.0 × 10−7 (arsenite), 1.0 × 10−4 (naphthalene), 1.0 × 10−4 (Cu2+), 3.8 × 10−4 (H2O2), 1.0 × 10−3 (Cd2+), 1.0 × 10−3 (Zn2+), 1.0 × 10−2 (menadione), 1.0 (triphenyltin), 1.56 (zinc pyrithione), 3.1 (selenite) and 6.3 (Pb2+), respectively. Heavy metal-induced oxidation showed unclear response patterns, whereas concentration-dependent sigmoid curves were observed for other compounds. In vivo GSH content and in vitro roGFP2 oxidation assays together with E. coli-roGFP2 results suggest that roGFP2 is sensitive to redox potential change and thiol modification induced by environmental stressors. Based on redox-sensitive technology, E. coli-roGFP2 provides a fast comprehensive detection system for toxicants that induce cellular oxidation. PMID:22163550

  20. Biochemical and cellular characterization of Helicobacter pylori RecA, a protein with high-level constitutive expression.

    PubMed

    Orillard, Emilie; Radicella, J Pablo; Marsin, Stéphanie

    2011-12-01

    Helicobacter pylori is a bacterial pathogen colonizing half of the world's human population. It has been implicated in a number of gastric diseases, from asymptomatic gastritis to cancer. It is characterized by an amazing genetic variability that results from high mutation rates and efficient DNA homologous recombination and transformation systems. Here, we report the characterization of H. pylori RecA (HpRecA), a protein shown to be involved in DNA repair, transformation, and mouse colonization. The biochemical characterization of the purified recombinase reveals activities similar to those of Escherichia coli RecA (EcRecA). We show that in H. pylori, HpRecA is present in about 80,000 copies per cell during exponential growth and decreases to about 50,000 copies in stationary phase. The amount of HpRecA remains unchanged after induction of DNA lesions, suggesting that HpRecA is always expressed at a high level in order to repair DNA damage or facilitate recombination. We performed HpRecA localization analysis by adding a Flag tag to the protein, revealing two different patterns of localization. During exponential growth, RecA-Flag presents a diffuse pattern, overlapping with the DAPI (4',6-diamidino-2-phenylindole) staining of DNA, whereas during stationary phase, the protein is present in more defined areas devoid of DAPI staining. These localizations are not affected by inactivation of competence or DNA recombination genes. Neither UV irradiation nor gamma irradiation modified HpRecA localization, suggesting the existence of a constitutive DNA damage adaptation system. PMID:21949074

  1. Constitutive expression of a Mg2+-inhibited K+ current and a TRPM7-like current in human erythroleukemia cells.

    PubMed

    Mason, Michael J; Schaffner, Catherine; Floto, R Andres; Teo, Quok An

    2012-03-15

    Whole cell patch-clamp experiments were undertaken to define the basal K(+) conductance(s) in human erythroleukemia cells and its contribution to the setting of resting membrane potential. Experiments revealed a non-voltage-activated, noninactivating K(+) current. The magnitude of the current recorded under whole cell conditions was inhibited by an increase in free intracellular Mg(2+) concentration. Activation or inactivation of the Mg(2+)-inhibited K(+) current (MIP) was paralleled by activation or inactivation of a Mg(2+)-inhibited TRPM7-like current displaying characteristics indistinguishable from those reported for molecularly identified TRPM7 current. The MIP and TRPM7 currents were inhibited by 5-lipoxygenase inhibitors. However, inhibition of the MIP current was temporally distinct from inhibition of TRPM7 current, allowing for isolation of the MIP current. Isolation of the MIP conductance revealed a current reversing near the K(+) equilibrium potential, indicative of a highly K(+)-selective conductance. Consistent with this finding, coactivation of the nonselective cation current TRPM7 and the MIP current following dialysis with nominally Mg(2+)-free pipette solution resulted in hyperpolarized whole cell reversal potentials, consistent with an important role for the MIP current in the setting of a negative resting membrane potential. The MIP and TRPM7-like conductances were constitutively expressed under in vivo conditions of intracellular Mg(2+), as judged by their initial detection and subsequent inactivation following dialysis with a pipette solution containing 5 mM free Mg(2+). The MIP current was blocked in a voltage-dependent fashion by extracellular Cs(+) and, to a lesser degree, by Ba(2+) and was blocked by extracellular La(3+) and 2-aminoethoxydiphenyl borate. MIP currents were unaffected by blockers of ATP-sensitive K(+) channels, human ether-à-go-go-related gene current, and intermediate-conductance Ca(2+)-activated K(+) channels. In addition

  2. Differential expression profiles and roles of inducible DUSPs and ERK1/2-specific constitutive DUSP6 and DUSP7 in microglia.

    PubMed

    Ham, Ji-Eun; Oh, Eun-Kyung; Kim, Dong-Hoon; Choi, Sang-Hyun

    2015-11-13

    Dual-specificity phosphatases (DUSPs) show distinct substrate preferences for specific MAPKs. DUSPs sharing a substrate preference for ERK1/2 may be classified as inducible or constitutive. In contrast to the inducible DUSPs which also dephosphorylate p38 MAPK and JNK in the major inflammatory pathways, constitutive DUSP6 and DUSP7 are specific to ERK1/2 and have not been studied in microglia and other immune cells to date. In the present study, we differentiated mRNA expression profiles of inducible and constitutive DUSPs that dephosphorylate ERK1/2 in microglia. Lipopolysaccharide (LPS) at 1 ng/ml induced prompt phosphorylation of ERK1/2 with peak induction at 30 min. LPS induced expression of DUSP1, DUSP2, and DUSP5 within 60 min, whereas DUSP4 expression was induced more slowly. DUSP6 and DUSP7 exhibited constitutive basal expression, which decreased immediately after LPS stimulation but subsequently returned to basal levels. The expression of DUSP6 and DUSP7 was regulated inverse to the phosphorylation of ERK1/2 in LPS-stimulated microglia. Therefore, we next investigated the correlation between DUSP6 and DUSP7 expression and ERK1/2 phosphorylation in resting and LPS-stimulated microglia. Inhibition of the ERK1/2 pathway by PD98059 and FR180204 resulted in a decrease in DUSP6 and DUSP7 expression, both in resting and LPS-stimulated microglia. These inhibitors partially blocked the LPS-induced expression of DUSP1, DUSP2, and DUSP4, but had no effect on DUSP5. Finally, we examined the role of DUSP6 activity in the downregulation of ERK1/2 phosphorylation. BCI, an inhibitor of DUSP6, increased the phosphorylation of ERK1/2. However, pretreatment with BCI inhibited the LPS-induced phosphorylation of ERK1/2. These results demonstrate that constitutive DUPS6 and DUSP7 expression was downregulated inverse to the expression of inducible DUSPs and the phosphorylation of ERK1/2 in LPS-stimulated microglia. The expression of DUPS6 and DUSP7 was mediated by ERK1

  3. Expression and assembly of functional bacterial luciferase in plants

    PubMed Central

    Koncz, Csaba; Olsson, Olof; Langridge, William H. R.; Schell, Jeff; Szalay, Aladar A.

    1987-01-01

    The luxA and luxB structural genes of Vibrio harveyi luciferase [alkanal,reduced FMN:oxygen oxidoreductase (1-hydroxylating, luminescing), EC 1.14.14.3] were introduced into a plant expression vector and transferred into tobacco and carrot cells by Agrobacterium-mediated or direct DNA transformation. Simultaneous expression of the luxA and luxB genes was monitored by protein immunoblot analysis. Luciferase-mediated light emission provided evidence for the assembly of the two protein subunits into a functional dimeric enzyme in plant protoplasts, in transformed calli, and in leaves of transformed plants. Bacterial luciferase may provide a useful marker-gene system for the quantitative assay of coordinate gene expression in transgenic plants. Images PMID:16593793

  4. Phytodetoxification of TNT by transgenic plants expressing a bacterial nitroreductase.

    PubMed

    Hannink, N; Rosser, S J; French, C E; Basran, A; Murray, J A; Nicklin, S; Bruce, N C

    2001-12-01

    There is major international concern over the wide-scale contamination of soil and associated ground water by persistent explosives residues. 2,4,6-Trinitrotoluene (TNT) is one of the most recalcitrant and toxic of all the military explosives. The lack of affordable and effective cleanup technologies for explosives contamination requires the development of better processes. Significant effort has recently been directed toward the use of plants to extract and detoxify TNT. To explore the possibility of overcoming the high phytotoxic effects of TNT, we expressed bacterial nitroreductase in tobacco plants. Nitroreductase catalyzes the reduction of TNT to hydroxyaminodinitrotoluene (HADNT), which is subsequently reduced to aminodinitrotoluene derivatives (ADNTs). Transgenic plants expressing nitroreductase show a striking increase in ability to tolerate, take up, and detoxify TNT. Our work suggests that expression of nitroreductase (NR) in plants suitable for phytoremediation could facilitate the effective cleanup of sites contaminated with high levels of explosives. PMID:11731787

  5. A promoter from sugarcane bacilliform badnavirus drives transgene expression in banana and other monocot and dicot plants.

    PubMed

    Schenk, P M; Sagi, L; Remans, T; Dietzgen, R G; Bernard, M J; Graham, M W; Manners, J M

    1999-04-01

    A 1369 bp DNA fragment (Sc) was isolated from a full-length clone of sugarcane bacilliform badnavirus (ScBV) and was shown to have promoter activity in transient expression assays using monocot (banana, maize, millet and sorghum) and dicot plant species (tobacco, sunflower, canola and Nicotiana benthamiana). This promoter was also tested for stable expression in transgenic banana and tobacco plants. These experiments showed that this promoter could drive high-level expression of the beta-glucuronidase (GUS) reporter gene in most plant cells. The expression level was comparable to the maize ubiquitin promoter in standardised transient assays in maize. In transgenic banana plants the expression levels were variable for different transgenic lines but was generally comparable with the activities of both the maize ubiquitin promoter and the enhanced cauliflower mosaic virus (CaMV) 35S promoter. The Sc promoter appears to express in a near-constitutive manner in transgenic banana and tobacco plants. The promoter from sugarcane bacilliform virus represents a useful tool for the high-level expression of foreign genes in both monocot and dicot transgenic plants that could be used similarly to the CaMV 35S or maize polyubiquitin promoter. PMID:10380808

  6. Constitutive and stress-inducible overexpression of a native aquaporin gene (MusaPIP2;6) in transgenic banana plants signals its pivotal role in salt tolerance.

    PubMed

    Sreedharan, Shareena; Shekhawat, Upendra K Singh; Ganapathi, Thumballi R

    2015-05-01

    High soil salinity constitutes a major abiotic stress and an important limiting factor in cultivation of crop plants worldwide. Here, we report the identification and characterization of a aquaporin gene, MusaPIP2;6 which is involved in salt stress signaling in banana. MusaPIP2;6 was firstly identified based on comparative analysis of stressed and non-stressed banana tissue derived EST data sets and later overexpression in transgenic banana plants was performed to study its tangible functions in banana plants. The overexpression of MusaPIP2;6 in transgenic banana plants using constitutive or inducible promoter led to higher salt tolerance as compared to equivalent untransformed control plants. Cellular localization assay performed using transiently transformed onion peel cells indicated that MusaPIP2;6 protein tagged with green fluorescent protein was translocated to the plasma membrane. MusaPIP2;6-overexpressing banana plants displayed better photosynthetic efficiency and lower membrane damage under salt stress conditions. Our results suggest that MusaPIP2;6 is involved in salt stress signaling and tolerance in banana. PMID:25757388

  7. Constitutive and stress-inducible overexpression of a native aquaporin gene (MusaPIP2;6) in transgenic banana plants signals its pivotal role in salt tolerance.

    PubMed

    Sreedharan, Shareena; Shekhawat, Upendra K Singh; Ganapathi, Thumballi R

    2015-05-01

    High soil salinity constitutes a major abiotic stress and an important limiting factor in cultivation of crop plants worldwide. Here, we report the identification and characterization of a aquaporin gene, MusaPIP2;6 which is involved in salt stress signaling in banana. MusaPIP2;6 was firstly identified based on comparative analysis of stressed and non-stressed banana tissue derived EST data sets and later overexpression in transgenic banana plants was performed to study its tangible functions in banana plants. The overexpression of MusaPIP2;6 in transgenic banana plants using constitutive or inducible promoter led to higher salt tolerance as compared to equivalent untransformed control plants. Cellular localization assay performed using transiently transformed onion peel cells indicated that MusaPIP2;6 protein tagged with green fluorescent protein was translocated to the plasma membrane. MusaPIP2;6-overexpressing banana plants displayed better photosynthetic efficiency and lower membrane damage under salt stress conditions. Our results suggest that MusaPIP2;6 is involved in salt stress signaling and tolerance in banana.

  8. Constitutive expression of a novel antimicrobial protein, Hcm1, confers resistance to both Verticillium and Fusarium wilts in cotton

    PubMed Central

    Zhang, Zhiyuan; Zhao, Jun; Ding, Lingyun; Zou, Lifang; Li, Yurong; Chen, Gongyou; Zhang, Tianzhen

    2016-01-01

    Fusarium and Verticillium wilts, two of the most important diseases in cotton, pose serious threats to cotton production. Here we introduced a novel antimicrobial protein Hcm1, which comprised harpin protein from Xanthomonas oryzae pv. oryzicola (Xoc), and the chimeric protein, cecropin A-melittin, into cotton. The transgenic cotton lines with stable Hcm1 expression showed a higher resistance to Verticillium and Fusarium wilts both in greenhouse and field trials compared to controls. Hcm1 enabled the transgenic cotton to produced a microscopic hypersensitive response (micro-HR), reactive oxygen species (ROS) burst, and caused the activation of pathogenesis-related (PR) genes in response to biotic stress, indicating that the transgenic cotton was in a primed state and ready to protect the host from pathogenic infection. Simultaneously, Hcm1 protein inhibited the growth of Verticillium dahliae (V. dahliae) and Fusarium oxysporum (F. oxysporum) in vitro. The spread of fungal biomass was also inhibited in vivo since the V. dahliae biomass was decreased dramatically in transgenic cotton plants after inoculation with V. dahliae. Together, these results demonstrate that Hcm1 could activate innate immunity and inhibit the growth of V. dahliae and F. oxysporum to protect cotton against Verticillium and Fusarium wilts. PMID:26856318

  9. Transient expression systems for plant-derived biopharmaceuticals.

    PubMed

    Komarova, Tatiana V; Baschieri, Selene; Donini, Marcello; Marusic, Carla; Benvenuto, Eugenio; Dorokhov, Yuri L

    2010-08-01

    In the molecular farming area, transient expression approaches for pharmaceutical proteins production, mainly recombinant monoclonal antibodies and vaccines, were developed almost two decades ago and, to date, these systems basically depend on Agrobacterium-mediated delivery and virus expression machinery. We survey here the current state-of-the-art of this research field. Several vectors have been designed on the basis of DNA- and RNA-based plant virus genomes and viral vectors are used both as single- and multicomponent expression systems in different combinations depending on the protein of interest. The obvious advantages of these systems are ease of manipulation, speed, low cost and high yield of proteins. In addition, Agrobacterium-mediated expression also allows the production in plants of complex proteins assembled from subunits. Currently, the transient expression methods are preferential over any other transgenic system for the exploitation of large and unrestricted numbers of plants in a contained environment. By designing optimal constructs and related means of delivery into plant cells, the overall technology plan considers scenarios that envisage high yield of bioproducts and ease in monitoring the whole spectrum of upstream production, before entering good manufacturing practice facilities. In this way, plant-derived bioproducts show promise of high competitiveness towards classical eukaryotic cell factory systems. PMID:20673010

  10. Arsenic and mercury tolerance and cadmium sensitivity in Arabidopsis plants expressing bacterial gamma-glutamylcysteine synthetase.

    PubMed

    Li, Yujing; Dhankher, Om Parkash; Carreira, Laura; Balish, Rebecca S; Meagher, Richard B

    2005-06-01

    Cysteine sulfhydryl-rich peptide thiols are believed to play important roles in the detoxification of many heavy metals and metalloids such as arsenic, mercury, and cadmium in plants. The gamma-glutamylcysteine synthetase (gamma-ECS) catalyzes the synthesis of the dipeptidethiol gamma-glu-cys (gamma-EC), the first step in the biosynthesis of phytochelatins (PCs). Arabidopsis thaliana, engineered to express the bacterial gamma-ECS gene under control of a strong constitutive actin regulatory sequence (A2), expressed gamma-ECS at levels approaching 0.1% of total protein. In response to arsenic, mercury, and cadmium stresses, the levels of gamma-EC and its derivatives, glutathione (GSH) and PCs, were increased in the A2::ECS transgenic plants to three- to 20-fold higher concentrations than the increases that occurred in wild-type (WT). Compared to cadmium and mercury treatments, arsenic treatment most significantly increased levels of gamma-EC and PCs in both the A2::ECS transgenic and WT plants. The A2::ECS transgenic plants were highly resistant to arsenic and weakly resistant to mercury. Although exposure to cadmium produced three- to fivefold increases in levels of gamma-EC-related peptides in the A2::ECS lines, these plants were significantly more sensitive to Cd(II) than WT and trace levels of Cd(II) blocked resistance to arsenic and mercury. A few possible mechanisms for gamma-ECS-enhanced arsenic and mercury resistance and cadmium hypersensitivity are discussed. PMID:16117113

  11. Arsenic and mercury tolerance and cadmium sensitivity in Arabidopsis plants expressing bacterial gamma-glutamylcysteine synthetase.

    PubMed

    Li, Yujing; Dhankher, Om Parkash; Carreira, Laura; Balish, Rebecca S; Meagher, Richard B

    2005-06-01

    Cysteine sulfhydryl-rich peptide thiols are believed to play important roles in the detoxification of many heavy metals and metalloids such as arsenic, mercury, and cadmium in plants. The gamma-glutamylcysteine synthetase (gamma-ECS) catalyzes the synthesis of the dipeptidethiol gamma-glu-cys (gamma-EC), the first step in the biosynthesis of phytochelatins (PCs). Arabidopsis thaliana, engineered to express the bacterial gamma-ECS gene under control of a strong constitutive actin regulatory sequence (A2), expressed gamma-ECS at levels approaching 0.1% of total protein. In response to arsenic, mercury, and cadmium stresses, the levels of gamma-EC and its derivatives, glutathione (GSH) and PCs, were increased in the A2::ECS transgenic plants to three- to 20-fold higher concentrations than the increases that occurred in wild-type (WT). Compared to cadmium and mercury treatments, arsenic treatment most significantly increased levels of gamma-EC and PCs in both the A2::ECS transgenic and WT plants. The A2::ECS transgenic plants were highly resistant to arsenic and weakly resistant to mercury. Although exposure to cadmium produced three- to fivefold increases in levels of gamma-EC-related peptides in the A2::ECS lines, these plants were significantly more sensitive to Cd(II) than WT and trace levels of Cd(II) blocked resistance to arsenic and mercury. A few possible mechanisms for gamma-ECS-enhanced arsenic and mercury resistance and cadmium hypersensitivity are discussed.

  12. Dynamic alternations in cellular and molecular components during blossom-end rot development in tomatoes expressing sCAX1, a constitutively active Ca2+/H+ antiporter from Arabidopsis.

    PubMed

    Tonetto de Freitas, Sergio; Padda, Malkeet; Wu, Qingyu; Park, Sunghun; Mitcham, Elizabeth J

    2011-06-01

    Although calcium (Ca) concentration in cellular compartments has been suggested to be tightly regulated, Ca deficiency disorders such as blossom-end rot (BER) in tomato (Solanum lycopersicum) fruit may be induced by abnormal regulation of Ca partitioning and distribution in the cell. The objectives of this work were to analyze the effects of high expression of the constitutively functional Arabidopsis (Arabidopsis thaliana) Ca(2+)/H(+) exchanger (sCAX1) tonoplast protein in tomato fruit on cellular Ca partitioning and distribution, membrane integrity, and the transcriptional profile of genes potentially involved in BER development. Wild-type and sCAX1-expressing tomato plants were grown in a greenhouse. Wild-type plants did not develop BER, whereas sCAX1-expressing plants reached 100% BER incidence at 15 d after pollination. The sCAX1-expressing fruit pericarp had higher total tissue and water-soluble Ca concentrations, lower apoplastic and cytosolic Ca concentrations, higher membrane leakage, and Ca accumulation in the vacuole of sCAX1-expressing cells. Microarray analysis of healthy sCAX1-expressing fruit tissue indicated down-regulation of genes potentially involved in BER development, such as genes involved in membrane structure and repair and cytoskeleton metabolism, as well as up-regulation of genes that may have limited BER damage expansion, such as genes coding for heat shock proteins, glutathione S-transferases, and peroxidases. The results indicate that the high expression of the sCAX1 gene reduces cytosolic and apoplastic Ca concentrations, affecting plasma membrane structure and leading to BER symptom development in the fruit tissue. PMID:21464475

  13. Expression of streptavidin gene in bacteria and plants

    SciTech Connect

    Guan, Xueni; Wurtele, E.S.; Nikolau, B.J. )

    1990-05-01

    Six biotin-containing proteins are present in plants, representing at least four different biotin enzymes. The physiological function of these biotin enzymes is not understood. Streptavidin, a protein from Streptomyces avidinii, binds tightly and specifically to biotin causing inactivation of biotin enzymes. One approach to elucidating the physiological function of biotin enzymes in plant metabolism is to create transgenic plants expressing the streptavidin gene. A plasmid containing a fused streptavidin-beta-galactosidase gene has been expressed in E. coli. We also have constructed various fusion genes that include an altered CaMV 35S promoter, signal peptides to target the streptavidin protein to specific organelles, and the streptavidin coding gene. We are examining the expression of these genes in cells of carrot.

  14. Let there be light: Regulation of gene expression in plants

    PubMed Central

    Petrillo, Ezequiel; Godoy Herz, Micaela A; Barta, Andrea; Kalyna, Maria; Kornblihtt, Alberto R

    2014-01-01

    Gene expression regulation relies on a variety of molecular mechanisms affecting different steps of a messenger RNA (mRNA) life: transcription, processing, splicing, alternative splicing, transport, translation, storage and decay. Light induces massive reprogramming of gene expression in plants. Differences in alternative splicing patterns in response to environmental stimuli suggest that alternative splicing plays an important role in plant adaptation to changing life conditions. In a recent publication, our laboratories showed that light regulates alternative splicing of a subset of Arabidopsis genes encoding proteins involved in RNA processing by chloroplast retrograde signals. The light effect on alternative splicing is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. These results point at alternative splicing regulation by retrograde signals as an important mechanism for plant adaptation to their environment. PMID:25590224

  15. GenoCAD Plant Grammar to Design Plant Expression Vectors for Promoter Analysis.

    PubMed

    Coll, Anna; Wilson, Mandy L; Gruden, Kristina; Peccoud, Jean

    2016-01-01

    With the rapid advances in prediction tools for discovery of new promoters and their cis-elements, there is a need to improve plant expression methodologies in order to facilitate a high-throughput functional validation of these promoters in planta. The promoter-reporter analysis is an indispensible approach for characterization of plant promoters. It requires the design of complex plant expression vectors, which can be challenging. Here, we describe the use of a plant grammar implemented in GenoCAD that will allow the users to quickly design constructs for promoter analysis experiments but also for other in planta functional studies. The GenoCAD plant grammar includes a library of plant biological parts organized in structural categories to facilitate their use and management and a set of rules that guides the process of assembling these biological parts into large constructs.

  16. GenoCAD Plant Grammar to Design Plant Expression Vectors for Promoter Analysis.

    PubMed

    Coll, Anna; Wilson, Mandy L; Gruden, Kristina; Peccoud, Jean

    2016-01-01

    With the rapid advances in prediction tools for discovery of new promoters and their cis-elements, there is a need to improve plant expression methodologies in order to facilitate a high-throughput functional validation of these promoters in planta. The promoter-reporter analysis is an indispensible approach for characterization of plant promoters. It requires the design of complex plant expression vectors, which can be challenging. Here, we describe the use of a plant grammar implemented in GenoCAD that will allow the users to quickly design constructs for promoter analysis experiments but also for other in planta functional studies. The GenoCAD plant grammar includes a library of plant biological parts organized in structural categories to facilitate their use and management and a set of rules that guides the process of assembling these biological parts into large constructs. PMID:27557770

  17. Constitutive expression of Campylobacter jejuni truncated hemoglobin CtrHb improves the growth of Escherichia coli cell under aerobic and anaerobic conditions.

    PubMed

    Yang, Jiang-Ke; Xiong, Wei; Xu, Li; Li, Jia; Zhao, Xiu-Ju

    2015-01-01

    Bacteria hemoglobin could bind to the oxygen, transfer it from the intracellular microenvironment to the respiration process and sustain the energy for the metabolism and reproduction of cells. Heterologous expression of bacteria hemoglobin gene could improve the capacity of the host on oxygen-capturing and allow it to grow even under microaerophilic condition. To develop a system based on hemoglobin to help bacteria cells overcome the oxygen shortage in fermentation, in this study, Campylobacter jejuni truncated hemoglobin (CtrHb) gene was synthesized and expressed under the control of constitutive expression promoters P2 and P(SPO1-II) in Escherichia coli. As showed by the growth curves of the two recombinants P2-CtrHb and P(SPO1-II)-CtrHb, constitutive expression of CtrHb improved cell growth under aerobic shaking-flasks, anaerobic capped-bottles and bioreactor conditions. According to the NMR analysis, this improvement might come from the expression of hemoglobin which could boost the metabolism of cells by supplying more oxygen to the respiratory chain processes. Through semi-quantitative RT-PCR and CO differential spectrum assays, we further discussed the connection between the growth patterns of the recombinants, the expression level of CtrHb and oxygen binding capacity of CtrHb in cells. Based on the growth patterns of these recombinants in bioreactor, a possible choice on different type of recombinants under specific fermentation conditions was also suggested in this study.

  18. Constitutive expression of Campylobacter jejuni truncated hemoglobin CtrHb improves the growth of Escherichia coli cell under aerobic and anaerobic conditions.

    PubMed

    Yang, Jiang-Ke; Xiong, Wei; Xu, Li; Li, Jia; Zhao, Xiu-Ju

    2015-01-01

    Bacteria hemoglobin could bind to the oxygen, transfer it from the intracellular microenvironment to the respiration process and sustain the energy for the metabolism and reproduction of cells. Heterologous expression of bacteria hemoglobin gene could improve the capacity of the host on oxygen-capturing and allow it to grow even under microaerophilic condition. To develop a system based on hemoglobin to help bacteria cells overcome the oxygen shortage in fermentation, in this study, Campylobacter jejuni truncated hemoglobin (CtrHb) gene was synthesized and expressed under the control of constitutive expression promoters P2 and P(SPO1-II) in Escherichia coli. As showed by the growth curves of the two recombinants P2-CtrHb and P(SPO1-II)-CtrHb, constitutive expression of CtrHb improved cell growth under aerobic shaking-flasks, anaerobic capped-bottles and bioreactor conditions. According to the NMR analysis, this improvement might come from the expression of hemoglobin which could boost the metabolism of cells by supplying more oxygen to the respiratory chain processes. Through semi-quantitative RT-PCR and CO differential spectrum assays, we further discussed the connection between the growth patterns of the recombinants, the expression level of CtrHb and oxygen binding capacity of CtrHb in cells. Based on the growth patterns of these recombinants in bioreactor, a possible choice on different type of recombinants under specific fermentation conditions was also suggested in this study. PMID:26047918

  19. Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming.

    PubMed

    Makhzoum, Abdullah; Benyammi, Roukia; Moustafa, Khaled; Trémouillaux-Guiller, Jocelyne

    2014-04-01

    Plant molecular pharming is a promising system to produce important recombinant proteins such as therapeutic antibodies, pharmaceuticals, enzymes, growth factors, and vaccines. The system provides an interesting alternative method to the direct extraction of proteins from inappropriate source material while offering the possibility to overcome problems related to product safety and source availability. Multiple factors including plant hosts, genes of interest, expression vector cassettes, and extraction and purification techniques play important roles in the plant molecular pharming. Plant species, as a biosynthesis platform, are a crucial factor in achieving high yields of recombinant protein in plant. The choice of recombinant gene and its expression strategy is also of great importance in ensuring a high amount of the recombinant proteins. Many studies have been conducted to improve expression, accumulation, and purification of the recombinant protein from molecular pharming systems. Re-engineered vectors and expression cassettes are also pivotal tools in enhancing gene expression at the transcription and translation level, and increasing protein accumulation, stability, retention and targeting of specific organelles. In this review, we report recent advances and strategies of plant molecular pharming while focusing on the choice of plant hosts and the role of some molecular pharming elements and approaches: promoters, codon optimization, signal sequences, and peptides used for upstream design, purification and downstream processing.

  20. Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming.

    PubMed

    Makhzoum, Abdullah; Benyammi, Roukia; Moustafa, Khaled; Trémouillaux-Guiller, Jocelyne

    2014-04-01

    Plant molecular pharming is a promising system to produce important recombinant proteins such as therapeutic antibodies, pharmaceuticals, enzymes, growth factors, and vaccines. The system provides an interesting alternative method to the direct extraction of proteins from inappropriate source material while offering the possibility to overcome problems related to product safety and source availability. Multiple factors including plant hosts, genes of interest, expression vector cassettes, and extraction and purification techniques play important roles in the plant molecular pharming. Plant species, as a biosynthesis platform, are a crucial factor in achieving high yields of recombinant protein in plant. The choice of recombinant gene and its expression strategy is also of great importance in ensuring a high amount of the recombinant proteins. Many studies have been conducted to improve expression, accumulation, and purification of the recombinant protein from molecular pharming systems. Re-engineered vectors and expression cassettes are also pivotal tools in enhancing gene expression at the transcription and translation level, and increasing protein accumulation, stability, retention and targeting of specific organelles. In this review, we report recent advances and strategies of plant molecular pharming while focusing on the choice of plant hosts and the role of some molecular pharming elements and approaches: promoters, codon optimization, signal sequences, and peptides used for upstream design, purification and downstream processing. PMID:23959796

  1. Expression of recombinant cellulase Cel5A from Trichoderma reesei in tobacco plants.

    PubMed

    Garvey, Megan; Klinger, Johannes; Klose, Holger; Fischer, Rainer; Commandeur, Ulrich

    2014-01-01

    Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems. PMID:24962636

  2. Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

    PubMed Central

    Garvey, Megan; Fischer, Rainer; Commandeur, Ulrich

    2014-01-01

    Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems. PMID:24962636

  3. Copper-controllable gene expression system for whole plants.

    PubMed

    Mett, V L; Lochhead, L P; Reynolds, P H

    1993-05-15

    We describe a system for gene expression in plants based on the regulation mechanism of the yeast metallothionein (MT) gene. The system consists of two elements: (i) the yeast ace1 (activating copper-MT expression) gene encoding a transcription factor under control of the cauliflower mosaic virus (CaMV) 35S RNA promoter, and (ii) a gene of interest under control of a chimeric promoter consisting of the 90-base-pair domain A of the CaMV 35S RNA promoter linked to the ACE1 transcription factor-binding site. At elevated copper ion concentrations, the ACE1 protein changes conformation, binds to, and activates transcription from the chimeric promoter. To test the functioning of the system in plants, a construct containing the beta-glucuronidase (GUS) reporter gene under control of the chimeric promoter was prepared, and transgenic tobacco plants were produced. It was shown that GUS activity in the leaves of transgenic plants increased up to 50-fold, either after addition of 50 microM CuSO4 to the nutrient solution or after application of 0.5 microM CuSO4 to the plants in a foliar spray. This GUS expression was repressed after the removal of copper ions. The results show that the activity of the described chimeric promoter directly depends on copper ion concentration and that this system can be used in experiments that demand precise timing of expression.

  4. Evolution, diversification, and expression of KNOX proteins in plants

    PubMed Central

    Gao, Jie; Yang, Xue; Zhao, Wei; Lang, Tiange; Samuelsson, Tore

    2015-01-01

    The KNOX (KNOTTED1-like homeobox) transcription factors play a pivotal role in leaf and meristem development. The majority of these proteins are characterized by the KNOX1, KNOX2, ELK, and homeobox domains whereas the proteins of the KNATM family contain only the KNOX domains. We carried out an extensive inventory of these proteins and here report on a total of 394 KNOX proteins from 48 species. The land plant proteins fall into two classes (I and II) as previously shown where the class I family seems to be most closely related to the green algae homologs. The KNATM proteins are restricted to Eudicots and some species have multiple paralogs of this protein. Certain plants are characterized by a significant increase in the number of KNOX paralogs; one example is Glycine max. Through the analysis of public gene expression data we show that the class II proteins of this plant have a relatively broad expression specificity as compared to class I proteins, consistent with previous studies of other plants. In G. max, class I protein are mainly distributed in axis tissues and KNATM paralogs are overall poorly expressed; highest expression is in the early plumular axis. Overall, analysis of gene expression in G. max demonstrates clearly that the expansion in gene number is associated with functional diversification. PMID:26557129

  5. The role of roots in sex expression in hemp plants.

    PubMed

    Chailakhyan, M K; Khryanin, V N

    1978-01-01

    When the shoots of young hemp (Cannabis sativa L.) plants were cut off the roots, cultured as cuttings, and regenerating (adventitious) roots were removed as soon as appearing, ca. 80-90% of the plants became male (had staminate flowers) whereas if the roots were allowed to develop a similar percentage became female (pistillate flowers). Treatment of de-rooted cuttings with 6-benzylaminopurine (15 mg/l) restored the percent of female plants to ca. 80. It is suggested that the root system plays an essential role in sex expression in hemp and that this role is related to cytokinin synthesis in the root.

  6. Functional genomics: Probing plant gene function and expression with transposons

    PubMed Central

    Martienssen, Robert A.

    1998-01-01

    Transposable elements provide a convenient and flexible means to disrupt plant genes, so allowing their function to be assessed. By engineering transposons to carry reporter genes and regulatory signals, the expression of target genes can be monitored and to some extent manipulated. Two strategies for using transposons to assess gene function are outlined here: First, the PCR can be used to identify plants that carry insertions into specific genes from among pools of heavily mutagenized individuals (site-selected transposon mutagenesis). This method requires that high copy transposons be used and that a relatively large number of reactions be performed to identify insertions into genes of interest. Second, a large library of plants, each carrying a unique insertion, can be generated. Each insertion site then can be amplified and sequenced systematically. These two methods have been demonstrated in maize, Arabidopsis, and other plant species, and the relative merits of each are discussed in the context of plant genome research. PMID:9482828

  7. Expression of rice thaumatin-like protein gene in transgenic banana plants enhances resistance to fusarium wilt.

    PubMed

    Mahdavi, F; Sariah, M; Maziah, M

    2012-02-01

    The possibility of controlling Fusarium wilt--caused by Fusarium oxysporum sp. cubensec (race 4)--was investigated by genetic engineering of banana plants for constitutive expression of rice thaumatin-like protein (tlp) gene. Transgene was introduced to cauliflower-like bodies' cluster, induced from meristemic parts of male inflorescences, using particle bombardment with plasmid carrying a rice tlp gene driving by the CaMV 35S promoter. Hygromycin B was used as the selection reagent. The presence and integration of rice tlp gene in genomic DNA confirmed by PCR and Southern blot analyses. RT-PCR revealed the expression of transgene in leaf and root tissues in transformants. Bioassay of transgenic banana plants challenged with Fusarium wilt pathogen showed that expression of TLP enhanced resistance to F. oxysporum sp. cubensec (race 4) compared to control plants.

  8. Regulation of oncogene expression in T-DNA-transformed host plant cells.

    PubMed

    Zhang, Yi; Lee, Chil-Woo; Wehner, Nora; Imdahl, Fabian; Svetlana, Veselova; Weiste, Christoph; Dröge-Laser, Wolfgang; Deeken, Rosalia

    2015-01-01

    Virulent Agrobacterium tumefaciens strains integrate their T-DNA into the plant genome where the encoded agrobacterial oncogenes are expressed and cause crown gall disease. Essential for crown gall development are IaaH (indole-3-acetamide hydrolase), IaaM (tryptophan monooxygenase) and Ipt (isopentenyl transferase), which encode enzymes for the biosynthesis of auxin (IaaH, IaaM) and cytokinin (Ipt). Although these oncogenes are well studied as the tumor-inducing principle, nothing is known about the regulation of oncogene expression in plant cells. Our studies show that the intergenic regions (IGRs) between the coding sequences (CDS) of the three oncogenes function as promoters in plant cells. These promoters possess a eukaryotic sequence organization and cis-regulatory elements for the binding of plant transcription factors. WRKY18, WRKY40, WRKY60 and ARF5 were identified as activators of the Ipt promoter whereas IaaH and IaaM is constitutively expressed and no transcription factor further activates their promoters. Consistent with these results, the wrky triple mutant plants in particular, develops smaller crown galls than wild-type and exhibits a reduced Ipt transcription, despite the presence of an intact ARF5 gene. WRKY40 and WRKY60 gene expression is induced by A. tumefaciens within a few hours whereas the ARF5 gene is transcribed later during crown gall development. The WRKY proteins interact with ARF5 in the plant nucleus, but only WRKY40 together with ARF5 synergistically boosts the activation of the Ipt promoter in an auxin-dependent manner. From our data, we propose that A. tumefaciens initially induces WRKY40 gene expression as a pathogen defense response of the host cell. The WRKY protein is recruited to induce Ipt expression, which initiates cytokinin-dependent host cell division. With increasing auxin levels triggered by ubiquitous expression of IaaH and IaaM, ARF5 is activated and interacts with WRKY40 to potentiate Ipt expression and balance

  9. Constitutive IDO expression in human cancer is sustained by an autocrine signaling loop involving IL-6, STAT3 and the AHR.

    PubMed

    Litzenburger, Ulrike M; Opitz, Christiane A; Sahm, Felix; Rauschenbach, Katharina J; Trump, Saskia; Winter, Marcus; Ott, Martina; Ochs, Katharina; Lutz, Christian; Liu, Xiangdong; Anastasov, Natasa; Lehmann, Irina; Höfer, Thomas; von Deimling, Andreas; Wick, Wolfgang; Platten, Michael

    2014-02-28

    Indoleamine-2,3-dioxygenase (IDO) inhibitors have entered clinical trials based on their ability to restore anti-tumor immunity in preclinical studies. However, the mechanisms leading to constitutive expression of IDO in human tumors are largely unknown. Here we analyzed the pathways mediating constitutive IDO expression in human cancer. IDO-positive tumor cells and tissues showed basal phosphorylation and acetylation of STAT3 as evidenced by western blotting and immunoprecipitation. Inhibition of IL-6 or STAT3 using siRNA and/or pharmacological inhibitors reduced IDO mRNA and protein expression as well as kynurenine formation. In turn, IDO enzymatic activity activated the AHR as shown by the induction of AHR target genes. IDO-mediated AHR activation induced IL-6 expression, while inhibition or knockdown of the AHR reduced IL-6 expression. IDO activity thus sustains its own expression via an autocrine AHR-IL-6-STAT3 signaling loop. Inhibition of the AHR-IL-6-STAT3 signaling loop restored T-cell proliferation in mixed leukocyte reactions performed in the presence of IDO-expressing human cancer cells. Identification of the IDO-AHR-IL-6-STAT3 signaling loop maintaining IDO expression in human cancers reveals novel therapeutic targets for the inhibition of this core pathway promoting immunosuppression of human cancers. The relevance of the IDO-AHR-IL-6-STAT3 transcriptional circuit is underscored by the finding that high expression of its members IDO, STAT3 and the AHR target gene CYP1B1 is associated with reduced relapse-free survival in lung cancer patients.

  10. Emerging Use of Gene Expression Microarrays in Plant Physiology

    DOE PAGES

    Wullschleger, Stan D.; Difazio, Stephen P.

    2003-01-01

    Microarrays have become an important technology for the global analysis of gene expression in humans, animals, plants, and microbes. Implemented in the context of a well-designed experiment, cDNA and oligonucleotide arrays can provide highthroughput, simultaneous analysis of transcript abundance for hundreds, if not thousands, of genes. However, despite widespread acceptance, the use of microarrays as a tool to better understand processes of interest to the plant physiologist is still being explored. To help illustrate current uses of microarrays in the plant sciences, several case studies that we believe demonstrate the emerging application of gene expression arrays in plant physiology weremore » selected from among the many posters and presentations at the 2003 Plant and Animal Genome XI Conference. Based on this survey, microarrays are being used to assess gene expression in plants exposed to the experimental manipulation of air temperature, soil water content and aluminium concentration in the root zone. Analysis often includes characterizing transcript profiles for multiple post-treatment sampling periods and categorizing genes with common patterns of response using hierarchical clustering techniques. In addition, microarrays are also providing insights into developmental changes in gene expression associated with fibre and root elongation in cotton and maize, respectively. Technical and analytical limitations of microarrays are discussed and projects attempting to advance areas of microarray design and data analysis are highlighted. Finally, although much work remains, we conclude that microarrays are a valuable tool for the plant physiologist interested in the characterization and identification of individual genes and gene families with potential application in the fields of agriculture, horticulture and forestry.« less

  11. Emerging Use of Gene Expression Microarrays in Plant Physiology

    PubMed Central

    Difazio, Stephen P.

    2003-01-01

    Microarrays have become an important technology for the global analysis of gene expression in humans, animals, plants, and microbes. Implemented in the context of a well-designed experiment, cDNA and oligonucleotide arrays can provide highthroughput, simultaneous analysis of transcript abundance for hundreds, if not thousands, of genes. However, despite widespread acceptance, the use of microarrays as a tool to better understand processes of interest to the plant physiologist is still being explored. To help illustrate current uses of microarrays in the plant sciences, several case studies that we believe demonstrate the emerging application of gene expression arrays in plant physiology were selected from among the many posters and presentations at the 2003 Plant and Animal Genome XI Conference. Based on this survey, microarrays are being used to assess gene expression in plants exposed to the experimental manipulation of air temperature, soil water content and aluminium concentration in the root zone. Analysis often includes characterizing transcript profiles for multiple post-treatment sampling periods and categorizing genes with common patterns of response using hierarchical clustering techniques. In addition, microarrays are also providing insights into developmental changes in gene expression associated with fibre and root elongation in cotton and maize, respectively. Technical and analytical limitations of microarrays are discussed and projects attempting to advance areas of microarray design and data analysis are highlighted. Finally, although much work remains, we conclude that microarrays are a valuable tool for the plant physiologist interested in the characterization and identification of individual genes and gene families with potential application in the fields of agriculture, horticulture and forestry. PMID:18629133

  12. Evaluation of constitutive iron reductase (AtFRO2) expression on mineral accumulation and distribution in soybean (Glycine max L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Iron is an important micronutrient in human and plant nutrition. Adequate iron nutrition during crop production is central for assuring appropriate iron concentrations in the harvestable organs, for human food or animal feed. The whole-plant movement of iron involves several processes, including the...

  13. Biodegradation of explosives by transgenic plants expressing pentaerythritol tetranitrate reductase.

    PubMed

    French, C E; Rosser, S J; Davies, G J; Nicklin, S; Bruce, N C

    1999-05-01

    Plants offer many advantages over bacteria as agents for bioremediation; however, they typically lack the degradative capabilities of specially selected bacterial strains. Transgenic plants expressing microbial degradative enzymes could combine the advantages of both systems. To investigate this possibility in the context of bioremediation of explosive residues, we generated transgenic tobacco plants expressing pentaerythritol tetranitrate reductase, an enzyme derived from an explosive-degrading bacterium that enables degradation of nitrate ester and nitroaromatic explosives. Seeds from transgenic plants were able to germinate and grow in the presence of 1 mM glycerol trinitrate (GTN) or 0.05 mM trinitrotoluene, at concentrations that inhibited germination and growth of wild-type seeds. Transgenic seedlings grown in liquid medium with 1 mM GTN showed more rapid and complete denitration of GTN than wild-type seedlings. This example suggests that transgenic plants expressing microbial degradative genes may provide a generally applicable strategy for bioremediation of organic pollutants in soil. PMID:10331811

  14. Transient Expression Systems in Plants: Potentialities and Constraints.

    PubMed

    Canto, Tomas

    2016-01-01

    Plants have been used from old to extract and isolate by different means the products of interest that they store. In recent years new techniques have emerged that allow the use of plants as factories to overexpress transiently and often efficiently, specific genes of interest, either endogenous or foreign, in their native form or modified. These techniques allow and facilitate the targeted purification of gene products for research and commercial purposes without resorting to lengthy, time-consuming and sometimes challenging plant stable transformations, while avoiding some of their associated regulatory constraints. In this chapter we describe the main strategies available for the transient expression of gene sequences and their encoded products in plants. We discuss biological issues affecting transient expression, including resistance responses elicited by the plant against sequences that it recognizes naturally as foreign, and ways to neutralize them. We also discuss the relative advantages of each expression strategy as well as their inherent drawbacks and technical limitations, and how to partially prevent or overcome them, whenever possible.

  15. [Methods for construction of transgenic plant expression vector: a review].

    PubMed

    Zhang, Yangpu; Yang, Shushen

    2015-03-01

    Construction of recombinant plasmid vector for gene expression is a key step in making transgenic plants and important to study gene function and plant genetic engineering. A right choice of gene construction method can be cost-effective and achieve more diverse recombinant plasmids. In addition to the traditional methods in construction of plant gene expression vectors, such as Gateway technology, three DNA method and one step cloning, a few novel methods have been developed in recent years. These methods include oligonucleotide synthesis-based construction of small fragment gene expression vectors via competitive connection; construction of small RNA expression vector using pre-microRNA; recombination-fusion PCR method which inserts DNA fragments of multiple restriction sites into the target vector; and insertion of a DNA fragment into any region of a linear vector via In-Fusion Kit. Construction of complex vectors with many fragments uses sequence and ligation-independent cloning method, Gibson isothermal assembly or Golden Gate assembly. This paper summarizes our working experience in the area of recombinant vector construction and reports from others with an intention to disseminate ideas about currently widely used DNA recombination methods for plant transformation.

  16. Systemic spread of an RNA insect virus in plants expressing plant viral movement protein genes

    PubMed Central

    Dasgupta, Ranjit; Garcia, Bradley H.; Goodman, Robert M.

    2001-01-01

    Flock house virus (FHV), a single-stranded RNA insect virus, has previously been reported to cross the kingdom barrier and replicate in barley protoplasts and in inoculated leaves of several plant species [Selling, B. H., Allison, R. F. & Kaesberg, P. (1990) Proc. Natl. Acad. Sci. USA 87, 434–438]. There was no systemic movement of FHV in plants. We tested the ability of movement proteins (MPs) of plant viruses to provide movement functions and cause systemic spread of FHV in plants. We compared the growth of FHV in leaves of nontransgenic and transgenic plants expressing the MP of tobacco mosaic virus or red clover necrotic mosaic virus (RCNMV). Both MPs mobilized cell-to-cell and systemic movement of FHV in Nicotiana benthamiana plants. The yield of FHV was more than 100-fold higher in the inoculated leaves of transgenic plants than in the inoculated leaves of nontransgenic plants. In addition, FHV accumulated in the noninoculated upper leaves of both MP-transgenic plants. RCNMV MP was more efficient in mobilizing FHV to noninoculated upper leaves. We also report here that FHV replicates in inoculated leaves of six additional plant species: alfalfa, Arabidopsis, Brassica, cucumber, maize, and rice. Our results demonstrate that plant viral MPs cause cell-to-cell and long-distance movement of an animal virus in plants and offer approaches to the study of the evolution of viruses and mechanisms governing mRNA trafficking in plants as well as to the development of promising vectors for transient expression of foreign genes in plants. PMID:11296259

  17. Using a periclinal chimera to unravel layer-specific gene expression in plants.

    PubMed

    Filippis, Ioannis; Lopez-Cobollo, Rosa; Abbott, James; Butcher, Sarah; Bishop, Gerard J

    2013-09-01

    Plant organs are made from multiple cell types, and defining the expression level of a gene in any one cell or group of cells from a complex mixture is difficult. Dicotyledonous plants normally have three distinct layers of cells, L1, L2 and L3. Layer L1 is the single layer of cells making up the epidermis, layer L2 the single cell sub-epidermal layer and layer L3 constitutes the rest of the internal cells. Here we show how it is possible to harvest an organ and characterise the level of layer-specific expression by using a periclinal chimera that has its L1 layer from Solanum pennellii and its L2 and L3 layers from Solanum lycopersicum. This is possible by measuring the level of the frequency of species-specific transcripts. RNA-seq analysis enabled the genome-wide assessment of whether a gene is expressed in the L1 or L2/L3 layers. From 13 277 genes that are expressed in both the chimera and the parental lines and with at least one polymorphism between the parental alleles, we identified 382 genes that are preferentially expressed in L1 in contrast to 1159 genes in L2/L3. Gene ontology analysis shows that many genes preferentially expressed in L1 are involved in cutin and wax biosynthesis, whereas numerous genes that are preferentially expressed in L2/L3 tissue are associated with chloroplastic processes. These data indicate the use of such chimeras and provide detailed information on the level of layer-specific expression of genes.

  18. Using a periclinal chimera to unravel layer-specific gene expression in plants

    PubMed Central

    Filippis, Ioannis; Lopez-Cobollo, Rosa; Abbott, James; Butcher, Sarah; Bishop, Gerard J

    2013-01-01

    Plant organs are made from multiple cell types, and defining the expression level of a gene in any one cell or group of cells from a complex mixture is difficult. Dicotyledonous plants normally have three distinct layers of cells, L1, L2 and L3. Layer L1 is the single layer of cells making up the epidermis, layer L2 the single cell sub-epidermal layer and layer L3 constitutes the rest of the internal cells. Here we show how it is possible to harvest an organ and characterise the level of layer-specific expression by using a periclinal chimera that has its L1 layer from Solanum pennellii and its L2 and L3 layers from Solanum lycopersicum. This is possible by measuring the level of the frequency of species-specific transcripts. RNA-seq analysis enabled the genome-wide assessment of whether a gene is expressed in the L1 or L2/L3 layers. From 13 277 genes that are expressed in both the chimera and the parental lines and with at least one polymorphism between the parental alleles, we identified 382 genes that are preferentially expressed in L1 in contrast to 1159 genes in L2/L3. Gene ontology analysis shows that many genes preferentially expressed in L1 are involved in cutin and wax biosynthesis, whereas numerous genes that are preferentially expressed in L2/L3 tissue are associated with chloroplastic processes. These data indicate the use of such chimeras and provide detailed information on the level of layer-specific expression of genes. PMID:23725542

  19. Constitutive expression of the embryonic stem cell marker OCT4 in bovine somatic donor cells influences blastocysts rate and quality after nucleus transfer.

    PubMed

    Rodríguez-Alvarez, Lleretny; Manriquez, Jose; Velasquez, Alejandra; Castro, Fidel Ovidio

    2013-10-01

    Nuclear transfer (NT) is associated with epigenetic reprogramming of donor cells. Expression of certain genes in these cells might facilitate their expression in the NT embryo. This research was aimed to investigate the effect of constitutive expression of OCT4 in bovine somatic cells used for NT on the developmental potential of derived cloned embryos as well as in the expression of pluripotency markers in the Day-7 resulting embryos. Cloned blastocysts were generated from five cell lines that expressed OCT4. Pools of blastocysts were screened to detect OCT4, SOX2, and NANOG by qPCR. In vitro-fertilized time-matched blastocysts were used as controls. The development potential was assessed on the basis of blastocysts rate; grading and total cell counts at Day 7. OCT4 expression in the cell lines positively correlates with blastocysts rate (r = 0.92; p = 0.02), number of grade I blastocysts (r = 0.96; p = 0.01), and total cell number (r = 0.98; p = 0.002). The high expression of OCT4 in the cell line did not improve the final outcome of cloning. Somatic expression of OCT4 lead to increased expression of OCT4 and SOX2 in cloned grade I blastocysts; however, there was a bigger variability in OCT4 and SOX2 (p = 0.03; p = 0.02) expression in the embryos generated from cells expressing highest levels of OCT4. Probably the higher variability in OCT4 expression in cloned embryos is due to incorrect reprogramming and incapability of the oocyte to correct for higher OCT4 levels. For that reason, we concluded that OCT4 expression in somatic cells is not a good prognosis marker for selecting cell lines. PMID:23846396

  20. Constitutive expression of IDO by dendritic cells of mesenteric lymph nodes: functional involvement of the CTLA-4/B7 and CCL22/CCR4 interactions.

    PubMed

    Onodera, Toshiharu; Jang, Myoung Ho; Guo, Zijin; Yamasaki, Mikako; Hirata, Takako; Bai, Zhongbin; Tsuji, Noriko M; Nagakubo, Daisuke; Yoshie, Osamu; Sakaguchi, Shimon; Takikawa, Osamu; Miyasaka, Masayuki

    2009-11-01

    Dendritic cells (DCs) express the immunoregulatory enzyme IDO in response to certain inflammatory stimuli, but it is unclear whether DCs express this enzyme under steady-state conditions in vivo. In this study, we report that the DCs in mesenteric lymph nodes (MLNs) constitutively express functional IDO, which metabolizes tryptophan to kynurenine. In line with a previous report that regulatory T cells (Tregs) can induce IDO in DCs via the CTLA-4/B7 interaction, a substantial proportion of the MLN DCs were located in juxtaposition to Tregs, whereas this tendency was not observed for splenic DCs, which do not express IDO constitutively. When CTLA-4 was selectively deleted in Tregs, the frequency of IDO-expressing DCs in MLNs decreased significantly, confirming CTLA-4's role in IDO expression by MLN DCs. We also found that the MLN DCs produced CCL22, which can attract Tregs via CCR4, and that the phagocytosis of autologous apoptotic cells induced CCL22 expression in CCL22 mRNA-negative DCs. Mice genetically deficient in the receptor for CCL22, CCR4, showed markedly reduced IDO expression in MLN-DCs, supporting the involvement of the CCL22/CCR4 axis in IDO induction. Together with our previous observation that MLN DCs contain much intracytoplasmic cellular debris in vivo, these results indicate that reciprocal interactions between the DCs and Tregs via both B7/CTLA-4 and CCL22/CCR4 lead to IDO induction in MLN DCs, which may be initiated and/or augmented by the phagocytosis of autologous apoptotic cells by intestinal DCs. Such a mechanism may help induce the specific milieu in MLNs that is required for the induction of oral tolerance.

  1. Identification, Expression, and Evolutionary Analyses of Plant Lipocalins1[W

    PubMed Central

    Frenette Charron, Jean-Benoit; Ouellet, François; Pelletier, Mélanie; Danyluk, Jean; Chauve, Cédric; Sarhan, Fathey

    2005-01-01

    Lipocalins are a group of proteins that have been characterized in bacteria, invertebrate, and vertebrate animals. However, very little is known about plant lipocalins. We have previously reported the cloning of the first true plant lipocalins. Here we report the identification and characterization of plant lipocalins and lipocalin-like proteins using an integrated approach of data mining, expression studies, cellular localization, and phylogenetic analyses. Plant lipocalins can be classified into two groups, temperature-induced lipocalins (TILs) and chloroplastic lipocalins (CHLs). In addition, violaxanthin de-epoxidases (VDEs) and zeaxanthin epoxidases (ZEPs) can be classified as lipocalin-like proteins. CHLs, VDEs, and ZEPs possess transit peptides that target them to the chloroplast. On the other hand, TILs do not show any targeting peptide, but localization studies revealed that the proteins are found at the plasma membrane. Expression analyses by quantitative real-time PCR showed that expression of the wheat (Triticum aestivum) lipocalins and lipocalin-like proteins is associated with abiotic stress response and is correlated with the plant's capacity to develop freezing tolerance. In support of this correlation, data mining revealed that lipocalins are present in the desiccation-tolerant red algae Porphyra yezoensis and the cryotolerant marine yeast Debaryomyces hansenii, suggesting a possible association with stress-tolerant organisms. Considering the plant lipocalin properties, tissue specificity, response to temperature stress, and their association with chloroplasts and plasma membranes of green leaves, we hypothesize a protective function of the photosynthetic system against temperature stress. Phylogenetic analyses suggest that TIL lipocalin members in higher plants were probably inherited from a bacterial gene present in a primitive unicellular eukaryote. On the other hand, CHLs, VDEs, and ZEPs may have evolved from a cyanobacterial ancestral gene

  2. The in vitro and in vivo effects of constitutive light expression on a bioluminescent strain of the mouse enteropathogen Citrobacter rodentium

    PubMed Central

    Read, Hannah M.; Mills, Grant; Johnson, Sarah; Tsai, Peter; Dalton, James; Barquist, Lars; Print, Cristin G.; Patrick, Wayne M.

    2016-01-01

    Bioluminescent reporter genes, such as those from fireflies and bacteria, let researchers use light production as a non-invasive and non-destructive surrogate measure of microbial numbers in a wide variety of environments. As bioluminescence needs microbial metabolites, tagging microorganisms with luciferases means only live metabolically active cells are detected. Despite the wide use of bioluminescent reporter genes, very little is known about the impact of continuous (also called constitutive) light expression on tagged bacteria. We have previously made a bioluminescent strain of Citrobacter rodentium, a bacterium which infects laboratory mice in a similar way to how enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infect humans. In this study, we compared the growth of the bioluminescent C. rodentium strain ICC180 with its non-bioluminescent parent (strain ICC169) in a wide variety of environments. To understand more about the metabolic burden of expressing light, we also compared the growth profiles of the two strains under approximately 2,000 different conditions. We found that constitutive light expression in ICC180 was near-neutral in almost every non-toxic environment tested. However, we also found that the non-bioluminescent parent strain has a competitive advantage over ICC180 during infection of adult mice, although this was not enough for ICC180 to be completely outcompeted. In conclusion, our data suggest that constitutive light expression is not metabolically costly to C. rodentium and supports the view that bioluminescent versions of microbes can be used as a substitute for their non-bioluminescent parents to study bacterial behaviour in a wide variety of environments. PMID:27366640

  3. The in vitro and in vivo effects of constitutive light expression on a bioluminescent strain of the mouse enteropathogen Citrobacter rodentium.

    PubMed

    Read, Hannah M; Mills, Grant; Johnson, Sarah; Tsai, Peter; Dalton, James; Barquist, Lars; Print, Cristin G; Patrick, Wayne M; Wiles, Siouxsie

    2016-01-01

    Bioluminescent reporter genes, such as those from fireflies and bacteria, let researchers use light production as a non-invasive and non-destructive surrogate measure of microbial numbers in a wide variety of environments. As bioluminescence needs microbial metabolites, tagging microorganisms with luciferases means only live metabolically active cells are detected. Despite the wide use of bioluminescent reporter genes, very little is known about the impact of continuous (also called constitutive) light expression on tagged bacteria. We have previously made a bioluminescent strain of Citrobacter rodentium, a bacterium which infects laboratory mice in a similar way to how enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infect humans. In this study, we compared the growth of the bioluminescent C. rodentium strain ICC180 with its non-bioluminescent parent (strain ICC169) in a wide variety of environments. To understand more about the metabolic burden of expressing light, we also compared the growth profiles of the two strains under approximately 2,000 different conditions. We found that constitutive light expression in ICC180 was near-neutral in almost every non-toxic environment tested. However, we also found that the non-bioluminescent parent strain has a competitive advantage over ICC180 during infection of adult mice, although this was not enough for ICC180 to be completely outcompeted. In conclusion, our data suggest that constitutive light expression is not metabolically costly to C. rodentium and supports the view that bioluminescent versions of microbes can be used as a substitute for their non-bioluminescent parents to study bacterial behaviour in a wide variety of environments. PMID:27366640

  4. Enhanced drought and heat stress tolerance of tobacco plants with ectopically enhanced cytokinin oxidase/dehydrogenase gene expression.

    PubMed

    Macková, Hana; Hronková, Marie; Dobrá, Jana; Turečková, Veronika; Novák, Ondřej; Lubovská, Zuzana; Motyka, Václav; Haisel, Daniel; Hájek, Tomáš; Prášil, Ilja Tom; Gaudinová, Alena; Štorchová, Helena; Ge, Eva; Werner, Tomáš; Schmülling, Thomas; Vanková, Radomíra

    2013-07-01

    Responses to drought, heat, and combined stress were compared in tobacco (Nicotiana tabacum L.) plants ectopically expressing the cytokinin oxidase/dehydrogenase CKX1 gene of Arabidopsis thaliana L. under the control of either the predominantly root-expressed WRKY6 promoter or the constitutive 35S promoter, and in the wild type. WRKY6:CKX1 plants exhibited high CKX activity in the roots under control conditions. Under stress, the activity of the WRKY6 promoter was down-regulated and the concomitantly reduced cytokinin degradation coincided with raised bioactive cytokinin levels during the early phase of the stress response, which might contribute to enhanced stress tolerance of this genotype. Constitutive expression of CKX1 resulted in an enlarged root system, a stunted, dwarf shoot phenotype, and a low basal level of expression of the dehydration marker gene ERD10B. The high drought tolerance of this genotype was associated with a relatively moderate drop in leaf water potential and a significant decrease in leaf osmotic potential. Basal expression of the proline biosynthetic gene P5CSA was raised. Both wild-type and WRKY6:CKX1 plants responded to heat stress by transient elevation of stomatal conductance, which correlated with an enhanced abscisic acid catabolism. 35S:CKX1 transgenic plants exhibited a small and delayed stomatal response. Nevertheless, they maintained a lower leaf temperature than the other genotypes. Heat shock applied to drought-stressed plants exaggerated the negative stress effects, probably due to the additional water loss caused by a transient stimulation of transpiration. The results indicate that modulation of cytokinin levels may positively affect plant responses to abiotic stress through a variety of physiological mechanisms.

  5. Light regulation of gene expression in higher plants

    SciTech Connect

    Tobin, E.M.; Silverthorne, J.

    1985-01-01

    In this review areas of currently active research are considered which have demonstrated that a plant's response to light involves changes in the expression of specific genes at the level of RNA. The regulation of gene expression by phytochrome and the UV-sensitive photoreceptor have been studied most extensively at the molecular level, and this review particularly focuses on such studies in higher plants. Some of the observations made on the differences in gene expression between light-grown and dark-grown plants are also included, although the photoreceptor(s) responsible for the differences may not have been ascertained. In some of these cases, phytochrome involvement has been or may be demonstrated in later studies, while in others the observed differences may be a result of the action of other photoreceptors or of multiple light-affected processes. One such process is the development of chloroplasts, a major developmental step triggered by light in angiosperms. In addition, many of the genes whose expression is changed by light and which have been studied at a molecular level encode chloroplast proteins. 156 references.

  6. Exogenous plant hormones and cyclotide expression in Viola uliginosa (Violaceae).

    PubMed

    Slazak, Blazej; Jacobsson, Erik; Kuta, Elżbieta; Göransson, Ulf

    2015-09-01

    Plants from Violaceae produce cyclotides, peptides characterized by a circular peptide backbone and a cystine knot. This signature motif gives stability that can harness a wide spectrum of biological activities, with implications in plant defense and with applications in medicine and biotechnology. In the current work, cyclotide expressing in vitro cultures were established from Viola uliginosa. These cultures are useful models for studying biosynthesis of cyclotides and can also be used in their production. The cyclotide expression pattern is shown to be dependent on exogenous plant growth regulators, both on peptide and gene expression levels. The highest yields of cyclotides were obtained on media containing only a cytokinin and were correlated with storage material accumulation. Exposure to auxins decreased cyclotide production and caused shifting of the biosynthesis pattern to root specific cyclotides. The response to stimuli in terms of cyclotide expression pattern appears to be developmental, and related to polar auxin transportation and the auxin/cytokinin ratio regulating tissue differentiation. By the use of whole transcriptome shotgun sequencing (WTSS) and peptidomics, 20 cyclotide sequences from V. uliginosa (including 12 new) and 12 complete precursor proteins could be identified. The most abundant cyclotides were cycloviolacin O3 (CyO3), CyO8 and CyO13. A suspension culture was obtained that grew exponentially with a doubling time of approximately 3 days. After ten days of growth, the culture provided a yield of more than 4 mg CyO13 per gram dry mass.

  7. Endophytic Herbaspirillum seropedicae expresses nif genes in gramineous plants.

    PubMed

    Roncato-Maccari, Lauren D B; Ramos, Humberto J O; Pedrosa, Fabio O; Alquini, Yedo; Chubatsu, Leda S; Yates, Marshall G; Rigo, Liu U; Steffens, Maria Berenice R; Souza, Emanuel M

    2003-07-01

    Abstract The interactions between maize, sorghum, wheat and rice plants and Herbaspirillum seropedicae were examined microscopically following inoculation with the H. seropedicae LR15 strain, a Nif(+) (Pnif::gusA) mutant obtained by the insertion of a gusA-kanamycin cassette into the nifH gene of the H. seropedicae wild-type strain. The expression of the Pnif::gusA fusion was followed during the association of the diazotroph with the gramineous species. Histochemical analysis of seedlings of maize, sorghum, wheat and rice grown in vermiculite showed that strain LR15 colonized root surfaces and inner tissues. In early steps of the endophytic association, H. seropedicae colonized root exudation sites, such as axils of secondary roots and intercellular spaces of the root cortex; it then occupied the vascular tissue and there expressed nif genes. The expression of nif genes occurred in roots, stems and leaves as detected by the GUS reporter system. The expression of nif genes was also observed in bacterial colonies located in the external mucilaginous root material, 8 days after inoculation. Moreover, the colonization of plant tissue by H. seropedicae did not depend on the nitrogen-fixing ability, since similar numbers of cells were isolated from roots or shoots of the plants inoculated with Nif(+) or Nif(-) strains.

  8. Exogenous plant hormones and cyclotide expression in Viola uliginosa (Violaceae).

    PubMed

    Slazak, Blazej; Jacobsson, Erik; Kuta, Elżbieta; Göransson, Ulf

    2015-09-01

    Plants from Violaceae produce cyclotides, peptides characterized by a circular peptide backbone and a cystine knot. This signature motif gives stability that can harness a wide spectrum of biological activities, with implications in plant defense and with applications in medicine and biotechnology. In the current work, cyclotide expressing in vitro cultures were established from Viola uliginosa. These cultures are useful models for studying biosynthesis of cyclotides and can also be used in their production. The cyclotide expression pattern is shown to be dependent on exogenous plant growth regulators, both on peptide and gene expression levels. The highest yields of cyclotides were obtained on media containing only a cytokinin and were correlated with storage material accumulation. Exposure to auxins decreased cyclotide production and caused shifting of the biosynthesis pattern to root specific cyclotides. The response to stimuli in terms of cyclotide expression pattern appears to be developmental, and related to polar auxin transportation and the auxin/cytokinin ratio regulating tissue differentiation. By the use of whole transcriptome shotgun sequencing (WTSS) and peptidomics, 20 cyclotide sequences from V. uliginosa (including 12 new) and 12 complete precursor proteins could be identified. The most abundant cyclotides were cycloviolacin O3 (CyO3), CyO8 and CyO13. A suspension culture was obtained that grew exponentially with a doubling time of approximately 3 days. After ten days of growth, the culture provided a yield of more than 4 mg CyO13 per gram dry mass. PMID:26246035

  9. Conserved Gene Expression Programs in Developing Roots from Diverse Plants.

    PubMed

    Huang, Ling; Schiefelbein, John

    2015-08-01

    The molecular basis for the origin and diversification of morphological adaptations is a central issue in evolutionary developmental biology. Here, we defined temporal transcript accumulation in developing roots from seven vascular plants, permitting a genome-wide comparative analysis of the molecular programs used by a single organ across diverse species. The resulting gene expression maps uncover significant similarity in the genes employed in roots and their developmental expression profiles. The detailed analysis of a subset of 133 genes known to be associated with root development in Arabidopsis thaliana indicates that most of these are used in all plant species. Strikingly, this was also true for root development in a lycophyte (Selaginella moellendorffii), which forms morphologically different roots and is thought to have evolved roots independently. Thus, despite vast differences in size and anatomy of roots from diverse plants, the basic molecular mechanisms employed during root formation appear to be conserved. This suggests that roots evolved in the two major vascular plant lineages either by parallel recruitment of largely the same developmental program or by elaboration of an existing root program in the common ancestor of vascular plants.

  10. Constitutive HIF-1α expression blunts the beneficial effects of cardiosphere-derived cell therapy in the heart by altering paracrine factor balance.

    PubMed

    Bonios, Michael; Chang, Connie Yachan; Terrovitis, John; Pinheiro, Aurelio; Barth, Andreas; Dong, Peihong; Santaularia, Miguel; Foster, D Brian; Raman, Venu; Abraham, Theodore P; Abraham, Maria Roselle

    2011-06-01

    Hypoxia-inducible factor-1alpha (HIF-1α) expression promotes angiogenesis and can influence stem cell engraftment. We investigated the effect of stable over-expression of constitutively active HIF-1α on cardiosphere-derived cell (CDC) engraftment and left ventricular function. CDCs were transduced with a lentivirus expressing a constitutively active mutant of human HIF-1α (LVHIF-1α). Two million male rat CDCs were injected into the infarct following ligation of the mid-LAD in female syngeneic rats. Left ventricular ejection fraction (EF) and circumferential strain were measured by echocardiography at 1 and 4 weeks post-MI in the following groups: PBS group (n = 7), CELL group (n = 7), and CELL-HIF group (n = 7). HIF-1α, VEGF, endothelin-1 expression, and CDC engraftment were measured by quantitative PCR. At 30 days, EF was unchanged in the CELL-HIF group (p = NS), increased in the CELL group (p = 0.025), and decreased in the PBS group (p = 0.021), but engraftment was similar (2.4% ± 3.3% vs 1.7% ± 0.8%, p = NS). Mean circumferential strain of the infarcted region was unchanged in the CELL-HIF group, but improved in the CELL group (p = 0.02). Endothelin-1 and VEGF expression were higher in HIF-CDCs exposed to hypoxia, compared with non-transduced CDCs. HIF-1α expression in CDCs blunted the beneficial functional effects of CDC transplantation, suggesting that paracrine factor balance may play an important role in cardiac regeneration. PMID:21538185

  11. Approaches for gene targeting and targeted gene expression in plants.

    PubMed

    Husaini, Amjad Masood; Rashid, Zerka; Mir, Reyaz-ul Rouf; Aquil, Bushra

    2011-01-01

    Transgenic science and technology are fundamental to state-of-the-art plant molecular genetics and crop improvement. The new generation of technology endeavors to introduce genes 'stably' into 'site-specific' locations and in 'single copy' without the integration of extraneous vector 'backbone' sequences or selectable markers and with a 'predictable and consistent' expression. Several similar strategies and technologies, which can push the development of 'smart' genetically modified plants with desirable attributes, as well as enhance their consumer acceptability, are discussed in this review.

  12. Expression of constitutively active alpha-PAK reveals effects of the kinase on actin and focal complexes.

    PubMed Central

    Manser, E; Huang, H Y; Loo, T H; Chen, X Q; Dong, J M; Leung, T; Lim, L

    1997-01-01

    The family of p21-activated protein kinases (PAKs) appear to be present in all organisms that have Cdc42-like GTPases. In mammalian cells, PAKs have been implicated in the activation of mitogen-activated protein kinase cascades, but there are no reported effects of these kinases on the cytoskeleton. Recently we have shown that a Drosophila PAK is enriched in the leading edge of embryonic epithelial cells undergoing dorsal closure (N. Harden, J. Lee, H.-Y. Loh, Y.-M. Ong, I. Tan, T. Leung, E. Manser, and L. Lim, Mol. Cell. Biol. 16:1896-1908, 1996), where it colocalizes with structures resembling focal complexes. We show here by transfection that in epithelial HeLa cells alpha-PAK is recruited from the cytoplasm to distinct focal complexes by both Cdc42(G12V) and Rac1(G12V), which themselves colocalize to these sites. By deletion analysis, the N terminus of PAK is shown to contain targeting sequences for focal adhesions which indicate that these complexes are the site of kinase function in vivo. Cdc42 and Rac1 cause alpha-PAK autophosphorylation and kinase activation. Mapping alpha-PAK autophosphorylation sites has allowed generation of a constitutively active kinase mutant. By fusing regions of Cdc42 to the C terminus of PAK, activated chimeras were also obtained. Plasmids encoding these different constitutively active alpha-PAKs caused loss of stress fibers when introduced into both HeLa cells and fibroblasts, which was similar to the effect of introducing Cdc42(G12V) or Rac1(G12V). Significantly dramatic losses of focal adhesions were also observed. These combined effects resulted in retraction of the cell periphery after plasmid microinjection. These data support our previous suggestions of a role for PAK downstream of both Cdc42 and Rac1 and indicate that PAK functions include the dissolution of stress fibers and reorganization of focal complexes. PMID:9032240

  13. Improved Shoot Regeneration, Salinity Tolerance and Reduced Fungal Susceptibility in Transgenic Tobacco Constitutively Expressing PR-10a Gene.

    PubMed

    Agarwal, Parinita; Dabi, Mitali; More, Prashant; Patel, Khantika; Jana, Kalyanashis; Agarwal, Pradeep K

    2016-01-01

    Plants in ecosystems are simultaneously exposed to abiotic and biotic stresses, which restrict plant growth and development. The complex responses to these stresses are largely regulated by plant hormones, which in turn, orchestrate the different biochemical and molecular pathways to maneuver stress tolerance. The PR-10 protein family is reported to be involved in defense regulation, stress response and plant growth and development. The JcPR-10a overexpression resulted in increased number of shoot buds in tobacco (Nicotiana tabacum), which could be due to high cytokinin to auxin ratio in the transgenics. The docking analysis shows the binding of three BAP molecules at the active sites of JcPR-10a protein. JcPR-10a transgenics showed enhanced salt tolerance, as was evident by increased germination rate, shoot and root length, relative water content, proline, soluble sugar and amino acid content under salinity. Interestingly, the transgenics also showed enhanced endogenous cytokinin level as compared to WT, which, further increased with salinity. Exposure of gradual salinity resulted in increased stomatal conductance, water use efficiency, photosynthesis rate and reduced transpiration rate. Furthermore, the transgenics also showed enhanced resistance against Macrophomina fungus. Thus, JcPR-10a might be working in co-ordination with cytokinin signaling in mitigating the stress induced damage by regulating different stress signaling pathways, leading to enhanced stress tolerance. PMID:26973666

  14. Improved Shoot Regeneration, Salinity Tolerance and Reduced Fungal Susceptibility in Transgenic Tobacco Constitutively Expressing PR-10a Gene

    PubMed Central

    Agarwal, Parinita; Dabi, Mitali; More, Prashant; Patel, Khantika; Jana, Kalyanashis; Agarwal, Pradeep K.

    2016-01-01

    Plants in ecosystems are simultaneously exposed to abiotic and biotic stresses, which restrict plant growth and development. The complex responses to these stresses are largely regulated by plant hormones, which in turn, orchestrate the different biochemical and molecular pathways to maneuver stress tolerance. The PR-10 protein family is reported to be involved in defense regulation, stress response and plant growth and development. The JcPR-10a overexpression resulted in increased number of shoot buds in tobacco (Nicotiana tabacum), which could be due to high cytokinin to auxin ratio in the transgenics. The docking analysis shows the binding of three BAP molecules at the active sites of JcPR-10a protein. JcPR-10a transgenics showed enhanced salt tolerance, as was evident by increased germination rate, shoot and root length, relative water content, proline, soluble sugar and amino acid content under salinity. Interestingly, the transgenics also showed enhanced endogenous cytokinin level as compared to WT, which, further increased with salinity. Exposure of gradual salinity resulted in increased stomatal conductance, water use efficiency, photosynthesis rate and reduced transpiration rate. Furthermore, the transgenics also showed enhanced resistance against Macrophomina fungus. Thus, JcPR-10a might be working in co-ordination with cytokinin signaling in mitigating the stress induced damage by regulating different stress signaling pathways, leading to enhanced stress tolerance. PMID:26973666

  15. [Optimization of the mineral nutrition of plants constituting the phototrophic component of closed biological life support systems].

    PubMed

    Tikhomirov, A A; Ushakova, S A; Golovko, T K; Velichko, V V; Tabalenkova, G N; Zakhozhiĭ, I G; Garmash, E V

    2012-01-01

    Applicability of a new substrate for crops cultivation in bioregenerative LSSs with a high degree of mass-exchange closure was tested. Optimization of leaf cabbage nutrition by supplementing the basic substrate fabricated of plant and animal residues with ion-exchange resins proved to have a success.

  16. Constitutive expression of IL-12Rβ2 on human multiple myeloma cells delineates a novel therapeutic target

    PubMed Central

    Cocco, Claudia; Giuliani, Nicola; Ferrarini, Marina; Colla, Simona; Ognio, Emanuela; Taverniti, Giuseppe; Di Carlo, Emma; Cutrona, Giovanna; Perfetti, Vittorio; Rizzoli, Vittorio; Ribatti, Domenico; Pistoia, Vito

    2008-01-01

    The interleukin-12 (IL-12) receptor (R) B2 gene acts as tumor suppressor in human acute and chronic B-cell leukemias/lymphomas and IL-12rb2–deficient mice develop spontaneously localized plasmacytomas. With this background, we investigated the role of IL-12Rβ2 in multiple myeloma (MM) pathogenesis. Here we show the following: (1) IL-12Rβ2 was expressed in primary MM cells but down-regulated compared with normal polyclonal plasmablastic cells and plasma cells (PCs). IL-6 dampened IL-12Rβ2 expression on polyclonal plasmablastic cells and MM cells. (2) IL-12 reduced the proangiogenic activity of primary MM cells in vitro and decreased significantly (P = .001) the tumorigenicity of the NCI-H929 cell line in SCID/NOD mice by inhibiting cell proliferation and angiogenesis. The latter phenomenon was found to depend on abolished expression of a wide panel of proangiogenic genes and up-regulated expression of the antiangiogenic genes IFN-γ, IFN-α, platelet factor-4, and TIMP-2. Inhibition of the angiogenic potential of primary MM cells was related to down-regulated expression of the proangiogenic genes CCL11, vascular endothelial-cadherin, CD13, and AKT and to up-regulation of an IFN-γ–related antiangiogenic pathway. Thus, IL-12Rβ2 directly restrains MM cell growth, and targeting of IL-12 to tumor cells holds promise as new therapeutic strategy. PMID:18474725

  17. Expression patterns of FLAGELLIN SENSING 2 map to bacterial entry sites in plant shoots and roots

    PubMed Central

    Beck, Martina; Wyrsch, Ines; Strutt, James; Wimalasekera, Rinukshi; Webb, Alex; Boller, Thomas; Robatzek, Silke

    2014-01-01

    Pathogens can colonize all plant organs and tissues. To prevent this, each cell must be capable of autonomously triggering defence. Therefore, it is generally assumed that primary sensors of the immune system are constitutively present. One major primary sensor against bacterial infection is the FLAGELLIN SENSING 2 (FLS2) pattern recognition receptor (PRR). To gain insights into its expression pattern, the FLS2 promoter activity in β-glucuronidase (GUS) reporter lines was monitored. The data show that pFLS2::GUS activity is highest in cells and tissues vulnerable to bacterial entry and colonization, such as stomata, hydathodes, and lateral roots. GUS activity is also high in the vasculature and, by monitoring Ca2+ responses in the vasculature, it was found that this tissue contributes to flg22-induced Ca2+ burst. The FLS2 promoter is also regulated in a tissue- and cell type-specific manner and is responsive to hormones, damage, and biotic stresses. This results in stimulus-dependent expansion of the FLS2 expression domain. In summary, a tissue- and cell type-specific map of FLS2 expression has been created correlating with prominent entry sites and target tissues of plant bacterial pathogens. PMID:25205577

  18. Effect of Drought on Herbivore-Induced Plant Gene Expression: Population Comparison for Range Limit Inferences

    PubMed Central

    Gill, Gunbharpur Singh; Haugen, Riston; Matzner, Steven L.; Barakat, Abdelali; Siemens, David H.

    2016-01-01

    Low elevation “trailing edge” range margin populations typically face increases in both abiotic and biotic stressors that may contribute to range limit development. We hypothesize that selection may act on ABA and JA signaling pathways for more stable expression needed for range expansion, but that antagonistic crosstalk prevents their simultaneous co-option. To test this hypothesis, we compared high and low elevation populations of Boechera stricta that have diverged with respect to constitutive levels of glucosinolate defenses and root:shoot ratios; neither population has high levels of both traits. If constraints imposed by antagonistic signaling underlie this divergence, one would predict that high constitutive levels of traits would coincide with lower plasticity. To test this prediction, we compared the genetically diverged populations in a double challenge drought-herbivory growth chamber experiment. Although a glucosinolate defense response to the generalist insect herbivore Spodoptera exigua was attenuated under drought conditions, the plastic defense response did not differ significantly between populations. Similarly, although several potential drought tolerance traits were measured, only stomatal aperture behavior, as measured by carbon isotope ratios, was less plastic as predicted in the high elevation population. However, RNAseq results on a small subset of plants indicated differential expression of relevant genes between populations as predicted. We suggest that the ambiguity in our results stems from a weaker link between the pathways and the functional traits compared to transcripts. PMID:27135233

  19. Effect of Drought on Herbivore-Induced Plant Gene Expression: Population Comparison for Range Limit Inferences.

    PubMed

    Gill, Gunbharpur Singh; Haugen, Riston; Matzner, Steven L; Barakat, Abdelali; Siemens, David H

    2016-01-01

    Low elevation "trailing edge" range margin populations typically face increases in both abiotic and biotic stressors that may contribute to range limit development. We hypothesize that selection may act on ABA and JA signaling pathways for more stable expression needed for range expansion, but that antagonistic crosstalk prevents their simultaneous co-option. To test this hypothesis, we compared high and low elevation populations of Boechera stricta that have diverged with respect to constitutive levels of glucosinolate defenses and root:shoot ratios; neither population has high levels of both traits. If constraints imposed by antagonistic signaling underlie this divergence, one would predict that high constitutive levels of traits would coincide with lower plasticity. To test this prediction, we compared the genetically diverged populations in a double challenge drought-herbivory growth chamber experiment. Although a glucosinolate defense response to the generalist insect herbivore Spodoptera exigua was attenuated under drought conditions, the plastic defense response did not differ significantly between populations. Similarly, although several potential drought tolerance traits were measured, only stomatal aperture behavior, as measured by carbon isotope ratios, was less plastic as predicted in the high elevation population. However, RNAseq results on a small subset of plants indicated differential expression of relevant genes between populations as predicted. We suggest that the ambiguity in our results stems from a weaker link between the pathways and the functional traits compared to transcripts.

  20. Bone morphogenetic protein-7 inhibits constitutive and interleukin-1 beta-induced monocyte chemoattractant protein-1 expression in human mesangial cells: role for JNK/AP-1 pathway.

    PubMed

    Lee, Myung-Ja; Yang, Chul Woo; Jin, Dong Chan; Chang, Yoon Sik; Bang, Byung Kee; Kim, Yong-Soo

    2003-03-01

    Bone morphogenetic protein-7 (BMP-7), which belongs to the TGF-beta superfamily, has been shown to reduce macrophage infiltration and tissue injury in animal models of inflammatory renal disease. To explore the mechanism involved in the anti-inflammatory effect, we investigated the effect of BMP-7 on monocyte chemoattractant protein-1 (MCP-1) expression in cultured human mesangial cells. BMP- 7 significantly inhibited constitutive and IL-1 beta-induced MCP-1 protein production and MCP-1 mRNA expression by mesangial cells in a time- and concentration-dependent manner. BMP-7 also inhibited IL-1 beta-induced monocyte chemotactic activity released from the mesangial cells. We examined the role of transcription factors NF-kappa B and AP-1 in BMP-7 inhibition of IL-1 beta-induced MCP-1 expression. IL-1 beta increased NF-kappa B and AP-1 activity and both transcription factors mediated IL-1 beta-induced MCP-1 expression in mesangial cells. BMP-7 inhibited IL-1 beta-induced AP-1 activity in a concentration-dependent manner. In contrast, IL-1 beta-induced NF-kappa B activity and I kappa B alpha degradation were not affected by BMP-7. Furthermore, IL-1 beta-induced phosphorylation of c-Jun N-terminal kinase was inhibited by BMP-7. These data suggest that BMP-7 inhibits constitutive and IL-1 beta-induced MCP-1 expression in human mesangial cells partly by inhibiting c-Jun N-terminal kinase activity and subsequent AP-1 activity, and provide new insight into the therapeutic potential of BMP-7 in the inflammatory renal diseases.

  1. Enhanced transformation of tnt by tobacco plants expressing a bacterial nitroreductase.

    PubMed

    Hannink, Nerissa K; Subramanian, Murali; Rosser, Susan J; Basran, Amrik; Murray, James A H; Shanks, Jacqueline V; Bruce, Neil C

    2007-01-01

    The manufacture, disposal, and detonation of explosives have resulted in the pollution of large tracts of land and groundwater. Historically, 2,4,6-trinitrotoluene (TNT) is the most widely used military explosive and is toxic to biological systems and recalcitrant to degradation. To examine the feasibility of enhancing the ability of plants to detoxify the explosive TNT, we created transgenic tobacco (Nicotiana tabacum) constitutively expressing the nsfI nitroreductase gene from Enterobacter cloacae. The product of TNT reduction by the nitroreductase was found to be 4-hydroxylamino-2,6-dinitrotoluene (4-HADNT). Characterization of the transgenic lines in sterile, aqueous conditions amended with TNT demonstrated that these plants were able to remove all of the TNT from the medium at an initial concentration of 0.5 mM (113 mg L(-1)) TNT. In contrast, growth was suppressed in wild-type plants at 0.1 mM (23 mg L(-1)). Following uptake, transgenic seedlings transformed TNT predominantly to 4-HADNT and its high levels appeared to correlate with enhanced tolerance and transformation of TNT. Transformation products of TNT were subsequently conjugated to plant macromolecules to a greater degree in transgenic tobacco, indicating enhanced detoxification compared to the wild type. PMID:18246725

  2. Transposon-mediated alteration of TaMATE1B expression in wheat confers constitutive citrate efflux from root apices.

    PubMed

    Tovkach, Andriy; Ryan, Peter R; Richardson, Alan E; Lewis, David C; Rathjen, Tina M; Ramesh, Sunita; Tyerman, Stephen D; Delhaize, Emmanuel

    2013-02-01

    The TaMATE1B gene (for multidrug and toxic compound extrusion) from wheat (Triticum aestivum) was isolated and shown to encode a citrate transporter that is located on the plasma membrane. TaMATE1B expression in roots was induced by iron deficiency but not by phosphorus deficiency or aluminum treatment. The coding region of TaMATE1B was identical in a genotype showing citrate efflux from root apices (cv Carazinho) to one that lacked citrate efflux (cv Egret). However, sequence upstream of the coding region differed between these two genotypes in two ways. The first difference was a single-nucleotide polymorphism located approximately 2 kb upstream from the start codon in cv Egret. The second difference was an 11.1-kb transposon-like element located 25 bp upstream of the start codon in cv Carazinho that was absent from cv Egret. The influence of these polymorphisms on TaMATE1B expression was investigated using fusions to green fluorescent protein expressed in transgenic lines of rice (Oryza sativa). Fluorescence measurements in roots of rice indicated that 1.5- and 2.3-kb regions upstream of TaMATE1B in cv Carazinho (which incorporated 3' regions of the transposon-like element) generated 20-fold greater expression in the apical 1 mm of root compared with the native promoter in cv Egret. By contrast, fluorescence in more mature tissues was similar in both cultivars. The presence of the single-nucleotide polymorphism alone consistently generated 2-fold greater fluorescence than the cv Egret promoter. We conclude that the transposon-like element in cv Carazinho extends TaMATE1B expression to the root apex, where it confers citrate efflux and enhanced aluminum tolerance.

  3. Alternative polyadenylation and gene expression regulation in plants.

    PubMed

    Xing, Denghui; Li, Qingshun Quinn

    2011-01-01

    Functioning as an essential step of pre-mRNA processing, polyadenylation has been realized in recent years to play an important regulatory role during eukaryotic gene expression. Such regulation occurs mostly through the use of alternative polyadenylation (APA) sites and generates different transcripts with altered coding capacity for proteins and/or RNA. However, the molecular mechanisms that underlie APAs are poorly understood. Besides APA cases demonstrated in animal embryo development, cancers, and other diseases, there are a number of APA examples reported in plants. The best-known ones are related to flowering time control pathways and stress responses. Genome-wide studies have revealed that plants use APA extensively to generate diversity in their transcriptomes. Although each transcript produced by RNA polymerase II has a poly(A) tail, over 50% of plant genes studied possess multiple APA sites in their transcripts. The signals defining poly(A) sites in plants were mostly studied through classical genetic means. Our understanding of these poly(A) signals is enhanced by the tallies of whole plant transcriptomes. The profiles of these signals have been used to build computer models that can predict poly(A) sites in newly sequenced genomes, potential APA sites in genes of interest, and/or to identify, and then mutate, unwanted poly(A) sites in target transgenes to facilitate crop improvements. In this review, we provide readers an update on recent research advances that shed light on the understanding of polyadenylation, APA, and its role in gene expression regulation in plants.

  4. Reduced toxicity and broad spectrum resistance to viral and fungal infection in transgenic plants expressing pokeweed antiviral protein II.

    PubMed

    Wang, P; Zoubenko, O; Tumer, N E

    1998-12-01

    Pokeweed antiviral protein II (PAPII), a 30 kDa protein isolated from leaves of Phytolacca americana, inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. The protein sequence of PAPII shows only 41% identity to PAP and PAP-S, two other antiviral proteins isolated from pokeweed. We isolated a cDNA corresponding to PAPII and introduced it into tobacco plants. PAPII expressed in transgenic tobacco was correctly processed to the mature form as in pokeweed and accumulated to at least 10-fold higher levels than wild-type PAP. We had previously observed a significant decrease in transformation frequency with PAP and recovered only two transgenic lines expressing 1-2 ng per mg protein. In contrast, eight different transgenic lines expressing up to 250 ng/mg PAPII were recovered, indicating that PAPII is less toxic than PAP. Two symptomless transgenic lines expressing PAPII were resistant to tobacco mosaic virus, potato virus X and the fungal pathogen Rhizoctonia solani. The level of viral and fungal resistance observed correlated well with the amount of PAPII protein accumulated. Pathogenesis-related protein PR1 was constitutively expressed in transgenic lines expressing PAPII. Although PR1 was constitutively expressed, no increase in salicylic acid levels was detected, indicating that PAPII may elicit a salicylic acid-independent signal transduction pathway.

  5. Plant biofarming: novel insights for peptide expression in heterologous systems.

    PubMed

    Viana, Antônio Américo Barbosa; Pelegrini, Patrícia Barbosa; Grossi-de-Sá, Maria Fátima

    2012-01-01

    Peptide expression methods have been widely studied and developed from many different biological sources. The cultivation ofprokaryotic and eukaryotic cells has proven to be efficient for the expression of foreign peptides in several heterologous systems, including bacteria, insects, yeasts, and mammals. Earlier reports brought up new insights for the improvement of expressed products to not only increase the production rate of desired peptides but also reproduce desirable post-translational modifications and even to reduce the risk of allergenicity when those products are aimed for human use. The development of bioreactor systems provided the optimization of cell growth conditions to scale up the amounts of expressed peptides. On the other hand, different cell systems and mutants provided a plethora of possible peptide modifications. Hence, in this report, we describe the many organisms and systems used for the large scale production of several macromolecules with relevance in health and agriculture. We also bring into discussion plant biofarming in the moss Physcomitrella patens and its recent adaptations, as a cost-effective and efficient approach in the production of more complex heterologous proteins, given the fact that its glycosylation pattern can be engineered to avoid allergenicity to humans (common to plant-derived glycoproteins). PMID:23193604

  6. Long noncoding RNA expression profiles in gut tissues constitute molecular signatures that reflect the types of microbes.

    PubMed

    Liang, Lunxi; Ai, Luoyan; Qian, Jin; Fang, Jing-Yuan; Xu, Jie

    2015-01-01

    The gut microbiota is commonly referred to as a hidden organ due to its pivotal effects on host physiology, metabolism, nutrition and immunity. The gut microbes may be shaped by environmental and host genetic factors, and previous studies have focused on the roles of protein-coding genes. Here we show a link between long non-coding RNA (lncRNA) expression and gut microbes. By repurposing exon microarrays and comparing the lncRNA expression profiles between germ-free, conventional and different gnotobiotic mice, we revealed subgroups of lncRNAs that were specifically enriched in each condition. A nearest shrunken centroid methodology was applied to obtain lncRNA-based signatures to identify mice in different conditions. The lncRNA-based prediction model successfully identified different gnotobiotic mice from conventional and germ-free mice, and also discriminated mice harboring transplanted microbes from fecal samples of mice or zebra fishes. To achieve optimal prediction accuracy, fewer lncRNAs were required in the prediction model than protein-coding genes. Taken together, our study demonstrated the effecacy of lncRNA expression profiles in discriminating the types of microbes in the gut. These results also provide a resource of gut microbe-associated lncRNAs for the development of lncRNA biomarkers and the identification of functional lncRNAs in host-microbes interactions. PMID:26123364

  7. ALK1 signalling analysis identifies angiogenesis related genes and reveals disparity between TGF-β and constitutively active receptor induced gene expression

    PubMed Central

    Lux, Andreas; Salway, Fiona; Dressman, Holly K; Kröner-Lux, Gabriele; Hafner, Mathias; Day, Philip JR; Marchuk, Douglas A; Garland, John

    2006-01-01

    Background TGF-β1 is an important angiogenic factor involved in the different aspects of angiogenesis and vessel maintenance. TGF-β signalling is mediated by the TβRII/ALK5 receptor complex activating the Smad2/Smad3 pathway. In endothelial cells TGF-β utilizes a second type I receptor, ALK1, activating the Smad1/Smad5 pathway. Consequently, a perturbance of ALK1, ALK5 or TβRII activity leads to vascular defects. Mutations in ALK1 cause the vascular disorder hereditary hemorrhagic telangiectasia (HHT). Methods The identification of ALK1 and not ALK5 regulated genes in endothelial cells, might help to better understand the development of HHT. Therefore, the human microvascular endothelial cell line HMEC-1 was infected with a recombinant constitutively active ALK1 adenovirus, and gene expression was studied by using gene arrays and quantitative real-time PCR analysis. Results After 24 hours, 34 genes were identified to be up-regulated by ALK1 signalling. Analysing ALK1 regulated gene expression after 4 hours revealed 13 genes to be up- and 2 to be down-regulated. Several of these genes, including IL-8, ET-1, ID1, HPTPη and TEAD4 are reported to be involved in angiogenesis. Evaluation of ALK1 regulated gene expression in different human endothelial cell types was not in complete agreement. Further on, disparity between constitutively active ALK1 and TGF-β1 induced gene expression in HMEC-1 cells and primary HUVECs was observed. Conclusion Gene array analysis identified 49 genes to be regulated by ALK1 signalling and at least 14 genes are reported to be involved in angiogenesis. There was substantial agreement between the gene array and quantitative real-time PCR data. The angiogenesis related genes might be potential HHT modifier genes. In addition, the results suggest endothelial cell type specific ALK1 and TGF-β signalling. PMID:16594992

  8. Constitutive Smad signaling and Smad-dependent collagen gene expression in mouse embryonic fibroblasts lacking peroxisome proliferator-activated receptor-{gamma}

    SciTech Connect

    Ghosh, Asish K Wei, Jun; Wu, Minghua; Varga, John

    2008-09-19

    Transforming growth factor-{beta} (TGF-{beta}), a potent inducer of collagen synthesis, is implicated in pathological fibrosis. Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is a nuclear hormone receptor that regulates adipogenesis and numerous other biological processes. Here, we demonstrate that collagen gene expression was markedly elevated in mouse embryonic fibroblasts (MEFs) lacking PPAR-{gamma} compared to heterozygous control MEFs. Treatment with the PPAR-{gamma} ligand 15d-PGJ{sub 2} failed to down-regulate collagen gene expression in PPAR-{gamma} null MEFs, whereas reconstitution of these cells with ectopic PPAR-{gamma} resulted in their normalization. Compared to control MEFs, PPAR-{gamma} null MEFs displayed elevated levels of the Type I TGF-{beta} receptor (T{beta}RI), and secreted more TGF-{beta}1 into the media. Furthermore, PPAR-{gamma} null MEFs showed constitutive phosphorylation of cellular Smad2 and Smad3, even in the absence of exogenous TGF-{beta}, which was abrogated by the ALK5 inhibitor SB431542. Constitutive Smad2/3 phosphorylation in PPAR-{gamma} null MEFs was associated with Smad3 binding to its cognate DNA recognition sequences, and interaction with coactivator p300 previously implicated in TGF-{beta} responses. Taken together, these results indicate that loss of PPAR-{gamma} in MEFs is associated with upregulation of collagen synthesis, and activation of intracellular Smad signal transduction, due, at least in part, to autocrine TGF-{beta} stimulation.

  9. Expression of a constitutively active calcineurin encoded by an intron-retaining mRNA in follicular keratinocytes.

    PubMed

    Fujimura, Atsushi; Michiue, Hiroyuki; Nishiki, Tei-ichi; Ohmori, Iori; Wei, Fan-Yan; Matsui, Hideki; Tomizawa, Kazuhito

    2011-03-14

    Hair growth is a highly regulated cyclical process. Immunosuppressive immunophilin ligands such as cyclosporin A (CsA) and FK506 are known as potent hair growth modulatory agents in rodents and humans that induce active hair growth and inhibit hair follicle regression. The immunosuppressive effectiveness of these drugs has been generally attributed to inhibition of T cell activation through well-characterized pathways. Specifically, CsA and FK506 bind to intracellular proteins, principally cyclophilin A and FKBP12, respectively, and thereby inhibit the phosphatase calcineurin (Cn). The calcineurin (Cn)/NFAT pathway has an important, but poorly understood, role in the regulation of hair follicle development. Here we show that a novel-splicing variant of calcineurin Aß CnAß-FK, which is encoded by an intron-retaining mRNA and is deficient in the autoinhibitory domain, is predominantly expressed in mature follicular keratinocytes but not in the proliferating keratinocytes of rodents. CnAß-FK was weakly sensitive to Ca(2+) and dephosphorylated NFATc2 under low Ca(2+) levels in keratinocytes. Inhibition of Cn/NFAT induced hair growth in nude mice. Cyclin G2 was identified as a novel target of the Cn/NFATc2 pathway and its expression in follicular keratinocytes was reduced by inhibition of Cn/NFAT. Overexpression of cyclin G2 arrested the cell cycle in follicular keratinocytes in vitro and the Cn inhibitor, cyclosporin A, inhibited nuclear localization of NFATc2, resulting in decreased cyclin G2 expression in follicular keratinocytes of rats in vivo. We therefore suggest that the calcineurin/NFAT pathway has a unique regulatory role in hair follicle development.

  10. Expression of a Constitutively Active Calcineurin Encoded by an Intron-Retaining mRNA in Follicular Keratinocytes

    PubMed Central

    Fujimura, Atsushi; Michiue, Hiroyuki; Nishiki, Tei-ichi; Ohmori, Iori; Wei, Fan-Yan; Matsui, Hideki; Tomizawa, Kazuhito

    2011-01-01

    Hair growth is a highly regulated cyclical process. Immunosuppressive immunophilin ligands such as cyclosporin A (CsA) and FK506 are known as potent hair growth modulatory agents in rodents and humans that induce active hair growth and inhibit hair follicle regression. The immunosuppressive effectiveness of these drugs has been generally attributed to inhibition of T cell activation through well-characterized pathways. Specifically, CsA and FK506 bind to intracellular proteins, principally cyclophilin A and FKBP12, respectively, and thereby inhibit the phosphatase calcineurin (Cn). The calcineurin (Cn)/NFAT pathway has an important, but poorly understood, role in the regulation of hair follicle development. Here we show that a novel-splicing variant of calcineurin Aß CnAß-FK, which is encoded by an intron-retaining mRNA and is deficient in the autoinhibitory domain, is predominantly expressed in mature follicular keratinocytes but not in the proliferating keratinocytes of rodents. CnAß-FK was weakly sensitive to Ca2+ and dephosphorylated NFATc2 under low Ca2+ levels in keratinocytes. Inhibition of Cn/NFAT induced hair growth in nude mice. Cyclin G2 was identified as a novel target of the Cn/NFATc2 pathway and its expression in follicular keratinocytes was reduced by inhibition of Cn/NFAT. Overexpression of cyclin G2 arrested the cell cycle in follicular keratinocytes in vitro and the Cn inhibitor, cyclosporin A, inhibited nuclear localization of NFATc2, resulting in decreased cyclin G2 expression in follicular keratinocytes of rats in vivo. We therefore suggest that the calcineurin/NFAT pathway has a unique regulatory role in hair follicle development. PMID:21423799

  11. Promoter/leader deletion analysis and plant expression vectors with the figwort mosaic virus (FMV) full length transcript (FLt) promoter containing single or double enhancer domains.

    PubMed

    Maiti, I B; Gowda, S; Kiernan, J; Ghosh, S K; Shepherd, R J

    1997-03-01

    The boundaries required for maximal expression from the promoter/leader region of the full length transcript of figwort mosaic virus (FLt promoter) coupled to reporter genes were defined by 5' and 3' deletion analyses. In transient expression assays using protoplasts of Nicotiana edwardsonii, a 314 bp FLt promoter fragment sequence (-249 to +65 from the transcription start site) was sufficient for strong expression activity. Plant expression vectors developed with modified FLt promoters were tested with GUS or CAT as reporter genes in transgenic plants. The FLt promoter is a strong constitutive promoter, with strength comparable to or greater than that of the CaMV 35S promoter. The FLt promoter with its double enhancer domain linked to GUS or CAT reporter genes provides an average 4-fold greater activity than the FLt promoter with a single enhancer domain (-55 to -249 bp upstream fragment) in tests with transgenic plants and in protoplast transient expression assays.

  12. Isolation and expression analysis of peanut chlorotic streak caulimovirus (PClSV) full-length transcript (FLt) promoter in transgenic plants.

    PubMed

    Maiti, I B; Shepherd, R J

    1998-03-17

    A promoter fragment from peanut chlorotic streak caulimovirus (PClSV) full-length transcript (FLt) was identified and later modified to have duplicated enhancer domain. The FLt promoter with its single or double enhancer domains, fused with the GUS reporter gene to form chimeric gene constructs, showed a high level of expression of these genes in cells and transgenic plants. The FLt promoter with its double enhancer domain gives an average threefold greater expression of genes compared to the FLt promoter with its single enhancer domain in transgenic plants. In young seedlings the expression was in the order root > leaf > stem. The histochemical GUS assay in young seedlings showed more activity in root tips and leaf midribs, veins, and other vascular tissues. The expression from the PClSV FLt promoter was compared with that from the figwort mosaic virus promoter in transgenic plants. These constitutive promoters were comparable in respect to GUS expression level.

  13. Shoot to root communication is necessary to control the expression of iron-acquisition genes in Strategy I plants.

    PubMed

    García, María J; Romera, Francisco J; Stacey, Minviluz G; Stacey, Gary; Villar, Eduardo; Alcántara, Esteban; Pérez-Vicente, Rafael

    2013-01-01

    Previous research showed that auxin, ethylene, and nitric oxide (NO) can activate the expression of iron (Fe)-acquisition genes in the roots of Strategy I plants grown with low levels of Fe, but not in plants grown with high levels of Fe. However, it is still an open question as to how Fe acts as an inhibitor and which pool of Fe (e.g., root, phloem, etc.) in the plant acts as the key regulator for gene expression control. To further clarify this, we studied the effect of the foliar application of Fe on the expression of Fe-acquisition genes in several Strategy I plants, including wild-type cultivars of Arabidopsis [Arabidopsis thaliana (L.) Heynh], pea [Pisum sativum L.], tomato [Solanum lycopersicon Mill.], and cucumber [Cucumis sativus L.], as well as mutants showing constitutive expression of Fe-acquisition genes when grown under Fe-sufficient conditions [Arabidopsis opt3-2 and frd3-3, pea dgl and brz, and tomato chln (chloronerva)]. The results showed that the foliar application of Fe blocked the expression of Fe-acquisition genes in the wild-type cultivars and in the frd3-3, brz, and chln mutants, but not in the opt3-2 and dgl mutants, probably affected in the transport of a Fe-related repressive signal in the phloem. Moreover, the addition of either ACC (ethylene precursor) or GSNO (NO donor) to Fe-deficient plants up-regulated the expression of Fe-acquisition genes, but this effect did not occur in Fe-deficient plants sprayed with foliar Fe, again suggesting the existence of a Fe-related repressive signal moving from leaves to roots.

  14. Constitutive Expression of the Cytochrome P450 EthABCD Monooxygenase System Enables Degradation of Synthetic Dialkyl Ethers in Aquincola tertiaricarbonis L108

    PubMed Central

    Schuster, Judith; Purswani, Jessica; Breuer, Uta; Pozo, Clementina; Harms, Hauke; Müller, Roland H.

    2013-01-01

    In Rhodococcus ruber IFP 2001, Rhodococcus zopfii IFP 2005, and Gordonia sp. strain IFP 2009, the cytochrome P450 monooxygenase EthABCD catalyzes hydroxylation of methoxy and ethoxy residues in the fuel oxygenates methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME). The expression of the IS3-type transposase-flanked eth genes is ETBE dependent and controlled by the regulator EthR (C. Malandain et al., FEMS Microbiol. Ecol. 72:289–296, 2010). In contrast, we demonstrated by reverse transcription-quantitative PCR (RT-qPCR) that the betaproteobacterium Aquincola tertiaricarbonis L108, which possesses the ethABCD genes but lacks ethR, constitutively expresses the P450 system at high levels even when growing on nonether substrates, such as glucose. The mutant strain A. tertiaricarbonis L10, which is unable to degrade dialkyl ethers, resulted from a transposition event mediated by a rolling-circle IS91-type element flanking the eth gene cluster in the wild-type strain L108. The constitutive expression of Eth monooxygenase is likely initiated by the housekeeping sigma factor σ70, as indicated by the presence in strain L108 of characteristic −10 and −35 binding sites upstream of ethA which are lacking in strain IFP 2001. This enables efficient degradation of diethyl ether, diisopropyl ether, MTBE, ETBE, TAME, and tert-amyl ethyl ether (TAEE) without any lag phase in strain L108. However, ethers with larger residues, n-hexyl methyl ether, tetrahydrofuran, and alkyl aryl ethers, were not attacked by the Eth system at significant rates in resting-cell experiments, indicating that the residue in the ether molecule which is not hydroxylated also contributes to the determination of substrate specificity. PMID:23354715

  15. Preferential expression and immunogenicity of HIV-1 Tat fusion protein expressed in tomato plant.

    PubMed

    Cueno, Marni E; Hibi, Yurina; Karamatsu, Katsuo; Yasutomi, Yasuhiro; Imai, Kenichi; Laurena, Antonio C; Okamoto, Takashi

    2010-10-01

    HIV-1 Tat plays a major role in viral replication and is essential for AIDS development making it an ideal vaccine target providing that both humoral and cellular immune responses are induced. Plant-based antigen production, due to its cheaper cost, appears ideal for vaccine production. In this study, we created a plant-optimized tat and mutant (Cys30Ala/Lys41Ala) tat (mtat) gene and ligated each into a pBI121 expression vector with a stop codon and a gusA gene positioned immediately downstream. The vector construct was bombarded into tomato leaf calli and allowed to develop. We thus generated recombinant tomato plants preferentially expressing a Tat-GUS fusion protein over a Tat-only protein. In addition, plants bombarded with either tat or mtat genes showed no phenotypic difference and produced 2-4 microg Tat-GUS fusion protein per milligram soluble plant protein. Furthermore, tomato extracts intradermally inoculated into mice were found to induce a humoral and, most importantly, cellular immunity. PMID:20072815

  16. Mitochondria of a human multidrug-resistant hepatocellular carcinoma cell line constitutively express inducible nitric oxide synthase in the inner membrane.

    PubMed

    Fantappiè, Ornella; Sassoli, Chiara; Tani, Alessia; Nosi, Daniele; Marchetti, Serena; Formigli, Lucia; Mazzanti, Roberto

    2015-06-01

    Mitochondria play a crucial role in pathways of stress conditions. They can be transported from one cell to another, bringing their features to the cell where they are transported. It has been shown in cancer cells overexpressing multidrug resistance (MDR) that mitochondria express proteins involved in drug resistance such as P-glycoprotein (P-gp), breast cancer resistant protein and multiple resistance protein-1. The MDR phenotype is associated with the constitutive expression of COX-2 and iNOS, whereas celecoxib, a specific inhibitor of COX-2 activity, reverses drug resistance of MDR cells by releasing cytochrome c from mitochondria. It is possible that COX-2 and iNOS are also expressed in mitochondria of cancer cells overexpressing the MDR phenotype. This study involved experiments using the human HCC PLC/PRF/5 cell line with and without MDR phenotype and melanoma A375 cells that do not express the MDR1 phenotype but they do iNOS. Western blot analysis, confocal immunofluorescence and immune electron microscopy showed that iNOS is localized in mitochondria of MDR1-positive cells, whereas COX-2 is not. Low and moderate concentrations of celecoxib modulate the expression of iNOS and P-gp in mitochondria of MDR cancer cells independently from inhibition of COX-2 activity. However, A375 cells that express iNOS also in mitochondria, were not MDR1 positive. In conclusion, iNOS can be localized in mitochondria of HCC cells overexpressing MDR1 phenotype, however this phenomenon appears independent from the MDR1 phenotype occurrence. The presence of iNOS in mitochondria of human HCC cells phenotype probably concurs to a more aggressive behaviour of cancer cells.

  17. Coordinately Co-opted Multiple Transposable Elements Constitute an Enhancer for wnt5a Expression in the Mammalian Secondary Palate

    PubMed Central

    Kimura-Yoshida, Chiharu; Yan, Kuo; Bormuth, Olga; Ding, Qiong; Nakanishi, Akiko; Sasaki, Takeshi; Hirakawa, Mika; Sumiyama, Kenta; Furuta, Yasuhide; Tarabykin, Victor; Matsuo, Isao; Okada, Norihiro

    2016-01-01

    Acquisition of cis-regulatory elements is a major driving force of evolution, and there are several examples of developmental enhancers derived from transposable elements (TEs). However, it remains unclear whether one enhancer element could have been produced via cooperation among multiple, yet distinct, TEs during evolution. Here we show that an evolutionarily conserved genomic region named AS3_9 comprises three TEs (AmnSINE1, X6b_DNA and MER117), inserted side-by-side, and functions as a distal enhancer for wnt5a expression during morphogenesis of the mammalian secondary palate. Functional analysis of each TE revealed step-by-step retroposition/transposition and co-option together with acquisition of a binding site for Msx1 for its full enhancer function during mammalian evolution. The present study provides a new perspective suggesting that a huge variety of TEs, in combination, could have accelerated the diversity of cis-regulatory elements involved in morphological evolution. PMID:27741242

  18. Constitutive expression of Wnt/β-catenin target genes promotes proliferation and invasion of liver cancer stem cells

    PubMed Central

    CHEN, WEI; ZHANG, YU-WEI; LI, YANG; ZHANG, JIAN-WEN; ZHANG, TONG; FU, BIN-SHENG; ZHANG, QI; JIANG, NAN

    2016-01-01

    Wnt/β-catenin is an important signaling pathways involved in the tumorgenesis, progression and maintenance of cancer stem cells (CSCs). In the present study, the role of Wnt/β-catenin signaling in CSC-mediated tumorigenesis and invasion in liver CSCs was investigated. A small population of cancer stem-like side population (SP) cells (3.6%) from liver cancer samples were identified. The cells were highly resistant to drug treatment due to the enhanced expression of drug efflux pumps, such as ABC subfamily G member 2, multidrug resistance protein 1 and ATP-binding cassette subfamily B member 5. Furthermore, using TOPflash and reverse transcription-quantitative polymerase chain reaction analysis, Wnt/β-catenin signaling and the transcriptional regulation of Wnt/β-catenin target genes including dickkopf Wnt signaling pathway inhibitor 1, axis inhibition protein 2 and cyclin D1 were observed to be markedly upregulated in liver cancer SP cells. As a consequence, SP cells possessed infinite cell proliferation potential and the ability to generating tumor spheres. In addition, upon reducing Wnt/β-catenin signaling, the rates of proliferation, tumor sphere formation and tumor invasion of SP cells were markedly reduced. Therefore, these data suggest that Wnt/β-catenin signaling is a potential therapeutic target to reduce CSC-mediated tumorigenicity and invasion in liver cancer. PMID:26956539

  19. Technical advance: An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants.

    PubMed

    Zuo, J; Niu, Q W; Chua, N H

    2000-10-01

    We have developed an estrogen receptor-based chemical-inducible system for use in transgenic plants. A chimeric transcription activator, XVE, was assembled by fusion of the DNA-binding domain of the bacterial repressor LexA (X), the acidic transactivating domain of VP16 (V) and the regulatory region of the human estrogen receptor (E; ER). The transactivating activity of the chimeric XVE factor, whose expression was controlled by the strong constitutive promoter G10-90, was strictly regulated by estrogens. In transgenic Arabidopsis and tobacco plants, estradiol-activated XVE can stimulate expression of a GFP reporter gene controlled by the target promoter, which consists of eight copies of the LexA operator fused upstream of the -46 35S minimal promoter. Upon induction by estradiol, GFP expression levels can be eightfold higher than that transcribed from a 35S promoter, whereas the uninduced controls have no detectable GFP transcripts, as monitored by Northern blot analysis. Neither toxic nor adverse physiological effects of the XVE system have been observed in transgenic Arabidopsis plants under all the conditions tested. The XVE system thus appears to be a reliable and efficient chemical-inducible system for regulating transgene expression in plants.

  20. Constitutively expressed Siglec-9 inhibits LPS-induced CCR7, but enhances IL-4-induced CD200R expression in human macrophages.

    PubMed

    Higuchi, Hiroshi; Shoji, Toru; Iijima, Shinji; Nishijima, Ken-Ichi

    2016-06-01

    Siglecs recognize the sialic acid moiety and regulate various immune responses. In the present study, we compared the expression levels of Siglecs in human monocytes and macrophages using a quantitative real-time reverse transcription-polymerase chain reaction analysis. The differentiation of monocytes into macrophages by macrophage colony-stimulating factor or granulocyte macrophage colony-stimulating factor enhanced the expression of Siglec-7 and Siglec-9. The differentiated macrophages were stimulated by lipopolysaccharide (LPS) plus interferon (IFN)-γ or interleukin (IL)-4. The expression of Siglec-10 was enhanced by IL-4, whereas that of Siglec-7 was reduced by LPS plus IFN-γ. The expression of Siglec-9 was not affected by these stimuli. The knockdown of Siglec-9 enhanced the expression of CCR7 induced by the LPS or the LPS plus IFN-γ stimulation, and decreased the IL-4-induced expression of CD200R. These results suggest that Siglec-9 is one of the main Siglecs in human blood monocytes/macrophages and modulates innate immunity. PMID:26923638

  1. Archetype JC virus efficiently replicates in COS-7 cells, simian cells constitutively expressing simian virus 40 T antigen.

    PubMed

    Hara, K; Sugimoto, C; Kitamura, T; Aoki, N; Taguchi, F; Yogo, Y

    1998-07-01

    JC polyomavirus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), is ubiquitous in humans, infecting children asymptomatically and then persisting in the kidney. Renal JCV is not latent but replicates to excrete progeny in the urine. The renal-urinary JCV DNAs carry the archetype regulatory region that generates various rearranged regulatory regions occurring in JCVs derived from the brains of PML patients. Tissue cultures that support the efficient growth of archetype JCV have not been reported. We studied whether archetype JCV could replicate in COS-7 cells, simian cells transformed with an origin-defective mutant of simian virus 40 (SV40). Efficient JCV replication, as detected by a hemagglutination assay, was observed in cultures transfected with five of the six archetype DNAs. The progeny JCVs could be passaged to fresh COS-7 cells. However, when the parental cells of COS-7 not expressing T antigen were transfected with archetype JCV DNAs, no viral replication was detected, indicating that SV40 T antigen is essential for the growth of JCV in COS-7 cells. The archetype regulatory region was conserved during viral growth in COS-7 cells, although a small proportion of JCV DNAs underwent rearrangements outside the regulatory region. We then attempted to recover archetype JCV from urine by viral culture in COS-7 cells. Efficient JCV production was observed in COS-7 cells infected with five of the six JCV-positive urine samples examined. Thus, COS-7 cells should be of use not only for the production of archetype JCV on a large scale but also for the isolation of archetype JCV from urine.

  2. Oral Rabies Vaccine Design for Expression in Plants.

    PubMed

    Singh, Ankit; Saxena, Gauri; Verma, Praveen C

    2016-01-01

    Vaccination is the sensitization process of the immune system against any pathogen. Generally, recombinant subunit vaccines are considered safer than attenuated vaccines. As whole pathogenic organisms are used in the immunization process, the attenuated vaccines are considered more risky than subunit vaccines. Rabies is the oldest known zoonosis which spreads through a neurotropic Lyssavirus primarily mediated through infected canine bites. Rabies causes worldwide loss of more than 60,000 human lives every year. Animal vaccination is equally important to check the transmission of rabies into humans. Rabies oral vaccination can be a good alternative where multiple booster and priming regimens are required while the painful vaccination process can continue for long durations. Introduction of oral vaccines was made to ease the discomfort associated with the mode of introduction of conventional vaccines into the body. Although the rabies oral vaccine can substantially reduce the cost of vaccination in the developing countries, mass immunization programs need larger quantities of vaccines which should be delivered at nominal cost. Expression of recombinant antigen proteins in E. coli is often not viable because of lack of post-translational modifications and folding requirements. Though yeast and insect cell line expression systems have post-translational processing and modifications, significantly different immunological response against their post-translational modification pattern limits their deployment as an expression system. As an alternative, plants are emerging as a promising system to express and deliver wide range of functionally active biopharmaceutical product at lower cost for mass immunization programs. As generation of vaccine antigenic proteins in plant systems are cheaper, the strategy will benefit developing countries where this disease causes thousands of deaths every year. In this chapter, we will discuss about our efforts toward development of oral

  3. Regulation of the Expression of Plant Resistance Gene SNC1 by a Protein with a Conserved BAT2 Domain1[C][W][OA

    PubMed Central

    Li, Yingzhong; Tessaro, Mark J.; Li, Xin; Zhang, Yuelin

    2010-01-01

    Plant Resistance (R) genes encode immune receptors that recognize pathogens and activate defense responses. Because of fitness costs associated with maintaining R protein-mediated resistance, expression levels of R genes have to be tightly regulated. However, mechanisms on how R-gene expression is regulated are poorly understood. Here we show that MODIFIER OF snc1, 1 (MOS1) regulates the expression of SUPPRESSOR OF npr1-1, CONSTITUTIVE1 (SNC1), which encodes a Toll/interleukin receptor-nucleotide binding site-leucine-rich repeat type of R protein in Arabidopsis (Arabidopsis thaliana). In the mos1 loss-of-function mutant plants, snc1 expression is repressed and constitutive resistance responses mediated by snc1 are lost. The repression of snc1 expression in mos1 is released by knocking out DECREASE IN DNA METHYLATION1. In mos1 mutants, DNA methylation in a region upstream of SNC1 is altered. Furthermore, expression of snc1 transgenes using the native promoter does not require MOS1, indicating that regulation of SNC1 expression by MOS1 is at the chromatin level. Map-based cloning of MOS1 revealed that it encodes a novel protein with a HLA-B ASSOCIATED TRANSCRIPT2 (BAT2) domain that is conserved in plants and animals. Our study on MOS1 suggests that BAT2 domain-containing proteins may function in regulation of gene expression at chromatin level. PMID:20439546

  4. Recruitment of CREB1 and Histone Deacetylase 2 (HDAC2) to the Mouse Ltbp-1 Promoter Regulates its Constitutive Expression in a Dioxin Receptor-dependent Manner

    PubMed Central

    Gomez-Duran, Aurea; Ballestar, Esteban; Carvajal-Gonzalez, Jose M.; Marlowe, Jennifer L.; Puga, Alvaro; Esteller, Manel; Fernandez-Salguero, Pedro M.

    2010-01-01

    Latent TGFβ-binding protein 1 (LTBP-1) is a key regulator of TGFβ targeting and activation in the extracellular matrix. LTBP-1 is recognized as a major docking molecule to localize, and possibly to activate, TGFβ in the extracellular matrix. Despite this relevant function, the molecular mechanisms regulating Ltbp-1 transcription remain largely unknown. Previous results from our laboratory revealed that mouse embryonic fibroblasts (MEF) lacking dioxin receptor (AhR) had increased Ltbp-1 mRNA expression and elevated TGFβ activity, suggesting that AhR repressed Ltbp-1 transcription. Here, we have cloned the mouse Ltbp-1 gene promoter and analysed its mechanism of transcriptional repression by AhR. Reporter gene assays, AhR over-expression and site-directed mutagenesis showed that basal Ltbp-1 transcription is AhR-dependent. Chromatin immunoprecipitation (ChIP) and RNA interference (RNAi) revealed that AhR regulates Ltbp-1 transcription by a mechanism involving recruitment of co-activators such as CREB1 and co-repressors such as HDAC2 to the Ltbp-1 promoter. In AhR-expressing (AhR+/+) MEF cells, the recruitment of HDAC1, 2 and 4 correlated with decreased K8H4 acetylation and impaired binding of pCREBSer133 to the Ltbp-1 promoter, likely maintaining a constitutive repressed state. AhR−/− MEF cells had the opposite pattern of HDACs and pCREB1Ser133 binding to Ltbp-1 promoter, and therefore, over-expressed Ltbp-1 mRNA. In agreement, siRNA for HDAC2 increased Ltbp-1 expression and K8H4 acetylation in AhR+/+ but not in AhR−/− MEF cells. We suggest that HDAC2 binding keeps Ltbp-1 promoter repressed in AhR+/+ MEF cells, whereas in AhR-null MEF cells the absence of HDAC2 and the binding of pCREBSer133 allow Ltbp-1 transcription. Thus, epigenetics can contribute to constitutive Ltbp-1 repression by a mechanism requiring AhR activity. PMID:18508077

  5. Constitutive Production of NF-κB2 p52 Is Not Tumorigenic but Predisposes Mice to Inflammatory Autoimmune Disease by Repressing Bim Expression*

    PubMed Central

    Wang, Zhe; Zhang, Baochun; Yang, Liqun; Ding, Jane; Ding, Han-Fei

    2008-01-01

    Normal development of the immune system requires regulated processing of NF-κB2 p100 to p52, which activates NF-κB2 signaling. Constitutive production of p52 has been suggested as a major mechanism underlying lymphomagenesis induced by NF-κB2 mutations, which occur recurrently in a variety of human lymphoid malignancies. To test the hypothesis, we generated transgenic mice with targeted expression of p52 in lymphocytes. In contrast to their counterparts expressing the tumor-derived NF-κB2 mutant p80HT, which develop predominantly B cell tumors, p52 transgenic mice are not prone to lymphomagenesis. However, they are predisposed to inflammatory autoimmune disease characterized by multiorgan infiltration of activated lymphocytes, high levels of autoantibodies in the serum, and immune complex glomerulonephritis. p52, but not p80HT, represses Bim expression, leading to defects in apoptotic processes critical for elimination of autoreactive lymphocytes and control of immune response. These findings reveal distinct signaling pathways for actions of NF-κB2 mutants and p52 and suggest a causal role for sustained NF-κB2 activation in the pathogenesis of autoimmunity. PMID:18281283

  6. Selection for low or high primary dormancy in Lolium rigidum Gaud seeds results in constitutive differences in stress protein expression and peroxidase activity

    PubMed Central

    Goggin, Danica E.; Powles, Stephen B.; Steadman, Kathryn J.

    2011-01-01

    Seed dormancy in wild Lolium rigidum Gaud (annual ryegrass) populations is highly variable and not well characterized at the biochemical level. To identify some of the determinants of dormancy level in these seeds, the proteomes of subpopulations selected for low and high levels of primary dormancy were compared by two-dimensional polyacrylamide gel electrophoresis of extracts from mature, dry seeds. High-dormancy seeds showed higher expression of small heat shock proteins, enolase, and glyoxalase I than the low-dormancy seeds. The functional relevance of these differences in protein expression was confirmed by the fact that high-dormancy seeds were more tolerant to high temperatures imposed at imbibition and had consistently higher glyoxalase I activity over 0–42 d dark stratification. Higher expression of a putative glutathione peroxidase in low-dormancy seeds was not accompanied by higher activity, but these seeds had a slightly more oxidized glutathione pool and higher total peroxidase activity. Overall, these biochemical and physiological differences suggest that L. rigidum seeds selected for low dormancy are more prepared for rapid germination via peroxidase-mediated cell wall weakening, whilst seeds selected for high dormancy are constitutively prepared to survive environmental stresses, even in the absence of stress during seed development. PMID:20974739

  7. Sp1 and Sp3 control constitutive expression of the human NHE2 promoter by interactions with the proximal promoter and the transcription initiation site

    PubMed Central

    Pearse, Ian; Zhu, Ying X.; Murray, Eleanor J.; Dudeja, Pradeep K.; Ramaswamy, Krishnamurthy; Malakooti, Jaleh

    2007-01-01

    We have previously cloned the human Na+/H+ exchanger NHE2 gene and its promoter region. In the present study, the regulatory elements responsible for the constitutive expression of NHE2 were studied. Transient transfection assays revealed that the −40/+150 promoter region contains the core promoter responsible for the optimal promoter activity. A smaller fragment, −10/+40, containing the TIS (transcription initiation site) showed minimal activity. We identified a palindrome that overlaps the TIS and binds to the transcription factors Sp1 and Sp3. Mutations in the 5′ flank of the palindrome abolished the Sp1/Sp3 interaction and reduced promoter activity by approx. 45%. In addition, a conserved GC-box centered at −25 was found to play a critical role in basal promoter activity and also interacted with Sp1 and Sp3. An internal deletion in the GC-box severely reduced the promoter activity. Sp1/Sp3 binding to these elements was established using gel-mobility shift assays, confirmed by chromatin immunoprecipitation and co-transfections in Drosophila SL2 cells. Furthermore, we identified two positive regulatory elements in the DNA region corresponding to the 5′-UTR (5′-untranslated region). The results in the present study indicate that Sp1 and Sp3 are required for constitutive NHE2 expression and that the positive regulatory elements of the 5′-UTR may co-operate with the 5′-flanking region to achieve the optimal promoter activity. PMID:17561809

  8. Constitutive salicylic acid accumulation in pi4kIIIβ1β2 Arabidopsis plants stunts rosette but not root growth.

    PubMed

    Sašek, Vladimír; Janda, Martin; Delage, Elise; Puyaubert, Juliette; Guivarc'h, Anne; López Maseda, Encarnación; Dobrev, Petre I; Caius, José; Bóka, Károly; Valentová, Olga; Burketová, Lenka; Zachowski, Alain; Ruelland, Eric

    2014-08-01

    Phospholipids have recently been found to be integral elements of hormone signalling pathways. An Arabidopsis thaliana double mutant in two type III phosphatidylinositol-4-kinases (PI4Ks), pi4kIIIβ1β2, displays a stunted rosette growth. The causal link between PI4K activity and growth is unknown. Using microarray analysis, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and multiple phytohormone analysis by LC-MS we investigated the mechanism responsible for the pi4kIIIβ1β2 phenotype. The pi4kIIIβ1β2 mutant accumulated a high concentration of salicylic acid (SA), constitutively expressed SA marker genes including PR-1, and was more resistant to Pseudomonas syringae. pi4kIIIβ1β2 was crossed with SA signalling mutants eds1 and npr1 and SA biosynthesis mutant sid2 and NahG. The dwarf phenotype of pi4kIIIβ1β2 rosettes was suppressed in all four triple mutants. Whereas eds1 pi4kIIIβ1β2, sid2 pi4kIIIβ1β2 and NahG pi4kIIIβ1β2 had similar amounts of SA as the wild-type (WT), npr1pi4kIIIβ1β2 had more SA than pi4kIIIβ1β2 despite being less dwarfed. This indicates that PI4KIIIβ1 and PI4KIIIβ2 are genetically upstream of EDS1 and need functional SA biosynthesis and perception through NPR1 to express the dwarf phenotype. The slow root growth phenotype of pi4kIIIβ1β2 was not suppressed in any of the triple mutants. The pi4kIIIβ1β2 mutations together cause constitutive activation of SA signalling that is responsible for the dwarf rosette phenotype but not for the short root phenotype.

  9. The Origins of the Constitution.

    ERIC Educational Resources Information Center

    Grindle, Diane

    1987-01-01

    Maintains that there were three forces operating in colonial American culture which led to the drafting of the Constitution. These were: English law and tradition, the lifestyle of the colonies, and the Enlightenment. Provides examples of how these forces were expressed in the events surrounding the birth of the United States Constitution. (JDH)

  10. The Constitution: Perspectives on Contemporary American Democracy.

    ERIC Educational Resources Information Center

    Close Up Foundation, Arlington, VA.

    Four articles expressing the views of nine prominent United States citizens about the Constitution provide a context for reflecting on the meaning of the Constitution in present-day America. In "Why Has the Constitution Endured So Long?" Don Edwards, chairman of the House Civil and Constitutional Rights Subcommittee, discusses why the Constitution…

  11. Omega-3 fatty acids attenuate constitutive and insulin-induced CD36 expression through a suppression of PPAR α/γ activity in microvascular endothelial cells.

    PubMed

    Madonna, Rosalinda; Salerni, Sara; Schiavone, Deborah; Glatz, Jan F; Geng, Yong-Jian; De Caterina, Raffaele

    2011-09-01

    Microvascular dysfunction occurs in insulin resistance and/or hyperinsulinaemia. Enhanced uptake of free fatty acids (FFA) and oxidised low-density lipoproteins (oxLDL) may lead to oxidative stress and microvascular dysfunction interacting with CD36, a PPARα/γ-regulated scavenger receptor and long-chain FFA transporter. We investigated CD36 expression and CD36-mediated oxLDL uptake before and after insulin treatment in human dermal microvascular endothelial cells (HMVECs), ± different types of fatty acids (FA), including palmitic, oleic, linoleic, arachidonic, eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids. Insulin (10(-8) and 10(-7) M) time-dependently increased DiI-oxLDL uptake and CD36 surface expression (by 30 ± 13%, p<0.05 vs. untreated control after 24 hours incubation), as assessed by ELISA and flow cytometry, an effect that was potentiated by the PI3-kinase inhibitor wortmannin and reverted by the ERK1/2 inhibitor PD98059 and the PPARα/γ antagonist GW9662. A ≥ 24 hour exposure to 50 μM DHA or EPA, but not other FA, blunted both the constitutive (by 23 ± 3% and 29 ± 2%, respectively, p<0.05 for both) and insulin-induced CD36 expressions (by 45 ± 27 % and 12 ± 3 %, respectively, p<0.05 for both), along with insulin-induced uptake of DiI-oxLDL and the downregulation of phosphorylated endothelial nitric oxide synthase (P-eNOS). At gel shift assays, DHA reverted insulin-induced basal and oxLDL-stimulated transactivation of PPRE and DNA binding of PPARα/γ and NF-κB. In conclusion, omega-3 fatty acids blunt the increased CD36 expression and activity promoted by high concentrations of insulin. Such mechanisms may be the basis for the use of omega-3 fatty acids in diabetic microvasculopathy. PMID:21727988

  12. Acetaminophen Modulates P-Glycoprotein Functional Expression at the Blood-Brain Barrier by a Constitutive Androstane Receptor–Dependent Mechanism

    PubMed Central

    Thompson, Brandon J.; Sanchez-Covarrubias, Lucy; Zhang, Yifeng; Laracuente, Mei-Li; Vanderah, Todd W.; Ronaldson, Patrick T.; Davis, Thomas P.

    2013-01-01

    Effective pharmacologic treatment of pain with opioids requires that these drugs attain efficacious concentrations in the central nervous system (CNS). A primary determinant of CNS drug permeation is P-glycoprotein (P-gp), an endogenous blood-brain barrier (BBB) efflux transporter that is involved in brain-to-blood transport of opioid analgesics (i.e., morphine). Recently, the nuclear receptor constitutive androstane receptor (CAR) has been identified as a regulator of P-gp functional expression at the BBB. This is critical to pharmacotherapy of pain/inflammation, as patients are often administered acetaminophen (APAP), a CAR-activating ligand, in conjunction with an opioid. Our objective was to investigate, in vivo, the role of CAR in regulation of P-gp at the BBB. Following APAP treatment, P-gp protein expression was increased up to 1.4–1.6-fold in a concentration-dependent manner. Additionally, APAP increased P-gp transport of BODIPY-verapamil in freshly isolated rat brain capillaries. This APAP-induced increase in P-gp expression and activity was attenuated in the presence of CAR pathway inhibitor okadaic acid or transcriptional inhibitor actinomycin D, suggesting P-gp regulation is CAR-dependent. Furthermore, morphine brain accumulation was enhanced by P-gp inhibitors in APAP-treated animals, suggesting P-gp–mediated transport. A warm-water (50°C) tail-flick assay revealed a significant decrease in morphine analgesia in animals treated with morphine 3 or 6 hours after APAP treatment, as compared with animals treated concurrently. Taken together, our data imply that inclusion of APAP in a pain treatment regimen activates CAR at the BBB and increases P-gp functional expression, a clinically significant drug-drug interaction that modulates opioid analgesic efficacy. PMID:24019224

  13. Crushed Salt Constitutive Model

    SciTech Connect

    Callahan, G.D.

    1999-02-01

    The constitutive model used to describe the deformation of crushed salt is presented in this report. Two mechanisms -- dislocation creep and grain boundary diffusional pressure solution -- are combined to form the basis for the constitutive model governing the deformation of crushed salt. The constitutive model is generalized to represent three-dimensional states of stress. Upon complete consolidation, the crushed-salt model reproduces the Multimechanism Deformation (M-D) model typically used for the Waste Isolation Pilot Plant (WIPP) host geological formation salt. New shear consolidation tests are combined with an existing database that includes hydrostatic consolidation and shear consolidation tests conducted on WIPP and southeastern New Mexico salt. Nonlinear least-squares model fitting to the database produced two sets of material parameter values for the model -- one for the shear consolidation tests and one for a combination of the shear and hydrostatic consolidation tests. Using the parameter values determined from the fitted database, the constitutive model is validated against constant strain-rate tests. Shaft seal problems are analyzed to demonstrate model-predicted consolidation of the shaft seal crushed-salt component. Based on the fitting statistics, the ability of the model to predict the test data, and the ability of the model to predict load paths and test data outside of the fitted database, the model appears to capture the creep consolidation behavior of crushed salt reasonably well.

  14. Spikelet-specific variation in ethylene production and constitutive expression of ethylene receptors and signal transducers during grain filling of compact- and lax-panicle rice (Oryza sativa) cultivars.

    PubMed

    Sekhar, Sudhanshu; Panda, Binay B; Mohapatra, Trupti; Das, Kaushik; Shaw, Birendra P; Kariali, Ekamber; Mohapatra, Pravat K

    2015-05-01

    Grain yields in modern super rice cultivars do not always meet the expectations because many spikelets are located on secondary branches in closely packed homogeneous distribution in these plants, and they do not fill properly. The factors limiting grain filling of such spikelets, especially in the lower panicle branches, are elusive. Two long-duration rice cultivars differing in panicle density, Mahalaxmi (compact) and Upahar (lax), were cultivated in an open field plot. Grain filling, ethylene production and constitutive expression of ethylene receptors and ethylene signal transducers in apical and basal spikelets of the panicle were compared during the early post-anthesis stage, which is the most critical period for grain development. In another experiment, a similar assessment was made for the medium-duration cultivars compact-panicle OR-1918 and lax-panicle Lalat. Grain weight of the apical spikelets was always higher than that of the basal spikelets. This gradient of grain weight was wide in the compact-panicle cultivars and narrow in the lax-panicle cultivars. Compared to apical spikelets, the basal spikelets produced more ethylene at anthesis and retained the capacity for post-anthesis expression of ethylene receptors and ethylene signal transducers longer. High ethylene production enhanced the expression of the RSR1 gene, but reduced expression of the GBSS1 gene. Ethylene inhibited the partitioning of assimilates of developing grains resulting in low starch biosynthesis and high accumulation of soluble carbohydrates. It is concluded that an increase in grain/spikelet density in rice panicles reduces apical dominance to the detriment of grain filling by production of ethylene and/or enhanced perception of the ethylene signal. Ethylene could be a second messenger for apical dominance in grain filling. The manipulation of the ethylene signal would possibly improve rice grain yield.

  15. Interleukin-22 binding protein (IL-22BP) is constitutively expressed by a subset of conventional dendritic cells and is strongly induced by retinoic acid

    PubMed Central

    Martin, JCJ; Bériou, G; Heslan, M; Chauvin, C; Utriainen, L; Aumeunier, A; Scott, CL; Mowat, A; Cerovic, V; Houston, SA; Leboeuf, M; Hubert, FX; Hémont, C; Merad, M; Milling, S; Josien, R

    2014-01-01

    IL-22 is mainly produced at barrier surfaces by T cells and innate lymphoid cells and is crucial to maintain epithelial integrity. However, dysregulated IL-22 action leads to deleterious inflammation and is involved in diseases such as psoriasis, intestinal inflammation and cancer. IL-22BP is a soluble inhibitory IL-22 receptor and may represent a crucial regulator of IL-22. We show both in rats and mice that, in the steady state, the main source of IL-22BP is constituted by a subset of conventional dendritic cells (DC) in lymphoid and non lymphoid tissues. In mouse intestine, IL-22BP was specifically expressed in lamina propria CD103+CD11b+ DC. In humans, IL-22BP was expressed in immature monocyte-derived DC (MDDC) and strongly induced by retinoic acid (RA) but dramatically reduced upon maturation. Our data suggest that a subset of immature DC may actively participate in the regulation of IL-22 activity in the gut by producing high levels of IL-22BP. PMID:23653115

  16. Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei

    PubMed Central

    Su, Shengchang; Bangar, Hansraj; Saldanha, Roland; Pemberton, Adin; Aronow, Bruce; Dean, Gary E; Lamkin, Thomas J; Hassett, Daniel J

    2014-01-01

    Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages. PMID:25044501

  17. Quantitative expression profiling of G-protein-coupled receptors (GPCRs) in metastatic melanoma: the constitutively active orphan GPCR GPR18 as novel drug target.

    PubMed

    Qin, Y; Verdegaal, E M E; Siderius, M; Bebelman, J P; Smit, M J; Leurs, R; Willemze, R; Tensen, C P; Osanto, S

    2011-02-01

    G-protein-coupled receptors (GPCRs) have been implicated in the tumorigenesis and metastasis of human cancers and are considered amongst the most desirable targets for drug development. Utilizing a robust quantitative PCR array, we quantified expression of 94 human GPCRs, including 75 orphan GPCRs and 19 chemokine receptors, and 36 chemokine ligands, in 40 melanoma metastases from different individuals and benign nevi. Inter-metastatic site comparison revealed that orphan GPR174 and CCL28 are statistically significantly overexpressed in subcutaneous metastases, while P2RY5 is overexpressed in brain metastases. Comparison between metastases (all three metastatic sites) and benign nevi revealed that 16 genes, including six orphan receptors (GPR18, GPR34, GPR119, GPR160, GPR183 and P2RY10) and chemokine receptors CCR5, CXCR4, and CXCR6, were statistically significantly differentially expressed. Subsequent functional experiments in yeast and melanoma cells indicate that GPR18, the most abundantly overexpressed orphan GPCR in all melanoma metastases, is constitutively active and inhibits apoptosis, indicating an important role for GPR18 in tumor cell survival. GPR18 and five other orphan GPCRs with yet unknown biological function may be considered potential novel anticancer targets in metastatic melanoma. PMID:20880198

  18. Bone-marrow derived hematopoietic stem/progenitor cells express multiple isoforms of NADPH oxidase and produce constitutively reactive oxygen species.

    PubMed

    Piccoli, Claudia; D'Aprile, Annamaria; Ripoli, Maria; Scrima, Rosella; Lecce, Lucia; Boffoli, Domenico; Tabilio, Antonio; Capitanio, Nazzareno

    2007-02-23

    Consolidated evidence highlights the importance of redox signalling in poising the balance between self-renewal and differentiation in adult stem cells. The present study shows that human hematopoietic stem/progenitor cells (HSCs) constitutively generate low levels of hydrogen peroxide whose production is inhibited by DPI, apocynin, catalase, and LY294002 and scarcely stimulated by PMA. Moreover, it is shown that HSCs express at the mRNA and protein levels the catalytic subunits of NOX1, NOX2, and NOX4 isoforms of the NADPH oxidase family along with the complete battery of the regulatory subunits p22, p40, p47, p67, rac1, rac2, NOXO1, and NOXA1 as well as the splicing variant NOX2s and that the three NOX isoforms are largely co-expressed in the same HSC. These findings are interpreted in terms of a positive feed-back mechanism of NOXs activation enabling a fine tuning of the ROS level to be possibly used in redox-mediated signalling for growth and differentiation of HSCs. PMID:17204244

  19. Bone-marrow derived hematopoietic stem/progenitor cells express multiple isoforms of NADPH oxidase and produce constitutively reactive oxygen species.

    PubMed

    Piccoli, Claudia; D'Aprile, Annamaria; Ripoli, Maria; Scrima, Rosella; Lecce, Lucia; Boffoli, Domenico; Tabilio, Antonio; Capitanio, Nazzareno

    2007-02-23

    Consolidated evidence highlights the importance of redox signalling in poising the balance between self-renewal and differentiation in adult stem cells. The present study shows that human hematopoietic stem/progenitor cells (HSCs) constitutively generate low levels of hydrogen peroxide whose production is inhibited by DPI, apocynin, catalase, and LY294002 and scarcely stimulated by PMA. Moreover, it is shown that HSCs express at the mRNA and protein levels the catalytic subunits of NOX1, NOX2, and NOX4 isoforms of the NADPH oxidase family along with the complete battery of the regulatory subunits p22, p40, p47, p67, rac1, rac2, NOXO1, and NOXA1 as well as the splicing variant NOX2s and that the three NOX isoforms are largely co-expressed in the same HSC. These findings are interpreted in terms of a positive feed-back mechanism of NOXs activation enabling a fine tuning of the ROS level to be possibly used in redox-mediated signalling for growth and differentiation of HSCs.

  20. Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei.

    PubMed

    Su, Shengchang; Bangar, Hansraj; Saldanha, Roland; Pemberton, Adin; Aronow, Bruce; Dean, Gary E; Lamkin, Thomas J; Hassett, Daniel J

    2014-10-01

    Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages.

  1. Vascular endothelial growth factor is constitutively expressed in normal human salivary glands and is secreted in the saliva of healthy individuals.

    PubMed

    Pammer, J; Weninger, W; Mildner, M; Burian, M; Wojta, J; Tschachler, E

    1998-10-01

    The expression of vascular endothelial growth factor (VEGF), a specific mitogen for endothelial cells, was examined in salivary glands and in normal saliva. In normal salivary glands, VEGF mRNA and protein were strongly present in acinar cells, whereas little or no VEGF was found in ductal cells. In chronically inflamed glands, VEGF protein was in addition present in ductal elements and in infiltrating mononuclear cells. No difference of VEGF expression was observed between benign and malignant salivary gland tumours. By ELISA, whole saliva of 24 healthy individuals contained up to 2.5 ng/ml (mean 1.4 ng/ml; SD 0.77 ng/ml) of VEGF, confirming the constitutive secretion of this cytokine by human salivary glands. Western blot analysis of normal saliva under non-reducing conditions detected anti-VEGF reactive protein moieties of approximately 46 kD, corresponding to VEGF secreted by cells in tissue culture. Additional anti-VEGF reactive proteins of approximately 60 and 90 kD were detected in the saliva of some individuals. The presence of considerable quantities of VEGF in normal human saliva suggests an important role for this cytokine in the maintenance of the homeostasis of mucous membranes, with rapid induction of neoangiogenesis by salivary VEGF helping to accelerate wound healing within the oral cavity. Moreover, salivary VEGF may permeabilize intraglandular capillaries and thus participate in the regulation of saliva production itself.

  2. Constitutive or Inducible Protective Mechanisms against UV-B Radiation in the Brown Alga Fucus vesiculosus? A Study of Gene Expression and Phlorotannin Content Responses

    PubMed Central

    Creis, Emeline; Delage, Ludovic; Charton, Sophie; Goulitquer, Sophie; Leblanc, Catherine; Potin, Philippe; Ar Gall, Erwan

    2015-01-01

    A role as UV sunscreens has been suggested for phlorotannins, the phenolic compounds that accumulate in brown algae in response to a number of external stimuli and take part in cell wall structure. After exposure of the intertidal brown alga Fucus vesiculosus to artificial UV-B radiation, we examined its physiological responses by following the transcript level of the pksIII gene encoding a phloroglucinol synthase, likely to be involved in the first step of phlorotannins biosynthesis. We also monitored the expression of three targeted genes, encoding a heat shock protein (hsp70), which is involved in global stress responses, an aryl sulfotransferase (ast), which could be involved in the sulfation of phlorotannins, and a vanadium bromoperoxidase (vbpo), which can potentially participate in the scavenging of Reactive Oxygen Species (ROS) and in the cross-linking and condensation of phlorotannins. We investigated whether transcriptional regulation of these genes is correlated with an induction of phlorotannin accumulation by establishing metabolite profiling of purified fractions of low molecular weight phlorotannins. Our findings demonstrated that a high dose of UV-B radiation induced a significant overexpression of hsp70 after 12 and 24 hours following the exposure to the UV-B treatment, compared to control treatment. The physiological performance of algae quantified by the photosynthetic efficiency (Fv/Fm) was slightly reduced. However UV-B treatment did not induce the accumulation of soluble phlorotannins in F. vesiculosus during the kinetics of four weeks, a result that may be related to the lack of induction of the pksIII gene expression. Taken together these results suggest a constitutive accumulation of phlorotannins occurring during the development of F.vesiculosus, rather than inducible processes. Gene expression studies and phlorotannin profiling provide here complementary approaches to global quantifications currently used in studies of phenolic compounds

  3. Constitutive expression of a COOH-terminal leucine mutant of lysosome-associated membrane protein-1 causes its exclusive localization in low density intracellular vesicles.

    PubMed

    Akasaki, Kenji; Shiotsu, Keiko; Michihara, Akihiro; Ide, Norie; Wada, Ikuo

    2014-07-01

    Lysosome-associated membrane protein-1 (LAMP-1) is a type I transmembrane protein with a short cytoplasmic tail that possesses a lysosome-targeting signal of GYQTI(382)-COOH. Wild-type (WT)-LAMP-1 was exclusively localized in high density lysosomes, and efficiency of LAMP-1's transport to lysosomes depends on its COOH-terminal amino acid residue. Among many different COOH-terminal amino acid substitution mutants of LAMP-1, a leucine-substituted mutant (I382L) displays the most efficient targeting to late endosomes and lysosomes [Akasaki et al. (2010) J. Biochem. 148: , 669-679]. In this study, we generated two human hepatoma cell lines (HepG2 cell lines) that stably express WT-LAMP-1 and I382L, and compared their intracellular distributions. The subcellular fractionation study using Percoll density gradient centrifugation revealed that WT-LAMP-1 had preferential localization in the high density secondary lysosomes where endogenous human LAMP-1 was enriched. In contrast, a major portion of I382L was located in a low density fraction. The low density fraction also contained approximately 80% of endogenous human LAMP-1 and significant amounts of endogenous β-glucuronidase and LAMP-2, which probably represents occurrence of low density lysosomes in the I382L-expressing cells. Double immunofluorescence microscopic analyses distinguished I382L-containing intracellular vesicles from endogenous LAMP-1-containing lysosomes and early endosomes. Altogether, constitutive expression of I382L causes its aberrant intracellular localization and generation of low density lysosomes, indicating that the COOH-terminal isoleucine is critical for normal localization of LAMP-1 in the dense lysosomes.

  4. A constitutively expressed pair of rpoE2-chrR2 in Azospirillum brasilense Sp7 is required for survival under antibiotic and oxidative stress.

    PubMed

    Gupta, Namrata; Kumar, Santosh; Mishra, Mukti Nath; Tripathi, Anil Kumar

    2013-02-01

    Extracytoplasmic function (ECF) sigma factors (σ(E)) are known to bring about changes in gene expression to enable bacteria to adapt to different stresses. The Azospirillum brasilense Sp245 genome harbours nine genes encoding σ(E), of which two are adjacent to the genes encoding ChrR-type zinc-binding anti-sigma (ZAS) factors. We describe here the role and regulation of a new pair of rpoE-chrR, which was found in the genome of A. brasilense Sp7 in addition to the previously described rpoE-chrR pair (designated rpoE1-chrR1). The rpoE2-chrR2 pair is also cotranscribed, and their products show protein-protein interaction. The -10 and -35 promoter elements of rpoE2-chrR2 and rpoE1-chrR1 were similar but not identical. Unlike the promoter of rpoE1-chrR1, the rpoE2-chrR2 promoter was neither autoregulated nor induced by oxidative stress. Inactivation of chrR2 or overexpression of rpoE2 in A. brasilense Sp7 resulted in an overproduction of carotenoids. It also conferred resistance to oxidative stresses and antibiotics. By controlling the synthesis of carotenoids, initiation and elongation of translation, protein folding and purine biosynthesis, RpoE2 seems to play a crucial role in preventing and repairing the cellular damage caused by oxidative stress. Lack of autoregulation and constitutive expression of rpoE2-chrR2 suggest that RpoE2-ChrR2 may provide a rapid mechanism to cope with oxidative stress, wherein singlet oxygen ((1)O(2))-mediated dissociation of the RpoE2-ChrR2 complex might release RpoE2 to drive the expression of its target genes.

  5. Abundant constitutive expression of the immediate-early 94K protein from cytomegalovirus (Colburn) in a DNA-transfected mouse cell line

    SciTech Connect

    Jeang, K.T.; Cho, M.S.; Hayward, G.S.

    1984-10-01

    A 94-kilodalton phosphoprotein known as IE94 is the only viral polypeptide synthesized in abundance under immediate-early conditions after infection by cytomegalovirus (CMV) strain Colburn in either permissive primate or nonpermissive rodent cells. The authors isolated a clonal Ltk/sup +/ cell line which expressed the /sup 35/methionine-labeled IE94 polypeptide in sufficient abundance to be visualized directly in autoradiographs after gel electrophoresis of total-cell-culture protein extracts. The IE94 polypeptide synthesized in the transfected cells was indistinguishable in size and overall net charge from that produced in virus-infected cells. In addition, the IE94 protein expressed in LH/sub 2/p198-3 cells was phosphorylated (presumably by a cellular protein kinase) and generated similar phosphopeptide patterns after partial tryptic digestion to those obtained with the CMV IE94 protein from infected cells. The cell line contained two to four stably integrated copies of the IE94 gene and synthesized a single virus-specific mRNA of 2.5 kilobases detectable on Northern blots. A new antigen, detectable by indirect anticomplement immunofluorescence with monoclonal antibody against the human CMV IE68 protein, was present in the nuclei of more than 95% of the LH/sub 2/l198-3 cells. This evidence suggests that (unlike most herpesvirus genes) the CMV IE94 gene, together with its complex promoter and spliced mRNA structure, may contain all of the regulatory elements necessary for strong constitutive expression in mammalian cells in the absence of other viral factors.

  6. Macrophage growth arrest by cyclic AMP defines a distinct checkpoint in the mid-G1 stage of the cell cycle and overrides constitutive c-myc expression.

    PubMed

    Rock, C O; Cleveland, J L; Jackowski, S

    1992-05-01

    Proliferation of a murine macrophage cell line (BAC1.2F5) in response to colony-stimulating factor 1 (CSF-1) is inhibited by prostaglandin E2 (PGE2)-mediated elevation of intracellular cyclic AMP (cAMP). When BAC1.2F5 cells were growth arrested in early G1 by CSF-1 starvation and stimulated to synchronously enter the cell cycle by readdition of growth factor, PGE2 inhibited [3H]thymidine incorporation when added before mid-G1, but its addition at later times did not block the onset of S phase. Reversible cell cycle arrest mediated by a cAMP analog required the presence of CSF-1 for cells to initiate DNA synthesis, whereas cells released from an aphidicolin block at the G1/S boundary entered S phase in the absence of CSF-1. PGE2 or cAMP analogs did not block the initial induction of c-myc mRNA by CSF-1 but abolished the CSF-1-dependent expression of c-myc mRNA in the mid-G1 stage of the cell cycle. The cAMP-mediated reduction in c-myc RNA levels was due to decreased c-myc transcription. However, CSF-1-dependent BAC1.2F5 clones infected with a c-myc retrovirus were growth arrested by cAMP analogs despite constitutive c-myc expression. Therefore, the reduction of endogenous c-myc expression by cAMP is neither necessary nor sufficient for growth inhibition.

  7. Constitutive or Inducible Protective Mechanisms against UV-B Radiation in the Brown Alga Fucus vesiculosus? A Study of Gene Expression and Phlorotannin Content Responses.

    PubMed

    Creis, Emeline; Delage, Ludovic; Charton, Sophie; Goulitquer, Sophie; Leblanc, Catherine; Potin, Philippe; Ar Gall, Erwan

    2015-01-01

    A role as UV sunscreens has been suggested for phlorotannins, the phenolic compounds that accumulate in brown algae in response to a number of external stimuli and take part in cell wall structure. After exposure of the intertidal brown alga Fucus vesiculosus to artificial UV-B radiation, we examined its physiological responses by following the transcript level of the pksIII gene encoding a phloroglucinol synthase, likely to be involved in the first step of phlorotannins biosynthesis. We also monitored the expression of three targeted genes, encoding a heat shock protein (hsp70), which is involved in global stress responses, an aryl sulfotransferase (ast), which could be involved in the sulfation of phlorotannins, and a vanadium bromoperoxidase (vbpo), which can potentially participate in the scavenging of Reactive Oxygen Species (ROS) and in the cross-linking and condensation of phlorotannins. We investigated whether transcriptional regulation of these genes is correlated with an induction of phlorotannin accumulation by establishing metabolite profiling of purified fractions of low molecular weight phlorotannins. Our findings demonstrated that a high dose of UV-B radiation induced a significant overexpression of hsp70 after 12 and 24 hours following the exposure to the UV-B treatment, compared to control treatment. The physiological performance of algae quantified by the photosynthetic efficiency (Fv/Fm) was slightly reduced. However UV-B treatment did not induce the accumulation of soluble phlorotannins in F. vesiculosus during the kinetics of four weeks, a result that may be related to the lack of induction of the pksIII gene expression. Taken together these results suggest a constitutive accumulation of phlorotannins occurring during the development of F.vesiculosus, rather than inducible processes. Gene expression studies and phlorotannin profiling provide here complementary approaches to global quantifications currently used in studies of phenolic compounds

  8. Nucleotide sequence and transcript organization of a region of the vaccinia virus genome which encodes a constitutively expressed gene required for DNA replication.

    PubMed

    Roseman, N A; Hruby, D E

    1987-05-01

    A vaccinia virus (VV) gene required for DNA replication has been mapped to the left side of the 16-kilobase (kb) VV HindIII D DNA fragment by marker rescue of a DNA- temperature-sensitive mutant, ts17, using cloned fragments of the viral genome. The region of VV DNA containing the ts17 locus (3.6 kb) was sequenced. This nucleotide sequence contains one complete open reading frame (ORF) and two incomplete ORFs reading from left to right. Analysis of this region at early times revealed that transcription from the incomplete upstream ORF terminates coincidentally with the complete ORF encoding the ts17 gene product, which is directly downstream. The predicted proteins encoded by this region correlate well with polypeptides mapped by in vitro translation of hybrid-selected early mRNA. The nucleotide sequences of a 1.3-kb BglII fragment derived from ts17 and from two ts17 revertants were also determined, and the nature of the ts17 mutation was identified. S1 nuclease protection studies were carried out to determine the 5' and 3' ends of the transcripts and to examine the kinetics of expression of the ts17 gene during viral infection. The ts17 transcript is present at both early and late times postinfection, indicating that this gene is constitutively expressed. Surprisingly, the transcriptional start throughout infection occurs at the proposed late regulatory element TAA, which immediately precedes the putative initiation codon ATG. Although the biological activity of the ts17-encoded polypeptide was not identified, it was noted that in ts17-infected cells, expression of a nonlinked VV immediate-early gene (thymidine kinase) was deregulated at the nonpermissive temperature. This result may indicate that the ts17 gene product is functionally required at an early step of the VV replicative cycle.

  9. Constitutive expression of McCHIT1-PAT enhances resistance to rice blast and herbicide, but does not affect grain yield in transgenic glutinous rice.

    PubMed

    Zeng, Xiao-Fang; Li, Lei; Li, Jian-Rong; Zhao, De-Gang

    2016-01-01

    To produce new rice blast- and herbicide-resistant transgenic rice lines, the McCHIT1 gene encoding the class I chitinase from Momordica charantia and the herbicide resistance gene PAT were introduced into Lailong (Oryza sativa L. ssp. Japonica), a glutinous local rice variety from Guizhou Province, People's Republic of China. Transgenic lines were identified by ß-glucuronidase (GUS) histochemical staining, PCR, and Southern blot analyses. Agronomic traits, resistance to rice blast and herbicide, chitinase activities, and transcript levels of McCHIT1 were assessed in the T2 progeny of three transgenic lines (L1, L8, and L10). The results showed that the introduction of McCHIT1-PAT into Lailong significantly enhanced herbicide and blast resistance. After infection with the blast fungus Magnaporthe oryzae, all of the T2 progeny exhibited less severe lesion symptoms than those of wild type. The disease indices were 100% for wild type, 65.66% for T2 transgenic line L1, 59.69% for T2 transgenic line L8, and 79.80% for T2 transgenic line L10. Transgenic lines expressing McCHIT1-PAT did not show a significant difference from wild type in terms of malondialdehyde (MDA) content, polyphenol oxidase (PPO) activity, and superoxide dismutase (SOD) activity in the leaves. However, after inoculation with M. oryzae, transgenic plants showed significantly higher SOD and PPO activities and lower MDA contents in leaves, compared with those in wild-type leaves. The transgenic and the wild-type plants did not show significant differences in grain yield parameters including plant height, panicles per plant, seeds per panicle, and 1000-grain weight. Therefore, the transgenic plants showed increased herbicide and blast resistance, with no yield penalty.

  10. The Ectopic Expression of CaRop1 Modulates the Response of Tobacco Plants to Ralstonia solanacearum and Aphids.

    PubMed

    Qiu, Ailian; Liu, Zhiqin; Li, Jiazhi; Chen, Yanshen; Guan, Deyi; He, Shuilin

    2016-01-01

    In plants, Rho-related GTPases (Rops) are versatile molecular switches that regulate various biological processes, although their exact roles are not fully understood. Herein, we provide evidence that the ectopic expression of a Rop derived from Capsicum annuum, designated CaRop1, in tobacco plants modulates the response of these plants to Ralstonia solanacearum or aphid attack. The deduced amino acid sequence of CaRop1 harbors a conserved Rho domain and is highly homologous to Rops of other plant species. Transient expression of a CaRop1-GFP fusion protein in Nicotiana benthamiana leaf epidermal cells revealed localization of the GFP signal to the plasma membrane, cytoplasm, and nucleus. Overexpression (OE) of the wild-type CaRop1 or its dominant-negative mutant (DN-CaRop1) conferred substantial resistance to R. solanacearum infection and aphid attack, and this effect was accompanied by enhanced transcriptional expression of the hypersensitive-reaction marker gene HSR201; the jasmonic acid (JA)-responsive PR1b and LOX1; the insect resistance-associated NtPI-I, NtPI-II, and NtTPI; the ethylene (ET) production-associated NtACS1; and NPK1, a mitogen-activated protein kinase kinase kinase (MAPKKK) that interferes with N-, Bs2-, and Rx-mediated disease resistance. In contrast, OE of the constitutively active mutant of CaRop1(CA-CaRop1) enhanced susceptibility of the transgenic tobacco plants to R. solanacearum infection and aphid attack and downregulated or sustained the expression of HSR201, PR1b, NPK1, NtACS1, NtPI-I, NtPI-II, and NtTPI. These results collectively suggest that CaRop1 acts as a signaling switch in the crosstalk between Solanaceaes's response to R. solanacearum infection and aphid attack possibly via JA/ET-mediated signaling machinery. PMID:27551287

  11. The Ectopic Expression of CaRop1 Modulates the Response of Tobacco Plants to Ralstonia solanacearum and Aphids.

    PubMed

    Qiu, Ailian; Liu, Zhiqin; Li, Jiazhi; Chen, Yanshen; Guan, Deyi; He, Shuilin

    2016-01-01

    In plants, Rho-related GTPases (Rops) are versatile molecular switches that regulate various biological processes, although their exact roles are not fully understood. Herein, we provide evidence that the ectopic expression of a Rop derived from Capsicum annuum, designated CaRop1, in tobacco plants modulates the response of these plants to Ralstonia solanacearum or aphid attack. The deduced amino acid sequence of CaRop1 harbors a conserved Rho domain and is highly homologous to Rops of other plant species. Transient expression of a CaRop1-GFP fusion protein in Nicotiana benthamiana leaf epidermal cells revealed localization of the GFP signal to the plasma membrane, cytoplasm, and nucleus. Overexpression (OE) of the wild-type CaRop1 or its dominant-negative mutant (DN-CaRop1) conferred substantial resistance to R. solanacearum infection and aphid attack, and this effect was accompanied by enhanced transcriptional expression of the hypersensitive-reaction marker gene HSR201; the jasmonic acid (JA)-responsive PR1b and LOX1; the insect resistance-associated NtPI-I, NtPI-II, and NtTPI; the ethylene (ET) production-associated NtACS1; and NPK1, a mitogen-activated protein kinase kinase kinase (MAPKKK) that interferes with N-, Bs2-, and Rx-mediated disease resistance. In contrast, OE of the constitutively active mutant of CaRop1(CA-CaRop1) enhanced susceptibility of the transgenic tobacco plants to R. solanacearum infection and aphid attack and downregulated or sustained the expression of HSR201, PR1b, NPK1, NtACS1, NtPI-I, NtPI-II, and NtTPI. These results collectively suggest that CaRop1 acts as a signaling switch in the crosstalk between Solanaceaes's response to R. solanacearum infection and aphid attack possibly via JA/ET-mediated signaling machinery.

  12. The Ectopic Expression of CaRop1 Modulates the Response of Tobacco Plants to Ralstonia solanacearum and Aphids

    PubMed Central

    Qiu, Ailian; Liu, Zhiqin; Li, Jiazhi; Chen, Yanshen; Guan, Deyi; He, Shuilin

    2016-01-01

    In plants, Rho-related GTPases (Rops) are versatile molecular switches that regulate various biological processes, although their exact roles are not fully understood. Herein, we provide evidence that the ectopic expression of a Rop derived from Capsicum annuum, designated CaRop1, in tobacco plants modulates the response of these plants to Ralstonia solanacearum or aphid attack. The deduced amino acid sequence of CaRop1 harbors a conserved Rho domain and is highly homologous to Rops of other plant species. Transient expression of a CaRop1-GFP fusion protein in Nicotiana benthamiana leaf epidermal cells revealed localization of the GFP signal to the plasma membrane, cytoplasm, and nucleus. Overexpression (OE) of the wild-type CaRop1 or its dominant-negative mutant (DN-CaRop1) conferred substantial resistance to R. solanacearum infection and aphid attack, and this effect was accompanied by enhanced transcriptional expression of the hypersensitive-reaction marker gene HSR201; the jasmonic acid (JA)-responsive PR1b and LOX1; the insect resistance-associated NtPI-I, NtPI-II, and NtTPI; the ethylene (ET) production-associated NtACS1; and NPK1, a mitogen-activated protein kinase kinase kinase (MAPKKK) that interferes with N-, Bs2-, and Rx-mediated disease resistance. In contrast, OE of the constitutively active mutant of CaRop1(CA-CaRop1) enhanced susceptibility of the transgenic tobacco plants to R. solanacearum infection and aphid attack and downregulated or sustained the expression of HSR201, PR1b, NPK1, NtACS1, NtPI-I, NtPI-II, and NtTPI. These results collectively suggest that CaRop1 acts as a signaling switch in the crosstalk between Solanaceaes’s response to R. solanacearum infection and aphid attack possibly via JA/ET-mediated signaling machinery. PMID:27551287

  13. The 5' flanking region of a barley B hordein gene controls tissue and developmental specific CAT expression in tobacco plants.

    PubMed

    Marris, C; Gallois, P; Copley, J; Kreis, M

    1988-07-01

    The 549 base pairs of the 5' flanking region of a barley seed storage protein (B1 hordein) gene were linked to the reporter gene encoding chloramphenicol acetyl transferase (CAT). The chimaeric gene was transferred into tobacco plants using Agrobacterium tumefaciens. CAT enzyme activity was detected in the seeds, but not in the leaves, of the transgenic plants. Furthermore, enzyme activity was found only in the endosperm, and only from fifteen days after pollination. In contrast, the constitutive 19S promoter from cauliflower mosaic virus (CaMV) directed the expression of the CAT gene in the leaves as well as in both the endosperm and embryo and at all stages in seed development.

  14. Selective expression of constitutively active pro-apoptotic protein BikDD gene in primary mammary tumors inhibits tumor growth and reduces tumor initiating cells.

    PubMed

    Rahal, Omar M; Nie, Lei; Chan, Li-Chuan; Li, Chia-Wei; Hsu, Yi-Hsin; Hsu, Jennifer; Yu, Dihua; Hung, Mien-Chie

    2015-01-01

    Our previous study showed that specifically delivering BikDD, a constitutive active mutant of pro-apoptotic protein Bik, to breast cancer cell xenografts in immunocompromised mice has a potent activity against tumor initiating cells (TICs), and that the combination between tyrosine kinase inhibitors (TKI) and BikDD gene therapy yielded synergistic effect on EGFR and HER2 positive breast cancer cells in immunodeficient nude mice. Those encouraging results have allowed us to propose a clinical trial using the liposome-complexing plasmid DNA expressing BikDD gene which has been approved by the NIH RAC Advisory committee. However, it is imperative to test whether systemic delivery of BikDD-expressing plasmid DNAs with liposomes into immunocompetent mice has therapeutic efficacy and tolerable side effects as what we observed in the nude mice model. In this study, we investigated the effects of BikDD gene-therapy on the primary mammary tumors, especially on tumor initiating cells (TICs), of a genetically engineered immunocompetent mouse harboring normal microenvironment and immune response. The effects on TIC population in tumors were determined by FACS analysis with different sets of murine specific TIC markers, CD49f(high)CD61(high) and CD24(+)Jagged1(-). First we showed in vitro that ectopic expression of BikDD in murine N202 cells derived from MMTV-HER2/Neu transgenic mouse tumors induced apoptosis and decreased the number of TICs. Consistently, systemic delivery of VISA-Claudin4-BikDD by liposome complexes significantly inhibited mammary tumor growth and slowed down residual tumor growth post cessation of therapy in MMTV-HER2/Neu transgenic mice compared to the controls. In addition, the anti-tumor effects of BikDD in vivo were consistent with decreased TIC population assessed by FACS analysis and in vitro tumorsphere formation assay of freshly isolated tumor cells. Importantly, systemic administration of BikDD did not cause significant cytotoxic response in

  15. Rice plants expressing the moss sodium pumping ATPase PpENA1 maintain greater biomass production under salt stress.

    PubMed

    Jacobs, Andrew; Ford, Kristina; Kretschmer, Jodie; Tester, Mark

    2011-10-01

    High cytosolic concentrations of Na+ inhibit plant growth and development. To maintain low cytosolic concentrations of Na+ , higher plants use membrane-bound transporters that drive the efflux of Na+ or partition Na+ ions from the cytosol, either to the extracellular compartment or into the vacuole. Bryophytes also use an energy-dependent Na+ pumping ATPase, not found in higher plants, to efflux Na+ . To investigate whether this transporter can increase the salt tolerance of crop plants, Oryza sativa has been transformed with the Physcomitrella patens Na+ pumping ATPase (PpENA1). When grown in solutions containing 50 mm NaCl, plants constitutively expressing the PpENA1 gene are more salt tolerant and produce greater biomass than controls. Transgenics and controls accumulate similar amounts of Na+ in leaf and root tissues under stress, which indicates that the observed tolerance is not because of Na+ exclusion. Moreover, inductively coupled plasma analysis reveals that the concentration of other ions in the transformants and the controls is similar. The transgenic lines are developmentally normal and fertile, and the transgene expression levels remain stable in subsequent generations. GFP reporter fusions, which do not alter the ability of PpENA1 to complement a salt-sensitive yeast mutant, indicate that when it is expressed in plant tissues, the PpENA1 protein is located in the plasma membrane. PpENA1 peptides are found in plasma membrane fractions supporting the plasma membrane targeting. The results of this study demonstrate the utility of PpENA1 as a potential tool for engineering salinity tolerance in important crop species.

  16. A set of fluorescent protein-based markers expressed from constitutive and arbuscular mycorrhiza-inducible promoters to label organelles, membranes and cytoskeletal elements in Medicago truncatula.

    PubMed

    Ivanov, Sergey; Harrison, Maria J

    2014-12-01

    Medicago truncatula is widely used for analyses of arbuscular mycorrhizal (AM) symbiosis and nodulation. To complement the genetic and genomic resources that exist for this species, we generated fluorescent protein fusions that label the nucleus, endoplasmic reticulum, Golgi apparatus, trans-Golgi network, plasma membrane, apoplast, late endosome/multivesicular bodies (MVB), transitory late endosome/ tonoplast, tonoplast, plastids, mitochondria, peroxisomes, autophagosomes, plasmodesmata, actin, microtubules, periarbuscular membrane (PAM) and periarbuscular apoplastic space (PAS) and expressed them from the constitutive AtUBQ10 promoter and the AM symbiosis-specific MtBCP1 promoter. All marker constructs showed the expected expression patterns and sub-cellular locations in M. truncatula root cells. As a demonstration of their utility, we used several markers to investigate AM symbiosis where root cells undergo major cellular alterations to accommodate their fungal endosymbiont. We demonstrate that changes in the position and size of the nuclei occur prior to hyphal entry into the cortical cells and do not require DELLA signaling. Changes in the cytoskeleton, tonoplast and plastids also occur in the colonized cells and in contrast to previous studies, we show that stromulated plastids are abundant in cells with developing and mature arbuscules, while lens-shaped plastids occur in cells with degenerating arbuscules. Arbuscule development and secretion of the PAM creates a periarbuscular apoplastic compartment which has been assumed to be continuous with apoplast of the cell. However, fluorescent markers secreted to the periarbuscular apoplast challenge this assumption. This marker resource will facilitate cell biology studies of AM symbiosis, as well as other aspects of legume biology.

  17. Constitutive expression of the DUR1,2 gene in an industrial yeast strain to minimize ethyl carbamate production during Chinese rice wine fermentation.

    PubMed

    Wu, Dianhui; Li, Xiaomin; Lu, Jian; Chen, Jian; Zhang, Liang; Xie, Guangfa

    2016-01-01

    Urea and ethanol are the main precursors of ethyl carbamate (EC) in Chinese rice wine. During fermentation, urea is generated from arginine by arginase in Saccharomyces cerevisiae, and subsequently cleaved by urea amidolyase or directly transported out of the cell into the fermentation liquor, where it reacts with ethanol to form EC. To reduce the amount of EC in Chinese rice wine, we metabolically engineered two yeast strains, N85(DUR1,2) and N85(DUR1,2)-c, from the wild-type Chinese rice wine yeast strain N85. Both new strains were capable of constitutively expressing DUR1,2 (encodes urea amidolyase) and thus enhancing urea degradation. The use of N85(DUR1,2) and N85(DUR1,2)-c reduced the concentration of EC in Chinese rice wine fermented on a small-scale by 49.1% and 55.3%, respectively, relative to fermentation with the parental strain. All of the engineered strains showed good genetic stability and minimized the production of urea during fermentation, with no exogenous genes introduced during genetic manipulation, and were therefore suitable for commercialization to increase the safety of Chinese rice wine.

  18. Characterization of plants expressing the human β1,4-galactosyltrasferase gene.

    PubMed

    Schneider, Jeannine; Castilho, Alexandra; Pabst, Martin; Altmann, Friedrich; Gruber, Clemens; Strasser, Richard; Gattinger, Pia; Seifert, Georg J; Steinkellner, Herta

    2015-07-01

    Modification of the plant N-glycosylation pathway towards human type structures is an important strategy to implement plants as expression systems for therapeutic proteins. Nevertheless, relatively little is known about the overall impact of non-plant glycosylation enzymes in stable transformed plants. Here, we analyzed transgenic lines (Nicotiana benthamiana and Arabidopsis thaliana) that stably express a modified version of human β1,4-galactosyltransferase ((ST)GalT). While some transgenic plants grew normally, other lines exhibited a severe phenotype associated with stunted growth and developmental retardation. The severity of the phenotype correlated with both increased (ST)GalT mRNA and protein levels but no differences were observed between N-glycosylation profiles of plants with and without the phenotype. In contrast to non-transgenic plants, all (ST)GalT expressing plants synthesized significant amounts of incompletely processed (largely depleted of core fucose) N-glycans with up to 40% terminally galactosylated structures. While transgenic plants showed no differences in nucleotide sugar composition and cell wall monosaccharide content, alterations in the reactivity of cell wall carbohydrate epitopes associated with arabinogalactan-proteins and pectic homogalacturonan were detected in (ST)GalT expressing plants. Notably, plants with phenotypic alterations showed increased levels of hydrogen peroxide, most probably a consequence of hypersensitive reactions. Our data demonstrate that unfavorable phenotypical modifications may occur upon stable in planta expression of non-native glycosyltransferases. Such important issues need to be taken into consideration in respect to stable glycan engineering in plants.

  19. Acinetobacter strains IH9 and OCI1, two rhizospheric phosphate solubilizing isolates able to promote plant growth, constitute a new genomovar of Acinetobacter calcoaceticus.

    PubMed

    Peix, Alvaro; Lang, Elke; Verbarg, Susanne; Spröer, Cathrin; Rivas, Raúl; Santa-Regina, Ignacio; Mateos, Pedro F; Martínez-Molina, Eustoquio; Rodríguez-Barrueco, Claudino; Velázquez, Encarna

    2009-08-01

    During a screening of phosphate solubilizing bacteria (PSB) in agricultural soils, two strains, IH9 and OCI1, were isolated from the rhizosphere of grasses in Spain, and they showed a high ability to solubilize phosphate in vitro. Inoculation experiments in chickpea and barley were conducted with both strains and the results demonstrated their ability to promote plant growth. The 16S rRNA gene sequences of these strains were nearly identical to each other and to those of Acinetobacter calcoaceticus DSM 30006(T), as well as the strain CIP 70.29 representing genomospecies 3. Their phenotypic characteristics also coincided with those of strains forming the A. calcoaceticus-baumannii complex. They differed from A. calcoaceticus in the utilization of l-tartrate as a carbon source and from genomospecies 3 in the use of d-asparagine as a carbon source. The 16S-23S intergenic spacer (ITS) sequences of the two isolates showed nearly 98% identities to those of A. calcoaceticus, confirming that they belong to this phylogenetic group. However, the isolates appeared as a separate branch from the A. calcoaceticus sequences, indicating their molecular separation from other A. calcoaceticus strains. The analysis of three housekeeping genes, recA, rpoD and gyrB, confirmed that IH9 and OCI1 form a distinct lineage within A. calcoaceticus. These results were congruent with those from DNA-DNA hybridization, indicating that strains IH9 and OCI1 constitute a new genomovar for which we propose the name A. calcoaceticus genomovar rhizosphaerae.

  20. Stress-Inducible Expression of an F-box Gene TaFBA1 from Wheat Enhanced the Drought Tolerance in Transgenic Tobacco Plants without Impacting Growth and Development.

    PubMed

    Kong, Xiangzhu; Zhou, Shumei; Yin, Suhong; Zhao, Zhongxian; Han, Yangyang; Wang, Wei

    2016-01-01

    E3 ligase plays an important role in the response to many environment stresses in plants. In our previous study, constitutive overexpression of an F-box protein gene TaFBA1 driven by 35S promoter improved the drought tolerance in transgenic tobacco plants, but the growth and development in transgenic plants was altered in normal conditions. In this study, we used stress-inducible promoter RD29A instead of 35S promoter, as a results, the stress-inducible transgenic tobacco plants exhibit a similar phenotype with wild type (WT) plants. However, the drought tolerance of the transgenic plants with stress-inducible expressed TaFBA1 was enhanced. The improved drought tolerance of transgenic plants was indicated by their higher seed germination rate and survival rate, greater biomass and photosynthesis than those of WT under water stress, which may be related to their greater water retention capability and osmotic adjustment. Moreover, the transgenic plants accumulated less reactive oxygen species, kept lower MDA content and membrane leakage under water stress, which may be related to their higher levels of antioxidant enzyme activity and upregulated gene expression of some antioxidant enzymes. These results suggest that stress induced expression of TaFBA1 confers drought tolerance via the improved water retention and antioxidative compete ability. Meanwhile, this stress-inducible expression strategy by RD29A promoter can minimize the unexpectable effects by 35S constitutive promoter on phenotypes of the transgenic plants. PMID:27656187

  1. Stress-Inducible Expression of an F-box Gene TaFBA1 from Wheat Enhanced the Drought Tolerance in Transgenic Tobacco Plants without Impacting Growth and Development

    PubMed Central

    Kong, Xiangzhu; Zhou, Shumei; Yin, Suhong; Zhao, Zhongxian; Han, Yangyang; Wang, Wei

    2016-01-01

    E3 ligase plays an important role in the response to many environment stresses in plants. In our previous study, constitutive overexpression of an F-box protein gene TaFBA1 driven by 35S promoter improved the drought tolerance in transgenic tobacco plants, but the growth and development in transgenic plants was altered in normal conditions. In this study, we used stress-inducible promoter RD29A instead of 35S promoter, as a results, the stress-inducible transgenic tobacco plants exhibit a similar phenotype with wild type (WT) plants. However, the drought tolerance of the transgenic plants with stress-inducible expressed TaFBA1 was enhanced. The improved drought tolerance of transgenic plants was indicated by their higher seed germination rate and survival rate, greater biomass and photosynthesis than those of WT under water stress, which may be related to their greater water retention capability and osmotic adjustment. Moreover, the transgenic plants accumulated less reactive oxygen species, kept lower MDA content and membrane leakage under water stress, which may be related to their higher levels of antioxidant enzyme activity and upregulated gene expression of some antioxidant enzymes. These results suggest that stress induced expression of TaFBA1 confers drought tolerance via the improved water retention and antioxidative compete ability. Meanwhile, this stress-inducible expression strategy by RD29A promoter can minimize the unexpectable effects by 35S constitutive promoter on phenotypes of the transgenic plants.

  2. Stress-Inducible Expression of an F-box Gene TaFBA1 from Wheat Enhanced the Drought Tolerance in Transgenic Tobacco Plants without Impacting Growth and Development

    PubMed Central

    Kong, Xiangzhu; Zhou, Shumei; Yin, Suhong; Zhao, Zhongxian; Han, Yangyang; Wang, Wei

    2016-01-01

    E3 ligase plays an important role in the response to many environment stresses in plants. In our previous study, constitutive overexpression of an F-box protein gene TaFBA1 driven by 35S promoter improved the drought tolerance in transgenic tobacco plants, but the growth and development in transgenic plants was altered in normal conditions. In this study, we used stress-inducible promoter RD29A instead of 35S promoter, as a results, the stress-inducible transgenic tobacco plants exhibit a similar phenotype with wild type (WT) plants. However, the drought tolerance of the transgenic plants with stress-inducible expressed TaFBA1 was enhanced. The improved drought tolerance of transgenic plants was indicated by their higher seed germination rate and survival rate, greater biomass and photosynthesis than those of WT under water stress, which may be related to their greater water retention capability and osmotic adjustment. Moreover, the transgenic plants accumulated less reactive oxygen species, kept lower MDA content and membrane leakage under water stress, which may be related to their higher levels of antioxidant enzyme activity and upregulated gene expression of some antioxidant enzymes. These results suggest that stress induced expression of TaFBA1 confers drought tolerance via the improved water retention and antioxidative compete ability. Meanwhile, this stress-inducible expression strategy by RD29A promoter can minimize the unexpectable effects by 35S constitutive promoter on phenotypes of the transgenic plants. PMID:27656187

  3. Stress-Inducible Expression of an F-box Gene TaFBA1 from Wheat Enhanced the Drought Tolerance in Transgenic Tobacco Plants without Impacting Growth and Development.

    PubMed

    Kong, Xiangzhu; Zhou, Shumei; Yin, Suhong; Zhao, Zhongxian; Han, Yangyang; Wang, Wei

    2016-01-01

    E3 ligase plays an important role in the response to many environment stresses in plants. In our previous study, constitutive overexpression of an F-box protein gene TaFBA1 driven by 35S promoter improved the drought tolerance in transgenic tobacco plants, but the growth and development in transgenic plants was altered in normal conditions. In this study, we used stress-inducible promoter RD29A instead of 35S promoter, as a results, the stress-inducible transgenic tobacco plants exhibit a similar phenotype with wild type (WT) plants. However, the drought tolerance of the transgenic plants with stress-inducible expressed TaFBA1 was enhanced. The improved drought tolerance of transgenic plants was indicated by their higher seed germination rate and survival rate, greater biomass and photosynthesis than those of WT under water stress, which may be related to their greater water retention capability and osmotic adjustment. Moreover, the transgenic plants accumulated less reactive oxygen species, kept lower MDA content and membrane leakage under water stress, which may be related to their higher levels of antioxidant enzyme activity and upregulated gene expression of some antioxidant enzymes. These results suggest that stress induced expression of TaFBA1 confers drought tolerance via the improved water retention and antioxidative compete ability. Meanwhile, this stress-inducible expression strategy by RD29A promoter can minimize the unexpectable effects by 35S constitutive promoter on phenotypes of the transgenic plants.

  4. Constitutive high-level expression of a codon-optimized β-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris.

    PubMed

    Menéndez, Carmen; Martínez, Duniesky; Trujillo, Luis E; Mazola, Yuliet; González, Ernesto; Pérez, Enrique R; Hernández, Lázaro

    2013-02-01

    Enzymes for use in the sugar industry are preferred to be thermotolerant. In this study, a synthetic codon-optimized gene encoding a highly thermostable β-fructosidase (BfrA, EC 3.2.1.26) from the bacterium Thermotoga maritima was expressed in the yeast Pichia pastoris. The gradual increase of the transgene dosage from one to four copies under the control of the constitutive glyceraldehyde 3-phosphate dehydrogenase promoter had an additive effect on BfrA yield without causing cell toxicity. Maximal values of cell biomass (115 g/l, dry weight) and overall invertase activity (241 U/ml) were reached at 72 h in fed-batch fermentations using cane sugar as the main carbon source for growth. Secretion driven by the Saccharomyces cerevisiae α-factor signal peptide resulted in periplasmic retention (44 %) and extracellular release (56 %) of BfrA. The presence of N-linked oligosaccharides did not influence the optimal activity, thermal stability, kinetic properties, substrate specificity, and exo-type action mode of the yeast-secreted BfrA in comparison to the native unglycosylated enzyme. Complete inversion of cane sugar at initial concentration of 60 % (w/v) was achieved by periplasmic BfrA in undisrupted cells reacting at pH 5.5 and 70 °C, with average productivity of 4.4 g of substrate hydrolyzed per grams of biomass (wet weight) per hour. The high yield of fully active glycosylated BfrA here attained by recombinant P. pastoris in a low-cost fermentation process appears to be attractive for the large-scale production of this thermostable enzyme useful for the manufacture of inverted sugar syrup.

  5. Constitutive high-level expression of a codon-optimized β-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris.

    PubMed

    Menéndez, Carmen; Martínez, Duniesky; Trujillo, Luis E; Mazola, Yuliet; González, Ernesto; Pérez, Enrique R; Hernández, Lázaro

    2013-02-01

    Enzymes for use in the sugar industry are preferred to be thermotolerant. In this study, a synthetic codon-optimized gene encoding a highly thermostable β-fructosidase (BfrA, EC 3.2.1.26) from the bacterium Thermotoga maritima was expressed in the yeast Pichia pastoris. The gradual increase of the transgene dosage from one to four copies under the control of the constitutive glyceraldehyde 3-phosphate dehydrogenase promoter had an additive effect on BfrA yield without causing cell toxicity. Maximal values of cell biomass (115 g/l, dry weight) and overall invertase activity (241 U/ml) were reached at 72 h in fed-batch fermentations using cane sugar as the main carbon source for growth. Secretion driven by the Saccharomyces cerevisiae α-factor signal peptide resulted in periplasmic retention (44 %) and extracellular release (56 %) of BfrA. The presence of N-linked oligosaccharides did not influence the optimal activity, thermal stability, kinetic properties, substrate specificity, and exo-type action mode of the yeast-secreted BfrA in comparison to the native unglycosylated enzyme. Complete inversion of cane sugar at initial concentration of 60 % (w/v) was achieved by periplasmic BfrA in undisrupted cells reacting at pH 5.5 and 70 °C, with average productivity of 4.4 g of substrate hydrolyzed per grams of biomass (wet weight) per hour. The high yield of fully active glycosylated BfrA here attained by recombinant P. pastoris in a low-cost fermentation process appears to be attractive for the large-scale production of this thermostable enzyme useful for the manufacture of inverted sugar syrup. PMID:22821437

  6. An OMV Vaccine Derived from a Capsular Group B Meningococcus with Constitutive FetA Expression: Preclinical Evaluation of Immunogenicity and Toxicity

    PubMed Central

    Norheim, Gunnstein; Sanders, Holly; Mellesdal, Jardar W.; Sundfør, Idunn; Chan, Hannah; Brehony, Carina; Vipond, Caroline; Dold, Chris; Care, Rory; Saleem, Muhammad; Maiden, Martin C. J.; Derrick, Jeremy P.; Feavers, Ian; Pollard, Andrew J.

    2015-01-01

    Following the introduction of effective protein-polysaccharide conjugate vaccines against capsular group C meningococcal disease in Europe, meningococci of capsular group B remain a major cause of death and can result in debilitating sequelae. The outer membrane proteins PorA and FetA have previously been shown to induce bactericidal antibodies in humans. Despite considerable antigenic variation among PorA and FetA OMPs in meningococci, systematic molecular epidemiological studies revealed this variation is highly structured so that a limited repertoire of antigenic types is congruent with the hyperinvasive meningococcal lineages that have caused most of the meningococcal disease in Europe in recent decades. Here we describe the development of a prototype vaccine against capsular group B meningococcal infection based on a N. meningitidis isolate genetically engineered to have constitutive expression of the outer membrane protein FetA. Deoxycholate outer membrane vesicles (dOMVs) extracted from cells cultivated in modified Frantz medium contained 21.8% PorA protein, 7.7% FetA protein and 0.03 μg LPS per μg protein (3%). The antibody response to the vaccine was tested in three mouse strains and the toxicological profile of the vaccine was tested in New Zealand white rabbits. Administration of the vaccine, MenPF-1, when given by intramuscular injection on 4 occasions over a 9 week period, was well tolerated in rabbits up to 50 μg/dose, with no evidence of systemic toxicity. These data indicated that the MenPF-1 vaccine had a toxicological profile suitable for testing in a phase I clinical trial. PMID:26390123

  7. Constitutive Activation of the G-Protein Subunit G[alpha]s within Forebrain Neurons Causes PKA-Dependent Alterations in Fear Conditioning and Cortical "Arc" mRNA Expression

    ERIC Educational Resources Information Center

    Kelly, Michele P.; Cheung, York-Fong; Favilla, Christopher; Siegel, Steven J.; Kanes, Stephen J.; Houslay, Miles D.; Abel, Ted

    2008-01-01

    Memory formation requires cAMP signaling; thus, this cascade has been of great interest in the search for cognitive enhancers. Given that medications are administered long-term, we determined the effects of chronically increasing cAMP synthesis in the brain by expressing a constitutively active isoform of the G-protein subunit G[alpha]s…

  8. Minimizing the unpredictability of transgene expression in plants: the role of genetic insulators

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genetic transformation of plants has become a necessary tool for fundamental plant biology research, as well as the generation of engineered plants exhibiting improved agronomic and industrial traits. However, this technology is significantly hindered by the fact that transgene expression is hi...

  9. Gene Expression in Plant Lipid Metabolism in Arabidopsis Seedlings

    PubMed Central

    Hsiao, An-Shan; Haslam, Richard P.; Michaelson, Louise V.; Liao, Pan; Napier, Johnathan A.; Chye, Mee-Len

    2014-01-01

    Events in plant lipid metabolism are important during seedling establishment. As it has not been experimentally verified whether lipid metabolism in 2- and 5-day-old Arabidopsis thaliana seedlings is diurnally-controlled, quantitative real-time PCR analysis was used to investigate the expression of target genes in acyl-lipid transfer, β-oxidation and triacylglycerol (TAG) synthesis and hydrolysis in wild-type Arabidopsis WS and Col-0. In both WS and Col-0, ACYL-COA-BINDING PROTEIN3 (ACBP3), DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) and DGAT3 showed diurnal control in 2- and 5-day-old seedlings. Also, COMATOSE (CTS) was diurnally regulated in 2-day-old seedlings and LONG-CHAIN ACYL-COA SYNTHETASE6 (LACS6) in 5-day-old seedlings in both WS and Col-0. Subsequently, the effect of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) from the core clock system was examined using the cca1lhy mutant and CCA1-overexpressing (CCA1-OX) lines versus wild-type WS and Col-0, respectively. Results revealed differential gene expression in lipid metabolism between 2- and 5-day-old mutant and wild-type WS seedlings, as well as between CCA1-OX and wild-type Col-0. Of the ACBPs, ACBP3 displayed the most significant changes between cca1lhy and WS and between CCA1-OX and Col-0, consistent with previous reports that ACBP3 is greatly affected by light/dark cycling. Evidence of oil body retention in 4- and 5-day-old seedlings of the cca1lhy mutant in comparison to WS indicated the effect of cca1lhy on storage lipid reserve mobilization. Lipid profiling revealed differences in primary lipid metabolism, namely in TAG, fatty acid methyl ester and acyl-CoA contents amongst cca1lhy, CCA1-OX, and wild-type seedlings. Taken together, this study demonstrates that lipid metabolism is subject to diurnal regulation in the early stages of seedling development in Arabidopsis. PMID:25264899

  10. Expression of complete metabolic pathways in transgenic plants.

    PubMed

    Krichevsky, Alexander; Zaltsman, Adi; King, Lisa; Citovsky, Vitaly

    2012-01-01

    Plant genetic engineering emerged as a methodology to introduce only few transgenes into the plant genome. Following fast-paced developments of the past few decades, engineering of much larger numbers of transgenes became a reality, allowing to introduce full metabolic pathways from other organisms into plants and generate transgenics with startling new traits. From the advent of the classical plant genetic engineering, the transgenes were introduced into the nuclear genome of the plant cell, and this strategy still is quite successful when applied to few transgenes. However, for introducing large number of transgenes, we advocate that the chloroplast genome is a superior choice, especially for engineering of new complete metabolic pathways into plants. The ability to genetically engineer plants with complex and fully functional metabolic pathways from other organisms bears a substantial promise in generation of pharmaceuticals, i.e., biopharming, and new agricultural crops with that traits never existed before, leading to enhancement in quality of human life. PMID:22616478

  11. Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants.

    PubMed

    Dugdale, Benjamin; Mortimer, Cara L; Kato, Maiko; James, Tess A; Harding, Robert M; Dale, James L

    2014-05-01

    Virus-based transgene expression systems have become particularly valuable for recombinant protein production in plants. The dual-module in-plant activation (INPACT) expression platform consists of a uniquely designed split-gene cassette incorporating the cis replication elements of Tobacco yellow dwarf geminivirus (TYDV) and an ethanol-inducible activation cassette encoding the TYDV Rep and RepA replication-associated proteins. The INPACT system is essentially tailored for recombinant protein production in stably transformed plants and provides both inducible and high-level transient transgene expression with the potential to be adapted to diverse crop species. The construction of a novel split-gene cassette, the inducible nature of the system and the ability to amplify transgene expression via rolling-circle replication differentiates this system from other DNA- and RNA-based virus vector systems used for stable or transient recombinant protein production in plants. Here we provide a detailed protocol describing the design and construction of a split-gene INPACT cassette, and we highlight factors that may influence optimal activation and amplification of gene expression in transgenic plants. By using Nicotiana tabacum, the protocol takes 6-9 months to complete, and recombinant proteins expressed using INPACT can accumulate to up to 10% of the leaf total soluble protein.

  12. Constitutively active UVR8 photoreceptor variant in Arabidopsis.

    PubMed

    Heijde, Marc; Binkert, Melanie; Yin, Ruohe; Ares-Orpel, Florence; Rizzini, Luca; Van De Slijke, Eveline; Persiau, Geert; Nolf, Jonah; Gevaert, Kris; De Jaeger, Geert; Ulm, Roman

    2013-12-10

    Arabidopsis thaliana UV RESISTANCE LOCUS 8 (UVR8) is a UV-B photoreceptor that initiates photomorphogenic responses underlying acclimation and UV-B tolerance in plants. UVR8 is a homodimer in its ground state, and UV-B exposure results in its instantaneous monomerization followed by interaction with CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), a major factor in UV-B signaling. UV-B photoreception by UVR8 is based on intrinsic tryptophan aromatic amino acid residues, with tryptophan-285 as the main chromophore. We generated transgenic plants expressing UVR8 with a single amino acid change of tryptophan-285 to alanine. UVR8(W285A) appears monomeric and shows UV-B-independent interaction with COP1. Phenotypically, the plants expressing UVR8(W285A) exhibit constitutive photomorphogenesis associated with constitutive activation of target genes, elevated levels of anthocyanins, and enhanced, acclimation-independent UV-B tolerance. Moreover, we have identified COP1, REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 and 2 (RUP1 and RUP2), and the SUPPRESSOR OF PHYA-105 (SPA) family as proteins copurifying with UVR8(W285A). Whereas COP1, RUP1, and RUP2 are known to directly interact with UVR8, we show that SPA1 interacts with UVR8 indirectly through COP1. We conclude that UVR8(W285A) is a constitutively active UVR8 photoreceptor variant in Arabidopsis, as is consistent with the crucial importance of monomer formation and COP1 binding for UVR8 activity.

  13. A stable cytosolic expression of VH antibody fragment directed against PVY NIa protein in transgenic potato plant confers partial protection against the virus.

    PubMed

    Bouaziz, Donia; Ayadi, Malika; Bidani, Amira; Rouis, Souad; Nouri-Ellouz, Oumèma; Jellouli, Raïda; Drira, Noureddine; Gargouri-Bouzid, Radhia

    2009-04-01

    The expression of recombinant antibodies in transgenic plants has been proved to be an efficient approach for large-scale production. However, the stability of these molecules and their accumulation level depend on their molecular properties and cellular targeting. The expression of single-domain antibody fragment (VH) can be advantageous since it offers small length, high expression, solubility and stability. It can therefore be preferred to other antibody derivatives avoiding the expression difficulties related to immunoglobulin domain folding via the formation of disulfide bridge. This report describes the production of transgenic potato plants expressing a VH antibody directed against the NIa protease of potato virus Y. The antibody was driven by the constitutive CaMV 35S RNA promoter. The expression cassette was transferred into potato plants via Agrobacterium tumefaciens mediated transformation. All transgenic lines showed detectable levels of VH protein confirming the efficient translation and stability of this protein. The cellular localisation of the VH antibody was investigated. Transgenic and control plants were transferred in the greenhouse and mechanically inoculated by PVY(o) suspension. Some of the transgenic lines showed delayed symptoms at the first period post inoculation and then displayed a recovery phenomenon while the virions were still detected in the leaves. PMID:26493138

  14. Differential gene expression according to race and host plant in the pea aphid.

    PubMed

    Eyres, Isobel; Jaquiéry, Julie; Sugio, Akiko; Duvaux, Ludovic; Gharbi, Karim; Zhou, Jing-Jiang; Legeai, Fabrice; Nelson, Michaela; Simon, Jean-Christophe; Smadja, Carole M; Butlin, Roger; Ferrari, Julia

    2016-09-01

    Host-race formation in phytophagous insects is thought to provide the opportunity for local adaptation and subsequent ecological speciation. Studying gene expression differences amongst host races may help to identify phenotypes under (or resulting from) divergent selection and their genetic, molecular and physiological bases. The pea aphid (Acyrthosiphon pisum) comprises host races specializing on numerous plants in the Fabaceae and provides a unique system for examining the early stages of diversification along a gradient of genetic and associated adaptive divergence. In this study, we examine transcriptome-wide gene expression both in response to environment and across pea aphid races selected to cover the range of genetic divergence reported in this species complex. We identify changes in expression in response to host plant, indicating the importance of gene expression in aphid-plant interactions. Races can be distinguished on the basis of gene expression, and higher numbers of differentially expressed genes are apparent between more divergent races; these expression differences between host races may result from genetic drift and reproductive isolation and possibly divergent selection. Expression differences related to plant adaptation include a subset of chemosensory and salivary genes. Genes showing expression changes in response to host plant do not make up a large portion of between-race expression differences, providing confirmation of previous studies' findings that genes involved in expression differences between diverging populations or species are not necessarily those showing initial plasticity in the face of environmental change. PMID:27474484

  15. Enhanced disease resistance in transgenic carrot (Daucus carota L.) plants over-expressing a rice cationic peroxidase.

    PubMed

    Wally, O; Punja, Z K

    2010-10-01

    Plant class III peroxidases are involved in numerous responses related to pathogen resistance including controlling hydrogen peroxide (H(2)O(2)) levels and lignin formation. Peroxidases catalyze the oxidation of organic compounds using H(2)O(2) as an oxidant. We examined the mechanisms of disease resistance in a transgenic carrot line (P23) which constitutively over-expresses the rice cationic peroxidase OsPrx114 (previously known as PO-C1) and which exhibits enhanced resistance to necrotrophic foliar pathogens. OsPrx114 over-expression led to a slight enhancement of constitutive transcript levels of pathogenesis-related (PR) genes. These transcript levels were dramatically increased in line P23 compared to controls [GUS construct under the control of 35S promoter (35S::GUS)] when tissues were treated with cell wall fragments of the fungal pathogen Sclerotinia sclerotiorum (SS-walls), and to a lesser extent with 2,6-dichloroisonicotinic acid. There was no basal increase in basal H(2)O(2) levels in tissues of the line P23. However, during an oxidative burst response elicited by SS-walls, H(2)O(2) accumulation was reduced in line P23 despite, typical media alkalinization associated with oxidative burst responses was observed, suggesting that OsPrx114 was involved in rapid H(2)O(2) consumption during the oxidative burst response. Tap roots of line P23 had increased lignin formation in the outer periderm tissues, which was further increased during challenge inoculation with Alternaria radicina. Plant susceptibility to a biotrophic pathogen, Erysiphe heraclei, was not affected. Disease resistance to necrotrophic pathogens in carrot as a result of OsPrx114 over-expression is manifested through increased PR transcript accumulation, rapid removal of H(2)O(2) during oxidative burst response and enhanced lignin formation.

  16. Molecular cloning and bacterial expression of cDNA encoding a plant cysteine synthase.

    PubMed Central

    Saito, K; Miura, N; Yamazaki, M; Hirano, H; Murakoshi, I

    1992-01-01

    Cysteine synthase (CSase) [O-acetyl-L-serine acetate-lyase (adding hydrogen sulfide), EC 4.2.99.8] catalyzes the formation of L-cysteine, the key step in sulfur assimilation in plants, from O-acetyl-L-serine and hydrogen sulfide. We report here the isolation and characterization of cDNA clones encoding cysteine synthase from spinach (Spinacia oleracea L.). Internal peptide sequences were obtained from V8 protease-digested fragments of purified CSase. A lambda gt10 cDNA library was constructed from poly(A)+ RNA of young green leaves of spinach. Screening with two synthetic mixed nucleotides encoding the partial peptide sequences revealed 19 positively hybridized clones among 2 x 10(5) clones. Nucleotide sequence analysis of two independent cDNA clones revealed a continuous open reading frame encoding a polypeptide of 325 amino acids with a calculated molecular mass of 34,185 Da. Sequence comparison of the deduced amino acids revealed 53% identity with CSases of Escherichia coli and Salmonella typhimurium. Sequence homology was also observed with other metabolic enzymes for amino acids in bacteria and yeast and with rat hemoprotein H-450. A bacterial expression vector was constructed and could genetically complement an E. coli auxotroph that lacks CSases. The accumulation of functionally active spinach CSase in E. coli was also demonstrated by immunoblotting and assaying enzymatic activity. Southern hybridization analysis showed the presence of two to three copies of the cDNA sequence in the genome of spinach. RNA blot hybridization suggested constitutive expression in leaves and roots of spinach. Images PMID:1518833

  17. Strategies and Methodologies for the Co-expression of Multiple Proteins in Plants.

    PubMed

    Ferrer, Albert; Arró, Monserrat; Manzano, David; Altabella, Teresa

    2016-01-01

    The first transgenes were introduced in a plant genome more than 30 years ago. Since then, the capabilities of the plant scientific community to engineer the genome of plants have progressed at an unparalleled speed. Plant genetic engineering has become a central technology that has dramatically incremented our basic knowledge of plant biology and has enabled the translation of this knowledge into a number of increasingly complex and sophisticated biotechnological applications, which in most cases rely on the simultaneous co-expression of multiple recombinant proteins from different origins. To meet the new challenges of modern plant biotechnology, the plant scientific community has developed a vast arsenal of innovative molecular tools and genome engineering strategies. In this chapter we review a variety of tools, technologies, and strategies developed to transfer and simultaneously co-express multiple transgenes and proteins in a plant host. Their potential advantages, disadvantages, and future prospects are also discussed. PMID:27165331

  18. Transgenic tobacco expressing a modified spider peptide inhibits the growth of plant pathogens and insect larvae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The gene encoding lycotoxin I, an amphipathic pore-forming peptide, was modified to increase oral toxicity to insects. One of the most active modified genes was then constitutively expressed in tobacco (Nicotiana tabacum) and transformants were evaluated for insect and disease resistance. Pathogenic...

  19. The Constitutional Amendment Process

    ERIC Educational Resources Information Center

    Chism, Kahlil

    2005-01-01

    This article discusses the constitutional amendment process. Although the process is not described in great detail, Article V of the United States Constitution allows for and provides instruction on amending the Constitution. While the amendment process currently consists of six steps, the Constitution is nevertheless quite difficult to change.…

  20. Expression of Functional Human Coagulation Factor XIII A-domain in Plant Cell Suspensions and Whole Plants

    SciTech Connect

    Gao, Johnway; Hooker, Brian S.; Anderson, Daniel B.

    2004-09-01

    Coagulation factor XIII, a zymogen present in blood as a tetramer (A2B2) of A- and B-domains, is one of the components of many ''wound sealants'' which are proposed for use or currently in use as effective hemostatic agents, sealants and tissue adhesives in surgery. After activation by ?-thrombin cleavage, coagulation factor XIII A-domain, a transglutaminase, is formed and catalyzes the covalent crosslinking of the ?- and ?-chains of linear fibrin to form homopolymers, which can quickly stop bleeding. We have successfully expressed the A-domain of factor XIII in both plant cell cultures and whole plants. Transgenic plant cell culture allows a rapid method for testing production feasibility while expression in whole plants demonstrates an economic production system for recombinant human plasma-based proteins. The expressed factor XIII A-domain had a similar size as that of human plasma-derived factor XIII. Crude plant extract containing recombinant factor XIII A-domain showed transglutaminase activity with monodansylcadaverine and casein as substrates and crosslinking activity in the presence of linear fibrin. The expression of factor XIII A-domain was not affected by plant leaf position.

  1. Structure Conservation and Differential Expression of Farnesyl Diphosphate Synthase Genes in Euphorbiaceous Plants

    PubMed Central

    Guo, Dong; Li, Hui-Liang; Peng, Shi-Qing

    2015-01-01

    Farnesyl diphosphate synthase (FPS) is a key enzyme of isoprenoids biosynthesis. However, knowledge of the FPSs of euphorbiaceous species is limited. In this study, ten FPSs were identified in four euphorbiaceous plants. These FPSs exhibited similar exon/intron structure. The deduced FPS proteins showed close identities and exhibited the typical structure of plant FPS. The members of the FPS family exhibit tissue expression patterns that vary among several euphorbiaceous plant species under normal growth conditions. The expression profiles reveal spatial and temporal variations in the expression of FPSs of different tissues from Euphorbiaceous plants. Our results revealed wide conservation of FPSs and diverse expression in euphorbiaceous plants during growth and development. PMID:26389894

  2. Structural and enzymatic insights into Lambda glutathione transferases from Populus trichocarpa, monomeric enzymes constituting an early divergent class specific to terrestrial plants.

    PubMed

    Lallement, Pierre-Alexandre; Meux, Edgar; Gualberto, José M; Prosper, Pascalita; Didierjean, Claude; Saul, Frederick; Haouz, Ahmed; Rouhier, Nicolas; Hecker, Arnaud

    2014-08-15

    GSTs represent a superfamily of multifunctional proteins which play crucial roles in detoxification processes and secondary metabolism. Instead of promoting the conjugation of glutathione to acceptor molecules as do most GSTs, members of the Lambda class (GSTLs) catalyse deglutathionylation reactions via a catalytic cysteine residue. Three GSTL genes (Pt-GSTL1, Pt-GSTL2 and Pt-GSTL3) are present in Populus trichocarpa, but two transcripts, differing in their 5' extremities, were identified for Pt-GSTL3. Transcripts for these genes were primarily found in flowers, fruits, petioles and buds, but not in leaves and roots, suggesting roles associated with secondary metabolism in these organs. The expression of GFP-fusion proteins in tobacco showed that Pt-GSTL1 is localized in plastids, whereas Pt-GSTL2 and Pt-GSTL3A and Pt-GSTL3B are found in both the cytoplasm and the nucleus. The resolution of Pt-GSTL1 and Pt-GSTL3 structures by X-ray crystallography indicated that, although these proteins adopt a canonical GST fold quite similar to that found in dimeric Omega GSTs, their non-plant counterparts, they are strictly monomeric. This might explain some differences in the enzymatic properties of both enzyme types. Finally, from competition experiments between aromatic substrates and a fluorescent probe, we determined that the recognition of glutathionylated substrates is favoured over non-glutathionylated forms.

  3. Plants that express a potyvirus proteinase gene are resistant to virus infection.

    PubMed Central

    Maiti, I B; Murphy, J F; Shaw, J G; Hunt, A G

    1993-01-01

    Transgenic tobacco plants that express the genome-linked protein/proteinase-coding region of the potyvirus tobacco vein mottling virus (TVMV) were produced and tested for their reaction to inoculation with TVMV and two other potyviruses. These plants did not develop disease symptoms after being inoculated with large doses of TVMV but were as susceptible to infection by the other potyviruses as were control plants. Lines of tobacco that express the coat protein- or the nonstructural cylindrical inclusion protein-coding regions were also produced. The coat protein transgenic plants were protected against all three potyviruses, and the cylindrical inclusion transgenic plants were susceptible to all three potyviruses. These results indicate that some, but not all, TVMV genes can be used to confer protection against potyviruses in plants. The results also suggest that combinations of viral genes in transgenic plants might improve protection against potyviruses. Images Fig. 2 PMID:8327491

  4. Express yourself: Transcriptional regulation of plant innate immunity.

    PubMed

    Garner, Christopher M; Kim, Sang Hee; Spears, Benjamin J; Gassmann, Walter

    2016-08-01

    The plant immune system is a complex network of components that function together to sense the presence and activity of potential biotic threats, and integrate these signals into an appropriate output, namely the transcription of genes that activate an immune response that is commensurate with the perceived threat. Given the variety of biotic threats a plant must face the immune response must be plastic, but because an immune response is costly to the plant in terms of energy expenditure and development it must also be under tight control. To meet these needs transcriptional control is exercised at multiple levels. In this article we will review some of the latest developments in understanding how the plant immune response is regulated at the level of transcription. New roles are being discovered for the long-studied WRKY and TGA transcription factor families, while additional critical defense functions are being attributed to TCPs and other transcription factors. Dynamically controlling access to DNA through post-translational modification of histones is emerging as an essential component of priming, maintaining, attenuating, and repressing transcription in response to biotic stress. Unsurprisingly, the plant's transcriptional response is targeted by pathogen effectors, and in turn resistance proteins stand guard over and participate in transcriptional regulation. Together, these multiple layers lead to the observed complexity of the plant transcriptional immune response, with different transcription factors or chromatin components playing a prominent role depending on the plant-pathogen interaction being studied. PMID:27174437

  5. Express yourself: Transcriptional regulation of plant innate immunity.

    PubMed

    Garner, Christopher M; Kim, Sang Hee; Spears, Benjamin J; Gassmann, Walter

    2016-08-01

    The plant immune system is a complex network of components that function together to sense the presence and activity of potential biotic threats, and integrate these signals into an appropriate output, namely the transcription of genes that activate an immune response that is commensurate with the perceived threat. Given the variety of biotic threats a plant must face the immune response must be plastic, but because an immune response is costly to the plant in terms of energy expenditure and development it must also be under tight control. To meet these needs transcriptional control is exercised at multiple levels. In this article we will review some of the latest developments in understanding how the plant immune response is regulated at the level of transcription. New roles are being discovered for the long-studied WRKY and TGA transcription factor families, while additional critical defense functions are being attributed to TCPs and other transcription factors. Dynamically controlling access to DNA through post-translational modification of histones is emerging as an essential component of priming, maintaining, attenuating, and repressing transcription in response to biotic stress. Unsurprisingly, the plant's transcriptional response is targeted by pathogen effectors, and in turn resistance proteins stand guard over and participate in transcriptional regulation. Together, these multiple layers lead to the observed complexity of the plant transcriptional immune response, with different transcription factors or chromatin components playing a prominent role depending on the plant-pathogen interaction being studied.

  6. Antimicrobial peptide production and plant-based expression systems for medical and agricultural biotechnology.

    PubMed

    Holaskova, Edita; Galuszka, Petr; Frebort, Ivo; Oz, M Tufan

    2015-11-01

    Antimicrobial peptides (AMPs) are vital components of the innate immune system of nearly all living organisms. They generally act in the first line of defense against various pathogenic bacteria, parasites, enveloped viruses and fungi. These low molecular mass peptides are considered prospective therapeutic agents due to their broad-spectrum rapid activity, low cytotoxicity to mammalian cells and unique mode of action which hinders emergence of pathogen resistance. In addition to medical use, AMPs can also be employed for development of innovative approaches for plant protection in agriculture. Conferred disease resistance by AMPs might help us surmount losses in yield, quality and safety of agricultural products due to plant pathogens. Heterologous expression in plant-based systems, also called plant molecular farming, offers cost-effective large-scale production which is regarded as one of the most important factors for clinical or agricultural use of AMPs. This review presents various types of AMPs as well as plant-based platforms ranging from cell suspensions to whole plants employed for peptide production. Although AMP production in plants holds great promises for medicine and agriculture, specific technical limitations regarding product yield, function and stability still remain. Additionally, establishment of particular stable expression systems employing plants or plant tissues generally requires extended time scale for platform development compared to certain other heterologous systems. Therefore, fast and promising tools for evaluation of plant-based expression strategies and assessment of function and stability of the heterologously produced AMPs are critical for molecular farming and plant protection.

  7. Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation.

    PubMed

    Shcherbak, N; Kishchenko, O; Sakhno, L; Komarnytsky, I; Kuchuk, M

    2013-01-01

    Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants. PMID:23821951

  8. Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation.

    PubMed

    Shcherbak, N; Kishchenko, O; Sakhno, L; Komarnytsky, I; Kuchuk, M

    2013-01-01

    Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.

  9. Symptoms induced by transgenic expression of p23 from Citrus tristeza virus in phloem-associated cells of Mexican lime mimic virus infection without the aberrations accompanying constitutive expression.

    PubMed

    Soler, Nuria; Fagoaga, Carmen; López, Carmelo; Moreno, Pedro; Navarro, Luis; Flores, Ricardo; Peña, Leandro

    2015-05-01

    Citrus tristeza virus (CTV) is phloem restricted in natural citrus hosts. The 23-kDa protein (p23) encoded by the virus is an RNA silencing suppressor and a pathogenicity determinant. The expression of p23, or its N-terminal 157-amino-acid fragment comprising the zinc finger and flanking basic motifs, driven by the constitutive 35S promoter of cauliflower mosaic virus, induces CTV-like symptoms and other aberrations in transgenic citrus. To better define the role of p23 in CTV pathogenesis, we compared the phenotypes of Mexican lime transformed with p23-derived transgenes from the severe T36 and mild T317 CTV isolates under the control of the phloem-specific promoter from Commelina yellow mottle virus (CoYMV) or the 35S promoter. Expression of the constructs restricted to the phloem induced a phenotype resembling CTV-specific symptoms (vein clearing and necrosis, and stem pitting), but not the non-specific aberrations (such as mature leaf epinasty and yellow pinpoints, growth cessation and apical necrosis) observed when p23 was ectopically expressed. Furthermore, vein necrosis and stem pitting in Mexican lime appeared to be specifically associated with p23 from T36. Phloem-specific accumulation of the p23Δ158-209(T36) fragment was sufficient to induce the same anomalies, indicating that the region comprising the N-terminal 157 amino acids of p23 is responsible (at least in part) for the vein clearing, stem pitting and, possibly, vein corking in this host.

  10. Development of Useful Recombinant Promoter and Its Expression Analysis in Different Plant Cells Using Confocal Laser Scanning Microscopy

    PubMed Central

    Kumar, Deepak; Sahoo, Dipak K.; Maiti, Indu B.; Dey, Nrisingha

    2011-01-01

    Background Designing functionally efficient recombinant promoters having reduced sequence homology and enhanced promoter activity will be an important step toward successful stacking or pyramiding of genes in a plant cell for developing transgenic plants expressing desired traits(s). Also basic knowledge regarding plant cell specific expression of a transgene under control of a promoter is crucial to assess the promoter's efficacy. Methodology/Principal Findings We have constructed a set of 10 recombinant promoters incorporating different up-stream activation sequences (UAS) of Mirabilis mosaic virus sub-genomic transcript (MS8, -306 to +27) and TATA containing core domains of Figwort mosaic virus sub-genomic transcript promoter (FS3, −271 to +31). Efficacies of recombinant promoters coupled to GUS and GFP reporter genes were tested in tobacco protoplasts. Among these, a 369-bp long hybrid sub-genomic transcript promoter (MSgt-FSgt) showed the highest activity in both transient and transgenic systems. In a transient system, MSgt-FSgt was 10.31, 2.86 and 2.18 times more active compared to the CaMV35S, MS8 and FS3 promoters, respectively. In transgenic tobacco (Nicotiana tabaccum, var. Samsun NN) and Arabidopsis plants, the MSgt-FSgt hybrid promoter showed 14.22 and 7.16 times stronger activity compared to CaMV35S promoter respectively. The correlation between GUS activity and uidA-mRNA levels in transgenic tobacco plants were identified by qRT-PCR. Both CaMV35S and MSgt-FSgt promoters caused gene silencing but the degree of silencing are less in the case of the MSgt-FSgt promoter compared to CaMV35S. Quantification of GUS activity in individual plant cells driven by the MSgt-FSgt and the CaMV35S promoter were estimated using confocal laser scanning microscopy and compared. Conclusion and Significance We propose strong recombinant promoter MSgt-FSgt, developed in this study, could be very useful for high-level constitutive expression of transgenes in a wide variety

  11. Stable heterologous expression of biologically active terpenoids in green plant cells

    PubMed Central

    Ikram, N. Kusaira B. K.; Zhan, Xin; Pan, Xi-Wu; King, Brian C.; Simonsen, Henrik T.

    2015-01-01

    Plants biosynthesize a great diversity of biologically active small molecules of interest for fragrances, flavors, and pharmaceuticals. Among specialized metabolites, terpenoids represent the greatest molecular diversity. Many terpenoids are very complex, and total chemical synthesis often requires many steps and difficult chemical reactions, resulting in a low final yield or incorrect stereochemistry. Several drug candidates with terpene skeletons are difficult to obtain by chemical synthesis due to their large number of chiral centers. Thus, biological production remains the preferred method for industrial production for many of these compounds. However, because these chemicals are often found in low abundance in the native plant, or are produced in plants which are difficult to cultivate, there is great interest in engineering increased production or expression of the biosynthetic pathways in heterologous hosts. Although there are many examples of successful engineering of microbes such as yeast or bacteria to produce these compounds, this often requires extensive changes to the host organism's metabolism. Optimization of plant gene expression, post-translational protein modifications, subcellular localization, and other factors often present challenges. To address the future demand for natural products used as drugs, new platforms are being established that are better suited for heterologous production of plant metabolites. Specifically, direct metabolic engineering of plants can provide effective heterologous expression for production of valuable plant-derived natural products. In this review, our primary focus is on small terpenoids and we discuss the benefits of plant expression platforms and provide several successful examples of stable production of small terpenoids in plants. PMID:25852702

  12. Homogalacturonan-modifying enzymes: structure, expression, and roles in plants

    PubMed Central

    Sénéchal, Fabien; Wattier, Christopher; Rustérucci, Christine; Pelloux, Jérôme

    2014-01-01

    Understanding the changes affecting the plant cell wall is a key element in addressing its functional role in plant growth and in the response to stress. Pectins, which are the main constituents of the primary cell wall in dicot species, play a central role in the control of cellular adhesion and thereby of the rheological properties of the wall. This is likely to be a major determinant of plant growth. How the discrete changes in pectin structure are mediated is thus a key issue in our understanding of plant development and plant responses to changes in the environment. In particular, understanding the remodelling of homogalacturonan (HG), the most abundant pectic polymer, by specific enzymes is a current challenge in addressing its fundamental role. HG, a polymer that can be methylesterified or acetylated, can be modified by HGMEs (HG-modifying enzymes) which all belong to large multigenic families in all species sequenced to date. In particular, both the degrees of substitution (methylesterification and/or acetylation) and polymerization can be controlled by specific enzymes such as pectin methylesterases (PMEs), pectin acetylesterases (PAEs), polygalacturonases (PGs), or pectate lyases-like (PLLs). Major advances in the biochemical and functional characterization of these enzymes have been made over the last 10 years. This review aims to provide a comprehensive, up to date summary of the recent data concerning the structure, regulation, and function of these fascinating enzymes in plant development and in response to biotic stresses. PMID:25056773

  13. Physiological and biochemical responses of transgenic potato plants with altered expression of PSII manganese stabilizing protein.

    PubMed

    Gururani, Mayank Anand; Upadhyaya, Chandrama Prakash; Strasser, Reto J; Woong, Yu Jae; Park, Se Won

    2012-09-01

    Manganese-stabilizing protein (MSP) represents a key component of the oxygen-evolving complex (OEC). Transgenic potato plants with both enhanced (sense) and reduced (anti-sense) MSP expression levels were generated to investigate the possible physiological role of MSP in overall plant growth, particularly in tuber development. MSP antisense plants exhibited both higher tuberization frequency and higher tuber yield with increased total soluble carbohydrates. The photosynthetic efficiencies of the plants were examined using the OJIP kinetics; MSP-antisense plants were photosynthetically more active than the MSP-sense and UT (untransformed) control plants. The oxygen measurements indicated that the relative oxygen evolution was directly proportional to the MSP expression, as MSP-antisense plants showed much lower oxygen evolution compared to MSP-sense as well as UT plants. MSP-sense plants behaved like the UT plants with respect to morphology, tuber yield, and photosynthetic performance. Chlorophyll a fluorescence analyses indicate a possible lack of intact Oxygen Evolving Complexes (OECs) in MSP antisense plants, which allow access to internal non-water electron donors (e.g., ascorbate and proline) and consequently increase the Photosystem II (PSII) activity of those plants. These findings further indicate that this altered photosynthetic machinery may be associated with early tuberization and increased tuberization frequency.

  14. Expression and Chloroplast Targeting of Cholesterol Oxidase in Transgenic Tobacco Plants

    PubMed Central

    Corbin, David R.; Grebenok, Robert J.; Ohnmeiss, Thomas E.; Greenplate, John T.; Purcell, John P.

    2001-01-01

    Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism. PMID:11457962

  15. Plant expression systems, a budding way to confront chikungunya and Zika in developing countries?

    PubMed Central

    Cardona-Ospina, Jaime A.; Sepúlveda-Arias, Juan C.; Mancilla, L.; Gutierrez-López, Luis G.

    2016-01-01

    Plant expression systems could be used as biofactories of heterologous proteins that have the potential to be used with biopharmaceutical aims and vaccine design. This technology is scalable, safe and cost-effective and it has been previously proposed as an option for vaccine and protein pharmaceutical development in developing countries. Here we present a proposal of how plant expression systems could be used to address Zika and chikungunya outbreaks through development of vaccines and rapid diagnostic kits. PMID:27781090

  16. Sustained expression of human cytomegalovirus glycoprotein B (UL55) in the seeds of homozygous rice plants.

    PubMed

    Tackaberry, Eilleen S; Prior, Fiona A; Rowlandson, Karen; Tocchi, Monika; Mehic, Jelica; Porter, Suzanne; Walsh, Mike; Schleiss, Mark R; Ganz, Peter R; Sardana, Ravinder K; Altosaar, Illimar; Dudani, Anil K

    2008-09-01

    Production of recombinant subunit vaccines in transgenic plants may be a means of reducing vaccine costs while increasing availability and safety. A plant-derived product found safe and effective for oral administration would provide additional advantages when used as a vaccine. Outstanding issues with the technology include transgene stability through successive generations and consistent bioproduction. We previously reported expression of glycoprotein B (gB) of human cytomegalovirus in seeds of transgenic tobacco. Here the goal was to determine if gB could be similarly expressed in rice, and if so, to examine expression over several plant generations. Results show that immunoreactive gB was successfully expressed in transgenic rice seeds, with sustained expression over three generations. The gB contained several neutralizing epitopes and was stable over 27 months.

  17. Ectopic Terpene Synthase Expression Enhances Sesquiterpene Emission in Nicotiana attenuata without Altering Defense or Development of Transgenic Plants or Neighbors1[W

    PubMed Central

    Schuman, Meredith C.; Palmer-Young, Evan C.; Schmidt, Axel; Gershenzon, Jonathan; Baldwin, Ian T.

    2014-01-01

    Sesquiterpenoids, with approximately 5,000 structures, are the most diverse class of plant volatiles with manifold hypothesized functions in defense, stress tolerance, and signaling between and within plants. These hypotheses have often been tested by transforming plants with sesquiterpene synthases expressed behind the constitutively active 35S promoter, which may have physiological costs measured as inhibited growth and reduced reproduction or may require augmentation of substrate pools to achieve enhanced emission, complicating the interpretation of data from affected transgenic lines. Here, we expressed maize (Zea mays) terpene synthase10 (ZmTPS10), which produces (E)-α-bergamotene and (E)-β-farnesene, or a point mutant ZmTPS10M, which produces primarily (E)-β-farnesene, under control of the 35S promoter in the ecological model plant Nicotiana attenuata. Transgenic N. attenuata plants had specifically enhanced emission of target sesquiterpene(s) with no changes detected in their emission of any other volatiles. Treatment with herbivore or jasmonate elicitors induces emission of (E)-α-bergamotene in wild-type plants and also tended to increase emission of (E)-α-bergamotene and (E)-β-farnesene in transgenics. However, transgenics did not differ from the wild type in defense signaling or chemistry and did not alter defense chemistry in neighboring wild-type plants. These data are inconsistent with within-plant and between-plant signaling functions of (E)-β-farnesene and (E)-α-bergamotene in N. attenuata. Ectopic sesquiterpene emission was apparently not costly for transgenics, which were similar to wild-type plants in their growth and reproduction, even when forced to compete for common resources. These transgenics would be well suited for field experiments to investigate indirect ecological effects of sesquiterpenes for a wild plant in its native habitat. PMID:25187528

  18. Transgenic alfalfa plants expressing AtNDPK2 exhibit increased growth and tolerance to abiotic stresses.

    PubMed

    Wang, Zhi; Li, Hongbing; Ke, Qingbo; Jeong, Jae Cheol; Lee, Haeng-Soon; Xu, Bingcheng; Deng, Xi-Ping; Lim, Yong Pyo; Kwak, Sang-Soo

    2014-11-01

    In this study, we generated and evaluated transgenic alfalfa plants (Medicago sativa L. cv. Xinjiang Daye) expressing the Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) gene under the control of the oxidative stress-inducible SWPA2 promoter (referred to as SN plants) to develop plants with enhanced tolerance to various abiotic stresses. We selected two SN plants (SN4 and SN7) according to the expression levels of AtNDPK2 and the enzyme activity of NDPK in response to methyl viologen (MV)-mediated oxidative stress treatment using leaf discs for further characterization. SN plants showed enhanced tolerance to high temperature, NaCl, and drought stress on the whole-plant level. When the plants were subjected to high temperature treatment (42 °C for 24 h), the non-transgenic (NT) plants were severely wilted, whereas the SN plants were not affected because they maintained high relative water and chlorophyll contents. The SN plants also showed significantly higher tolerance to 250 mM NaCl and water stress treatment than the NT plants. In addition, the SN plants exhibited better plant growth through increased expression of auxin-related indole acetic acid (IAA) genes (MsIAA3, MsIAA5, MsIAA6, MsIAA7, and MsIAA16) under normal growth conditions compared to NT plants. The results suggest that induced overexpression of AtNDPK2 in alfalfa will be useful for increasing biomass production under various abiotic stress conditions.

  19. Increased expression of a plant actin gene during a biotrophic interaction between round-leaved mallow, Malva pusilla, and Colletotrichum gloeosporioides f. sp. malvae.

    PubMed

    Jin, S; Xu, R; Wei, Y; Goodwin, P H

    1999-10-01

    Two actin genes, actA from the hemibiotrophic anthracnose fungus, Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. f. sp. malvae, and act1 from its host, Malva pusilla (Sm.) were cloned from a cDNA library developed from infected host tissue. The actin gene, actA, of C. gloeosporioides f. sp. malvae, which is similar to that of other euascomycetes, appears to be expressed constitutively. The actin gene of M. pusilla is most similar to one of the actin genes of Arabidopsis thaliana that is unique in being responsive to environmental stimuli such as wounding. Expression of actA was used to follow the growth of the fungus in the plant tissue. Low actA expression occurred until 72-96 h after inoculation and then increased rapidly, corresponding with the timing of the shift from slower biotrophic fungal growth to much more rapid necrotrophic growth. In contrast, expression of act1 approximately doubled during the biotrophic phase and then rapidly declined during the necrotrophic phase. Increased host actin expression could be due to host cytoskeleton rearrangement in response to biotrophic infection, and the subsequent decrease in host actin expression could be due to host cell disruption resulting from tissue maceration during necrosis. This is the first report of a host actin gene that can increase in expression during a compatible plant-pathogen interaction.

  20. Differential allelopathic expression of different plant parts of Achillea biebersteinii.

    PubMed

    Abu-Romman, Saeid

    2016-06-01

    Achillea biebersteinii (Asteraceae) is a perennial medicinal plant and has a wide distributional range in the Mediterranean region. The present study investigated the inhibitory effects of different plant parts of A. biebersteinii on germination characteristics and seedling growth of wild barley (Hordeum spontaneum). Water extracts were prepared by incubating separately five grams of dried powder of roots, stems, leaves and flowers of A. biebersteinii in 100 ml of distilled water for 24 h and distilled water was used as the control. The water extracts from different plant parts of A. biebersteinii differed in their effects on the germination and seedling growth of wild barley. Water extracts prepared from leaves and flowers were more suppressive to germination of wild barley than root and stem extracts. The maximum inhibition in radical and plumule growth of germinating caryopses and in root and shoot growth of greenhouse-grown wild barley was recorded for leaf extract followed by flower extract. The lowest Chl a, Chl b and total chlorophyll and protein contents were resulted after exposure to leaf extracts. According to these results, the inhibitory effects of different A. biebersteinii plant parts can be arranged in the order: leaf > flower > stem > root. PMID:27165527

  1. SCOF-1-expressing transgenic sweetpotato plants show enhanced tolerance to low-temperature stress.

    PubMed

    Kim, Yun-Hee; Kim, Myoung Duck; Park, Sung-Chul; Yang, Kyoung-Sil; Jeong, Jae Cheol; Lee, Haeng-Soon; Kwak, Sang-Soo

    2011-12-01

    Low-temperature stress represents one of the principal limitations affecting the distribution and productivity of many plant species, including crops such as sweetpotato. Transgenic sweetpotato (Ipomoea batatas L. cv. Yulmi) plants expressing the soybean cold-inducible zinc finger protein (SCOF-1) under control of an oxidative stress-inducible peroxidase (SWPA2) promoter (referred to as SF plants), were developed and evaluated for enhanced tolerance to low-temperature conditions. Following 4 °C treatment of SF plants, SCOF-1 expression correlated positively with tolerance to low-temperature stress at the leaf disc level. Increased SCOF-1 expression also correlated with enhanced tolerance to different low-temperature treatments at the whole plant level. SF plants treated with low-temperature stress (4 or 10 °C for 30 h) exhibited less of a reduction in photosynthetic activity and lipid peroxidation levels than non-transgenic (NT) plants. Furthermore, the photosynthetic activity and lipid peroxidation levels of SF plants recovered to near pre-stress levels after 12 h of recovery at 25 °C. In contrast, these activities remained at a reduced level in NT plants after the same recovery period. Thus, this study has shown that low-temperature stress in sweetpotato can be efficiently modulated by overexpression of SCOF-1.

  2. The combination of plant translational enhancers and terminator increase the expression of human glucocerebrosidase in Nicotiana benthamiana plants.

    PubMed

    Limkul, Juthamard; Misaki, Ryo; Kato, Ko; Fujiyama, Kazuhito

    2015-11-01

    Gaucher's disease is a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCase). It is currently treated by enzyme replacement therapy using recombinant GCase expressed in mammalian cells. Plant production systems are among the most attractive alternatives for pharmaceutical protein production due to such advantages as low-cost, high-scalability, and safety from human pathogen contamination. Because of its high biomass yield, Nicotiana benthamiana could be an economical recombinant GCase production system. In this study, a translational enhancer and suitable terminator were utilized to obtain a powerful expression system for GCase production in N. benthamiana plants. Six plasmid constructs were used. The highest activity of 44.5units/mg protein (after subtraction of endogenous glucosidase activity of the wild-type plant) was observed in transgenic plants transformed with pAt-GC-HSP combined with a 5' untranslated region of the Arabidopsis alcohol dehydrogenase gene with the Arabidopsis heat shock protein terminator. These transgenic plant lines could pave the way to a stable plant-production system for low-cost, high-yield human GCase production. PMID:26475186

  3. The combination of plant translational enhancers and terminator increase the expression of human glucocerebrosidase in Nicotiana benthamiana plants.

    PubMed

    Limkul, Juthamard; Misaki, Ryo; Kato, Ko; Fujiyama, Kazuhito

    2015-11-01

    Gaucher's disease is a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCase). It is currently treated by enzyme replacement therapy using recombinant GCase expressed in mammalian cells. Plant production systems are among the most attractive alternatives for pharmaceutical protein production due to such advantages as low-cost, high-scalability, and safety from human pathogen contamination. Because of its high biomass yield, Nicotiana benthamiana could be an economical recombinant GCase production system. In this study, a translational enhancer and suitable terminator were utilized to obtain a powerful expression system for GCase production in N. benthamiana plants. Six plasmid constructs were used. The highest activity of 44.5units/mg protein (after subtraction of endogenous glucosidase activity of the wild-type plant) was observed in transgenic plants transformed with pAt-GC-HSP combined with a 5' untranslated region of the Arabidopsis alcohol dehydrogenase gene with the Arabidopsis heat shock protein terminator. These transgenic plant lines could pave the way to a stable plant-production system for low-cost, high-yield human GCase production.

  4. A R2R3-MYB transcription factor, GmMYB12B2, affects the expression levels of flavonoid biosynthesis genes encoding key enzymes in transgenic Arabidopsis plants.

    PubMed

    Li, Xiao-Wei; Li, Jing-Wen; Zhai, Ying; Zhao, Yan; Zhao, Xu; Zhang, Hai-Jun; Su, Lian-Tai; Wang, Ying; Wang, Qing-Yu

    2013-12-10

    Isoflavones play diverse roles in plant-microbe interactions and are potentially important for human nutrition and health. To study the regulation of isoflavonoid synthesis in soybean, the R2R3-MYB transcription factor GmMYB12B2 was isolated and characterized. Yeast expression experiments demonstrated that GmMYB12B2 showed transcriptional activity. GmMYB12B2 was localized in the nucleus when it was transiently expressed in onion epidermal cells. Real-time quantitative PCR analysis revealed that GmMYB12B2 transcription was increased in roots and mature seeds compared with other organs. The gene expression level in immature embryos was consistent with the accumulation of isoflavones. CHS8 is a key enzyme in plant flavonoid biosynthesis. Transient expression experiments in soybean calli demonstrated that CHS8 was regulated by GmMYB12B2 and produced more fluorescence. The expression levels of some key enzymes in flavonoid biosynthesis were examined in transgenic Arabidopsis lines. The results showed that the expression levels of PAL1, CHS and FLS in transgenic plants were significantly higher than those in wild type plants. However, the expression level of DFR was lower, and the expression levels of CHI, F3H and F3'H were the same in all lines. GmMYB12B2 expression caused a constitutive increase in the accumulation of flavonoids in transgenic Arabidopsis lines compared with wild type plants. PMID:24060295

  5. A R2R3-MYB transcription factor, GmMYB12B2, affects the expression levels of flavonoid biosynthesis genes encoding key enzymes in transgenic Arabidopsis plants.

    PubMed

    Li, Xiao-Wei; Li, Jing-Wen; Zhai, Ying; Zhao, Yan; Zhao, Xu; Zhang, Hai-Jun; Su, Lian-Tai; Wang, Ying; Wang, Qing-Yu

    2013-12-10

    Isoflavones play diverse roles in plant-microbe interactions and are potentially important for human nutrition and health. To study the regulation of isoflavonoid synthesis in soybean, the R2R3-MYB transcription factor GmMYB12B2 was isolated and characterized. Yeast expression experiments demonstrated that GmMYB12B2 showed transcriptional activity. GmMYB12B2 was localized in the nucleus when it was transiently expressed in onion epidermal cells. Real-time quantitative PCR analysis revealed that GmMYB12B2 transcription was increased in roots and mature seeds compared with other organs. The gene expression level in immature embryos was consistent with the accumulation of isoflavones. CHS8 is a key enzyme in plant flavonoid biosynthesis. Transient expression experiments in soybean calli demonstrated that CHS8 was regulated by GmMYB12B2 and produced more fluorescence. The expression levels of some key enzymes in flavonoid biosynthesis were examined in transgenic Arabidopsis lines. The results showed that the expression levels of PAL1, CHS and FLS in transgenic plants were significantly higher than those in wild type plants. However, the expression level of DFR was lower, and the expression levels of CHI, F3H and F3'H were the same in all lines. GmMYB12B2 expression caused a constitutive increase in the accumulation of flavonoids in transgenic Arabidopsis lines compared with wild type plants.

  6. Antisense-mediated depletion of GMPase gene expression in tobacco decreases plant tolerance to temperature stresses and alters plant development.

    PubMed

    Wang, Hua-Sen; Zhu, Zhu-Jun; Feng, Zhen; Zhang, Shi-Gang; Yu, Chao

    2012-12-01

    In our previous work [1] we investigated the role of tomato GDP-mannose pyrophosphorylase (EC 2.7.7.22) in plants by overexpressing its gene in tobacco leaves and showed its function in AsA metabolism and detoxification of reactive oxygen species under temperature stresses. In this study, we use the antisense technique to block the endogenous GMPase gene expression in tobacco in order to further investigate its function. Northern and western blot analysis confirmed that the expression of endogenous tobacco GMPase mRNA and protein was inhibited by this antisense expression. Consequently, the activity of GMPase and the content of AsA in the leaves of antisense transgenic plants were markedly decreased. This was also the case for the activities of both chloroplastic SOD (superoxide dismutase EC 1.15.1.1), APX (ascorbate peroxidase EC 1.11.1.7) and the content of AsA in leaves of the transgenic plants. On the contrary, the contents of H(2)O(2) and O(2) (-•) were increased. Meanwhile, the net photosynthetic rate (Pn) and the maximal photochemical efficiency of PSII (Fv/Fm) also declined in the leaves of antisense plants. Under high or low temperature stresses, the seed germination rate of the antisense transgenic plants was significantly decreased in comparison with that of the wild-type tobacco. Interestingly, the antisense plants had smaller leaves and an earlier onset of flowering. In conclusion, the depletion of GMPase decreased the content of AsA, resulting in the plants susceptible to the oxidative damage caused by temperature stresses and subjected to developmental alternations.

  7. Gene silencing and gene expression in phytopathogenic fungi using a plant virus vector

    PubMed Central

    Mascia, Tiziana; Nigro, Franco; Abdallah, Alì; Ferrara, Massimo; De Stradis, Angelo; Faedda, Roberto; Palukaitis, Peter; Gallitelli, Donato

    2014-01-01

    RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi. PMID:24594602

  8. An Oomycete CRN Effector Reprograms Expression of Plant HSP Genes by Targeting their Promoters

    PubMed Central

    Song, Tianqiao; Ma, Zhenchuan; Shen, Danyu; Li, Qi; Li, Wanlin; Su, Liming; Ye, Tingyue; Zhang, Meixiang; Wang, Yuanchao; Dou, Daolong

    2015-01-01

    Oomycete pathogens produce a large number of CRN effectors to manipulate plant immune responses and promote infection. However, their functional mechanisms are largely unknown. Here, we identified a Phytophthora sojae CRN effector PsCRN108 which contains a putative DNA-binding helix-hairpin-helix (HhH) motif and acts in the plant cell nucleus. Silencing of the PsCRN108 gene reduced P. sojae virulence to soybean, while expression of the gene in Nicotiana benthamiana and Arabidopsis thaliana enhanced plant susceptibility to P. capsici. Moreover, PsCRN108 could inhibit expression of HSP genes in A. thaliana, N. benthamiana and soybean. Both the HhH motif and nuclear localization signal of this effector were required for its contribution to virulence and its suppression of HSP gene expression. Furthermore, we found that PsCRN108 targeted HSP promoters in an HSE- and HhH motif-dependent manner. PsCRN108 could inhibit the association of the HSE with the plant heat shock transcription factor AtHsfA1a, which initializes HSP gene expression in response to stress. Therefore, our data support a role for PsCRN108 as a nucleomodulin in down-regulating the expression of plant defense-related genes by directly targeting specific plant promoters. PMID:26714171

  9. An Oomycete CRN Effector Reprograms Expression of Plant HSP Genes by Targeting their Promoters.

    PubMed

    Song, Tianqiao; Ma, Zhenchuan; Shen, Danyu; Li, Qi; Li, Wanlin; Su, Liming; Ye, Tingyue; Zhang, Meixiang; Wang, Yuanchao; Dou, Daolong

    2015-12-01

    Oomycete pathogens produce a large number of CRN effectors to manipulate plant immune responses and promote infection. However, their functional mechanisms are largely unknown. Here, we identified a Phytophthora sojae CRN effector PsCRN108 which contains a putative DNA-binding helix-hairpin-helix (HhH) motif and acts in the plant cell nucleus. Silencing of the PsCRN108 gene reduced P. sojae virulence to soybean, while expression of the gene in Nicotiana benthamiana and Arabidopsis thaliana enhanced plant susceptibility to P. capsici. Moreover, PsCRN108 could inhibit expression of HSP genes in A. thaliana, N. benthamiana and soybean. Both the HhH motif and nuclear localization signal of this effector were required for its contribution to virulence and its suppression of HSP gene expression. Furthermore, we found that PsCRN108 targeted HSP promoters in an HSE- and HhH motif-dependent manner. PsCRN108 could inhibit the association of the HSE with the plant heat shock transcription factor AtHsfA1a, which initializes HSP gene expression in response to stress. Therefore, our data support a role for PsCRN108 as a nucleomodulin in down-regulating the expression of plant defense-related genes by directly targeting specific plant promoters. PMID:26714171

  10. Wound healing response and xylem differentiation in tobacco plants over-expressing a fungal endopolygalacturonase is mediated by copper amine oxidase activity.

    PubMed

    Cona, Alessandra; Tisi, Alessandra; Ghuge, Sandip Annasaheb; Franchi, Stefano; De Lorenzo, Giulia; Angelini, Riccardo

    2014-09-01

    In this work, we have investigated the involvement of copper amine oxidase (CuAO; EC 1.4.3.21) in wound healing and xylem differentiation of Nicotiana tabacum plants over-expressing a fungal endopolygalacturonase (PG plants), which show constitutively activated defence responses. In petioles and stems of PG plants, we found higher CuAO activity and lower polyamine (PA) levels, particularly putrescine (Put), with respect to wild-type (WT) plants. Upon wounding, a more intense autofluorescence of cell wall phenolics was observed in correspondence of wound surface, extending to epidermis and cortical parenchima only in PG plants. This response was mostly dependent on CuAO activity, as suggested by the reversion of autofluorescence upon supply of 2-bromoethylamine (2-BrEt), a CuAO specific inhibitor. Moreover, in unwounded plants, histochemical analysis revealed a tissue-specific expression of the enzyme in the vascular cambium and neighboring derivative cells of both petioles and stems of PG plants, whereas the corresponding WT tissues appeared unstained or faintly stained. A higher histochemical CuAO activity was also observed in xylem cells of PG plants as compared to WT xylem tissues suggesting a peculiar role of CuAO activity in xylem differentiation in PG plants. Indeed, roots of PG plants exhibited early xylem differentiation, a phenotype consistent with both the higher CuAO and the lower Put levels observed and supported by the 2-BrEt-mediated reversion of early root xylem differentiation and H2O2 accumulation. These results strongly support the relevance of PA-catabolism derived H2O2 in defence responses, such as those signaled by a compromised status of cell wall pectin integrity.

  11. Expression and affinity purification of recombinant proteins from plants

    NASA Technical Reports Server (NTRS)

    Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun

    2002-01-01

    With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

  12. Capsicum annuum tobacco mosaic virus-induced clone 1 expression perturbation alters the plant's response to ethylene and interferes with the redox homeostasis.

    PubMed

    Shin, Ryoung; An, Jong-Min; Park, Chang-Jin; Kim, Young Jin; Joo, Sunjoo; Kim, Woo Taek; Paek, Kyung-Hee

    2004-05-01

    Capsicum annuum tobacco mosaic virus (TMV)-induced clone 1 (CaTin1) gene was expressed early during incompatible interaction of hot pepper (Caspsicum annuum) plants with TMV and Xanthomonas campestris. RNA-blot analysis showed that CaTin1 gene was expressed only in roots in untreated plants and induced mainly in leaf in response to ethylene, NaCl, and methyl viologen but not by salicylic acid and methyl jasmonate. The ethylene dependence of CaTin1 induction upon TMV inoculation was demonstrated by the decrease of CaTin1 expression in response to several inhibitors of ethylene biosynthesis or its action. Transgenic tobacco (Nicotiana tabacum) plants expressing CaTin1 gene in sense- or antisense-orientation showed interesting characteristics such as the accelerated growth and the enhanced resistance to biotic as well as abiotic stresses. Such characteristics appear to be caused by the elevated level of ethylene and H2O2. Moreover, in transgenic plants expressing antisense CaTin1 gene, the expression of some pathogenesis-related genes was enhanced constitutively, which may be mainly due to the increased ethylene level. The promoter of CaTin1 has four GCC-boxes, two AT-rich regions, and an elicitor-inducible W-box. The induction of the promoter activity by ethylene depends on GCC-boxes and by TMV on W-box. Taken together, we propose that the CaTin1 up-regulation or down-regulation interferes with the redox balance of plants leading to the altered response to ethylene and biotic as well as abiotic stresses.

  13. Expression of Active Subunit of Nitrogenase via Integration into Plant Organelle Genome

    PubMed Central

    Groat, Jeanna; Staub, Jeffrey M.; Stephens, Michael

    2016-01-01

    Nitrogen availability is crucial for crop yield with nitrogen fertilizer accounting for a large percentage of farmers’ expenses. However, an untimely or excessive application of fertilizer can increase risks of negative environmental effects. These factors, along with the environmental and energy costs of synthesizing nitrogen fertilizer, led us to seek out novel biotechnology-driven approaches to supply nitrogen to plants. The strategy we focused on involves transgenic expression of nitrogenase, a bacterial multi-subunit enzyme that can capture atmospheric nitrogen. Here we report expression of the active Fe subunit of nitrogenase via integration into the tobacco plastid genome of bacterial gene sequences modified for expression in plastid. Our study suggests that it will be possible to engineer plants that are able to produce their own nitrogen fertilizer by expressing nitrogenase genes in plant plastids. PMID:27529475

  14. Expression of Active Subunit of Nitrogenase via Integration into Plant Organelle Genome.

    PubMed

    Ivleva, Natalia B; Groat, Jeanna; Staub, Jeffrey M; Stephens, Michael

    2016-01-01

    Nitrogen availability is crucial for crop yield with nitrogen fertilizer accounting for a large percentage of farmers' expenses. However, an untimely or excessive application of fertilizer can increase risks of negative environmental effects. These factors, along with the environmental and energy costs of synthesizing nitrogen fertilizer, led us to seek out novel biotechnology-driven approaches to supply nitrogen to plants. The strategy we focused on involves transgenic expression of nitrogenase, a bacterial multi-subunit enzyme that can capture atmospheric nitrogen. Here we report expression of the active Fe subunit of nitrogenase via integration into the tobacco plastid genome of bacterial gene sequences modified for expression in plastid. Our study suggests that it will be possible to engineer plants that are able to produce their own nitrogen fertilizer by expressing nitrogenase genes in plant plastids. PMID:27529475

  15. A Fungal Symbiont of Plant-Roots Modulates Mycotoxin Gene Expression in the Pathogen Fusarium sambucinum

    PubMed Central

    Ismail, Youssef; McCormick, Susan; Hijri, Mohamed

    2011-01-01

    Fusarium trichothecenes are fungal toxins that cause disease on infected plants and, more importantly, health problems for humans and animals that consume infected fruits or vegetables. Unfortunately, there are few methods for controlling mycotoxin production by fungal pathogens. In this study, we isolated and characterized sixteen Fusarium strains from naturally infected potato plants in the field. Pathogenicity tests were carried out in the greenhouse to evaluate the virulence of the strains on potato plants as well as their trichothecene production capacity, and the most aggressive strain was selected for further studies. This strain, identified as F. sambucinum, was used to determine if trichothecene gene expression was affected by the symbiotic Arbuscular mycorrhizal fungus (AMF) Glomus irregulare. AMF form symbioses with plant roots, in particular by improving their mineral nutrient uptake and protecting plants against soil-borne pathogens. We found that that G. irregulare significantly inhibits F. sambucinum growth. We also found, using RT-PCR assays to assess the relative expression of trichothecene genes, that in the presence of the AMF G. irregulare, F. sambucinum genes TRI5 and TRI6 were up-regulated, while TRI4, TRI13 and TRI101 were down-regulated. We conclude that AMF can modulate mycotoxin gene expression by a plant fungal pathogen. This previously undescribed effect may be an important mechanism for biological control and has fascinating implications for advancing our knowledge of plant-microbe interactions and controlling plant pathogens. PMID:21455305

  16. Physiological and gene expression responses of sunflower (Helianthus annuus L.) plants differ according to irrigation placement.

    PubMed

    Aguado, Ana; Capote, Nieves; Romero, Fernando; Dodd, Ian C; Colmenero-Flores, José M

    2014-10-01

    To investigate effects of soil moisture heterogeneity on plant physiology and gene expression in roots and leaves, three treatments were implemented in sunflower plants growing with roots split between two compartments: a control (C) treatment supplying 100% of plant evapotranspiration, and two treatments receiving 50% of plant evapotranspiration, either evenly distributed to both compartments (deficit irrigation - DI) or unevenly distributed to ensure distinct wet and dry compartments (partial rootzone drying - PRD). Plants receiving the same amount of water responded differently under the two irrigation systems. After 3 days, evapotranspiration was similar in C and DI, but 20% less in PRD, concomitant with decreased leaf water potential (Ψleaf) and increased leaf xylem ABA concentration. Six water-stress responsive genes were highly induced in roots growing in the drying soil compartment of PRD plants, and their expression was best correlated with local soil water content. On the other hand, foliar gene expression differed significantly from that of the root and correlated better with xylem ABA concentration and Ψleaf. While the PRD irrigation strategy triggered stronger physiological and molecular responses, suggesting a more intense and systemic stress reaction due to local dehydration of the dry compartment of PRD plants, the DI strategy resulted in similar water savings without strongly inducing these responses. Correlating physiological and molecular responses in PRD/DI plants may provide insights into the severity and location of water deficits and may enable a better understanding of long-distance signalling mechanisms.

  17. A fungal symbiont of plant-roots modulates mycotoxin gene expression in the pathogen Fusarium sambucinum.

    PubMed

    Ismail, Youssef; McCormick, Susan; Hijri, Mohamed

    2011-01-01

    Fusarium trichothecenes are fungal toxins that cause disease on infected plants and, more importantly, health problems for humans and animals that consume infected fruits or vegetables. Unfortunately, there are few methods for controlling mycotoxin production by fungal pathogens. In this study, we isolated and characterized sixteen Fusarium strains from naturally infected potato plants in the field. Pathogenicity tests were carried out in the greenhouse to evaluate the virulence of the strains on potato plants as well as their trichothecene production capacity, and the most aggressive strain was selected for further studies. This strain, identified as F. sambucinum, was used to determine if trichothecene gene expression was affected by the symbiotic Arbuscular mycorrhizal fungus (AMF) Glomus irregulare. AMF form symbioses with plant roots, in particular by improving their mineral nutrient uptake and protecting plants against soil-borne pathogens. We found that that G. irregulare significantly inhibits F. sambucinum growth. We also found, using RT-PCR assays to assess the relative expression of trichothecene genes, that in the presence of the AMF G. irregulare, F. sambucinum genes TRI5 and TRI6 were up-regulated, while TRI4, TRI13 and TRI101 were down-regulated. We conclude that AMF can modulate mycotoxin gene expression by a plant fungal pathogen. This previously undescribed effect may be an important mechanism for biological control and has fascinating implications for advancing our knowledge of plant-microbe interactions and controlling plant pathogens.

  18. Physiological and gene expression responses of sunflower (Helianthus annuus L.) plants differ according to irrigation placement.

    PubMed

    Aguado, Ana; Capote, Nieves; Romero, Fernando; Dodd, Ian C; Colmenero-Flores, José M

    2014-10-01

    To investigate effects of soil moisture heterogeneity on plant physiology and gene expression in roots and leaves, three treatments were implemented in sunflower plants growing with roots split between two compartments: a control (C) treatment supplying 100% of plant evapotranspiration, and two treatments receiving 50% of plant evapotranspiration, either evenly distributed to both compartments (deficit irrigation - DI) or unevenly distributed to ensure distinct wet and dry compartments (partial rootzone drying - PRD). Plants receiving the same amount of water responded differently under the two irrigation systems. After 3 days, evapotranspiration was similar in C and DI, but 20% less in PRD, concomitant with decreased leaf water potential (Ψleaf) and increased leaf xylem ABA concentration. Six water-stress responsive genes were highly induced in roots growing in the drying soil compartment of PRD plants, and their expression was best correlated with local soil water content. On the other hand, foliar gene expression differed significantly from that of the root and correlated better with xylem ABA concentration and Ψleaf. While the PRD irrigation strategy triggered stronger physiological and molecular responses, suggesting a more intense and systemic stress reaction due to local dehydration of the dry compartment of PRD plants, the DI strategy resulted in similar water savings without strongly inducing these responses. Correlating physiological and molecular responses in PRD/DI plants may provide insights into the severity and location of water deficits and may enable a better understanding of long-distance signalling mechanisms. PMID:25219304

  19. Data on keratin expression in human cells cultured with Australian native plant extracts

    PubMed Central

    Adams, Damian H.; Shou, Qingyao; Wohlmuth, Hans; Cowin, Allison J.

    2016-01-01

    Australian native plants have a long history of therapeutic use in indigenous cultures particularly for the treatment of wounds. We analysed 14 plant derived compounds from the species Pilidiostigma glabrum, Myoporum montanum, Geijera parviflora, and Rhodomyrtus psidioides for keratin 1, 5, 10 and 14 supporting the research article “Native Australian plant extracts differentially induce Collagen I and Collagen III in vitro and could be important targets for the development of new wound healing therapies” [5]. An in situ immunofluorescence assay was used in a 96 well tissue culture plate format to measure keratin expression in immortalised human keratinocytes (HaCaTs) exposed Australian native plant compounds to NMR spectra for the plant extracts are included in this article as is quantitative fluorescent intensity data of keratin 1, 5, 10 and 14 expression. PMID:27077086

  20. Transgenic plants expressing ω-ACTX-Hv1a and snowdrop lectin (GNA) fusion protein show enhanced resistance to aphids.

    PubMed

    Nakasu, Erich Y T; Edwards, Martin G; Fitches, Elaine; Gatehouse, John A; Gatehouse, Angharad M R

    2014-01-01

    Recombinant fusion proteins containing arthropod toxins have been developed as a new class of biopesticides. The recombinant fusion protein Hv1a/GNA, containing the spider venom toxin ω-ACTX-Hv1a linked to snowdrop lectin (GNA) was shown to reduce survival of the peach-potato aphid Myzus persicae when delivered in artificial diet, with survival <10% after 8 days exposure to fusion protein at 1 mg/ml. Although the fusion protein was rapidly degraded by proteases in the insect, Hv1a/GNA oral toxicity to M. persicae was significantly greater than GNA alone. A construct encoding the fusion protein, including the GNA leader sequence, under control of the constitutive CaMV 35S promoter was transformed into Arabidopsis; the resulting plants contained intact fusion protein in leaf tissues at an estimated level of 25.6 ± 4.1 ng/mg FW. Transgenic Arabidopsis expressing Hv1a/GNA induced up to 40% mortality of M. persicae after 7 days exposure in detached leaf bioassays, demonstrating that transgenic plants can deliver fusion proteins to aphids. Grain aphids (Sitobion avenae) were more susceptible than M. persicae to the Hv1a/GNA fusion protein in artificial diet bioassays (LC50 = 0.73 mg/ml after 2 days against LC50 = 1.81 mg/ml for M. persicae), as they were not able to hydrolyze the fusion protein as readily as M. persicae. Expression of this fusion protein in suitable host plants for the grain aphid is likely to confer higher levels of resistance than that shown with the M. persicae/Arabidopsis model system.

  1. PathoPlant: a platform for microarray expression data to analyze co-regulated genes involved in plant defense responses.

    PubMed

    Bülow, Lorenz; Schindler, Martin; Hehl, Reinhard

    2007-01-01

    Plants react to pathogen attack by expressing specific proteins directed toward the infecting pathogens. This involves the transcriptional activation of specific gene sets. PathoPlant, a database on plant-pathogen interactions and signal transduction reactions, has now been complemented by microarray gene expression data from Arabidopsis thaliana subjected to pathogen infection and elicitor treatment. New web tools enable identification of plant genes regulated by specific stimuli. Sets of genes co-regulated by multiple stimuli can be displayed as well. A user-friendly web interface was created for the submission of gene sets to be analyzed. This results in a table, listing the stimuli that act either inducing or repressing on the respective genes. The search can be restricted to certain induction factors to identify, e.g. strongly up- or down-regulated genes. Up to three stimuli can be combined with the option of induction factor restriction to determine similarly regulated genes. To identify common cis-regulatory elements in co-regulated genes, a resulting gene list can directly be exported to the AthaMap database for analysis. PathoPlant is freely accessible at http://www.pathoplant.de. PMID:17099232

  2. Genes Expressed Differentially in Hessian Fly Larvae Feeding in Resistant and Susceptible Plants.

    PubMed

    Chen, Ming-Shun; Liu, Sanzhen; Wang, Haiyan; Cheng, Xiaoyan; El Bouhssini, Mustapha; Whitworth, R Jeff

    2016-01-01

    The Hessian fly, Mayetiola destructor, is a destructive pest of wheat worldwide and mainly controlled by deploying resistant cultivars. In this study, we investigated the genes that were expressed differentially between larvae in resistant plants and those in susceptible plants through RNA sequencing on the Illumina platform. Informative genes were 11,832, 14,861, 15,708, and 15,071 for the comparisons between larvae in resistant versus susceptible plants for 0.5, 1, 3, and 5 days, respectively, after larvae had reached the feeding site. The transcript abundance corresponding to 5401, 6902, 8457, and 5202 of the informative genes exhibited significant differences (p ≤ 0.05) in the respective paired comparisons. Overall, genes involved in nutrient metabolism, RNA and protein synthesis exhibited lower transcript abundance in larvae from resistant plants, indicating that resistant plants inhibited nutrient metabolism and protein production in larvae. Interestingly, the numbers of cytochrome P450 genes with higher transcript abundance in larvae from resistant plants were comparable to, or higher than those with lower transcript abundance, indicating that toxic chemicals from resistant plants may have played important roles in Hessian fly larval death. Our study also identified several families of genes encoding secreted salivary gland proteins (SSGPs) that were expressed at early stage of 1(st) instar larvae and with more genes with higher transcript abundance in larvae from resistant plants. Those SSGPs are candidate effectors with important roles in plant manipulation. PMID:27529231

  3. Genes Expressed Differentially in Hessian Fly Larvae Feeding in Resistant and Susceptible Plants

    PubMed Central

    Chen, Ming-Shun; Liu, Sanzhen; Wang, Haiyan; Cheng, Xiaoyan; El Bouhssini, Mustapha; Whitworth, R. Jeff

    2016-01-01

    The Hessian fly, Mayetiola destructor, is a destructive pest of wheat worldwide and mainly controlled by deploying resistant cultivars. In this study, we investigated the genes that were expressed differentially between larvae in resistant plants and those in susceptible plants through RNA sequencing on the Illumina platform. Informative genes were 11,832, 14,861, 15,708, and 15,071 for the comparisons between larvae in resistant versus susceptible plants for 0.5, 1, 3, and 5 days, respectively, after larvae had reached the feeding site. The transcript abundance corresponding to 5401, 6902, 8457, and 5202 of the informative genes exhibited significant differences (p ≤ 0.05) in the respective paired comparisons. Overall, genes involved in nutrient metabolism, RNA and protein synthesis exhibited lower transcript abundance in larvae from resistant plants, indicating that resistant plants inhibited nutrient metabolism and protein production in larvae. Interestingly, the numbers of cytochrome P450 genes with higher transcript abundance in larvae from resistant plants were comparable to, or higher than those with lower transcript abundance, indicating that toxic chemicals from resistant plants may have played important roles in Hessian fly larval death. Our study also identified several families of genes encoding secreted salivary gland proteins (SSGPs) that were expressed at early stage of 1st instar larvae and with more genes with higher transcript abundance in larvae from resistant plants. Those SSGPs are candidate effectors with important roles in plant manipulation. PMID:27529231

  4. Transgenic sweet orange plants expressing a dermaseptin coding sequence show reduced symptoms of citrus canker disease.

    PubMed

    Furman, Nicolás; Kobayashi, Ken; Zanek, Maria Cecilia; Calcagno, Javier; Garcia, Maria Laura; Mentaberry, Alejandro

    2013-09-20

    Citrus canker provoked by Xanthomonas axonopodis pv. citri is a bacterial disease causing severe losses in all citrus-producing areas around the world. Xanthomonas infection is considered as an endemic disease in Northeast and Northwest Argentina, affecting as much as 10% of commercial citrus plantations. There is not known natural resistance neither in orange varieties nor in rootstocks used for grafting of commercial cultivars. To introduce resistance to this disease, plants of Pineapple sweet orange were transformed with a genetic construct allowing constitutive accumulation of dermaseptin. In comparison with non-transformed plants, transgenic plants showed symptom reduction levels of up to 50% in in planta assays performed under controlled conditions. PMID:23896218

  5. Sex Differences in Constitutive Autophagy

    PubMed Central

    Oliván, Sara; Calvo, Ana Cristina; Manzano, Raquel; Zaragoza, Pilar

    2014-01-01

    Sex bias has been described nowadays in biomedical research on animal models, although sexual dimorphism has been confirmed widely under pathological and physiological conditions. The main objective of our work was to study the sex differences in constitutive autophagy in spinal cord and skeletal muscle tissue from wild type mice. To examine the influence of sex on autophagy, mRNA and proteins were extracted from male and female mice tissues. The expressions of microtubule-associated protein 1 light chain 3 (LC3) and sequestosome 1 (p62), markers to monitor autophagy, were analyzed at 40, 60, 90, and 120 days of age. We found significant sex differences in the expression of LC3 and p62 in both tissues at these ages. The results indicated that sex and tissue specific differences exist in constitutive autophagy. These data underlined the need to include both sexes in the experimental groups to minimize any sex bias. PMID:24719882

  6. Inbreeding compromises host plant defense gene expression and improves herbivore survival.

    PubMed

    Portman, Scott L; Kariyat, Rupesh R; Johnston, Michelle A; Stephenson, Andrew G; Marden, James H

    2015-01-01

    Inbreeding commonly occurs in flowering plants and often results in a decline in the plant's defense response. Insects prefer to feed and oviposit on inbred plants more than outbred plants--suggesting that selecting inbred host plants offers them fitness benefits. Until recently, no studies have examined the effects of host plant inbreeding on insect fitness traits such as growth and dispersal ability. In a recent article, we documented that tobacco hornworm (Manduca sexta L.) larvae that fed on inbred horsenettle (Solanum carolinense L.) plants exhibited accelerated larval growth and increased adult flight capacity compared to larvae that fed on outbred plants. Here we report that M. sexta mortality decreased by 38.2% when larvae were reared on inbred horsenettle plants compared to larvae reared on outbreds. Additionally, inbred plants showed a notable reduction in the average relative expression levels of lipoxygenease-D (LoxD) and 12-oxophytodienoate reductase-3 (OPR3), two genes in the jasmonic acid signaling pathway that are upregulated in response to herbivore damage. Our study presents evidence that furthers our understanding of the biochemical mechanism responsible for differences in insect performance on inbred vs. outbred host plants. PMID:26039489

  7. Inbreeding compromises host plant defense gene expression and improves herbivore survival

    PubMed Central

    Portman, Scott L; Kariyat, Rupesh R; Johnston, Michelle A; Stephenson, Andrew G; Marden, James H

    2015-01-01

    Inbreeding commonly occurs in flowering plants and often results in a decline in the plant's defense response. Insects prefer to feed and oviposit on inbred plants more than outbred plants – suggesting that selecting inbred host plants offers them fitness benefits. Until recently, no studies have examined the effects of host plant inbreeding on insect fitness traits such as growth and dispersal ability. In a recent article, we documented that tobacco hornworm (Manduca sexta L.) larvae that fed on inbred horsenettle (Solanum carolinense L.) plants exhibited accelerated larval growth and increased adult flight capacity compared to larvae that fed on outbred plants. Here we report that M. sexta mortality decreased by 38.2% when larvae were reared on inbred horsenettle plants compared to larvae reared on outbreds. Additionally, inbred plants showed a notable reduction in the average relative expression levels of LIPOXYGENEASE-D (LoxD) and 12-OXOPHYTODIENOATE REDUCTASE-3 (OPR3), two genes in the jasmonic acid signaling pathway that are upregulated in response to herbivore damage. Our study presents evidence that furthers our understanding of the biochemical mechanism responsible for differences in insect performance on inbred vs. outbred host plants. PMID:26039489

  8. Role of plant expression systems in antibody production for passive immunization.

    PubMed

    Virdi, Vikram; Depicker, Ann

    2013-01-01

    Passive immunization is a method to achieve immediate protection against infectious agents by administering pathogen-specific antibodies. It has proven to be lifesaving for many acute infections, and it is now also used for cancer treatment. Passive immunization therapies, however, are extremely expensive because they require large amounts of specific antibodies that are produced predominantly in mammalian expression systems. The cost for manufacturing plant-made antibodies is estimated to be comparatively low since plant production systems require relatively less capital investments. In addition, they are not prone to mammalian pathogens, which also eases downstream processing along with making it a safe expression system. Moreover, some of the recent developments in transient expression have enabled rapid, cGMP (current Good Manufacturing Practices) compliant manufacturing of antibodies. Whether lower production costs will be reflected in a lower market price for purified antibodies will be known when more plant-produced antibodies come to the market. Promisingly, the current molecular techniques in the field of in planta expression have enabled high-level production of a variety of antibodies in different plant organs, like roots/tubers/fruits, leaves and seeds, of a variety of plants, like potato, tobacco, maize, rice, tomato and pea, providing a very wide range of possible plant-based passive immunization therapies. For instance, the production of antibodies in edible tissues would allow for a unique, convenient, needle-less, oral passive immunization at the gastric mucosal surface. The technological advances, together with the innate capacity of plant tissues to assemble complex antibodies, will enable carving a niche in the antibody market. This non-exhaustive review aims to shed light on the role of plants as a flexible expression system for passive immunotherapy, which we envisage to progress alongside the conventional production platforms to manufacture

  9. Post-synthetic modification of plant cell walls by expression of microbial hydrolases in the apoplast.

    PubMed

    Pogorelko, Gennady; Fursova, Oksana; Lin, Ming; Pyle, Eric; Jass, Johanna; Zabotina, Olga A

    2011-11-01

    The systematic creation of defined cell wall modifications in the model plant Arabidopsis thaliana by expression of microbial hydrolases with known specific activities is a promising approach to examine the impacts of cell wall composition and structure on both plant fitness and cell wall recalcitrance. Moreover, this approach allows the direct evaluation in living plants of hydrolase specificity, which can differ from in vitro specificity. To express genes encoding microbial hydrolases in A. thaliana, and target the hydrolases to the apoplast compartment, we constructed an expression cassette composed of the Cauliflower Mosaic Virus 35S RNA promoter, the A. thaliana β-expansin signal peptide, and the fluorescent marker protein YFP. Using this construct we successfully introduced into Colombia-0 plants three Aspergillus nidulans hydrolases, β-xylosidase/α-arabinosidase, feruloyl esterase, acetylxylan esterase, and a Xanthomonas oryzae putative a-L: -arabinofuranosidase. Fusion with YFP permitted quick and easy screening of transformants, detection of apoplastic localization, and protein size confirmation. Compared to wild-type Col-0, all transgenic lines showed a significant increase in the corresponding hydrolytic activity in the apoplast and changes in cell wall composition. Examination of hydrolytic activity in the transgenic plants also showed, for the first time, that the X. oryzae gene indeed encoded an enzyme with α-L: -arabinofuranosidase activity. None of the transgenic plants showed a visible phenotype; however, the induced compositional changes increased the degradability of biomass from plants expressing feruloyl esterase and β-xylosidase/α-arabinosidase. Our results demonstrate the viability of creating a set of transgenic A. thaliana plants with modified cell walls to use as a toolset for investigation of how cell wall composition contributes to recalcitrance and affects plant fitness.

  10. Functional network analysis of genes differentially expressed during xylogenesis in soc1ful woody Arabidopsis plants.

    PubMed

    Davin, Nicolas; Edger, Patrick P; Hefer, Charles A; Mizrachi, Eshchar; Schuetz, Mathias; Smets, Erik; Myburg, Alexander A; Douglas, Carl J; Schranz, Michael E; Lens, Frederic

    2016-06-01

    Many plant genes are known to be involved in the development of cambium and wood, but how the expression and functional interaction of these genes determine the unique biology of wood remains largely unknown. We used the soc1ful loss of function mutant - the woodiest genotype known in the otherwise herbaceous model plant Arabidopsis - to investigate the expression and interactions of genes involved in secondary growth (wood formation). Detailed anatomical observations of the stem in combination with mRNA sequencing were used to assess transcriptome remodeling during xylogenesis in wild-type and woody soc1ful plants. To interpret the transcriptome changes, we constructed functional gene association networks of differentially expressed genes using the STRING database. This analysis revealed functionally enriched gene association hubs that are differentially expressed in herbaceous and woody tissues. In particular, we observed the differential expression of genes related to mechanical stress and jasmonate biosynthesis/signaling during wood formation in soc1ful plants that may be an effect of greater tension within woody tissues. Our results suggest that habit shifts from herbaceous to woody life forms observed in many angiosperm lineages could have evolved convergently by genetic changes that modulate the gene expression and interaction network, and thereby redeploy the conserved wood developmental program. PMID:26952251

  11. Methods for analysis of gene expression in plants using MPSS.

    PubMed

    Nobuta, Kan; Vemaraju, Kalyan; Meyers, Blake C

    2007-01-01

    Massively parallel signature sequencing (MPSS) is a technology capable of sequencing simultaneously almost all the DNA molecules in a sample. This technology is well suited for deep profiling of mRNA and small RNA by the sequencing of cDNA tags. A series of mRNA MPSS databases has been created from various libraries derived from four different species (Arabidopsis, rice, grape, and Magnaporthe grisea, the rice blast fungus). Our mRNA MPSS databases measure the absolute expression level of most genes in the sample and provide information about potentially novel transcripts (antisense transcripts, alternative splice isoforms, and regulatory intergenic transcripts). In addition to these data, we have recently built an extensive database from MPSS-derived Arabidopsis small RNA samples. All the databases are accessible thorough our Web interface (http://mpss.udel.edu), and the individual pages are equipped with various graphical and analytical tools. Here, we focus on a subset of these tools (e.g., Gene Analysis [GA], Chromosome Viewer [CV], and Library Analysis [LIBAN]) and describe how the users can analyze and interpret our MPSS expression data.

  12. Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma

    PubMed Central

    Hallberg, Andrea R; Vorrink, Sabine U; Hudachek, Danielle R; Cramer-Morales, Kimberly; Milhem, Mohammed M; Cornell, Robert A; Domann, Frederick E

    2014-01-01

    Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs. PMID:25625848

  13. Studies on gene expressions analyses for Arabidopsis thaliana plants stimulated by space flight condition

    NASA Astrophysics Data System (ADS)

    Lu, Jinying; Liu, Min; Pan, Yi; Li, Huasheng

    We carried out whole-genome microarray to screen the transcript profile of Arabidopsis thaliana seedlings after three treatment: space microgravity condition( Seedlings grown in microgravity state of space flight of SIMBOX on Shenzhou-8), 1g centrifugal force in space(Seedlings grown in 1g centrifugal force state of space flight of SIMBOX on Shenzhou-8) and ground control. The result of microarray analysis is as followed: There were 368 genes significantly differentially expressed in space microgravity condition compared with that in 1g centrifuge space condition. Space radiation caused 246 genes significantly differentially expressed between seedlings in 1g centrifuge space condition and ground control. Space conditions (including microgravity and radiation) caused 621 genes significantly differentially expressed between seedlings in space microgravity condition and ground control. Microgravity and radiation as a single factor can cause plant gene expression change, but two factors synergism can produce some new effects on plant gene expression. The function of differential expression genes were analyst by bioinformatics, and we found the expression of genes related with stress were more different, such as the dehydration of protein (dehydrin Xero2) expression is up-regulated 57 times; low-temperature-induced protein expression is up-regulated in 49 times; heat shock protein expression is up-regulated 20 times; transcription factor DREB2A expression increase 25 times; protein phosphatase 2C expression is up-regulated 14 times; transcription factor NAM-like protein expression is up-regulated 13 times; cell wall metabolism related genes (xyloglucan, endo-1, 4-beta-D-glucanase) expression is down-regulated in 15 times. The results provide scientific data for the mechanism of space mutation.

  14. A Novel Moderate Constitutive Promoter Derived from Poplar (Populus tomentosa Carrière)

    PubMed Central

    Chen, Zhong; Wang, Jia; Ye, Mei-Xia; Li, Hao; Ji, Le-Xiang; Li, Ying; Cui, Dong-Qing; Liu, Jun-Mei; An, Xin-Min

    2013-01-01

    A novel sequence that functions as a promoter element for moderate constitutive expression of transgenes, designated as the PtMCP promoter, was isolated from the woody perennial Populus tomentosa. The PtMCP promoter was fused to the GUS reporter gene to characterize its expression pattern in different species. In stable Arabidopsis transformants, transcripts of the GUS reporter gene could be detected by RT-PCR in the root, stem, leaf, flower and silique. Further histochemical and fluorometric GUS activity assays demonstrated that the promoter could direct transgene expression in all tissues and organs, including roots, stems, rosette leaves, cauline leaves and flowers of seedlings and maturing plants. Its constitutive expression pattern was similar to that of the CaMV35S promoter, but the level of GUS activity was significantly lower than in CaMV35S promoter::GUS plants. We also characterized the promoter through transient expression in transgenic tobacco and observed similar expression patterns. Histochemical GUS staining and quantitative analysis detected GUS activity in all tissues and organs of tobacco, including roots, stems, leaves, flower buds and flowers, but GUS activity in PtMCP promoter::GUS plants was significantly lower than in CaMV35S promoter::GUS plants. Our results suggested that the PtMCP promoter from poplar is a constitutive promoter with moderate activity and that its function is presumably conserved in different species. Therefore, the PtMCP promoter may provide a practical choice to direct moderate level constitutive expression of transgenes and could be a valuable new tool in plant genetic engineering. PMID:23507754

  15. Loss of aquaporin 3 protein expression constitutes an independent prognostic factor for progression-free survival: an immunohistochemical study on stage pT1 urothelial bladder cancer

    PubMed Central

    2012-01-01

    Background Treatment of patients with stage pT1 urothelial bladder cancer (UBC) continues to be a challenge due to its unpredictable clinical course. Reliable molecular markers that help to determine appropriate individual treatment are still lacking. Loss of aquaporin (AQP) 3 protein expression has previously been shown in muscle-invasive UBC. The aim of the present study was to investigate the prognostic value of AQP3 protein expression with regard to the prognosis of stage pT1 UBC. Method AQP 3 protein expression was investigated by immunohistochemistry in specimens of 87 stage T1 UBC patients, who were diagnosed by transurethral resection of the bladder (TURB) and subsequent second resection at a high-volume urological centre between 2002 and 2009. Patients underwent adjuvant instillation therapy with Bacillus Calmette-Guérin (BCG). Loss of AQP3 protein expression was defined as complete absence of the protein within the whole tumour. Expression status was correlated retrospectively with clinicopathological and follow-up data (median: 31 months). Multivariate Cox regression analysis was used to assess the value of AQP3 tumour expression with regard to recurrence-free (RFS), progression-free (PFS) and cancer-specific survival (CSS). RFS, PFS and CSS were calculated by Kaplan-Meier analysis and Log rank test. Results 59% of patients were shown to exhibit AQP3-positive tumours, whereas 41% of tumours did not express the marker. Loss of AQP3 protein expression was associated with a statistically significantly worse PFS (20% vs. 72%, p=0.020). This finding was confirmed by multivariate Cox regression analysis (HR 7.58, CI 1.29 – 44.68; p=0.025). Conclusions Loss of AQP3 protein expression in pT1 UBC appears to play a key role in disease progression and is associated with worse PFS. Considering its potential prognostic value, assessment of AQP3 protein expression could be used to help stratify the behavior of patients with pT1 UBC. PMID:23043286

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

    PubMed

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

    2015-09-01

    Production of novel value-added compounds in transgenic crops has become an increasingly viable approach in recent years. However, in many cases, product yield still falls short of the levels necessary for optimal profitability. Determination of the limiting factors is thus of supreme importance for the long-term viability of this approach. A significant challenge to most metabolic engineering projects is the need for strong coordinated co-expression of multiple transgenes. Strong constitutive promoters have been well-characterized during the >30 years since plant transformation techniques were developed. However, organ- or tissue-specific promoters are poorly characterized in many cases. Oilseeds are one such example. Reports spanning at least 20 years have described the use of certain seed-specific promoters to drive expression of individual transgenes. Multi-gene engineering strategies are often hampered by sub-optimal expression levels or improper tissue-specificity of particular promoters, or rely on the use of multiple copies of the same promoter, which can result in DNA instability or transgene silencing. We describe here a flexible system of plasmids that allows for expression of 1-7 genes per binary plasmid, and up to 18 genes altogether after multiple rounds of transformation or sexual crosses. This vector system includes six seed-specific promoters and two constitutive promoters. Effective constitutive and seed-specific RNA interference gene-suppression cloning vectors were also constructed for silencing of endogenous genes. Taken together, this molecular toolkit allows combinatorial cloning for multiple transgene expression in seeds, vegetative organs, or both simultaneously, while also providing the means to coordinately overexpress some genes while silencing others. PMID:26254605

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

    PubMed

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

    2015-09-01

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

  18. cpg15 and cpg15-2 constitute a family of activity-regulated ligands expressed differentially in the nervous system to promote neurite growth and neuronal survival.

    PubMed

    Fujino, Tadahiro; Wu, Zhen; Lin, Walter C; Phillips, Marnie A; Nedivi, Elly

    2008-04-10

    Many ligands that affect nervous system development are members of gene families that function together to coordinate the assembly of complex neural circuits. cpg15/neuritin encodes an extracellular ligand that promotes neurite growth, neuronal survival, and synaptic maturation. Here we identify cpg15-2 as the only paralogue of cpg15 in the mouse and human genome. Both genes are expressed predominantly in the nervous system, where their expression is regulated by activity. cpg15-2 expression increases by more than twofold in response to kainate-induced seizures and nearly fourfold in the visual cortex in response to 24 hours of light exposure following dark adaptation. cpg15 and cpg15-2 diverge in their spatial and temporal expression profiles. cpg15-2 mRNA is most abundant in the retina and the olfactory bulb, as opposed to the cerebral cortex and the hippocampus for cpg15. In the retina, they differ in their cell-type specificity. cpg15 is expressed in retinal ganglion cells, whereas cpg15-2 is predominantly in bipolar cells. Developmentally, onset of cpg15-2 expression is delayed compared with cpg15 expression. CPG15-2 is glycosylphosphatidylinositol (GPI) anchored to the cell membrane and, like CPG15, can be released in a soluble-secreted form, but with lower efficiency. CPG15 and CPG15-2 were found to form homodimers and heterodimers with each other. In hippocampal explants and dissociated cultures, CPG15 and CPG15-2 promote neurite growth and neuronal survival with similar efficacy. Our findings suggest that CPG15 and CPG15-2 perform similar cellular functions but may play distinct roles in vivo through their cell-type- and tissue-specific transcriptional regulation. PMID:18265009

  19. Expression of plant ferredoxin-like protein (PFLP) enhances tolerance to heat stress in Arabidopsis thaliana.

    PubMed

    Lin, Yi-Hsien; Huang, Li-Fen; Hase, Tashiharu; Huang, Hsiang-En; Feng, Teng-Yung

    2015-03-25

    Under adverse environments, plants produce reactive oxygen species (ROS), which can trigger cell death when their accumulation surpasses the antioxidant capacity of ROS scavenging systems. These systems function in chloroplasts mainly through the ascorbate-mediated water-water cycle, in which ascorbate is photoreduced by ferredoxin in the photosynthetic system. Our previous study showed that the fraction of the reduced form of ascorbate was increased in ferredoxin-transgenic Arabidopsis (CPF) plants which overexpressed plant ferredoxin-like protein (PFLP) in their chloroplasts. Thus, we hypothesized that expression of PFLP could alter the tolerance of plants to abiotic stresses through increasing reduced form of ascorbate. In this study, we found that two CPF lines exhibited lower mortality rates at five days, following two days of heat treatment. Compared to non-transgenic wild type (Col-0) plants, CPF plants exhibited decreased H2O2 content, MDA accumulation, and ion leakage after heat treatment. To confirm the efficacy of ferredoxin against heat stress in chloroplasts, we evaluated two RNA interference (RNAi) lines on two endogenous ferredoxin isoforms, Atfd1 or Atfd2, of Arabidopsis plants. Both lines not only decreased their amounts of ascorbate, but also exhibited adverse reactions following heat treatment. Based on these results, we conclude that expression of PFLP in chloroplasts can confer tolerance to heat stress. This tolerance might be associated with the increasing of ascorbate in plants.

  20. Using HEK293T Expression System to Study Photoactive Plant Cryptochromes

    PubMed Central

    Yang, Liang; Wang, Xu; Deng, Weixian; Mo, Weiliang; Gao, Jie; Liu, Qing; Zhang, Chuanyu; Wang, Qin; Lin, Chentao; Zuo, Zecheng

    2016-01-01

    Cryptochromes are photolyase-like blue light receptors that are conserved in plants and animals. Although the light-dependent catalytic mechanism of photolyase is well studied, the photochemical mechanism of cryptochromes remains largely unknown. Lack of an appropriate protein expression system to obtain photochemically active cryptochrome holoproteins is a technical obstacle for the study of plant cryptochromes. We report here an easy-to-use method to express and study Arabidopsis cryptochrome in HEK293T cells. Our results indicate that Arabidopsis cryptochromes expressed in HEK293T are photochemically active. We envision a broad use of this method in the functional investigation of plant proteins, especially in the large-scale analyses of photochemical activities of cryptochromes such as blue light-dependent protein–protein interactions. PMID:27446167

  1. Constitutively overexpressing a tomato fructokinase gene (lefrk1) in cotton (Gossypium hirsutum L. cv. coker 312) positively affects plant vegetative growth, boll number and seed cotton yield.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Increasing fructokinase (FRK) activity in cotton (Gossypium hirsutum L.) plants may reduce fructose inhibition of sucrose synthase (Sus) and lead to improved fibre yield and quality. Cotton was transformed with a tomato (Solanum lycopersicum L.) fructokinase gene (LeFRK1) under the control of the C...

  2. Genetically stable expression of functional miraculin, a new type of alternative sweetener, in transgenic tomato plants.

    PubMed

    Sun, Hyeon-Jin; Kataoka, Hiroshi; Yano, Megumu; Ezura, Hiroshi

    2007-11-01

    Miraculin is a taste-modifying protein isolated from the red berries of Richadella dulcifica, a shrub native to West Africa. Miraculin by itself is not sweet, but it is able to turn a sour taste into a sweet taste. This unique property has led to increasing interest in this protein. In this article, we report the high-yield production of miraculin in transgenic tomato plants. High and genetically stable expression of miraculin was confirmed by Western blot analysis and enzyme-linked immunosorbent assay. Recombinant miraculin accumulated to high levels in leaves and fruits, up to 102.5 and 90.7 microg/g fresh weight, respectively. Purified recombinant miraculin expressed in transgenic tomato plants showed strong sweetness-inducing activity, similar to that of native miraculin. These results demonstrate that recombinant miraculin was correctly processed in transgenic tomato plants, and that this production system could be a good alternative to production from the native plant. PMID:17692073

  3. Delay of Disease Development in Transgenic Plants that Express the Tobacco Mosaic Virus Coat Protein Gene

    NASA Astrophysics Data System (ADS)

    Powell Abel, Patricia; Nelson, Richard S.; de, Barun; Hoffmann, Nancy; Rogers, Stephen G.; Fraley, Robert T.; Beachy, Roger N.

    1986-05-01

    A chimeric gene containing a cloned cDNA of the coat protein (CP) gene of tobacco mosaic virus (TMV) was introduced into tobacco cells on a Ti plasmid of Agrobacterium tumefaciens from which tumor inducing genes had been removed. Plants regenerated from transformed cells expressed TMV mRNA and CP as a nuclear trait. Seedlings from self-fertilized transgenic plants were inoculated with TMV and observed for development of disease symptoms. The seedlings that expressed the CP gene were delayed in symptom development and 10 to 60 percent of the transgenic plants failed to develop symptoms for the duration of the experiments. Increasing the concentration of TMV in the inoculum shortened the delay in appearance of symptoms. The results of these experiments indicate that plants can be genetically transformed for resistance to virus disease development.

  4. The Constitution by Cell

    ERIC Educational Resources Information Center

    Greenhut, Stephanie; Jones, Megan

    2010-01-01

    On their visit to the National Archives Experience in Washington, D.C., students in Jenni Ashley and Gay Brock's U.S. history classes at the Potomac School in McLean, Virginia, participated in a pilot program called "The Constitution by Cell." Armed with their cell phones, a basic understanding of the Constitution, and a willingness to participate…

  5. Interpreting the Constitution.

    ERIC Educational Resources Information Center

    Brennan, William J., Jr.

    1987-01-01

    Discusses constitutional interpretations relating to capital punishment and protection of human dignity. Points out the document's effectiveness in creating a new society by adapting its principles to current problems and needs. Considers two views of the Constitution that lead to controversy over the legitimacy of judicial decisions. (PS)

  6. Teaching the Constitution.

    ERIC Educational Resources Information Center

    Weatherman, Donald V.

    1987-01-01

    Courses on the Constitution must focus on the principles of government. Those principles and how the understanding of those principles shaped the document are appropriate subjects for consideration. The best sources for an examination of the Constitution are "The Records of the Federal Convention of 1787" and "The Federalist." (MLW)

  7. Rat mammary carcinogenesis induced by in situ expression of constitutive Raf kinase activity is prevented by tethering Raf to the plasma membrane.

    PubMed

    McFarlin, Daniel R; Gould, Michael N

    2003-06-01

    Mammary carcinogenesis induced through expression of activated Raf was investigated using a model in which retroviral vectors were infused into the central ducts of rat mammary glands. This model allows efficient expression of experimental proteins in a small fraction of endogenous mammary epithelial cells in situ. We previously reported that Raf is the dominant oncogenic signaling pathway from activated Ras in rat mammary glands. We show here that mammary gland carcinogenesis is rapidly induced by the expression of c-Raf-1 kinase that is activated by N-terminal truncation (Delta-Raf). Interestingly, targeting Raf to the plasma membrane via C-terminal fusion with Ras membrane localization signals (Raf-Caax) induces Raf kinase activity that transforms 3T3 cells more frequently than Delta-Raf, yet in situ expression of Raf-Caax does not induce mammary carcinomas. To investigate these contrasting results and begin elucidating the mechanisms of Raf-induced mammary carcinogenesis, we combined both activating mutations (N-terminal truncation and C-terminal membrane localization motifs) in one Raf construct (Delta-Raf-Caax). While Delta-Raf-Caax transforms 3T3 cells more efficiently than Delta-Raf or Raf-Caax, in situ expression of Delta-Raf-Caax does not induce carcinomas in vivo, demonstrating that lipid modification on the C-terminus of Delta-Raf negates its oncogenic potential in rat mammary gland.

  8. Survival relative to new and ancestral host plants, phytoplasma infection, and genetic constitution in host races of a polyphagous insect disease vector.

    PubMed

    Maixner, Michael; Albert, Andreas; Johannesen, Jes

    2014-08-01

    Dissemination of vectorborne diseases depends strongly on the vector's host range and the pathogen's reservoir range. Because vectors interact with pathogens, the direction and strength of a vector's host shift is vital for understanding epidemiology and is embedded in the framework of ecological specialization. This study investigates survival in host-race evolution of a polyphagous insect disease vector, Hyalesthes obsoletus, whether survival is related to the direction of the host shift (from field bindweed to stinging nettle), the interaction with plant-specific strains of obligate vectored pathogens/symbionts (stolbur phytoplasma), and whether survival is related to genetic differentiation between the host races. We used a twice repeated, identical nested experimental design to study survival of the vector on alternative hosts and relative to infection status. Survival was tested with Kaplan-Meier analyses, while genetic differentiation between vector populations was quantified with microsatellite allele frequencies. We found significant direct effects of host plant (reduced survival on wrong hosts) and sex (males survive longer than females) in both host races and relative effects of host (nettle animals more affected than bindweed animals) and sex (males more affected than females). Survival of bindweed animals was significantly higher on symptomatic than nonsymptomatic field bindweed, but in the second experiment only. Infection potentially had a positive effect on survival in nettle animals but due to low infection rates the results remain suggestive. Genetic differentiation was not related to survival. Greater negative plant-transfer effect but no negative effect of stolbur in the derived host race suggests preadaptation to the new pathogen/symbiont strain before strong diversifying selection during the specialization process. Physiological maladaptation or failure to accept the ancestral plant will have similar consequences, namely positive assortative

  9. Gene expression from plants grown on the International Space Station

    NASA Astrophysics Data System (ADS)

    Stimpson, Alexander; Pereira, Rhea; Kiss, John Z.; Correll, Melanie

    Three experiments were performed on the International Space Station (ISS) in 2006 as part of the TROPI experiments. These experiments were performed to study graviTROPIsm and photoTROPIsm responses of Arabidopsis in microgravity (µg). Seedlings were grown with a variety of light and gravitational treatments for approximately five days. The frozen samples were returned to Earth during three space shuttle missions in 2007 and stored at -80° C. Due to the limited amount of plant biomass returned, new protocols were developed to minimize the amount of material needed for RNA extraction as a preparation for microarray analysis. Using these new protocols, RNA was extracted from several sets of seedlings grown in red light followed by blue light with one sample from 1.0g treatment and the other at µg. Using a 2-fold change criterion, microarray (Affymetrix, GeneChip) results showed that 613 genes were upregulated in the µg sample while 757 genes were downregulated. Upregulated genes in response to µg included transcription factors from the WRKY (15 genes), MYB (3) and ZF (8) families as well as those that are involved in auxin responses (10). Downregulated genes also included transcription factors such as MYB (5) and Zinc finger (10) but interestingly only two WRKY family genes were down-regulated during the µg treatment. Studies are underway to compare these results with other samples to identify the genes involved in the gravity and light signal transduction pathways (this project is Supported By: NASA NCC2-1200).

  10. Determinants of nucleosome positioning and their influence on plant gene expression.

    PubMed

    Liu, Ming-Jung; Seddon, Alexander E; Tsai, Zing Tsung-Yeh; Major, Ian T; Floer, Monique; Howe, Gregg A; Shiu, Shin-Han

    2015-08-01

    Nucleosome positioning influences the access of transcription factors (TFs) to their binding sites and gene expression. Studies in plant, animal, and fungal models demonstrate similar nucleosome positioning patterns along genes and correlations between occupancy and expression. However, the relationships among nucleosome positioning, cis-regulatory element accessibility, and gene expression in plants remain undefined. Here we showed that plant nucleosome depletion occurs on specific 6-mer motifs and this sequence-specific nucleosome depletion is predictive of expression levels. Nucleosome-depleted regions in Arabidopsis thaliana tend to have higher G/C content, unlike yeast, and are centered on specific G/C-rich 6-mers, suggesting that intrinsic sequence properties, such as G/C content, cannot fully explain plant nucleosome positioning. These 6-mer motif sites showed higher DNase I hypersensitivity and are flanked by strongly phased nucleosomes, consistent with known TF binding sites. Intriguingly, this 6-mer-specific nucleosome depletion pattern occurs not only in promoter but also in genic regions and is significantly correlated with higher gene expression level, a phenomenon also found in rice but not in yeast. Among the 6-mer motifs enriched in genes responsive to treatment with the defense hormone jasmonate, there are no significant changes in nucleosome occupancy, suggesting that these sites are potentially preconditioned to enable rapid response without changing chromatin state significantly. Our study provides a global assessment of the joint contribution of nucleosome occupancy and motif sequences that are likely cis-elements to the control of gene expression in plants. Our findings pave the way for further understanding the impact of chromatin state on plant transcriptional regulatory circuits.

  11. Phylogenetic analyses and expression studies reveal two distinct groups of calreticulin isoforms in higher plants.

    PubMed

    Persson, Staffan; Rosenquist, Magnus; Svensson, Karin; Galvão, Rafaelo; Boss, Wendy F; Sommarin, Marianne

    2003-11-01

    Calreticulin (CRT) is a multifunctional protein mainly localized to the endoplasmic reticulum in eukaryotic cells. Here, we present the first analysis, to our knowledge, of evolutionary diversity and expression profiling among different plant CRT isoforms. Phylogenetic studies and expression analysis show that higher plants contain two distinct groups of CRTs: a CRT1/CRT2 group and a CRT3 group. To corroborate the existence of these isoform groups, we cloned a putative CRT3 ortholog from Brassica rapa. The CRT3 gene appears to be most closely related to the ancestral CRT gene in higher plants. Distinct tissue-dependent expression patterns and stress-related regulation were observed for the isoform groups. Furthermore, analysis of posttranslational modifications revealed differences in the glycosylation status among members within the CRT1/CRT2 isoform group. Based on evolutionary relationship, a new nomenclature for plant CRTs is suggested. The presence of two distinct CRT isoform groups, with distinct expression patterns and posttranslational modifications, supports functional specificity among plant CRTs and could account for the multiple functional roles assigned to CRTs.

  12. Synthetic pheromones and plant volatiles alter the expression of chemosensory genes in Spodoptera exigua

    PubMed Central

    Wan, Xinlong; Qian, Kai; Du, Yongjun

    2015-01-01

    Pheromone and plant odorants are important for insect mating, foraging food sources and oviposition. To understand the molecular mechanisms regulating pheromone and odorant signaling, we employed qRT-PCR to study the circadian rhythms of ABP, OBP, PBP, and OR gene expression in the beet armyworm, Spodoptera exigua and their responses after a pre-exposure to sex pheromone compounds or plant volatiles. The neuronal responses of male S. exigua to 20 chemical compounds were recorded at three specific time periods using the electroantennogram. The results showed a circadian rhythm in the expression profiles of some chemosensory genes in the antennae similar to their behavioral rhythm. The expression profiles of OR3, OR6, OR11, OR13, OR16, OR18, Orco, ABP2, OBP1, OBP7, and PBP1, and EAG responses to chemical compounds, as well as their circadian rhythm were significantly affected after exposure to synthetic sex pheromones and plant volatiles. These findings provide the first evidence that the gene expression of chemosensory genes and olfactory sensitivity to sex pheromones are affected by pre-exposing insects to pheromone compounds and plant volatiles. It helps to understand the molecular mechanisms underlying pheromone activity, and the application of sex pheromones and plant volatiles in mating disruption or mass trapping. PMID:26611815

  13. A biomarker based on gene expression indicates plant water status in controlled and natural environments.

    PubMed

    Marchand, Gwenaëlle; Mayjonade, Baptiste; Varès, Didier; Blanchet, Nicolas; Boniface, Marie-Claude; Maury, Pierre; Nambinina Andrianasolo, Fety; Nambinina, Fety Andrianasolo; Burger, Philippe; Debaeke, Philippe; Casadebaig, Pierre; Vincourt, Patrick; Langlade, Nicolas B

    2013-12-01

    Plant or soil water status is required in many scientific fields to understand plant responses to drought. Because the transcriptomic response to abiotic conditions, such as water deficit, reflects plant water status, genomic tools could be used to develop a new type of molecular biomarker. Using the sunflower (Helianthus annuus L.) as a model species to study the transcriptomic response to water deficit both in greenhouse and field conditions, we specifically identified three genes that showed an expression pattern highly correlated to plant water status as estimated by the pre-dawn leaf water potential, fraction of transpirable soil water, soil water content or fraction of total soil water in controlled conditions. We developed a generalized linear model to estimate these classical water status indicators from the expression levels of the three selected genes under controlled conditions. This estimation was independent of the four tested genotypes and the stage (pre- or post-flowering) of the plant. We further validated this gene expression biomarker under field conditions for four genotypes in three different trials, over a large range of water status, and we were able to correct their expression values for a large diurnal sampling period.

  14. Studies of plant gene expression and function stimulated by space microgravity

    NASA Astrophysics Data System (ADS)

    Lu, Jinying; Liu, Min; Li, Huasheng; Zhao, Hui

    2016-07-01

    One of the important questions in space biology is how plants respond to an outer space environment i.e., how genetic expression is altered in space microgravity. In this study, the transcriptome of Arabidopsis thaliana seedlings was analyzed as part of the Germany SIMBOX (Science in Microgravity Box) spaceflight experiment on Shenzhou 8. A gene chip was used to screen gene expression differences in Arabidopsis thaliana seedlings between microgravity and 1g centrifugal force in space. Microarray analysis revealed that 368 genes were differentially expressed. Gene Ontology (GO) analysis indicated that these genes were involved in the plant's response to stress, secondary metabolism, hormone metabolism, transcription, protein phosphorylation, lipid metabolism, transport and cell wall metabolism processes. Real time PCR was used to analyzed the miRNA expression including Arabidopsis miR160,miR161, miR394, miR402, miR403, and miR408. MiR408 was significantly upregulated. An overexpression vector of Arabidopsis miR408 was constructed and transferred to Arabidopsis plant. The roots of plants over expressing miR408 exhibited a slower reorientation upon gravistimulation in comparison with those of wild-type. This result indicated that miR408 could play a role in root gravitropic response.

  15. Development of Plant Gene Vectors for Tissue-Specific Expression Using GFP as a Reporter Gene

    NASA Technical Reports Server (NTRS)

    Jackson, Jacquelyn; Egnin, Marceline; Xue, Qi-Han; Prakash, C. S.

    1997-01-01

    Reporter genes are widely employed in plant molecular biology research to analyze gene expression and to identify promoters. Gus (UidA) is currently the most popular reporter gene but its detection requires a destructive assay. The use of jellyfish green fluorescent protein (GFP) gene from Aequorea Victoria holds promise for noninvasive detection of in vivo gene expression. To study how various plant promoters are expressed in sweet potato (Ipomoea batatas), we are transcriptionally fusing the intron-modified (mGFP) or synthetic (modified for codon-usage) GFP coding regions to these promoters: double cauliflower mosaic virus 35S (CaMV 35S) with AMV translational enhancer, ubiquitin7-intron-ubiquitin coding region (ubi7-intron-UQ) and sporaminA. A few of these vectors have been constructed and introduced into E. coli DH5a and Agrobacterium tumefaciens EHA105. Transient expression studies are underway using protoplast-electroporation and particle bombardment of leaf tissues.

  16. Hateful Expression and First Amendment Values: Toward a Theory of Constitutional Constraint on Hate Speech at Colleges and Universities after R.A.V. v. St. Paul.

    ERIC Educational Resources Information Center

    Hartman, Rhonda G.

    1993-01-01

    Discussion of hate speech on college campuses focuses on whether the concept of hate speech encompasses expression deserving full First Amendment protection and thus virtual immunity from college regulation. It is argued that the Supreme Court could sustain some form of hate-speech policy on campus based on precedent elsewhere. (MSE)

  17. Dehydrin2 is a stress-inducible, whereas Dehydrin1 is constitutively expressed but up-regulated gene under varied cues in tea [Camellia sinensis (L.) O. Kuntze].

    PubMed

    Paul, Asosii; Kumar, Sanjay

    2013-05-01

    Two expressed sequence tags with similarity to dehydrin were cloned to full-length (CsDHN1 and CsDHN2) through rapid amplification of cDNA ends. CsDHN1 and CsDHN2 were 1,027 and 992 base pair long and encoded for predicted polypeptides of 260 and 201 amino acids, respectively. Deduced CsDHN1 protein had a S-segment and three lysine-rich consensus motifs (K-segments). The Y-segment was, however, absent in the deduced CsDHN1. CsDHN2 had three Y motifs, one S-segment and two K-segments. Expression of CsDHN1 was visible at all the time points of study, though up-regulation was observed in response to winter dormancy (WD) as well as abiotic stresses [low temperature, sodium chloride, polyethylene glycol, and hydrogen peroxide]. Expression of CsDHN2 was strongly up-regulated within 1 h of exposure to abiotic stresses as well in the tissue harvested during WD in contrast to the respective "control" for abiotic stresses and in tissue during the period of active growth, when the expression was not detectable.

  18. Plant pharming of a full-sized, tumour-targeting antibody using different expression strategies.

    PubMed

    Villani, Maria Elena; Morgun, Bogdan; Brunetti, Patrizia; Marusic, Carla; Lombardi, Raffaele; Pisoni, Ivan; Bacci, Camilla; Desiderio, Angiola; Benvenuto, Eugenio; Donini, Marcello

    2009-01-01

    The aims of this work were to obtain a human antibody against the tumour-associated antigen tenascin-C (TNC) and to compare the yield and quality of plant-produced antibody in either stable transgenics or using a transient expression system. To this end, the characterization of a full-sized human immunoglobulin G (IgG) [monoclonal antibody H10 (mAb H10)], derived from a selected single-chain variable fragment (scFv) and produced in plants, is presented. The human mAb gene was engineered for plant expression, and Nicotiana tabacum transgenic lines expressing both heavy (HC) and light (LC) chain were obtained and evaluated for antibody expression levels, in vivo assembly and functionality. Affinity-purified H10 from transgenics (yield, 0.6-1.1 mg/kg fresh weight) revealed that more than 90% of HC was specifically degraded, leading to the formation of functional antigen-binding fragments (Fab). Consequently, H10 was transiently expressed in Nicotiana benthamiana plants through an Agrobacterium-mediated gene-transfer system. Moreover, the use of the p19 silencing suppressor gene from artichoke mottled crinkle virus raised antibody expression levels by an order of magnitude (yields of purified H10, 50-100 mg/kg fresh weight). Approximately 75% of purified protein consisted of full-sized antibody functionally binding to TNC (K(D) = 14 nm), and immunohistochemical analysis on tumour tissues revealed specific accumulation around tumour blood vessels. The data indicate that the purification yields of mAb H10, using a transient expression system boosted by the p19 silencing suppressor, are exceptionally high when compared with the results reported previously, providing a technique for the over-expression of anticancer mAbs by a rapid, cost-effective, molecular farming approach.

  19. Plant pharming of a full-sized, tumour-targeting antibody using different expression strategies.

    PubMed

    Villani, Maria Elena; Morgun, Bogdan; Brunetti, Patrizia; Marusic, Carla; Lombardi, Raffaele; Pisoni, Ivan; Bacci, Camilla; Desiderio, Angiola; Benvenuto, Eugenio; Donini, Marcello

    2009-01-01

    The aims of this work were to obtain a human antibody against the tumour-associated antigen tenascin-C (TNC) and to compare the yield and quality of plant-produced antibody in either stable transgenics or using a transient expression system. To this end, the characterization of a full-sized human immunoglobulin G (IgG) [monoclonal antibody H10 (mAb H10)], derived from a selected single-chain variable fragment (scFv) and produced in plants, is presented. The human mAb gene was engineered for plant expression, and Nicotiana tabacum transgenic lines expressing both heavy (HC) and light (LC) chain were obtained and evaluated for antibody expression levels, in vivo assembly and functionality. Affinity-purified H10 from transgenics (yield, 0.6-1.1 mg/kg fresh weight) revealed that more than 90% of HC was specifically degraded, leading to the formation of functional antigen-binding fragments (Fab). Consequently, H10 was transiently expressed in Nicotiana benthamiana plants through an Agrobacterium-mediated gene-transfer system. Moreover, the use of the p19 silencing suppressor gene from artichoke mottled crinkle virus raised antibody expression levels by an order of magnitude (yields of purified H10, 50-100 mg/kg fresh weight). Approximately 75% of purified protein consisted of full-sized antibody functionally binding to TNC (K(D) = 14 nm), and immunohistochemical analysis on tumour tissues revealed specific accumulation around tumour blood vessels. The data indicate that the purification yields of mAb H10, using a transient expression system boosted by the p19 silencing suppressor, are exceptionally high when compared with the results reported previously, providing a technique for the over-expression of anticancer mAbs by a rapid, cost-effective, molecular farming approach. PMID:18793269

  20. Another player joins the complex field of sugar-regulated gene expression in plants

    SciTech Connect

    Gibson, Susan I.; Graham, Ian A.

    1999-04-01

    This article summarizes recent progress in understanding the molecular mechanisms by which soluble sugar levels affect plant development and gene expression. The article focuses on the role played by a newly identified protein, the PRL1 protein. The PRL1 protein has been found to interact with the SNF1 protein. Previously, SNF1 was shown to function in sugar-regulated gene expression in yeast. Mutations in the gene encoding PRL1 confer increased sensitivity to sugar and to several phytohormones.

  1. Transposon-Mediated Alteration of TaMATE1B Expression in Wheat Confers Constitutive Citrate Efflux from Root Apices[W

    PubMed Central

    Tovkach, Andriy; Ryan, Peter R.; Richardson, Alan E.; Lewis, David C.; Rathjen, Tina M.; Ramesh, Sunita; Tyerman, Stephen D.; Delhaize, Emmanuel

    2013-01-01

    The TaMATE1B gene (for multidrug and toxic compound extrusion) from wheat (Triticum aestivum) was isolated and shown to encode a citrate transporter that is located on the plasma membrane. TaMATE1B expression in roots was induced by iron deficiency but not by phosphorus deficiency or aluminum treatment. The coding region of TaMATE1B was identical in a genotype showing citrate efflux from root apices (cv Carazinho) to one that lacked citrate efflux (cv Egret). However, sequence upstream of the coding region differed between these two genotypes in two ways. The first difference was a single-nucleotide polymorphism located approximately 2 kb upstream from the start codon in cv Egret. The second difference was an 11.1-kb transposon-like element located 25 bp upstream of the start codon in cv Carazinho that was absent from cv Egret. The influence of these polymorphisms on TaMATE1B expression was investigated using fusions to green fluorescent protein expressed in transgenic lines of rice (Oryza sativa). Fluorescence measurements in roots of rice indicated that 1.5- and 2.3-kb regions upstream of TaMATE1B in cv Carazinho (which incorporated 3′ regions of the transposon-like element) generated 20-fold greater expression in the apical 1 mm of root compared with the native promoter in cv Egret. By contrast, fluorescence in more mature tissues was similar in both cultivars. The presence of the single-nucleotide polymorphism alone consistently generated 2-fold greater fluorescence than the cv Egret promoter. We conclude that the transposon-like element in cv Carazinho extends TaMATE1B expression to the root apex, where it confers citrate efflux and enhanced aluminum tolerance. PMID:23204428

  2. Expression of foreign genes in regenerated plants and in their progeny

    PubMed Central

    De Block, Marc; Herrera-Estrella, Luis; Van Montagu, Marc; Schell, Jeff; Zambryski, Patricia

    1984-01-01

    Chimeric genes comprised of the nopaline synthase promoter and bacterial coding sequences specifying resistance to kanamycin, chloramphenicol or methotrexate, were inserted into the non-oncogenic Ti plasmid vector pGV3850 by recombination (through homologous pBR322 sequences present in the chimeric gene constructs and pGV3850). These co-integrates in Agrobacterium were used to infect single plant protoplasts of Nicotiana by co-cultivation. The resistance traits allowed the selection of transformed calli in tissue culture in the presence of the appropriate antibiotic. Furthermore, as a non-oncogenic Ti plasmid was used for the protoplast transformation, phenotypically normal and fertile plants could be regenerated from the resistant calli. We have shown that these fully differentiated plant tissues exhibit functional expression of resistance traits (KmR and CmR). All plants carrying the chimeric genes developed normally, flowered, and set seeds. The inheritance of several of these resistance traits was analyzed and shown to be Mendelian. These results are model experiments to demonstrate that genes of interest can be systematically transferred to the genome of plants using non-oncogenic Ti plasmid derivatives; and that transformed plants are capable of normal growth and differentiation, thus providing a natural environment for the study of gene expression and development of plant cells. ImagesFig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6. PMID:16453538

  3. Calcium-dependent protein kinases in plants: evolution, expression and function.

    PubMed

    Valmonte, Gardette R; Arthur, Kieren; Higgins, Colleen M; MacDiarmid, Robin M

    2014-03-01

    Calcium-dependent protein kinases (CPKs) are plant proteins that directly bind calcium ions before phosphorylating substrates involved in metabolism, osmosis, hormone response and stress signaling pathways. CPKs are a large multigene family of proteins that are present in all plants studied to date, as well as in protists, oomycetes and green algae, but are not found in animals and fungi. Despite the increasing evidence of the importance of CPKs in developmental and stress responses from various plants, a comprehensive genome-wide analysis of CPKs from algae to higher plants has not been undertaken. This paper describes the evolution of CPKs from green algae to plants using a broadly sampled phylogenetic analysis and demonstrates the functional diversification of CPKs based on expression and functional studies in different plant species. Our findings reveal that CPK sequence diversification into four major groups occurred in parallel with the terrestrial transition of plants. Despite significant expansion of the CPK gene family during evolution from green algae to higher plants, there is a high level of sequence conservation among CPKs in all plant species. This sequence conservation results in very little correlation between CPK evolutionary groupings and functional diversity, making the search for CPK functional orthologs a challenge.

  4. Cotton WRKY1 mediates the plant defense-to-development transition during infection of cotton by Verticillium dahliae by activating JASMONATE ZIM-DOMAIN1 expression.

    PubMed

    Li, Chao; He, Xin; Luo, Xiangyin; Xu, Li; Liu, Linlin; Min, Ling; Jin, Li; Zhu, Longfu; Zhang, Xianlong

    2014-12-01

    Plants have evolved an elaborate signaling network to ensure an appropriate level of immune response to meet the differing demands of developmental processes. Previous research has demonstrated that DELLA proteins physically interact with JASMONATE ZIM-DOMAIN1 (JAZ1) and dynamically regulate the interaction of the gibberellin (GA) and jasmonate (JA) signaling pathways. However, whether and how the JAZ1-DELLA regulatory node is regulated at the transcriptional level in plants under normal growth conditions or during pathogen infection is not known. Here, we demonstrate multiple functions of cotton (Gossypium barbadense) GbWRKY1 in the plant defense response and during development. Although GbWRKY1 expression is induced rapidly by methyl jasmonate and infection by Verticillium dahliae, our results show that GbWRKY1 is a negative regulator of the JA-mediated defense response and plant resistance to the pathogens Botrytis cinerea and V. dahliae. Under normal growth conditions, GbWRKY1-overexpressing lines displayed GA-associated phenotypes, including organ elongation and early flowering, coupled with the down-regulation of the putative targets of DELLA. We show that the GA-related phenotypes of GbWRKY1-overexpressing plants depend on the constitutive expression of Gossypium hirsutum GhJAZ1. We also show that GhJAZ1 can be transactivated by GbWRKY1 through TGAC core sequences, and the adjacent sequences of this binding site are essential for binding specificity and affinity to GbWRKY1, as revealed by dual-luciferase reporter assays and electrophoretic mobility shift assays. In summary, our data suggest that GbWRKY1 is a critical regulator mediating the plant defense-to-development transition during V. dahliae infection by activating JAZ1 expression.

  5. Regulation of Gene Expression in Plants through miRNA Inactivation

    PubMed Central

    Zhang, Yuanji; Ziegler, Todd E.; Roberts, James K.; Heck, Gregory R.

    2011-01-01

    Eukaryotic organisms possess a complex RNA-directed gene expression regulatory network allowing the production of unique gene expression patterns. A recent addition to the repertoire of RNA-based gene regulation is miRNA target decoys, endogenous RNA that can negatively regulate miRNA activity. miRNA decoys have been shown to be a valuable tool for understanding the function of several miRNA families in plants and invertebrates. Engineering and precise manipulation of an endogenous RNA regulatory network through modification of miRNA activity also affords a significant opportunity to achieve a desired outcome of enhanced plant development or response to environmental stresses. Here we report that expression of miRNA decoys as single or heteromeric non-cleavable microRNA (miRNA) sites embedded in either non-protein-coding or within the 3′ untranslated region of protein-coding transcripts can regulate the expression of one or more miRNA targets. By altering the sequence of the miRNA decoy sites, we were able to attenuate miRNA inactivation, which allowed for fine regulation of native miRNA targets and the production of a desirable range of plant phenotypes. Thus, our results demonstrate miRNA decoys are a flexible and robust tool, not only for studying miRNA function, but also for targeted engineering of gene expression in plants. Computational analysis of the Arabidopsis transcriptome revealed a number of potential miRNA decoys, suggesting that endogenous decoys may have an important role in natural modulation of expression in plants. PMID:21731706

  6. Fall Armyworm (Lepidoptera: Noctuidae) Development, Survivorship, and Damage on Cotton Plants Expressing Insecticidal Plant-Incorporated Protectants.

    PubMed

    Hardke, Jarrod T; Jackson, Ryan E; Leonard, B Rogers; Temple, Joshua H

    2015-06-01

    Cotton, Gossypium hirsutum (L.), plants expressing insecticidal crystal (Cry) proteins of Bacillus thuringiensis (Bt) Berliner are planted on significant acreage across the southern region of the United States. Fall armyworm, Spodoptera frugiperda (J. E. Smith), can be a significant cotton pest in some years, but this species has not been a primary target of Bt cotton technologies. The objective of this study was to quantify fall armyworm larval survivorship and fruiting form injury on transgenic cotton lines expressing Cry1Ac (Bollgard), Cry1Ac+Cry2Ab (Bollgard II), and Cry1Ac+Cry1F (WideStrike) Bt proteins. Larval survivorship and fruiting form damage of fall armyworm on Bollgard, Bollgard II, WideStrike, and non-Bt (control) cotton lines were evaluated in no-choice field studies. Fall armyworm (third instars) were placed on flower buds (squares), white flowers, and bolls, enclosed within a nylon mesh exclusion cage, and evaluated at selected intervals after infestation. Exposure of fall armyworm larvae to Bollgard cotton lines generally resulted in no significant effects on survivorship compared with larvae exposed to the non-Bt cotton line. Survivorship and plant injury by fall armyworm on Bollgard II cotton lines was variable compared with that on non-Bt cotton lines, and significant differences between treatments were inconsistent. Fall armyworm had significantly lower survivorship and caused less plant injury on WideStrike cotton lines than on non-Bt cotton lines across all plant structures. Development and survivorship of fall armyworm larvae on these cotton lines also were evaluated in no-choice laboratory assays by offering the previously described fruiting forms to third instars. Bollgard II and WideStrike cotton lines significantly reduced fall armyworm development and survivorship compared with those larvae offered non-Bt tissue. These results suggest that differences exist among selected Bt cotton technologies in their performance against fall

  7. Antisense expression of the peptide transport gene AtPTR2-B delays flowering and arrests seed development in transgenic Arabidopsis plants.

    PubMed Central

    Song, W; Koh, S; Czako, M; Marton, L; Drenkard, E; Becker, J M; Stacey, G

    1997-01-01

    Previously, we identified a peptide transport gene, AtPTR2-B, from Arabidopsis thaliana that was constitutively expressed in all plant organs, suggesting an important physiological role in plant growth and development. To evaluate the function of this transporter, transgenic Arabidopsis plants were constructed expressing antisense or sense AtPTR2-B. Genomic Southern analysis indicated that four independent antisense and three independent sense AtPTR2-B transgenic lines were obtained, which was confirmed by analysis of the segregation of the kanamycin resistance gene carried on the T-DNA. RNA blot data showed that the endogenous AtPTR2-B mRNA levels were significantly reduced in transgenic leaves and flowers, but not in transgenic roots. Consistent with this reduction in endogenous AtPTR2-B mRNA levels, all four antisense lines and one sense line exhibited significant phenotypic changes, including late flowering and arrested seed development. These phenotypic changes could be explained by a defect in nitrogen nutrition due to the reduced peptide transport activity conferred by AtPTR2-B. These results suggest that AtPTR2-B may play a general role in plant nutrition. The AtPTR2-B gene was mapped to chromosome 2, which is closely linked to the restriction fragment length polymorphism marker m246. PMID:9232875

  8. Antisense expression of the peptide transport gene AtPTR2-B delays flowering and arrests seed development in transgenic Arabidopsis plants.

    PubMed

    Song, W; Koh, S; Czako, M; Marton, L; Drenkard, E; Becker, J M; Stacey, G

    1997-07-01

    Previously, we identified a peptide transport gene, AtPTR2-B, from Arabidopsis thaliana that was constitutively expressed in all plant organs, suggesting an important physiological role in plant growth and development. To evaluate the function of this transporter, transgenic Arabidopsis plants were constructed expressing antisense or sense AtPTR2-B. Genomic Southern analysis indicated that four independent antisense and three independent sense AtPTR2-B transgenic lines were obtained, which was confirmed by analysis of the segregation of the kanamycin resistance gene carried on the T-DNA. RNA blot data showed that the endogenous AtPTR2-B mRNA levels were significantly reduced in transgenic leaves and flowers, but not in transgenic roots. Consistent with this reduction in endogenous AtPTR2-B mRNA levels, all four antisense lines and one sense line exhibited significant phenotypic changes, including late flowering and arrested seed development. These phenotypic changes could be explained by a defect in nitrogen nutrition due to the reduced peptide transport activity conferred by AtPTR2-B. These results suggest that AtPTR2-B may play a general role in plant nutrition. The AtPTR2-B gene was mapped to chromosome 2, which is closely linked to the restriction fragment length polymorphism marker m246. PMID:9232875

  9. Increasing plant growth by modulating omega-amidase expression in plants

    DOEpatents

    Unkefer, Pat J.; Anderson, Penelope S.; Knight, Thomas J.

    2015-06-30

    The present disclosure relates to compositions and methods for increasing the leaf-to-root ratio of the signal metabolite 2-oxoglutaramate and related proline molecules in plants by modulating levels of .omega.-amidase to increase nitrogen use efficiency, resulting in enhanced growth, faster growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, increased tolerance to high salt conditions, and increased biomass yields.

  10. Evidence for cross-pathway regulation of metabolic gene expression in plants.

    PubMed Central

    Guyer, D; Patton, D; Ward, E

    1995-01-01

    In Arabidopsis thaliana, blocking histidine biosynthesis with a specific inhibitor of imidazoleglycerol-phosphate dehydratase caused increased expression of eight genes involved in the biosynthesis of aromatic amino acids, histidine, lysine, and purines. A decrease in expression of glutamine synthetase was also observed. Addition of histidine eliminated the gene-regulating effects of the inhibitor, demonstrating that the changes in gene expression resulted from histidine-pathway blockage. These results show that plants are capable of cross-pathway metabolic regulation. Images Fig. 2 Fig. 3 Fig. 4 PMID:7761437

  11. Constitution and Bylaws.

    ERIC Educational Resources Information Center

    Journal of Dental Education, 1981

    1981-01-01

    Constitutions and bylaws of the American Association of Dental Schools are presented, covering organization name, statement of purpose, administrative organization, offices, membership, legislative and administrative bodies, elected and appointed officers, regular and special sessions, sections, publications, election, and finance. (MSE)

  12. The 5th Symposium on Post-Transcriptional Regulation of Plant Gene Expression (PTRoPGE)

    SciTech Connect

    Karen S. Browning; Marie Petrocek; Bonnie Bartel

    2006-06-01

    The 5th Symposium on Post-Transcriptional Regulation of Plant Gene Expression (PTRoPGE) will be held June 8-12, 2005 at the University of Texas at Austin. Exciting new and ongoing discoveries show significant regulation of gene expression occurs after transcription. These post-transcriptional control events in plants range from subtle regulation of transcribed genes and phosphorylation, to the processes of gene regulation through small RNAs. This meeting will focus on the regulatory role of RNA, from transcription, through translation and finally degradation. The cross-disciplinary design of this meeting is necessary to encourage interactions between researchers that have a common interest in post-transcriptional gene expression in plants. By bringing together a diverse group of plant molecular biologist and biochemists at all careers stages from across the world, this meeting will bring about more rapid progress in understanding how plant genomes work and how genes are finely regulated by post-transcriptional processes to ultimately regulate cells.

  13. Changes in endogenous gene transcript and protein levels in maize plants expressing the soybean ferritin transgene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic agricultural crops with increased nutritive value present prospects for contributing to public health. However, their acceptance is poor in many countries due to the perception that genetic modification may cause unintended effects on expression of native genes in the host plant. Here, w...

  14. Highly effective expression of glutamine synthetase genes GS1 and GS2 in transgenic rice plants increases nitrogen-deficiency tolerance.

    PubMed

    Sun, Hui; Huang, Qi-Man; Su, Jin

    2005-10-01

    Glutamine synthetase (GS, EC6.3.1.2) is a key enzyme in ammonia assimilation both in plants and in Gram-negative microorganisms. It plays an important role in efficient use of nitrogen sources and nitrogen metabolism in organisms. Two groups of GS isoenzymes, plastidic (GS2) and cytosolic (GS1), have been identified in higher plants. A plant constitutive expression vector p2GS harboring GS1 and GS2 under the control of rice actin 1 (Act1) and maize ubiquitin (Ubi) promoters was constructed for the first time in a single plasmid, and 3 rounds of ligation and transformation were performed. There has been no report about studies on rice transformation with the two GS enzymes (GS1 and GS2). The p2GS thus constructed was introduced into rice var. Zhonghua 10 by Agrobacterium-mediated transfer method, and transgenic plants with resistance to hygromycin (Hyg) were obtained. Results of PCR and Southern blot analysis showed that the foreign genes have been integrated into the rice genome. The transcription of GS1-GS2 genes in the transformants was also confirmed by Northern blot analysis. The transgenic rice plants thus obtained can grow well in an MS medium in which the nitrogen source was replaced by (NH(4))(2)SO(4) 0.7 mmol/L, and fresh weight of the transformants was significantly higher than the control rice plants. The result suggests that expression of p2GS makes the transgenic rice plants tolerant to nitrogen-deficiency. PMID:16222091

  15. Abiotic Stresses: Insight into Gene Regulation and Protein Expression in Photosynthetic Pathways of Plants.

    PubMed

    Nouri, Mohammad-Zaman; Moumeni, Ali; Komatsu, Setsuko

    2015-01-01

    Global warming and climate change intensified the occurrence and severity of abiotic stresses that seriously affect the growth and development of plants,especially, plant photosynthesis. The direct impact of abiotic stress on the activity of photosynthesis is disruption of all photosynthesis components such as photosystem I and II, electron transport, carbon fixation, ATP generating system and stomatal conductance. The photosynthetic system of plants reacts to the stress differently, according to the plant type, photosynthetic systems (C₃ or C₄), type of the stress, time and duration of the occurrence and several other factors. The plant responds to the stresses by a coordinate chloroplast and nuclear gene expression. Chloroplast, thylakoid membrane, and nucleus are the main targets of regulated proteins and metabolites associated with photosynthetic pathways. Rapid responses of plant cell metabolism and adaptation to photosynthetic machinery are key factors for survival of plants in a fluctuating environment. This review gives a comprehensive view of photosynthesis-related alterations at the gene and protein levels for plant adaptation or reaction in response to abiotic stress.

  16. Abiotic Stresses: Insight into Gene Regulation and Protein Expression in Photosynthetic Pathways of Plants

    PubMed Central

    Nouri, Mohammad-Zaman; Moumeni, Ali; Komatsu, Setsuko

    2015-01-01

    Global warming and climate change intensified the occurrence and severity of abiotic stresses that seriously affect the growth and development of plants, especially, plant photosynthesis. The direct impact of abiotic stress on the activity of photosynthesis is disruption of all photosynthesis components such as photosystem I and II, electron transport, carbon fixation, ATP generating system and stomatal conductance. The photosynthetic system of plants reacts to the stress differently, according to the plant type, photosynthetic systems (C3 or C4), type of the stress, time and duration of the occurrence and several other factors. The plant responds to the stresses by a coordinate chloroplast and nuclear gene expression. Chloroplast, thylakoid membrane, and nucleus are the main targets of regulated proteins and metabolites associated with photosynthetic pathways. Rapid responses of plant cell metabolism and adaptation to photosynthetic machinery are key factors for survival of plants in a fluctuating environment. This review gives a comprehensive view of photosynthesis-related alterations at the gene and protein levels for plant adaptation or reaction in response to abiotic stress. PMID:26343644

  17. Abiotic Stresses: Insight into Gene Regulation and Protein Expression in Photosynthetic Pathways of Plants.

    PubMed

    Nouri, Mohammad-Zaman; Moumeni, Ali; Komatsu, Setsuko

    2015-01-01

    Global warming and climate change intensified the occurrence and severity of abiotic stresses that seriously affect the growth and development of plants,especially, plant photosynthesis. The direct impact of abiotic stress on the activity of photosynthesis is disruption of all photosynthesis components such as photosystem I and II, electron transport, carbon fixation, ATP generating system and stomatal conductance. The photosynthetic system of plants reacts to the stress differently, according to the plant type, photosynthetic systems (C₃ or C₄), type of the stress, time and duration of the occurrence and several other factors. The plant responds to the stresses by a coordinate chloroplast and nuclear gene expression. Chloroplast, thylakoid membrane, and nucleus are the main targets of regulated proteins and metabolites associated with photosynthetic pathways. Rapid responses of plant cell metabolism and adaptation to photosynthetic machinery are key factors for survival of plants in a fluctuating environment. This review gives a comprehensive view of photosynthesis-related alterations at the gene and protein levels for plant adaptation or reaction in response to abiotic stress. PMID:26343644

  18. Sequence Architecture Downstream of the Initiator Codon Enhances Gene Expression and Protein Stability in Plants1

    PubMed Central

    Sawant, Samir V.; Kiran, Kanti; Singh, Pradhyumna K.; Tuli, Rakesh

    2001-01-01

    Nucleotide positions conserved on the 3′ side of the initiator codon ATG and the corresponding N-terminal amino acid residues in a number of highly abundant plant proteins were identified by computational analysis of a dataset of highly expressed plant genes. The reporter genes uidA and gfp were modified to introduce these features. Insertion of GCT TCC TCC after the initiator codon ATG augmented expression for both the reporter genes. The insertion of each successive codon improved the expression of β-glucuronidase (GUS) in an incremental fashion in transient transformation of tobacco (Nicotiana tabacum) leaves. The insertion of alanine-serine (Ser)-Ser resulted in about a 2-fold increase in the stability of GUS. However, this did not account for the 30- to 40-fold increase in GUS activity between the constructs coding for methionine-alanine-Ser-Ser-GUS and the native enzyme. Substitution of the codon for Ser at the third amino acid residue with synonymous codons reduced GUS expression. The results suggest a role for the conserved nucleotides in the +4 to +11 region in augmenting posttranscriptional events in the expression of genes in plants. PMID:11500561

  19. BAR expressolog identification: expression profile similarity ranking of homologous genes in plant species.

    PubMed

    Patel, Rohan V; Nahal, Hardeep K; Breit, Robert; Provart, Nicholas J

    2012-09-01

    Large numbers of sequences are now readily available for many plant species, allowing easy identification of homologous genes. However, orthologous gene identification across multiple species is made difficult by evolutionary events such as whole-genome or segmental duplications. Several developmental atlases of gene expression have been produced in the past couple of years, and it may be possible to use these transcript abundance data to refine ortholog predictions. In this study, clusters of homologous genes between seven plant species - Arabidopsis, soybean, Medicago truncatula, poplar, barley, maize and rice - were identified. Following this, a pipeline to rank homologs within gene clusters by both sequence and expression profile similarity was devised by determining equivalent tissues between species, with the best expression profile match being termed the 'expressolog'. Five electronic fluorescent pictograph (eFP) browsers were produced as part of this effort, to aid in visualization of gene expression data and to complement existing eFP browsers at the Bio-Array Resource (BAR). Within the eFP browser framework, these expression profile similarity rankings were incorporated into an Expressolog Tree Viewer to allow cross-species homolog browsing by both sequence and expression pattern similarity. Global analyses showed that orthologs with the highest sequence similarity do not necessarily exhibit the highest expression pattern similarity. Other orthologs may show different expression patterns, indicating that such genes may require re-annotation or more specific annotation. Ultimately, it is envisaged that this pipeline will aid in improvement of the functional annotation of genes and translational plant research.

  20. Maize Adh-1 promoter sequences control anaerobic regulation: addition of upstream promoter elements from constitutive genes is necessary for expression in tobacco

    PubMed Central

    Ellis, J.G.; Llewellyn, D.J.; Dennis, E.S.; Peacock, W.J.

    1987-01-01

    The promoter region of a maize alcohol dehydrogenase gene (Adh-1) was linked to a reporter gene encoding chloramphenicol acetyl transferase (CAT) and transformed stably into tobacco cells using T-DNA vectors. No CAT enzyme activity could be detected in transgenic tobacco plants unless upstream promoter elements from the octopine synthase gene or the cauliflower mosaic virus 35S promoter were supplied in addition to the maize promoter region. CAT enzyme activity and transcription of the chimaeric gene were then readily detected after anaerobic induction. The first 247 bp upstream of the translation initiation codon of the maize Adh-1 gene were sufficient to impose anaerobic regulation on the hybrid gene and S1 nuclease mapping confirmed mRNA initiation is from the normal maize Adh-1 transcription start point. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6. PMID:15981329

  1. Expressed sequence tag analysis in Cycas, the most primitive living seed plant

    PubMed Central

    Brenner, Eric D; Stevenson, Dennis W; McCombie, Richard W; Katari, Manpreet S; Rudd, Stephen A; Mayer, Klaus FX; Palenchar, Peter M; Runko, Suzan J; Twigg, Richard W; Dai, Guangwei; Martienssen, Rob A; Benfey, Phillip N; Coruzzi, Gloria M

    2003-01-01

    Background Cycads are ancient seed plants (living fossils) with origins in the Paleozoic. Cycads are sometimes considered a 'missing link' as they exhibit characteristics intermediate between vascular non-seed plants and the more derived seed plants. Cycads have also been implicated as the source of 'Guam's dementia', possibly due to the production of S(+)-beta-methyl-alpha, beta-diaminopropionic acid (BMAA), which is an agonist of animal glutamate receptors. Results A total of 4,200 expressed sequence tags (ESTs) were created from Cycas rumphii and clustered into 2,458 contigs, of which 1,764 had low-stringency BLAST similarity to other plant genes. Among those cycad contigs with similarity to plant genes, 1,718 cycad 'hits' are to angiosperms, 1,310 match genes in gymnosperms and 734 match lower (non-seed) plants. Forty-six contigs were found that matched only genes in lower plants and gymnosperms. Upon obtaining the complete sequence from the clones of 37/46 contigs, 14 still matched only gymnosperms. Among those cycad contigs common to higher plants, ESTs were discovered that correspond to those involved in development and signaling in present-day flowering plants. We purified a cycad EST for a glutamate receptor (GLR)-like gene, as well as ESTs potentially involved in the synthesis of the GLR agonist BMAA. Conclusions Analysis of cycad ESTs has uncovered conserved and potentially novel genes. Furthermore, the presence of a glutamate receptor agonist, as well as a glutamate receptor-like gene in cycads, supports the hypothesis that such neuroactive plant products are not merely herbivore deterrents but may also serve a role in plant signaling. PMID:14659015

  2. Transgenic potato plants expressing cry3A gene confer resistance to Colorado potato beetle.

    PubMed

    Mi, Xiaoxiao; Ji, Xiangzhuo; Yang, Jiangwei; Liang, Lina; Si, Huaijun; Wu, Jiahe; Zhang, Ning; Wang, Di

    2015-07-01

    The Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is a fatal pest, which is a quarantine pest in China. The CPB has now invaded the Xinjiang Uygur Autonomous Region and is constantly spreading eastward in China. In this study, we developed transgenic potato plants expressing cry3A gene. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the cry3A gene expressed in leaves, stems and roots of the transgenic plants under the control of CaMV 35S promoter, while they expressed only in leaves and stems under the control of potato leaf and stem-specific promoter ST-LS1. The mortality of the larvae was higher (28% and 36%) on the transgenic plant line 35S1 on the 3rd and 4th days, and on ST3 (48%) on the 5th day after inoculation with instar larvae. Insect biomass accumulation on the foliage of the transgenic plant lines 35S1, 35S2 and ST3 was significantly lower (0.42%, 0.43% and 0.42%). Foliage consumption was lowest on transgenic lines 35S8 and ST2 among all plant foliage (7.47 mg/larvae/day and 12.46 mg/larvae/day). The different transgenic plant foliages had varied inhibition to larval growth. The survivors on the transgenic lines obviously were smaller than their original size and extremely weak. The transgenic potato plants with CPB resistance could be used to develop germplasms or varieties for controlling CPB damage and halting its spread in China. PMID:26025753

  3. Transgenic potato plants expressing cry3A gene confer resistance to Colorado potato beetle.

    PubMed

    Mi, Xiaoxiao; Ji, Xiangzhuo; Yang, Jiangwei; Liang, Lina; Si, Huaijun; Wu, Jiahe; Zhang, Ning; Wang, Di

    2015-07-01

    The Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is a fatal pest, which is a quarantine pest in China. The CPB has now invaded the Xinjiang Uygur Autonomous Region and is constantly spreading eastward in China. In this study, we developed transgenic potato plants expressing cry3A gene. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the cry3A gene expressed in leaves, stems and roots of the transgenic plants under the control of CaMV 35S promoter, while they expressed only in leaves and stems under the control of potato leaf and stem-specific promoter ST-LS1. The mortality of the larvae was higher (28% and 36%) on the transgenic plant line 35S1 on the 3rd and 4th days, and on ST3 (48%) on the 5th day after inoculation with instar larvae. Insect biomass accumulation on the foliage of the transgenic plant lines 35S1, 35S2 and ST3 was significantly lower (0.42%, 0.43% and 0.42%). Foliage consumption was lowest on transgenic lines 35S8 and ST2 among all plant foliage (7.47 mg/larvae/day and 12.46 mg/larvae/day). The different transgenic plant foliages had varied inhibition to larval growth. The survivors on the transgenic lines obviously were smaller than their original size and extremely weak. The transgenic potato plants with CPB resistance could be used to develop germplasms or varieties for controlling CPB damage and halting its spread in China.

  4. Caspase-resistant VirD2 protein provides enhanced gene delivery and expression in plants.

    PubMed

    Reavy, Brian; Bagirova, Svetlana; Chichkova, Nina V; Fedoseeva, Svetlana V; Kim, Sang Hyon; Vartapetian, Andrey B; Taliansky, Michael E

    2007-08-01

    Agrobacterium tumefaciens VirD2 protein is one of the key elements of Agrobacterium-mediated plant transformation, a process of transfer of T-DNA sequence from the Agrobacterium tumour inducing plasmid into the nucleus of infected plant cells and its integration into the host genome. The VirD2 protein has been shown to be a substrate for a plant caspase-like protease activity (PCLP) in tobacco. We demonstrate here that mutagenesis of the VirD2 protein to prevent cleavage by PCLP increases the efficiency of reporter gene transfer and expression. These results indicate that PCLP cleavage of the Agrobacterium VirD2 protein acts to limit the effectiveness of T-DNA transfer and is a novel resistance mechanism that plants utilise to combat Agrobacterium infection. PMID:17370074

  5. Anticancer Activities of Medicinal Plants: Modulation of p53 Expression and Induction of Apoptosis.

    PubMed

    Parveen, Amna; Akash, Muhammad Sajid Hamid; Rehman, Kanwal; Kyunn, Whang Wan

    2016-01-01

    For the treatment of several types of cancers, tumors and malignancies, scientists are investigating natural sources to discover novel therapeutic agents from medicinal plants having diverse anticancer properties. Research on natural products is being conducted to identify unexplored phytochemical constituents that have been proven to have diverse pharmacological activities. Several medicinal plants have been reported to regulate the progression of different types of cancers, tumors, and malignancies. In this article, we briefly summarize the recent progress in exploring the anticancer properties of various medicinal plants reported to modulate the expression of p53 and the induction of apoptosis. These plants provide a rich source of chemo-protective agents that can ultimately be used to manage cancer progression. PMID:27650989

  6. Flexible control of plant architecture and yield via switchable expression of Arabidopsis gai.

    PubMed

    Ait-ali, Tahar; Rands, Carley; Harberd, Nicholas P

    2003-09-01

    The growth of plants is repressed by DELLA proteins, nuclear regulators whose activities are opposed by the growth-promoting phytohormone gibberellin (GA). Mutations affecting DELLA protein function were previously used by plant breeders to create the high-yielding semidwarf wheat varieties of the green revolution. gai is an Arabidopsis mutant DELLA protein-encoding orthologue of the wheat semidwarfing genes. Here we describe the development of a transgene that confers ethanol-inducible gai expression. Transient induction of gai causes transient growth repression: growth prior to and after treatment is unaffected. Appropriate ethanol treatments result in dwarf plants that produce the same numbers of seeds as untreated controls. This new technology represents a substantial advance in the applicability of genes encoding mutant DELLA proteins to agricultural and horticultural improvement, enhancing the flexibity with which these genes can be used for the sustainable achievement of increased crop plant yields. PMID:17166132

  7. Creation of transgenic Brassica napus L. plants expressing human alpha 2b interferon gene.

    PubMed

    Sakhno, L O; Kvasko, O Y; Olevinska, Z M; Spivak, M Y; Kuchuk, M V

    2012-01-01

    Spring rapeseed transgenic lines expressing human interferon alpha 2b were created by Agrobacterium-mediated transformation of aseptic plant leaf explants. The maximum antiviral activity of the leaf extracts reached 4500 IU/g fresh weight. It was determined that the antioxidant activity and the activity of an enzyme of plant antioxidant system--superoxide dismutase (SOD)--in the leaf tissues of transgenic plants increased compared to controls. There were no correlations between the interferon and antioxidant activities, as well as between SOD and interferon activities. Using the obtained transgenic rapeseed plants with high interferon and antioxidant activities as a feed additive for animals might have preventive effect on their body, increasing resistance to infections of various origins. PMID:23285745

  8. Flexible control of plant architecture and yield via switchable expression of Arabidopsis gai.

    PubMed

    Ait-ali, Tahar; Rands, Carley; Harberd, Nicholas P

    2003-09-01

    The growth of plants is repressed by DELLA proteins, nuclear regulators whose activities are opposed by the growth-promoting phytohormone gibberellin (GA). Mutations affecting DELLA protein function were previously used by plant breeders to create the high-yielding semidwarf wheat varieties of the green revolution. gai is an Arabidopsis mutant DELLA protein-encoding orthologue of the wheat semidwarfing genes. Here we describe the development of a transgene that confers ethanol-inducible gai expression. Transient induction of gai causes transient growth repression: growth prior to and after treatment is unaffected. Appropriate ethanol treatments result in dwarf plants that produce the same numbers of seeds as untreated controls. This new technology represents a substantial advance in the applicability of genes encoding mutant DELLA proteins to agricultural and horticultural improvement, enhancing the flexibity with which these genes can be used for the sustainable achievement of increased crop plant yields.

  9. Stimulation of human thyroid growth via the inhibitory guanine nucleotide binding (G) protein Gi: constitutive expression of the G-protein alpha subunit Gi alpha-1 in autonomous adenoma.

    PubMed Central

    Selzer, E; Wilfing, A; Schiferer, A; Hermann, M; Grubeck-Loebenstein, B; Freissmuth, M

    1993-01-01

    The alpha subunits of the stimulatory and inhibitory G proteins, Gs alpha and Gi alpha, activate transmembrane-signaling systems involved in the control of cell proliferation. We have investigated the pattern of expression of Gi alpha subtypes and Gi alpha-mediated proliferative responses in the human thyroid. Human thyroid membranes contain two subtypes of Gi alpha, Gi alpha-1 and Gi alpha-2, as assessed by using specific antibodies. The expression of Gi alpha-1 is under tight control by thyrotropin in vivo and in primary cultures of thyroid epithelial cells. In contrast, Gi alpha-1 is expressed in the absence of thyrotropin in thyroid autonomous adenoma, an endocrine-active tumor, and its levels are not regulated by thyrotropin in thyroid epithelial cells prepared from these tumors. If thyroid epithelial cells are treated with pertussis toxin to block signal transduction via Gi, the mitogenic response to serum factors is reduced. These observations demonstrate that Gi subtypes transmit growth stimuli in the human thyroid. The constitutive expression of Gi alpha-1 in autonomous adenoma may allow for the unregulated stimulation of thyroid cell proliferation by a yet unidentified signaling pathway and, thus, be causally related to autonomous growth of thyroid cells. Images PMID:8434024

  10. Eskimo1 mutants of Arabidopsis are constitutively freezing-tolerant.

    PubMed

    Xin, Z; Browse, J

    1998-06-23

    Temperate plants develop a greater ability to withstand freezing in response to a period of low but nonfreezing temperatures through a complex, adaptive process of cold acclimation. Very little is known about the signaling processes by which plants perceive the low temperature stimulus and transduce it into the nucleus to activate genes needed for increased freezing tolerance. To help understand the signaling processes, we have isolated mutants of Arabidopsis that are constitutively freezing-tolerant in the absence of cold acclimation. Freezing tolerance of wild-type Arabidopsis was increased from -5.5 degreesC to -12.6 degreesC by cold acclimation whereas the freezing tolerance of 26 mutant lines ranged from -6.8 degreesC to -10.6 degreesC in the absence of acclimation. Plants with mutations at the eskimo1 (esk1) locus accumulated high levels of proline, a compatible osmolyte, but did not exhibit constitutively increased expression of several cold-regulated genes involved in freezing tolerance. RNA gel blot analysis suggested that proline accumulation in esk1 plants was mediated by regulation of transcript levels of genes involved in proline synthesis and degradation. The characterization of esk1 mutants and results from other mutants suggest that distinct signaling pathways activate different aspects of cold acclimation and that activation of one pathway can result in considerable freezing tolerance without activation of other pathways.

  11. Over-expression of a pepper plastid lipid-associated protein in tobacco leads to changes in plastid ultrastructure and plant development upon stress.

    PubMed

    Rey, P; Gillet, B; Römer, S; Eymery, F; Massimino, J; Peltier, G; Kuntz, M

    2000-03-01

    Proteins homologous to fibrillin, a pepper plastid lipid-associated protein involved in carotenoid storage in fruit chromoplasts, have been recently identified in leaf chloroplasts from several species and shown to be induced upon environmental stress. To further investigate the role of the protein, transgenic Nicotiana tabacum plants over-expressing fibrillin using a constitutive promoter were generated. Transgenics grown under standard light intensities (300 micromol photons m-2 sec-1) were found to contain substantial amounts of fibrillin in flowers and leaves. In leaves, the protein was immunolocalized within chloroplasts in both stromal and thylakoid subfractions. No change was noticed in thylakoid structures from transgenics, but chloroplasts contained an increased number of plastoglobules organized in clusters. In petals, leucoplasts were also found to contain more agglutinated plastoglobules. The effects of environmental factors on fibrillin gene expression and protein localization were studied in tobacco leaves. Less fibrillin was present in plants grown under low light intensities, which can be explained by the involvement of a light-dependent splicing step in the control of fibrillin gene expression in leaves. Analysis of protein subfractions from plants subjected to drought or high light showed that both stresses resulted in fibrillin association with thylakoids. Whereas no growth difference between wild-type (WT) and transgenic plants was noticed under low light conditions, transgenics exhibit a longer main stem, enhanced development of lateral stems and accelerated floral development under higher light intensities. These data suggest that fibrillin-related proteins fulfil an important function in plant development in relation to environmental constraints. PMID:10758499

  12. Constitution, 15 August 1982.

    PubMed

    1987-01-01

    This document reprints major provisions of the 1982 Constitution of Equatorial Guinea. The Constitution calls for protection of the family as the basic building block of society.