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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. Constitutive expression of transgenes encoding derivatives of the synthetic antimicrobial peptide BP100: impact on rice host plant fitness

    PubMed Central

    2012-01-01

    Background The Biopeptide BP100 is a synthetic and strongly cationic α-helical undecapeptide with high, specific antibacterial activity against economically important plant-pathogenic bacteria, and very low toxicity. It was selected from a library of synthetic peptides, along with other peptides with activities against relevant bacterial and fungal species. Expression of the BP100 series of peptides in plants is of major interest to establish disease-resistant plants and facilitate molecular farming. Specific challenges were the small length, peptide degradation by plant proteases and toxicity to the host plant. Here we approached the expression of the BP100 peptide series in plants using BP100 as a proof-of-concept. Results Our design considered up to three tandemly arranged BP100 units and peptide accumulation in the endoplasmic reticulum (ER), analyzing five BP100 derivatives. The ER retention sequence did not reduce the antimicrobial activity of chemically synthesized BP100 derivatives, making this strategy possible. Transformation with sequences encoding BP100 derivatives (bp100der) was over ten-fold less efficient than that of the hygromycin phosphotransferase (hptII) transgene. The BP100 direct tandems did not show higher antimicrobial activity than BP100, and genetically modified (GM) plants constitutively expressing them were not viable. In contrast, inverted repeats of BP100, whether or not elongated with a portion of a natural antimicrobial peptide (AMP), had higher antimicrobial activity, and fertile GM rice lines constitutively expressing bp100der were produced. These GM lines had increased resistance to the pathogens Dickeya chrysanthemi and Fusarium verticillioides, and tolerance to oxidative stress, with agronomic performance comparable to untransformed lines. Conclusions Constitutive expression of transgenes encoding short cationic α-helical synthetic peptides can have a strong negative impact on rice fitness. However, GM plants expressing, for

  3. 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

  4. Constitutively High Expression of the Histidine Biosynthetic Pathway Contributes to Nickel Tolerance in Hyperaccumulator PlantsW⃞

    PubMed Central

    Ingle, Robert A.; Mugford, Sam T.; Rees, Jonathan D.; Campbell, Malcolm M.; Smith, J. Andrew C.

    2005-01-01

    Plants that hyperaccumulate Ni exhibit an exceptional degree of Ni tolerance and the ability to translocate Ni in large amounts from root to shoot. In hyperaccumulator plants in the genus Alyssum, free His is an important Ni binding ligand that increases in the xylem proportionately to root Ni uptake. To determine the molecular basis of the His response and its contribution to Ni tolerance, transcripts representing seven of the eight enzymes involved in His biosynthesis were investigated in the hyperaccumulator species Alyssum lesbiacum by RNA gel blot analysis. None of the transcripts changed in abundance in either root or shoot tissue when plants were exposed to Ni, but transcript levels were constitutively higher in A. lesbiacum than in the congeneric nonaccumulator A. montanum, especially for the first enzyme in the biosynthetic pathway, ATP-phosphoribosyltransferase (ATP-PRT). Comparison with the weak hyperaccumulator A. serpyllifolium revealed a close correlation between Ni tolerance, root His concentration, and ATP-PRT transcript abundance. Overexpression of an A. lesbiacum ATP-PRT cDNA in transgenic Arabidopsis thaliana increased the pool of free His up to 15-fold in shoot tissue, without affecting the concentration of any other amino acid. His-overproducing lines also displayed elevated tolerance to Ni but did not exhibit increased Ni concentrations in either xylem sap or shoot tissue, suggesting that additional factors are necessary to recapitulate the complete hyperaccumulator phenotype. These results suggest that ATP-PRT expression plays a major role in regulating the pool of free His and contributes to the exceptional Ni tolerance of hyperaccumulator Alyssum species. PMID:15923352

  5. 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

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

    PubMed

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

    2004-09-01

    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

  7. 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

  8. Constitutive expression of pathogen-inducible OsWRKY31 enhances disease resistance and affects root growth and auxin response in transgenic rice plants.

    PubMed

    Zhang, Juan; Peng, Youliang; Guo, Zejian

    2008-04-01

    WRKY transcription factors have many regulatory roles in response to biotic and abiotic stresses. In this study, we isolated a rice WRKY gene (OsWRKY31) that is induced by the rice blast fungus Magnaporthe grisea and auxin. This gene encodes a polypeptide of 211 amino-acid residues and belongs to a subgroup of the rice WRKY gene family that probably originated after the divergence of monocot and dicot plants. OsWRKY31 was found to be localized to the nucleus of onion epidermis cells to transiently express OsWRKY31-eGFP fusion protein. Analysis of OsWRKY31 and its mutants fused with a Gal4 DNA-binding domain indicated that OsWRKY31 has transactivation activity in yeast. Overexpression of the OsWRKY31 gene was found to enhance resistance against infection with M. grisea, and the transgenic lines exhibited reduced lateral root formation and elongation compared with wild-type and RNAi plants. The lines with overexpression showed constitutive expression of many defense-related genes, such as PBZ1 and OsSci2, as well as early auxin-response genes, such as OsIAA4 and OsCrl1 genes. Furthermore, the plants with overexpression were less sensitive to exogenously supplied IBA, NAA and 2,4-D at high concentrations, suggesting that overexpression of the OsWRKY31 gene might alter the auxin response or transport. These results also suggest that OsWRKY31 might be a common component in the signal transduction pathways of the auxin response and the defense response in rice. PMID:18071364

  9. 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. PMID:26025521

  10. Constitutive Expression of a miR319 Gene Alters Plant Development and Enhances Salt and Drought Tolerance in Transgenic Creeping Bentgrass1[W][OA

    PubMed Central

    Zhou, Man; Li, Dayong; Li, Zhigang; Hu, Qian; Yang, Chunhua; Zhu, Lihuang; Luo, Hong

    2013-01-01

    MicroRNA319 (miR319) is one of the first characterized and conserved microRNA families in plants and has been demonstrated to target TCP (for TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTORS [PCF]) genes encoding plant-specific transcription factors. MiR319 expression is regulated by environmental stimuli, suggesting its involvement in plant stress response, although experimental evidence is lacking and the underlying mechanism remains elusive. This study investigates the role that miR319 plays in the plant response to abiotic stress using transgenic creeping bentgrass (Agrostis stolonifera) overexpressing a rice (Oryza sativa) miR319 gene, Osa-miR319a. We found that transgenic plants overexpressing Osa-miR319a displayed morphological changes and exhibited enhanced drought and salt tolerance associated with increased leaf wax content and water retention but reduced sodium uptake. Gene expression analysis indicated that at least four putative miR319 target genes, AsPCF5, AsPCF6, AsPCF8, and AsTCP14, and a homolog of the rice NAC domain gene AsNAC60 were down-regulated in transgenic plants. Our results demonstrate that miR319 controls plant responses to drought and salinity stress. The enhanced abiotic stress tolerance in transgenic plants is related to significant down-regulation of miR319 target genes, implying their potential for use in the development of novel molecular strategies to genetically engineer crop species for enhanced resistance to environmental stress. PMID:23292790

  11. Constitutive expression of a miR319 gene alters plant development and enhances salt and drought tolerance in transgenic creeping bentgrass.

    PubMed

    Zhou, Man; Li, Dayong; Li, Zhigang; Hu, Qian; Yang, Chunhua; Zhu, Lihuang; Luo, Hong

    2013-03-01

    MicroRNA319 (miR319) is one of the first characterized and conserved microRNA families in plants and has been demonstrated to target TCP (for TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTORS [PCF]) genes encoding plant-specific transcription factors. MiR319 expression is regulated by environmental stimuli, suggesting its involvement in plant stress response, although experimental evidence is lacking and the underlying mechanism remains elusive. This study investigates the role that miR319 plays in the plant response to abiotic stress using transgenic creeping bentgrass (Agrostis stolonifera) overexpressing a rice (Oryza sativa) miR319 gene, Osa-miR319a. We found that transgenic plants overexpressing Osa-miR319a displayed morphological changes and exhibited enhanced drought and salt tolerance associated with increased leaf wax content and water retention but reduced sodium uptake. Gene expression analysis indicated that at least four putative miR319 target genes, AsPCF5, AsPCF6, AsPCF8, and AsTCP14, and a homolog of the rice NAC domain gene AsNAC60 were down-regulated in transgenic plants. Our results demonstrate that miR319 controls plant responses to drought and salinity stress. The enhanced abiotic stress tolerance in transgenic plants is related to significant down-regulation of miR319 target genes, implying their potential for use in the development of novel molecular strategies to genetically engineer crop species for enhanced resistance to environmental stress. PMID:23292790

  12. 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

  13. 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.

  14. Constitutive expression of the poplar WRKY transcription factor PtoWRKY60 enhances resistance to Dothiorella gregaria Sacc. in transgenic plants.

    PubMed

    Ye, Shenglong; Jiang, Yuanzhong; Duan, Yanjiao; Karim, Abdul; Fan, Di; Yang, Li; Zhao, Xin; Yin, Jia; Luo, Keming

    2014-10-01

    WRKY proteins are involved in various physiological processes in plants, especially in coping with diverse biotic and abiotic stresses. However, limited information is available on the roles of specific WRKY transcription factors in poplar defense. In this study, we reported the characterization of PtoWRKY60, a Group IIa WRKY member, from Populus tomentosa Carr. The gene expression profile of PtoWRKY60 in various tissues showed that it significantly accumulated in old leaves. Phylogenetic analyses revealed that PtoWRKY60 had a close relationship with AtWRKY18, AtWRKY40 and AtWRKY60. PtoWRKY60 was induced mainly by salicylic acid (SA) and slightly by Dothiorella gregaria Sacc., jasmonic acid, wounding treatment, low temperature and salinity stresses. Overexpression of PtoWRKY60 in poplar resulted in increased resistance to D. gregaria. The defense-associated genes, such as PR5.1, PR5.2, PR5.4, PR5.5 and CPR5, were markedly up-regulated in transgenic plants overexpressing PtoWRKY60. These results indicate that PtoWRKY60 might be partly involved in the signal transduction pathway initiated by SA in Populus. PMID:25281841

  15. Constitutive Expression of Rice MicroRNA528 Alters Plant Development and Enhances Tolerance to Salinity Stress and Nitrogen Starvation in Creeping Bentgrass1[OPEN

    PubMed Central

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

    2015-01-01

    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

  16. 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

  17. Tradeoffs associated with constitutive and induced plant resistance against herbivory

    PubMed Central

    Kempel, Anne; Schädler, Martin; Chrobock, Thomas; Fischer, Markus; van Kleunen, Mark

    2011-01-01

    Several prominent hypotheses have been posed to explain the immense variability among plant species in defense against herbivores. A major concept in the evolutionary ecology of plant defenses is that tradeoffs of defense strategies are likely to generate and maintain species diversity. In particular, tradeoffs between constitutive and induced resistance and tradeoffs relating these strategies to growth and competitive ability have been predicted. We performed three independent experiments on 58 plant species from 15 different plant families to address these hypotheses in a phylogenetic framework. Because evolutionary tradeoffs may be altered by human-imposed artificial selection, we used 18 wild plant species and 40 cultivated garden-plant species. Across all 58 plant species, we demonstrate a tradeoff between constitutive and induced resistance, which was robust to accounting for phylogenetic history of the species. Moreover, the tradeoff was driven by wild species and was not evident for cultivated species. In addition, we demonstrate that more competitive species—but not fast growing ones—had lower constitutive but higher induced resistance. Thus, our multispecies experiments indicate that the competition–defense tradeoff holds for constitutive resistance and is complemented by a positive relationship of competitive ability with induced resistance. We conclude that the studied genetically determined tradeoffs are indeed likely to play an important role in shaping the high diversity observed among plant species in resistance against herbivores and in life history traits. PMID:21389269

  18. 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…

  19. Constitutive expression of Botrytis aclada laccase in Pichia pastoris.

    PubMed

    Kittl, Roman; Gonaus, Christoph; Pillei, Christian; Haltrich, Dietmar; Ludwig, Roland

    2012-01-01

    The heterologous expression of laccases is important for their large-scale production and genetic engineering--a prerequisite for industrial application. Pichia pastoris is the preferred expression host for fungal laccases. The recently cloned laccase from the ascomycete Botrytis aclada (BaLac) has been efficiently expressed in P. pastoris under the control of the inducible alcohol oxidase (AOX1) promoter. In this study, we compare these results to the constitutive expression in the same organism using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The results show that the amounts of BaLac produced with the GAP system (517 mgL(-1)) and the AOX1 system (495 mgL(-1)) are comparable. The constitutive expression is, however, faster, and the specific activity of BaLac in the culture supernatant is higher (41.3 Umg(-1) GAP, 14.2 Umg(-1) AOX1). In microtiter plates, the constitutive expression provides a clear advantage due to easy manipulation (simple medium, no methanol feeding) and fast enzyme production (high-throughput screening assays can already be performed after 48 h). PMID:22705842

  20. Constitutive expression of Botrytis aclada laccase in Pichia pastoris

    PubMed Central

    Kittl, Roman; Gonaus, Christoph; Pillei, Christian; Haltrich, Dietmar; Ludwig, Roland

    2012-01-01

    The heterologous expression of laccases is important for their large-scale production and genetic engineering—a prerequisite for industrial application. Pichia pastoris is the preferred expression host for fungal laccases. The recently cloned laccase from the ascomycete Botrytis aclada (BaLac) has been efficiently expressed in P. pastoris under the control of the inducible alcohol oxidase (AOX1) promoter. In this study, we compare these results to the constitutive expression in the same organism using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The results show that the amounts of BaLac produced with the GAP system (517 mgL-1) and the AOX1 system (495 mgL-1) are comparable. The constitutive expression is, however, faster, and the specific activity of BaLac in the culture supernatant is higher (41.3 Umg-1 GAP, 14.2 Umg-1 AOX1). In microtiter plates, the constitutive expression provides a clear advantage due to easy manipulation (simple medium, no methanol feeding) and fast enzyme production (high-throughput screening assays can already be performed after 48 h). PMID:22705842

  1. Plant chemical defenses: are all constitutive antimicrobial metabolites phytoanticipins?

    PubMed

    Pedras, M Soledade C; Yaya, Estifanos E

    2015-01-01

    A critical perspective on phytoanticipins, constitutive plant secondary metabolites with defensive roles against microbes is presented. This mini-review focuses on the chemical groups and structural types of defensive plant metabolites thus far not reviewed from the phytoanticipin perspective: i) fatty acid derivatives and polyketides, ii) terpenoids, iii) shikimates, phenylpropanoids and derivatives, and iv) benzylisoquinoline and pyrrolizidine alkaloids. The more traditional groups of phytoanticipins are briefly summarized, with particular focus on the latest results: i) benzoxazinoids, ii) cyanogenic glycosides, iii) glucosinolates and their metabolic products, and iv) saponins. Current evidence suggests that a better understanding of the functions of plant metabolites will drive their application to protect crops against microbial diseases. PMID:25920246

  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. PMID:15517994

  3. 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

  4. 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. PMID:25716695

  5. 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

  6. 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

  7. 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.

  8. Constitutive telomerase expression promotes mammary carcinomas in aging mice

    PubMed Central

    Artandi, Steven E.; Alson, Scott; Tietze, Maja K.; Sharpless, Norman E.; Ye, Siqin; Greenberg, Roger A.; Castrillon, Diego H.; Horner, James W.; Weiler, Sarah R.; Carrasco, Ruben D.; DePinho, Ronald A.

    2002-01-01

    Telomerase is up-regulated in the vast majority of human cancers and serves to halt the progressive telomere shortening that ultimately blocks would-be cancer cells from achieving a full malignant phenotype. In contrast to humans, the laboratory mouse possesses long telomeres and, even in early generation telomerase-deficient mice, the level of telomere reserve is sufficient to avert telomere-based checkpoint responses and to permit full malignant progression. These features in the mouse provide an opportunity to determine whether enforced high-level telomerase activity can serve functions that extend beyond its ability to sustain telomere length and function. Here, we report the generation and characterization of transgenic mice that express the catalytic subunit of telomerase (mTERT) at high levels in a broad variety of tissues. Expression of mTERT conferred increased telomerase enzymatic activity in several tissues, including mammary gland, splenocytes, and cultured mouse embryonic fibroblasts. In mouse embryonic fibroblasts, mTERT overexpression extended telomere lengths but did not prevent culture-induced replicative arrest, thus reinforcing the view that this phenomenon is not related to occult telomere shortening. Robust telomerase activity, however, was associated with the spontaneous development of mammary intraepithelial neoplasia and invasive mammary carcinomas in a significant proportion of aged females. These data indicate that enforced mTERT expression can promote the development of spontaneous cancers even in the setting of ample telomere reserve. PMID:12034875

  9. Constitutive expression of ciliary neurotrophic factor in mouse hypothalamus

    PubMed Central

    Severi, Ilenia; Carradori, Maria Rita; Lorenzi, Teresa; Amici, Adolfo; Cinti, Saverio; Giordano, Antonio

    2012-01-01

    Ciliary neurotrophic factor (CNTF) is a potent survival molecule for a large number of neuronal and glial cells in culture; its expression in glial cells is strongly upregulated after a variety of nerve tissue injuries. Exogenously administered CNTF produces an anorectic effect via activation of hypothalamic neurons and stimulates neurogenesis in mouse hypothalamus. To determine whether CNTF is produced endogenously in the hypothalamus, we sought cellular sources and examined their distribution in adult mouse hypothalamus by immunohistochemistry. CNTF immunoreactivity (IR) was predominantly detected in the ependymal layer throughout the rostrocaudal extension of the third ventricle, where numerous ependymocytes and tanycytes exhibited specific staining. Some astrocytes in the grey matter of the anterior hypothalamus and in the median eminence of the hypothalamic tuberal region were also positive. Stimulation of cells bearing CNTF receptor α (CNTFRα) induces specific activation of the signal transducer and activator of transcription 3 (STAT3) signalling system. Treatment with recombinant CNTF and detection of the nuclear expression of phospho-STAT3 (P-STAT3) showed that CNTF-producing ependymal cells and tanycytes were intermingled with, or very close to, P-STAT3-positive, CNTFRα-bearing cells. A fraction of CNTF-producing ependymal cells and tanycytes and some median eminence astrocytes also exhibited P-STAT3 IR. Thus, in normal adult mice the ependyma of the third ventricle is both a source of and a target for CNTF, which may play hitherto unknown roles in hypothalamic function in physiological conditions. PMID:22458546

  10. Heterelogous Expression of Plant Genes

    PubMed Central

    Yesilirmak, Filiz; Sayers, Zehra

    2009-01-01

    Heterologous expression allows the production of plant proteins in an organism which is simpler than the natural source. This technology is widely used for large-scale purification of plant proteins from microorganisms for biochemical and biophysical analyses. Additionally expression in well-defined model organisms provides insights into the functions of proteins in complex pathways. The present review gives an overview of recombinant plant protein production methods using bacteria, yeast, insect cells, and Xenopus laevis oocytes and discusses the advantages of each system for functional studies and protein characterization. PMID:19672459

  11. 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

  12. 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. PMID:25120219

  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. Constitutive and induced defenses to herbivory in above- and belowground plant tissues.

    PubMed

    Kaplan, Ian; Halitschke, Rayko; Kessler, André; Sardanelli, Sandra; Denno, Robert F

    2008-02-01

    A recent surge in attention devoted to the ecology of soil biota has prompted interest in quantifying similarities and differences between interactions occurring in above- and belowground communities. Furthermore, linkages that interconnect the dynamics of these two spatially distinct ecosystems are increasingly documented. We use a similar approach in the context of understanding plant defenses to herbivory, including how they are allocated between leaves and roots (constitutive defenses), and potential cross-system linkages (induced defenses). To explore these issues we utilized three different empirical approaches. First, we manipulated foliar and root herbivory on tobacco (Nicotiana tabacum) and measured changes in the secondary chemistry of above- and belowground tissues. Second, we reviewed published studies that compared levels of secondary chemistry between leaves and roots to determine how plants distribute putative defense chemicals across the above- and belowground systems. Last, we used meta-analysis to quantify the impact of induced responses across plant tissue types. In the tobacco system, leaf-chewing insects strongly induced higher levels of secondary metabolites in leaves but had no impact on root chemistry. Nematode root herbivores, however, elicited changes in both leaves and roots. Virtually all secondary chemicals measured were elevated in nematode-induced galls, whereas the impact of root herbivory on foliar chemistry was highly variable and depended on where chemicals were produced within the plant. Importantly, nematodes interfered with aboveground metabolites that have biosynthetic sites located in roots (e.g., nicotine) but had the opposite effect (i.e., nematodes elevated foliar expression) on chemicals produced in shoots (e.g., phenolics and terpenoids). Results from our literature review suggest that, overall, constitutive defense levels are extremely similar when comparing leaves with roots, although certain chemical classes (e

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

    PubMed

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

    2016-08-01

    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 LacI(q) 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

  16. 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

  17. 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.

  18. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  19. 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

  20. Whole genome expression and biochemical correlates of extreme constitutional types defined in Ayurveda

    PubMed Central

    Prasher, Bhavana; Negi, Sapna; Aggarwal, Shilpi; Mandal, Amit K; Sethi, Tav P; Deshmukh, Shailaja R; Purohit, Sudha G; Sengupta, Shantanu; Khanna, Sangeeta; Mohammad, Farhan; Garg, Gaurav; Brahmachari, Samir K; Mukerji, Mitali

    2008-01-01

    Background Ayurveda is an ancient system of personalized medicine documented and practiced in India since 1500 B.C. According to this system an individual's basic constitution to a large extent determines predisposition and prognosis to diseases as well as therapy and life-style regime. Ayurveda describes seven broad constitution types (Prakritis) each with a varying degree of predisposition to different diseases. Amongst these, three most contrasting types, Vata, Pitta, Kapha, are the most vulnerable to diseases. In the realm of modern predictive medicine, efforts are being directed towards capturing disease phenotypes with greater precision for successful identification of markers for prospective disease conditions. In this study, we explore whether the different constitution types as described in Ayurveda has molecular correlates. Methods Normal individuals of the three most contrasting constitutional types were identified following phenotyping criteria described in Ayurveda in Indian population of Indo-European origin. The peripheral blood samples of these individuals were analysed for genome wide expression levels, biochemical and hematological parameters. Gene Ontology (GO) and pathway based analysis was carried out on differentially expressed genes to explore if there were significant enrichments of functional categories among Prakriti types. Results Individuals from the three most contrasting constitutional types exhibit striking differences with respect to biochemical and hematological parameters and at genome wide expression levels. Biochemical profiles like liver function tests, lipid profiles, and hematological parameters like haemoglobin exhibited differences between Prakriti types. Functional categories of genes showing differential expression among Prakriti types were significantly enriched in core biological processes like transport, regulation of cyclin dependent protein kinase activity, immune response and regulation of blood coagulation. A

  1. Genetically engineered maize plants reveal distinct costs and benefits of constitutive volatile emissions in the field.

    PubMed

    Robert, Christelle Aurélie Maud; Erb, Matthias; Hiltpold, Ivan; Hibbard, Bruce Elliott; Gaillard, Mickaël David Philippe; Bilat, Julia; Degenhardt, Jörg; Cambet-Petit-Jean, Xavier; Turlings, Ted Christiaan Joannes; Zwahlen, Claudia

    2013-06-01

    Genetic manipulation of plant volatile emissions is a promising tool to enhance plant defences against herbivores. However, the potential costs associated with the manipulation of specific volatile synthase genes are unknown. Therefore, we investigated the physiological and ecological effects of transforming a maize line with a terpene synthase gene in field and laboratory assays, both above- and below ground. The transformation, which resulted in the constitutive emission of (E)-β-caryophyllene and α-humulene, was found to compromise seed germination, plant growth and yield. These physiological costs provide a possible explanation for the inducibility of an (E)-β-caryophyllene-synthase gene in wild and cultivated maize. The overexpression of the terpene synthase gene did not impair plant resistance nor volatile emission. However, constitutive terpenoid emission increased plant apparency to herbivores, including adults and larvae of the above ground pest Spodoptera frugiperda, resulting in an increase in leaf damage. Although terpenoid overproducing lines were also attractive to the specialist root herbivore Diabrotica virgifera virgifera below ground, they did not suffer more root damage in the field, possibly because of the enhanced attraction of entomopathogenic nematodes. Furthermore, fewer adults of the root herbivore Diabrotica undecimpunctata howardii were found to emerge near plants that emitted (E)-β-caryophyllene and α-humulene. Yet, overall, under the given field conditions, the costs of constitutive volatile production overshadowed its benefits. This study highlights the need for a thorough assessment of the physiological and ecological consequences of genetically engineering plant signals in the field to determine the potential of this approach for sustainable pest management strategies. PMID:23425633

  2. Achieving efficient protein expression in Trichoderma reesei by using strong constitutive promoters

    PubMed Central

    2012-01-01

    Backgrounds The fungus Trichoderma reesei is an important workhorse for expression of homologous or heterologous genes, and the inducible cbh1 promoter is generally used. However, constitutive expression is more preferable in some cases than inducible expression that leads to production of unwanted cellulase components. In this work, constitutive promoters of T. reesei were screened and successfully used for high level homologous expression of xylanase II. Results The transcriptional profiles of 13 key genes that participate in glucose metabolism in T. reesei were analyzed by quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR). The results indicated that the mRNA levels of pdc (encoding pyruvate decarboxylase) and eno (encoding enolase) genes were much higher than other genes under high glucose conditions. Recombinant T. reesei strains that homologously expressed xylanase II were constructed by using the promoters of the pdc and eno genes, and they respectively produced 9266 IU/ml and 8866 IU/ml of xylanase activities in the cultivation supernatant in a medium with high glucose concentration. The productivities of xylanase II were 1.61 g/L (with the pdc promoter) and 1.52 g/L (with the eno promoter), approximately accounted for 83% and 82% of the total protein secreted by T. reesei, respectively. Conclusions This work demonstrates the screening of constitutive promoters by using RT-qPCR in T. reesei, and has obtained the highest expression of recombinant xylanase II to date by using these promoters. PMID:22709462

  3. Constitutive Expression of a Chromosomal Class A (BJM Group 2) β-Lactamase in Xanthomonas campestris

    PubMed Central

    Weng, Shu-Fen; Lin, Juey-Wen; Chen, Chih-Hung; Chen, Yih-Yuan; Tseng, Yi-Hsuan; Tseng, Yi-Hsiung

    2004-01-01

    Sequencing of the upstream region of the β-lactamase gene from Xanthomonas campestris pv. campestris 11 (blaXCC-1) revealed the cognate ampR1 gene (289 amino acids, 31 kDa). It runs divergently from blaXCC-1 with a 100-bp intergenic region (IG) containing partially overlapped promoters with structural features typical of the bla-ampR IG. The deduced AmpR1 protein shows significant identity in amino acid sequence and conserved motifs with AmpR proteins of other species, e.g., of Pseudomonas aeruginosa (58.2% amino acid identity). Results of insertional mutation, complementation tests, and β-lactamase assays suggested that expression of blaXCC-1 was constitutive and dependent on AmpR1. Four bla genes and two ampR genes are present in the fully sequenced X. campestris pv. campestris ATCC 33913 genome, with XCC3039 and XCC3040 considered the analogues of blaXCC-1 and ampR1, respectively. An ampR1 homologue was detected by Southern hybridization in the ampicillin-resistant Xanthomonas strains, which appear to express β-lactamase constitutively. Although the significance remains to be studied, constitutive expression of β-lactamase by a widespread bacterial genus raises environmental concerns regarding the dissemination of resistance genes. PMID:14693541

  4. 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

  5. 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

  6. Constitutive expression of human keratin 14 gene in mouse lung induces premalignant lesions and squamous differentiation.

    PubMed

    Dakir, E L Habib; Feigenbaum, Lionel; Linnoila, R Ilona

    2008-12-01

    Squamous cell carcinoma accounts for 20% of all human lung cancers and is strongly linked to cigarette smoking. It develops through premalignant changes that are characterized by high levels of keratin 14 (K14) expression in the airway epithelium and evolve through basal cell hyperplasia, squamous metaplasia and dysplasia to carcinoma in situ and invasive carcinoma. In order to explore the impact of K14 in the pulmonary epithelium that normally lacks both squamous differentiation and K14 expression, human keratin 14 gene hK14 was constitutively expressed in mouse airway progenitor cells using a mouse Clara cell specific 10 kDa protein (CC10) promoter. While the lungs of CC10-hK14 transgenic mice developed normally, we detected increased expression of K14 and the molecular markers of squamous differentiation program such as involucrin, loricrin, small proline-rich protein 1A, transglutaminase 1 and cholesterol sulfotransferase 2B1. In contrast, wild-type lungs were negative. Aging CC10-hK14 mice revealed multifocal airway cell hyperplasia, occasional squamous metaplasia and their lung tumors displayed evidence for multidirectional differentiation. We conclude that constitutive expression of hK14 initiates squamous differentiation program in the mouse lung, but fails to promote squamous maturation. Our study provides a novel model for assessing the mechanisms of premalignant lesions in vivo by modifying differentiation and proliferation of airway progenitor cells. PMID:18701433

  7. 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

  8. 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

  9. Rhizoremediation of Trichloroethylene by a Recombinant, Root-Colonizing Pseudomonas fluorescens Strain Expressing Toluene ortho-Monooxygenase Constitutively

    PubMed Central

    Yee, Dennis C.; Maynard, Jennifer A.; Wood, Thomas K.

    1998-01-01

    Trichloroethylene (TCE) was removed from soils by using a wheat rhizosphere established by coating seeds with a recombinant, TCE-degrading Pseudomonas fluorescens strain that expresses the tomA+ (toluene o-monooxygenase) genes from Burkholderia cepacia PR123(TOM23C). A transposon integration vector was used to insert tomA+ into the chromosome of P. fluorescens 2-79, producing a stable strain that expressed constitutively the monooxygenase at a level of 1.1 nmol/min · mg of protein (initial TCE concentration, 10 μM, assuming that all of the TCE was in the liquid) for more than 280 cell generations (36 days). We also constructed a salicylate-inducible P. fluorescens strain that degraded TCE at an initial rate of 2.6 nmol/min · mg of protein in the presence of 10 μM TCE [cf. B. cepacia G4 PR123(TOM23C), which degraded TCE at an initial rate of 2.5 nmol/min · mg of protein]. A constitutive strain, P. fluorescens 2-79TOM, grew (maximum specific growth rate, 0.78 h−1) and colonized wheat (3 × 106 CFU/cm of root) as well as wild-type P. fluorescens 2-79 (maximum specific growth rate, 0.77 h−1; level of colonization, 4 × 106 CFU/cm of root). Rhizoremediation of TCE was demonstrated by using microcosms containing the constitutive monooxygenase-expressing microorganism, soil, and wheat. These closed microcosms degraded an average of 63% of the initial TCE in 4 days (20.6 nmol of TCE/day · plant), compared to the 9% of the initial TCE removed by negative controls consisting of microcosms containing wild-type P. fluorescens 2-79-inoculated wheat, uninoculated wheat, or sterile soil. PMID:9435067

  10. Enhanced expression of constitutive and inducible forms of nitric oxide synthase in autoimmune encephalomyelitis.

    PubMed

    Kim, S; Moon, C; Wie, M B; Kim, H; Tanuma, N; Matsumoto, Y; Shin, T

    2000-06-01

    To elucidate the role of nitric oxide synthase (NOS) in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), we analyzed the expression of constitutive neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) in the spinal cords of rats with EAE. We further examined the structural interaction between apoptotic cells and spinal cord cells including neurons and astrocytes, which are potent cell types of nitric oxide (NO) production in the brain. Western blot analysis showed that three forms of NOS significantly increased in the spinal cords of rats at the peak stage of EAE, while small amounts of these enzymes were identified in the spinal cords of rats without EAE. Immunohistochemical study showed that the expression of either nNOS or eNOS increased in the brain cells including neurons and astrocytes during the peak and recovery stages of EAE, while the expression of iNOS was found mainly in the inflammatory macrophages in the perivascular EAE lesions. Double labeling showed that apoptotic cells had intimate contacts with either neurons or astrocytes, which are major cell types to express nNOS and eNOS constitutively. Our results suggest that the three NOS may play an important role in the recovery of EAE. PMID:14612615

  11. 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

  12. 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

  13. 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.

  14. Systematic study of constitutive cyclooxygenase-2 expression: Role of NF-κB and NFAT transcriptional pathways

    PubMed Central

    Kirkby, Nicholas S.; Chan, Melissa V.; Zaiss, Anne K.; Garcia-Vaz, Eliana; Jiao, Jing; Berglund, Lisa M.; Verdu, Elena F.; Ahmetaj-Shala, Blerina; Wallace, John L.; Herschman, Harvey R.; Gomez, Maria F.; Mitchell, Jane A.

    2016-01-01

    Cyclooxygenase-2 (COX-2) is an inducible enzyme that drives inflammation and is the therapeutic target for widely used nonsteroidal antiinflammatory drugs (NSAIDs). However, COX-2 is also constitutively expressed, in the absence of overt inflammation, with a specific tissue distribution that includes the kidney, gastrointestinal tract, brain, and thymus. Constitutive COX-2 expression is therapeutically important because NSAIDs cause cardiovascular and renal side effects in otherwise healthy individuals. These side effects are now of major concern globally. However, the pathways driving constitutive COX-2 expression remain poorly understood. Here we show that in the kidney and other sites, constitutive COX-2 expression is a sterile response, independent of commensal microorganisms and not associated with activity of the inflammatory transcription factor NF-κB. Instead, COX-2 expression in the kidney but not other regions colocalized with nuclear factor of activated T cells (NFAT) transcription factor activity and was sensitive to inhibition of calcineurin-dependent NFAT activation. However, calcineurin/NFAT regulation did not contribute to constitutive expression elsewhere or to inflammatory COX-2 induction at any site. These data address the mechanisms driving constitutive COX-2 and suggest that by targeting transcription it may be possible to develop antiinflammatory therapies that spare the constitutive expression necessary for normal homeostatic functions, including those important to the cardiovascular-renal system. PMID:26712011

  15. 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

  16. 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...

  17. Cloning, constitutive activity and expression profiling of two receptors related to relaxin receptors in Drosophila melanogaster.

    PubMed

    Van Hiel, Matthias B; Vandersmissen, Hans Peter; Proost, Paul; Vanden Broeck, Jozef

    2015-06-01

    Leucine-rich repeat containing G protein-coupled receptors (LGRs) comprise a cluster of transmembrane proteins, characterized by the presence of a large N-terminal extracellular domain. This receptor group can be classified into three subtypes. Belonging to the subtype C LGRs are the mammalian relaxin receptors LGR7 (RXFP1) and LGR8 (RXFP2), which mediate important reproductive and other processes. We identified two related receptors in the genome of the fruit fly and cloned their open reading frames into an expression vector. Interestingly, dLGR3 demonstrated constitutive activity at very low doses of transfected plasmid, whereas dLGR4 did not show any basal activity. Both receptors exhibited a similar expression pattern during development, with relatively high transcript levels during the first larval stage. In addition, both receptors displayed higher expression in male adult flies as compared to female flies. Analysis of the tissue distribution of both receptor transcripts revealed a high expression of dLGR3 in the female fat body, while the expression of dLGR4 peaked in the midgut of both the wandering and adult stage. PMID:25064813

  18. 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.

  19. 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

  20. Regions flanking exon 1 regulate constitutive expression of the human antithrombin gene.

    PubMed

    Fernandez-Rachubinski, F A; Weiner, J H; Blajchman, M A

    1996-11-15

    We have identified cis-acting elements and trans-acting factors that regulate constitutive expression of the human antithrombin gene. The activity of the sequences flanking the first exon of the gene was investigated using a luciferase-based reporter assay in transiently transfected HepG2, COS1, BSC40, and HeLa cells. Deletion analysis allowed the mapping of two elements able to promote antithrombin gene transcription in HepG2 and COS1 cells. The first element is located upstream of the first exon (-150/+68 nucleotides). The second element is in the first intervening sequence (+300/+700 nucleotides) and functions in an orientation opposite to that of the first. Footprint analysis showed three protected areas in the 5' upstream element at -92/-68 (element A), -14/+37 (element B), and -126/-100 nucleotides (element C). These elements acted as enhancers in luciferase reporter assays. Gel retardation analysis demonstrated that two liver-enriched transcription factors, hepatocyte nuclear factor 4 (HNF4) and CCAAT enhancer-binding protein (C/EBPa), bound to the 5' upstream element. HNF4 bound to elements A and C, whereas C/EBPa bound to element B. Element A also interacted with the ubiquitous nuclear hormone receptors chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1), thyroid hormone receptor alpha (TRalpha), peroxisome proliferator-activated receptor alpha(PPARalpha), and retinoid X receptor alpha (RXRalpha). In HepG2 and BSC40 cells, HNF4, C/EBPalpha, and RXRalpha activated luciferase expression from a reporter construct containing the 5'-upstream minimal antithrombin gene promoter, while COUP-TF1, TRalpha, and HNF3 (alpha or beta) repressed such expression. Our results show that constitutive expression of the human antithrombin gene depends in part upon the interplay of these transcription factors and suggest that signaling pathways regulated by these factors can modulate antithrombin gene transcription. PMID:8910619

  1. 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.

  2. 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. PMID:26743044

  3. 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

  4. Constitutive expression of multidrug resistance in human colorectal tumours and cell lines.

    PubMed Central

    Kramer, R.; Weber, T. K.; Morse, B.; Arceci, R.; Staniunas, R.; Steele, G.; Summerhayes, I. C.

    1993-01-01

    In this study we report detection of mdr1 gene expression in the liver metastases of 7/11 patients with colon carcinoma and characterise the MDR phenotype associated with a panel of 19 human colon carcinoma cell lines. Within this panel, mdr1 mRNA biosynthesis and surface localisation of Pgp were assessed with respect to MDR functionality where the cell lines are representative of different clinical stages of tumour progression, metastatic potential and differentiation. The data indicates that constitutive levels of mdr1 mRNA/Pgp expression may not necessarily result in the functional expression of the MDR phenotype. While low levels of mdr1 mRNA/Pgp were detected in 5/8 well differentiated colon cell lines, only 2/8 were functionally MDR. In contrast, 10/11 moderate and poorly differentiated lines expressed mdr1 mRNA/Pgp and of these, 9/11 were functionally MDR. The phosphorylation status of the mature 170 kD P-glycoprotein and the surface localisation of this glycoprotein showed the strongest correlation with functionality. Analysis of cell lines for cross-resistance and chemosensitivity profiles against a battery of chemotherapeutic drugs suggests multiple mechanisms, in addition to Pgp, contribute to the overall resistance of colorectal cancer. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8098614

  5. Effect of constitutive expression of bacterial phytoene desaturase CRTI on photosynthetic electron transport in Arabidopsis thaliana.

    PubMed

    Galzerano, Denise; Feilke, Kathleen; Schaub, Patrick; Beyer, Peter; Krieger-Liszkay, Anja

    2014-03-01

    The constitutive expression of the bacterial carotene desaturase (CRTI) in Arabidopsis thaliana leads to increased susceptibility of leaves to light-induced damage. Changes in the photosynthetic electron transport chain rather than alterations of the carotenoid composition in the antenna were responsible for the increased photoinhibition. A much higher level of superoxide/hydrogen peroxide was generated in the light in thylakoid membranes from the CRTI expressing lines than in wild-type while the level of singlet oxygen generation remained unchanged. The increase in reactive oxygen species was related to the activity of plastid terminal oxidase (PTOX) since their generation was inhibited by the PTOX-inhibitor octyl gallate, and since the protein level of PTOX was increased in the CRTI-expressing lines. Furthermore, cyclic electron flow was suppressed in these lines. We propose that PTOX competes efficiently with cyclic electron flow for plastoquinol in the CRTI-expressing lines and that it plays a crucial role in the control of the reduction state of the plastoquinone pool. PMID:24378845

  6. Constitutive expression of barley α-amylase in Pichia pastoris by high-density cell culture.

    PubMed

    Liu, Z W; Yin, H X; Yi, X P; Zhang, A L; Luo, J X; Zhang, T Y; Fu, C Y; Zhang, Z H; Shen, J C; Chen, L P

    2012-05-01

    α-amy gene amplified from barley genome was cloned into MCS of pGAP9K to generate pGAP9K-α-amy which was then transformed into Pichia pastoris GS115 by electroporation. Transformants with multi-copies and high expression for the foreign gene were selected on G418 containing plate and expression analysis. The fermentation was carried out in a 50 l bioreactor with 20 l working volume, using a high-density cell culture method by continuously feeding with 50% glycerol-0.8% PTM4 to the growing culture for 54 h at 30°C. Under the control of GAP promoter (pGAP), α-amy gene was constitutively expressed. At the end of the fermentation, the α-AMY expression reached 125 mg/l, while the biomass growth was 186 as measured by absorption of 600 nm. The secreted α-AMY was purified to 97.5% by SP-Sepharose FF ion-exchange chromatography and affinity purification. The recombinant α-AMY showed activity on hydrolysis of starch. PMID:22201022

  7. 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. PMID:25768292

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

    DOE PAGESBeta

    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

  9. 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

  10. Constitutive Expression of Enniatin Synthetase during Fermentative Growth of Fusarium scirpi

    PubMed Central

    Billich, Andreas; Zocher, Rainer

    1988-01-01

    The production of enniatins by Fusarium scirpi during fermentative growth in submerged cultures was measured. The fungus produced the antibiotic during mycelial growth, but not during the stationary phase of cultivation. By contrast, enniatin synthetase, the enzyme responsible for enniatin synthesis, was present during growth, during the stationary phase, and even in spores. Similarly, the enniatin synthetase mRNA was present at every stage of the cultivation of the fungus. Therefore, this multifunctional peptide synthetase is a constitutive enzyme, the expression of which is not regulated by any specific mechanism. The findings stand in contrast to the common assumption that production of secondary metabolites underlies regulatory control, leading to separation of the trophophase and the idiophase. Images PMID:16347758

  11. Premature lethality, hyperactivity, and aberrant phosphorylation in transgenic mice expressing a constitutively active form of Fyn

    PubMed Central

    Xia, Di; Götz, Jürgen

    2014-01-01

    The kinase Fyn, the microtubule-associated protein tau and the peptide amyloid-β (Aβ) constitute a toxic triad in Alzheimer's disease (AD). Tau's subcellular localization is mainly regulated by phosphorylation whereas Fyn's localization is dictated by palmitoylation targeting it to the plasma membrane in a reversible manner. We have previously shown that tau is required for Fyn to be targeted to the dendritic spine. We had also shown that a truncated form of tau (Δtau) that accumulates in the cell soma is capable of trapping Fyn and preventing it from entering the spine. Here we determined that palmitoylation is required for Fyn's membrane and spine localization. We further evaluated the functional consequences of neuronal over-expression of the constitutively active Y531F mutant form of Fyn (FynCA) in transgenic mice. We found that the FynCA transgenic mice displayed a reduced weight, a massively reduced lifespan and a high level of hyperactivity. The lifespan of the FynCA mice was only slightly extended by crossing them with Δtau transgenic mice, possibly reflecting differences in expression patterns of the transgenes and high levels of transgenic FynCA compared to endogenous Fyn. Analysis of synaptosomes revealed that FynCA accumulated at high levels in the spine, resulting in increased levels of the NMDA receptor subunit NR2b phosphorylated at residue Y1472. Tau was strongly phosphorylated at the AT8 epitope S202/T205 as shown by Western blot and immunohistochemistry indicating that an increased tyrosine kinase activity of Fyn has down-stream consequences for serine/threonine-directed phosphorylation. PMID:24860422

  12. 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.

  13. Plant Virus Expression Vector Development: New Perspectives

    PubMed Central

    Hefferon, Kathleen

    2014-01-01

    Plant made biologics have elicited much attention over recent years for their potential in assisting those in developing countries who have poor access to modern medicine. Additional applications such as the stockpiling of vaccines against pandemic infectious diseases or potential biological warfare agents are also under investigation. Plant virus expression vectors represent a technology that enables high levels of pharmaceutical proteins to be produced in a very short period of time. Recent advances in research and development have brought about the generation of superior virus expression systems which can be readily delivered to the host plant in a manner that is both efficient and cost effective. This review presents recent innovations in plant virus expression systems and their uses for producing biologics from plants. PMID:24745025

  14. Basic Equations and Constitutive Equations of Micropolar Magnetic Fluids with E-BAnalogy and the Abraham Expression of Electromagnetic Momentum

    NASA Astrophysics Data System (ADS)

    Ido, Yasushi

    A complete set of basic equations for magnetic fluids with internal rotation is proposed in this paper. The basic equations are derived from the conservation laws of mass, linear momentum, angular momentum and energy, while the constitutive equations are obtained by the thermodynamical method that is based on the free energy and the dissipation function. The concrete expression of constitutive equations are determined by the principle of material frame indifference and the principle of maximal dissipation rate. The Abraham expression of the electromagnetic momentum and E-Banalogy are adopted in this theory. It is shown that the difference in the basic equations in case of adopting the Abraham expression and the Minkowski expression, respectively. The constitutive equation of magnetization which includes the Shliomis relaxation equation (1972) is proposed.

  15. Constitutive expression of murine c-FLIPR causes autoimmunity in aged mice.

    PubMed

    Ewald, F; Annemann, M; Pils, M C; Plaza-Sirvent, C; Neff, F; Erck, C; Reinhold, D; Schmitz, I

    2014-01-01

    Death receptor-mediated apoptosis is a key mechanism for the control of immune responses and dysregulation of this pathway may lead to autoimmunity. Cellular FLICE-inhibitory proteins (c-FLIPs) are known as inhibitors of death receptor-mediated apoptosis. The only short murine c-FLIP splice variant is c-FLIPRaji (c-FLIPR). To investigate the functional role of c-FLIPR in the immune system, we used the vavFLIPR mouse model constitutively expressing murine c-FLIPR in all hematopoietic compartments. Lymphocytes from these mice are protected against CD95-mediated apoptosis and activation-induced cell death. Young vavFLIPR mice display normal lymphocyte compartments, but the lymphocyte populations alter with age. We identified reduced levels of T cells and slightly higher levels of B cells in 1-year-old vavFLIPR mice compared with wild-type (WT) littermates. Moreover, both B and T cells from aged vavFLIPR animals show activated phenotypes. Sera from 1-year-old WT and transgenic animals were analysed for anti-nuclear antibodies. Notably, elevated titres of these autoantibodies were detected in vavFLIPR sera. Furthermore, tissue damage in kidneys and lungs from aged vavFLIPR animals was observed, indicating that vavFLIPR mice develop a systemic lupus erythematosus-like phenotype with age. Taken together, these data suggest that c-FLIPR is an important modulator of apoptosis and enforced expression leads to autoimmunity. PMID:24722293

  16. Conditional Derepression of Ferritin Synthesis in Cells Expressing a Constitutive IRP1 Mutant

    PubMed Central

    Wang, Jian; Pantopoulos, Kostas

    2002-01-01

    Iron regulatory protein 1 (IRP1), a major posttranscriptional regulator of cellular iron and energy metabolism, is controlled by an iron-sulfur cluster switch. Cysteine-437 is critical for coordinating the cluster, and its replacement yields mutants that do not respond to iron perturbations and constitutively bind to cognate mRNA iron-responsive elements (IREs). The expression of IRP1C437S in cells has been associated with aberrations in iron homeostasis and toxicity. We have established clones of human lung (H1299) and breast (MCF7) cancer cells that express high levels of IRP1C437S in a tetracycline-inducible manner. As expected, IRP1C437S stabilizes transferrin receptor mRNA and inhibits translation of ferritin mRNA in both cell types by binding to their respective IREs. However, H1299 transfectants grown at high densities are able to overcome the IRP1C437S-mediated inhibition in ferritin synthesis. The mechanism involves neither alteration in ferritin mRNA levels nor utilization of alternative transcription start sites to eliminate the IRE or relocate it in less inhibitory downstream positions. The derepression of ferritin mRNA translation occurs under conditions where global protein synthesis appears to be impaired, as judged by a significant enrichment in the expression of the underphosphorylated form of the translational regulator 4E-BP1. Collectively, these data document an example where ferritin mRNA translation evades control of the IRE-IRP system. The physiological implications of this response are reflected in protection against iron-mediated toxicity, oxidative stress, and apoptosis. PMID:12052872

  17. 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

  18. The embryo MADS domain factor AGL15 acts postembryonically. Inhibition of perianth senescence and abscission via constitutive expression.

    PubMed

    Fernandez, D E; Heck, G R; Perry, S E; Patterson, S E; Bleecker, A B; Fang, S C

    2000-02-01

    AGL15 (AGAMOUS-like 15), a member of the MADS domain family of regulatory factors, accumulates preferentially throughout the early stages of the plant life cycle. In this study, we investigated the expression pattern and possible roles of postembryonic accumulation of AGL15. Using a combination of reporter genes, RNA gel blot analysis, and immunochemistry, we found that the AGL15 protein accumulates transiently in the shoot apex in young Arabidopsis and Brassica seedlings and that promoter activity is associated with the shoot apex and the base of leaf petioles throughout the vegetative phase. During the reproductive phase, AGL15 accumulates transiently in floral buds. When AGL15 was expressed in Arabidopsis under the control of a strong constitutive promoter, we noted a striking increase in the longevity of the sepals and petals as well as delays in a selected set of age-dependent developmental processes, including the transition to flowering and fruit maturation. Although ethylene has been implicated in many of these same processes, the effects of AGL15 could be clearly distinguished from the effects of the ethylene resistant1-1 mutation, which confers dominant insensitivity to ethylene. By comparing the petal breakstrength (the force needed to remove petals) for flowers of different ages, we determined that ectopic AGL15 had a novel effect: the breakstrength of petals initially declined, as occurs in the wild type, but was then maintained at an intermediate value over a prolonged period. Abscission-associated gene expression and structural changes were also altered in the presence of ectopic AGL15. PMID:10662856

  19. 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

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

    PubMed

    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

  1. Constitutive homologous expression of phosphoglucomutase and transaldolase increases the metabolic flux of Fusarium oxysporum

    PubMed Central

    2014-01-01

    Background Fusarium oxysporum is among the few filamentous fungi that have been reported of being able to directly ferment biomass to ethanol in a consolidated bioprocess. Understanding its metabolic pathways and their limitations can provide some insights on the genetic modifications required to enhance its growth and subsequent fermentation capability. In this study, we investigated the hypothesis reported previously that phosphoglucomutase and transaldolase are metabolic bottlenecks in the glycolysis and pentose phosphate pathway of the F. oxysporum metabolism. Results Both enzymes were homologously overexpressed in F. oxysporum F3 using the gpdA promoter of Aspergillus nidulans for constitutive expression. Transformants were screened for their phosphoglucomutase and transaldolase genes expression levels with northern blot. The selected transformant exhibited high mRNA levels for both genes, as well as higher specific activities of the corresponding enzymes, compared to the wild type. It also displayed more than 20 and 15% higher specific growth rate upon aerobic growth on glucose and xylose, respectively, as carbon sources and 30% higher biomass to xylose yield. The determination of the relative intracellular amino and non-amino organic acid concentrations at the end of growth on glucose revealed higher abundance of most determined metabolites between 1.5- and 3-times in the recombinant strain compared to the wild type. Lower abundance of the determined metabolites of the Krebs cycle and an 68-fold more glutamate were observed at the end of the cultivation, when xylose was used as carbon source. Conclusions Homologous overexpression of phosphoglucomutase and transaldolase in F. oxysporum was shown to enhance the growth characteristics of the strain in both xylose and glucose in aerobic conditions. The intracellular metabolites profile indicated how the changes in the metabolome could have resulted in the observed growth characteristics. PMID:24649884

  2. Constitutive expression and anticancer potency of a novel immunotoxin onconase-DV3.

    PubMed

    Sun, Miaonan; Tang, Huichun; Gao, Yan; Dai, Xinxuan; Yuan, Yue; Zhang, Chunmei; Sun, Dejun

    2016-04-01

    Onconase is an RNase of the ribonuclease A superfamily that is purified from the Northern leopard frog (Rana pipiens). It targets several types of malignant tumors, digests cytoplasmic transfer RNA (tRNA), and causes tumor cell apoptosis. Onconase has been employed in clinical trials as an antitumor drug, and has revealed its valuable clinical activity in several types of tumors, particularly pleural mesothelioma. However, its inefficiency in targeting tumor cells and its non‑specific toxicity in normal tissues have diminished its clinical benefits. Furthermore, cyclization of the N-terminal glutamine residue (Gln), possesses more RNase activity than the structure of Met ahead of Glu in the N-terminal (99:1), which is more difficult for producing onconase by Pichia pastoris. Under the guidance of α-mating factor-pre (α-MF-pre) secretion signal, the secretion of the recombinant protein can reach a high level. In the present study, we constructed a constitutive expression vector for onconase-(DV3)2 (Onc-DV3) production in yeast Pichia pastoris with the GAP promoter, in which the Onc-DV3 gene is inserted downstream of the truncated Saccharomyces cerevisiae α-mating factor-pre (α-MF-pre) secretion signal. The immuno-RNase Onc-DV3 expressed a high level of production and bioactivity and possessed enhanced capability to deliver the Onc molecule to tumor cell monomeric counterparts. Notably, Onc-DV3 showed strong cytotoxicity to highly metastatic tumor cells, weak cytotoxicity to lowly metastatic tumor cells and no toxicity to normal cells. These results demonstrate that the specific toxicity to highly metastatic tumor cells has made Onc-DV3 a promising antitumor drug by using two copies of DV3 for the targeted delivery of onconase. PMID:26782924

  3. Alkyl Hydroperoxide Reductases C and D Are Major Antigens Constitutively Expressed by Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Olsen, Ingrid; Reitan, Liv J.; Holstad, Gudmund; Wiker, Harald G.

    2000-01-01

    Antigens characteristic for Mycobacterium avium subspecies paratuberculosis were identified by crossed immunoelectrophoresis (CIE) and by absorbing out cross-reactive antigens by using a polyclonal and polyvalent Mycobacterium avium subspecies avium antiserum. Two antigens were present in M. avium subsp. paratuberculosis and not detected in Mycobacterium avium subsp. avium. They were identified as antigens 17 and 20 in a CIE reference system for M. avium subsp. paratuberculosis antigens. Purified antigen 20 was identified as alkyl hydroperoxide reductase C (AhpC) while the N-terminal part of purified antigen 17 showed 80% homology with alkyl hydroperoxide reductase D (AhpD) of Mycobacterium tuberculosis. AhpC had a nonreduced mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 45 kDa and is probably a homodimer linked with disulfide bridges in its native form. AhpD had a mobility corresponding to 19 kDa. Monospecific rabbit antiserum against AhpC and AhpD reacted with 9 strains of M. avium subsp. paratuberculosis but not with 20 other mycobacterial strains except for a Mycobacterium gordonae strain, against which a weak cross-reactive band was produced. Goats experimentally infected with M. avium subsp. paratuberculosis had strong gamma interferon (IFN-γ) responses toward both AhpC and AhpD, and they also had antibodies against AhpC. The ability of AhpC and AhpD to induce IFN-γ production shows that these proteins potentially could be used in future vaccines or in diagnostic assays. These results further show that AhpC and AhpD are immunologically important proteins which are constitutively and highly expressed in M. avium subsp. paratuberculosis without the bacteria being submitted to oxidative stress and that the specificities of antigens can be a matter of different levels of protein expression in various species as well as distinct structural differences. PMID:10639449

  4. CONSTITUTIVE AND STIMULATED MCP-1, GROA, B, AND Y EXPRESSION IN HUMAN A AIRWAY EPITHELIUM AND BRONCHOALVEOLAR MACROPHAGES

    EPA Science Inventory

    Constitutive expression of mRNAs for GROa, GROB, GROY, and MCP-1, belonging to the chemokine family of 8-10 kD cytokines with chemotactic properties for granulocytes and monocytes, has been identified in freshly isolated human nasal and bronchial epithelium, and in bronchoalveola...

  5. On the nature of facultative and constitutive CAM: environmental and developmental control of CAM expression during early growth of Clusia, Kalanchöe, and Opuntia.

    PubMed

    Winter, Klaus; Garcia, Milton; Holtum, Joseph A M

    2008-01-01

    The capacity to induce crassulacean acid metabolism developmentally (constitutive CAM) and to up-regulate CAM expression in response to drought stress (facultative CAM) was studied in whole shoots of seven species by measuring net CO(2) gas exchange for up to 120 day-night cycles during early growth. In Clusia rosea, CAM was largely induced developmentally. Well-watered seedlings began their life cycle as C(3) plants and developed net dark CO(2) fixation indicative of CAM after the initiation of the fourth leaf pair following the cotyledons. Thereafter, CAM activity increased progressively and drought stress led to only small additional, reversible increases in dark CO(2) fixation. In contrast, CAM expression was overwhelmingly under environmental control in seedlings and mature plants of Clusia pratensis. C(3)-type CO(2) exchange was maintained under well-watered conditions, but upon drought stress, CO(2) exchange shifted, in a fully reversible manner, to a CAM-type pattern. Clusia minor showed CO(2) exchange reponses intermediate to those of C. rosea and C. pratensis. Clusia cretosa operated in the C(3) mode at all times. Notably, reversible stress-induced increases of dark CO(2) fixation were also observed during the developmental progression to pronounced CAM in young Kalanchoë daigremontiana and Kalanchoë pinnata, two species considered constitutive CAM species. Drought-induced up-regulation of CAM was even detected in young cladodes of a cactus, Opuntia ficus-indica, an archetypal constitutive CAM species. Evidently, the defining characteristics of constitutive and facultative CAM are shared, to variable degrees, by all CAM species. PMID:18440928

  6. Mutations in SIN4 and RGR1 cause constitutive expression of MAL structural genes in Saccharomyces cerevisiae.

    PubMed

    Wang, Xin; Michels, Corinne A

    2004-10-01

    Transcription of the Saccharomyces MAL structural genes is induced 40-fold by maltose and requires the MAL-activator and maltose permease. To identify additional players involved in regulating MAL gene expression, we carried out a genetic selection for MAL constitutive mutants. Strain CMY4000 containing MAL1 and integrated copies of MAL61promoter-HIS3 and MAL61promoter-lacZ reporter genes was used to select constitutive mutants. The 29 recessive mutants fall into at least three complementation groups. Group 1 and group 2 mutants exhibit pleiotropic phenotypes and represent alleles of Mediator component genes RGR1 and SIN4, respectively. The rgr1 and sin4 constitutive phenotype does not require either the MAL-activator or maltose permease, indicating that Mediator represses MAL basal expression. Further genetic analysis demonstrates that RGR1 and SIN4 work in a common pathway and each component of the Mediator Sin4 module plays a distinct role in regulating MAL gene expression. Additionally, the Swi/Snf chromatin-remodeling complex is required for full induction, suggesting a role for chromatin remodeling in the regulation of MAL gene expression. A sin4Delta mutation is unable to suppress the defects in MAL gene expression resulting from loss of the Swi/Snf complex component Snf2p. The role of the Mediator in MAL gene regulation is discussed. PMID:15514050

  7. 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

  8. Constitutive expression of exogenous myc in myelomonocytic cells: acquisition of a more transformed phenotype and inhibition of differentiation induction.

    PubMed

    Chisholm, O; Stapleton, P; Symonds, G

    1992-09-01

    The effects of deregulated expression of the human c-myc and MC29 v-myc oncogenes have been examined in a murine myelomonocytic cell line J774 (c-myc) and in a variety of myelomonocytic cell lines of different degrees of maturity generated from primary hematopoietic tissue (v-myc). Introduction of a Moloney murine leukemia virus long terminal repeat (LTR) c-myc construct into J774 cells resulted in constitutive expression of the exogenous myc gene and a concomitant increase in the degree of transformation and tumorigenicity of the cells. In addition, constitutive expression of exogenous myc inhibited induced differentiation of these cells by a variety of treatments including addition to the medium of lipopolysaccharide (LPS) or the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) as well as complete withdrawal of serum from the medium. The degree of increased transformation, tumorigenicity and inhibition of terminal differentiation was dependent upon the level of exogenous myc expression. For the v-myc-generated myelomonocytic cell lines, introduction of v-myc resulted in a high degree of transformation and, irrespective of the differentiation status of the cells, a block of induced differentiation. These results indicate that the level of constitutive myc expression can affect the transformed phenotype, tumorigenicity and differentiation inducibility of myelomonocytic cells. PMID:1501891

  9. 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

  10. Constitutive expression of cytochrome P450 in foetal and adult porcine livers-Effects of body weight.

    PubMed

    Rasmussen, Martin Krøyer; Theil, Peter Kappel; Oksbjerg, Niels

    2016-09-01

    The liver hosts a great number of enzymatically driven processes, including detoxification. The super-family of enzymes named cytochrome P450 (CYP) is the major participant in that process. The expression of CYPs is affected by several factors including life-stage (foetal vs. adult). In the present study we investigated the impact of birth-weight (high or low birth weight) and life-stage on constitutive expression of porcine hepatic CYP1A1, CYP1A2, CYP2A19, CYP2B22, CYP2C33, CYP2D25, CYP2E1 and CYP3A29, as well as the transcription factors controlling their expression; aryl hydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, C/EBP and hepatocyte nuclear factors 1 and 4. Both RT-PCR and western blotting showed a marked increase in the expression of the adult pigs compared with prenatal pigs. Moreover, CYP2E1 mRNA expression was 7.5 fold higher in foetuses with low birth weight compared with foetuses with high birth weight. Gender did not affect the mRNA expression within the different life-stages. These results indicate a similarity to what is observed in humans and porcine foetuses may therefore be a model for humans when studying expression of CYPs. PMID:27320961

  11. A dual cis-regulatory code links IRF8 to constitutive and inducible gene expression in macrophages

    PubMed Central

    Mancino, Alessandra; Termanini, Alberto; Barozzi, Iros; Ghisletti, Serena; Ostuni, Renato; Prosperini, Elena; Ozato, Keiko

    2015-01-01

    The transcription factor (TF) interferon regulatory factor 8 (IRF8) controls both developmental and inflammatory stimulus-inducible genes in macrophages, but the mechanisms underlying these two different functions are largely unknown. One possibility is that these different roles are linked to the ability of IRF8 to bind alternative DNA sequences. We found that IRF8 is recruited to distinct sets of DNA consensus sequences before and after lipopolysaccharide (LPS) stimulation. In resting cells, IRF8 was mainly bound to composite sites together with the master regulator of myeloid development PU.1. Basal IRF8–PU.1 binding maintained the expression of a broad panel of genes essential for macrophage functions (such as microbial recognition and response to purines) and contributed to basal expression of many LPS-inducible genes. After LPS stimulation, increased expression of IRF8, other IRFs, and AP-1 family TFs enabled IRF8 binding to thousands of additional regions containing low-affinity multimerized IRF sites and composite IRF–AP-1 sites, which were not premarked by PU.1 and did not contribute to the basal IRF8 cistrome. While constitutively expressed IRF8-dependent genes contained only sites mediating basal IRF8/PU.1 recruitment, inducible IRF8-dependent genes contained variable combinations of constitutive and inducible sites. Overall, these data show at the genome scale how the same TF can be linked to constitutive and inducible gene regulation via distinct combinations of alternative DNA-binding sites. PMID:25637355

  12. 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.

  13. 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.

  14. 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

  15. PLEXdb: gene expression resources for plants and plant pathogens.

    PubMed

    Dash, Sudhansu; Van Hemert, John; Hong, Lu; Wise, Roger P; Dickerson, Julie A

    2012-01-01

    PLEXdb (http://www.plexdb.org), 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 facilitate the interpretation of structure, function and regulation of genes in economically important plants. A list of Gene Atlas experiments highlights data sets that give responses across different developmental stages, conditions and tissues. Tools at PLEXdb allow users to perform complex analyses quickly and easily. The Model Genome Interrogator (MGI) tool supports mapping gene lists onto corresponding genes from model plant organisms, including rice and Arabidopsis. MGI predicts homologies, displays gene structures and supporting information for annotated genes and full-length cDNAs. The gene list-processing wizard guides users through PLEXdb functions for creating, analyzing, annotating and managing gene lists. Users can upload their own lists or create them from the output of PLEXdb tools, and then apply diverse higher level analyses, such as ANOVA and clustering. PLEXdb also provides methods for users to track how gene expression changes across many different experiments using the Gene OscilloScope. This tool can identify interesting expression patterns, such as up-regulation under diverse conditions or checking any gene's suitability as a steady-state control. PMID:22084198

  16. 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

  17. Constitutive expression of fibronectin binding in Streptococcus pyogenes as a result of anaerobic activation of rofA.

    PubMed Central

    Fogg, G C; Caparon, M G

    1997-01-01

    Protein F is a fibronectin-binding surface protein of Streptococcus pyogenes (group A streptococcus) that mediates adherence to host cells. A gene product encoded by rofA activates transcription of the gene that encodes protein F (prtF) and was identified in a strain of S. pyogenes that expressed high levels of protein F under all conditions tested. Insertional inactivation of rofA in this strain results in a phenotype similar to that of other strains where high-level transcription of prtF occurs only in response to increased oxygen tension. In this study, we have compared the regulation of prtF and rofA in O2-regulated and constitutive strains in order to gain further insight into the function of rofA. Comparison of the prtF and rofA transcripts by S1 nuclease and primer extension assays indicated that the same promoters for each transcript are used in both O2-regulated and constitutive strains. However, analyses of rofA-lacZ reporter alleles revealed that a key difference between strains involves regulation of rofA itself. In O2-regulated strains, expression of rofA was elevated following culture under conditions of reduced O2 tension. However, a much more robust activation of rofA expression was observed when constitutive strains were grown under similar conditions. Exchange of reporter and rofA alleles between strains demonstrated that host genetic background, and not the sequence of the respective rofA allele or regulatory region, dictates the expression phenotype. Activation of rofA required RofA, and RofA was shown to bind specifically to DNA containing the promoters for rofA and prtF. Finally, overexpression of either allele of rofA caused constitutive expression of prtF regardless of host background. These data suggest a model where anaerobic expression of prtF in constitutive hosts is controlled at the level of transcription of rofA and implicate additional factors in this regulatory pathway. PMID:9324268

  18. A strong constitutive positive element is essential for the ammonium-regulated expression of a soybean gene encoding cytosolic glutamine synthetase.

    PubMed

    Tercé-Laforgue, T; Carrayol, E; Cren, M; Desbrosses, G; Hecht, V; Hirel, B

    1999-02-01

    In order to identify important promoter elements controlling the ammonium-regulated expression of the soybean gene GS15 encoding cytosolic glutamine synthetase, a series of 5' promoter deletions were fused to the GUS reporter gene. To allow the detection of positive and negative regulatory elements, a series of 3' deletions were fused to a -90 CaMV 35S promoter fragment placed upstream of the GUS gene. Both types of construct were introduced into Lotus corniculatus plants and soybean roots via Agrobacterium rhizogenes-mediated transformation. Both spectrophotometric enzymatic analysis and histochemical localization of GUS activity in roots, root nodules and shoots of transgenic plants revealed that a strong constitutive positive element (SCPE) of 400 bp, located in the promoter distal region is indispensable for the ammonium-regulated expression of GS15. Interestingly, this SCPE was able to direct constitutive expression in both a legume and non-legume background to a level similar to that driven by the CaMV 35S full-length promoter. In addition, results showed that separate proximal elements, located in the first 727 bp relative to the transcription start site, are essential for root- and root nodule-specific expression. This proximal region contains an AAAGAT and two TATTTAT consensus sequences characteristic of nodulin or nodule-enhanced gene promoters. A putative silencer region containing the same TATTTAT consensus sequence was identified between the SCPE and the organ-specific elements. The presence of positive, negative and organ-specific elements together with the three TATTTAT consensus sequences within the promoter strongly suggest that these multiple promoter fragments act in a cooperative manner, depending on the spatial conformation of the DNA for trans-acting factor accessibility. PMID:10092182

  19. Constitutive and cytokine-induced expression of human leukocyte antigens and cell adhesion molecules by human myotubes.

    PubMed Central

    Michaelis, D.; Goebels, N.; Hohlfeld, R.

    1993-01-01

    Understanding the immunobiology of muscle is relevant to muscular autoimmune diseases and to gene therapies based on myoblast transfer. We have investigated the constitutive and cytokine-induced intra- and extracellular expression of histocompatibility human leukocyte antigens (HLA) and cell adhesion molecules by multinucleated human myotubes using immunofluorescence microscopy. Myotubes constitutively expressed HLA class I but not HLA class II. Exposure to interferon-gamma, but not tumor necrosis factor-alpha, induced HLA-DR in the cytoplasm and on the surface membrane of approximately 40 to 95% of cultured myotubes. Surface expression was strongest in perinuclear membrane areas, and cytoplasmic expression was strongest at branching points and at the tips of myotubes. HLA-DP and HLA-DQ were not expressed in detectable amounts. Both interferon-gamma and tumor necrosis factor-alpha induced the intercellular adhesion molecule-1 (CD54) in the cytoplasm and on the surface of nearly all myotubes. The distribution of intercellular adhesion molecule-1 and HLA-DR was similar but not identical in double-positive myotubes. The leukocyte function-associated (LFA) adhesion molecules LFA-1 (CD11a/CD18), LFA-2 (CD2), and LFA-3 (CD58) could not be detected in the cytoplasm or on the surface. Our results indicate that cytokine-induced myotubes can participate in immune interactions with T lymphocytes. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8214008

  20. Consequences of constitutive and induced variation in plant nutritional quality for immune defence of a herbivore against parasitism.

    PubMed

    Bukovinszky, Tibor; Poelman, Erik H; Gols, Rieta; Prekatsakis, Georgios; Vet, Louise E M; Harvey, Jeffrey A; Dicke, Marcel

    2009-05-01

    The mechanisms through which trophic interactions between species are indirectly mediated by distant members in a food web have received increasing attention in the field of ecology of multitrophic interactions. Scarcely studied aspects include the effects of varying plant chemistry on herbivore immune defences against parasitoids. We investigated the effects of constitutive and herbivore-induced variation in the nutritional quality of wild and cultivated populations of cabbage (Brassica oleracea) on the ability of small cabbage white Pieris rapae (Lepidoptera, Pieridae) larvae to encapsulate eggs of the parasitoid Cotesia glomerata (Hymenoptera, Braconidae). Average encapsulation rates in caterpillars parasitised as first instars were low and did not differ among plant populations, with caterpillar weight positively correlating with the rates of encapsulation. When caterpillars were parasitised as second instar larvae, encapsulation of eggs increased. Caterpillars were larger on the cultivated Brussels sprouts plants and exhibited higher levels of encapsulation compared with caterpillars on plants of either of the wild cabbage populations. Observed differences in encapsulation rates between plant populations could not be explained exclusively by differences in host growth on the different Brassica populations. Previous herbivore damage resulted in a reduction in the larval weight of subsequent herbivores with a concomitant reduction in encapsulation responses on both Brussels sprouts and wild cabbage plants. To our knowledge this is the first study demonstrating that constitutive and herbivore-induced changes in plant chemistry act in concert, affecting the immune response of herbivores to parasitism. We argue that plant-mediated immune responses of herbivores may be important in the evaluation of fitness costs and benefits of herbivore diet on the third trophic level. PMID:19271243

  1. 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

  2. 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

  3. Analysis of the tumorigenic potential of common marmoset lymphoblastoid cells expressing a constitutively activated c-myc gene.

    PubMed Central

    Hotchin, N. A.; Wedderburn, N.; Roberts, I.; Thomas, J. A.; Bungey, J. A.; Naylor, B.; Crawford, D. H.

    1993-01-01

    The respective roles of Epstein-Barr virus (EBV) and c-myc in the pathogenesis of endemic Burkitt's lymphoma (BL) are unclear. In order to help resolve the question whether constitutive expression of the c-myc gene in an EBV-immortalised B cell is sufficient to induce a tumorigenic phenotype, B cells from a common marmoset (Callithrix jacchus) were immortalised with EBV, transfected with a constitutively activated c-myc gene and inoculated into the host animals. Despite the cell line transfected with c-myc displaying enhanced growth characteristics, in vitro and in vivo experiments demonstrated that this was not sufficient to induce a tumorigenic phenotype. This supports our previous findings with EBV-immortalised human B cells transfected with an activated c-myc gene (Hotchin et al., 1990). Images Figure 1 Figure 2 Figure 4 PMID:8388232

  4. Genetically engineered maize plants reveal distinct costs and benefits of constitutive volatile emissions in the field

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic manipulation of plant volatile emissions is a promising tool to enhance plant defences against herbivores. However, the potential costs associated with the manipulation of specific volatile synthase genes are unknown. Therefore, we investigated the physiological and ecological effects of tra...

  5. Constitutive expression of major histocompatibility complex class II antigens in pulmonary epithelium and endothelium varies among different species.

    PubMed

    Houser, Stuart L; Benjamin, Louis C; Wain, John C; Madsen, Joren C; Allan, James S

    2004-02-27

    We have observed high constitutive levels of class II antigen expression on porcine and human coronary endothelium, but not on the endothelium of rats and mice. This study examines whether a similar interspecies difference exists in the expression of class II molecules on pulmonary epithelium and endothelium. Lung tissues from naïve human, porcine, and rodent sources were stained with the monoclonal antibody ISCR3 and examined by light microscopy. Immunoperoxidase staining of class II molecules was observed on human and porcine pulmonary epithelium and endothelium, but was absent in rats and mice. By using an antibody with cross-species reactivity, we demonstrated that naïve swine pulmonary epithelium and endothelium, unlike those of rodent species, express basal levels of class II antigens in a manner similar to that observed in human lung tissue. These interspecies differences may explain experimental differences observed between murine and large-animal constructs. PMID:15084944

  6. 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

  7. Expression of alternansucrase in potato plants

    PubMed Central

    Kok-Jacon, Géraldine A.; Vincken, Jean-Paul; Suurs, Luc C. J. M.; Wang, Denong; Liu, Shaoyi

    2007-01-01

    Alternan, which consists of alternating α-(1→3)/α-(1→6)-linked glucosyl residues, was produced in potato tubers by expressing a mature alternansucrase (Asr) gene from Leuconostoc mesenteroides NRRL B-1355 in potato. Detection of alternan was performed by enzyme-linked immunosorbent assay in tuber juices, revealing a concentration between 0.3 and 1.2 mg g-1 fresh wt. The Asr transcript levels correlated well with alternan accumulation in tuber juices. It appeared that the expression of sucrose-regulated starch-synthesizing genes (ADP-glucose pyrophosphorylase subunit S and granule-bound starch synthase I) was down-regulated. Despite this, the physico-chemical properties of the transgenic starches were unaltered. These results are compared to those obtained with other transgenic potato plants producing mutan [α-(1→3)-linked glucosyl residues] and dextran [α-(1→6)-linked glucosyl residues]. PMID:17380272

  8. Measurement of tomato plant gene expression on a genomic scale for tomato plants that over express peroxidase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We examined the gene expression of transgenic tomato plants that over-express the plant defense peroxidase in comparison to the control tomato plants with normal levels of peroxidase. In general, jasmonate-related plant defenses such as putative protease inhibitors were suppressed in peroxidase-rel...

  9. Constitutive expression of various xenobiotic and endobiotic transporter mRNAs in the choroid plexus of rats.

    PubMed

    Choudhuri, Supratim; Cherrington, Nathan J; Li, Ning; Klaassen, Curtis D

    2003-11-01

    The aim of this study was to quantitatively determine the constitutive expression levels of various transporter mRNAs in rat choroid plexus. To provide a reference for the relative expression levels, the expression of various transporter mRNAs in choroid plexus were compared with that in liver, kidney, and ileum. The mRNA levels of multidrug resistance protein (Mrp)1, 2, 3, 4, 5, and 6; multidrug resistance (Mdr)1a, 1b, and 2; organic anion transporting polypeptide (Oatp)1, 2, 3, 4, 5, 9, 12, and Oat-K (1/2); organic anion transporter (Oat)1, 2, and 3; organic cation transporter (Oct)1, 2, 3, N1, and N2; bile acid transporters sodium taurocholate cotransporting polypeptide (Ntcp), bile salt excretory protein (Bsep), and ileal bile acid transporter (Ibat); divalent metal transporter 1 (DMT1), Menke's and Wilson's metal transporters; equilibrative nucleotide transporters (Ent) 1 and 2, and constitutive nucleotide transporters (Cnt)1 and 2; peptide transporters (Pept)1 and 2; as well as ATP-binding cassette (Abc)G5 and 8 were measured in choroid plexus by the branched DNA signal amplification method. Mrp1, 4, and 5, Oatp3, Menke's transporter, DMT1, Ent1, and Pept2 mRNAs were expressed in choroid plexus at higher levels than in liver, kidney, or ileum. OctN1 and N2, Oatp2, Oat2 and 3, and Cnt1 and 2 mRNAs expressions were detectable in choroid plexus, but the levels were lower compared with that in liver, kidney, or ileum. The remaining transporters [Mrp2, Mrp3, Oct1, Oct2, Oatp1, Oatp4, Oatp5, Oatp12, Oat-K (1/2), Ntcp, Bsep, Ibat, Mdr1a, Mdr1b, Mdr2, Oat1, Ent2, Pept1, AbcG5, AbcG8] were expressed at very low levels in choroid plexus. The constitutive expression levels of different transporters in choroid plexus may provide an insight into the range of xenobiotics that can potentially be transported by the choroid plexus, thereby providing a means of xenobiotic detoxification in the brain. PMID:14570765

  10. 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

  11. 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

  12. Constitutive heterologous expression of avrXa27 in rice containing the R gene Xa27 confers enhanced resistance to compatible Xanthomonas oryzae strains.

    PubMed

    Tian, Dongsheng; Yin, Zhongchao

    2009-01-01

    The vascular pathogen Xanthomonas oryzae pv. oryzae (Xoo) and nonvascular pathogen Xanthomonas oryzae pv. oryzicola (Xoc) cause bacterial blight (BB) and bacterial leaf streak (BLS) diseases of rice, respectively. We have previously identified the avirulence gene avrXa27 from Xoo PXO99(A), which specifically induces the expression of the rice resistance gene Xa27, ultimately leading to resistance against BB disease in rice. In this study, we have generated a transgenic rice line (L24) that expresses avrXa27 constitutively under the control of the PR1 promoter, and have examined its role in the host-pathogen interaction. L24 is not more susceptible to BB, indicating that avrXa27 does not contribute to virulence. AvrXa27 retains avirulence activity in L24 and, after crossing with a line containing Xa27, progeny display phenotypic changes including inhibition of tillering, delay in flowering, stiff leaves, early leaf senescence and activation of pathogenesis-related (PR) genes. On challenge with a variety of compatible strains of Xoo and Xoc strain L8, lines with both avrXa27 and Xa27 also show enhanced resistance to bacterial infection. The induction of Xa27 and subsequent inhibition of Xoc growth in Xa27 plants are observed on inoculation with Xoc L8 harbouring avrXa27. Our results indicate that the heterologous expression of avrXa27 in rice containing Xa27 triggers R gene-specific resistance and, at the same time, confers enhanced resistance to compatible strains of Xoo and Xoc. The expression of AvrXa27 and related proteins in plants has the potential to generate broad resistance in plants. PMID:19161350

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. GUS expression driven by constitutive and vascular specific promoters in citrus hybrid US-802

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic solutions are being widely explored to develop huanglongbing (HLB) resistance in citrus, and a critical component of the transgenic construct is the promoter, which determines tissue specificity and level of target gene expression. This study compares the characteristics of five promoters...

  1. 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...

  2. 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

  3. 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

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

    PubMed

    Tseng, Hua-Pin; Hseu, Tzong-Hsiung; Buhler, Donald R; Wang, Wen-Der; Hu, Chin-Hwa

    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. PMID:15922010

  5. 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.

  6. Increased ability of transgenic plants expressing the bacterial enzyme ACC deaminase to accumulate Cd, Co, Cu, Ni, Pb, and Zn.

    PubMed

    Grichko, V P; Filby, B; Glick, B R

    2000-07-28

    Transgenic tomato plants Lycopersicon esculentum (Solanaceae) cv. Heinz 902 expressing the bacterial gene 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, under the transcriptional control of either two tandem 35S cauliflower mosaic virus promoters (constitutive expression), the rolD promoter from Agrobacterium rhizogenes (root specific expression) or the pathogenesis related PRB-1b promoter from tobacco, were compared to non-transgenic tomato plants in their ability to grow in the presence of Cd, Co, Cu, Mg, Ni, Pb, or Zn and to accumulate these metals. Parameters that were examined include metal concentration and ACC deaminase activity in both plant shoots and roots; root and shoot development; and leaf chlorophyll content. In general, transgenic tomato plants expressing ACC deaminase, especially those controlled by the PRB-1b promoter, acquired a greater amount of metal within the plant tissues, and were less subject to the deleterious effects of the metals on plant growth than were non-transgenic plants. PMID:10936659

  7. Construction of a constitutively expressed homo-fermentative pathway in Lactobacillus brevis.

    PubMed

    Guo, Wei; He, Ronglin; Ma, Lijuan; Jia, Wendi; Li, Demao; Chen, Shulin

    2014-08-01

    Lactobacillus brevis is a promising lactic acid producing strain that simultaneously utilizes glucose and xylose from lignocellulosic hydrolysate without carbon catabolic repression and inhibition. The production of by-products acetic acid and ethanol has been the major drawback of this strain. Two genes, pfkA (fructose-6-phosphate kinase [PFK]) and fbaA (fructose-1,6-biphosphate aldolase [FBA]), that encode the key enzymes of the EMP/glycolytic pathway from Lactobacillus rhamnosus, were fused to the downstream of the strong promoter P32 and expressed in L. brevis s3f4 as a strategy to minimize the formation of by-products. By expressing the two enzymes, a homo-fermentative pathway for lactic acid production was constructed. The lactic acid yields achieved from glucose in the transformants were 1.12 and 1.16 mol/mol, which is higher than that of the native strain (0.74 mol/mol). However, the lactic acid yield from xylose in the transformants stayed the same as that of the native strain. Enzyme assay indicated that the activity of the foreign protein FBA in the transformants was much higher than that of the native strains, but was ten times lower than that in L. rhamnosus. This result was consistent with the metabolic flux analysis, which indicated that the conversion efficiency of the expressed PFK and FBA was somewhat low. Less than 20 % of the carbons accumulated in the form of fructose-6-phosphate were converted into glyceraldehyde-3-phosphate (GAP) by the expressed PFK and FBA. Metabolic flux analysis also indicated that the enzyme phosphoketolase (XPK) played an important role in splitting the carbon flow from the pentose phosphate pathway to the phosphoketolase pathway. This study suggested that the lactic acid yield of L. brevis could be improved by constructing a homo-fermentative pathway. PMID:24728715

  8. 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

  9. 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

  10. 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

  11. 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

  12. Constitutive expression of the Vi polysaccharide capsular antigen in attenuated Salmonella enterica serovar typhi oral vaccine strain CVD 909.

    PubMed

    Wang, J Y; Noriega, F R; Galen, J E; Barry, E; Levine, M M

    2000-08-01

    Live oral Ty21a and parenteral Vi polysaccharide vaccines provide significant protection against typhoid fever, albeit by distinct immune mechanisms. Vi stimulates serum immunoglobulin G Vi antibodies, whereas Ty21a, which does not express Vi, elicits humoral and cell-mediated immune responses other than Vi antibodies. Protection may be enhanced if serum Vi antibody as well as cell-mediated and humoral responses can be stimulated. Disappointingly, several new attenuated Salmonella enterica serovar Typhi oral vaccines (e.g., CVD 908-htrA and Ty800) that elicit serum O and H antibody and cell-mediated responses following a single dose do not stimulate serum Vi antibody. Vi expression is regulated in response to environmental signals such as osmolarity by controlling the transcription of tviA in the viaB locus. To investigate if Vi antibodies can be stimulated if Vi expression is rendered constitutive, we replaced P(tviA) in serovar Typhi vaccine CVD 908-htrA with the constitutive promoter P(tac), resulting in CVD 909. CVD 909 expresses Vi even under high-osmolarity conditions and is less invasive for Henle 407 cells. In mice immunized with a single intranasal dose, CVD 909 was more immunogenic than CVD 908-htrA in eliciting serum Vi antibodies (geometric mean titer of 160 versus 49, P = 0.0007), whereas O antibody responses were virtually identical (geometric mean titer of 87 versus 80). In mice challenged intraperitoneally with wild-type serovar Typhi 4 weeks after a single intranasal immunization, the mortality of those immunized with CVD 909 (3 of 8) was significantly lower than that of control mice (10 of 10, P = 0.043) or mice given CVD 908-htrA (9 of 10, P = 0.0065). PMID:10899868

  13. Constitutive expression of pathogenesis-related proteins and antioxydant enzyme activities triggers maize resistance towards Fusarium verticillioides.

    PubMed

    Maschietto, Valentina; Lanubile, Alessandra; Leonardis, Silvana De; Marocco, Adriano; Paciolla, Costantino

    2016-08-01

    Fusarium verticillioides is a fungal pathogen of maize that causes ear rot and contaminates the grains with fumonisin mycotoxins. Breeding for resistance to Fusarium emerged as the most economic and environmentally safe strategy; therefore the discovery of resistant sources and effective molecular markers are a priority. Ears of resistant (CO441 and CO433) and susceptible (CO354 and CO389) maize lines were inoculated with F. verticillioides and the expression of pathogenesis-related (PR) genes (PR1, PR5, PRm3, PRm6) and genes that protect from oxidative stress (peroxidase, catalase, superoxide dismutase and ascorbate peroxidase) were evaluated in the kernels at 72h post inoculation. In addition, the oxidation level and the enzymatic activity of ascorbate-glutathione cycle, catalase, superoxide dismutase and cytosolic and wall peroxidases were investigated. The uninoculated kernels of the resistant lines showed higher gene expression and enzymatic activities, highlighting the key role of constitutive resistance in limiting pathogen attack. In contrast, the susceptible lines activated defensive genes only after pathogen inoculation, resulting in increased levels of H2O2 and lipid peroxidation, as well as lower enzymatic activities. The constitutive defenses observed in this study from seed could be profitably exploited to develop markers to speed up conventional breeding programs in the selection of resistant genotypes. PMID:27340858

  14. Constitutively active Akt1 expression in mouse pancreas requires S6 kinase 1 for insulinoma formation

    PubMed Central

    Alliouachene, Samira; Tuttle, Robyn L.; Boumard, Stephanie; Lapointe, Thomas; Berissi, Sophie; Germain, Stephane; Jaubert, Francis; Tosh, David; Birnbaum, Morris J.; Pende, Mario

    2008-01-01

    Factors that promote pancreatic β cell growth and function are potential therapeutic targets for diabetes mellitus. In mice, genetic experiments suggest that signaling cascades initiated by insulin and IGFs positively regulate β cell mass and insulin secretion. Akt and S6 kinase (S6K) family members are activated as part of these signaling cascades, but how the interplay between these proteins controls β cell growth and function has not been determined. Here, we found that although transgenic mice overexpressing the constitutively active form of Akt1 under the rat insulin promoter (RIP-MyrAkt1 mice) had enlarged β cells and high plasma insulin levels, leading to improved glucose tolerance, a substantial proportion of the mice developed insulinomas later in life, which caused decreased viability. This oncogenic transformation tightly correlated with nuclear exclusion of the tumor suppressor PTEN. To address the role of the mammalian target of rapamycin (mTOR) substrate S6K1 in the MyrAkt1-mediated phenotype, we crossed RIP-MyrAkt1 and S6K1-deficient mice. The resulting mice displayed reduced insulinemia and glycemia compared with RIP-MyrAkt1 mice due to a combined effect of improved insulin secretion and insulin sensitivity. Importantly, although the increase in β cell size in RIP-MyrAkt1 mice was not affected by S6K1 deficiency, the hyperplastic transformation required S6K1. Our results therefore identify S6K1 as a critical element for MyrAkt1-induced tumor formation and suggest that it may represent a useful target for anticancer therapy downstream of mTOR. PMID:18846252

  15. Constitutively expressed ERF-VII transcription factors redundantly activate the core anaerobic response in Arabidopsis thaliana.

    PubMed

    Bui, Liem T; Giuntoli, Beatrice; Kosmacz, Monika; Parlanti, Sandro; Licausi, Francesco

    2015-07-01

    Plant adaptation to hypoxic conditions is mediated by the transcriptional activation of genes involved in the metabolic reprogramming of plant cells to cope with reduced oxygen availability. Recent studies indicated that members of the group VII of the Ethylene Responsive Transcription Factor (ERFs) family act as positive regulators of this molecular response. In the current study, the five ERF-VII transcription factors of Arabidopsis thaliana were compared to infer a hierarchy in their role with respect to the anaerobic response. When the activity of each transcription factor was tested on a set of hypoxia-responsive promoters, RAP2.2, RAP2.3 and RAP2.12 appeared to be the most powerful activators. RAP2.12 was further dissected in transactivation assays in Arabidopsis protoplasts to identify responsible regions for transcriptional activation. An ultimate C-terminal motif was identified as sufficient to drive gene transcription. Finally, using realtime RT-PCR in single and double mutants for the corresponding genes, we confirmed that RAP2.2 and RAP2.12 exert major control upon the anaerobic response. PMID:26025519

  16. 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

  17. Resistance to simian immunodeficiency virus low dose rectal challenge is associated with higher constitutive TRIM5α expression in PBMC

    PubMed Central

    2014-01-01

    Background At least six host-encoded restriction factors (RFs), APOBEC3G, TRIM5α, tetherin, SAMHD1, schlafen 11, and Mx2 have now been shown to inhibit HIV and/or SIV replication in vitro. To determine their role in vivo in the resistance of macaques to mucosally-acquired SIV, we quantified both pre-exposure (basal) and post-exposure mRNA levels of these RFs, Mx1, and IFNγ in PBMC, lymph nodes, and duodenum of rhesus macaques undergoing weekly low dose rectal exposures to the primary isolate, SIV/DeltaB670. Results Repetitive challenge divided the monkeys into two groups with respect to their susceptibility to infection: highly susceptible (2–3 challenges, 5 monkeys) and poorly susceptible (≥6 challenges, 3 monkeys). Basal RF and Mx1 expression varied among the three tissues examined, with the lowest expression generally detected in duodenal tissues, and the highest observed in PBMC. The one exception was A3G whose basal expression was greatest in lymph nodes. Importantly, significantly higher basal expression of TRIM5α and Mx1 was observed in PBMC of animals more resistant to mucosal infection. Moreover, individual TRIM5α levels were stable throughout a year prior to infection. Post-exposure induction of these genes was also observed after virus appearance in plasma, with elevated levels in PBMC and duodenum transiently occurring 7–10 days post infection. They did not appear to have an effect on control of viremia. Interestingly, minimal to no induction was observed in the resistant animal that became an elite controller. Conclusions These results suggest that constitutively expressed TRIM5α appears to play a greater role in restricting mucosal transmission of SIV than that associated with type I interferon induction following virus entry. Surprisingly, this association was not observed with the other RFs. The higher basal expression of TRIM5α observed in PBMC than in duodenal tissues emphasizes the understated role of the second barrier to systemic

  18. Constitutive hepcidin expression prevents iron overload in a mouse model of hemochromatosis.

    PubMed

    Nicolas, Gaël; Viatte, Lydie; Lou, Dan-Qing; Bennoun, Myriam; Beaumont, Carole; Kahn, Axel; Andrews, Nancy C; Vaulont, Sophie

    2003-05-01

    Hereditary hemochromatosis is a prevalent genetic disorder of iron hyperabsorption leading to hyperferremia, tissue iron deposition and complications including cirrhosis, hepatocarcinoma, cardiomyopathy and diabetes. Most individuals affected with hereditary hemochromatosis are homozygous with respect to a missense mutation that disrupts the conformation of HFE, an atypical HLA class I molecule (ref. 1; OMIM 235200). Mice lacking Hfe or producing a C282Y mutant Hfe protein develop hyperferremia and have high hepatic iron levels. In both humans and mice, hereditary hemochromatosis is associated with a paucity of iron in reticuloendothelial cells. It has been suggested that HFE modulates uptake of transferrin-bound iron by undifferentiated intestinal crypt cells, thereby programming the absorptive capacity of enterocytes derived from these cells; however, this model is unproven and controversial. Hepcidin, a peptide hormone (HAMP; OMIM 606464), seems to act in the same regulatory pathway as HFE. Although expression of mouse Hamp is normally greater during iron overload, Hfe-/- mice have inappropriately low expression of Hamp. We crossed Hfe-/- mice with transgenic mice overexpressing Hamp and found that Hamp inhibited the iron accumulation normally observed in the Hfe-/- mice. This argues against the crypt programming model and suggests that failure of Hamp induction contributes to the pathogenesis of hemochromatosis, providing a rationale for the use of HAMP in the treatment of this disease. PMID:12704388

  19. Biomedicine in the environment: cyclotides constitute potent natural toxins in plants and soil bacteria.

    PubMed

    Ovesen, Rikke Gleerup; Brandt, Kristian Koefoed; Göransson, Ulf; Nielsen, John; Hansen, Hans Christian Bruun; Cedergreen, Nina

    2011-05-01

    Bioactive compounds produced by plants are easily transferred to soil and water and may cause adverse ecosystem effects. Cyclotides are gene-encoded, circular, cystine-rich mini-proteins produced in Violaceae and Rubiaceae in high amounts. Based on their biological activity and stability, cyclotides have promising pharmaceutical and agricultural applications. We report the toxicity of the cyclotides: kalata B1, kalata B2, and cycloviolacin O2 extracted from plants to green algae (Pseudokirchneriella subcapitata), duckweed (Lemna minor L.), lettuce (Lactuca sativa L.), and bacteria extracted from soil measured as [³H]leucine incorporation. Quantification by liquid chromatography-mass spectrometry demonstrated up to 98% loss of cyclotides from aqueous solutions because of sorption to test vials. Sorption was prevented by adding bovine serum albumin (BSA) to the aqueous media. Cyclotides were toxic to all test organisms with EC50 values of 12 through 140 µM (algae), 9 through 40 µM (duckweed), 4 through 54 µM (lettuce), and 7 through 26 µM (bacteria). Cycloviolacin O2 was the most potent cyclotide in all assays examined. This report is the first to document toxic effects of cyclotides in plants and soil bacteria and to demonstrate that cyclotides are as toxic as commonly used herbicides and biocides. Hence, cyclotides may adversely affect soil and aquatic environments, which needs to be taken into account in future risk assessment of cropping systems for production of these highly bioactive compounds. PMID:21337607

  20. Human fallopian tube epithelium constitutively expresses integrin endometrial receptivity markers: no evidence for a tubal implantation window.

    PubMed

    Brown, J K; Shaw, J L V; Critchley, H O D; Horne, A W

    2012-03-01

    Understanding of ectopic implantation within the Fallopian tube (FT) is limited. In the human uterus, the putative 'window of implantation' in the mid-luteal phase of the menstrual cycle is accompanied by increased endometrial epithelial expression of the integrins α(1)β(1), α(4)β(1) and α(v)β(3) and its ligand osteopontin. Similar cyclical changes in FT integrin expression have been proposed to contribute to ectopic implantation, but supporting data are limited. In the current study, we present quantitative data on human FT transcription and translation of the integrin subunits α(1), α(4), α(V), β(1) and β(3) during the follicular and mid-luteal phases of the menstrual cycle, together with a supporting immuocytochemical analysis of their spatial distribution within the FT, and that of osteopontin. In contrast to previous studies, our data indicate that all five integrin receptivity markers are constitutively transcribed and translated in the FT, with no evidence for changes in their expression or distribution during the window of implantation in the mid-luteal phase of the cycle. Furthermore, we could find no evidence for cyclic redistribution of the integrin α(v)β(3) ligand osteopontin within the FT. Although we do not rule out the involvement of integrin endometrial receptivity markers in the establishment of ectopic pregnancy, our findings do not support their differential expression during a tubal implantation window. PMID:22002573

  1. Colon epithelial cell differentiation is inhibited by constitutive c-myb expression or mutant APC plus activated RAS.

    PubMed

    Ramsay, Robert G; Ciznadija, Daniel; Sicurella, Catherine; Reyes, Nancy; Mitchelhill, Ken; Darcy, Phillip K; D'Abaco, Giovanna; Mantamadiotis, Theo

    2005-01-01

    Blocked differentiation is a hallmark of cancer cells and the restoration of differentiation programs in vivo is an actively pursued clinical aim. Understanding the key regulators of cyto-differentiation may focus therapies on molecules that reactivate this process. c-myb expression declines rapidly when human colon cancer epithelial cells are induced to differentiate with the physiologically relevant short-chain fatty acid, sodium butyrate. These cells show increased expression of alkaline phosphatase and cytokeratin 8. Similarly, murine Immorto-epithelial cells derived from wild-type colon cells also show c-myb mRNA declines when induced to differentiate with sodium butyrate. Immorto-cells harboring a single APC mutation are indistinguishable from wild-type cells with regard to differentiation, while addition of activated RAS alone markedly enhances differentiation. In marked contrast, complete differentiation arrest occurs when both APC and RAS are mutated. Expression of MybER, a 4-hydroxytamoxifen-activatable form of c-Myb, blocks differentiation in wildtype and APC mutant Immorto-cell lines as well as LIM1215 human colon carcinoma cells. These data identify two pathways of oncogenic change that lead to retarded epithelial cell differentiation, one involving the presence of a single APC mutation in conjunction with activated RAS or alternatively constitutive c-myb expression. PMID:15684716

  2. Expression of a plant defensin in rice confers resistance to fungal phytopathogens.

    PubMed

    Jha, Sanjay; Chattoo, Bharat B

    2010-06-01

    Transgenic rice (Oryza sativa L. cv. Pusa basmati 1), overexpressing the Rs-AFP2 defensin gene from the Raphanus sativus was generated by Agrobacterium tumefaciens-mediated transformation. Expression levels of Rs-AFP2 ranged from 0.45 to 0.53% of total soluble protein in transgenic plants. It was observed that constitutive expression of Rs-AFP2 suppresses the growth of Magnaporthe oryzae and Rhizoctonia solani by 77 and 45%, respectively. No effect on plant morphology was observed in the Rs-AFP2 expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of Rs-AFP2 plants on the in vitro growth of M. oryzae indicated that the Rs-AFP2 protein produced by transgenic rice plants was biologically active. Transgene expression of Rs-AFP2 was not accompanied by an induction of pathogenesis-related (PR) gene expression, suggesting that the expression of Rs-AFP2 directly inhibits the pathogens. Here, we demonstrate that transgenic rice plants expressing the Rs-AFP2 gene show enhanced resistance to M. oryzae and R. solani, two of the most important pathogens of rice. PMID:19690975

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

    PubMed

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

    2016-07-01

    Mast cells are evolutionarily ancient sentinel cells. Like basophils, mast cells express the high-affinity receptor for immunoglobulin E (IgE) and have been linked to host defense and diverse immune-system-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 shared more overlap with other circulating granulocytes than with mast cells. The derivation of mast-cell and basophil transcriptional signatures underscores their differential capacities to detect environmental signals and influence the inflammatory milieu. PMID:27135604

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

    DOEpatents

    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.

  5. Constitutive gene expression profile segregates toxicity in locally advanced breast cancer patients treated with high-dose hyperfractionated radical radiotherapy

    PubMed Central

    Henríquez Hernández, Luis Alberto; Lara, Pedro Carlos; Pinar, Beatriz; Bordón, Elisa; Gallego, Carlos Rodríguez; Bilbao, Cristina; Pérez, Leandro Fernández; Morales, Amílcar Flores

    2009-01-01

    Breast cancer patients show a wide variation in normal tissue reactions after radiotherapy. The individual sensitivity to x-rays limits the efficiency of the therapy. Prediction of individual sensitivity to radiotherapy could help to select the radiation protocol and to improve treatment results. The aim of this study was to assess the relationship between gene expression profiles of ex vivo un-irradiated and irradiated lymphocytes and the development of toxicity due to high-dose hyperfractionated radiotherapy in patients with locally advanced breast cancer. Raw data from microarray experiments were uploaded to the Gene Expression Omnibus Database (GEO accession GSE15341). We obtained a small group of 81 genes significantly regulated by radiotherapy, lumped in 50 relevant pathways. Using ANOVA and t-test statistical tools we found 20 and 26 constitutive genes (0 Gy) that segregate patients with and without acute and late toxicity, respectively. Non-supervised hierarchical clustering was used for the visualization of results. Six and 9 pathways were significantly regulated respectively. Concerning to irradiated lymphocytes (2 Gy), we founded 29 genes that separate patients with acute toxicity and without it. Those genes were gathered in 4 significant pathways. We could not identify a set of genes that segregates patients with and without late toxicity. In conclusion, we have found an association between the constitutive gene expression profile of peripheral blood lymphocytes and the development of acute and late toxicity in consecutive, unselected patients. These observations suggest the possibility of predicting normal tissue response to irradiation in high-dose non-conventional radiation therapy regimens. Prospective studies with higher number of patients are needed to validate these preliminary results. PMID:19497124

  6. 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

  7. 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

  8. 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

  9. Constitutive expression of the Poplar FD-like basic leucine zipper transcription factor alters growth and bud development.

    PubMed

    Parmentier-Line, Cécile M; Coleman, Gary D

    2016-01-01

    In poplar, the CO/FT regulatory module mediates seasonal growth cessation. Although FT interacts with the basic leucine zipper transcription factor FD, surprisingly little is known about the possible role of FD in bud development and growth cessation in trees. In this study, we examined the expression and localization of the poplar FD homolog, PtFD1, during short-day (SD)-induced bud development, and the consequences of overexpressing PtFD1 on bud development and shoot growth. PtFD1 was primarily expressed in apical and axillary buds and exhibited a transient increase in expression during the initial stages of SD-induced bud development. This transient increase declined with continued SD treatment. When PtFD1 was overexpressed in poplar, SD-induced growth cessation and bud formation were abolished. PTFD1 overexpression also resulted in precocious flowering of juvenile plants in long-day (LD) photoperiods. Because the phenotypes associated with overexpression of PtFD1 are similar to those observe when poplar FT1 is overexpressed (Science, 312, 2006, 1040), the expression and diurnal patterns of expression of both poplar FT1 and FT2 were characterized in PtFD1 overexpression poplars and found to be altered. DNA microarray analysis revealed few differences in gene expression between PtFD1 overexpressing poplars in LD conditions while extensive levels of differential gene expression occur in SD-treated plants. These results enforce the connection between the regulation of flowering and the regulation of growth cessation and bud development in poplar. PMID:25915693

  10. Constitutive expression of SMAR1 confers susceptibility to Mycobacterium tuberculosis infection in a transgenic mouse model

    PubMed Central

    Yadav, Bhawna; Malonia, Sunil K.; Majumdar, Subeer S.; Gupta, Pushpa; Wadhwa, Neerja; Badhwar, Archana; Gupta, Umesh D.; Katoch, Vishwa M.; Chattopadhyay, Samit

    2015-01-01

    Background & objectives: Studies involving animal models of experimental tuberculosis have elucidated the predominant role of cytokines secreted by T cells and macrophages to be an essential component of the immune response against Mycobacterium tuberculosis infection. The immune activities of CD4+ T cells are mediated in part by Th1 cytokine interferon gamma (IFN-γ) which is produced primarily by T cells and natural killer (NK) cells and critical for initiating the immune response against intracellular pathogen such as M. tuberculosis. Nuclear matrix protein SMAR1 plays an important role in V(D)J recombination, T helper cell differentiation and inflammatory diseases. In this study a transgenic mouse model was used to study the role of SMAR1 in M. tuberculosis infection. Methods: Wild type BALB/c, C57BL/6, BALB/c-EGFP-SMAR1 and C57BL/6-SMAR1 transgenic mice were infected with M. tuberculosis (H37Rv). A dose of 100 bacilli was used for infection via respiratory route. Bacterial load in lung and spleen of infected mice was determined at 2, 4, 6 and 8 wk post-infection. Gene expression analysis for Th1 cytokines and inducible nitric oxide synthase (iNOS) was performed in infected lung tissues by quantitative reverse transcription (RT)-PCR. Results: SMAR1 transgenic mice from both BALB/c and C57BL/6 genetic background displayed higher bacillary load and susceptibility to M. tuberculosis infection compared to wild type mice. This susceptibility was attributed due to compromised of Th1 response exhibited by transgenic mice. Interpretation & conclusions: SMAR1 transgenic mice exhibited susceptibility to M. tuberculosis infection in vivo irrespective of genetic background. This susceptibility was attributed to downregulation of Th1 response and its hallmark cytokine IFN-γ. Hence, SMAR1 plays an important role in modulating host immune response after M. tuberculosis infection. PMID:26831422

  11. 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

  12. What constitutes a population for the plant parasitic nematode Globodera pallida in its native area (Peru)?

    PubMed

    Picard, Damien; Plantard, Olivier

    2006-01-01

    Although numerous species are distributed in discrete populations easily recognised by geographical barriers, continuous populations are a common feature of plants or marine organisms. This is particularly true for soil organisms as their habitat is continuous and their range cannot easily be assessed as they are buried below ground. In the case of organisms for which standard methods such as Capture/Mark/Recapture cannot be used, population genetics provide a straightforward approach to delimitate populations. In this study, we have pursued this topic with a soil-dwelling nematode (Globodera pallida), which parasitises potato roots and is indigenous to South America. Potential barriers to gene flow were identified using the analysis of the F(ST)/(1-F(ST)) ratio against geographical distance and spatial autocorrelation combined with model-based clustering algorithm. Inside regions, neither genetic differentiation nor isolation by distance (IBD) occur among fields less than 50 km distant. We hypothesise that the large amount of gene flow revealed by the absence of genetic structure of this organism could be due to large passive dispersion inside an agronomic area where G. pallida has a continuous distribution and is found at high density. The first evidence of genetic differentiation appeared when a field was separated from others by an area free of farms (where G. pallida is absent or rare). Among regions, a high genetic structure coupled with an IBD pattern occurs as the consequences of the limitations of passive dispersal across deep valleys or high mountains. To our knowledge, this is the first study identifying the spatial limit of a population for a plant nematode parasite. PMID:16239004

  13. A Constitutive Expressed Phosphate Transporter, OsPht1;1, Modulates Phosphate Uptake and Translocation in Phosphate-Replete Rice1[W][OA

    PubMed Central

    Sun, Shubin; Gu, Mian; Cao, Yue; Huang, Xinpeng; Zhang, Xiao; Ai, Penghui; Zhao, Jianning; Fan, Xiaorong; Xu, Guohua

    2012-01-01

    A number of phosphate (Pi) starvation- or mycorrhiza-regulated Pi transporters belonging to the Pht1 family have been functionally characterized in several plant species, whereas functions of the Pi transporters that are not regulated by changes in Pi supply are lacking. In this study, we show that rice (Oryza sativa) Pht1;1 (OsPT1), one of the 13 Pht1 Pi transporters in rice, was expressed abundantly and constitutively in various cell types of both roots and shoots. OsPT1 was able to complement the proton-coupled Pi transporter activities in a yeast mutant defective in Pi uptake. Transgenic plants of OsPT1 overexpression lines and RNA interference knockdown lines contained significantly higher and lower phosphorus concentrations, respectively, compared with the wild-type control in Pi-sufficient shoots. These responses of the transgenic plants to Pi supply were further confirmed by the changes in depolarization of root cell membrane potential, root hair occurrence, 33P uptake rate and transportation, as well as phosphorus accumulation in young leaves at Pi-sufficient levels. Furthermore, OsPT1 expression was strongly enhanced by the mutation of Phosphate Overaccumulator2 (OsPHO2) but not by Phosphate Starvation Response2, indicating that OsPT1 is involved in the OsPHO2-regulated Pi pathway. These results indicate that OsPT1 is a key member of the Pht1 family involved in Pi uptake and translocation in rice under Pi-replete conditions. PMID:22649273

  14. 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

  15. Expression of constitutively active FoxO3 in murine forebrain leads to a loss of neural progenitors.

    PubMed

    Schmidt-Strassburger, Uta; Schips, Tobias G; Maier, Harald J; Kloiber, Katharina; Mannella, Francesca; Braunstein, Kerstin E; Holzmann, Karlheinz; Ushmorov, Alexey; Liebau, Stefan; Boeckers, Tobias M; Wirth, Thomas

    2012-12-01

    Inactivation of FoxO proteins by phosphorylation is the result of a number of stimuli, including the insulin/IGF pathway. We were interested in the consequence of blunting this pathway by employing transgenic mice with tetracycline-controllable conditional expression of a constitutively active allele of FOXO3 under the control of the forebrain-specific CaMKIIα promoter. Although transgene-expressing mice were viable, brain weight was reduced by 30% in adult animals. Brains showed an isocortex compression with normal cortical layering, and a size reduction in regions known to depend on adult neurogenesis, i.e., the olfactory bulbs and the dentate gyrus. On postnatal activation of the transgene, adult neurogenesis was also severely affected. Investigating the molecular basis of this phenotype, we observed enhanced apoptosis starting from embryonic day E10.5 and a subsequent loss of progenitors in the ventricular/subventricular zones, but not in the isocortex or the striatum of adult mice. The enhanced apoptosis was accompanied by increased expression of PIK3IP1, which we identified as a direct transcriptional target of FOXO3. Transfection of Pik3ip1 into differentiating neural progenitors resulted in a significant reduction of viable cells. We therefore conclude that neural progenitors are particularly vulnerable to FOXO3-induced apoptosis, which is mediated by PIK3IP1, a negative PI3 kinase regulator. PMID:22935140

  16. Molecular characterization of Haemophilus parasuis ferric hydroxamate uptake (fhu) genes and constitutive expression of the FhuA receptor.

    PubMed

    del Río, Maria Luisa; Navas, Jesús; Martín, Ana Judith; Gutiérrez, César B; Rodríguez-Barbosa, José-Ignacio; Rodríguez Ferri, Elías F

    2006-01-01

    Bacteria have evolved a set of highly specialized proteins to capture iron in iron-depleted environments. The acquisition and uptake of iron present in the extracellular milieu of eukaryotic organisms is indispensable for the growth and survival of microbial pathogens in the course of infection. Haemophilus parasuis is the causative agent of Glässer disease, which is responsible for considerable financial losses in pig-rearing worldwide. To gain insight into the mechanisms involved in siderophore-mediated iron uptake in H. parasuis, genes in the H. parasuis ferric hydroxamate uptake (Fhu) region were amplified in the work being reported here. As has been described in A. pleuropneumoniae, an Fhu genomic region was also present in H. parasuis, being composed of four potential consecutive open reading frames (ORF) designated as fhuC, fhuD, fhuB, and fhuA, respectively. By immunoblotting, using a cross-reactive polyclonal antibody raised against Actinobacillus pleuropneumoniae FhuA protein, it was demonstrated that this protein was constitutively expressed in H. parasuis and its level of expression was not modified under conditions of restricted iron availability. This is the first report describing the presence of the fhu genes in H. parasuis. Our results indicate that FhuA protein expression is not affected under iron-restricted conditions, however, it is one of the targets of the humoral immune response. PMID:16336924

  17. Constitutive TL1A Expression under Colitogenic Conditions Modulates the Severity and Location of Gut Mucosal Inflammation and Induces Fibrostenosis

    PubMed Central

    Barrett, Robert; Zhang, Xiaolan; Koon, Hon Wai; Vu, Michelle; Chang, Jyh-Yau; Yeager, Nicole; Nguyen, Mary Ann; Michelsen, Kathrin S.; Berel, Dror; Pothoulakis, Charalabos; Targan, Stephan R.; Shih, David Q.

    2012-01-01

    Intestinal fibrostenosis is a hallmark of severe Crohn's disease and can lead to multiple surgeries. Patients with certain TNFSF15 variants overexpress TL1A. The aim of this study was to determine the effect of TL1A overexpression on intestinal inflammation and the development of fibrostenosis. We assessed the in vivo consequences of constitutive TL1A expression on gut mucosal inflammation and fibrostenosis using two murine models of chronic colitis. In the dextran sodium sulfate (DSS) and adoptive T-cell transfer models, there was proximal migration of colonic inflammation, worsened patchy intestinal inflammation, and long gross intestinal strictures in Tl1a transgenic compared to wild-type littermates. In the DSS model, myeloid- and T-cell–expressing Tl1a transgenic mice had increased T-cell activation markers and interleukin-17 expression compared to wild-type mice. In the T-cell transfer model, Rag1−/− mice receiving Tl1a transgenic T cells had increased interferon-γ expression but reduced T-helper 17 cells and IL-17 production. Narrowed ureters with hydronephrosis were found only in the Tl1a transgenic mice in all chronic colitis models. In human translational studies, Crohn's disease patients with higher peripheral TL1A expression also exhibited intestinal fibrostenosis and worsened ileocecal inflammation with relative sparing of rectosigmoid inflammation. These data show that TL1A is an important cytokine that not only modulates the location and severity of mucosal inflammation, but also induces fibrostenosis. PMID:22138299

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. Plant enolase: gene structure, expression, and evolution.

    PubMed Central

    Van der Straeten, D; Rodrigues-Pousada, R A; Goodman, H M; Van Montagu, M

    1991-01-01

    Enolase genes were cloned from tomato and Arabidopsis. Comparison of their primary structures with other enolases revealed a remarkable degree of conservation, except for the presence of an insertion of 5 amino acids unique to plant enolases. Expression of the enolase genes was studied under various conditions. Under normal growth conditions, steady-state messenger and enzyme activity levels were significantly higher in roots than in green tissue. Large inductions of mRNA, accompanied by a moderate increase in enzyme activity, were obtained by an artificial ripening treatment in tomato fruits. However, there was little effect of anaerobiosis on the abundance of enolase messenger. In heat shock conditions, no induction of enolase mRNA was observed. We also present evidence that, at least in Arabidopsis, the hypothesis that there exists a complete set of glycolytic enzymes in the chloroplast is not valid, and we propose instead the occurrence of a substrate shuttle in Arabidopsis chloroplasts for termination of the glycolytic cycle. PMID:1841726

  4. 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.

  5. Plant origin and ploidy influence gene expression and life cycle characteristics in an invasive weed

    PubMed Central

    Broz, Amanda K; Manter, Daniel K; Bowman, Gillianne; Müller-Schärer, Heinz; Vivanco, Jorge M

    2009-01-01

    Background Ecological, evolutionary and physiological studies have thus far provided an incomplete picture of why some plants become invasive; therefore we used genomic resources to complement and advance this field. In order to gain insight into the invasive mechanism of Centaurea stoebe we compared plants of three geo-cytotypes, native Eurasian diploids, native Eurasian tetraploids and introduced North American tetraploids, grown in a common greenhouse environment. We monitored plant performance characteristics and life cycle habits and characterized the expression of genes related to constitutive defense and genome stability using quantitative PCR. Results Plant origin and ploidy were found to have a significant effect on both life cycle characteristics and gene expression, highlighting the importance of comparing appropriate taxonomic groups in studies of native and introduced plant species. We found that introduced populations of C. stoebe exhibit reduced expression of transcripts related to constitutive defense relative to their native tetraploid counterparts, as might be expected based on ideas of enemy release and rapid evolution. Measurements of several vegetative traits were similar for all geo-cytotypes; however, fecundity of tetraploids was significantly greater than diploids, due in part to their polycarpic nature. A simulation of seed production over time predicts that introduced tetraploids have the highest fecundity of the three geo-cytotypes. Conclusion Our results suggest that characterizing gene expression in an invasive species using populations from both its native and introduced range can provide insight into the biology of plant invasion that can complement traditional measurements of plant performance. In addition, these results highlight the importance of using appropriate taxonomic units in ecological genomics investigations. PMID:19309502

  6. 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

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

    PubMed

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

    2015-08-01

    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

  8. 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

  9. Site-specific integration and constitutive expression of key genes into Escherichia coli chromosome increases shikimic acid yields.

    PubMed

    Liu, Xianglei; Lin, Jun; Hu, Haifeng; Zhou, Bin; Zhu, Baoquan

    2016-01-01

    As the key starting material for the chemical synthesis of Oseltamivir, shikimic acid (SA) has captured worldwide attention. Many researchers have tried to improve SA production by metabolic engineering, yet expression plasmids were used generally. In recent years, site-specific integration of key genes into chromosome to increase the yield of metabolites showed considerable advantages. The genes could maintain stably and express constitutively without induction. Herein, crucial genes aroG, aroB, tktA, aroE (encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, dehydroquinate synthase, transketolase and shikimate dehydrogenase, respectively) of SA pathway and glk, galP (encoding glucokinase and galactose permease) were integrated into the locus of ptsHIcrr (phosphoenolpyruvate: carbohydrate phosphotransferase system operon) in a shikimate kinase genetic defect strain Escherichia coli BW25113 (ΔaroL/aroK, DE3). Furthermore, another key gene ppsA (encoding phosphoenolpyruvate synthase) was integrated into tyrR (encoding Tyr regulator protein). As a result, SA production of the recombinant (SA5/pGBAE) reached to 4.14 g/L in shake flask and 27.41 g/L in a 5-L bioreactor. These data suggested that integration of key genes increased SA yields effectively. This strategy is environmentally friendly for no antibiotic is added, simple to handle without induction, and suitable for industrial production. PMID:26672454

  10. Molecular characterization of the constitutive expression of the plasma platelet-activating factor acetylhydrolase gene in macrophages.

    PubMed Central

    Wu, Xiaoqing; McIntyre, Thomas M; Zimmerman, Guy A; Prescott, Stephen M; Stafforini, Diana M

    2003-01-01

    Plasma platelet-activating factor acetylhydrolase (PAF-AH) is a phospholipase that inactivates platelet-activating factor (PAF) and PAF-like lipids to generate products with little or no biological activity. The levels of circulating PAF-AH correlate with several disease syndromes. We previously reported that mediators of inflammation regulate the expression of the human PAF-AH gene at the transcriptional level. In the present paper, we characterize the constitutive expression of plasma PAF-AH using the mouse gene as a model system, and we report comparative results obtained using human and mouse promoter constructs. We first cloned, sequenced and analysed the promoter region of the murine plasma PAF-AH (mPAF-AH) gene and found that this gene lacks a canonical TATA box. We demonstrated that the cis -elements required for basal transcription are localized within the -316 to -68 bp region. In vitro band-shift and supershift assays showed that Sp1 and Sp3 transcription factors from RAW264.7 and J774A.1 macrophage nuclear extracts bound strongly to a distal GC-rich site within -278/-243 [specificity protein (Sp-A)] and to a proximal TC-rich motif within -150/-114 (Sp-B). In addition, we observed weak binding to a GA-rich site within -110/-82 (Sp-C). The regions containing Sp-B and Sp-C are highly conserved between the human and mouse genes. Forced expression of Sp1 or Sp3 in Sp-lacking Drosophila SL2 cells induced markedly the activity of the exogenous mPAF-AH promoter in a dose-dependent manner, and this induction was dependent on the presence of intact Sp-A and Sp-B. Interestingly, we found that the Sp1- and Sp3-associated DNA-binding activities increased during the maturation of primary human monocytes into macrophages in cell culture. These results demonstrate that Sp1 and Sp3 are key factors that contribute to the basal, constitutive transcription of the plasma PAF-AH gene in macrophages. PMID:12854969

  11. beta-Glucosidase as a reporter for the gene expression studies in Thermus thermophilus and constitutive expression of DNA repair genes.

    PubMed

    Ohta, Toshihiro; Tokishita, Shin-Ichi; Imazuka, Reiko; Mori, Ichiro; Okamura, Jin; Yamagata, Hideo

    2006-07-01

    Thermus thermophilus is an extremely thermophilic eubacterium that grows optimally at 70-75 degrees C. Because the frequency of DNA damage, such as deamination, depurination and single-strand breaks, increases as the temperature rises, the regulation of expression as well as the specificities and activities of T.thermophilus DNA repair systems are of particular interest. To study those systems, we developed a gene expression vector using the T.thermophilus beta-glucosidase gene (bgl) with host strain JOS9 (Deltabgl) derived from the T.thermophilus wild-type strain HB27. Since HB27 has two putative beta-galactosidase genes, the use of a single bgl gene as a reporter in combination with a Deltabgl host strain permits the study of gene expression against a low background level. We assayed Bgl activity with 2-nitrophenyl-beta-d-glucopyranoside as the substrate at 80 degrees C. We measured the expression of seven genes involved in DNA repair--three nucleotide excision repair genes (uvrA, uvrB and uvrC) and four recombinational repair genes (recA, ruvA, ruvB and ruvC). Expression levels of uvrA and uvrB were about three times those of uvrC, while those of ruvA, ruvB and ruvC were almost equal. Both ruvA and ruvC formed an operon with their adjacent 5'-upstream gene paaG and ftsQAZ, respectively. recA was transcribed as an operon of four genes, amt-cinA-ligT-recA. All seven DNA repair genes were expressed constitutively, and the DNA damaging agent mitomycin C did not increase their expression. PMID:16777922

  12. Resistance to geminivirus infection by virus-induced expression of dianthin in transgenic plants.

    PubMed

    Hong, Y; Saunders, K; Hartley, M R; Stanley, J

    1996-06-01

    Ribosome-inactivating proteins (RIPs) are naturally occurring plant toxins that exhibit antiviral activity against a diverse range of plant and animal viruses. Here, the action of dianthin, a potent RIP isolated from Dianthus caryophyllus, has been exploited to engineer resistance to a plant DNA virus, African cassava mosaic virus (ACMV), in transgenic Nicotiana benthamiana. To achieve this, dianthin has been expressed from the ACMV virion-sense promoter that is transactivated by the product of viral gene AC2. This avoids the need for constitutive expression of the RIP, facilitating the regeneration of phenotypically normal plants, and ensures transgene expression is localized to virus-infected cells. When challenged with ACMV, transgenic plants produce atypical necrotic lesions on inoculated leaves, indicative of dianthin expression, viral DNA accumulation is significantly reduced in these tissues, and plants exhibit attenuated systemic symptoms from which they recover. This phenotype holds for isolates of ACMV but not for other geminiviruses, suggesting that AC2 homologues from the latter are unable to efficiently transactivate the ACMV promoter. PMID:8659104

  13. Constitutive expression of the ZmZIP7 in Arabidopsis alters metal homeostasis and increases Fe and Zn content.

    PubMed

    Li, Suzhen; Zhou, Xiaojin; Zhao, Yongfeng; Li, Hongbo; Liu, Yuanfeng; Zhu, Liying; Guo, Jinjie; Huang, Yaqun; Yang, Wenzhu; Fan, Yunliu; Chen, Jingtang; Chen, Rumei

    2016-09-01

    Iron (Fe) and zinc (Zn) are important micronutrients for plant growth and development. Zinc-regulated transporters and the iron-regulated transporter-like protein (ZIP) are necessary for the homeostatic regulation of these metal micronutrients. In this study, the physiological function of ZmZIP7 which encodes a ZIP family transporter was characterized. We detected the expression profiles of ZmZIP7 in maize, and found that the accumulation of ZmZIP7 in root, stem, leaf, and seed was relatively higher than tassel and young ear. ZmZIP7 overexpression transgenic Arabidopsis lines were generated and the metal contents in transgenic and wild-type (WT) plants were examined using inductively coupled plasma atomic emission spectroscopy (ICP-OES) and Zinpyr-1 staining. Fe and Zn concentrations were elevated in the roots and shoots of ZmZIP7-overexpressing plants, while only Fe content was elevated in the seeds. We also analyzed the expression profiles of endogenous genes associated with metal homeostasis. Both endogenic Fe-deficiency inducible genes and the genes responsible for Zn and Fe transport and storage were stimulated in ZmZIP7 transgenic plants. In conclusion, ZmZIP7 encodes a functional Zn and Fe transporter, and ectopic overexpression of ZmZIP7 in Arabidopsis stimulate endogenous Fe and Zn uptake mechanisms, thereby facilitating both metal uptake and homeostasis. Our results contribute to improved understanding of ZIP family transporter functions and suggest that ZmZIP7 could be used to enhance Fe levels in grains. PMID:27135812

  14. 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.

  15. 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

  16. Constitutive expression of ectopic c-Myc delays glucocorticoid-evoked apoptosis of human leukemic CEM-C7 cells

    PubMed Central

    Medh, Rheem D; Wang, Aixia; Zhou, Feng; Thompson, E Brad

    2009-01-01

    Sensitivity to glucocorticoid (GC)-evoked apoptosis in lymphoid cell lines correlates closely with GC-mediated suppression of c-Myc expression. To establish a functional role for c-Myc in GC-mediated apoptosis, we have stably expressed MycER™, the human c-Myc protein fused to the modified ligand-binding domain of the murine estrogen receptor α, in GC-sensitive CEM-C7-14 cells. In CEM-C7-14 cells, MycER™ constitutively imparts c-Myc functions. Cells expressing MycER™ (C7-MycER™) exhibited a marked reduction in cell death after 72 h in 100 nM dexamethasone (Dex), with 10 – 20-fold more viable cells when compared to the parental CEM-C7-14 clone. General GC responsiveness was not compromised, as evidenced by Dex-mediated suppression of endogenous c-Myc and cyclin D3, and induction of c-Jun and the glucocorticoid receptor. MycER™ also blunted Dex-mediated upregulation of p27kip1 and suppression of the Myc target p53. In comparison to parental CEM-C7-14 cells, Dex-evoked DNA strand breaks were negligible and caspase activation was delayed, but the extent of G1 cell cycle arrest was similar in C7-MycER™ cells. Myc-ER™ did not result in permanent, complete resistance to GC however, and the GC-treated cells eventually died, indicative of redundant or interactive mechanisms in the GC-evoked lytic response of lymphoid cells. Our results emphasize the importance of c-Myc suppression in GC-evoked apoptosis of CEM-C7-14 cells. PMID:11498786

  17. Preparation and characterization of a stable BHK-21 cell line constitutively expressing the Schmallenberg virus nucleocapsid protein.

    PubMed

    Zhang, Yongning; Wu, Shaoqiang; Song, Shanshan; Lv, Jizhou; Feng, Chunyan; Lin, Xiangmei

    2015-08-01

    Schmallenberg virus (SBV) is a newly emerged orthobunyavirus that predominantly infects livestock such as cattle, sheep, and goats. Its nucleocapsid (N) protein is an ideal target antigen for SBV diagnosis. In this study, a stable BHK-21 cell line, BHK-21-EGFP-SBV-N, constitutively expressing the SBV N protein was obtained using a lentivector-mediated gene transfer system combined with puromycin selection. To facilitate the purification of recombinant SBV N protein, the coding sequence for a hexa-histidine tag was introduced into the C-terminus of the SBV N gene during construction of the recombinant lentivirus vector pLV-EGFP-SBV-N. The BHK-21-EGFP-SBV-N cell line was demonstrated to spontaneously emit strong enhanced green fluorescent protein (EGFP) signals that exhibited a discrete punctate distribution throughout the cytoplasm. SBV N mRNA and protein expression in this cell line were detected by real-time RT-PCR and western blot, respectively. The expressed recombinant SBV N protein carried an N-terminal EGFP tag, and was successfully purified using Ni-NTA agarose by means of its C-terminal His tag. The purified SBV N protein could be recognized by SBV antisera and an anti-SBV monoclonal antibody (mAb) 2C8 in an indirect enzyme-linked immunosorbent assay and western blot analyses. Indirect immunofluorescence assays further demonstrated that the stable cell line reacts with SBV antisera and mAb 2C8. These results suggest that the generated cell line has the potential to be used in the serological diagnosis of SBV. PMID:26013296

  18. Effect of constitutive expression of porcine IGFBP-3 on proliferation and differentiation of L6 myogenic cells.

    PubMed

    Xi, G; Kamanga-Sollo, E; Hathaway, M R; Dayton, W R; White, M E

    2006-07-01

    We have previously shown that exogenous recombinant porcine IGFBP-3 (rpIGFBP-3) suppresses proliferation and differentiation of L6 myogenic cells in an IGF-I-dependent manner and suppresses proliferation of L6 myogenic cells via an IGF-I-independent mechanism. In order to assess the effects of endogenously produced IGFBP-3, we have transfected L6 myogenic cells with a pEF6/V5 vector containing pIGFBP-3 cDNA under the control of the human elongation factor 1alpha (hEF-1alpha) promoter and with the empty vector. We have isolated a cell population that constitutively produces porcine IGFBP-3 (tL6 cells) and a stable mock transfected cell population containing the empty vector (mtL6 cells). Constitutive expression of IGFBP-3 slightly reduced the expression of IGFBP-5 but had no effect on IGFBP-4 production by L6 myogenic cells. Immunoneutralization of IGFBP-3 increased both IGF-I- and Long-R3-IGF-I-stimulated proliferation of tL6 cells (58 and 33%, respectively) (P<0.01). These data indicate endogenous pIGFBP-3, like exogenous rpIGFBP-3, suppresses the proliferation of L6 myogenic cells via both IGF-I-dependent and -independent pathways. Immunoneutralization of IGFBP-3 also increased IGF-I-stimulated differentiation (21%, P<0.05) but had no effect on Long-R3-IGF-I stimulated differentiation of tL6 myogenic cells. Results indicate that exogenous and endogenous IGFBP-3 affect proliferation and differentiation of L6 myogenic cells in a similar way. Immunohistochemical localization data reveal that pre-incubation with anti-pIGFBP-3 dramatically reduces the level of intracellular IGFBP-3 in tL6 myogenic cells indicating that endogenously produced IGFBP-3 must first be secreted before it is internalized and that anti-pIGFBP-3 prevents internalization of IGFBP-3. TL6 and mtL6 cells provide a good system to further investigate the mechanisms by which IGFBP-3 affects proliferation and differentiation of myogenic cells. PMID:16233971

  19. Expression of tobacco mosaic virus RNA in transgenic plants.

    PubMed

    Yamaya, J; Yoshioka, M; Meshi, T; Okada, Y; Ohno, T

    1988-03-01

    Tobacco mosaic virus (TMV) is a message-sense, single-stranded RNA virus that infects many Solanaceae plants. A full-length cDNA copy of TMV genomic RNA was constructed and introduced into the genomic DNA of tobacco plants using a disarmed Ti plasmid vector. Transformed plants showed typical symptoms of TMV infection, and their leaves contained infectious TMV particles. This is the first example of the expression of RNA virus genomic RNAs in plants. PMID:2835637

  20. Constitutive modulation of Raf-1 protein kinase is associated with differential gene expression of several known and unknown genes.

    PubMed Central

    Patel, S.; Wang, F. H.; Whiteside, T. L.; Kasid, U.

    1997-01-01

    BACKGROUND: Raf-1, a cytoplasmic serine/threonine protein kinase, plays an important role in mitogen- and damage-responsive cellular signal transduction pathways. Consistent with this notion is the fact that constitutive modulation of expression and/or activity of Raf-1 protein kinase modifies cell growth, proliferation, and cell survival. Although these effects are controlled at least in part by transcriptional mechanisms, the role of Raf-1 in the regulation of specific gene expression is unclear. MATERIALS AND METHODS: Differential display of mRNA was used to identify the genes differentially expressed in human head and neck squamous carcinoma cells (PCI-06A) transfected with either the antisense c-raf-1 cDNA (PCI-06A-Raf(AS)), or a portion of cDNA coding for the kinase domain of Raf-1 (PCI-06A-Raf(K)). The differentially expressed fragments were cloned and sequenced, and they were used as probes to compare the expression patterns in parent transfectants by Northern blot analysis. In addition, expression patterns of the novel genes were examined in normal tissues and cancer cell lines. RESULTS: Six differentially expressed cDNA fragments were identified and sequenced. Northern blot analysis revealed that four of these fragments representing human alpha 1-antichymotrypsin (alpha 1-ACT), mitochondrial cytochrome c oxidase subunit II (COX-II), and two as-yet unidentified cDNAs (KAS-110 and KAS-111) were relatively overexpressed in PCI-06A-Raf(AS) transfectants compared with PCI-06A-Raf(K) transfectants. The other two cDNA fragments representing human elongation factor-1 alpha (HEF-1 alpha) and ornithine decarboxylase antizyme (OAz) were overexpressed in PCI-06A-Raf(K) transfectants compared with PCI-06A-Raf(AS) transfectants. The KAS-110 (114 bp) and KAS-111 (202 bp) cDNAs did not show significant matches with sequences in the GenEMBL, TIGR, and HGS DNA databases, and these may represent novel genes. The KAS-110 and KAS-111 transcripts, approximately 0.9 kb and

  1. 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.

  2. 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

  3. 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

  4. Increased skin barrier disruption by sodium lauryl sulfate in mice expressing a constitutively active STAT6 in T cells.

    PubMed

    DaSilva, Sonia C; Sahu, Ravi P; Konger, Raymond L; Perkins, Susan M; Kaplan, Mark H; Travers, Jeffrey B

    2012-01-01

    Atopic dermatitis (AD) is a pruritic, chronic inflammatory skin disease that affects 10-20% of children and 1-3% of adults worldwide. Recent studies have indicated that the ability of Th2 cytokines, such as interleukin-4 (IL-4) to regulate skin barrier function may be a predisposing factor for AD development. The present studies examined the ability of increased Th2 activity to affect cutaneous barrier function in vivo and epidermal thickening. Mice that express a constitutively active Signal Transducer and Activator of Transcription 6 (STAT6VT) have increased Th2 cells and a predisposition to allergic inflammation were used in these studies, they demonstrate that topical treatment with the irritant sodium lauryl sulfate (SLS) caused increased transepidermal water loss and epidermal thickening in STAT6VT mice over similarly treated wild-type mice. The proliferation marker Ki-67 was increased in the epidermis of STAT6VT compared to the wild-type mice. However, these differences do not appear to be linked to the addition of an irritant as control-treated STAT6VT skin also exhibited elevated Ki-67 levels, suggesting that the increased epidermal thickness in SLS-treated STAT6VT mice is primarily driven by epidermal cell hypertrophy rather than an increase in cellular proliferation. Our results suggest that an environment with increased Th2 cytokines results in abnormal responses to topical irritants. PMID:21959772

  5. 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

  6. Regulation of Expansin Gene Expression Affects Growth and Development in Transgenic Rice Plants

    PubMed Central

    Choi, Dongsu; Lee, Yi; Cho, Hyung-Taeg; Kende, Hans

    2003-01-01

    To investigate the in vivo functions of expansins, we generated transgenic rice plants that express sense and antisense constructs of the expansin gene OsEXP4. In adult plants with constitutive OsEXP4 expression, 12% of overexpressors were taller and 88% were shorter than the average control plants, and most overexpressors developed at least two additional leaves. Antisense plants were shorter and flowered earlier than the average control plants. In transgenic plants with inducible OsEXP4 expression, we observed a close correlation between OsEXP4 protein levels and seedling growth. Coleoptile and mesocotyl length increased by up to 31 and 97%, respectively, in overexpressors, whereas in antisense seedlings, they decreased by up to 28 and 43%, respectively. The change in seedling growth resulted from corresponding changes in cell size, which in turn appeared to be a function of altered cell wall extensibility. Our results support the hypothesis that expansins are involved in enhancing growth by mediating cell wall loosening. PMID:12782731

  7. 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

  8. Separation and purification of the tonoplast ATPase and pyrophosphatase from plants with constitutive and inducible Crassulacean acid metabolism.

    PubMed

    Bremberger, C; Haschke, H P; Lüttge, U

    1988-10-01

    Tonoplast vesicles were isolated from Kalanchoe daigremontiana Hamet et Pierrer de la Bâthie and Mesembryanthemum crystallinum L., exhibiting constitutive and inducible crassulacean acid metabolism (CAM), respectively. Membrane-bound proteins were detergent-solubilized with 2% of Triton X-100. During CAM induction in M. crystallinum, ATPase activity increases four-fold, whereas pyrophosphatase activity decreases somewhat. With all plants, ATPase and pyrophosphatase could be separated by size-exclusion chromatography (SEC, Sephacryl S 400), and the ATPase was further purified by diethylaminoethyl-ion-exchange chromatography. Sodium-dodecyl-sulfate electrophoresis of the SEC fractions from K. daigremontiana containing maximum ATPase activity separates several protein bands, indicating subunits of 72, 56, 48, 42, 28, and 16 kDa. Purified ATPase from M. crystallinum in the C3 and CAM states shows a somewhat different protein pattern. With M. crystallinum, an increase in ATP-hydrolysis and changes in the subunit composition of the native enzyme indicate that the change from the C3 to the CAM state is accompanied by de-novo synthesis and by structural changes of the tonoplast ATPase. PMID:24221927

  9. 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. PMID:23384757

  10. 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.

  11. TRANSGENIC PLANTS EXPRESSING BACILLUS THURINGIENSIS DELTA-ENDOTOXINS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Commercial varieties of transgenic Bacillus thuringiensis (Bt) plants have been developed in many countries to control target pests. Initially, the expression of native Bt genes in plants was low due to mRNA instability, improper splicing, and post-translation modifications. Subsequently, modificati...

  12. Leu343Phe substitution in the Malx3 protein of Saccharomyces cerevisiae increases the constitutivity and glucose insensitivity of MAL gene expression.

    PubMed

    Higgins, V J; Braidwood, M; Bissinger, P; Dawes, I W; Attfield, P V

    1999-06-01

    To utilise maltose as a carbon source Saccharomyces cerevisiae needs one or more functional MAL loci that contain the MALx1 gene encoding maltose permease, MALx2 encoding maltase, and MALx3 encoding a transcriptional activator. Maltose causes a rapid MALx3-dependent induction of MAL gene transcription, and glucose represses this activation via Mig1p. A MALx3 gene conveying high MAL gene expression in the absence of maltose in a malx3 laboratory mutant strain has been isolated from baker's yeast. The construction of hybrid genes between the isolated gene and a highly regulated MALx3 gene showed that constitutivity was the result of multiple amino-acid alterations throughout the structural gene. The combined effect of these amino-acid alterations was shown to be stronger than the sum of their individual effects on constitutivity. Analysis in glucose-repressed conditions confirmed that increased MALx3 transcript levels increased the glucose insensitivity of MAL gene expression but did not affect constitutivity. Analysis of four mutations between aa 343 and 375, lying within a proposed negative regulatory domain, showed that the single mutation of Leu343Phe increased the glucose insensitivity of MAL gene expression by 30-fold. These results demonstrate that not only Mig1p modulation of MALx3 expression, but also the MALx3 protein structure, is involved in the glucose-insensitive expression of the MAL genes. PMID:10369955

  13. 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

  14. 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

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

    PubMed

    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

  16. 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

  17. 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

  18. 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

  19. 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

  20. Inducible virus-mediated expression of a foreign protein in suspension-cultured plant cells.

    PubMed

    Dohi, K; Nishikiori, M; Tamai, A; Ishikawa, M; Meshi, T; Mori, M

    2006-06-01

    Although suspension-cultured plant cells have many potential merits as sources of useful proteins, the lack of an efficient expression system has prevented using this approach. In this study, we established an inducible tomato mosaic virus (ToMV) infection system in tobacco BY-2 suspension-cultured cells to inducibly and efficiently produce a foreign protein. In this system, a modified ToMV encoding a foreign protein as replacement of the coat protein is expressed from stably transformed cDNA under the control of an estrogen-inducible promoter in transgenic BY-2 cells. Estrogen added to the culture activates an estrogen-inducible transactivator expressed constitutively from the transgene and induces transcription and replication of viral RNA. In our experiments, accumulation of viral RNA and expression of green fluorescent protein (GFP) encoded in the virus were observed within 24 h after induction. The amount of GFP reached approximately 10% of total soluble protein 4 d after induction. In contrast, neither viral RNA nor GFP were detected in uninduced cells. The inducible virus infection system established here should be utilized not only for the expression of foreign proteins, but also for investigations into the viral replication process in cultured plant cells. PMID:16421635

  1. 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

  2. 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

  3. 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

  4. In Vivo Electroporation of Constitutively Expressed HIF-1α Plasmid DNA Improves Neovascularization in a Mouse Model of Limb Ischemia

    PubMed Central

    Ouma, Geoffrey O.; Rodriguez, Eduardo; Muthumani, Karuppiah; Weiner, David B.; Wilensky, Robert L.; Mohler, Emile R.

    2013-01-01

    Objectives Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor that stimulates angiogenesis during tissue ischemia. In vivo electroporation (EP) enhances tissue DNA transfection. We hypothesized that in vivo EP of plasmid DNA encoding a constitutively expressed HIF-1α gene enhances neovascularization compared to intramuscular (IM) injection alone. Methods Left femoral artery ligation was performed in mice assigned to three groups: (1) HIF-EP (n=13); (2) HIF-IM (n=14); and (3) pVAX-EP (n=12). A single dose of HIF-1α or empty plasmid (pVAX) DNA (20 μl of 5 μg/μl each) was injected into the ischemic adductor muscle followed by EP (group 1 & 3). Mice in group 2 received IM injection of HIF-1α plasmid DNA alone. From pre-ligation to days 0, 3, 7, 14, & 21 post ligation, limb perfusion recovery quantified by Laser Doppler Perfusion Imager (LDPI), limb function and limb necrosis were measured. On day 21, the surviving mice (4 – 5 per group) were sacrificed and adductor muscle tissues stained for necrosis using hematoxylin and eosin (H&E); capillary density (anti-CD31 antibodies); and collateral vessels via anti-α-smooth muscle actin (α-SMA) antibodies. Results In vivo EP of HIF-1α DNA significantly improved limb perfusion (HIF-EP: 1.03 ± 0.15 vs. HIF-IM: 0.78 ± 0.064; P < .05, vs. pVAX-EP: 0.41 ± 0.019; P < .001), limb functional recovery (HIF-EP: 3.5 ± 0.58 vs. HIF-IM, 2.4 ± 1.14; p < 0.05, vs. pVAX-EP: 2.4 ± 1.14; P < .001) and limb auto-amputation on day 21 (HIF-EP: 77% ± 12% vs. HIF-IM: 43% ± 14%; P <.05 vs. pVAX-EP: 17% ± 11%; P < .01). Adductor muscle tissue necrosis decreased (HIF-EP: 20.7% ± 1.75% vs. HIF-IM: 44% ± 3.73; P < .001, vs. pVAX-EP: 60.05% ± 2.17%; P < .0001), capillary density increased (HIF-EP: 96.83 ± 5.72 vessels/hpf vs. HIF-IM: 62.87 ± 2.0 vessels/hpf; P < .001, vs. pVAX-EP: 39.37 ± 2.76 vessels/hpf; P < .0001), collateral vessel formation increased (HI-EP: 76.33 ± 1.94 vessels/hpf vs. HIF-IM: 37.5 ± 1

  5. 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

  6. Subdomains of the octopine synthase upstream activating element direct cell-specific expression in transgenic tobacco plants.

    PubMed Central

    Kononowicz, H; Wang, Y E; Habeck, L L; Gelvin, S B

    1992-01-01

    Previous work has shown that the octopine synthase (ocs) gene encoded by the Agrobacterium tumefaciens Ti-plasmid contains an upstream activating sequence necessary for its expression in plant cells. This sequence is composed of an essential 16-bp palindrome and flanking sequences that modulate the level of expression of the ocs promoter in transgenic tobacco calli. In this study, we have used RNA gel blot analysis of RNA extracted from transgenic tobacco plants to show that the octopine synthase gene is not constitutively expressed in all plant tissues and organs. This tissue-specific pattern of expression is determined, to a large extent, by the 16-bp palindrome. Histochemical analysis, using an ocs-lacZ fusion gene, has indicated that the 16-bp palindrome directs the expression of the ocs promoter in specific cell types in the leaves, stems, and roots of transgenic tobacco plants. This expression is especially strong in the vascular tissue of the leaves, leaf mesophyll cells, leaf and stem guard cells, and the meristematic regions of the shoots and roots. Sequences surrounding the palindrome in the upstream activating sequence restrict the expression of the ocs promoter to fewer cell types, resulting in a reduced level of expression of beta-galactosidase activity in the central vascular tissue of leaves, certain types of leaf trichomes, and the leaf primordia. PMID:1525561

  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. PMID:22183565

  8. 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. PMID:24657910

  9. Emerging Use of Gene Expression Microarrays in Plant Physiology

    DOE PAGESBeta

    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

  10. Brg-1 mediates the constitutive and fenretinide-induced expression of SPARC in mammary carcinoma cells via its interaction with transcription factor Sp1

    PubMed Central

    2010-01-01

    Background Secreted protein, acidic and rich in cysteine (SPARC) is a matricellular protein that mediates cell-matrix interactions. It has been shown, depending on the type of cancer, to possess either pro- or anti-tumorigenic properties. The transcriptional regulation of the SPARC gene expression has not been fully elucidated and the effects of anti-cancer drugs on this process have not been explored. Results In the present study, we demonstrated that chromatin remodeling factor Brg-1 is recruited to the proximal SPARC promoter region (-130/-56) through an interaction with transcription factor Sp1. We identified Brg-1 as a critical regulator for the constitutive expression levels of SPARC mRNA and protein in mammary carcinoma cell lines and for SPARC secretion into culture media. Furthermore, we found that Brg-1 cooperates with Sp1 to enhance SPARC promoter activity. Interestingly, fenretinide [N-4(hydroxyphenyl) retinamide, 4-HPR], a synthetic retinoid with anti-cancer properties, was found to up-regulate the transcription, expression and secretion of SPARC via induction of the Brg-1 in a dose-dependent manner. Finally, our results demonstrated that fenretinide-induced expression of SPARC contributes significantly to a decreased invasion of mammary carcinoma cells. Conclusions Overall, our results reveal a novel cooperative role of Brg-1 and Sp1 in mediating the constitutive and fenretinide-induced expression of SPARC, and provide new insights for the understanding of the anti-cancer effects of fenretinide. PMID:20687958

  11. Isolation and expression in transgenic tobacco and rice plants, of the cassava vein mosaic virus (CVMV) promoter.

    PubMed

    Verdaguer, B; de Kochko, A; Beachy, R N; Fauquet, C

    1996-09-01

    The cassava vein mosaic virus (CVMV) is a double stranded DNA virus which infects cassava plants (Manihot esculenta Crantz) and has been characterized as a plant pararetrovirus belonging to the caulimovirus subgroup. Two DNA fragments, CVP1 of 388 nucleotides from position -368 to +20 and CVP2 of 511 nucleotides from position -443 to +72, were isolated from the viral genome and fused to the uidA reporter gene to test promoter expression. The transcription start site of the viral promoter was determined using RNA isolated from transgenic plants containing the CVMV promoter:uidA fusion gene. Both promoter fragments were able to cause high levels of gene expression in protoplasts isolated from cassava and tobacco cell suspensions. The expression pattern of the CVMV promoters was analyzed in transgenic tobacco and rice plants, and revealed that the GUS staining pattern was similar for each construct and in both plants. The two promoter fragments were active in all plant organs tested and in a variety of cell types, suggesting a near constitutive pattern of expression. In both tobacco and rice plants, GUS activity was highest in vascular elements, in leaf mesophyll cells, and in root tips. PMID:8914529

  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. 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

  14. [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. PMID:26204753

  15. 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. PMID:27165332

  16. Inducible but Not Constitutive Expression of PD-L1 in Human Melanoma Cells Is Dependent on Activation of NF-κB

    PubMed Central

    Gowrishankar, Kavitha; Gunatilake, Dilini; Gallagher, Stuart J.; Tiffen, Jessamy; Rizos, Helen; Hersey, Peter

    2015-01-01

    Monoclonal antibodies against immune checkpoint blockade have proven to be a major success in the treatment of melanoma. The programmed death receptor-1 ligand-1 (PD-L1) expression on melanoma cells is believed to have an inhibitory effect on T cell responses and to be an important escape mechanism from immune attack. Previous studies have shown that PD-L1 can be expressed constitutively or can be induced by IFN-γ secreted by infiltrating lymphocytes. In the present study we have investigated the mechanism underlying these two modes of PD-L1 expression in melanoma cells including cells that had acquired resistance to the BRAF inhibitor vemurafenib. PD-L1 expression was examined by flow cytometry and immunoblotting. Specific inhibitors and siRNA knockdown approaches were used to examine the roles of the RAF/ MEK, PI3K, NF-κB, STAT3 and AP1/ c-Jun pathways. IFN-γ inducible expression of PD-L1 was dependent on NF-κB as shown by inhibition with BMS-345541, an inhibitor of IκB and the BET protein inhibitor I-BET151, as well as by siRNA knockdown of NF-κB subunits. We were unable to implicate the BRAF/MEK pathway as major regulators in PD-L1 expression on vemurafenib resistant cells. Similarly the PI3K/AKT pathway and the transcription factors STAT3 and c-Jun had only minor roles in IFN-γ induced expression of PD-L1. The mechanism underlying constitutive expression remains unresolved. We suggest these results have significance in selection of treatments that can be used in combination with monoclonal antibodies against PD1, to enhance their effectiveness and to reduce inhibitory effects melanoma cells have against cytotoxic T cell activity. PMID:25844720

  17. In plants, expression breadth and expression level distinctly and non-linearly correlate with gene structure

    PubMed Central

    2009-01-01

    Background Compactness of highly/broadly expressed genes in human has been explained as selection for efficiency, regional mutation biases or genomic design. However, highly expressed genes in flowering plants were shown to be less compact than lowly expressed ones. On the other hand, opposite facts have also been documented that pollen-expressed Arabidopsis genes tend to contain shorter introns and highly expressed moss genes are compact. This issue is important because it provides a chance to compare the selectionism and the neutralism views about genome evolution. Furthermore, this issue also helps to understand the fates of introns, from the angle of gene expression. Results In this study, I used expression data covering more tissues and employ new analytical methods to reexamine the correlations between gene expression and gene structure for two flowering plants, Arabidopsis thaliana and Oryza sativa. It is shown that, different aspects of expression pattern correlate with different parts of gene sequences in distinct ways. In detail, expression level is significantly negatively correlated with gene size, especially the size of non-coding regions, whereas expression breadth correlates with non-coding structural parameters positively and with coding region parameters negatively. Furthermore, the relationships between expression level and structural parameters seem to be non-linear, with the extremes of structural parameters possibly scale as power-laws or logrithmic functions of expression levels. Conclusion In plants, highly expressed genes are compact, especially in the non-coding regions. Broadly expressed genes tend to contain longer non-coding sequences, which may be necessary for complex regulations. In combination with previous studies about other plants and about animals, some common scenarios about the correlation between gene expression and gene structure begin to emerge. Based on the functional relationships between extreme values of structural

  18. Constitutional Conservatism

    ERIC Educational Resources Information Center

    Berkowitz, Peter

    2009-01-01

    After their dismal performance in election 2008, conservatives are taking stock. As they examine the causes that have driven them into the political wilderness and as they explore paths out, they should also take heart. After all, election 2008 shows that America's constitutional order is working as designed. Indeed, while sorting out their errors…

  19. 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

  20. 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

  1. Prostacyclin Synthase: Upregulation during Renal Development and in Glomerular Disease as well as Its Constitutive Expression in Cultured Human Mesangial Cells

    PubMed Central

    Klaus, Günther

    2015-01-01

    Prostacyclin (PGI2) plays a critical role in nephrogenesis and renal physiology. However, our understanding of how prostacyclin release in the kidney is regulated remains poorly defined. We studied expression of prostacyclin synthase (PGIS) in developing and adult human kidneys, and also in selected pediatric renal diseases. We also examined PGI2 formation in human mesangial cells in vitro. We observed abundant expression of PGIS in the nephrogenic cortex in humans and in situ hybridization revealed an identical pattern in mice. In the normal adult kidney, PGIS-immunoreactive protein and mRNA appear to localize to mesangial fields and endothelial and smooth muscle cells of arteries and peritubular capillaries. In kidney biopsies taken from pediatric patients, enhanced expression of PGIS-immunoreactive protein was noted mainly in endothelial cells of patients with IgA-nephropathy. Cultured human mesangial cells produce primarily PGI2 and prostaglandin E2, followed by prostaglandin F2α Cytokine stimulation increased PGI2 formation 24-fold. Under these conditions expression of PGIS mRNA and protein remained unaltered whereas mRNA for cyclooxygenase-2 was markedly induced. In contrast to its constitutive expression in vitro, renal expression of prostacyclin-synthase appears to be regulated both during development and in glomerular disease. Further research is needed to identify the factors involved in regulation of PGIS-expression. PMID:25684863

  2. 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

  3. 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.

  4. Consolidated pretreatment and hydrolysis of plant biomass expressing cell wall degrading enzymes

    DOEpatents

    Raab, R. Michael; Zhang, Dongcheng; Bougri, Oleg

    2016-02-02

    Methods for consolidated pretreatment and hydrolysis of genetically engineered plants expressing cell wall degrading enzymes are provided. Expression cassettes and vectors for making transgenic plants are described. Plants engineered to express one or more cell wall degrading enzymes using expression cassettes and vectors of the invention are also provided.

  5. High levan accumulation in transgenic tobacco plants expressing the Gluconacetobacter diazotrophicus levansucrase gene.

    PubMed

    Banguela, Alexander; Arrieta, Juan G; Rodríguez, Raisa; Trujillo, Luis E; Menéndez, Carmen; Hernández, Lázaro

    2011-06-10

    Bacterial levansucrase (EC 2.4.1.10) converts sucrose into non-linear levan consisting of long β(2,6)-linked fructosyl chains with β(2,1) branches. Bacterial levan has wide food and non-food applications, but its production in industrial reactors is costly and low yielding. Here, we report the constitutive expression of Gluconacetobacter diazotrophicus levansucrase (LsdA) fused to the vacuolar targeting pre-pro-peptide of onion sucrose:sucrose 1-fructosyltransferase (1-SST) in tobacco, a crop that does not naturally produce fructans. In the transgenic plants, levan with degree of polymerization above 10(4) fructosyl units was detected in leaves, stem, root, and flowers, but not in seeds. High levan accumulation in leaves led to gradual phenotypic alterations that increased with plant age through the flowering stage. In the transgenic lines, the fructan content in mature leaves varied from 10 to 70% of total dry weight. No oligofructans were stored in the plant organs, although the in vitro reaction of transgenic LsdA with sucrose yielded β(2,1)-linked FOS and levan. Transgenic lines with levan representing up to 30mgg(-1) of fresh leaf weight produced viable seeds and the polymer accumulation remained stable in the tested T1 and T2 progenies. The lsdA-expressing tobacco represents an alternative source of highly polymerized levan. PMID:21540065

  6. Expression of functionally active sialylated human erythropoietin in plants

    PubMed Central

    Jez, Jakub; Castilho, Alexandra; Grass, Josephine; Vorauer-Uhl, Karola; Sterovsky, Thomas; Altmann, Friedrich; Steinkellner, Herta

    2013-01-01

    Recombinant human erythropoietin (rhEPO), a glycohormone, is one of the leading biopharmaceutical products. The production of rhEPO is currently restricted to mammalian cell expression systems because of rhEPO's highly complex glycosylation pattern, which is a major determinant for drug-efficacy. Here we evaluate the ability of plants to produce different glycoforms of rhEPO. cDNA constructs were delivered to Nicotiana benthamiana (N. benthamiana) and transiently expressed by a viral based expression system. Expression levels up to 85 mg rhEPO/kg fresh leaf material were achieved. Moreover, co-expression of rhEPO with six mammalian genes required for in planta protein sialylation resulted in the synthesis of rhEPO decorated mainly with bisialylated N-glycans (NaNa), the most abundant glycoform of circulating hEPO in patients with anemia. A newly established peptide tag (ELDKWA) fused to hEPO was particularly well-suited for purification of the recombinant hormone based on immunoaffinity. Subsequent lectin chromatography allowed enrichment of exclusively sialylated rhEPO. All plant-derived glycoforms exhibited high biological activity as determined by a cell-based receptor-binding assay. The generation of rhEPO carrying largely homogeneous glycosylation profiles (GnGnXF, GnGn, and NaNa) will facilitate further investigation of functionalities with potential implications for medical applications. PMID:23325672

  7. Galactinol and Raffinose Constitute a Novel Function to Protect Plants from Oxidative Damage1[W][OA

    PubMed Central

    Nishizawa, Ayako; Yabuta, Yukinori; Shigeoka, Shigeru

    2008-01-01

    Galactinol synthase (GolS) is a key enzyme in the synthesis of raffinose family oligosaccharides that function as osmoprotectants in plant cells. In leaves of Arabidopsis (Arabidopsis thaliana) plants overexpressing heat shock transcription factor A2 (HsfA2), the transcription of GolS1, -2, and -4 and raffinose synthase 2 (RS2) was highly induced; thus, levels of galactinol and raffinose increased compared with those in wild-type plants under control growth conditions. In leaves of the wild-type plants, treatment with 50 μm methylviologen (MV) increased the transcript levels of not only HsfA2, but also GolS1, -2, -3, -4, and -8 and RS2, -4, -5, and -6, the total activities of GolS isoenzymes, and the levels of galactinol and raffinose. GolS1- or GolS2-overexpressing Arabidopsis plants (Ox-GolS1-11, Ox-GolS2-8, and Ox-GolS2-29) had increased levels of galactinol and raffinose in the leaves compared with wild-type plants under control growth conditions. High intracellular levels of galactinol and raffinose in the transgenic plants were correlated with increased tolerance to MV treatment and salinity or chilling stress. Galactinol and raffinose effectively protected salicylate from attack by hydroxyl radicals in vitro. These findings suggest the possibility that galactinol and raffinose scavenge hydroxyl radicals as a novel function to protect plant cells from oxidative damage caused by MV treatment, salinity, or chilling. PMID:18502973

  8. An ATF/CREB binding motif is required for aberrant constitutive expression of the MHC class II DR alpha promoter and activation by SV40 T-antigen.

    PubMed Central

    Cox, P M; Goding, C R

    1992-01-01

    Constitutive expression of major histocompatibility complex class II (MHC II) antigens normally occurs in B-lymphocytes and antigen presenting cells of the monocyte/macrophage lineage. However, many malignant tumours and transformed cells express these proteins aberrantly. We demonstrate here that the MHC II DR alpha promoter is constitutively active both in the SV40 large T antigen transformed cell line, COS, and in CV1 cells from which they are derived. As an approach to understanding the molecular mechanisms underlying aberrant DR alpha expression we have examined the cis- and trans-acting requirements for DR alpha transcription in these cell types. Electrophoretic mobility shift assays showed that the region immediately 3' to the X-box was bound by a member of the ATF/CREB family of transcription factors. Using deletions and point mutations in the DR alpha promoter we demonstrate that, in contrast to B-cells, the octamer motif and conserved X- and Y-boxes make only a minor contribution to promoter function while single point mutations in the ATF/CREB motif reduced transcription up to 20-fold. In addition, we show that the DR alpha promoter is activated by SV40 large T-antigen and that activation requires an intact ATF/CREB motif. Similar data were obtained using B16 melanoma cells. These results suggest that the ATF/CREB motif may be a target for transcription deregulation in several transformed cell types. Images PMID:1329030

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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. PMID:27135233

  14. Cold stress regulation of gene expression in plants.

    PubMed

    Chinnusamy, Viswanathan; Zhu, Jianhua; Zhu, Jian-Kang

    2007-10-01

    Cold stress adversely affects plant growth and development. Most temperate plants acquire freezing tolerance by a process called cold acclimation. Here, we focus on recent progress in transcriptional, post-transcriptional and post-translational regulation of gene expression that is critical for cold acclimation. Transcriptional regulation is mediated by the inducer of C-repeat binding factor (CBF) expression 1 (ICE1), the CBF transcriptional cascade and CBF-independent regulons during cold acclimation. ICE1 is negatively regulated by ubiquitination-mediated proteolysis and positively regulated by SUMO (small ubiquitin-related modifier) E3 ligase-catalyzed sumoylation. Post-transcriptional regulatory mechanisms, such as pre-mRNA splicing, mRNA export and small RNA-directed mRNA degradation, also play important roles in cold stress responses. PMID:17855156

  15. Exogenous isoprene modulates gene expression in unstressed Arabidopsis thaliana plants.

    PubMed

    Harvey, Christopher M; Sharkey, Thomas D

    2016-06-01

    Isoprene is a well-studied volatile hemiterpene that protects plants from abiotic stress through mechanisms that are not fully understood. The antioxidant and membrane stabilizing potential of isoprene are the two most commonly invoked mechanisms. However, isoprene also affects phenylpropanoid metabolism, suggesting an additional role as a signalling molecule. In this study, microarray-based gene expression profiling reveals transcriptional reprogramming of Arabidopsis thaliana plants fumigated for 24 h with a physiologically relevant concentration of isoprene. Functional enrichment analysis of fumigated plants revealed enhanced heat- and light-stress-responsive processes in response to isoprene. Isoprene induced a network enriched in ERF and WRKY transcription factors, which may play a role in stress tolerance. The isoprene-induced up-regulation of phenylpropanoid biosynthetic genes was specifically confirmed using quantitative reverse transcription polymerase chain reaction. These results support a role for isoprene as a signalling molecule, in addition to its possible roles as an antioxidant and membrane thermoprotectant. PMID:26477606

  16. Regulation of Rubisco gene expression in C4 plants.

    PubMed

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

    2016-06-01

    Ribulose-1,5-bisphosphate-carboxylase/oxygenase (Rubisco) incorporates inorganic carbon into an organic form, making this chloroplastic enzyme one of the most essential factors for all life on earth. Despite its central role in photosynthesis, research into regulation of the chloroplast rbcL and nuclear RbcS genes that encode this enzyme has lagged behind other plant gene systems. A major characteristic of kranz-type C4 plants is the accumulation of Rubisco only within chloroplasts of internalized bundle sheath cells that surround the leaf vascular centers. In plants that utilize the less common single cell C4 system, Rubisco accumulates only within one type of dimorphic chloroplasts localized to a specific region of leaf chlorenchyma cells. Understanding regulatory processes that restrict Rubisco gene expression to only one cell type or chloroplast type is a major focus of C4 research. Regulatory steps may include transcriptional, post-transcriptional, and post-translational processes. PMID:27026038

  17. Vulnerability of the human airway epithelium to hyperoxia. Constitutive expression of the catalase gene in human bronchial epithelial cells despite oxidant stress.

    PubMed

    Yoo, J H; Erzurum, S C; Hay, J G; Lemarchand, P; Crystal, R G

    1994-01-01

    Although catalase is a major intracellular antioxidant, the expression of the human catalase gene appears to be limited in the airway epithelium, making these cells vulnerable to oxidant stress. The basis for this limited gene expression was examined by evaluation of the expression of the endogenous gene in human bronchial epithelial cells in response to hyperoxia. Hyperoxia failed to upregulate endogenous catalase gene expression, in contrast to a marked increase in expression of the heat shock protein gene. Sequence analysis of 1.7 kb of the 5'-flanking region of the human catalase gene showed features of a "house-keeping" gene (no TATA box, high GC content, multiple CCAAT boxes, and transcription start sites). Transfection of human bronchial epithelial cells with fusion genes composed of various lengths of the catalase 5'-flanking region and luciferase as a reporter gene showed low level constitutive promoter activity that did not change after exposure to hyperoxia. Importantly, using a replication-deficient recombinant adenoviral vector containing the human catalase cDNA, levels of catalase were significantly increased in human airway epithelial cells and this was associated with increased survival of the cells when exposed to hyperoxia. These observations provide a basis for understanding the sensitivity of the human airway epithelium to oxidant stress and a strategy for protecting the epithelium from such injury. PMID:8282800

  18. Methylosinus trichosporium OB3b Mutants Having Constitutive Expression of Soluble Methane Monooxygenase in the Presence of High Levels of Copper

    PubMed Central

    Phelps, Patricia A.; Agarwal, Sandeep K.; Speitel, Gerald E.; Georgiou, George

    1992-01-01

    The methanotrophic bacterium Methylosinus trichosporium OB3b is unusually active in degrading recalcitrant haloalkanes such as trichloroethylene (TCE). The first and rate-limiting step in the degradation of TCE is catalyzed by a soluble methane monooxygenase (sMMO). This enzyme is not expressed when the cells are grown in the presence of copper at concentrations typically found in polluted groundwater. Under these conditions, M. trichosporium OB3b expresses a particulate form of the enzyme (pMMO), which has a narrow substrate specificity and does not degrade TCE at any significant rate. We have isolated M. trichosporium OB3b mutants that are deficient in pMMO and express sMMO constitutively in the presence of elevated concentrations of copper. One mutant (PP358) exhibited a TCE degradation rate which was almost twice as high as that of the wild-type strain grown under optimal conditions (without copper). All of the mutants lost the ability to express pMMO activity and to form stacked intracellular membranes characteristic of wild-type cells expressing pMMO. Images PMID:16348810

  19. 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

  20. Cytochrome P450 2A5 constitutive expression and induction by heavy metals is dependent on redox-sensitive transcription factor Nrf2 in liver.

    PubMed

    Lämsä, Virpi; Levonen, Anna-Liisa; Leinonen, Hanna; Ylä-Herttuala, Seppo; Yamamoto, Masayuki; Hakkola, Jukka

    2010-05-17

    Mouse cytochrome P450 2A5 (CYP2A5) is upregulated in various pathophysiological liver diseases and induced by structurally variable hepatotoxic chemicals. A putative common feature for all of these conditions is altered cellular redox status. Nuclear factor erythroid 2-like 2 (Nrf2) is a transcription factor that is post-translationally regulated by oxidative stress and controls the transcription of numerous protective target genes. In the present study, we have extensively characterized the regulation of Cyp2a5 by Nrf2 and compared it to a well-characterized target gene Hmox1. The treatment of mouse primary hepatocytes with lead chloride, methylmercury chloride, or phenethyl isothiocyanate all leads to nuclear accumulation of Nrf2. Both CYP2A5 and HMOX1 were induced by all three compounds; however, HMOX1 responded more rapidly and transiently as compared to CYP2A5. Experiments in Nrf2(-/-) primary hepatocytes showed that Nrf2 is crucial for CYP2A5 induction but not for elevation of HMOX1. Both CYP2A5 and HMOX1 were upregulated by Nrf2 overexpression and downregulated by Keap1 or Bach1 overexpression. However, in all cases, CYP2A5 responded much more potently. Results in Nrf2-deficient animals showed that CYP2A5 expression is significantly attenuated in the absence of Nrf2, while expression of HMOX1 was unaffected. Therefore, Cyp2a5 joins the group of genes constitutively regulated by Nrf2. Our current results unequivocally show that expression of CYP2A5 is tightly controlled by Nrf2 in liver. Nrf2 is needed for constitutive expression of CYP2A5, and CYP2A5 is also sensitively upregulated by an increased level of Nrf2 protein. Therefore, CYP2A5 upregulation could be a useful indicator for hepatic activation of the Nrf2 pathway. PMID:20402460

  1. 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.

  2. Cloning and constitutive expression of His-tagged xylanase GH 11 from Penicillium occitanis Pol6 in Pichia pastoris X33: purification and characterization.

    PubMed

    Driss, Dorra; Bhiri, Fatma; Ghorbel, Raoudha; Chaabouni, Semia Ellouz

    2012-05-01

    High-level constitutive expression of xylanase GH11 from Penicillium occitanis Pol6 termed PoXyn2 was achieved using the methylotrophic yeast Pichia pastoris. The PoXyn2 cDNA encoding for a mature xylanase of 320 amino acids was subcloned into the pGAPZαA vector, to construct recombinant xylanse with six histidine residues at the N-terminal and further integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAP) constitutive promoter. Activity assay and SDS-PAGE demonstrate that the His-tagged xylanase was extracellularly expressed in P. pastoris and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography (Ni-NTA resin). The purified PoXyn2 showed a single band on SDS-PAGE with an apparent molecular weight of 30 kDa. The xylanase activity was optimal at pH 3.0 and 50°C. The specific activity measured for Oat Spelt Xylan was 8549.85 U mg(-1). The apparent The K(M) and V(max) values were 8.33±0.7 mg ml(-1)and 58.82±0.9 μmol min(-1) ml(-1), respectively, as measured on Oat Spelt Xylan. This is the first report demonstrating the possibility of mass production of P. occitanis xylanase using P. pastoris. PMID:22402470

  3. Constitutive heat shock protein 70 (HSC70) expression in rainbow trout hepatocytes: effect of heat shock and heavy metal exposure.

    PubMed

    Boone, Adrienne N; Vijayan, Mathilakath M

    2002-06-01

    The 70-kDa family of heat shock proteins plays an important role as molecular chaperones in unstressed and stressed cells. The constitutive member of the 70 family (hsc70) is crucial for the chaperoning function of unstressed cells, whereas the inducible form (hsp70) is important for allowing cells to cope with acute stressor insult, especially those affecting the protein machinery. In fish, the role of hsc70 in the cellular stress response process is less clear primarily because of the lack of a fish-specific antibody for hsc70 detection. In this study, we purified hsc70 to homogeneity from trout liver using a three-step purification protocol with differential centrifugation, ATP-agarose affinity chromatography and electroelution. Polyclonal antibodies to trout hsc70 generated in rabbits cross-reacted strongly with both purified trout hsc70 protein and also purified recombinant bovine hsc70. Two-dimensional electrophoresis followed by Western blotting confirmed that the isoelectric point of rainbow trout hsc70 was more acidic than hsp70. Using this antibody, we detected hsc70 content in the liver, heart, gill and skeletal muscle of unstressed rainbow trout. Primary cultures of trout hepatocytes subjected to a heat shock (+15 degrees C for 1 h) or exposed to either CuSO(4) (200 microM for 24 h), CdCl(2) (10 microM for 24 h) or NaAsO(2) (50 microM for 1 h) resulted in higher hsp70 accumulation over a 24-h period. However, hsc70 content showed no change with either heat shock or heavy metal exposure suggesting that hsc70 is not modulated by sublethal acute stressors in trout hepatocytes. Taken together, we have for the first time generated polyclonal antibodies specific to rainbow trout hsc70 and this antibody will allow for the characterization of the role of hsc70 in the cellular stress response process in fish. PMID:12106899

  4. 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. PMID:9090062

  5. 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. PMID:9514942

  6. A dominant-negative variant of SNAP-23 decreases the cell surface expression of the neuronal glutamate transporter EAAC1 by slowing constitutive delivery.

    PubMed

    Fournier, Keith M; Robinson, Michael B

    2006-01-01

    A family of high-affinity transporters controls the extracellular concentration of glutamate in the brain, ensuring appropriate excitatory signaling and preventing excitotoxicity. There is evidence that one of the neuronal glutamate transporters, EAAC1, is rapidly recycled on and off the plasma membrane with a half-life of no more than 5-7 min in both C6 glioma cells and cortical neurons. Syntaxin 1A has been implicated in the trafficking of several neurotransmitter transporters and in the regulation of EAAC1, but it has not been determined if this SNARE protein is required for EAAC1 trafficking. Expression of two different sets of SNARE proteins was examined in C6 glioma with Western blotting. These cells did not express syntaxin 1A, vesicle-associated membrane protein-1 (VAMP1), or synaptosomal-associated protein of 25 kDa (SNAP-25), but did express a family of SNARE proteins that has been implicated in glucose transporter trafficking, including syntaxin 4, vesicle-associated membrane protein-2 (VAMP2), and synaptosomal-associated protein of 23 kDa (SNAP-23). cDNAs encoding variants of SNAP-23 were co-transfected with Myc-tagged EAAC1 to determine if SNAP-23 function was required for maintenance of EAAC1 surface expression. Expression of a dominant-negative variant of SNAP-23 that lacks a domain required for SNARE complex assembly decreased the fraction of EAAC1 found on the cell surface and decreased total EAAC1 expression, while two control constructs had no effect. The dominant-negative variant of SNAP-23 also slowed the rate of EAAC1 delivery to the plasma membrane. These data strongly suggest that syntaxin 1A is not required for EAAC1 trafficking and provide evidence that SNAP-23 is required for constitutive recycling of EAAC1. PMID:16516346

  7. 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

  8. 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

  9. Constitutive induction of intestinal Tc17 cells in the absence of hematopoietic cell-specific MHC class II expression.

    PubMed

    Rubino, Stephen J; Geddes, Kaoru; Magalhaes, Joao G; Streutker, Catherine; Philpott, Dana J; Girardin, Stephen E

    2013-11-01

    The enteric pathogen Citrobacter rodentium induces a mucosal IL-17 response in CD4(+) T helper (Th17) cells that is dependent on the Nod-like receptors Nod1 and Nod2. Here, we sought to determine whether this early Th17 response required antigen presentation by major histocompatibility complex class II (MHCII) for full induction. At early phases of C. rodentium infection, we observed that the intestinal mucosal Th17 response was fully blunted in irradiated mice reconstituted with MHCII-deficient (MHCII(-/-) →WT) hematopoietic cells. Surprisingly, we also observed a substantial increase in the relative frequency of IL-17(+) CD8(+) CD4(-) TCR-β(+) cells (Tc17 cells) and FOXP3(+) CD8(+) CD4(-) TCR-β(+) cells in the lamina propria and intraepithelial lymphocyte compartment of MHCII(-/-) →WT mice compared with that in WT→WT counterparts. Moreover, MHCII(-/-) →WT mice displayed increased susceptibility, increased bacterial translocation to deeper organs, and more severe colonic histopathology after infection with C. rodentium. Finally, a similar phenotype was observed in mice deficient for CIITA, a transcriptional regulator of MHCII expression. Together, these results indicate that MHCII is required to mount early mucosal Th17 responses to an enteric pathogen, and that MHCII regulates the induction of atypical CD8(+) T-cell subsets, such as Tc17 cells and FOXP3(+) CD8(+) cells, in vivo. PMID:23881368

  10. 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