Endoribonuclease-Based Two-Component Repressor Systems for Tight Gene Expression Control in Plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, Yan; Richardson, Sarah; Yan, Jingwei

Tight control and multifactorial regulation of gene expression are important challenges in genetic engineering and are critical for the development of regulatory circuits. In meeting these challenges we will facilitate transgene expression regulation and support the fine-tuning of metabolic pathways to avoid the accumulation of undesired intermediates. By employing the endoribonuclease Csy4 and its recognition sequence from Pseudomonas aeruginosa and manipulating 5'UTR of mRNA, we developed a two-component expression–repression system to tightly control synthesis of transgene products. We demonstrated that this regulatory device was functional in monocotyledonous and dicotyledonous plant species, and showed that it can be used to repressmore » transgene expression by >400-fold and to synchronize transgene repression. In addition to tissue-specific transgene repression, this system offers stimuli-dependent expression control. Here, we identified 54 orthologous systems from various bacteria, using a bioinformatics approach and then validated in planta the activity for a few of those systems, demonstrating the potential diversity of such a two-component repressor system.« less

Transgenic Expression of ZBP1 in Neurons Suppresses Cocaine-Associated Conditioning

ERIC Educational Resources Information Center

Lapidus, Kyle A. B.; Nwokafor, Chiso; Scott, Daniel; Baroni, Timothy E.; Tenenbaum, Scott A.; Hiroi, Noboru; Singer, Robert H.; Czaplinski, Kevin

2012-01-01

To directly address whether regulating mRNA localization can influence animal behavior, we created transgenic mice that conditionally express Zipcode Binding Protein 1 (ZBP1) in a subset of neurons in the brain. ZBP1 is an RNA-binding protein that regulates the localization, as well as translation and stability of target mRNAs in the cytoplasm. We…

A novel positive transcriptional feedback loop in midbrain-hindbrain boundary development is revealed through analysis of the zebrafish pax2.1 promoter in transgenic lines.

PubMed

Picker, Alexander; Scholpp, Steffen; Böhli, Heike; Takeda, Hiroyuki; Brand, Michael

2002-07-01

The pax2.1 gene encodes a paired-box transcription factor that is one of the earliest genes to be specifically activated in development of the midbrain and midbrain-hindbrain boundary (MHB), and is required for the development and organizer activity of this territory. To understand how this spatially restricted transcriptional activity of pax2.1 is achieved, we have isolated and characterized the pax2.1-promoter using a lacZ and a GFP reporter gene in transient injection assays and transgenic lines. Stable transgenic expression of this reporter gene shows that a 5.3-kb fragment of the 5' region contains most, but not all, elements required for driving pax2.1 expression. The expressing tissues include the MHB, hindbrain, spinal cord, ear and pronephros. Transgene activation in the pronephros and developing ear suggests that these pax2.1-expressing tissues are composed of independently regulated subdomains. In addition, ectopic but spatially restricted activation of the reporter genes in rhombomeres 3 and 5 and in the forebrain, which do not normally express endogenous pax2.1, demonstrates the importance of negative regulation of pax2.1. Comparison of transgene expression in wild-type and homozygous pax2.1 mutant no isthmus (noi) embryos reveals that the transgene contains control element(s) for a novel, positive transcriptional feedback loop in MHB development. Transcription of endogenous pax2.1 at the MHB is known to be initially Pax2.1 independent, during activation in late gastrulation. In contrast, transgene expression requires the endogenous Pax2.1 function. Transplantations, mRNA injections and morpholino knock-down experiments show that this feedback regulation of pax2.1 transcription occurs cell-autonomously, and that it requires eng2 and eng3 as known targets for Pax2.1 regulation. We suggest that this novel feedback loop may allow continuation of pax2.1 expression, and hence development of the MHB organizer, to become independent of the patterning machinery of the gastrula embryo.

Selective rescue of selenoprotein expression in mice lacking a highly specialized methyl group in selenocysteine tRNA.

PubMed

Carlson, Bradley A; Xu, Xue-Ming; Gladyshev, Vadim N; Hatfield, Dolph L

2005-02-18

Selenocysteine (Sec) is the 21st amino acid in the genetic code. Its tRNA is variably methylated on the 2'-O-hydroxyl site of the ribosyl moiety at position 34 (Um34). Herein, we identified a role of Um34 in regulating the expression of some, but not all, selenoproteins. A strain of knock-out transgenic mice was generated, wherein the Sec tRNA gene was replaced with either wild type or mutant Sec tRNA transgenes. The mutant transgene yielded a tRNA that lacked two base modifications, N(6)-isopentenyladenosine at position 37 (i(6)A37) and Um34. Several selenoproteins, including glutathione peroxidases 1 and 3, SelR, and SelT, were not detected in mice rescued with the mutant transgene, whereas other selenoproteins, including thioredoxin reductases 1 and 3 and glutathione peroxidase 4, were expressed in normal or reduced levels. Northern blot analysis suggested that other selenoproteins (e.g. SelW) were also poorly expressed. This novel regulation of protein expression occurred at the level of translation and manifested a tissue-specific pattern. The available data suggest that the Um34 modification has greater influence than the i(6)A37 modification in regulating the expression of various mammalian selenoproteins and Um34 is required for synthesis of several members of this protein class. Many proteins that were poorly rescued appear to be involved in responses to stress, and their expression is also highly dependent on selenium in the diet. Furthermore, their mRNA levels are regulated by selenium and are subject to nonsense-mediated decay. Overall, this study described a novel mechanism of regulation of protein expression by tRNA modification that is in turn regulated by levels of the trace element, selenium.

A Primer for Using Transgenic Insecticidal Cotton in Developing Countries

PubMed Central

Showalter, Ann M.; Heuberger, Shannon; Tabashnik, Bruce E.; Carrière, Yves

2009-01-01

Many developing countries face the decision of whether to approve the testing and commercial use of insecticidal transgenic cotton and the task of developing adequate regulations for its use. In this review, we outline concepts and provide information to assist farmers, regulators and scientists in making decisions concerning this technology. We address seven critical topics: 1) molecular and breeding techniques used for the development of transgenic cotton cultivars, 2) properties of transgenic cotton cultivars and their efficacy against major insect pests, 3) agronomic performance of transgenic cotton in developing countries, 4) factors affecting transgene expression, 5) impact of gene flow between transgenic and non-transgenic cotton, 6) non-target effects of transgenic cotton, and 7) management of pest resistance to transgenic cotton. PMID:19613464

Mifepristone-inducible transgene expression in neural progenitor cells in vitro and in vivo

PubMed Central

Hjelm, BE; Grunseich, C; Gowing, G; Avalos, P; Tian, J; Shelley, BC; Mooney, M; Narwani, K; Shi, Y; Svendsen, CN; Wolfe, JH; Fischbeck, KH; Pierson, TM

2016-01-01

Numerous gene and cell therapy strategies are being developed for the treatment of neurodegenerative disorders. Many of these strategies use constitutive expression of therapeutic transgenic proteins, and although functional in animal models of disease, this method is less likely to provide adequate flexibility for delivering therapy to humans. Ligand-inducible gene expression systems may be more appropriate for these conditions, especially within the central nervous system (CNS). Mifepristone’s ability to cross the blood–brain barrier makes it an especially attractive ligand for this purpose. We describe the production of a mifepristone-inducible vector system for regulated expression of transgenes within the CNS. Our inducible system used a lentivirus-based vector platform for the ex vivo production of mifepristone-inducible murine neural progenitor cells that express our transgenes of interest. These cells were processed through a series of selection steps to ensure that the cells exhibited appropriate transgene expression in a dose-dependent and temporally controlled manner with minimal background activity. Inducible cells were then transplanted into the brains of rodents, where they exhibited appropriate mifepristone-inducible expression. These studies detail a strategy for regulated expression in the CNS for use in the development of safe and efficient gene therapy for neurological disorders. PMID:26863047

Transgenic expression of medicago truncatula PR10 and PR5 promoters in alfalfa shows pathogen-induced up-regulation of transgene expression

USDA-ARS?s Scientific Manuscript database

Genetic modification of alfalfa to introduce novel traits requires promoters for controlling gene expression. Promoters that are constitutively activated for expression of genes that enhance disease resistance pose a great energy load on the plant and exert a strong selective pressure on the pathoge...

Influence of human lactoferrin expression on iron homeostasis, flavonoids, and antioxidants in transgenic tobacco.

PubMed

Kumar, Vinay; Gill, Tejpal; Grover, Sunita; Ahuja, Paramvir Singh; Yadav, Sudesh Kumar

2013-02-01

This study was aimed at to check the influence of human lactoferrin (hLF) expression on iron homeostasis, flavonoids, and antioxidants in transgenic tobacco. Transgenic tobacco expressing hLF cDNA under the control of a CaMV 35S promoter was produced. The iron content as well as chlorophyll content of transgenic tobacco was lower compared to mock and untransformed wild plants. Interestingly, hLF transgenic tobacco showed higher level of transcript expression for genes related to iron content regulation like iron transporter and metal transporter. While expression of genes related to iron storage such as ferritin 1 and ferritin 2 was downregulated. The transcript expression of genes encoding antioxidant enzymes such as glutathione reductase, glutathione-S-transferase, ascorbate peroxidase, and catalase was downregulated in hLF transgenic tobacco compared to controls. Further, the transcript expression of two important genes encoding dihydroflavonol reductase (DFR) and phenylalanine ammonia lyase regulatory enzymes of flavonoid biosynthesis pathway was analyzed. The expression of DFR was found to be downregulated, while PAL expression was upregulated in hLF transgenic tobacco compared to mock and untransformed wild plant. Total phenolics, flavonoids, and proanthocyanidins contents were found to be higher in hLF transgenic tobacco than the mock and untransformed wild plant. Results suggest that hLF expression in transgenic tobacco leads to iron deficiency, downregulation of antioxidant enzymes, and increase in total flavonoids.

A heterologous hormone response element enhances expression of rat beta-casein promoter-driven chloramphenicol acetyltransferase fusion genes in the mammary gland of transgenic mice.

PubMed

Greenberg, N M; Reding, T V; Duffy, T; Rosen, J M

1991-10-01

Previous studies have demonstrated that the entire rat beta-casein (R beta C) gene and a -524/+490 R beta C fragment-chloramphenicol acetyltransferase (CAT) fusion gene are expressed preferentially in the mammary gland of transgenic mice in a developmentally regulated fashion. However, transgene expression was infrequent, less than 1% of that observed for the endogenous gene, and varied as much as 500-fold, presumably due to the site of chromosomal integration. To determine whether a heterologous hormone-responsive enhancer could be used to increase both the level and frequency of expression in the mammary gland, a fragment derived from the mouse mammary tumor virus long terminal repeat containing four hormone response elements (HREs) was inserted into the R beta C promoter at a site not known to contain transcriptional regulatory elements. Transgenic mice generated which carried HRE-enhanced R beta C-CAT fusion genes expressed CAT activity in the mammary glands of all founder lines examined at levels that were on average 13-fold greater than for lines generated with similar constructs not carrying HREs. In the highest expressing line, the level of HRE-enhanced transgene expression was found to be developmentally regulated, increasing 14-fold in the mammary gland from virgin to day 10 of lactation. In this line, expression was also observed in the thymus and spleen; however, the level of CAT activity was 4-fold lower than in the mammary gland and was not developmentally regulated. In adrenalectomized mice, the administration of dexamethasone stimulated CAT expression in the mammary gland but not in the thymus and spleen. These studies demonstrate that in the context of the R beta C promoter, the HRE functions in the mammary gland to increase both the frequency and level of transgene expression.

Comparative Transcriptome Analyses Reveal Potential Mechanisms of Enhanced Drought Tolerance in Transgenic Salvia Miltiorrhiza Plants Expressing AtDREB1A from Arabidopsis.

PubMed

Wei, Tao; Deng, Kejun; Wang, Hongbin; Zhang, Lipeng; Wang, Chunguo; Song, Wenqin; Zhang, Yong; Chen, Chengbin

2018-03-12

In our previous study, drought-resistant transgenic plants of Salvia miltiorrhiza were produced via overexpression of the transcription factor AtDREB1A. To unravel the molecular mechanisms underpinning elevated drought tolerance in transgenic plants, in the present study we compared the global transcriptional profiles of wild-type (WT) and AtDREB1A -expressing transgenic plants using RNA-sequencing (RNA-seq). Using cluster analysis, we identified 3904 differentially expressed genes (DEGs). Compared with WT plants, 423 unigenes were up-regulated in pRD29A::AtDREB1A-31 before drought treatment, while 936 were down-regulated and 1580 and 1313 unigenes were up- and down-regulated after six days of drought. COG analysis revealed that the 'signal transduction mechanisms' category was highly enriched among these DEGs both before and after drought stress. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation, DEGs associated with "ribosome", "plant hormone signal transduction", photosynthesis", "plant-pathogen interaction", "glycolysis/gluconeogenesis" and "carbon fixation" are hypothesized to perform major functions in drought resistance in AtDREB1A -expressing transgenic plants. Furthermore, the number of DEGs associated with different transcription factors increased significantly after drought stress, especially the AP2/ERF, bZIP and MYB protein families. Taken together, this study substantially expands the transcriptomic information for S. miltiorrhiza and provides valuable clues for elucidating the mechanism of AtDREB1A-mediated drought tolerance in transgenic plants.

Persistence of transgene expression influences CD8+ T-cell expansion and maintenance following immunization with recombinant adenovirus.

PubMed

Finn, Jonathan D; Bassett, Jennifer; Millar, James B; Grinshtein, Natalie; Yang, Teng Chih; Parsons, Robin; Evelegh, Carole; Wan, Yonghong; Parks, Robin J; Bramson, Jonathan L

2009-12-01

Previous studies determined that the CD8(+) T-cell response elicited by recombinant adenovirus exhibited a protracted contraction phase that was associated with long-term presentation of antigen. To gain further insight into this process, a doxycycline-regulated adenovirus was constructed to enable controlled extinction of transgene expression in vivo. We investigated the impact of premature termination of transgene expression at various time points (day 3 to day 60) following immunization. When transgene expression was terminated before the maximum response had been attained, overall expansion was attenuated, yielding a small memory population. When transgene expression was terminated between day 13 and day 30, the memory population was not sustained, demonstrating that the early memory population was antigen dependent. Extinction of transgene expression at day 60 had no obvious impact on memory maintenance, indicating that maintenance of the memory population may ultimately become independent of transgene expression. Premature termination of antigen expression had significant but modest effects on the phenotype and cytokine profile of the memory population. These results offer new insights into the mechanisms of memory CD8(+) T-cell maintenance following immunization with a recombinant adenovirus.

Regulated Expression of a Calmodulin Isoform Alters Growth and Development in Potato

NASA Technical Reports Server (NTRS)

Poovaiah, B. W.; Takezawa, D.; An, G.; Han, T.-J.

1996-01-01

A transgene approach was taken to study the consequences of altered expression of a calmodutin iso-form on plant growth and development. Eight genomic clones of potato calmodulin (PCM 1 to 8) have been isolated and characterized. Among the potato calmodulin isoforms studied, PCM 1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM 1 fused to the CAMV 35S promoter. Transgenic plants showing a moderate increase in PCM 1 MRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM 1 MRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM 1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM 1 MRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM 1 protein in transgenic plants, indicating that the expression of both MRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM 1 alters growth and development in potato plants.

Transgenic expression of BRCA1 disturbs hematopoietic stem and progenitor cells quiescence and function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bai, Lin; Shi, Guiying; Zhang, Xu

The balance between quiescence and proliferation of HSCs is an important regulator of hematopoiesis. Loss of quiescence frequently results in HSCs exhaustion, which underscores the importance of tight regulation of proliferation in these cells. Studies have indicated that cyclin-dependent kinases are involved in the regulation of quiescence in HSCs. BRCA1 plays an important role in the repair of DNA double-stranded breaks, cell cycle, apoptosis and transcription. BRCA1 is expressed in the bone marrow. However, the function of BRCA1 in HSCs is unknown. In our study, we generated BRCA1 transgenic mice to investigate the effects of BRCA1 on the mechanisms ofmore » quiescence and differentiation in HSCs. The results demonstrate that over-expression of BRCA1 in the bone marrow impairs the development of B lymphocytes. Furthermore, BRCA1 induced an increase in the number of LSKs, LT-HSCs, ST-HSCs and MPPs. A competitive transplantation assay found that BRCA1 transgenic mice failed to reconstitute hematopoiesis. Moreover, BRCA1 regulates the expression of p21{sup waf1}/cip1 and p57{sup kip2}, which results in a loss of quiescence in LSKs. Together, over-expression of BRCA1 in bone marrow disrupted the quiescent of LSKs, induced excessive accumulation of LSKs, and disrupted differentiation of the HSCs, which acts through the down-regulated of p21{sup waf1}/cip1 and p57{sup kip2}. - Highlights: • Over-expression of BRCA1 results in impaired B lymphocyte development. • BRCA1 transgenic mice disrupted the quiescent of LSKs, induced excessive accumulation of LSKs. • BRCA1 impairs the function of HSCs through the down-regulated of p21{sup waf1/cip1} and p57{sup kip2}.« less

Chrysanthemum WRKY gene DgWRKY5 enhances tolerance to salt stress in transgenic chrysanthemum.

PubMed

Liang, Qian-Yu; Wu, Yin-Huan; Wang, Ke; Bai, Zhen-Yu; Liu, Qing-Lin; Pan, Yuan-Zhi; Zhang, Lei; Jiang, Bei-Bei

2017-07-06

WRKY transcription factors play important roles in plant growth development, resistance and substance metabolism regulation. However, the exact function of the response to salt stress in plants with specific WRKY transcription factors remains unclear. In this research, we isolated a new WRKY transcription factor DgWRKY5 from chrysanthemum. DgWRKY5 contains two WRKY domains of WKKYGQK and two C 2 H 2 zinc fingers. The expression of DgWRKY5 in chrysanthemum was up-regulated under various treatments. Meanwhile, we observed higher expression levels in the leaves contrasted with other tissues. Under salt stress, the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) enzymes in transgenic chrysanthemum were significantly higher than those in WT, whereas the accumulation of H 2 O 2 , O 2 - and malondialdehyde (MDA) was reduced in transgenic chrysanthemum. Several parameters including root length, root length, fresh weight, chlorophyll content and leaf gas exchange parameters in transgenic chrysanthemum were much better compared with WT under salt stress. Moreover, the expression of stress-related genes DgAPX, DgCAT, DgNCED3A, DgNCED3B, DgCuZnSOD, DgP5CS, DgCSD1 and DgCSD2 was up-regulated in DgWRKY5 transgenic chrysanthemum compared with that in WT. These results suggested that DgWRKY5 could function as a positive regulator of salt stress in chrysanthemum.

Ectopic expression of a fruit phytoene synthase from Citrus paradisi Macf. promotes abiotic stress tolerance in transgenic tobacco.

PubMed

Cidade, Luciana C; de Oliveira, Tahise M; Mendes, Amanda F S; Macedo, Amanda F; Floh, Eny I S; Gesteira, Abelmon S; Soares-Filho, Walter S; Costa, Marcio G C

2012-12-01

Abscisic acid (ABA) is an important regulator of plant responses to environmental stresses and an absolute requirement for stress tolerance. Recently, a third phytoene synthase (PSY3) gene paralog was identified in monocots and demonstrated to play a specialized role in stress-induced ABA formation, thus suggesting that the first committed step in carotenogenesis is a key limiting step in ABA biosynthesis. To examine whether the ectopic expression of PSY, other than PSY3, would similarly affect ABA level and stress tolerance, we have produced transgenic tobacco containing a fruit-specific PSY (CpPSY) of grapefruit (Citrus paradisi Macf.). The transgenic plants contained a single- or double-locus insertion and expressed CpPSY at varying transcript levels. In comparison with the wild-type plants, the CpPSY expressing transgenic plants showed a significant increase on root length and shoot biomass under PEG-, NaCl- and mannitol-induced osmotic stress. The enhanced stress tolerance of transgenic plants was correlated with the increased endogenous ABA level and expression of stress-responsive genes, which in turn was correlated with the CpPSY copy number and expression level in different transgenic lines. Collectively, these results provide further evidence that PSY is a key enzyme regulating ABA biosynthesis and that the altered expression of other PSYs in transgenic plants may provide a similar function to that of the monocot's PSY3 in ABA biosynthesis and stress tolerance. The results also pave the way for further use of CpPSY, as well as other PSYs, as potential candidate genes for engineering tolerance to drought and salt stress in crop plants.

Genomic Methylation Inhibits Expression of Hepatitis B Virus Envelope Protein in Transgenic Mice: A Non-Infectious Mouse Model to Study Silencing of HBV Surface Antigen Genes.

PubMed

Graumann, Franziska; Churin, Yuri; Tschuschner, Annette; Reifenberg, Kurt; Glebe, Dieter; Roderfeld, Martin; Roeb, Elke

2015-01-01

The Hepatitis B virus genome persists in the nucleus of virus infected hepatocytes where it serves as template for viral mRNA synthesis. Epigenetic modifications, including methylation of the CpG islands contribute to the regulation of viral gene expression. The present study investigates the effects of spontaneous age dependent loss of hepatitis B surface protein- (HBs) expression due to HBV-genome specific methylation as well as its proximate positive effects in HBs transgenic mice. Liver and serum of HBs transgenic mice aged 5-33 weeks were analyzed by Western blot, immunohistochemistry, serum analysis, PCR, and qRT-PCR. From the third month of age hepatic loss of HBs was observed in 20% of transgenic mice. The size of HBs-free area and the relative number of animals with these effects increased with age and struck about 55% of animals aged 33 weeks. Loss of HBs-expression was strongly correlated with amelioration of serum parameters ALT and AST. In addition lower HBs-expression went on with decreased ER-stress. The loss of surface protein expression started on transcriptional level and appeared to be regulated epigenetically by DNA methylation. The amount of the HBs-expression correlated negatively with methylation of HBV DNA in the mouse genome. Our data suggest that methylation of specific CpG sites controls gene expression even in HBs-transgenic mice with truncated HBV genome. More important, the loss of HBs expression and intracellular aggregation ameliorated cell stress and liver integrity. Thus, targeted modulation of HBs expression may offer new therapeutic approaches. Furthermore, HBs-transgenic mice depict a non-infectious mouse model to study one possible mechanism of HBs gene silencing by hypermethylation.

Developing tTA Transgenic Rats for Inducible and Reversible Gene Expression

PubMed Central

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

2009-01-01

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

Expression of hsrω-RNAi transgene prior to heat shock specifically compromises accumulation of heat shock-induced Hsp70 in Drosophila melanogaster.

PubMed

Singh, Anand K; Lakhotia, Subhash C

2016-01-01

A delayed organismic lethality was reported in Drosophila following heat shock when developmentally active and stress-inducible noncoding hsrω-n transcripts were down-regulated during heat shock through hs-GAL4-driven expression of the hsrω-RNAi transgene, despite the characteristic elevation of all heat shock proteins (Hsp), including Hsp70. Here, we show that hsrω-RNAi transgene expression prior to heat shock singularly prevents accumulation of Hsp70 in all larval tissues without affecting transcriptional induction of hsp70 genes and stability of their transcripts. Absence of the stress-induced Hsp70 accumulation was not due to higher levels of Hsc70 in hsrω-RNAi transgene-expressing tissues. Inhibition of proteasomal activity during heat shock restored high levels of the induced Hsp70, suggesting very rapid degradation of the Hsp70 even during the stress when hsrω-RNAi transgene was expressed ahead of heat shock. Unexpectedly, while complete absence of hsrω transcripts in hsrω (66) homozygotes (hsrω-null) did not prevent high accumulation of heat shock-induced Hsp70, hsrω-RNAi transgene expression in hsrω-null background blocked Hsp70 accumulation. Nonspecific RNAi transgene expression did not affect Hsp70 induction. These observations reveal that, under certain conditions, the stress-induced Hsp70 can be selectively and rapidly targeted for proteasomal degradation even during heat shock. In the present case, the selective degradation of Hsp70 does not appear to be due to down-regulation of the hsrω-n transcripts per se; rather, this may be an indirect effect of the expression of hsrω-RNAi transgene whose RNA products may titrate away some RNA-binding proteins which may also be essential for stability of the induced Hsp70.

Calreticulin Induces Dilated Cardiomyopathy

PubMed Central

Lee, Dukgyu; Oka, Tatsujiro; Hunter, Beth; Robinson, Alison; Papp, Sylvia; Nakamura, Kimitoshi; Srisakuldee, Wattamon; Nickel, Barbara E.; Light, Peter E.; Dyck, Jason R. B.; Lopaschuk, Gary D.; Kardami, Elissavet; Opas, Michal; Michalak, Marek

2013-01-01

Background Calreticulin, a Ca2+-buffering chaperone of the endoplasmic reticulum, is highly expressed in the embryonic heart and is essential for cardiac development. After birth, the calreticulin gene is sharply down regulated in the heart, and thus, adult hearts have negligible levels of calreticulin. In this study we tested the role of calreticulin in the adult heart. Methodology/Principal Findings We generated an inducible transgenic mouse in which calreticulin is targeted to the cardiac tissue using a Cre/loxP system and can be up-regulated in adult hearts. Echocardiography analysis of hearts from transgenic mice expressing calreticulin revealed impaired left ventricular systolic and diastolic function and impaired mitral valve function. There was altered expression of Ca2+ signaling molecules and the gap junction proteins, Connexin 43 and 45. Sarcoplasmic reticulum associated Ca2+-handling proteins (including the cardiac ryanodine receptor, sarco/endoplasmic reticulum Ca2+-ATPase, and cardiac calsequestrin) were down-regulated in the transgenic hearts with increased expression of calreticulin. Conclusions/Significance We show that in adult heart, up-regulated expression of calreticulin induces cardiomyopathy in vivo leading to heart failure. This is due to an alternation in changes in a subset of Ca2+ handling genes, gap junction components and left ventricle remodeling. PMID:23437120

Genetic modification of alternative respiration in Nicotiana benthamiana affects basal and salicylic acid-induced resistance to potato virus X

PubMed Central

2011-01-01

Background Salicylic acid (SA) regulates multiple anti-viral mechanisms, including mechanism(s) that may be negatively regulated by the mitochondrial enzyme, alternative oxidase (AOX), the sole component of the alternative respiratory pathway. However, studies of this mechanism can be confounded by SA-mediated induction of RNA-dependent RNA polymerase 1, a component of the antiviral RNA silencing pathway. We made transgenic Nicotiana benthamiana plants in which alternative respiratory pathway capacity was either increased by constitutive expression of AOX, or decreased by expression of a dominant-negative mutant protein (AOX-E). N. benthamiana was used because it is a natural mutant that does not express a functional RNA-dependent RNA polymerase 1. Results Antimycin A (an alternative respiratory pathway inducer and also an inducer of resistance to viruses) and SA triggered resistance to tobacco mosaic virus (TMV). Resistance to TMV induced by antimycin A, but not by SA, was inhibited in Aox transgenic plants while SA-induced resistance to this virus appeared to be stronger in Aox-E transgenic plants. These effects, which were limited to directly inoculated leaves, were not affected by the presence or absence of a transgene constitutively expressing a functional RNA-dependent RNA polymerase (MtRDR1). Unexpectedly, Aox-transgenic plants infected with potato virus X (PVX) showed markedly increased susceptibility to systemic disease induction and virus accumulation in inoculated and systemically infected leaves. SA-induced resistance to PVX was compromised in Aox-transgenic plants but plants expressing AOX-E exhibited enhanced SA-induced resistance to this virus. Conclusions We conclude that AOX-regulated mechanisms not only play a role in SA-induced resistance but also make an important contribution to basal resistance against certain viruses such as PVX. PMID:21356081

EARLY BUD-BREAK 1 (EBB1) is a regulator of release from seasonal dormancy in poplar trees

PubMed Central

Yordanov, Yordan S.; Ma, Cathleen; Strauss, Steven H.; Busov, Victor B.

2014-01-01

Trees from temperate latitudes transition between growth and dormancy to survive dehydration and freezing stress during winter months. We used activation tagging to isolate a dominant mutation affecting release from dormancy and identified the corresponding gene EARLY BUD-BREAK 1 (EBB1). We demonstrate through positioning of the tag, expression analysis, and retransformation experiments that EBB1 encodes a putative APETALA2/Ethylene responsive factor transcription factor. Transgenic up-regulation of the gene caused early bud-flush, whereas down-regulation delayed bud-break. Native EBB1 expression was highest in actively growing apices, undetectable during the dormancy period, but rapidly increased before bud-break. The EBB1 transcript was localized in the L1/L2 layers of the shoot meristem and leaf primordia. EBB1-overexpressing transgenic plants displayed enlarged shoot meristems, open and poorly differentiated buds, and a higher rate of cell division in the apex. Transcriptome analyses of the EBB1 transgenics identified 971 differentially expressed genes whose expression correlated with the EBB1 expression changes in the transgenic plants. Promoter analysis among the differentially expressed genes for the presence of a canonical EBB1-binding site identified 65 putative target genes, indicative of a broad regulatory context of EBB1 function. Our results suggest that EBB1 has a major and integrative role in reactivation of meristem activity after winter dormancy. PMID:24951507

A transgenic model of transactivation by the Tax protein of HTLV-I.

PubMed

Bieberich, C J; King, C M; Tinkle, B T; Jay, G

1993-09-01

The human T-lymphotropic virus type I (HTLV-I) Tax protein is a transcriptional regulatory protein that has been suggested to play a causal role in the development of several HTLV-I-associated diseases. Tax regulates expression of its own LTR and of certain cellular promoters perhaps by usurping the function of the host transcriptional machinery. We have established a transgenic mouse model system to define the spectrum of tissues in vivo that are capable of supporting Tax-mediated transcriptional transactivation. Transgenic mice carrying the HTLV-I LTR driving expression of the Escherichia coli beta-galactosidase (beta gal) gene were generated, and this LTR-beta gal gene was transcriptionally inactive in all tissues. When LTR-beta gal mice were mated to transgenic mice carrying the same LTR driving expression of the HTLV-I tax gene, mice that carried both transgenes showed restricted expression of the beta gal reporter gene in several tissues including muscle, bone, salivary glands, skin, and nerve. In addition, a dramatic increase in the number of beta gal-expressing cells was seen in response to wounding. These observations provide direct evidence for viral transactivation in vivo, delimit the tissues capable of supporting that transactivation, and provide a model system to study the mechanism of gene regulation by Tax.

Technical advance: stringent control of transgene expression in Arabidopsis thaliana using the Top10 promoter system

NASA Technical Reports Server (NTRS)

Love, J.; Scott, A. C.; Thompson, W. F.; Brown, C. S. (Principal Investigator)

2000-01-01

We show that the tightly regulated tetracycline-sensitive Top10 promoter system (Weinmann et al. Plant J. 1994, 5, 559-569) is functional in Arabidopsis thaliana. A pure breeding A. thaliana line (JL-tTA/8) was generated which expressed a chimeric fusion of the tetracycline repressor and the activation domain of Herpes simplex virus (tTA), from a single transgenic locus. Plants from this line were crossed with transgenics carrying the ER-targeted green fluorescent protein coding sequence (mGFP5) under control of the Top10 promoter sequence. Progeny from this cross displayed ER-targeted GFP fluorescence throughout the plant, indicating that the tTA-Top10 promoter interaction was functional in A. thaliana. GFP expression was repressed by 100 ng ml-1 tetracycline, an order of magnitude lower than the concentration used previously to repress expression in Nicotiana tabacum. Moreover, the level of GFP expression was controlled by varying the concentration of tetracycline in the medium, allowing a titred regulation of transgenic activity that was previously unavailable in A. thaliana. The kinetics of GFP activity were determined following de-repression of the Top10:mGFP5 transgene, with a visible ER-targeted GFP signal appearing from 24 to 48 h after de-repression.

Increased abscisic acid levels in transgenic maize overexpressing AtLOS5 mediated root ion fluxes and leaf water status under salt stress

PubMed Central

Zhang, Juan; Yu, Haiyue; Zhang, Yushi; Wang, Yubing; Li, Maoying; Zhang, Jiachang; Duan, Liusheng; Zhang, Mingcai; Li, Zhaohu

2016-01-01

Abscisic acid (ABA) is a vital cellular signal in plants, and effective ABA signalling is pivotal for stress tolerance. AtLOS5 encoding molybdenum cofactor sulphurase is a key regulator of ABA biosynthesis. Here, transgenic AtLOS5 plants were generated to explore the role of AtLOS5 in salt tolerance in maize. AtLOS5 overexpression significantly up-regulated the expression of ZmVp14-2, ZmAO, and ZmMOCO, and increased aldehyde oxidase activities, which enhanced ABA accumulation in transgenic plants under salt stress. Concurrently, AtLOS5 overexpression induced the expression of ZmNHX1, ZmCBL4, and ZmCIPK16, and enhanced the root net Na+ efflux and H+ influx, but decreased net K+ efflux, which maintained a high cytosolic K+/Na+ ratio in transgenic plants under salt stress. However, amiloride or sodium orthovanadate could significantly elevate K+ effluxes and decrease Na+ efflux and H+ influx in salt-treated transgenic roots, but the K+ effluxes were inhibited by TEA, suggesting that ion fluxes regulated by AtLOS5 overexpression were possibly due to activation of Na+/H+ antiport and K+ channels across the plasma membrane. Moreover, AtLOS5 overexpression could up-regulate the transcripts of ZmPIP1:1, ZmPIP1:5, and ZmPIP2:4, and enhance root hydraulic conductivity. Thus transgenic plants had higher leaf water potential and turgor, which was correlated with greater biomass accumulation under salt stress. Thus AtLOS5 overexpression induced the expression of ABA biosynthetic genes to promote ABA accumulation, which activated ion transporter and PIP aquaporin gene expression to regulate root ion fluxes and water uptake, thus maintaining high cytosolic K+ and Na+ homeostasis and better water status in maize exposed to salt stress. PMID:26743432

Rescue of the colony-stimulating factor 1 (CSF-1)-nullizygous mouse (Csf1(op)/Csf1(op)) phenotype with a CSF-1 transgene and identification of sites of local CSF-1 synthesis.

PubMed

Ryan, G R; Dai, X M; Dominguez, M G; Tong, W; Chuan, F; Chisholm, O; Russell, R G; Pollard, J W; Stanley, E R

2001-07-01

Colony-stimulating factor 1 (CSF-1) regulates the survival, proliferation, and differentiation of mononuclear phagocytes. It is expressed as a secreted glycoprotein or proteoglycan found in the circulation or as a biologically active cell-surface glycoprotein. To investigate tissue CSF-1 regulation, CSF-1-null Csf1(op)/Csf1(op) mice expressing transgenes encoding the full-length membrane-spanning CSF-1 precursor driven by 3.13 kilobases of the mouse CSF-1 promoter and first intron were characterized. Transgene expression corrected the gross osteopetrotic, neurologic, weight, tooth, and reproductive defects of Csf1(op)/Csf1(op) mice. Detailed analysis of one transgenic line revealed that circulating CSF-1, tissue macrophage numbers, hematopoietic tissue cellularity, and hematopoietic parameters were normalized. Tissue CSF-1 levels were normal except for elevations in 4 secretory tissues. Skin fibroblasts from the transgenic mice secreted normal amounts of CSF-1 but also expressed some cell-surface CSF-1. Also, lacZ driven by the same promoter/first intron revealed beta-galactosidase expression in hematopoietic, reproductive, and other tissue locations proximal to CSF-1 cellular targets, consistent with local regulation by CSF-1 at these sites. These studies indicate that the 3.13-kilobase promoter/first intron confers essentially normal CSF-1 expression. They also pinpoint new cellular sites of CSF-1 expression, including ovarian granulosa cells, mammary ductal epithelium, testicular Leydig cells, serous acinar cells of salivary gland, Paneth cells of the small intestine, as well as local sites in several other tissues.

Silencing the HaHR3 Gene by Transgenic Plant-mediated RNAi to Disrupt Helicoverpa armigera Development

PubMed Central

Xiong, Yehui; Zeng, Hongmei; Zhang, Yuliang; Xu, Dawei; Qiu, Dewen

2013-01-01

RNA interference (RNAi) caused by exogenous double-stranded RNA (dsRNA) has developed into a powerful technique in functional genomics, and to date it is widely used to down-regulate crucial physiology-related genes to control pest insects. A molt-regulating transcription factor gene, HaHR3, of cotton bollworm (Helicoverpa armigera) was selected as the target gene. Four different fragments covering the coding sequence (CDS) of HaHR3 were cloned into vector L4440 to express dsRNAs in Escherichia coli. The most effective silencing fragment was then cloned into a plant over-expression vector to express a hairpin RNA (hpRNA) in transgenic tobacco (Nicotiana tabacum). When H. armigera larvae were fed the E. coli or transgenic plants, the HaHR3 mRNA and protein levels dramatically decreased, resulting developmental deformity and larval lethality. The results demonstrate that both recombinant bacteria and transgenic plants could induce HaHR3 silence to disrupt H. armigera development, transgenic plant-mediated RNAi is emerging as a powerful approach for controlling insect pests. PMID:23630449

Regulated expression of a calmodulin isoform alters growth and development in potato.

PubMed

Poovaiah, B W; Takezawa, D; An, G; Han, T J

1996-01-01

A transgene approach was taken to study the consequences of altered expression of a calmodulin isoform on plant growth and development. Eight genomic clones of potato calmodulin (PCM1 to 8) have been isolated and characterized (Takezawa et al., 1995). Among the potato calmodulin isoforms studied, PCM1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM1 fused to the CaMV 35S promoter. Transgenic plants showing a moderate increase in PCM1 mRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM1 mRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM1 mRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM1 protein in transgenic plants, indicating that the expression of both mRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM1 alters growth and development in potato plants.

Creation of an In vivo cytosensor using engineered mesangial cells. Automatic sensing of glomerular inflammation controls transgene activity.

PubMed

Kitamura, M; Kawachi, H

1997-09-15

Automatic control over exogenous gene expression in response to the activity of disease is a crucial hurdle for gene transfer-based therapies. Towards achieving this goal, we created a "cytosensor" that perceives local inflammatory states and subsequently regulates foreign gene expression. alpha-Smooth muscle actin is known to be expressed in glomerular mesangial cells exclusively in pathologic situations. CArG box element, the crucial regulatory sequence of the alpha-smooth muscle actin promoter, was used as a sensor for glomerular inflammation. Rat mesangial cells were stably transfected with an expression plasmid that introduces a beta-galactosidase gene under the control of CArG box elements. In vitro, the established cells expressed beta-galactosidase exclusively after stimulation with serum. To examine whether the cells are able to automatically control transgene activity in vivo, serum-stimulated or unstimulated cells were transferred into normal rat glomeruli or glomeruli subjected to anti-Thy 1 glomerulonephritis. When stimulated cells were transferred into the normal glomeruli, beta-galactosidase expression was switched off in vivo within 3 d. In contrast, when unstimulated cells were transferred into the nephritic glomeruli, transgene expression was substantially induced. These data indicate the feasibility of using the CArG box element as a molecular sensor for glomerular injury. In the context of advanced forms of gene therapy, this approach provides a novel concept for automatic regulation of local transgene expression where the transgene is required to be activated during inflammation and deactivated when the inflammation has subsided.

Development of Transgenic Minipigs with Expression of Antimorphic Human Cryptochrome 1

PubMed Central

Liu, Chunxin; Bolund, Lars; Vajta, Gábor; Dou, Hongwei; Yang, Wenxian; Xu, Ying; Luan, Jing; Wang, Jun; Yang, Huanming; Staunstrup, Nicklas Heine; Du, Yutao

2013-01-01

Minipigs have become important biomedical models for human ailments due to similarities in organ anatomy, physiology, and circadian rhythms relative to humans. The homeostasis of circadian rhythms in both central and peripheral tissues is pivotal for numerous biological processes. Hence, biological rhythm disorders may contribute to the onset of cancers and metabolic disorders including obesity and type II diabetes, amongst others. A tight regulation of circadian clock effectors ensures a rhythmic expression profile of output genes which, depending on cell type, constitute about 3–20% of the transcribed mammalian genome. Central to this system is the negative regulator protein Cryptochrome 1 (CRY1) of which the dysfunction or absence has been linked to the pathogenesis of rhythm disorders. In this study, we generated transgenic Bama-minipigs featuring expression of the Cys414-Ala antimorphic human Cryptochrome 1 mutant (hCRY1AP). Using transgenic donor fibroblasts as nuclear donors, the method of handmade cloning (HMC) was used to produce reconstructed embryos, subsequently transferred to surrogate sows. A total of 23 viable piglets were delivered. All were transgenic and seemingly healthy. However, two pigs with high transgene expression succumbed during the first two months. Molecular analyzes in epidermal fibroblasts demonstrated disturbances to the expression profile of core circadian clock genes and elevated expression of the proinflammatory cytokines IL-6 and TNF-α, known to be risk factors in cancer and metabolic disorders. PMID:24146819

Expression of geminiviral AC2 RNA silencing suppressor changes sugar and jasmonate responsive gene expression in transgenic tobacco plants

PubMed Central

2012-01-01

Background RNA-silencing is a conserved gene regulation and surveillance machinery, which in plants, is also used as major defence mechanism against viruses. Various virus-specific dsRNA structures are recognized by the silencing machinery leading to degradation of the viral RNAs or, as in case of begomoviruses, to methylation of their DNA genomes. Viruses produce specific RNA silencing suppressor (RSS) proteins to prevent these host defence mechanisms, and as these interfere with the silencing machinery they also disturb the endogenous silencing reactions. In this paper, we describe how expression of AC2 RSS, derived from African cassava mosaic geminivirus changes transcription profile in tobacco (Nicotiana tabacum) leaves and in flowers. Results Expression of AC2 RSS in transgenic tobacco plants induced clear phenotypic changes both in leaves and in flowers. Transcriptomes of these plants were strongly altered, with total of 1118 and 251 differentially expressed genes in leaves and flowers, respectively. The three most up-regulated transcript groups were related to stress, cell wall modifications and signalling, whereas the three most down-regulated groups were related to translation, photosynthesis and transcription. It appears that many of the gene expression alterations appeared to be related to enhanced biosynthesis of jasmonate and ethylene, and consequent enhancement of the genes and pathways that are regulated by these hormones, or to the retrograde signalling caused by the reduced photosynthetic activity and sugar metabolism. Comparison of these results to a previous transcriptional profiling of HC-Pro RSS-expressing plants revealed that some of same genes were induced by both RSSs, but their expression levels were typically higher in AC2 than in HC-Pro RSS expressing plants. All in all, a large number of transcript alterations were found to be specific to each of the RSS expressing transgenic plants. Conclusions AC2 RSS in transgenic tobacco plants interferes with the silencing machinery. It causes stress and defence reactions for instance via induction of the jasmonate and ethylene biosynthesis, and by consequent gene expression alteration regulated by these hormones. The changed sugar metabolism may cause significant down-regulation of genes encoding ribosomal proteins, thus reducing the general translation level. PMID:23130567

The rescue of dentin matrix protein 1 (DMP1)-deficient tooth defects by the transgenic expression of dentin sialophosphoprotein (DSPP) indicates that DSPP is a downstream effector molecule of DMP1 in dentinogenesis.

PubMed

Gibson, Monica Prasad; Zhu, Qinglin; Wang, Suzhen; Liu, Qilin; Liu, Ying; Wang, Xiaofang; Yuan, Baozhi; Ruest, L Bruno; Feng, Jian Q; D'Souza, Rena N; Qin, Chunlin; Lu, Yongbo

2013-03-08

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are essential for the formation of dentin. Previous in vitro studies have indicated that DMP1 might regulate the expression of DSPP during dentinogenesis. To examine whether DMP1 controls dentinogenesis through the regulation of DSPP in vivo, we cross-bred transgenic mice expressing normal DSPP driven by a 3.6-kb rat Col1a1 promoter with Dmp1 KO mice to generate mice expressing the DSPP transgene in the Dmp1 KO genetic background (referred to as "Dmp1 KO/DSPP Tg mice"). We used morphological, histological, and biochemical techniques to characterize the dentin and alveolar bone of Dmp1 KO/DSPP Tg mice compared with Dmp1 KO and wild-type mice. Our analyses showed that the expression of endogenous DSPP was remarkably reduced in the Dmp1 KO mice. Furthermore, the transgenic expression of DSPP rescued the tooth and alveolar bone defects of the Dmp1 KO mice. In addition, our in vitro analyses showed that DMP1 and its 57-kDa C-terminal fragment significantly up-regulated the Dspp promoter activities in a mesenchymal cell line. In contrast, the expression of DMP1 was not altered in the Dspp KO mice. These results provide strong evidence that DSPP is a downstream effector molecule that mediates the roles of DMP1 in dentinogenesis.

Tissue- and agonist-specific regulation of human and murine plasminogen activator inhibitor-1 promoters in transgenic mice.

PubMed

Eren, M; Painter, C A; Gleaves, L A; Schoenhard, J A; Atkinson, J B; Brown, N J; Vaughan, D E

2003-11-01

Numerous studies have described regulatory factors and sequences that control transcriptional responses in vitro. However, there is a paucity of information on the qualitative and quantitative regulation of heterologous promoters using transgenic strategies. In order to investigate the physiological regulation of human plasminogen activator inhibitor type-1 (hPAI-1) expression in vivo compared to murine PAI-1 (mPAI-1) and to test the physiological relevance of regulatory mechanisms described in vitro, we generated transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the proximal -2.9 kb of the hPAI-1 promoter. Transgenic animals were treated with Ang II, TGF-beta1 and lipopolysaccharide (LPS) to compare the relative activation of the human and murine PAI-1 promoters. Ang II increased EGFP expression most effectively in brain, kidney and spleen, while mPAI-1 expression was quantitatively enhanced most prominently in heart and spleen. TGF-beta1 failed to induce activation of the hPAI-1 promoter but potently stimulated mPAI-1 in kidney and spleen. LPS administration triggered robust expression of mPAI-1 in liver, kidney, pancreas, spleen and lung, while EGFP was induced only modestly in heart and kidney. These results indicate that the transcriptional response of the endogenous mPAI-1 promoter varies widely in terms of location and magnitude of response to specific stimuli. Moreover, the physiological regulation of PAI-1 expression likely involves a complex interaction of transcription factors and DNA sequences that are not adequately replicated by in vitro functional studies focused on the proximal -2.9 kb promoter.

Mouse and human BAC transgenes recapitulate tissue-specific expression of the vitamin D receptor in mice and rescue the VDR-null phenotype.

PubMed

Lee, Seong Min; Bishop, Kathleen A; Goellner, Joseph J; O'Brien, Charles A; Pike, J Wesley

2014-06-01

The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the vitamin D receptor (VDR), which is expressed in numerous target tissues in a cell type-selective manner. Recent studies using genomic analyses and recombineered bacterial artificial chromosomes (BACs) have defined the specific features of mouse and human VDR gene loci in vitro. In the current study, we introduced recombineered mouse and human VDR BACs as transgenes into mice and explored their expression capabilities in vivo. Individual transgenic mouse strains selectively expressed BAC-derived mouse or human VDR proteins in appropriate vitamin D target tissues, thereby recapitulating the tissue-specific expression of endogenous mouse VDR. The mouse VDR transgene was also regulated by 1,25(OH)2D3 and dibutyryl-cAMP. When crossed into a VDR-null mouse background, both transgenes restored wild-type basal as well as 1,25(OH)2D3-inducible gene expression patterns in the appropriate tissues. This maneuver resulted in the complete rescue of the aberrant phenotype noted in the VDR-null mouse, including systemic features associated with altered calcium and phosphorus homeostasis and disrupted production of parathyroid hormone and fibroblast growth factor 23, and abnormalities associated with the skeleton, kidney, parathyroid gland, and the skin. This study suggests that both mouse and human VDR transgenes are capable of recapitulating basal and regulated expression of the VDR in the appropriate mouse tissues and restore 1,25(OH)2D3 function. These results provide a baseline for further dissection of mechanisms integral to mouse and human VDR gene expression and offer the potential to explore the consequence of selective mutations in VDR proteins in vivo.

Selectivity and Efficiency of Late Transgene Expression by Transcriptionally Targeted Oncolytic Adenoviruses Are Dependent on the Transgene Insertion Strategy

PubMed Central

Quirin, Christina; Rohmer, Stanimira; Fernández-Ulibarri, Inés; Behr, Michael; Hesse, Andrea; Engelhardt, Sarah; Erbs, Philippe; Enk, Alexander H.

2011-01-01

Abstract Key challenges facing cancer therapy are the development of tumor-specific drugs and potent multimodal regimens. Oncolytic adenoviruses possess the potential to realize both aims by restricting virus replication to tumors and inserting therapeutic genes into the virus genome, respectively. A major effort in this regard is to express transgenes in a tumor-specific manner without affecting virus replication. Using both luciferase as a sensitive reporter and genetic prodrug activation, we show that promoter control of E1A facilitates highly selective expression of transgenes inserted into the late transcription unit. This, however, required multistep optimization of late transgene expression. Transgene insertion via internal ribosome entry site (IRES), splice acceptor (SA), or viral 2A sequences resulted in replication-dependent expression. Unexpectedly, analyses in appropriate substrates and with matching control viruses revealed that IRES and SA, but not 2A, facilitated indirect transgene targeting via tyrosinase promoter control of E1A. Transgene expression via SA was more selective (up to 1,500-fold) but less effective than via IRES. Notably, we also revealed transgene-dependent interference with splicing. Hence, the prodrug convertase FCU1 (a cytosine deaminase–uracil phosphoribosyltransferase fusion protein) was expressed only after optimizing the sequence surrounding the SA site and mutating a cryptic splice site within the transgene. The resulting tyrosinase promoter-regulated and FCU1-encoding adenovirus combined effective oncolysis with targeted prodrug activation therapy of melanoma. Thus, prodrug activation showed potent bystander killing and increased cytotoxicity of the virus up to 10-fold. We conclude that armed oncolytic viruses can be improved substantially by comparing and optimizing strategies for targeted transgene expression, thereby implementing selective and multimodal cancer therapies. PMID:20939692

Transgenic mice expressing human fibroblast growth factor-19 display increased metabolic rate and decreased adiposity.

PubMed

Tomlinson, Elizabeth; Fu, Ling; John, Linu; Hultgren, Bruce; Huang, Xiaojian; Renz, Mark; Stephan, Jean Philippe; Tsai, Saio Ping; Powell-Braxton, Lyn; French, Dorothy; Stewart, Timothy A

2002-05-01

The fibroblast growth factors (FGFs), and the corresponding receptors, are implicated in more than just the regulation of epithelial cell proliferation and differentiation. Specifically, FGF23 is a regulator of serum inorganic phosphate levels, and mice deficient in FGF receptor-4 have altered cholesterol metabolism. The recently described FGF19 is unusual in that it is nonmitogenic and appears to interact only with FGF receptor-4. Here, we report that FGF19 transgenic mice had a significant and specific reduction in fat mass that resulted from an increase in energy expenditure. Further, the FGF19 transgenic mice did not become obese or diabetic on a high fat diet. The FGF19 transgenic mice had increased brown adipose tissue mass and decreased liver expression of acetyl coenzyme A carboxylase 2, providing two mechanisms by which FGF19 may increase energy expenditure. Consistent with the reduction in expression of acetyl CoA carboxylase 2, liver triglyceride levels were reduced.

Enhanced seed oil production in canola by conditional expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in developing seeds.

PubMed

Tan, Helin; Yang, Xiaohui; Zhang, Fengxia; Zheng, Xiu; Qu, Cunmin; Mu, Jinye; Fu, Fuyou; Li, Jiana; Guan, Rongzhan; Zhang, Hongsheng; Wang, Guodong; Zuo, Jianru

2011-07-01

The seed oil content in oilseed crops is a major selection trait to breeders. In Arabidopsis (Arabidopsis thaliana), LEAFY COTYLEDON1 (LEC1) and LEC1-LIKE (L1L) are key regulators of fatty acid biosynthesis. Overexpression of AtLEC1 and its orthologs in canola (Brassica napus), BnLEC1 and BnL1L, causes an increased fatty acid level in transgenic Arabidopsis plants, which, however, also show severe developmental abnormalities. Here, we use truncated napin A promoters, which retain the seed-specific expression pattern but with a reduced expression level, to drive the expression of BnLEC1 and BnL1L in transgenic canola. Conditional expression of BnLEC1 and BnL1L increases the seed oil content by 2% to 20% and has no detrimental effects on major agronomic traits. In the transgenic canola, expression of a subset of genes involved in fatty acid biosynthesis and glycolysis is up-regulated in developing seeds. Moreover, the BnLEC1 transgene enhances the expression of several genes involved in Suc synthesis and transport in developing seeds and the silique wall. Consistently, the accumulation of Suc and Fru is increased in developing seeds of the transgenic rapeseed, suggesting the increased carbon flux to fatty acid biosynthesis. These results demonstrate that BnLEC1 and BnL1L are reliable targets for genetic improvement of rapeseed in seed oil production.

The WRKY transcription factor OsWRKY78 regulates stem elongation and seed development in rice.

PubMed

Zhang, Chang-Quan; Xu, Yong; Lu, Yan; Yu, Heng-Xiu; Gu, Ming-Hong; Liu, Qiao-Quan

2011-09-01

WRKY proteins are a large super family of transcriptional regulators primarily involved in various plant physiological programs. In present study, the expression profile and putative function of the WRKY transcriptional factor, WRKY78, in rice were identified. Real-time RT-PCR analysis showed that OsWRKY78 transcript was most abundant in elongating stems though its expression was detected in all the tested organs. The expression profiles were further confirmed by using promoter-GUS analysis in transgenic rice. OsWRKY78::GFP fusion gene transient expression analysis demonstrated that OsWRKY78 targeted to the nuclei of onion epidermal cell. Furthermore, OsWRKY78 RNAi and overexpression transgenic rice lines were generated. Transgenic plants with OsWRKY78 overexpression exhibited a phenotype identical to the wild type, whereas inhibition of OsWRKY78 expression resulted in a semi-dwarf and small kernel phenotype due to reduced cell length in transgenic plants. In addition, a T-DNA insertion mutant line oswrky78 was identified and a phenotype similar to that of RNAi plants was also observed. Grain quality analysis data showed no significant differences, with the exception of minor changes in endosperm starch crystal structure in RNAi plants. Taken together, these results suggest that OsWRKY78 may acts as a stem elongation and seed development regulator in rice.

The maize WRKY transcription factor ZmWRKY17 negatively regulates salt stress tolerance in transgenic Arabidopsis plants.

PubMed

Cai, Ronghao; Dai, Wei; Zhang, Congsheng; Wang, Yan; Wu, Min; Zhao, Yang; Ma, Qing; Xiang, Yan; Cheng, Beijiu

2017-12-01

We cloned and characterized the ZmWRKY17 gene from maize. Overexpression of ZmWRKY17 in Arabidopsis led to increased sensitivity to salt stress and decreased ABA sensitivity through regulating the expression of some ABA- and stress-responsive genes. The WRKY transcription factors have been reported to function as positive or negative regulators in many different biological processes including plant development, defense regulation and stress response. This study isolated a maize WRKY gene, ZmWRKY17, and characterized its role in tolerance to salt stress by generating transgenic Arabidopsis plants. Expression of the ZmWRKY17 was up-regulated by drought, salt and abscisic acid (ABA) treatments. ZmWRKY17 was localized in the nucleus with no transcriptional activation in yeast. Yeast one-hybrid assay showed that ZmWRKY17 can specifically bind to W-box, and it can activate W-box-dependent transcription in planta. Heterologous overexpression of ZmWRKY17 in Arabidopsis remarkably reduced plant tolerance to salt stress, as determined through physiological analyses of the cotyledons greening rate, root growth, relative electrical leakage and malondialdehyde content. Additionally, ZmWRKY17 transgenic plants showed decreased sensitivity to ABA during seed germination and early seedling growth. Transgenic plants accumulated higher content of ABA than wild-type (WT) plants under NaCl condition. Transcriptome and quantitative real-time PCR analyses revealed that some stress-related genes in transgenic seedlings showed lower expression level than that in the WT when treated with NaCl. Taken together, these results suggest that ZmWRKY17 may act as a negative regulator involved in the salt stress responses through ABA signalling.

MicroRNA Silencing Improves the Tumor Specificity of Adenoviral Transgene Expression

PubMed Central

Card, Paul B.; Hogg, Richard T.; del Alcazar, Carlos Gil

2012-01-01

Adenoviral technology has been thoroughly evaluated for delivering genetic material to tumor tissue and the surrounding microenvironment. Almost any gene can be cloned into an adenovirus (Ad) vector, which when combined with strong, constitutively active promoters permit up to a million-fold amplification of the transgene in a single adenoviral particle, thus facilitating their use in cancer therapy and imaging. However, widespread infection of the liver and other non-targeted tissues by Ad vectors is a substantial problem that often results in significant liver inflammation and hepatotoxicity at doses required to achieve efficient tumor transduction. miR-122 is a highly expressed liver-specific microRNA that provides a unique opportunity for down-regulating adenoviral transgene expression in liver tissue. The binding of endogenous miRNAs to complementary miRNA targeting elements (miRTs) incorporated into the 3′ untranslated region of adenoviral transgenes interferes with message stability and/or protein translation, and miRT elements against miR-122 (miRT-122) can selectively reduce adenoviral transgene expression in the liver. Previous studies using miR-122-based regulation, with and without other types of transcriptional targeting, have yielded promising preliminary results. However, investigations to date evaluating miRT-122 elements for improving tumor specificity have used either non-tumor bearing animals or direct intratumoral injection as the mode of delivery. In the present study, we confirmed the ability of miRT-122 sequences to selectively down-regulate adenoviral luciferase expression in the liver in vitro and in vivo, and show that this strategy can improve tumor specific transgene expression in a HT1080 human fibrosarcoma model. Rapid growth and the inefficient flow of blood through tumor neovasculature often results in profound hypoxia, which provides additional opportunities for targeting solid tumors and their microenvironment using vectors incorporating hypoxia-responsive promoters to drive transgene expression. We therefore employed a combinatorial approach using miRT-122 elements with hypoxia-responsive transcriptional targeting to further improve the tumor specific expression of an adenoviral reporter gene. Results from this investigation reveal that miRT122 elements alone decrease off-target liver expression and improve tumor specificity of adenoviral vectors. Furthermore, increased tumor specificity can be achieved by combining miRT-122 elements with hypoxia-responsive promoters. PMID:22555510

An alternative approach in regulation of expression of a transgene by endogenous miR-145 in carcinoma and normal breast cell lines.

PubMed

Ghanbari Safari, Maryam; Baesi, Kazem; Hosseinkhani, Saman

2017-03-01

MicroRNAs are small noncoding RNAs that regulate gene expression by repressing translation of target cellular transcripts. Increasing evidences indicate that miRNAs have different expression profiles and play crucial roles in numerous cellular processes. Delivery and expression of transgenes for cancer therapy must be specific for tumors to avoid killing of healthy tissues. Many investigators have shown that transgene expression can be suppressed in normal cells using vectors that are responsive to microRNA regulation. To overcome this problem, miR-145 that exhibits downregulation in many types of cancer cells was chosen for posttranscriptional regulatory systems mediated by microRNAs. In this study, a psiCHECK-145T vector carrying four tandem copies of target sequences of miR-145 into 3'-UTR of the Renilla luciferase gene was constructed. Renilla luciferase activity from the psiCHECK-145T vector was 57% lower in MCF10A cells with high miR-145 expression as compared to a control condition. Additionally, overexpression of miR-145 in MCF-7 cells with low expression level of miR-145 showed more than 76% reduction in the Renilla luciferase activity from the psiCHECK-145T vector. Inclusion of miR-145 target sequences into the 3'-UTR of the Renilla luciferase gene is a feasible strategy for restricting transgene expression in a breast cancer cell line while sparing a breast normal cell line. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

Over-expression of Stat5b confers protection against diabetes in the non-obese diabetic (NOD) mice via up-regulation of CD4{sup +}CD25{sup +} regulatory T cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin, Yulan; Purohit, Sharad; Department of Pathology, Medical College of Georgia, Georgia Health Sciences University, GA

Highlights: Black-Right-Pointing-Pointer This is the first study to provide direct evidence of the role of Stat5b in NOD mice. Black-Right-Pointing-Pointer Over-expression of wild type Stat5b transgene protects NOD mice against diabetes. Black-Right-Pointing-Pointer This protection may be mediated by the up-regulation of CD4{sup +}CD25{sup +} Tregs. -- Abstract: The signal transducers and activators of transcription (STAT) family of proteins play a critical role in cytokine signaling required for fine tuning of immune regulation. Previous reports showed that a mutation (L327M) in the Stat5b protein leads to aberrant cytokine signaling in the NOD mice. To further elaborate the role of Stat5b inmore » diabetes, we established a NOD transgenic mouse that over-expresses the wild type Stat5b gene. The incidences of spontaneous diabetes as well as cyclophosphamide-induced diabetes were significantly reduced and delayed in the Stat5b transgenic NOD mice compared to their littermate controls. The total cell numbers of CD4{sup +} T cells and especially CD8{sup +} T cells in the spleen and pancreatic lymph node were increased in the Stat5b transgenic NOD mice. Consistent with these findings, CD4{sup +} and CD8{sup +} T cells from the Stat5b transgenic NOD mice showed a higher proliferation capacity and up-regulation of multiple cytokines including IL-2, IFN-{gamma}, TNF-{alpha} and IL-10 as well as anti-apoptotic gene Bcl-xl. Furthermore, the number and proportion of CD4{sup +}CD25{sup +} regulatory T cells were significantly increased in transgenic mice although in vitro suppression ability of the regulatory T-cells was not affected by the transgene. Our results suggest that Stat5b confers protection against diabetes in the NOD mice by regulating the numbers and function of multiple immune cell types, especially by up-regulating CD4{sup +}CD25{sup +} regulatory T cells.« less

Selection of differently temporally regulated African swine fever virus promoters with variable expression activities and their application for transient and recombinant virus mediated gene expression.

PubMed

Portugal, Raquel S; Bauer, Anja; Keil, Guenther M

2017-08-01

African swine fever virus threatens pig production worldwide due to the lack of vaccines, for which generation of both deletion and insertion mutants is considered. For development of the latter, operational ASFV promoters of different temporal regulation and strengths are desirable. We therefore compared the capacities of putative promoter sequences from p72, CD2v, p30, viral DNA polymerase and U104L genes to mediate expression of luciferase from transfected plasmids after activation in trans, or p30-, DNA polymerase- and U104L promoters in cis, using respective ASFV recombinants. We identified sequences with promoter activities upstream the viral ORFs, and showed that they differ in both their expression intensity regulating properties and in their temporal regulation. In summary, p30 and DNA polymerase promoters are recommended for high level early regulated transgene expression. For late expression, the p72, CD2v and U104L promoter are suitable. The latter however, only if low level transgene expression is aimed. Copyright © 2017 Elsevier Inc. All rights reserved.

Sugar beet proteinase inhibitor (BvSTI) gene promoter is regulated by insects and wounding in transgenic Nicotiana benthamiana

USDA-ARS?s Scientific Manuscript database

A regulatory sequence from a serine proteinase inhibitor gene (BvSTIpro) shown to be up-regulated in resistant interactions with a root pest of sugar beet, the sugar beet root maggot, was fused to the ß-glucuronidase (GUS) reporter gene to characterize its expression patterns in transgenic Nicotiana...

Identification of Secretory Odontoblasts Using DMP1-GFP Transgenic Mice

PubMed Central

Balic, Anamaria; Mina, Mina

2011-01-01

Terminal differentiation of odontoblasts from dental papilla is a long process involving several intermediate steps and changes in the transcriptional profile and expression of proteins secreted by cells in the odontoblast lineage. Transgenic mouse lines in which GFP expression is under the control of tissue-and stage specific promoters have provided powerful experimental tools for identification and isolation of cells at specific stages of differentiation along a lineage. Our previous studies showed utilization of pOBCol3.6GFP and pOBCol2.3GFP animals for identification of odontoblasts at early and late stages of polarization respectively. In the present study we used the DMP1-GFP transgenic animal as an experimental model to examine its expression during the differentiation of odontoblasts from progenitor cells in vivo and in vitro. Our observations showed that DMP1-GFP transgene is first activated in secretory/functional odontoblasts engaged in secretion of predentin and then transiently expressed at high levels in newly differentiated odontoblasts. Expression of DMP1-GFP was down-regulated in highly differentiated odontoblasts. The temporal and spatial pattern of expression of DMP1-GFP transgene closely mimics the expression of endogenous DMP1. This transgenic animal will facilitate studies of gene expression and biological functions in secretory/functional odontoblasts. PMID:21172466

IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN THE KIDNEYS OF GROWTH HORMONE TRANSGENIC MICE

PubMed Central

Coschigano, K.T.; Wetzel, A.N.; Obichere, N.; Sharma, A.; Lee, S.; Rasch, R.; Guigneaux, M.M.; Flyvbjerg, A.; Wood, T.G.; Kopchick, J.J.

2010-01-01

Objective Bovine growth hormone (bGH) transgenic mice develop severe kidney damage. This damage may be due, at least in part, to changes in gene expression. Identification of genes with altered expression in the bGH kidney may identify mechanisms leading to damage in this system that may also be relevant to other models of kidney damage. Design cDNA subtraction libraries, northern blot analyses, microarray analyses and real-time reverse transcription polymerase chain reaction (RT/PCR) assays were used to identify and verify specific genes exhibiting differential RNA expression between kidneys of bGH mice and their non-transgenic (NT) littermates. Results Immunoglobulins were the vast majority of genes identified by the cDNA subtractions and the microarray analyses as being up-regulated in bGH. Several glycoprotein genes and inflammation-related genes also showed increased RNA expression in the bGH kidney. In contrast, only a few genes were identified as being significantly down-regulated in the bGH kidney. The most notable decrease in RNA expression was for the gene encoding kidney androgen-regulated protein. Conclusions A number of genes were identified as being differentially expressed in the bGH kidney. Inclusion of two groups, immunoglobulins and inflammation-related genes, suggests a role of the immune system in bGH kidney damage. PMID:20655258

Expression of an Arabidopsis molybdenum cofactor sulphurase gene in soybean enhances drought tolerance and increases yield under field conditions.

PubMed

Li, Yajun; Zhang, Jiachang; Zhang, Juan; Hao, Ling; Hua, Jinping; Duan, Liusheng; Zhang, Mingcai; Li, Zhaohu

2013-08-01

LOS5/ABA3 gene encoding molybdenum cofactor sulphurase is involved in aldehyde oxidase (AO) activity in Arabidopsis, which indirectly regulates ABA biosynthesis and increased stress tolerance. Here, we used a constitutive super promoter to drive LOS5/ABA3 overexpression in soybean (Glycine max L.) to enhance drought tolerance in growth chamber and field conditions. Expression of LOS5/ABA3 was up-regulated by drought stress, which led to increasing AO activity and then a notable increase in ABA accumulation. Transgenic soybean under drought stress had reduced water loss by decreased stomatal aperture size and transpiration rate, which alleviated leaf wilting and maintained higher relative water content. Exposed to drought stress, transgenic soybean exhibited reduced cell membrane damage by reducing electrolyte leakage and production of malondialdehyde and promoting proline accumulation and antioxidant enzyme activities. Also, overexpression of LOS5/ABA3 enhanced expression of stress-up-regulated genes. Furthermore, the seed yield of transgenic plants is at least 21% higher than that of wide-type plants under drought stress conditions in the field. These data suggest that overexpression of LOS5/ABA3 could improve drought tolerance in transgenic soybean via enhanced ABA accumulation, which could activate expression of stress-up-regulated genes and cause a series of physiological and biochemical resistant responses. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Ectopic expression of wheat expansin gene TaEXPA2 improved the salt tolerance of transgenic tobacco by regulating Na+ /K+ and antioxidant competence.

PubMed

Chen, Yanhui; Han, Yangyang; Kong, Xiangzhu; Kang, Hanhan; Ren, Yuanqing; Wang, Wei

2017-02-01

High salinity is one of the most serious environmental stresses that limit crop growth. Expansins are cell wall proteins that regulate plant development and abiotic stress tolerance by mediating cell wall expansion. We studied the function of a wheat expansin gene, TaEXPA2, in salt stress tolerance by overexpressing it in tobacco. Overexpression of TaEXPA2 enhanced the salt stress tolerance of transgenic tobacco plants as indicated by the presence of higher germination rates, longer root length, more lateral roots, higher survival rates and more green leaves under salt stress than in the wild type (WT). Further, when leaf disks of WT plants were incubated in cell wall protein extracts from the transgenic tobacco plants, their chlorophyll content was higher under salt stress, and this improvement from TaEXPA2 overexpression in transgenic tobacco was inhibited by TaEXPA2 protein antibody. The water status of transgenic tobacco plants was improved, perhaps by the accumulation of osmolytes such as proline and soluble sugar. TaEXPA2-overexpressing tobacco lines exhibited lower Na + but higher K + accumulation than WT plants. Antioxidant competence increased in the transgenic plants because of the increased activity of antioxidant enzymes. TaEXPA2 protein abundance in wheat was induced by NaCl, and ABA signaling was involved. Gene expression regulation was involved in the enhanced salt stress tolerance of the TaEXPA2 transgenic plants. Our results suggest that TaEXPA2 overexpression confers salt stress tolerance on the transgenic plants, and this is associated with improved water status, Na + /K + homeostasis, and antioxidant competence. ABA signaling participates in TaEXPA2-regulated salt stress tolerance. © 2016 Scandinavian Plant Physiology Society.

Effects of growth hormone over-expression on reproduction in the common carp Cyprinus carpio L.

PubMed

Cao, Mengxi; Chen, Ji; Peng, Wei; Wang, Yaping; Liao, Lanjie; Li, Yongming; Trudeau, Vance L; Zhu, Zuoyan; Hu, Wei

2014-01-01

To study the complex interaction between growth and reproduction we have established lines of transgenic common carp (Cyprinus carpio) carrying a grass carp (Ctenopharyngodon idellus) growth hormone (GH) transgene. The GH-transgenic fish showed delayed gonadal development compared with non-transgenic common carp. To gain a better understanding of the phenomenon, we studied body growth, gonad development, changes of reproduction related genes and hormones of GH-transgenic common carp for 2years. Over-expression of GH elevated peripheral gh transcription, serum GH levels, and inhibited endogenous GH expression in the pituitary. Hormone analyses indicated that GH-transgenic common carp had reduced pituitary and serum level of luteinizing hormone (LH). Among the tested genes, pituitary lhβ was inhibited in GH-transgenic fish. Further analyses in vitro showed that GH inhibited lhβ expression. Localization of ghr with LH indicates the possibility of direct regulation of GH on gonadotrophs. We also found that GH-transgenic common carp had reduced pituitary sensitivity to stimulation by co-treatments with a salmon gonadotropin-releasing hormone (GnRH) agonist and a dopamine antagonist. Together these results suggest that the main cause of delayed reproductive development in GH transgenic common carp is reduced LH production and release. Copyright © 2013 Elsevier Inc. All rights reserved.

Endothelial function in pigs transgenic for human complement regulating factor.

PubMed

Warnecke, Gregor; Severson, Sandra R; Ugurlu, Mustafa M; Taner, Cemal B; Logan, John S; Diamond, Lisa E; Miller, Virginia M; McGregor, Christopher G A

2002-04-15

Expression of human complement regulating factor (hCRF) in porcine organs prevents hyperacute rejection of these organs after xenotransplantation to nonhuman primates. Experiments were designed to characterize endothelial and smooth muscle function of arteries from pigs transgenic for hCD46. Arterial blood from outbred pigs transgenic for hCD46 expression and nontransgenic animals of the same lineage was analyzed for angiotensin-converting enzyme (ACE), C-type natriuretic peptide (CNP), and nitric oxide. Aortic endothelial cells were prepared for measurement of mRNA or activity for nitric oxide synthase (NOS). Rings cut from femoral and pulmonary arteries were suspended in organ chambers for measurement of isometric tension. CNP was significantly greater, ACE was similar, and nitric oxide was significantly less in plasma from transgenic compared with nontransgenic pigs. Neither mRNA nor activity of NOS differed between the groups. Endothelium-dependent relaxations to bradykinin and acetylcholine but not the calcium ionophore were shifted significantly to the left in femoral and pulmonary arteries from hCD46 transgenic pigs compared with nontransgenic pigs. The ACE-inhibitor captopril augmented relaxations similarly in both groups, but NG-monomethyl-L-arginine (L-NMMA) did not inhibit relaxations in rings from transgenic pigs. Data suggest that expression of hCD46 on endothelium of pigs selectively augments endothelium-dependent relaxations to bradykinin by increased release of endothelium-derived factors other than nitric oxide. There does not seem to be any change in activity of ACE or NOS with expression of the human protein. Increased relaxations to bradykinin may be beneficial in lowering vascular resistance when transgenic organs are used for xenotransplantation.

Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys.

PubMed

Petersen, Björn; Ramackers, Wolf; Lucas-Hahn, Andrea; Lemme, Erika; Hassel, Petra; Queisser, Anna-Lisa; Herrmann, Doris; Barg-Kues, Brigitte; Carnwath, Joseph W; Klose, Johannes; Tiede, Andreas; Friedrich, Lars; Baars, Wiebke; Schwinzer, Reinhard; Winkler, Michael; Niemann, Heiner

2011-01-01

The major immunological hurdle to successful porcine-to-human xenotransplantation is the acute vascular rejection (AVR), characterized by endothelial cell (EC) activation and perturbation of coagulation. Heme oxygenase-1 (HO-1) and its derivatives have anti-apoptotic, anti-inflammatory effects and protect against reactive oxygen species, rendering HO-1 a promising molecule to control AVR. Here, we report the production and characterization of pigs transgenic for human heme oxygenase-1 (hHO-1) and demonstrate significant protection in porcine kidneys against xenograft rejection in ex vivo perfusion with human blood and transgenic porcine aortic endothelial cells (PAEC) in a TNF-α-mediated apoptosis assay. Transgenic and non-transgenic PAEC were tested in a TNF-α-mediated apoptosis assay. Expression of adhesion molecules (ICAM-1, VCAM-1, and E-selectin) was measured by real-time PCR. hHO-1 transgenic porcine kidneys were perfused with pooled and diluted human AB blood in an ex vivo perfusion circuit. MHC class-II up-regulation after induction with IFN-γ was compared between wild-type and hHO-1 transgenic PAEC. Cloned hHO-1 transgenic pigs expressed hHO-1 in heart, kidney, liver, and in cultured ECs and fibroblasts. hHO-1 transgenic PAEC were protected against TNF-α-mediated apoptosis. Real-time PCR revealed reduced expression of adhesion molecules like ICAM-1, VCAM-1, and E-selectin. These effects could be abrogated by the incubation of transgenic PAECs with the specific HO-1 inhibitor zinc protoporphorine IX (Zn(II)PPIX, 20 μm). IFN-γ induced up-regulation of MHC class-II molecules was significantly reduced in PAECs from hHO-1 transgenic pigs. hHO-1 transgenic porcine kidneys could successfully be perfused with diluted human AB-pooled blood for a maximum of 240 min (with and without C1 inh), while in wild-type kidneys, blood flow ceased after ∼60 min. Elevated levels of d-Dimer and TAT were detected, but no significant consumption of fibrinogen and antithrombin was determined. Microthrombi could not be detected histologically. These results are encouraging and warrant further studies on the biological function of heme oxygenase-I expression in hHO-1 transgenic pigs in the context of xenotransplantation. © 2011 John Wiley & Sons A/S.

AN APPROACH TO TRANSGENIC CROP MONITORING

EPA Science Inventory

Remote sensing by aerial or satellite images may provide a method of identifying transgenic pesticidal crop distribution in the landscape. Genetically engineered crops containing bacterial gene(s) that express an insecticidal protein from Bacillus thuringiensis (Bt) are regulated...

Impacts on silkworm larvae midgut proteomics by transgenic Trichoderma strain and analysis of glutathione S-transferase sigma 2 gene essential for anti-stress response of silkworm larvae.

PubMed

Li, Yingying; Dou, Kai; Gao, Shigang; Sun, Jianan; Wang, Meng; Fu, Kehe; Yu, Chuanjin; Wu, Qiong; Li, Yaqian; Chen, Jie

2015-08-03

Lepidoptera is a large order of insects that have major impacts on humans as agriculture pests. The midgut is considered an important target for insect control. In the present study, 10 up-regulated, 18 down-regulated, and one newly emerged protein were identified in the transgenic Trichoderma-treated midgut proteome. Proteins related to stress response, biosynthetic process, and metabolism process were further characterized through quantitative real-time PCR (qPCR). Of all the identified proteins, the glutathione S-transferase sigma 2 (GSTs2) gene displayed enhanced expression when larvae were fed with Trichoderma wild-type or transgenic strains. Down regulation of GSTs2 expression by RNA interference (RNAi) resulted in inhibition of silkworm growth when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. Weight per larva decreased by 18.2%, 11.9%, and 10.7% in the untreated control, ddH2O, and GFP dsRNA groups, respectively, at 24h, while the weight decrease was higher at 42.4%, 28.8% and 32.4% at 72 h after treatment. Expression of glutathione S-transferase omega 2 (GSTo2) was also enhanced when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. These results indicated that there was indeed correlation between enhanced expression of GSTs2 and the anti-stress response of silkworm larvae against Trichoderma. This study represents the first attempt at understanding the effects of transgenic organisms on the midgut proteomic changes in silkworm larvae. Our findings could not only broaden the biological control targets of insect at the molecular level, but also provide a theoretical foundation for biological safety evaluation of the transgenic Trichoderma strain. Copyright © 2015 Elsevier B.V. All rights reserved.

Expression of Arabidopsis glycine-rich RNA-binding protein AtGRP2 or AtGRP7 improves grain yield of rice (Oryza sativa) under drought stress conditions.

PubMed

Yang, Deok Hee; Kwak, Kyung Jin; Kim, Min Kyung; Park, Su Jung; Yang, Kwang-Yeol; Kang, Hunseung

2014-01-01

Although posttranscriptional regulation of RNA metabolism is increasingly recognized as a key regulatory process in plant response to environmental stresses, reports demonstrating the importance of RNA metabolism control in crop improvement under adverse environmental stresses are severely limited. To investigate the potential use of RNA-binding proteins (RBPs) in developing stress-tolerant transgenic crops, we generated transgenic rice plants (Oryza sativa) that express Arabidopsis thaliana glycine-rich RBP (AtGRP) 2 or 7, which have been determined to harbor RNA chaperone activity and confer stress tolerance in Arabidopsis, and analyzed the response of the transgenic rice plants to abiotic stresses. AtGRP2- or AtGRP7-expressing transgenic rice plants displayed similar phenotypes comparable with the wild-type plants under high salt or cold stress conditions. By contrast, AtGRP2- or AtGRP7-expressing transgenic rice plants showed much higher recovery rates and grain yields compared with the wild-type plants under drought stress conditions. The higher grain yield of the transgenic rice plants was due to the increases in filled grain numbers per panicle. Collectively, the present results show the importance of posttranscriptional regulation of RNA metabolism in plant response to environmental stress and suggest that GRPs can be utilized to improve the yield potential of crops under stress conditions. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Genetic manipulation of the metabolism of polyamines in poplar cells. The regulation of putrescine catabolism

Treesearch

Pratiksha Bhatnagar; Rakesh Minocha; Subhash C. Minocha

2002-01-01

We investigated the catabolism of putrescine (Put) in a non-transgenic (NT) and a transgenic cell line of poplar (Populus nigra x maximowiczii) expressing a mouse (Mus musculus) ornithine (Orn) decarboxylase (odc) cDNA. The transgenic cells produce 3- to 4-fold higher amounts of Put than the NT...

Epithelial reticulon 4B (Nogo-B) is an endogenous regulator of Th2-driven lung inflammation

PubMed Central

Wright, Paulette L.; Yu, Jun; Di, Y.P. Peter; Homer, Robert J.; Chupp, Geoffrey; Elias, Jack A.; Cohn, Lauren

2010-01-01

Nogo-B is a member of the reticulon family of proteins (RTN-4B) that is highly expressed in lung tissue; however, its function remains unknown. We show that mice with Th2-driven lung inflammation results in a loss of Nogo expression in airway epithelium and smooth muscle compared with nonallergic mice, a finding which is replicated in severe human asthma. Mice lacking Nogo-A/B (Nogo-KO) display an exaggerated asthma-like phenotype, and epithelial reconstitution of Nogo-B in transgenic mice blunts Th2-mediated lung inflammation. Microarray analysis of lungs from Nogo-KO mice reveals a marked reduction in palate lung and nasal clone (PLUNC) gene expression, and the levels of PLUNC are enhanced in epithelial Nogo-B transgenic mice. Finally, transgenic expression of PLUNC into Nogo-KO mice rescues the enhanced asthmatic-like responsiveness in these KO mice. These data identify Nogo-B as a novel protective gene expressed in lung epithelia, and its expression regulates the levels of the antibacterial antiinflammatory protein PLUNC. PMID:20975041

Utrophin Up-Regulation by an Artificial Transcription Factor in Transgenic Mice

PubMed Central

Mattei, Elisabetta; Corbi, Nicoletta; Di Certo, Maria Grazia; Strimpakos, Georgios; Severini, Cinzia; Onori, Annalisa; Desantis, Agata; Libri, Valentina; Buontempo, Serena; Floridi, Aristide; Fanciulli, Maurizio; Baban, Dilair; Davies, Kay E.; Passananti, Claudio

2007-01-01

Duchenne Muscular Dystrophy (DMD) is a severe muscle degenerative disease, due to absence of dystrophin. There is currently no effective treatment for DMD. Our aim is to up-regulate the expression level of the dystrophin related gene utrophin in DMD, complementing in this way the lack of dystrophin functions. To this end we designed and engineered several synthetic zinc finger based transcription factors. In particular, we have previously shown that the artificial three zinc finger protein named Jazz, fused with the appropriate effector domain, is able to drive the transcription of a test gene from the utrophin promoter “A”. Here we report on the characterization of Vp16-Jazz-transgenic mice that specifically over-express the utrophin gene at the muscular level. A Chromatin Immunoprecipitation assay (ChIP) demonstrated the effective access/binding of the Jazz protein to active chromatin in mouse muscle and Vp16-Jazz was shown to be able to up-regulate endogenous utrophin gene expression by immunohistochemistry, western blot analyses and real-time PCR. To our knowledge, this is the first example of a transgenic mouse expressing an artificial gene coding for a zinc finger based transcription factor. The achievement of Vp16-Jazz transgenic mice validates the strategy of transcriptional targeting of endogenous genes and could represent an exclusive animal model for use in drug discovery and therapeutics. PMID:17712422

Regulation of animal biotechnology: research needs.

PubMed

Rexroad, C E; Green, R D; Wall, R J

2007-09-01

Livestock that result from biotechnology have been a part of agricultural science for over 30 years but have not entered the market place as food or fiber. Two biotechnologies are at the forefront as challenges to the world's systems for regulating the market place: animal clones and transgenic animals. Both technologies have come before the Food and Drug Administration in the United States and it appears that action is imminent for clones. The FDA has asserted principles for evaluation of clones and asserts that "... remaining hazard(s) from cloning are likely to be subtle in nature." The science-based principles recognize that in some areas related to developmental biology and gene expression in clones, additional scientific information would be useful. The role of science then is to use the genomic tools that we have available to answer questions about epigenetic regulation of development and reprogramming of genes to the state found in germ cells. Transgenics pose additional challenges to regulators. If the transgenics are produced using cloning from modified cells then the additional scientific information needed will be related to the effects of insertion and expression of the transgenes. Other approaches such as retrovirally vectored transgenesis will elicit additional questions. These questions will be challenging because the science will have to be related to the expression and function of each gene or class of genes. For the promises of animal biotechnology to be fulfilled, scientists will have to resolve many questions for regulators and the public but tools to answer those questions are rapidly becoming available.

Both coding exons of the c-myc gene contribute to its posttranscriptional regulation in the quiescent liver and regenerating liver and after protein synthesis inhibition.

PubMed Central

Lavenu, A; Pistoi, S; Pournin, S; Babinet, C; Morello, D

1995-01-01

In vivo, the steady-state level of c-myc mRNA is mainly controlled by posttranscriptional mechanisms. Using a panel of transgenic mice in which various versions of the human c-myc proto-oncogene were under the control of major histocompatibility complex H-2Kb class I regulatory sequences, we have shown that the 5' and the 3' noncoding sequences are dispensable for obtaining a regulated expression of the transgene in adult quiescent tissues, at the start of liver regeneration, and after inhibition of protein synthesis. These results indicated that the coding sequences were sufficient to ensure a regulated c-myc expression. In the present study, we have pursued this analysis with transgenes containing one or the other of the two c-myc coding exons either alone or in association with the c-myc 3' untranslated region. We demonstrate that each of the exons contains determinants which control c-myc mRNA expression. Moreover, we show that in the liver, c-myc exon 2 sequences are able to down-regulate an otherwise stable H-2K mRNA when embedded within it and to induce its transient accumulation after cycloheximide treatment and soon after liver ablation. Finally, the use of transgenes with different coding capacities has allowed us to postulate that the primary mRNA sequence itself and not c-Myc peptides is an important component of c-myc posttranscriptional regulation. PMID:7623834

The UT-A Urea Transporter Promoter, UT-Aα, Targets Principal Cells of the Renal Inner Medullary Collecting Duct

PubMed Central

Fenton, Robert A.; Shodeinde, Adetola; Knepper, Mark A.

2006-01-01

The urea transporters, UT-A1 and UT-A3, two members of the UT-A gene family, are localized to the terminal portion of the inner medullary collecting duct (IMCD). In this manuscript, we demonstrate that 4.2-kb of the 5′-flanking region of the UT-A gene (UT-Aα promoter) is sufficient to drive the IMCD-specific expression of a heterologous reporter gene, β-galactosidase (β-Gal), in transgenic mice. RT-PCR, immunoblotting and immunohistochemistry demonstrate that within the kidney, transgene expression is confined to the terminal portion of the IMCD. Co-localization studies with aquaporin 2 show that expression is localized to the principal cells of the IMCD2 and IMCD3 regions. Utilizing β-Gal activity assays, we further show that within the kidney, the β-Gal transgene can be regulated by both water restriction and glucocorticoids, similar to the regulation of the endogenous UT-A gene. These results demonstrate that 4.2-kb of the UT-Aα promoter is sufficient to drive expression of a heterologous reporter gene in a tissue-specific and cell-specific fashion in transgenic mice PMID:16091580

MusTRD can regulate postnatal fiber-specific expression.

PubMed

Issa, Laura L; Palmer, Stephen J; Guven, Kim L; Santucci, Nicole; Hodgson, Vanessa R M; Popovic, Kata; Joya, Josephine E; Hardeman, Edna C

2006-05-01

Human MusTRD1alpha1 was isolated as a result of its ability to bind a critical element within the Troponin I slow upstream enhancer (TnIslow USE) and was predicted to be a regulator of slow fiber-specific genes. To test this hypothesis in vivo, we generated transgenic mice expressing hMusTRD1alpha1 in skeletal muscle. Adult transgenic mice show a complete loss of slow fibers and a concomitant replacement by fast IIA fibers, resulting in postural muscle weakness. However, developmental analysis demonstrates that transgene expression has no impact on embryonic patterning of slow fibers but causes a gradual postnatal slow to fast fiber conversion. This conversion was underpinned by a demonstrable repression of many slow fiber-specific genes, whereas fast fiber-specific gene expression was either unchanged or enhanced. These data are consistent with our initial predictions for hMusTRD1alpha1 and suggest that slow fiber genes contain a specific common regulatory element that can be targeted by MusTRD proteins.

Amino Acids Regulate Transgene Expression in MDCK Cells

PubMed Central

Torrente, Marta; Guetg, Adriano; Sass, Jörn Oliver; Arps, Lisa; Ruckstuhl, Lisa; Camargo, Simone M. R.; Verrey, François

2014-01-01

Gene expression and cell growth rely on the intracellular concentration of amino acids, which in metazoans depends on extracellular amino acid availability and transmembrane transport. To investigate the impact of extracellular amino acid concentrations on the expression of a concentrative amino acid transporter, we overexpressed the main kidney proximal tubule luminal neutral amino acid transporter B0AT1-collectrin (SLC6A19-TMEM27) in MDCK cell epithelia. Exogenously expressed proteins co-localized at the luminal membrane and mediated neutral amino acid uptake. However, the transgenes were lost over few cell culture passages. In contrast, the expression of a control transgene remained stable. To test whether this loss was due to inappropriately high amino acid uptake, freshly transduced MDCK cell lines were cultivated either with physiological amounts of amino acids or with the high concentration found in standard cell culture media. Expression of exogenous transporters was unaffected by physiological amino acid concentration in the media. Interestingly, mycoplasma infection resulted in a significant increase in transgene expression and correlated with the rapid metabolism of L-arginine. However, L-arginine metabolites were shown to play no role in transgene expression. In contrast, activation of the GCN2 pathway revealed by an increase in eIF2α phosphorylation may trigger transgene derepression. Taken together, high extracellular amino acid concentration provided by cell culture media appears to inhibit the constitutive expression of concentrative amino acid transporters whereas L-arginine depletion by mycoplasma induces the expression of transgenes possibly via stimulation of the GCN2 pathway. PMID:24797296

Hepatocellular carcinomas of the albumin SV40 T-antigen transgenic rat display fetal-like re-expression of lgf2 and deregulation of H19.

PubMed

Czarny, Matthew J; Babcock, Karlee; Baus, Rebecca M; Manoharan, Herbert; Pitot, Henry C

2007-09-01

Previous studies in our laboratory have shown that one of the earliest events during hepatocarcinogenesis in the albumin SV40 T antigen (Alb SV40 T Ag) transgenic rat is the duplication of chromosome 1q3.7-4.3, a region which contains the imprinted and coordinately regulated genes Igf2 and H19. We have also shown that this duplication is associated with the biallelic expression of the normally monoallelically-expressed H19. These results, however, are seemingly at odds with studies in the mouse that have shown a conservation of fetal regulatory patterns of these two genes in hepatic neoplasms. We therefore aimed in this study to determine the allelic origin of Igf2 expression in hepatocellular carcinomas of the Alb SV40 T Ag transgenic rat. Sprague-Dawley Alb SV40 T Ag transgenic rats and Brown Norway rats were reciprocally mated and the expression of Igf2 in hepatocellular carcinomas of the resulting F(1) transgene-positive female rats was analyzed by Northern blotting and RT-PCR. We determined that Igf2 was expressed exclusively from the paternal allele, which prompted the study (by the same methods) of the allelic origin of H19 in the same hepatocellular carcinomas in order to determine if the two genes remained coordinately regulated. Our results demonstrate fetal-like re-expression of Igf2 and deregulation of H19 in singular hepatocellular carcinomas of the rat. These results imply that another regulatory mechanism other than the generally accepted ICR/CTCF mechanism may play a role in the control of Igf2 and H19 expression. (c) 2007 Wiley-Liss, Inc.

Recent advances in development of marker-free transgenic plants: regulation and biosafety concern.

PubMed

Tuteja, Narendra; Verma, Shiv; Sahoo, Ranjan Kumar; Raveendar, Sebastian; Reddy, I N Bheema Lingeshwara

2012-03-01

During the efficient genetic transformation of plants with the gene of interest, some selectable marker genes are also used in order to identify the transgenic plant cells or tissues. Usually, antibiotic- or herbicide-selective agents and their corresponding resistance genes are used to introduce economically valuable genes into crop plants. From the biosafety authority and consumer viewpoints, the presence of selectable marker genes in released transgenic crops may be transferred to weeds or pathogenic microorganisms in the gastrointestinal tract or soil, making them resistant to treatment with herbicides or antibiotics, respectively. Sexual crossing also raises the problem of transgene expression because redundancy of transgenes in the genome may trigger homology-dependent gene silencing. The future potential of transgenic technologies for crop improvement depends greatly on our abilities to engineer stable expression of multiple transgenic traits in a predictable fashion and to prevent the transfer of undesirable transgenic material to non-transgenic crops and related species. Therefore, it is now essential to develop an efficient marker-free transgenic system. These considerations underline the development of various approaches designed to facilitate timely elimination of transgenes when their function is no longer needed. Due to the limiting number of available selectable marker genes, in future the stacking of transgenes will be increasingly desirable. The production of marker-free transgenic plants is now a critical requisite for their commercial deployment and also for engineering multiple and complex trait. Here we describe the current technologies to eliminate the selectable marker genes (SMG) in order to develop marker-free transgenic plants and also discuss the regulation and biosafety concern of genetically modified (GM) crops.

Gibberellin 3-oxidase Gene Expression Patterns Influence Gibberellin Biosynthesis, Growth, and Development in Pea1[W][OPEN

PubMed Central

Reinecke, Dennis M.; Wickramarathna, Aruna D.; Ozga, Jocelyn A.; Kurepin, Leonid V.; Jin, Alena L.; Good, Allen G.; Pharis, Richard P.

2013-01-01

Gibberellins (GAs) are key modulators of plant growth and development. PsGA3ox1 (LE) encodes a GA 3β-hydroxylase that catalyzes the conversion of GA20 to biologically active GA1. To further clarify the role of GA3ox expression during pea (Pisum sativum) plant growth and development, we generated transgenic pea lines (in a lele background) with cauliflower mosaic virus-35S-driven expression of PsGA3ox1 (LE). PsGA3ox1 transgene expression led to higher GA1 concentrations in a tissue-specific and development-specific manner, altering GA biosynthesis and catabolism gene expression and plant phenotype. PsGA3ox1 transgenic plants had longer internodes, tendrils, and fruits, larger stipules, and displayed delayed flowering, increased apical meristem life, and altered vascular development relative to the null controls. Transgenic PsGA3ox1 overexpression lines were then compared with lines where endogenous PsGA3ox1 (LE) was introduced, by a series of backcrosses, into the same genetic background (BC LEle). Most notably, the BC LEle plants had substantially longer internodes containing much greater GA1 levels than the transgenic PsGA3ox1 plants. Induction of expression of the GA deactivation gene PsGA2ox1 appears to make an important contribution to limiting the increase of internode GA1 to modest levels for the transgenic lines. In contrast, PsGA3ox1 (LE) expression driven by its endogenous promoter was coordinated within the internode tissue to avoid feed-forward regulation of PsGA2ox1, resulting in much greater GA1 accumulation. These studies further our fundamental understanding of the regulation of GA biosynthesis and catabolism at the tissue and organ level and demonstrate that the timing/localization of GA3ox expression within an organ affects both GA homeostasis and GA1 levels, and thereby growth. PMID:23979969

Gibberellin 3-oxidase gene expression patterns influence gibberellin biosynthesis, growth, and development in pea.

PubMed

Reinecke, Dennis M; Wickramarathna, Aruna D; Ozga, Jocelyn A; Kurepin, Leonid V; Jin, Alena L; Good, Allen G; Pharis, Richard P

2013-10-01

Gibberellins (GAs) are key modulators of plant growth and development. PsGA3ox1 (LE) encodes a GA 3β-hydroxylase that catalyzes the conversion of GA20 to biologically active GA1. To further clarify the role of GA3ox expression during pea (Pisum sativum) plant growth and development, we generated transgenic pea lines (in a lele background) with cauliflower mosaic virus-35S-driven expression of PsGA3ox1 (LE). PsGA3ox1 transgene expression led to higher GA1 concentrations in a tissue-specific and development-specific manner, altering GA biosynthesis and catabolism gene expression and plant phenotype. PsGA3ox1 transgenic plants had longer internodes, tendrils, and fruits, larger stipules, and displayed delayed flowering, increased apical meristem life, and altered vascular development relative to the null controls. Transgenic PsGA3ox1 overexpression lines were then compared with lines where endogenous PsGA3ox1 (LE) was introduced, by a series of backcrosses, into the same genetic background (BC LEle). Most notably, the BC LEle plants had substantially longer internodes containing much greater GA1 levels than the transgenic PsGA3ox1 plants. Induction of expression of the GA deactivation gene PsGA2ox1 appears to make an important contribution to limiting the increase of internode GA1 to modest levels for the transgenic lines. In contrast, PsGA3ox1 (LE) expression driven by its endogenous promoter was coordinated within the internode tissue to avoid feed-forward regulation of PsGA2ox1, resulting in much greater GA1 accumulation. These studies further our fundamental understanding of the regulation of GA biosynthesis and catabolism at the tissue and organ level and demonstrate that the timing/localization of GA3ox expression within an organ affects both GA homeostasis and GA1 levels, and thereby growth.

Exogenous expression of Drp1 plays neuroprotective roles in the Alzheimer's disease in the Aβ42 transgenic Drosophila model.

PubMed

Lv, Fengshou; Yang, Xiaopeng; Cui, Chuanju; Su, Chunhe

2017-01-01

Alzheimer's disease (AD) is one of the most common neurodegenerative disorders. Recent studies have shown that mitochondrial dysfunction is a causative factor of AD. Drp1 (Dynamin-related protein 1), a regulator of mitochondrial fission, shows neuroprotective effects on Parkinson's disease. In this study, we investigate the effect and mechanism of Drp1 on Aβ42 transgenic Drosophila. Elav-gal4/UAS>Aβ42 transgenic Drosophila model was constructed using Elav-gal4 promoter. The effects of Drp1 on the lifespan, motor ability and neuronal degeneration of the transgenic Drosophila were explored by over-expressing Drp1 in the Aβ42 transgenic Drosophila. ATP levels in the brain tissues of Aβ42 transgenic Drosophila were detected using high performance liquid chromatography (HPLC). Exogenous expression of Drp1 promoted crawling ability, reduced the levels of ATP in Drosophila brain and suppressed the neuronal degeneration. The protective effect of Drp1 on the Aβ42 transgenic Drosophila was achieved by protecting the mitochondrial function, suggesting that Drp1 may be a potential therapeutic strategies for AD.

Transgenic Cotton Plants Expressing the HaHR3 Gene Conferred Enhanced Resistance to Helicoverpa armigera and Improved Cotton Yield

PubMed Central

Han, Qiang; Wang, Zhenzhen; He, Yunxin; Xiong, Yehui; Lv, Shun; Li, Shupeng; Zhang, Zhigang; Qiu, Dewen; Zeng, Hongmei

2017-01-01

RNA interference (RNAi) has been developed as an efficient technology. RNAi insect-resistant transgenic plants expressing double-stranded RNA (dsRNA) that is ingested into insects to silence target genes can affect the viability of these pests or even lead to their death. HaHR3, a molt-regulating transcription factor gene, was previously selected as a target expressed in bacteria and tobacco plants to control Helicoverpa armigera by RNAi technology. In this work, we selected the dsRNA-HaHR3 fragment to silence HaHR3 in cotton bollworm for plant mediated-RNAi research. A total of 19 transgenic cotton lines expressing HaHR3 were successfully cultivated, and seven generated lines were used to perform feeding bioassays. Transgenic cotton plants expressing dsHaHR3 were shown to induce high larval mortality and deformities of pupation and adult eclosion when used to feed the newly hatched larvae, and 3rd and 5th instar larvae of H. armigera. Moreover, HaHR3 transgenic cotton also demonstrated an improved cotton yield when compared with controls. PMID:28867769

Characterization of diabetic nephropathy in CaM kinase IIalpha (Thr286Asp) transgenic mice.

PubMed

Suzuki, Hikari; Kato, Ichiro; Usui, Isao; Takasaki, Ichiro; Tabuchi, Yoshiaki; Oya, Takeshi; Tsuneyama, Koichi; Kawaguchi, Hiroshi; Hiraga, Koichi; Takasawa, Shin; Okamoto, Hiroshi; Tobe, Kazuyuki; Sasahara, Masakiyo

2009-01-30

Detailed studies were performed on diabetic kidneys derived from transgenic mice overexpressing the mutant form (Thr286Asp) of Ca(2+)/calmodulin-dependent protein kinase IIalpha (CaM kinase IIalpha) in pancreatic beta-cells. Kidney weight/body weight ratio, urinary albumin/creatinine ratio, serum BUN level, and mesangial/glomerular area ratio were all significantly higher in transgenic mice than in wild-type mice. cDNA microarray analysis revealed 17 up-regulated genes and 12 down-regulated genes in transgenic kidney. Among up-regulated genes, cyclin D2 (6.70-fold) and osteopontin (2.35-fold) were thought to play important roles in the progression of diabetic nephropathy. Transgenic glomeruli and tubular epithelial cells were strongly stained for osteopontin, a molecule which induces immune response. In quantitative real-time RT-PCR analyses, expressions of not only M1 macrophage marker genes but also M2 macrophage marker genes were elevated in renal cortex of transgenic mice. Overall results indicate that CaM kinase IIalpha (Thr286Asp) transgenic mice serve as an excellent model for diabetic nephropathy.

The suppression of inflammatory macrophage-mediated cytotoxicity and proinflammatory cytokine production by transgenic expression of HLA-E.

PubMed

Maeda, Akira; Kawamura, Takuji; Ueno, Takehisa; Usui, Noriaki; Eguchi, Hiroshi; Miyagawa, Shuji

2013-12-01

Macrophages participate in xenogenic rejection and represent a major biological obstacle to successful xenotransplantation. The signal inhibitory regulatory protein α (SIRPα) receptor was reported to be a negative regulator of macrophage phagocytic activity via interaction with CD47, its ligand. Because a majority of human macrophages express the inhibitory receptor CD94/NKG2A, which binds specifically to the human leukocyte antigen (HLA)-E and contains immunoreceptor tyrosine-based inhibition motifs (ITIMs), the inhibitory function of HLA class I molecules, HLA-E, on macrophage-mediated cytolysis was examined. The suppressive effect against proinflammatory cytokine production by macrophages was also examined. Complementary DNA (cDNA) of HLA-E, and CD47 were prepared and transfected into swine endothelial cells (SEC). The expression of the modified genes was evaluated by flow cytometry and macrophage-mediated cytolysis was assessed using in vitro generated macrophages. Transgenic expression of HLA-E significantly suppressed the macrophage-mediated cytotoxicity. HLA-E transgenic expression demonstrated a significant suppression equivalent to CD47 transgenic expression. Furthermore, transgenic HLA-E suppressed the production of pro-inflammatory cytokines by inflammatory macrophages. These results indicate that generating transgenic HLA-E pigs might protect porcine grafts from, not only NK cytotoxicity, but also macrophage-mediated cytotoxicity. © 2013 Elsevier B.V. All rights reserved.

Enhanced Tolerance of Transgenic Potato Plants Over-Expressing Non-specific Lipid Transfer Protein-1 (StnsLTP1) against Multiple Abiotic Stresses

PubMed Central

Gangadhar, Baniekal H.; Sajeesh, Kappachery; Venkatesh, Jelli; Baskar, Venkidasamy; Abhinandan, Kumar; Yu, Jae W.; Prasad, Ram; Mishra, Raghvendra K.

2016-01-01

Abiotic stresses such as heat, drought, and salinity are major environmental constraints that limit potato (Solanum tuberosum L.) production worldwide. Previously, we found a potential thermo-tolerance gene, named StnsLTP1 from potato using yeast functional screening. Here, we report the functional characterization of StnsLTP1 and its role in multiple abiotic stresses in potato plants. Computational analysis of StnsLTP1 with other plant LTPs showed eight conserved cysteine residues, and four α-helices stabilized by four disulfide bridges. Expression analysis of StnsLTP1 gene showed differential expression under heat, water-deficit and salt stresses. Transgenic potato lines over-expressing StnsLTP1 gene displayed enhanced cell membrane integrity under stress conditions, as indicated by reduced membrane lipid per-oxidation, and hydrogen peroxide content relative to untransformed (UT) control plants. In addition, transgenic lines over-expressing StLTP1 also exhibited increased antioxidant enzyme activity with enhanced accumulation of ascorbates, and up-regulation of stress-related genes including StAPX, StCAT, StSOD, StHsfA3, StHSP70, and StsHSP20 compared with the UT plants. These results suggests that StnsLTP1 transgenic plants acquired improved tolerance to multiple abiotic stresses through enhanced activation of antioxidative defense mechanisms via cyclic scavenging of reactive oxygen species and regulated expression of stress-related genes. PMID:27597854

Fish as bioreactors: transgene expression of human coagulation factor VII in fish embryos.

PubMed

Hwang, Gyulin; Müller, Ferenc; Rahman, M Aziz; Williams, Darren W; Murdock, Paul J; Pasi, K John; Goldspink, Geoffrey; Farahmand, Hamid; Maclean, Norman

2004-01-01

A plasmid containing human coagulation factor VII (hFVII) complementary DNA regulated by a cytomegalovirus promoter was microinjected into fertilized eggs of zebrafish, African catfish, and tilapia. The active form of hFVll was detected in the fish embryos by various assays. This positive expression of human therapeutic protein in fish embryos demonstrates the possibility of exploitation of transgenic fish as bioreactors.

A 3'-untranslated region (3'UTR) induces organ adhesion by regulating miR-199a* functions.

PubMed

Lee, Daniel Y; Shatseva, Tatiana; Jeyapalan, Zina; Du, William W; Deng, Zhaoqun; Yang, Burton B

2009-01-01

Mature microRNAs (miRNAs) are single-stranded RNAs of 18-24 nucleotides that repress post-transcriptional gene expression. However, it is unknown whether the functions of mature miRNAs can be regulated. Here we report that expression of versican 3'UTR induces organ adhesion in transgenic mice by modulating miR-199a* activities. The study was initiated by the hypothesis that the non-coding 3'UTR plays a role in the regulation of miRNA function. Transgenic mice expressing a construct harboring the 3'UTR of versican exhibits the adhesion of organs. Computational analysis indicated that a large number of microRNAs could bind to this fragment potentially including miR-199a*. Expression of versican and fibronectin, two targets of miR-199a*, are up-regulated in transgenic mice, suggesting that the 3'UTR binds and modulates miR-199a* activities, freeing mRNAs of versican and fibronectin from being repressed by miR-199a*. Confirmation of the binding was performed by PCR using mature miR-199a* as a primer and the targeting was performed by luciferase assays. Enhanced adhesion by expression of the 3'UTR was confirmed by in vitro assays. Our results demonstrated that upon arrival in cytoplasm, miRNA activities can be modulated locally by the 3'UTR. Our assay may be developed as sophisticated approaches for studying the mutual regulation of miRNAs and mRNAs in vitro and in vivo. We anticipate that expression of the 3'UTR may be an approach in the development of gene therapy.

Retransformation of marker-free potato for enhanced resistance against fungal pathogens by pyramiding chitinase and wasabi defensin genes.

PubMed

Khan, Raham Sher; Darwish, Nader Ahmed; Khattak, Bushra; Ntui, Valentine Otang; Kong, Kynet; Shimomae, Kazuki; Nakamura, Ikuo; Mii, Masahiro

2014-09-01

Multi-auto-transformation vector system has been one of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators and thus facilitating transgene stacking. In the study reported here, retransformation was carried out in marker-free transgenic potato CV. May Queen containing ChiC gene (isolated from Streptomyces griseus strain HUT 6037) with wasabi defensin (WD) gene (isolated from Wasabia japonica) to pyramid the two disease resistant genes. Molecular analyses of the developed shoots confirmed the existence of both the genes of interest (ChiC and WD) in transgenic plants. Co-expression of the genes was confirmed by RT-PCR, northern blot, and western blot analyses. Disease resistance assay of in vitro plants showed that the transgenic lines co-expressing both the ChiC and WD genes had higher resistance against the fungal pathogens, Fusarium oxysporum (Fusarium wilt) and Alternaria solani (early blight) compared to the non-transformed control and the transgenic lines expressing either of the ChiC or WD genes. The disease resistance potential of the transgenic plants could be increased by transgene stacking or multiple transformations.

Ectopic Expression of JcWRKY Confers Enhanced Resistance in Transgenic Tobacco Against Macrophomina phaseolina.

PubMed

Agarwal, Parinita; Patel, Khantika; Agarwal, Pradeep K

2018-04-01

Plants possess an innate immune system comprising of a complex network of closely regulated defense responses involving differential gene expression mediated by transcription factors (TFs). The WRKYs comprise of an important plant-specific TF family, which is involved in regulation of biotic and abiotic defenses. The overexpression of JcWRKY resulted in improved resistance in transgenic tobacco against Macrophomina phaseolina. The production of reactive oxygen species (ROS) and its detoxification through antioxidative system in the transgenics facilitates defense against Macrophomina. The enhanced catalase activity on Macrophomina infection limits the spread of infection. The transcript expression of antioxidative enzymes gene (CAT and SOD) and salicylic acid (SA) biosynthetic gene ICS1 showed upregulation during Macrophomina infection and combinatorial stress. The enhanced transcript of pathogenesis-related genes PR-1 indicates the accumulation of SA during different stresses. The PR-2 and PR-5 highlight the activation of defense responses comprising of activation of hydrolytic cleavage of glucanases and thaumatin-like proteins causing disruption of fungal cells. The ROS homeostasis in coordination with signaling molecules regulate the defense responses and inhibit fungal growth.

The physiological response of Populus tremula x alba leaves to the down-regulation of PIP1 aquaporin gene expression under no water stress

PubMed Central

Secchi, Francesca; Zwieniecki, Maciej A.

2013-01-01

In order to study the role of PIP1 aquaporins in leaf water and CO2 transport, several lines of PIP1-deficient transgenic Populus tremula x alba were generated using a reverse genetic approach. These transgenic lines displayed no visible developmental or morphological phenotypes when grown under conditions of no water stress. Major photosynthetic parameters were also not affected by PIP1 down regulation. However, low levels of PIP1 expression resulted in greater leaf hydraulic resistance (an increase of 27%), which effectively implicated PIP1 role in water transport. Additionally, the expression level of PIP1 genes in the various transgenic lines was correlated with reductions in mesophyll conductance to CO2 (gm), suggesting that in poplar, these aquaporins influenced membrane permeability to CO2. Overall, although analysis showed that PIP1 genes contributed to the mass transfer of water and CO2 in poplar leaves, their down-regulation did not dramatically impair the physiological needs of this fast growing tree when cultivated under conditions of no stress. PMID:24379822

Ectopic expression of an apple cytochrome P450 gene MdCYPM1 negatively regulates plant photomorphogenesis and stress response in Arabidopsis.

PubMed

An, Jian-Ping; Li, Rui; Qu, Feng-Jia; You, Chun-Xiang; Wang, Xiao-Fei; Hao, Yu-Jin

2017-01-29

Cytochrome P450s play an important role in plant growth and are involved in multiple stresses response. However, little is known about the functions of cytochrome P450s in apple. Here, a Malus × domestica cytochrome P450 monooxygenase 1 gene, MdCYPM1, was identified and subsequently cloned from apple 'Gala' (Malus × domestica). To verify the functions of MdCYPM1, we generated transgenic Arabidopsis plants expressing the apple MdCYPM1 gene under the control of the Cauliflower mosaic virus 35S promoter. Four transgenic lines (#3, #5, #7 and #8) were selected for further study. The transgenic plants exhibited a series of skotomorphogenesis phenotypes relative to wild-type controls, such as reduction of the chlorophyll, anthocyanins content and hypocotyls elongation. In addition, overexpression of MdCYPM1 influenced auxin transport and flowering time in transgenic Arabidopsis. Furthermore, MdCYPM1 expression was induced by salt and mannitol treatments, and the transgenic plants were negatively regulated by salinity and osmotic stresses during germination. These results suggest that MdCYPM1 plays a vital role in plant growth and development. Copyright © 2017 Elsevier Inc. All rights reserved.

Skeletal Muscle-Specific Overexpression of PGC-1α Induces Fiber-Type Conversion through Enhanced Mitochondrial Respiration and Fatty Acid Oxidation in Mice and Pigs.

PubMed

Zhang, Lin; Zhou, Ying; Wu, Wangjun; Hou, Liming; Chen, Hongxing; Zuo, Bo; Xiong, Yuanzhu; Yang, Jinzeng

2017-01-01

Individual skeletal muscles in the animal body are heterogeneous, as each is comprised of different fiber types. Type I muscle fibers are rich with mitochondria, and have high oxidative metabolisms while type IIB fibers have few mitochondria and high glycolytic metabolic capacity. Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), a transcriptional co-activator that regulates mitochondrial biogenesis and respiratory function, is implicated in muscle fiber-type switching. Over-expression of PGC-1α in transgenic mice increased the proportion of red/oxidative type I fiber. During pig muscle growth, an increased number of type I fibers can give meat more red color. To explore the roles of PGC-1α in regulation of muscle fiber type conversion, we generated skeletal muscle-specific PGC-1α transgenic mice and pig. Ectopic over-expression of PGC-1α was detected in both fast and slow muscle fibers. The transgenic animals displayed a remarkable amount of red/oxidative muscle fibers in major skeletal muscle tissues. Skeletal muscles from transgenic mice and pigs have increased expression levels of oxidative fiber markers such as MHC1, MHC2x, myoglobin and Tnni1, and decreased expressions of glycolytic fiber genes (MHC2a, MHC2b, CASQ-1 and Tnni2). The genes responsible for the TCA cycle and oxidative phosphorylation, cytochrome coxidase 2 and 4, and citrate synthase were also increased in the transgenic mice and pigs. These results suggested that transgenic over-expressed PGC-1α significantly increased muscle mitochondrial biogenesis, resulting in qualitative changes from glycolytic to oxidative energy generation. The transgenic animals also had elevated levels of PDK4 and PPARγ proteins in muscle tissue, which can lead to increased glycogen deposition and fatty acid oxidation. Therefore, the results support a significant role of PGC-1α in conversion of fast glycolytic fibers to slow and oxidative fiber through enhanced mitochondrial respiration and fatty acid oxidation, and transgenic over-expression of PGC-1α in skeletal muscle leads to more red meat production in pigs.

Proteomic profiling of brain cortex tissues in a Tau transgenic mouse model of Alzheimer's disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Seong-Hun; Jung, In-Soo; Han, Gi-Yeon

Highlights: Black-Right-Pointing-Pointer A transgenic mouse model expressing NSE-htau23 was used. Black-Right-Pointing-Pointer 2D-gel electrophoresis to analyze the cortex proteins of transgenic mice was used. Black-Right-Pointing-Pointer Differentially expressed spots in different stages of AD were identified. Black-Right-Pointing-Pointer GSTP1 and CAII were downregulated with the progression of AD. Black-Right-Pointing-Pointer SCRN1 and ATP6VE1 were up regulated and down regulated differentially. -- Abstract: Alzheimer's disease (AD) involves regionalized neuronal death, synaptic loss, and an accumulation of intracellular neurofibrillary tangles and extracellular senile plaques. Although there have been numerous studies on tau proteins and AD in various stages of neurodegenerative disease pathology, the relationship between taumore » and AD is not yet fully understood. A transgenic mouse model expressing neuron-specific enolase (NSE)-controlled human wild-type tau (NSE-htau23), which displays some of the typical Alzheimer-associated pathological features, was used to analyze the brain proteome associated with tau tangle deposition. Two-dimensional electrophoresis was performed to compare the cortex proteins of transgenic mice (6- and 12-month-old) with those of control mice. Differentially expressed spots in different stages of AD were identified with ESI-Q-TOF (electrospray ionization quadruple time-of-flight) mass spectrometry and liquid chromatography/tandem mass spectrometry. Among the identified proteins, glutathione S-transferase P 1 (GSTP1) and carbonic anhydrase II (CAII) were down-regulated with the progression of AD, and secerin-1 (SCRN1) and V-type proton ATPase subunit E 1 (ATP6VE1) were up-regulated only in the early stages, and down-regulated in the later stages of AD. The proteins, which were further confirmed by RT-PCR at the mRNA level and with western blotting at the protein level, are expected to be good candidates as drug targets for AD. The study of up- and down-regulation of proteins during the progression of AD helps to explain the mechanisms associated with neuronal degeneration in AD.« less

Bacteriophage 5' untranslated regions for control of plastid transgene expression.

PubMed

Yang, Huijun; Gray, Benjamin N; Ahner, Beth A; Hanson, Maureen R

2013-02-01

Expression of foreign proteins from transgenes incorporated into plastid genomes requires regulatory sequences that can be recognized by the plastid transcription and translation machinery. Translation signals harbored by the 5' untranslated region (UTR) of plastid transcripts can profoundly affect the level of accumulation of proteins expressed from chimeric transgenes. Both endogenous 5' UTRs and the bacteriophage T7 gene 10 (T7g10) 5' UTR have been found to be effective in combination with particular coding regions to mediate high-level expression of foreign proteins. We investigated whether two other bacteriophage 5' UTRs could be utilized in plastid transgenes by fusing them to the aadA (aminoglycoside-3'-adenyltransferase) coding region that is commonly used as a selectable marker in plastid transformation. Transplastomic plants containing either the T7g1.3 or T4g23 5' UTRs fused to Myc-epitope-tagged aadA were successfully obtained, demonstrating the ability of these 5' UTRs to regulate gene expression in plastids. Placing the Thermobifida fusca cel6A gene under the control of the T7g1.3 or T4g23 5' UTRs, along with a tetC downstream box, resulted in poor expression of the cellulase in contrast with high-level accumulation while using the T7g10 5' UTR. However, transplastomic plants with the bacteriophage 5' UTRs controlling the aadA coding region exhibited fewer undesired recombinant species than plants containing the same marker gene regulated by the Nicotiana tabacum psbA 5' UTR. Furthermore, expression of the T7g1.3 and T4g23 5' UTR::aadA fusions downstream of the cel6A gene provided sufficient spectinomycin resistance to allow selection of homoplasmic transgenic plants and had no effect on Cel6A accumulation.

Selective loss of mouse embryos due to the expression of transgenic major histocompatibility class I molecules early in embryogenesis.

PubMed

Aït-Azzouzene, D; Langkopf, A; Cohen, J; Bleux, C; Gendron, M C; Kanellopoulos-Langevin, C

1998-05-01

Among the numerous hypotheses proposed to explain the absence of fetal rejection by the mother in mammals, it has been suggested that regulation of expression of the polymorphic major histocompatibility complex (MHC) at the fetal-maternal interface plays a major role. In addition to a lack of MHC gene expression in the placenta throughout gestation, the absence of polymorphic MHC molecules on the early embryo, as well as their low level of expression after midgestation, could contribute to this important biologic phenomenon. In order to test this hypothesis, we have produced transgenic mice able to express polymorphic MHC class I molecules early in embryogenesis. We have placed the MHC class la gene H-2Kb under the control of a housekeeping gene promoter, the hydroxy-methyl-glutaryl coenzyme A reductase (HMG) gene minimal promoter. This construct has been tested for functionality after transfection into mouse fibroblast L cells. The analysis of three founder transgenic mice and their progeny suggested that fetoplacental units that could express the H-2Kb heavy chains are unable to survive in utero beyond midgestation. We have shown further that a much higher resorption rate, on days 11 to 13 of embryonic development, is observed among transgenic embryos developing from eggs microinjected at the one-cell stage with the pHMG-Kb construct than in control embryos. This lethality is not due to immune phenomena, since it is observed in histocompatible combinations between mother and fetus. These results are discussed in the context of what is currently known about the regulation of MHC expression at the fetal-maternal interface and in various transgenic mouse models.

Studies on the Expression of Sesquiterpene Synthases Using Promoter-β-Glucuronidase Fusions in Transgenic Artemisia annua L

PubMed Central

Wang, Hongzhen; Han, Junli; Kanagarajan, Selvaraju; Lundgren, Anneli; Brodelius, Peter E.

2013-01-01

In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for β-caryophyllene (CPS), epi-cedrol (ECS) and β-farnesene (FS) synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production. PMID:24278301

Co-expression of NCED and ALO improves vitamin C level and tolerance to drought and chilling in transgenic tobacco and stylo plants.

PubMed

Bao, Gegen; Zhuo, Chunliu; Qian, Chunmei; Xiao, Ting; Guo, Zhenfei; Lu, Shaoyun

2016-01-01

Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses, while L-ascorbic acid (AsA) that is also named vitamin C is an important antioxidant and involves in plant stress tolerance and the immune system in domestic animals. Transgenic tobacco (Nicotiana tabacum L.) and stylo [Stylosanthes guianensis (Aublet) Swartz], a forage legume, plants co-expressing stylo 9-cis-epoxycarotenoid dioxygenase (SgNCED1) and yeast D-arabinono-1,4-lactone oxidase (ALO) genes were generated in this study, and tolerance to drought and chilling was analysed in comparison with transgenic tobacco overexpressing SgNCED1 or ALO and the wild-type plants. Compared to the SgNCED1 or ALO transgenic plants, in which only ABA or AsA levels were increased, both ABA and AsA levels were increased in transgenic tobacco and stylo plants co-expressing SgNCED1 and ALO genes. Compared to the wild type, an enhanced drought tolerance was observed in SgNCED1 transgenic tobacco plants with induced expression of drought-responsive genes, but not in ALO plants, while an enhanced chilling tolerance was observed in ALO transgenic tobaccos with induced expression of cold-responsive genes, but not in SgNCED1 plants. Co-expression of SgNCED1 and ALO genes resulted in elevated tolerance to both drought and chilling in transgenic tobacco and stylo plants with induced expression of both drought and cold-responsive genes. Our result suggests that co-expression of SgNCED1 and ALO genes is an effective way for use in forage plant improvement for increased tolerance to drought and chilling and nutrition quality. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

LARGE AREA MONITORING FOR PESTICIDAL TRANSGENIC CROPS: HOW SPECTRAL IMAGING MAY PLAY A ROLE

EPA Science Inventory

Crops genetically engineered to contain a bacterial gene that expresses an insecticidal protein from Bacillus thuringiensis are regulated by EPA under the Federal Insecticide Fungicide and Rodenticide Act (FIFRA). EPA has declared crops containing transgenic pesticidal traits to...

Modification of flower colour by suppressing β-ring carotene hydroxylase genes in Oncidium.

PubMed

Wang, H-M; To, K-Y; Lai, H-M; Jeng, S-T

2016-03-01

Oncidium 'Gower Ramsey' (Onc. GR) is a popular cut flower, but its colour is limited to bright yellow. The β-ring carotene hydroxylase (BCH2) gene is involved in carotenoid biogenesis for pigment formation. However, the role of BCH2 in Onc. GR is poorly understood. Here, we investigated the functions of three BCH2 genes, BCH-A2, BCH-B2 and BCH-C2 isolated from Onc. GR, to analyse their roles in flower colour. RT-PCR expression profiling suggested that BCH2 was mainly expressed in flowers. The expression of BCH-B2 remained constant while that of BCH-A2 gradually decreased during flower development. Using Agrobacterium tumefaciens to introduce BCH2 RNA interference (RNAi), we created transgenic Oncidium plants with down-regulated BCH expression. In the transgenic plants, flower colour changed from the bright yellow of the wild type to light and white-yellow. BCH-A2 and BCH-B2 expression levels were significantly reduced in the transgenic flower lips, which make up the major portion of the Oncidium flower. Sectional magnification of the flower lip showed that the amount of pigmentation in the papillate cells of the adaxial epidermis was proportional to the intensity of yellow colouration. HPLC analyses of the carotenoid composition of the transgenic flowers suggested major reductions in neoxanthin and violaxanthin. In conclusion, BCH2 expression regulated the accumulation of yellow pigments in the Oncidium flower, and the down-regulation of BCH-A2 and BCH-B2 changed the flower colour from bright yellow to light and white-yellow. © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.

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.

Transgenic mouse model harboring the transcriptional fusion ccl20-luciferase as a novel reporter of pro-inflammatory response.

PubMed

Crispo, Martina; Van Maele, Laurye; Tabareau, Julien; Cayet, Delphine; Errea, Agustina; Ferreira, Ana María; Rumbo, Martin; Sirard, Jean Claude

2013-01-01

The chemokine CCL20, the unique ligand of CCR6 functions as an attractant of immune cells. Expression of CCL20 is induced by Toll-like Receptor (TLR) signaling or proinflammatory cytokine stimulation. However CCL20 is also constitutively produced at specific epithelial sites of mucosa. This expression profile is achieved by transcriptional regulation. In the present work we characterized regulatory features of mouse Ccl20 gene. Transcriptional fusions between the mouse Ccl20 promoter and the firefly luciferase (luc) encoding gene were constructed and assessed in in vitro and in vivo assays. We found that liver CCL20 expression and luciferase activity were upregulated by systemic administration of the TLR5 agonist flagellin. Using shRNA and dominant negative form specific for mouse TLR5, we showed that this expression was controlled by TLR5. To address in situ the regulation of gene activity, a transgenic mouse line harboring a functional Ccl20-luc fusion was generated. The luciferase expression was highly concordant with Ccl20 expression in different tissues. Our data indicate that the transgenic mouse model can be used to monitor activation of innate response in vivo.

Transgenic Mouse Model Harboring the Transcriptional Fusion Ccl20-Luciferase as a Novel Reporter of Pro-Inflammatory Response

PubMed Central

Crispo, Martina; Van Maele, Laurye; Tabareau, Julien; Cayet, Delphine; Errea, Agustina; Ferreira, Ana María; Rumbo, Martin; Sirard, Jean Claude

2013-01-01

The chemokine CCL20, the unique ligand of CCR6 functions as an attractant of immune cells. Expression of CCL20 is induced by Toll-like Receptor (TLR) signaling or proinflammatory cytokine stimulation. However CCL20 is also constitutively produced at specific epithelial sites of mucosa. This expression profile is achieved by transcriptional regulation. In the present work we characterized regulatory features of mouse Ccl20 gene. Transcriptional fusions between the mouse Ccl20 promoter and the firefly luciferase (luc) encoding gene were constructed and assessed in in vitro and in vivo assays. We found that liver CCL20 expression and luciferase activity were upregulated by systemic administration of the TLR5 agonist flagellin. Using shRNA and dominant negative form specific for mouse TLR5, we showed that this expression was controlled by TLR5. To address in situ the regulation of gene activity, a transgenic mouse line harboring a functional Ccl20-luc fusion was generated. The luciferase expression was highly concordant with Ccl20 expression in different tissues. Our data indicate that the transgenic mouse model can be used to monitor activation of innate response in vivo. PMID:24265691

Optical modulation of transgene expression in retinal pigment epithelium

NASA Astrophysics Data System (ADS)

Palanker, D.; Lavinsky, D.; Chalberg, T.; Mandel, Y.; Huie, P.; Dalal, R.; Marmor, M.

2013-03-01

Over a million people in US alone are visually impaired due to the neovascular form of age-related macular degeneration (AMD). The current treatment is monthly intravitreal injections of a protein which inhibits Vascular Endothelial Growth Factor, thereby slowing progression of the disease. The immense financial and logistical burden of millions of intravitreal injections signifies an urgent need to develop more long-lasting and cost-effective treatments for this and other retinal diseases. Viral transfection of ocular cells allows creation of a "biofactory" that secretes therapeutic proteins. This technique has been proven successful in non-human primates, and is now being evaluated in clinical trials for wet AMD. However, there is a critical need to down-regulate gene expression in the case of total resolution of retinal condition, or if patient has adverse reaction to the trans-gene products. The site for genetic therapy of AMD and many other retinal diseases is the retinal pigment epithelium (RPE). We developed and tested in pigmented rabbits, an optical method to down-regulate transgene expression in RPE following vector delivery, without retinal damage. Microsecond exposures produced by a rapidly scanning laser vaporize melanosomes and destroy a predetermined fraction of the RPE cells selectively. RPE continuity is restored within days by migration and proliferation of adjacent RPE, but since the transgene is not integrated into the nucleus it is not replicated. Thus, the decrease in transgene expression can be precisely determined by the laser pattern density and further reduced by repeated treatment without affecting retinal structure and function.

ThPP1 gene, encodes an inorganic pyrophosphatase in Thellungiella halophila, enhanced the tolerance of the transgenic rice to alkali stress.

PubMed

He, Rui; Yu, Guohong; Han, Xiaori; Han, Jiao; Li, Wei; Wang, Bing; Huang, Shengcai; Cheng, Xianguo

2017-12-01

An inorganic pyrophosphorylase gene, ThPP1 , modulated the accumulations of phosphate and osmolytes by up-regulating the differentially expression genes, thus enhancing the tolerance of the transgenic rice to alkali stress (AS). Inorganic pyrophosphorylase is essential in catalyzing the hydrolysis of pyrophosphate to inorganic phosphate during plant growth. Here, we report the changes of physiological osmolytes and differentially expression genes in the transgenic rice overexpressing a soluble inorganic pyrophosphatase gene ThPP1 of Thellungiella halophila in response to AS. Analyses showed that the ThPP1 gene was a PPase family I member which is located to the cytoplasm. Data showed that the transgenic lines revealed an enhanced tolerance to AS compared to the wild type, and effectively increased the accumulations of inorganic phosphate and organic small molecules starch, sucrose, proline and chlorophyll, and maintained the balance of osmotic potential by modulating the ratio of Na + /K + in plant cells. Under AS, total 379 of differentially expression genes were up-regulated in the leaves of the transgenic line compared with control, and the enhanced tolerance of the transgenic rice to the AS seemed to be associated with the up-regulations of the osmotic stress-related genes such as the L-type lectin-domain containing receptor kinase (L-type LecRK), the cation/H + antiporter gene and the vacuolar cation/proton exchanger 1 gene (CAX1), which conferred the involvements in the biosynthesis and metabolic pathways. Protein interaction showed that the ThPP1 protein specifically interacted with a 16# target partner of the photosystem II light-harvesting-Chl-binding protein. This study suggested that the ThPP1 gene plays an important regulatory role in conferring the tolerance of the transgenic rice to AS, and is an effective candidate in molecular breeding for crop cultivation of the alkali tolerance.

Expression of OsMYB55 in maize activates stress-responsive genes and enhances heat and drought tolerance.

PubMed

Casaretto, José A; El-Kereamy, Ashraf; Zeng, Bin; Stiegelmeyer, Suzy M; Chen, Xi; Bi, Yong-Mei; Rothstein, Steven J

2016-04-29

Plant response mechanisms to heat and drought stresses have been considered in strategies for generating stress tolerant genotypes, but with limited success. Here, we analyzed the transcriptome and improved tolerance to heat stress and drought of maize plants over-expressing the OsMYB55 gene. Over-expression of OsMYB55 in maize decreased the negative effects of high temperature and drought resulting in improved plant growth and performance under these conditions. This was evidenced by the higher plant biomass and reduced leaf damage exhibited by the transgenic lines compared to wild type when plants were subjected to individual or combined stresses and during or after recovery from stress. A global transcriptomic analysis using RNA sequencing revealed that several genes induced by heat stress in wild type plants are constitutively up-regulated in OsMYB55 transgenic maize. In addition, a significant number of genes up-regulated in OsMYB55 transgenic maize under control or heat treatments have been associated with responses to abiotic stresses including high temperature, dehydration and oxidative stress. The latter is a common and major consequence of imposed heat and drought conditions, suggesting that this altered gene expression may be associated with the improved stress tolerance in these transgenic lines. Functional annotation and enrichment analysis of the transcriptome also pinpoint the relevance of specific biological processes for stress responses. Our results show that expression of OsMYB55 can improve tolerance to heat stress and drought in maize plants. Enhanced expression of stress-associated genes may be involved in OsMYB55-mediated stress tolerance. Possible implications for the improved tolerance to heat stress and drought of OsMYB55 transgenic maize are discussed.

RNAi-mediated down-regulation of SHATTERPROOF gene in transgenic oilseed rape.

PubMed

Kord, Hadis; Shakib, Ali Mohammad; Daneshvar, Mohammad Hossein; Azadi, Pejman; Bayat, Vahid; Mashayekhi, Mohsen; Zarea, Mahboobeh; Seifi, Alireza; Ahmad-Raji, Mana

2015-06-01

Oilseed rape is one of the important oil plants. Pod shattering is one of the problems in oilseed rape production especially in regions with dry conditions. One of the important genes in Brassica pod opening is SHATTERPROOF1 (SHP1). Down-regulation of BnSHP1 expression by RNAi can increase resistance to pod shattering. A 470 bp of the BnSHP1 cDNA sequence constructed in an RNAi-silencing vector was transferred to oilseed rape cv. SLM046. Molecular analysis of T2 transgenic plants by RT-PCR and Real-time PCR showed that expression of the BnSHP alleles was highly decreased in comparison with control plants. Morphologically, transgenic plants were normal and produced seeds at greenhouse conditions. At ripening, stage pods failed to shatter, and a finger pressure was needed for pod opening.

Nogo receptor 1 regulates formation of lasting memories.

PubMed

Karlén, Alexandra; Karlsson, Tobias E; Mattsson, Anna; Lundströmer, Karin; Codeluppi, Simone; Pham, Therese M; Bäckman, Cristina M; Ogren, Sven Ove; Aberg, Elin; Hoffman, Alexander F; Sherling, Michael A; Lupica, Carl R; Hoffer, Barry J; Spenger, Christian; Josephson, Anna; Brené, Stefan; Olson, Lars

2009-12-01

Formation of lasting memories is believed to rely on structural alterations at the synaptic level. We had found that increased neuronal activity down-regulates Nogo receptor-1 (NgR1) in brain regions linked to memory formation and storage, and postulated this to be required for formation of lasting memories. We now show that mice with inducible overexpression of NgR1 in forebrain neurons have normal long-term potentiation and normal 24-h memory, but severely impaired month-long memory in both passive avoidance and swim maze tests. Blocking transgene expression normalizes these memory impairments. Nogo, Lingo-1, Troy, endogenous NgR1, and BDNF mRNA expression levels were not altered by transgene expression, suggesting that the impaired ability to form lasting memories is directly coupled to inability to down-regulate NgR1. Regulation of NgR1 may therefore serve as a key regulator of memory consolidation. Understanding the molecular underpinnings of synaptic rearrangements that carry lasting memories may facilitate development of treatments for memory dysfunction.

Nogo receptor 1 regulates formation of lasting memories

PubMed Central

Karlén, Alexandra; Karlsson, Tobias E.; Mattsson, Anna; Lundströmer, Karin; Codeluppi, Simone; Pham, Therese M.; Bäckman, Cristina M.; Ögren, Sven Ove; Åberg, Elin; Hoffman, Alexander F.; Sherling, Michael A.; Lupica, Carl R.; Hoffer, Barry J.; Spenger, Christian; Josephson, Anna; Brené, Stefan; Olson, Lars

2009-01-01

Formation of lasting memories is believed to rely on structural alterations at the synaptic level. We had found that increased neuronal activity down-regulates Nogo receptor-1 (NgR1) in brain regions linked to memory formation and storage, and postulated this to be required for formation of lasting memories. We now show that mice with inducible overexpression of NgR1 in forebrain neurons have normal long-term potentiation and normal 24-h memory, but severely impaired month-long memory in both passive avoidance and swim maze tests. Blocking transgene expression normalizes these memory impairments. Nogo, Lingo-1, Troy, endogenous NgR1, and BDNF mRNA expression levels were not altered by transgene expression, suggesting that the impaired ability to form lasting memories is directly coupled to inability to down-regulate NgR1. Regulation of NgR1 may therefore serve as a key regulator of memory consolidation. Understanding the molecular underpinnings of synaptic rearrangements that carry lasting memories may facilitate development of treatments for memory dysfunction. PMID:19915139

Arm-Gal4 inheritance influences development and lifespan in Drosophila melanogaster.

PubMed

Slade, F A; Staveley, B E

2015-10-19

The UAS-Gal4 ectopic expression system is a widely used and highly valued tool that allows specific gene expression in Drosophila melanogaster. Yeast transcription factor Gal4 can be directed using D. melanogaster transcriptional control elements, and is often assumed to have little effect on the organism. By evaluation of the consequences of maternal and paternal inheritance of a Gal4 transgene under the transcriptional regulation of armadillo control elements (arm-Gal4), we demonstrated that Gal4 expression could be detrimental to development and longevity. Male progeny expressing arm-Gal4 in the presence of UAS-lacZ transgene had reduced numbers and size of ommatidia, compared to flies expressing UAS-lacZ transgene under the control of other Gal4 transgenes. Aged at 25°C, the median life span of male flies with maternally inherited elav-Gal4 was 70 days, without a responding transgene or with UAS-lacZ. The median life span of maternally inherited arm-Gal4 male flies without a responding transgene was 48 days, and 40 days with the UAS-lacZ transgene. A partial rescue of this phenotype was observed with the expression of UAS-lacZ under paternal arm-Gal4 control, having an average median lifespan of 60 days. This data suggests that arm-Gal4 has detrimental effects on Drosophila development and lifespan that are directly dependent upon parental inheritance, and that the benign responder and reporter gene UAS-lacZ may influence D. melanogaster development. These findings should be taken into consideration during the design and execution of UAS-Gal4 expression experiments.

Chickpea WRKY70 Regulates the Expression of a Homeodomain-Leucine Zipper (HD-Zip) I Transcription Factor CaHDZ12, which Confers Abiotic Stress Tolerance in Transgenic Tobacco and Chickpea.

PubMed

Sen, Senjuti; Chakraborty, Joydeep; Ghosh, Prithwi; Basu, Debabrata; Das, Sampa

2017-11-01

Drought and salinity are the two major environmental constraints that severely affect global agricultural productivity. Plant-specific HD-Zip transcription factors are involved in plant growth, development and stress responses. In the present study, we explored the functional characteristics and regulation of a novel HD-Zip (I) gene from chickpea, CaHDZ12, in response to water-deficit and salt-stress conditions. Transgenic tobacco lines over-expressing CaHDZ12 exhibited improved tolerance to osmotic stresses and increased sensitivity to abscisic acid (ABA). Physiological compatibility of transgenic lines was found to be more robust compared to the wild-type plants under drought and salinity stress. Additionally, expression of several stress-responsive genes was significantly induced in CaHDZ12 transgenic plants. On the other hand, silencing of CaHDZ12 in chickpea resulted in increased sensitivity to salt and drought stresses. Analysis of different promoter deletion mutants identified CaWRKY70 transcription factor as a transcriptional regulator of CaHDZ12 expression. In vivo and in vitro interaction studies detected an association between CaWRKY70 and CaHDZ12 promoter during stress responses. Epigenetic modifications underlying histone acetylation at the CaHDZ12 promoter region play a significant role in stress-induced activation of this gene. Collectively, our study describes a crucial and unique mechanistic link between two distinct transcription factors in regulating plant adaptive stress response. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

Overexpression of Arabidopsis AnnAt8 Alleviates Abiotic Stress in Transgenic Arabidopsis and Tobacco

PubMed Central

Yadav, Deepanker; Ahmed, Israr; Shukla, Pawan; Boyidi, Prasanna; Kirti, Pulugurtha Bharadwaja

2016-01-01

Abiotic stress results in massive loss of crop productivity throughout the world. Because of our limited knowledge of the plant defense mechanisms, it is very difficult to exploit the plant genetic resources for manipulation of traits that could benefit multiple stress tolerance in plants. To achieve this, we need a deeper understanding of the plant gene regulatory mechanisms involved in stress responses. Understanding the roles of different members of plant gene families involved in different stress responses, would be a step in this direction. Arabidopsis, which served as a model system for the plant research, is also the most suitable system for the functional characterization of plant gene families. Annexin family in Arabidopsis also is one gene family which has not been fully explored. Eight annexin genes have been reported in the genome of Arabidopsis thaliana. Expression studies of different Arabidopsis annexins revealed their differential regulation under various abiotic stress conditions. AnnAt8 (At5g12380), a member of this family has been shown to exhibit ~433 and ~175 fold increase in transcript levels under NaCl and dehydration stress respectively. To characterize Annexin8 (AnnAt8) further, we have generated transgenic Arabidopsis and tobacco plants constitutively expressing AnnAt8, which were evaluated under different abiotic stress conditions. AnnAt8 overexpressing transgenic plants exhibited higher seed germination rates, better plant growth, and higher chlorophyll retention when compared to wild type plants under abiotic stress treatments. Under stress conditions transgenic plants showed comparatively higher levels of proline and lower levels of malondialdehyde compared to the wild-type plants. Real-Time PCR analyses revealed that the expression of several stress-regulated genes was altered in AnnAt8 over-expressing transgenic tobacco plants, and the enhanced tolerance exhibited by the transgenic plants can be correlated with altered expressions of these stress-regulated genes. Our findings suggest a role for AnnAt8 in enhancing abiotic stress tolerance at different stages of plant growth and development. PMID:27135239

Overexpression of Brassica rapa SHI-RELATED SEQUENCE genes suppresses growth and development in Arabidopsis thaliana.

PubMed

Hong, Joon Ki; Kim, Jin A; Kim, Jung Sun; Lee, Soo In; Koo, Bon Sung; Lee, Yeon-Hee

2012-08-01

S HI-R ELATED SEQUENCE (SRS) genes are plant-specific transcription factors containing a zinc-binding RING finger motif, which play a critical role in plant growth and development. We have characterized six SRS genes in Brassica rapa. Overexpression of the SRSs BrSTY1, BrSRS7, and BrLRP1 induced dwarf and compact plants, and significantly decreased primary root elongation and lateral root formation. Additionally, the transgenic plants had upward-curled leaves of narrow widths and with short petioles, and had shorter siliques and low fertility. In stems, hypocotyls, and styles, epidermal cell lengths were also significantly reduced in transgenic plants. RT-PCR analysis of transgenic plants revealed that BrSTY1, BrSRS7, and BrLRP1 regulate expression of several gibberellin (GA)- and auxin-related genes involved in morphogenesis in shoot apical regions. We conclude that BrSTY1, BrSRS7, and BrLRP1 regulate plant growth and development by regulating expression of GA- and auxin-related genes.

A 3′-Untranslated Region (3′UTR) Induces Organ Adhesion by Regulating miR-199a* Functions

PubMed Central

Lee, Daniel Y.; Shatseva, Tatiana; Jeyapalan, Zina; Du, William W.; Deng, Zhaoqun; Yang, Burton B.

2009-01-01

Mature microRNAs (miRNAs) are single-stranded RNAs of 18–24 nucleotides that repress post-transcriptional gene expression. However, it is unknown whether the functions of mature miRNAs can be regulated. Here we report that expression of versican 3′UTR induces organ adhesion in transgenic mice by modulating miR-199a* activities. The study was initiated by the hypothesis that the non-coding 3′UTR plays a role in the regulation of miRNA function. Transgenic mice expressing a construct harboring the 3′UTR of versican exhibits the adhesion of organs. Computational analysis indicated that a large number of microRNAs could bind to this fragment potentially including miR-199a*. Expression of versican and fibronectin, two targets of miR-199a*, are up-regulated in transgenic mice, suggesting that the 3′UTR binds and modulates miR-199a* activities, freeing mRNAs of versican and fibronectin from being repressed by miR-199a*. Confirmation of the binding was performed by PCR using mature miR-199a* as a primer and the targeting was performed by luciferase assays. Enhanced adhesion by expression of the 3′UTR was confirmed by in vitro assays. Our results demonstrated that upon arrival in cytoplasm, miRNA activities can be modulated locally by the 3′UTR. Our assay may be developed as sophisticated approaches for studying the mutual regulation of miRNAs and mRNAs in vitro and in vivo. We anticipate that expression of the 3′UTR may be an approach in the development of gene therapy. PMID:19223980

Functional repression of PtSND2 represses growth and development by disturbing auxin biosynthesis, transport and signaling in transgenic poplar.

PubMed

Wang, Haihai; Tang, Renjie; Wang, Cuiting; Qi, Qi; Gai, Ying; Jiang, Xiangning; Zhang, Hongxia

2015-01-01

Using chimeric repressor silencing technology, we previously reported that functional repression of PtSND2 severely arrested wood formation in transgenic poplar (Populus). Here, we provide further evidence that auxin biosynthesis, transport and signaling were disturbed in these transgenic plants, leading to pleiotropic defects in their growth patterns, including inhibited leaf enlargement and vascular tissue development in the leaf central vein, suppressed cambial growth and fiber elongation in the stem, and arrested growth in the root system. Two transgenic lines, which displayed the most remarkable phenotypic deviation from the wild-type, were selected for detailed studies. In both transgenic lines, expression of genes for auxin biosynthesis, transport and signaling was down-regulated, and indole-3-acetic acid distribution was severely disturbed in the apical buds, leaves, stems and roots of field-grown transgenic plants. Transient transcription dual-luciferase assays of ProPtTYDC2::LUC, ProPttLAX2::LUC and ProPoptrIAA20.2::LUC in poplar protoplasts revealed that expression of auxin-related genes might be regulated by PtSND2 at the transcriptional level. All these results indicate that functional repression of PtSND2 altered auxin biosynthesis, transport and signaling, and thereby disturbed the normal growth and development of transgenic plants. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

CREB activity in dopamine D1 receptor expressing neurons regulates cocaine-induced behavioral effects

PubMed Central

Bilbao, Ainhoa; Rieker, Claus; Cannella, Nazzareno; Parlato, Rosanna; Golda, Slawomir; Piechota, Marcin; Korostynski, Michal; Engblom, David; Przewlocki, Ryszard; Schütz, Günther; Spanagel, Rainer; Parkitna, Jan R.

2014-01-01

It is suggested that striatal cAMP responsive element binding protein (CREB) regulates sensitivity to psychostimulants. To test the cell-specificity of this hypothesis we examined the effects of a dominant-negative CREB protein variant expressed in dopamine receptor D1 (D1R) neurons on cocaine-induced behaviors. A transgenic mouse strain was generated by pronuclear injection of a BAC-derived transgene harboring the A-CREB sequence under the control of the D1R gene promoter. Compared to wild-type, drug-naïve mutants showed moderate alterations in gene expression, especially a reduction in basal levels of activity-regulated transcripts such as Arc and Egr2. The behavioral responses to cocaine were elevated in mutant mice. Locomotor activity after acute treatment, psychomotor sensitization after intermittent drug injections and the conditioned locomotion after saline treatment were increased compared to wild-type littermates. Transgenic mice had significantly higher cocaine conditioned place preference, displayed normal extinction of the conditioned preference, but showed an augmented cocaine-seeking response following priming-induced reinstatement. This enhanced cocaine-seeking response was associated with increased levels of activity-regulated transcripts and prodynorphin. The primary reinforcing effects of cocaine were not altered in the mutant mice as they did not differ from wild-type in cocaine self-administration under a fixed ratio schedule at the training dose. Collectively, our data indicate that expression of a dominant-negative CREB variant exclusively in neurons expressing D1R is sufficient to recapitulate the previously reported behavioral phenotypes associated with virally expressed dominant-negative CREB. PMID:24966820

Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane

PubMed Central

2014-01-01

Background Down-regulation or silencing of transgene expression can be a major hurdle to both molecular studies and biotechnology applications in many plant species. Sugarcane is particularly effective at silencing introduced transgenes, including reporter genes such as the firefly luciferase gene. Synthesizing transgene coding sequences optimized for usage in the host plant is one method of enhancing transgene expression and stability. Using specified design rules we have synthesised new coding sequences for both the firefly luciferase and Renilla luciferase reporter genes. We have tested these optimized versions for enhanced levels of luciferase activity and for increased steady state luciferase mRNA levels in sugarcane. Results The synthetic firefly luciferase (luc*) and Renilla luciferase (Renluc*) coding sequences have elevated G + C contents in line with sugarcane codon usage, but maintain 75% identity to the native firefly or Renilla luciferase nucleotide sequences and 100% identity to the protein coding sequences. Under the control of the maize pUbi promoter, the synthetic luc* and Renluc* genes yielded 60x and 15x higher luciferase activity respectively, over the native firefly and Renilla luciferase genes in transient assays on sugarcane suspension cell cultures. Using a novel transient assay in sugarcane suspension cells combining co-bombardment and qRT-PCR, we showed that synthetic luc* and Renluc* genes generate increased transcript levels compared to the native firefly and Renilla luciferase genes. In stable transgenic lines, the luc* transgene generated significantly higher levels of expression than the native firefly luciferase transgene. The fold difference in expression was highest in the youngest tissues. Conclusions We developed synthetic versions of both the firefly and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. These transgenes will be particularly useful for evaluating the expression patterns conferred by existing and newly isolated promoters in sugarcane tissues. The strategies used to design the synthetic luciferase transgenes could be applied to other transgenes that are aggressively silenced in sugarcane. PMID:24708613

Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane.

PubMed

Chou, Ting-Chun; Moyle, Richard L

2014-04-08

Down-regulation or silencing of transgene expression can be a major hurdle to both molecular studies and biotechnology applications in many plant species. Sugarcane is particularly effective at silencing introduced transgenes, including reporter genes such as the firefly luciferase gene.Synthesizing transgene coding sequences optimized for usage in the host plant is one method of enhancing transgene expression and stability. Using specified design rules we have synthesised new coding sequences for both the firefly luciferase and Renilla luciferase reporter genes. We have tested these optimized versions for enhanced levels of luciferase activity and for increased steady state luciferase mRNA levels in sugarcane. The synthetic firefly luciferase (luc*) and Renilla luciferase (Renluc*) coding sequences have elevated G + C contents in line with sugarcane codon usage, but maintain 75% identity to the native firefly or Renilla luciferase nucleotide sequences and 100% identity to the protein coding sequences.Under the control of the maize pUbi promoter, the synthetic luc* and Renluc* genes yielded 60x and 15x higher luciferase activity respectively, over the native firefly and Renilla luciferase genes in transient assays on sugarcane suspension cell cultures.Using a novel transient assay in sugarcane suspension cells combining co-bombardment and qRT-PCR, we showed that synthetic luc* and Renluc* genes generate increased transcript levels compared to the native firefly and Renilla luciferase genes.In stable transgenic lines, the luc* transgene generated significantly higher levels of expression than the native firefly luciferase transgene. The fold difference in expression was highest in the youngest tissues. We developed synthetic versions of both the firefly and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. These transgenes will be particularly useful for evaluating the expression patterns conferred by existing and newly isolated promoters in sugarcane tissues. The strategies used to design the synthetic luciferase transgenes could be applied to other transgenes that are aggressively silenced in sugarcane.

Expression of grape ACS1 in tomato decreases ethylene and alters the balance between auxin and ethylene during shoot and root formation.

PubMed

Ye, Xia; Fu, Mengmeng; Liu, Yu; An, Dongliang; Zheng, Xianbo; Tan, Bin; Li, Jidong; Cheng, Jun; Wang, Wei; Feng, Jiancan

2018-05-04

Ethylene plays an important role in the grape rachis, where its production can be 10 times higher than in the berry. VvACS1 is the only rachis-specific ACC synthase (ACS) gene, and its expression is coincident with ethylene production in the rachis of Vitis vinifera 'Thompson seedless'. VvACS1 was cloned and ectopically expressed in tomato (Solanum lycopersicum 'Moneymaker'). Lateral buds were increased in two- or four-week-old 35s∷VvACS1 transgenic tomato plants after transplanting. Compared with wild-type (WT) plants, the transgenic tomato plants showed higher expression of the VvACS1 gene in the flowers, leaves, rachis, and fruits. There was no obvious difference of ACS activity in the fruit of tomato, and only increased ACS activity in the rachis of tomato. Ethylene production was decreased in flowers, leaves, and fruits (seven weeks after full bloom), while the relative expression of endogenous tomato ACS1 and ACS6 genes was not down-regulated by the ectopic expression of VvACS1. These results imply that post-transcriptional or post-translational regulation of ACS may occur, resulting in lower ethylene production in the transgenic tomato plants. Moreover, expression of VvACS1 in tomato resulted in decreased auxin and increased zeatin contents in the lateral buds, as well as reduced or delayed formation of adventitious roots in lateral bud cuttings. RNA-Seq and qRT-PCR analyses of rooted lateral bud cuttings indicated that the relative expression levels of the genes for zeatin O-glucosyltransferase-like, auxin repressed/dormancy-associated protein, and ERF transcription factors were higher in transgenic tomatoes than in WT, suggesting that ethylene may regulate auxin transport and distribution in shoots and that adventitious root formation employs coordination between auxin and ethylene. Copyright © 2018 Elsevier GmbH. All rights reserved.

A novel model for development, organization, and function of gonadotropes in fish pituitary.

PubMed

Golan, Matan; Biran, Jakob; Levavi-Sivan, Berta

2014-01-01

The gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are key regulators of the reproductive axis in vertebrates. Despite the high popularity of zebrafish as a model organism for studying reproductive functions, to date no transgenic zebrafish with labeled gonadotropes have been introduced. Using gonadotropin regulatory elements from tilapia, we generated two transgenic zebrafish lines with labeled gonadotropes. The tilapia and zebrafish regulatory sequences were highly divergent but several conserved elements allowed the tilapia promoters to correctly drive the transgenes in zebrafish pituitaries. FSH cells reacted to stimulation with gonadotropin releasing hormone by proliferating and showing increased transgene fluorescence, whereas estrogen exposure caused a decrease in cell number and transgene fluorescence. Transgene fluorescence reflected the expression pattern of the endogenous fshb gene. Ontogenetic expression of the transgenes followed typical patterns, with FSH cells appearing early in development, and LH cells appearing later and increasing dramatically in number with the onset of puberty. Our transgenic lines provide a powerful tool for investigating the development, anatomy, and function of the reproductive axis in lower vertebrates.

The homeodomain transcription factor Cdx1 does not behave as an oncogene in normal mouse intestine.

PubMed

Crissey, Mary Ann S; Guo, Rong-Jun; Fogt, Franz; Li, Hong; Katz, Jonathan P; Silberg, Debra G; Suh, Eun Ran; Lynch, John P

2008-01-01

The Caudal-related homeobox genes Cdx1 and Cdx2 are intestine-specific transcription factors that regulate differentiation of intestinal cell types. Previously, we have shown Cdx1 to be antiproliferative and to promote cell differentiation. However, other studies have suggested that Cdx1 may be an oncogene. To test for oncogenic behavior, we used the murine villin promoter to ectopically express Cdx1 in the small intestinal villi and colonic surface epithelium. No changes in intestinal architecture, cell differentiation, or lineage selection were observed with expression of the transgene. Classic oncogenes enhance proliferation and induce tumors when ectopically expressed. However, the Cdx1 transgene neither altered intestinal proliferation nor induced spontaneous intestinal tumors. In a murine model for colitis-associated cancer, the Cdx1 transgene decreased, rather than increased, the number of adenomas that developed. In the polyps, the expression of the endogenous and the transgenic Cdx1 proteins was largely absent, whereas endogenous Villin expression was retained. This suggests that transgene silencing was specific and not due to a general Villin inactivation. In conclusion, neither the ectopic expression of Cdx1 was associated with changes in intestinal cell proliferation or differentiation nor was there increased intestinal cancer susceptibility. Our results therefore suggest that Cdx1 is not an oncogene in normal intestinal epithelium.

Targeting expression of a transforming growth factor beta 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation.

PubMed Central

Jhappan, C; Geiser, A G; Kordon, E C; Bagheri, D; Hennighausen, L; Roberts, A B; Smith, G H; Merlino, G

1993-01-01

Transforming growth factor-beta 1 (TGF-beta 1) possesses highly potent, diverse and often opposing cell-specific activities, and has been implicated in the regulation of a variety of physiologic and developmental processes. To determine the effects of in vivo overexpression of TGF-beta 1 on mammary gland function, transgenic mice were generated harboring a fusion gene consisting of the porcine TGF-beta 1 cDNA placed under the control of regulatory elements of the pregnancy-responsive mouse whey-acidic protein (WAP) gene. Females from two of four transgenic lines were unable to lactate due to inhibition of the formation of lobuloalveolar structures and suppression of production of endogenous milk protein. In contrast, ductal development of the mammary glands was not overtly impaired. There was a complete concordance in transgenic mice between manifestation of the lactation-deficient phenotype and expression of RNA from the WAP/TGF-beta 1 transgene, which was present at low levels in the virgin gland, but was greatly induced at mid-pregnancy. TGF-beta 1 was localized to numerous alveoli and to the periductal extracellular matrix in the mammary gland of transgenic females late in pregnancy by immunohistochemical analysis. Glands reconstituted from cultured transgenic mammary epithelial cells duplicated the inhibition of lobuloalveolar development observed in situ in the mammary glands of pregnant transgenic mice. Results from this transgenic model strongly support the hypothesis that TGF-beta 1 plays an important in vivo role in regulating the development and function of the mammary gland. Images PMID:8491177

Creating Transgenic shRNA Mice by Recombinase-Mediated Cassette Exchange

PubMed Central

Premsrirut, Prem K.; Dow, Lukas E.; Park, Youngkyu; Hannon, Gregory J.; Lowe, Scott W.

2014-01-01

RNA interference (RNAi) enables sequence-specific, experimentally induced silencing of virtually any gene by tapping into innate regulatory mechanisms that are conserved among most eukaryotes. The principles that enable transgenic RNAi in cell lines can also be used to create transgenic animals, which express short-hairpin RNAs (shRNAs) in a regulated or tissue-specific fashion. However, RNAi in transgenic animals is somewhat more challenging than RNAi in cultured cells. The activities of promoters that are commonly used for shRNA expression in cell culture can vary enormously in different tissues, and founder lines also typically vary in transgene expression due to the effects of their single integration sites. There are many ways to produce mice carrying shRNA transgenes and the method described here uses recombinase-mediated cassette exchange (RMCE). RMCE permits insertion of the shRNA transgene into a well-characterized locus that gives reproducible and predictable expression in each founder and enhances the probability of potent expression in many cell types. This procedure is more involved and complex than simple pronuclear injection, but if even a few shRNA mice are envisioned, for example, to probe the functions of several genes, the effort of setting up the processes outlined below are well worthwhile. Note that when creating a transgenic mouse, one should take care to use the most potent shRNA possible. As a rule of thumb, the sequence chosen should provide >90% knockdown when introduced into cultured cells at single copy (e.g., on retroviral infection at a multiplicity of ≤0.3). PMID:24003198

OsDREB2A, a Rice Transcription Factor, Significantly Affects Salt Tolerance in Transgenic Soybean

PubMed Central

Ma, Qi-bin; Yang, Cun-yi; Mu, Ying-hui; Suo, Hai-cui; Luo, Lai-hui; Nian, Hai

2013-01-01

The dehydration responsive element binding (DREB) transcription factors play an important role in regulating stress-related genes. OsDREB2A, a member of the DREBP subfamily of AP2/ERF transcription factors in rice (Oryza sativa), is involved in the abiotic stress response. OsDREB2A expression is induced by drought, low-temperature and salt stresses. Here, we report the ability of OsDREB2A to regulate high-salt response in transgenic soybean. Overexpressing OsDREB2A in soybeans enhanced salt tolerance by accumulating osmolytes, such as soluble sugars and free proline, and improving the expression levels of some stress-responsive transcription factors and key genes. The phenotypic characterization of transgenic soybean were significantly better than those of wild-type (WT). Electrophoresis mobility shift assay (EMSA) revealed that the OsDREB2A can bind to the DRE core element in vitro. These results indicate that OsDREB2A may participate in abiotic stress by directly binding with DRE element to regulate the expression of downstream genes. Overexpression of OsDREB2A in soybean might be used to improve tolerance to salt stress. PMID:24376625

Liver tumor formation by a mutant retinoblastoma protein in the transgenic mice is caused by an upregulation of c-Myc target genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Bo; Hikosaka, Keisuke; Sultana, Nishat

2012-01-06

Highlights: Black-Right-Pointing-Pointer Fifty percent of the mutant Rb transgenic mice produced liver tumors. Black-Right-Pointing-Pointer In the tumor, Foxm1, Skp2, Bmi1 and AP-1 mRNAs were up-regulated. Black-Right-Pointing-Pointer No increase in expression of the Myc-target genes was observed in the non-tumorous liver. Black-Right-Pointing-Pointer Tumor formation depends on up-regulation of the Myc-target genes. -- Abstract: The retinoblastoma (Rb) tumor suppressor encodes a nuclear phosphoprotein that regulates cellular proliferation, apoptosis and differentiation. In order to adapt itself to these biological functions, Rb is subjected to modification cycle, phosphorylation and dephosphorylation. To directly determine the effect of phosphorylation-resistant Rb on liver development and function, wemore » generated transgenic mice expressing phosphorylation-resistant human mutant Rb (mt-Rb) under the control of the rat hepatocyte nuclear factor-1 gene promoter/enhancer. Expression of mt-Rb in the liver resulted in macroscopic neoplastic nodules (adenomas) with {approx}50% incidence within 15 months old. Interestingly, quantitative reverse transcriptase-PCR analysis showed that c-Myc was up-regulated in the liver of mt-Rb transgenic mice irrespective of having tumor tissues or no tumor. In tumor tissues, several c-Myc target genes, Foxm1, c-Jun, c-Fos, Bmi1 and Skp2, were also up-regulated dramatically. We determined whether mt-Rb activated the Myc promoter in the HTP9 cells and demonstrated that mt-Rb acted as an inhibitor of wild-type Rb-induced repression on the Myc promoter. Our results suggest that continued upregulation of c-Myc target genes promotes the liver tumor formation after about 1 year of age.« less

Transgenic Muscle-Specific Nor-1 Expression Regulates Multiple Pathways That Effect Adiposity, Metabolism, and Endurance

PubMed Central

Pearen, Michael A.; Goode, Joel M.; Fitzsimmons, Rebecca L.; Eriksson, Natalie A.; Thomas, Gethin P.; Cowin, Gary J.; Wang, S.-C. Mary; Tuong, Zewen K.

2013-01-01

The mRNA encoding Nor-1/NR4A3 is rapidly and strikingly induced by β2-adrenergic signaling in glycolytic and oxidative skeletal muscle. In skeletal muscle cells, Nor-1 expression is important for the regulation of oxidative metabolism. Transgenic skeletal muscle-specific expression of activated Nor-1 resulted in the acquisition of an endurance phenotype, an increase in type IIA/X oxidative muscle fibers, and increased numbers of mitochondria. In the current study, we used dual-energy x-ray absorptiometry and magnetic resonance imaging analysis to demonstrate decreased adiposity in transgenic (Tg) Nor-1 mice relative to that in wild-type littermates. Furthermore, the Tg-Nor-1 mice were resistant to diet-induced weight gain and maintained fasting glucose at normoglycemic levels. Expression profiling and RT-quantitative PCR analysis revealed significant increases in genes involved in glycolysis, the tricarboxylic acid cycle, oxidative phosphorylation, fatty acid oxidation, and glycogen synthesis, in concordance with the lean phenotype. Moreover, expression profiling identified several Z-disc and sarcomeric binding proteins that modulate fiber type phenotype and endurance, eg, α-actinin-3. In addition, we demonstrated that the Tg-Nor-1 mouse line has significantly higher glycogen content in skeletal muscle relative to that in wild-type littermates. Finally, we identified a decreased NAD+/NADH ratio with a concordant increase in peroxisome proliferator-activated receptor γ coactivator-1α1 protein/mRNA expression. Increased NADH was associated with an induction of the genes involved in the malate-aspartate shuttle and a decrease in the glycerol 3-phosphate shuttle, which maximizes aerobic ATP production. In conclusion, skeletal muscle-specific Nor-1 expression regulates genes and pathways that regulate adiposity, muscle fiber type metabolic capacity, and endurance. PMID:24065705

Regulated expression of the Ren-2 gene in transgenic mice derived from parental strains carrying only the Ren-1 gene.

PubMed Central

Tronik, D; Dreyfus, M; Babinet, C; Rougeon, F

1987-01-01

The Ren-2 gene encoding the mouse submaxillary gland (SMG) renin was microinjected into the pronuclei of fertilized eggs from mice carrying only the Ren-1 gene. In addition to the whole transcription unit, the injected DNA contained 2.5 and 3 kb of upstream and downstream flanking sequences, respectively. Three independent transgenic mice lines were obtained; two of them had integrated one copy of the Ren-2 gene, the last one had integrated five and eleven copies at two independent sites. Independently of the number of Ren-2 copies integrated, the pattern of Ren-2 gene expression in all the transgenic mice was identical to that observed in wild-type animals in which Ren-1 and Ren-2 are closely linked on chromosome 1. In particular, the exogenous Ren-2 gene was only transcribed in the kidney and in the SMG. In the kidney, Ren-1 and Ren-2 mRNAs were present at a comparable level, whereas in the SMG Ren-2 mRNA was at least 100-fold more abundant than Ren-1 mRNA. Moreover, Ren-2 expression in the SMG was positively regulated by androgens. Only one difference between transgenic mice and wild-type mice carrying the Ren-2 gene has been observed: the basal level of Ren-2 transcription in the SMG of transgenic females was lower than in two-gene strain females. Androgen treatment of transgenic females induced SMG renin mRNA to a level identical to that of transgenic males. This suggests that the basal level of SMG renin mRNA is dependent upon cis-acting elements which are not present in the microinjected fragment. Images Fig. 1. Fig. 2. Fig. 3. PMID:3297677

Hepatitis B surface antigen gene expression is regulated by sex steroids and glucocorticoids in transgenic mice.

PubMed Central

Farza, H; Salmon, A M; Hadchouel, M; Moreau, J L; Babinet, C; Tiollais, P; Pourcel, C

1987-01-01

We have investigated the basis for liver-specific and sex-linked expression of hepatitis B surface antigen (HBsAg) gene in transgenic mice by monitoring the level of liver HBsAg mRNA and serum HBsAg at different stages of development and in response to sex-hormone regulation. Transcription of the HBsAg gene starts at day 15 of development, together with that of the albumin gene, and reaches a comparable level at birth. HBsAg mRNA level and HBsAg production are parallel in males and females during prenatal development and until the first month of life, but HBsAg gene expression increases 5-10 times in males at puberty. After castration, the level of expression decreases dramatically in both males and females and is subsequently increased by injection of testosterone or estradiol. Glucocorticoids also regulated positively expression of the HBsAg gene. Our results suggest that sex hormones play a role in hepatitis B virus gene expression during natural infection and could explain the difference in incidence of chronic carriers between men and women. Images PMID:3469661

CTCF Mediates Effect of Insulin On Glucagon Expression

PubMed Central

Tsui, Shanli; Gao, Jie; Wang, Charles; Lu, Luo

2013-01-01

Pancreatic islet α-cell development and glucagon production are mainly regulated by Pax6 in the homeobox gene families. However, the molecular mechanism fine-tuning the regulation of these events in α-cell still remains unclear. In ocular cells, Pax6 transcription is regulated by CTCF through its binding to specific sites in Pax6 promoter. In this study, CTCF-mediated regulations of islet α-cell development and glucagon production were investigated in both CTCF transgenic mice and α-TC-1-6 cells. Over-expression of CTCF in transgenic mice affected development of pancreatic islets by significantly suppressing α-cell population in both embryonic and adult pancreases. The effect of CTCF on Pax6 gene expression and subsequently, on pro-glucagon production was however, examined in pancreatic islet α-cells. Over-expression and knock-down of CTCF directly affected Pax6 expression. More importantly, the CTCF binding sites upstream from Pax6 p0 promoter were required for regulating p0 promoter activity in islet α-cells. Stimulation of α-cells with insulin resulted in a significant increase in CTCF expression and a decrease in Pax6 expression, and consequently suppressed pro-glucagon expression. In contrast, these insulin-induced effects were blocked by knockdown of CTCF mRNA with specific siRNA in α-cells. Altogether, our results demonstrated for the first time that CTCF functions as a switch-like molecule between the insulin signaling and the regulations of Pax6 and glucagon expression in pancreatic islet α-cells. PMID:22426149

Expression of Folate Pathway Genes in the Cartilage of Hoxd4 and Hoxc8 Transgenic Mice

PubMed Central

Kruger, Claudia; Talmadge, Catherine; Kappen, Claudia

2014-01-01

BACKGROUND Hox transcription factors are well known for their role in skeletal patterning in vertebrates. They regulate gene expression during the development of cartilage, the precursor to mature bone. We previously reported that overexpression of the homeobox genes Hoxc8 and Hoxd4 results in severe cartilage defects, reduced proteoglycan content, accumulation of immature chondrocytes, and decreased maturation to hypertrophy. We have also shown that Hoxd4 transgenic mice whose diets were supplemented with folate had their skeletal development restored. Since folate is required for growth and differentiation of chondrocytes, we hypothesized that the beneficial effect of folate in Hoxd4 transgenic mice might indicate a local deficiency in folate utilization, possibly caused by deregulation of genes encoding folate transport proteins or folate metabolic enzymes. METHODS We assayed the prevalence of transcripts for 22 folate transport proteins and metabolizing enzymes, here collectively referred to as folate pathway genes. Quantitative real-time PCR was performed on cDNA samples derived from RNA isolated from primary chondrocytes of individual rib cartilages from Hoxd4 and Hoxc8 transgenic mice, respectively. RESULTS This study shows that the Hox transgenes produce overexpression of Hoxd4 and Hoxc8 in primary chondrocytes from perinatal transgenic mice. However, no differences were found in expression levels of the folate pathway genes in transgenic cells compared to littermate controls. CONCLUSIONS Our results provide evidence that folate pathway genes are only indirect targets of Hox transgene overexpression in our transgenic animals. These expression studies provide a baseline for future studies into the role of folate metabolism in chondrocyte differentiation. PMID:16586448

Polycythemia in transgenic mice expressing the human erythropoietin gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Semenza, G.L.; Traystman, M.D.; Gearhart, J.D.

1989-04-01

Erythropoietin is a glycoprotein hormone that regulates mammalian erythropoiesis. To study the expression of the human erythropoietin gene, EPO, 4 kilobases of DNA encompassing the gene with 0.4 kilobase of 5{prime} flanking sequence and 0.7 kilobase of 3{prime} flanking sequence was microinjected into fertilized mouse eggs. Transgenic mice were generated that are polycythemic, with increased erythrocytic indices in peripheral blood, increased numbers of erythroid precursors in hematopoietic tissue, and increased serum erythropoietin levels. Transgenic homozygotes show a greater degree of polycythemia than do heterozygotes as well as striking extramedullary erythropoiesis. Human erythropoietin RNA was found not only in fetal liver,more » adult liver, and kidney but also in all other transgenic tissues analyzed. Anemia induced increased human erythropoietin RNA levels in liver but not kidney. These transgenic mice represent a unique model of polycythemia due to increased erythropoietin levels.« less

Roles of a maize phytochrome-interacting factors protein ZmPIF3 in regulation of drought stress responses by controlling stomatal closure in transgenic rice without yield penalty.

PubMed

Gao, Yong; Wu, Meiqin; Zhang, Menjiao; Jiang, Wei; Liang, Enxing; Zhang, Dongping; Zhang, Changquan; Xiao, Ning; Chen, Jianmin

2018-06-05

ZmPIF3 plays an important role in ABA-mediated regulation of stomatal closure in the control of water loss, and can improve both drought tolerance and did not affect the grain yield in the transgenic rice. Phytochrome-interacting factors (PIFs) are a subfamily of basic helix-loop-helix (bHLH) transcription factors and play important roles in regulating plant growth and development. In our previous study, overexpression of a maize PIFs family gene, ZmPIF3, improved drought tolerance in transgenic rice. In this study, measurement of water loss rate, transpiration rate, stomatal conductance, guard cell aperture, density and length of ZmPIF3 transgenic plants showed that ZmPIF3 can enhance water-saving and drought-resistance by decreasing stomatal aperture and reducing transpiration in both transgenic rice and transgenic Arabidopsis. Scrutiny of sensitivity to ABA showed that ZmPIF3 transgenic rice was hypersensitive to ABA, while the endogenous ABA level was not significantly changed. These results indicate that ZmPIF3 plays a major role in the ABA signaling pathway. In addition, DGE results further suggest that ZmPIF3 participates in the ABA signaling pathway and regulates stomatal aperture in rice. Comparison analysis of the phenotype, physiology, and transcriptome of ZmPIF3 transgenic rice compared to control plants further suggests that ZmPIF3 is a positive regulator of ABA signaling and enhances water-saving and drought-resistance traits by reducing stomatal openings to control water loss. Moreover, investigation of the agronomic traits of ZmPIF3 transgenic rice from four cultivating seasons showed that ZmPIF3 expression increased the tiller and panicle number and did not affect the grain yield in the transgenic rice. These results demonstrate that ZmPIF3 is a promising candidate gene in the transgenic breeding of water-saving and drought-resistant rice plants and crop improvement.

CaPUB1, a Hot Pepper U-box E3 Ubiquitin Ligase, Confers Enhanced Cold Stress Tolerance and Decreased Drought Stress Tolerance in Transgenic Rice (Oryza sativa L.).

PubMed

Min, Hye Jo; Jung, Ye Jin; Kang, Bin Goo; Kim, Woo Taek

2016-03-01

Abiotic stresses such as drought and low temperature critically restrict plant growth, reproduction, and productivity. Higher plants have developed various defense strategies against these unfavorable conditions. CaPUB1 (Capsicum annuum Putative U-box protein 1) is a hot pepper U-box E3 Ub ligase. Transgenic Arabidopsis plants that constitutively expressed CaPUB1 exhibited drought-sensitive phenotypes, suggesting that it functions as a negative regulator of the drought stress response. In this study, CaPUB1 was over-expressed in rice (Oryza sativa L.), and the phenotypic properties of transgenic rice plants were examined in terms of their drought and cold stress tolerance. Ubi:CaPUB1 T3 transgenic rice plants displayed phenotypes hypersensitive to dehydration, suggesting that its role in the negative regulation of drought stress response is conserved in dicot Arabidopsis and monocot rice plants. In contrast, Ubi:CaPUB1 progeny exhibited phenotypes markedly tolerant to prolonged low temperature (4°C) treatment, compared to those of wild-type plants, as determined by survival rates, electrolyte leakage, and total chlorophyll content. Cold stress-induced marker genes, including DREB1A, DREB1B, DREB1C, and Cytochrome P450, were more up-regulated by cold treatment in Ubi:CaPUB1 plants than in wild-type plants. These results suggest that CaPUB1 serves as both a negative regulator of the drought stress response and a positive regulator of the cold stress response in transgenic rice plants. This raises the possibility that CaPUB1 participates in the cross-talk between drought and low-temperature signaling pathways.

MicroRNA858 Is a Potential Regulator of Phenylpropanoid Pathway and Plant Development1

PubMed Central

Sharma, Deepika; Tiwari, Manish; Pandey, Ashutosh; Bhatia, Chitra; Sharma, Ashish; Trivedi, Prabodh Kumar

2016-01-01

MicroRNAs (miRNAs) are endogenous, noncoding small RNAs that function as critical regulators of gene expression. In plants, miRNAs have shown their potential as regulators of growth, development, signal transduction, and stress tolerance. Although the miRNA-mediated regulation of several processes is known, the involvement of miRNAs in regulating secondary plant product biosynthesis is poorly understood. In this study, we functionally characterized Arabidopsis (Arabidopsis thaliana) miR858a, which putatively targets R2R3-MYB transcription factors involved in flavonoid biosynthesis. Overexpression of miR858a in Arabidopsis led to the down-regulation of several MYB transcription factors regulating flavonoid biosynthesis. In contrast to the robust growth and early flowering of miR858OX plants, reduction of plant growth and delayed flowering were observed in Arabidopsis transgenic lines expressing an artificial miRNA target mimic (MIM858). Genome-wide expression analysis using transgenic lines suggested that miR858a targets a number of regulatory factors that modulate the expression of downstream genes involved in plant development and hormonal and stress responses. Furthermore, higher expression of MYBs in MIM858 lines leads to redirection of the metabolic flux towards the synthesis of flavonoids at the cost of lignin synthesis. Altogether, our study has established the potential role of light-regulated miR858a in flavonoid biosynthesis and plant growth and development. PMID:27208307

Regulation of trichome development in tobacco by JcZFP8, a C2H2 zinc finger protein gene from Jatropha curcas L.

PubMed

Shi, Xiaodong; Gu, Yuxi; Dai, Tingwei; Wu, Yang; Wu, Peng; Xu, Ying; Chen, Fang

2018-06-05

Trichomes are epidermal outgrowths of plant tissues that can secrete or store large quantities of secondary metabolites, which contribute to plant defense responses against stress. The use of bioengineering methods for regulating the development of trichomes and metabolism is a widely researched topic. In the present study, we demonstrate that JcZFP8, a C2H2 zinc finger protein gene from Jatropha curcas L., can regulate trichome development in transgenic tobacco. To understand the underlying mechanisms, we performed transcriptome profiling of overexpression JcZFP8 transgenic plants and wild-type tobacco. Based on the analysis of differentially expressed genes, we determined that genes of the plant hormone signal transduction pathway was significantly enriched, suggesting that these pathways were modulated in the transgenic plants. In addition, the transcript levels of the known trichome-related genes in Arabidopsis were not significantly changed, whereas CycB2 and MYB genes were differentially expressed in the transgenic plants. Despite tobacco and Arabidopsis have different types of trichomes, all the pathways were associated with C2H2 zinc finger protein genes. Our findings help us to understand the regulation of multicellular trichome formation and suggest a new metabolic engineering method for the improvement of plants. Copyright © 2018 Elsevier B.V. All rights reserved.

Gene regulation in amphioxus: An insight from transgenic studies in amphioxus and vertebrates.

PubMed

Kozmikova, Iryna; Kozmik, Zbynek

2015-12-01

Cephalochordates, commonly known as amphioxus or lancelets, are the most basal subphylum of chordates. Cephalochordates are thus key to understanding the origin of vertebrates and molecular mechanisms underlying vertebrate evolution. The evolution of developmental control mechanisms during invertebrate-to-vertebrate transition involved not only gene duplication events, but also specific changes in spatial and temporal expression of many genes. To get insight into the spatiotemporal regulation of gene expression during invertebrate-to-vertebrate transition, functional studies of amphioxus gene regulatory elements are highly warranted. Here, we review transgenic studies performed in amphioxus and vertebrates using promoters and enhancers derived from the genome of Branchiostoma floridae. We describe the current methods of transgenesis in amphioxus, provide evidence of Tol2 transposon-generated transgenic embryos of Branchiostoma lanceolatum and discuss possible future directions. We envision that comparative transgenic analysis of gene regulatory sequences in the context of amphioxus and vertebrate embryos will likely provide an important mechanistic insight into the evolution of vertebrate body plan. Copyright © 2015 Elsevier B.V. All rights reserved.

Production of an active feline interferon in the cocoon of transgenic silkworms using the fibroin H-chain expression system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kurihara, H.; Sezutsu, H.; Tamura, T.

2007-04-20

We constructed the fibroin H-chain expression system to produce recombinant proteins in the cocoon of transgenic silkworms. Feline interferon (FeIFN) was used for production and to assess the quality of the product. Two types of FeIFN fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, were designed to be secreted into the lumen of the posterior silk glands. The expression of the FeIFN/H-chain fusion gene was regulated by the fibroin H-chain promoter domain. The transgenic silkworms introduced these constructs with the piggyBac transposon-derived vector, which produced the normal sized cocoons containing each FeIFN/H-chain fusion protein. Although themore » native-protein produced by transgenic silkworms have almost no antiviral activity, the proteins after the treatment with PreScission protease to eliminate fibroin H-chain derived N- and C-terminal sequences from the products, had very high antiviral activity. This H-chain expression system, using transgenic silkworms, could be an alternative method to produce an active recombinant protein and silk-based biomaterials.« less

A WRKY transcription factor, PcWRKY33, from Polygonum cuspidatum reduces salt tolerance in transgenic Arabidopsis thaliana.

PubMed

Bao, Wenqi; Wang, Xiaowei; Chen, Mo; Chai, Tuanyao; Wang, Hong

2018-07-01

PcWRKY33 is a transcription factor which can reduce salt tolerance by decreasing the expression of stress-related genes and increasing the cellular levels of reactive oxygen species (ROS). WRKY transcription factors play important roles in the regulation of biotic and abiotic stresses. Here, we report a group I WRKY gene from Polygonum cuspidatum, PcWRKY33, that encodes a nucleoprotein, which specifically binds to the W-box in the promoter of target genes to regulate their expression. The results from qPCR and promoter analysis show that expression of PcWRKY33 can be induced by various abiotic stresses, including NaCl and plant hormones. Overexpression of PcWRKY33 in Arabidopsis thaliana reduced tolerance to salt stress. More specifically, several physiological parameters (such as root length, seed germination rate, seedling survival rate, and chlorophyll concentration) of the transgenic lines were significantly lower than those of the wild type under salt stress. In addition, following exposure to salt stress, transgenic plants showed decreased expression of stress-related genes, a weakened ability to maintain Na + /K + homeostasis, decreased activities of reactive oxygen species- (ROS-) scavenging enzymes, and increased accumulation of ROS. Taken together, these results suggest that PcWRKY33 negatively regulates the salt tolerance in at least two ways: by down-regulating the induction of stress-related genes and by increasing the level of cellular ROS. In sum, our results indicate that PcWRKY33 is a group I WRKY transcription factor involved in abiotic stress regulation.

DREAM regulates BDNF-dependent spinal sensitization

PubMed Central

2010-01-01

Background The transcriptional repressor DREAM (downstream regulatory element antagonist modulator) controls the expression of prodynorphin and has been involved in the modulation of endogenous responses to pain. To investigate the role of DREAM in central mechanisms of pain sensitization, we used a line of transgenic mice (L1) overexpressing a Ca2+- and cAMP-insensitive DREAM mutant in spinal cord and dorsal root ganglia. Results L1 DREAM transgenic mice showed reduced expression in the spinal cord of several genes related to pain, including prodynorphin and BDNF (brain-derived neurotrophic factor) and a state of basal hyperalgesia without change in A-type currents. Peripheral inflammation produced enhancement of spinal reflexes and increased expression of BDNF in wild type but not in DREAM transgenic mice. The enhancement of the spinal reflexes was reproduced in vitro by persistent electrical stimulation of C-fibers in wild type but not in transgenic mice. Exposure to exogenous BDNF produced a long-term enhancement of dorsal root-ventral root responses in transgenic mice. Conclusions Our results indicate that endogenous BDNF is involved in spinal sensitization following inflammation and that blockade of BDNF induction in DREAM transgenic mice underlies the failure to develop spinal sensitization. PMID:21167062

Production of Multiple Transgenic Yucatan Miniature Pigs Expressing Human Complement Regulatory Factors, Human CD55, CD59, and H-Transferase Genes

PubMed Central

Jang, Gun-Hyuk; Jeong, Yeun-Ik; Hwang, In-Sung; Jeong, Yeon-woo; Kim, Yu-Kyung; Shin, Taeyoung; Kim, Nam-Hyung; Hyun, Sang-Hwan; Jeung, Eui-Bae; Hwang, Woo-Suk

2013-01-01

The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT) using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR) when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT) and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC) were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC) compared with the human umbilical vein endothelial cells (HUVEC). Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative for the generation of transgenic large animals with multiple genetic modifications. PMID:23704897

Studies on the expression of an H-2K/human growth hormone fusion gene in giant transgenic mice.

PubMed Central

Morello, D; Moore, G; Salmon, A M; Yaniv, M; Babinet, C

1986-01-01

Transgenic mice carrying the H-2K/human growth hormone (hGH) fusion gene were produced by microinjecting into the pronucleus of fertilized eggs DNA molecules containing 2 kb of the 5' flanking sequences (including promoter) of the class I H-2Kb gene joined to the coding sequences of the hGH gene. Thirteen transgenic mice were obtained which all contained detectable levels of hGH hormone in their blood. Nine grew larger than their control litter-mates. Endogenous H-2Kb and exogenous hGH mRNA levels were analysed by S1 nuclease digestion experiments. hGH transcripts were found in all the tissues examined and the pattern of expression paralleled that of endogenous H-2K gene expression, being high in liver and lymphoid organs and low in muscle and brain. Thus 2 kb of the 5' promoter/regulatory region of the H-2K gene are sufficient to ensure regulated expression of hGH in transgenic mice. This promoter may therefore be of use to target the expression of different exogenous genes in most tissues of transgenic mice and to study the biological role of the corresponding proteins in different cellular environments. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:3019667

The homeodomain transcription factor TaHDZipI-2 from wheat regulates frost tolerance, flowering time and spike development in transgenic barley.

PubMed

Kovalchuk, Nataliya; Chew, William; Sornaraj, Pradeep; Borisjuk, Nikolai; Yang, Nannan; Singh, Rohan; Bazanova, Natalia; Shavrukov, Yuri; Guendel, Andre; Munz, Eberhard; Borisjuk, Ljudmilla; Langridge, Peter; Hrmova, Maria; Lopato, Sergiy

2016-07-01

Homeodomain leucine zipper class I (HD-Zip I) transcription factors (TFs) play key roles in the regulation of plant growth and development under stresses. Functions of the TaHDZipI-2 gene isolated from the endosperm of developing wheat grain were revealed. Molecular characterization of TaHDZipI-2 protein included studies of its dimerisation, protein-DNA interactions and gene activation properties using pull-down assays, in-yeast methods and transient expression assays in wheat cells. The analysis of TaHDZipI-2 gene functions was performed using transgenic barley plants. It included comparison of developmental phenotypes, yield components, grain quality, frost tolerance and the levels of expression of potential target genes in transgenic and control plants. Transgenic TaHDZipI-2 lines showed characteristic phenotypic features that included reduced growth rates, reduced biomass, early flowering, light-coloured leaves and narrowly elongated spikes. Transgenic lines produced 25-40% more seeds per spike than control plants, but with 50-60% smaller grain size. In vivo lipid imaging exposed changes in the distribution of lipids between the embryo and endosperm in transgenic seeds. Transgenic lines were significantly more tolerant to frost than control plants. Our data suggest the role of TaHDZipI-2 in controlling several key processes underlying frost tolerance, transition to flowering and spike development. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Down-Regulation of TM29, a Tomato SEPALLATA Homolog, Causes Parthenocarpic Fruit Development and Floral Reversion1

PubMed Central

Ampomah-Dwamena, Charles; Morris, Bret A.; Sutherland, Paul; Veit, Bruce; Yao, Jia-Long

2002-01-01

We have characterized the tomato (Lycopersicon esculentum Mill.) MADS box gene TM29 that shared a high amino acid sequence homology to the Arabidopsis SEP1, 2, and 3 (SEPALLATA1, 2, and 3) genes. TM29 showed similar expression profiles to SEP1, with accumulation of mRNA in the primordia of all four whorls of floral organs. In addition, TM29 mRNA was detected in inflorescence and vegetative meristems. To understand TM29 function, we produced transgenic tomato plants in which TM29 expression was down-regulated by either cosuppression or antisense techniques. These transgenic plants produced aberrant flowers with morphogenetic alterations in the organs of the inner three whorls. Petals and stamens were green rather than yellow, suggesting a partial conversion to a sepalloid identity. Stamens and ovaries were infertile, with the later developing into parthenocarpic fruit. Ectopic shoots with partially developed leaves and secondary flowers emerged from the fruit. These shoots resembled the primary transgenic flowers and continued to produce parthenocarpic fruit and additional ectopic shoots. Based on the temporal and spatial expression pattern and transgenic phenotypes, we propose that TM29 functions in floral organ development, fruit development, and maintenance of floral meristem identity in tomato. PMID:12376628

Functional characterization of a novel Brassica LEAFY homolog from Indian mustard: Expression pattern and gain-of-function studies.

PubMed

Dhakate, Priyanka; Tyagi, Shikha; Singh, Anupama; Singh, Anandita

2017-05-01

LEAFY plays a central role in regulation of flowering time and floral meristem identity in plants. Unfortunately, LFY function remains uncharacterized in agronomicaly important Brassicas. Herein, we illustrate fine-mapping of expression domains of LFY in 15 cultivars of 6 Brassica species and describe gain-of-function phenotypes in Arabidopsis and Brassica. We depict early flowering and altered fatty-acid composition in transgenic seed. The cDNA encoding BjuLFY (417aa) shared only 85% identity with reported homolog of B.juncea implying distinctness. Quantitative RT-PCR based coarse expression mapping of BjuLFY in tissue samples representing 3 time points at specific days after sowing (DAS), pre-flowering (30 DAS), flowering (75 DAS) and post-flowering (110 DAS), depicted an intense pulse of BjuLFY expression restricted to primary floral buds (75 DAS) which subsided in secondary floral buds (110 DAS); expression in root samples was also recorded implying neo-functionalization. Fine-mapping of expression during flowering confirmed tightly regulated LFY expression during early stages of bud development in 15 cultivars of 6 Brassica species implying functional conservation. Ectopic expression of BjuLFY in A. thaliana and B. juncea caused floral meristem defects and precocious flowering. B. juncea transgenics (T 1 ) over-expressing BjuLFY flowered 20days earlier produced normal flowers. GC-MS analysis of mature seed from Brassica transgenics showed an altered fatty-acid profile suggestive of seed maturation occurring at lower temperatures vis-à-vis control. Our findings implicate BjuLFY as a regulator of flowering in B. juncea and suggest its application in developing climate resilient crops. Copyright © 2017 Elsevier B.V. All rights reserved.

Hepatic oxidative stress in ovariectomized transgenic mice expressing the hepatitis C virus polyprotein is augmented through suppression of adenosine monophosphate-activated protein kinase/proliferator-activated receptor gamma co-activator 1 alpha signaling.

PubMed

Tomiyama, Yasuyuki; Nishina, Sohji; Hara, Yuichi; Kawase, Tomoya; Hino, Keisuke

2014-10-01

Oxidative stress plays an important role in hepatocarcinogenesis of hepatitis C virus (HCV)-related chronic liver diseases. Despite the evidence of an increased proportion of females among elderly patients with HCV-related hepatocellular carcinoma (HCC), it remains unknown whether HCV augments hepatic oxidative stress in postmenopausal women. The aim of this study was to determine whether oxidative stress was augmented in ovariectomized (OVX) transgenic mice expressing the HCV polyprotein and to investigate its underlying mechanisms. OVX and sham-operated female transgenic mice expressing the HCV polyprotein and non-transgenic littermates were assessed for the production of reactive oxygen species (ROS), expression of inflammatory cytokines and antioxidant potential in the liver. Compared with OVX non-transgenic mice, OVX transgenic mice showed marked hepatic steatosis and ROS production without increased induction of inflammatory cytokines, but there was no increase in ROS-detoxifying enzymes such as superoxide dismutase 2 and glutathione peroxidase 1. In accordance with these results, OVX transgenic mice showed less activation of peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α), which is required for the induction of ROS-detoxifying enzymes, and no activation of adenosine monophosphate-activated protein kinase-α (AMPKα), which regulates the activity of PGC-1α. Our study demonstrated that hepatic oxidative stress was augmented in OVX transgenic mice expressing the HCV polyprotein by attenuation of antioxidant potential through inhibition of AMPK/PGC-1α signaling. These results may account in part for the mechanisms by which HCV-infected women are at high risk for HCC development when some period has passed after menopause. © 2013 The Japan Society of Hepatology.

Comparative proteomic analysis of rd29A:RdreB1BI transgenic and non-transgenic strawberries exposed to low temperature.

PubMed

Gu, Xianbin; Gao, Zhihong; Zhuang, Weibing; Qiao, Yushan; Wang, Xiuyun; Mi, Lin; Zhang, Zhen; Lin, Zhilin

2013-05-01

Low-temperature stress is one of the major abiotic stresses in plants worldwide, and the dehydration responsive element binding protein (DREB) transcription factor induces expression of genes involved in environmental stress tolerance in plants. A proteomic approach based on two-dimensional gel electrophoresis (2-DE) and subsequent mass spectrometric identification was used to study the changes in the leaf proteome profiles of rd29A:RdreB1BI transgenic and non-transgenic strawberries exposed to low-temperature conditions. By comparing the proteomic profiles, we located 21 protein spots that were reproducibly up- or down-regulated by more than twofold between transgenic and non-transgenic strawberries. Eight identified proteins function in energy and metabolism, four in biosynthetic processes, four were stress and defense related, three spots were identified as cold-stress related expressed sequence tags (ESTs), and two were unknown proteins. The change patterns of low-temperature tolerance proteins, including photosynthetic proteins (RuBisCO large subunit and RuBisCO activase), cytoplasmic Cu/Zn-superoxide dismutase (Cu/Zn-SOD), late embryogenesis abundant protein 14-A (Lea14-A), eukaryotic translation initiation factor 5A (eIF5A), and cold-stress related ESTs, were differentially regulated between non-transgenic and rd29A:RdreB1BI transgenic strawberries. They are likely important gene products in the regulatory network of the RdreB1BI gene. Consequently, this study provides the first characterization of the transgenic strawberry proteome and the predicted target proteins of the RdreB1BI gene by using proteomic approaches. Copyright © 2013 Elsevier GmbH. All rights reserved.

Tight regulation of plant immune responses by combining promoter and suicide exon elements

DOE PAGES

Gonzalez, Tania L.; Liang, Yan; Nguyen, Bao N.; ...

2015-07-02

Effector-triggered immunity (ETI) is activated when plant disease resistance (R) proteins recognize the presence of pathogen effector proteins delivered into host cells. The ETI response generally encompasses a defensive ‘hypersensitive response’ (HR) that involves programmed cell death at the site of pathogen recognition. While many R protein and effector protein pairs are known to trigger HR, other components of the ETI signaling pathway remain elusive. Effector genes regulated by inducible promoters cause background HR due to leaky protein expression, preventing the generation of relevant transgenic plant lines. By employing the HyP5SM suicide exon, we have developed a strategy to tightlymore » regulate effector proteins such that HR is chemically inducible and non-leaky. This alternative splicing-based gene regulation system was shown to successfully control Bs2/AvrBs2-dependent and RPP1/ATR1Δ51-dependent HR in Nicotiana benthamiana and Nicotiana tabacum, respectively. It was also used to generate viable and healthy transgenic Arabidopsis thaliana plants that inducibly initiate HR. In conclusion, beyond enabling studies on the ETI pathway, our regulatory strategy is generally applicable to reduce or eliminate undesired background expression of transgenes.« less

Tight regulation of plant immune responses by combining promoter and suicide exon elements

PubMed Central

Gonzalez, Tania L.; Liang, Yan; Nguyen, Bao N.; Staskawicz, Brian J.; Loqué, Dominique; Hammond, Ming C.

2015-01-01

Effector-triggered immunity (ETI) is activated when plant disease resistance (R) proteins recognize the presence of pathogen effector proteins delivered into host cells. The ETI response generally encompasses a defensive ‘hypersensitive response’ (HR) that involves programmed cell death at the site of pathogen recognition. While many R protein and effector protein pairs are known to trigger HR, other components of the ETI signaling pathway remain elusive. Effector genes regulated by inducible promoters cause background HR due to leaky protein expression, preventing the generation of relevant transgenic plant lines. By employing the HyP5SM suicide exon, we have developed a strategy to tightly regulate effector proteins such that HR is chemically inducible and non-leaky. This alternative splicing-based gene regulation system was shown to successfully control Bs2/AvrBs2-dependent and RPP1/ATR1Δ51-dependent HR in Nicotiana benthamiana and Nicotiana tabacum, respectively. It was also used to generate viable and healthy transgenic Arabidopsis thaliana plants that inducibly initiate HR. Beyond enabling studies on the ETI pathway, our regulatory strategy is generally applicable to reduce or eliminate undesired background expression of transgenes. PMID:26138488

Tight regulation of plant immune responses by combining promoter and suicide exon elements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gonzalez, Tania L.; Liang, Yan; Nguyen, Bao N.

Effector-triggered immunity (ETI) is activated when plant disease resistance (R) proteins recognize the presence of pathogen effector proteins delivered into host cells. The ETI response generally encompasses a defensive ‘hypersensitive response’ (HR) that involves programmed cell death at the site of pathogen recognition. While many R protein and effector protein pairs are known to trigger HR, other components of the ETI signaling pathway remain elusive. Effector genes regulated by inducible promoters cause background HR due to leaky protein expression, preventing the generation of relevant transgenic plant lines. By employing the HyP5SM suicide exon, we have developed a strategy to tightlymore » regulate effector proteins such that HR is chemically inducible and non-leaky. This alternative splicing-based gene regulation system was shown to successfully control Bs2/AvrBs2-dependent and RPP1/ATR1Δ51-dependent HR in Nicotiana benthamiana and Nicotiana tabacum, respectively. It was also used to generate viable and healthy transgenic Arabidopsis thaliana plants that inducibly initiate HR. In conclusion, beyond enabling studies on the ETI pathway, our regulatory strategy is generally applicable to reduce or eliminate undesired background expression of transgenes.« less

Transgenic expression of cyclooxygenase-2 (COX2) causes premature aging phenotypes in mice.

PubMed

Kim, Joohwee; Vaish, Vivek; Feng, Mingxiao; Field, Kevin; Chatzistamou, Ioulia; Shim, Minsub

2016-10-07

Cyclooxygenase (COX) is a key enzyme in the biosynthesis of prostanoids, lipid signaling molecules that regulate various physiological processes. COX2, one of the isoforms of COX, is highly inducible in response to a wide variety of cellular and environmental stresses. Increased COX2 expression is thought to play a role in the pathogenesis of many age-related diseases. COX2 expression is also reported to be increased in the tissues of aged humans and mice, which suggests the involvement of COX2 in the aging process. However, it is not clear whether the increased COX2 expression is causal to or a result of aging. We have now addressed this question by creating an inducible COX2 transgenic mouse model. Here we show that post-natal expression of COX2 led to a panel of aging-related phenotypes. The expression of p16, p53, and phospho-H2AX was increased in the tissues of COX2 transgenic mice. Additionally, adult mouse lung fibroblasts from COX2 transgenic mice exhibited increased expression of the senescence-associated β-galactosidase. Our study reveals that the increased COX2 expression has an impact on the aging process and suggests that modulation of COX2 and its downstream signaling may be an approach for intervention of age-related disorders.

Transgenic Arabidopsis Plants Expressing the Type 1 Inositol 5-Phosphatase Exhibit Increased Drought Tolerance and Altered Abscisic Acid Signaling[W

PubMed Central

Perera, Imara Y.; Hung, Chiu-Yueh; Moore, Candace D.; Stevenson-Paulik, Jill; Boss, Wendy F.

2008-01-01

The phosphoinositide pathway and inositol-1,4,5-trisphosphate (InsP3) are implicated in plant responses to stress. To determine the downstream consequences of altered InsP3-mediated signaling, we generated transgenic Arabidopsis thaliana plants expressing the mammalian type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), which specifically hydrolyzes soluble inositol phosphates and terminates the signal. Rapid transient Ca2+ responses to a cold or salt stimulus were reduced by ∼30% in these transgenic plants. Drought stress studies revealed, surprisingly, that the InsP 5-ptase plants lost less water and exhibited increased drought tolerance. The onset of the drought stress was delayed in the transgenic plants, and abscisic acid (ABA) levels increased less than in the wild-type plants. Stomatal bioassays showed that transgenic guard cells were less responsive to the inhibition of opening by ABA but showed an increased sensitivity to ABA-induced closure. Transcript profiling revealed that the drought-inducible ABA-independent transcription factor DREB2A and a subset of DREB2A-regulated genes were basally upregulated in the InsP 5-ptase plants, suggesting that InsP3 is a negative regulator of these DREB2A-regulated genes. These results indicate that the drought tolerance of the InsP 5-ptase plants is mediated in part via a DREB2A-dependent pathway and that constitutive dampening of the InsP3 signal reveals unanticipated interconnections between signaling pathways. PMID:18849493

The Homeodomain Transcription Factor Cdx1 Does Not Behave as an Oncogene in Normal Mouse Intestine1

PubMed Central

Crissey, Mary Ann S; Guo, Rong-Jun; Fogt, Franz; Li, Hong; Katz, Jonathan P; Silberg, Debra G; Suh, Eun Ran; Lynch, John P

2008-01-01

The Caudal-related homeobox genes Cdx1 and Cdx2 are intestine-specific transcription factors that regulate differentiation of intestinal cell types. Previously, we have shown Cdx1 to be antiproliferative and to promote cell differentiation. However, other studies have suggested that Cdx1 may be an oncogene. To test for oncogenic behavior, we used the murine villin promoter to ectopically express Cdx1 in the small intestinal villi and colonic surface epithelium. No changes in intestinal architecture, cell differentiation, or lineage selection were observed with expression of the transgene. Classic oncogenes enhance proliferation and induce tumors when ectopically expressed. However, the Cdx1 transgene neither altered intestinal proliferation nor induced spontaneous intestinal tumors. In a murine model for colitis-associated cancer, the Cdx1 transgene decreased, rather than increased, the number of adenomas that developed. In the polyps, the expression of the endogenous and the transgenic Cdx1 proteins was largely absent, whereas endogenous Villin expression was retained. This suggests that transgene silencing was specific and not due to a general Villin inactivation. In conclusion, neither the ectopic expression of Cdx1 was associated with changes in intestinal cell proliferation or differentiation nor was there increased intestinal cancer susceptibility. Our results therefore suggest that Cdx1 is not an oncogene in normal intestinal epithelium. PMID:18231635

Muscle fiber-type conversion in the transgenic pigs with overexpression of PGC1α gene in muscle.

PubMed

Ying, Fei; Zhang, Liang; Bu, Guowei; Xiong, Yuanzhu; Zuo, Bo

2016-11-25

The peroxisome proliferator-activated receptor gamma, co-activator 1 alpha(PGC1α) effectively induced the biosynthesis of the mitochondria and the energy metabolism, and also regulated the muscle fiber-type shift. Overexpression of PGC1α gene in mice led to higher oxidative muscle fiber composition in muscle. However, no researches about the significant differences of muscle fiber phenotype in pigs after PGC1α overexpression had been reported. The composition of muscle fiber-types which were distinguished by four myosin heavy chain(MYHC) isoforms, can significantly affect the muscle functions. In our study, we generated the transgenic pigs to investigate the effect of overexpression of PGC1α gene on muscle fiber-type conversion. The results showed that the number of oxidative muscle fiber(type1 muscle fiber) was increased and the number of glycolytic muscle fiber(type2b muscle fiber) was decreased in the transgenic pigs. Furthermore, we found that PGC1α overexpression up-regulated the expression of MYHC1 and MYHC2a and down-regulated the expression of MYHC2b.The analysis of genes expression demonstrated the main differentially expressed genes were MSTN, Myog and FOXO1. In conclusion, the overexpression of PGC1α gene can promote the glycolytic muscle fiber transform to the oxidative muscle fiber in pigs. Copyright © 2016 Elsevier Inc. All rights reserved.

Muscle fiber-type conversion in the transgenic pigs with overexpression of PGC1α gene in muscle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ying, Fei; Zhang, Liang; Bu, Guowei

The peroxisome proliferator-activated receptor gamma, co-activator 1 alpha(PGC1α) effectively induced the biosynthesis of the mitochondria and the energy metabolism, and also regulated the muscle fiber-type shift. Overexpression of PGC1α gene in mice led to higher oxidative muscle fiber composition in muscle. However, no researches about the significant differences of muscle fiber phenotype in pigs after PGC1α overexpression had been reported. The composition of muscle fiber-types which were distinguished by four myosin heavy chain(MYHC) isoforms, can significantly affect the muscle functions. In our study, we generated the transgenic pigs to investigate the effect of overexpression of PGC1α gene on muscle fiber-typemore » conversion. The results showed that the number of oxidative muscle fiber(type1 muscle fiber) was increased and the number of glycolytic muscle fiber(type2b muscle fiber) was decreased in the transgenic pigs. Furthermore, we found that PGC1α overexpression up-regulated the expression of MYHC1 and MYHC2a and down-regulated the expression of MYHC2b.The analysis of genes expression demonstrated the main differentially expressed genes were MSTN, Myog and FOXO1. In conclusion, the overexpression of PGC1α gene can promote the glycolytic muscle fiber transform to the oxidative muscle fiber in pigs.« less

Overexpression of AtAP1M3 regulates flowering time and floral development in Arabidopsis and effects key flowering-related genes in poplar.

PubMed

Chen, Zhong; Ye, Meixia; Su, Xiaoxing; Liao, Weihua; Ma, Huandi; Gao, Kai; Lei, Bingqi; An, Xinmin

2015-08-01

APETALA1 plays a crucial role in the transition from vegetative to reproductive phase and in floral development. In this study, to determine the effect of AP1 expression on flowering time and floral organ development, transgenic Arabidopsis and poplar overexpressing of AtAP1M3 (Arabidopsis AP1 mutant by dominant negative mutation) were generated. Transgenic Arabidopsis with e35Spro::AtAP1M3 displayed phenotypes with delayed-flowering compared to wild-type and flowers with abnormal sepals, petals and stamens. In addition, transgenic Arabidopsis plants exhibited reduced growth vigor compared to the wild-type plants. Ectopic expression of AtAP1M3 in poplar resulted in up- or down-regulation of some endogenous key flowering-related genes, including floral meristems identity gene LFY, B-class floral organ identity genes AP3 and PI, flowering pathway integrator FT1 and flower repressors TFL1 and SVP. These results suggest that AtAP1M3 regulates flowering time and floral development in plants.

Generation and characterization of gsuα:EGFP transgenic zebrafish for evaluating endocrine-disrupting effects

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, Xiaoxia; University of Chinese Academy of Sciences, Beijing; Chen, Xiaowen

The glycoprotein subunit α (gsuα) gene encodes the shared α subunit of the three pituitary heterodimeric glycoprotein hormones: follicle-stimulating hormone β (Fshβ), luteinizing hormone β (Lhβ) and thyroid stimulating hormone β (Tshβ). In our current study, we identified and characterized the promoter region of zebrafish gsuα and generated a stable gsuα:EGFP transgenic line, which recapitulated the endogenous gsuα expression in the early developing pituitary gland. A relatively conserved regulatory element set is presented in the promoter regions of zebrafish and three other known mammalian gsuα promoters. Our results also demonstrated that the expression patterns of the gsuα:EGFP transgene were allmore » identical to those expression patterns of the endogenous gsuα expression in the pituitary tissue when our transgenic fish were treated with various endocrine chemicals, including forskolin (FSK), SP600125, trichostatin A (TSA), KClO{sub 4}, dexamethasone (Dex), β-estradiol and progesterone. Thus, this gsuα:EGFP transgenic fish reporter line provides another valuable tool for investigating the lineage development of gsuα-expressing gonadotrophins and the coordinated regulation of various glycoprotein hormone subunit genes. These reporter fish can serve as a novel platform to perform screenings of endocrine-disrupting chemicals (EDCs) in vivo as well. - Highlights: • Identification of the promoter of zebrafish glycoprotein subunit α (gsuα) gene • Generation of stable transmission gsuα:EGFP transgenic zebrafish reporter • Demonstration of the recapitulation of the gsuα:EGFP and endogenous gsuα expression • Suggestion of the gsuα:EGFP transgenic zebrafish as a novel platform for EDC study.« less

Development of Cre-loxP technology in zebrafish to study the regulation of fish reproduction.

PubMed

Lin, Heng-Ju; Lee, Shu-Hua; Wu, Jen-Leih; Duann, Yeh-Fang; Chen, Jyh-Yih

2013-12-01

One cannot seek permission to market transgenic fish mainly because there is no field test or any basic research on technological developments for evaluating their biosafety. Infertility is a necessary adjunct to exploiting transgenic fish unless completely secure land-locked facilities are available. In this study, we report the generation of a Cre transgenic zebrafish line using a cytomegalovirus promoter. We also produced fish carrying the Bax1 and Bax2 plasmids; these genes were separated by two loxP sites under a zona pellucida C promoter or were driven by an anti-Müllerian hormone promoter. We inserted a red fluorescent protein gene between the two loxP sites. After obtaining transgenic lines with the two transgenic fish crossed with each other (Cre transgenic zebrafish x loxP transgenic zebrafish), the floxed DNA was found to be specifically eliminated from the female or male zebrafish, and apoptosis gene expressions caused ovarian and testicular growth cessation and degeneration. Overexpression of the Bax1 and Bax2 genes caused various expression levels of apoptosis-related genes. Accordingly, this transgenic zebrafish model system provides a method to produce infertile fish and may be useful for application to genetically modified fish.

Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine β-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination.

PubMed

Jeong, Young-Hee; Kim, Yeong Ji; Kim, Eun Young; Kim, Se Eun; Kim, Jiwoo; Park, Min Jee; Lee, Hong-Gu; Park, Se Pill; Kang, Man-Jong

2016-06-01

Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine β-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the β-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine β-casein gene.

The zinc finger gene Krox20 regulates HoxB2 (Hox2.8) during hindbrain segmentation.

PubMed

Sham, M H; Vesque, C; Nonchev, S; Marshall, H; Frain, M; Gupta, R D; Whiting, J; Wilkinson, D; Charnay, P; Krumlauf, R

1993-01-29

The zinc finger gene Krox20 and many Hox homeobox genes are expressed in segment-restricted domains in the hindbrain. The restricted expression patterns appear before morphological segmentation, suggesting that these transcription factors may play an early role in the establishment and identity of rhombomeric segments. In this paper, we show that the HoxB2 (Hox2.8) gene is normally upregulated in rhombomeres (r) 3, 4, and 5, and we identify an enhancer region upstream of the gene that imposes r3/r5 expression in transgenic mice. This enhancer contains three Krox20-binding sites required in vitro for complex formation with Krox20 protein and in vivo for rhombomere-restricted expression. In transgenic mice, Krox20 expressed in ectopic domains can transactivate a reporter construct containing the HoxB2 r3/r5 enhancer. These data demonstrate that Krox20 is a part of the upstream transcriptional cascade that directly regulates HoxB2 expression during hindbrain segmentation.

Analysis of high iron rice lines reveals new miRNAs that target iron transporters in roots

PubMed Central

Paul, Soumitra; Gayen, Dipak; Datta, Swapan K.; Datta, Karabi

2016-01-01

The present study highlights the molecular regulation of iron transport in soyFER1-overexpressing transgenic rice. Accumulation of iron in three different seed developmental stages, milk, dough, and mature, has been examined. The transgenic seeds of the milk stage showed significant augmentation of iron and zinc levels compared with wild-type seeds, and similar results were observed throughout the dough and mature stages. To investigate the regulation of iron transport, the role of miRNAs was studied in roots of transgenic rice. Sequencing of small RNA libraries revealed 153 known and 41 novel miRNAs in roots. Among them, 59 known and 14 novel miRNAs were found to be significantly expressed. miR166, miR399, and miR408 were identified as playing a vital role in iron uptake in roots of transgenic plants . Most importantly, four putative novel miRNAs, namely miR11, miR26, miR30, and miR31, were found to be down-regulated in roots of transgenic plants. For all these four novel miRNAs, natural resistance-associated macrophage protein 4 (NRAMP4), encoding a metal transporter, was predicted as a target gene. It is hypothesized that the NRAMP4 transporter is activated in roots of transgenic plants due to the lower abundance of its corresponding putative novel miRNAs. The relative transcript level of the NRAMP4 transcript was increased from 0.107 in the wild type to 65.24 and 55.39 in transgenic plants, which demonstrates the elevated amount of iron transport in transgenic plants. In addition, up-regulation of OsYSL15, OsFRO2, and OsIRT1 in roots also facilitates iron loading in transgenic seeds. PMID:27729476

RNA interference of GhPEPC2 enhanced seed oil accumulation and salt tolerance in Upland cotton.

PubMed

Zhao, Yanpeng; Huang, Yi; Wang, Yumei; Cui, Yupeng; Liu, Zhengjie; Hua, Jinping

2018-06-01

Phosphoenolpyruvate carboxylase (PEPCase) mainly produces oxaloacetic acid for tricarboxylic acid (TCA) cycle. Here we reported that GhPEPC2 silencing with PEPC2-RNAi vector could regulate oil and protein accumulation in cottonseeds. In GhPEPC2 transgenic plants, PEPCase activities in immature embryos were significantly reduced, and the oil content in seed kernel was increased 7.3 percentages, whereas total proteins decreased 5.65 percentages. Compared to wild type, agronomical traits of transgenic plant were obviously unaffected. Furthermore, gene expression profile of GhPEPC2 transgenic seeds were investigated using RNA-seq, most lipid synthesis related genes were up-regulated, but amino acid metabolic related genes were down-regulated. In addition, the GhPEPC2 transgenic cotton seedlings were stressed using sodium salts at seedling stage, and the salt tolerance was significantly enhanced. Our observations of GhPEPC2 in cotton would shade light on understanding the regulation of oil content, protein accumulation and salt tolerance enhancement in other plants. Copyright © 2018 Elsevier B.V. All rights reserved.

The transcription factor AREB1 regulates primary metabolic pathways in tomato fruits

PubMed Central

Bastías, Adriana; Osorio, Sonia; Casaretto, José A.

2014-01-01

Tomato fruit development is regulated both by the action of plant hormones and by tight genetic control. Recent studies suggest that abscisic acid (ABA) signalling may affect different aspects of fruit maturation. Previously, it was shown that SlAREB1, an ABA-regulated transcription factor involved in stress-induced responses, is expressed in seeds and in fruit tissues in tomato. Here, the role of SlAREB1 in regulating the expression of genes relevant for primary metabolic pathways and affecting the metabolic profile of the fruit was investigated using transgenic tomato lines. Metabolite profiling using gas chromatography–time of flight mass spectrometry (GC-TOF-MS) and non-targeted liquid chromatography–mass spectrometry (LC-MS) was performed on pericarp tissue from fruits harvested at three stages of fruit development. Principal component analysis of the data could distinguish the metabolite profiles of non-transgenic fruits from those that overexpress and down-regulate SlAREB1. Overexpression of SlAREB1 resulted in increased content of organic acids, hexoses, hexose-phosphates, and amino acids in immature green, mature green, and red ripe fruits, and these modifications correlated with the up-regulation of enzyme-encoding genes involved in primary carbohydrate and amino acid metabolism. A non-targeted LC-MS analysis indicated that the composition of secondary metabolites is also affected in transgenic lines. In addition, gene expression data revealed that some genes associated with fruit ripening are also up-regulated in SlAREB1-overexpressing lines compared with wild-type and antisense lines. Taken together, the results suggest that SlAREB1 participates in the regulation of the metabolic programming that takes place during fruit ripening and that may explain part of the role of ABA in fruit development in tomato. PMID:24659489

Prediction and characterisation of a highly conserved, remote and cAMP responsive enhancer that regulates Msx1 gene expression in cardiac neural crest and outflow tract.

PubMed

Miller, Kerry Ann; Davidson, Scott; Liaros, Angela; Barrow, John; Lear, Marissa; Heine, Danielle; Hoppler, Stefan; MacKenzie, Alasdair

2008-05-15

Double knockouts of the Msx1 and Msx2 genes in the mouse result in severe cardiac outflow tract malformations similar to those frequently found in newborn infants. Despite the known role of the Msx genes in cardiac formation little is known of the regulatory systems (ligand receptor, signal transduction and protein-DNA interactions) that regulate the tissue-specific expression of the Msx genes in mammals during the formation of the outflow tract. In the present study we have used a combination of multi-species comparative genomics, mouse transgenic analysis and in-situ hybridisation to predict and validate the existence of a remote ultra-conserved enhancer that supports the expression of the Msx1 gene in migrating mouse cardiac neural crest and the outflow tract primordia. Furthermore, culturing of embryonic explants derived from transgenic lines with agonists of the PKC and PKA signal transduction systems demonstrates that this remote enhancer is influenced by PKA but not PKC dependent gene regulatory systems. These studies demonstrate the efficacy of combining comparative genomics and transgenic analyses and provide a platform for the study of the possible roles of Msx gene mis-regulation in the aetiology of congenital heart malformation.

Functional Assessment of the Role of BORIS in Ovarian Cancer Using a Novel in Vivo Model System

DTIC Science & Technology

2015-12-01

iv) we obtained founder BORIS-Tg mice and crossed into the FVB/N strain to fully characterize the transgenic gene configuration, v) we conducted...models, transgenic mice 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON USAMRMC a...genes, and wildtype p53 is a negative regulator of BORIS expression. To test these hypotheses, we will develop and utilize a murine transgenic model

Nuclear hormone retinoid X receptor (RXR) negatively regulates the glucose-stimulated insulin secretion of pancreatic ß-cells.

PubMed

Miyazaki, Satsuki; Taniguchi, Hidenori; Moritoh, Yusuke; Tashiro, Fumi; Yamamoto, Tsunehiko; Yamato, Eiji; Ikegami, Hiroshi; Ozato, Keiko; Miyazaki, Jun-ichi

2010-11-01

Retinoid X receptors (RXRs) are members of the nuclear hormone receptor superfamily and are thought to be key regulators in differentiation, cellular growth, and gene expression. Although several experiments using pancreatic β-cell lines have shown that the ligands of nuclear hormone receptors modulate insulin secretion, it is not clear whether RXRs have any role in insulin secretion. To elucidate the function of RXRs in pancreatic β-cells, we generated a double-transgenic mouse in which a dominant-negative form of RXRβ was inducibly expressed in pancreatic β-cells using the Tet-On system. We also established a pancreatic β-cell line from an insulinoma caused by the β-cell-specific expression of simian virus 40 T antigen in the above transgenic mouse. In the transgenic mouse, expression of the dominant-negative RXR enhanced the insulin secretion with high glucose stimulation. In the pancreatic β-cell line, the suppression of RXRs also enhanced glucose-stimulated insulin secretion at a high glucose concentration, while 9-cis-retinoic acid, an RXR agonist, repressed it. High-density oligonucleotide microarray analysis showed that expression of the dominant-negative RXR affected the expression levels of a number of genes, some of which have been implicated in the function and/or differentiation of β-cells. These results suggest that endogenous RXR negatively regulates the glucose-stimulated insulin secretion. Given these findings, we propose that the modulation of endogenous RXR in β-cells may be a new therapeutic approach for improving impaired insulin secretion in type 2 diabetes.

Evaluation of an FRDA-EGFP genomic reporter assay in transgenic mice.

PubMed

Sarsero, Joseph P; Holloway, Timothy P; Li, Lingli; McLenachan, Samuel; Fowler, Kerry J; Bertoncello, Ivan; Voullaire, Lucille; Gazeas, Sophie; Ioannou, Panos A

2005-04-01

Friedreich ataxia is an autosomal recessive neurodegenerative disorder caused by a GAA trinucleotide expansion in the first intron of the Friedreich ataxia gene (FRDA) that causes reduced synthesis of frataxin, a mitochondrial protein likely to be involved in biosynthesis of iron-sulfur clusters. This leads to increased oxidative stress, progressive loss of large sensory neurons, and hypertrophic cardiomyopathy. To elucidate the mechanisms regulating FRDA expression and to develop an in vivo assay for agents that might upregulate FRDA expression in a therapeutically relevant manner, we have generated transgenic mice with a BAC genomic reporter construct consisting of an in-frame fusion between FRDA and the gene coding for enhanced green fluorescent protein (EGFP). Production of full-length frataxin-EGFP fusion protein was demonstrated by immunoblotting. EGFP expression was observed as early as day E3.5 of development. Most tissues of adult transgenic mice were fluorescent. The level of FRDA-EGFP expression in peripheral blood, bone marrow, and cells obtained from enzymatically disaggregated tissues was quantitated by flow cytometry. There was a twofold increase in EGFP expression in mice homozygous for the transgene when compared to hemizygous mice. These transgenic mice are a valuable tool for the examination of spatial and temporal aspects of FRDA gene expression and for the preclinical evaluation of pharmacological inducers of FRDA expression in a whole-animal model. In addition, tissues from these mice should also be valuable for stem cell transplantation studies.

Regulation of expression of transgenes in developing fish.

PubMed

Moav, B; Liu, Z; Caldovic, L D; Gross, M L; Faras, A J; Hackett, P B

1993-05-01

The transcriptional regulatory elements of the beta-actin gene of carp (Cyprinus carpio) have been examined in zebrafish and goldfish harbouring transgenes. The high sequence conservation of the putative regulatory elements in the beta-actin genes of animals suggested that their function would be conserved, so that transgenic constructs with the same transcriptional control elements would promote similar levels of transgene expression in different species of transgenic animals. To test this assumption, we analysed the temporal expression of a reporter gene under the control of transcriptional control sequences from the carp beta-actin gene in zebrafish (Brachydanio rerio) and goldfish (Carrasius auratus). Our results indicated that, contrary to expectations, combinations of different transcriptional control elements affected the level, duration, and onset of gene expression differently in developing zebrafish and goldfish. The major differences in expression of beta-actin/CAT (chloramphenicol acetyltransferase) constructs in zebrafish and goldfish were: (1) overall expression was almost 100-fold higher in goldfish than in zebrafish embryos, (2) the first intron had an enhancing effect on gene expression in zebrafish but not in goldfish, and (3) the serum-responsive/CArG-containing regulatory element in the proximal promoter was not always required for maximal CAT activity in goldfish, but was required in zebrafish. These results suggest that in the zebrafish, but not in the goldfish, there may be interactions between motifs in the proximal promoter and the first intron which appear to be required for maximal enhancement of transcription.

Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat.

PubMed

Nemeth, Csilla; Freeman, Jackie; Jones, Huw D; Sparks, Caroline; Pellny, Till K; Wilkinson, Mark D; Dunwell, Jim; Andersson, Annica A M; Aman, Per; Guillon, Fabienne; Saulnier, Luc; Mitchell, Rowan A C; Shewry, Peter R

2010-03-01

(1,3;1,4)-beta-d-Glucan (beta-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative beta-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total beta-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable beta-glucan was also reduced by about 50% in the transgenic lines, and the M(r) distribution of the fraction was decreased from an average of 79 to 85 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

Transformation of miniature potted rose (Rosa hybrida cv. Linda) with P( SAG12 )-ipt gene delays leaf senescence and enhances resistance to exogenous ethylene.

PubMed

Zakizadeh, Hedayat; Lütken, Henrik; Sriskandarajah, Sridevy; Serek, Margrethe; Müller, Renate

2013-02-01

KEY MESSAGE : The P ( SAG12 ) -ipt gene was transferred to miniature rose, as the first woody species, resulting in increased ethylene resistance due to specific up-regulation of the ipt gene under senescence promoting conditions. Transgenic plants of Rosa hybrida 'Linda' were obtained via transformation with Agrobacterium tumefaciens strain harboring the binary vector pSG529(+) containing the P( SAG12 )-ipt construct. A. tumefaciens strains AGL1, GV3850 and LBA4404 (containing P(35S)-INTGUS gene) were used for transformation of embryogenic callus, but transgenic shoots were obtained only when AGL1 was applied. The highest transformation frequency was 10 % and it was achieved when half MS medium was used for the dilution of overnight culture of Agrobacterium. Southern blot confirmed integration of 1-6 copies of the nptII gene into the rose genome in the tested lines. Four transgenic lines were obtained which were morphologically true-to-type and indistinguishable from Wt shoots while they were in in vitro cultures. Adventitious root induction was more difficult in transgenic shoots compared to the Wt shoots, however, one of the transgenic lines (line 6) was rooted and subsequently analyzed phenotypically. The ipt expression levels were determined in this line after exposure to exogenous ethylene (3.5 μl l(-1)) and/or darkness. Darkness resulted in twofold up-regulation of ipt expression, whereas darkness combined with ethylene caused eightfold up-regulation in line 6 compared to Wt plants. The transgenic line had significantly higher content of chlorophyll at the end of the treatment period compared to Wt plants.

Regulation of the gut-specific carboxypeptidase: a study using the binary Gal4/UAS system in the mosquito Aedes aegypti

PubMed Central

Zhao, Bo; Kokoza, Vladimir A.; Saha, Tusar T.; Wang, Stephanie; Roy, Sourav; Raikhel, Alexander S.

2015-01-01

Pathogen transmission by mosquitoes is tightly linked to blood feeding which, in turn, is required for egg development. Studies of these processes would greatly benefit from genetic methods, such as the binary Gal4/UAS system. The latter has been well established for model organisms, but its availability is limited for mosquitoes. The objective of this study was to develop the blood-meal-activated, gut-specific Gal4/UAS system for the yellow-fever mosquito Aedes aegypti and utilize it to investigate the regulation of gut-specific gene expression. A 1.1-kb, 5' upstream region of the carboxypeptidase A (CP) gene was used to genetically engineer the CP-Gal4 driver mosquito line. The CP-Gal4 specifically activated the Enhanced Green Fluorescent Protein (EGFP) reporter only after blood feeding in the gut of the CP-Gal4>UAS-EGFP female Ae. aegypti. We used this system to study the regulation of CP gene expression. In vitro treatments with either amino acids (AAs) or insulin stimulated expression of the CP-Gal4>UAS-EGFP transgene; no effect was observed with 20-hydroxyecdysone (20E) treatments. The transgene activation by AAs and insulin was blocked by rapamycin, the inhibitor of the Target-of-Rapamycin kinase (TOR). RNA interference (RNAi) silence of the insulin receptor (IR) reduced the expression of the CP-Gal4>UAS-EGFP transgene. Thus, in vitro and in vivo experiments have revealed that insulin and TOR pathways control expression of the digestive enzyme CP. In contrast, 20E, the major regulator of post-blood-meal vitellogenic events in female mosquitoes, has no role in regulating the expression of this gene. This novel CP-Gal4/UAS system permits functional testing of midgut-specific genes that are involved in blood digestion and interaction with pathogens in Ae. aegypti mosquitoes. PMID:25152428

c-myc, c-fos, and c-jun regulation in the regenerating livers of normal and H-2K/c-myc transgenic mice.

PubMed Central

Morello, D; Fitzgerald, M J; Babinet, C; Fausto, N

1990-01-01

We investigated the mechanisms of regulation of c-myc, c-fos, and c-jun at the early stages of liver regeneration in mice. We show that the transient increase in steady-state levels of c-myc mRNA at the start of liver regeneration is most probably regulated by posttranscriptional mechanisms. Although there was a marked increase in c-myc transcriptional initiation shortly after partial hepatectomy, a block in elongation prevented the completion of most transcripts. To gain further information on the mechanism of regulation of c-myc expression during liver regeneration, we used transgenic mice harboring the human c-myc gene driven by the H-2K promoter. In these animals, the murine c-myc responded to the growth stimulus generated by partial hepatectomy, whereas the expression of the transgene was constitutive and did not change in the regenerating liver. However, the mRNA from both genes increased markedly after cycloheximide injection, suggesting that the regulation of c-myc mRNA abundance in the regenerating liver differs from that occurring after protein synthesis inhibition. Furthermore, we show that in normal mice c-fos and c-jun mRNA levels and transcriptional rates increase within 30 min after partial hepatectomy. c-fos transcriptional elongation was restricted in nongrowing liver, but the block was partially relieved in the regenerating liver. Nevertheless, for both c-fos and c-jun, changes in steady-state mRNA detected after partial hepatectomy were much greater than the transcriptional increase. In the regenerating liver of H-2K/c-myc mice, c-fos and c-jun expression was diminished, whereas mouse c-myc expression was enhanced in comparison with that in nontransgenic animals. Images PMID:2111449

Stress Inducible Expression of AtDREB1A Transcription Factor in Transgenic Peanut (Arachis hypogaea L.) Conferred Tolerance to Soil-Moisture Deficit Stress

PubMed Central

Sarkar, Tanmoy; Thankappan, Radhakrishnan; Kumar, Abhay; Mishra, Gyan P.; Dobaria, Jentilal R.

2016-01-01

Peanut, an important oilseed crop, is gaining priority for the development of drought tolerant genotypes in recent times, since the area under drought is constantly on the rise. To achieve this, one of the important strategies is to genetically engineer the ruling peanut varieties using transcription factor regulating the expression of several downstream, abiotic-stress responsive gene(s). In this study, eight independent transgenic peanut (cv. GG20) lines were developed using AtDREB1A gene, encoding for a transcription factor, through Agrobacterium-mediated genetic transformation. The transgene insertion was confirmed in (T0) using PCR and Dot-blot analysis, while copy-number(s) was ascertained using Southern-blot analysis. The inheritance of AtDREB1A gene in individual transgenic plants (T1 and T2) was confirmed using PCR. In homozygous transgenic plants (T2), under soil-moisture deficit stress, elevated level of AtDREB1A transgene expression was observed by RT-PCR assay. The transgenic plants at 45-d or reproductive growth stage showed tolerance to severe soil-moisture deficit stress. Physio-biochemical parameters such as proline content, osmotic potential, relative water content, electrolytic leakage, and total-chlorophyll content were found positively correlated with growth-related traits without any morphological abnormality, when compared to wild-type. qPCR analysis revealed consistent increase in expression of AtDREB1A gene under progressive soil-moisture deficit stress in two homozygous transgenic plants. The transgene expression showed significant correlation with improved physio-biochemical traits. The improvement of drought-stress tolerance in combination with improved growth-related traits is very essential criterion for a premium peanut cultivar like GG20, so that marginal farmers of India can incur the economic benefits during seasonal drought and water scarcity. PMID:27446163

Transient development of ovotestes in XX Sox9 transgenic mice

PubMed Central

Gregoire, Elodie P.; Lavery, Rowena; Chassot, Anne-Amandine; Akiyama, Haruhiko; Treier, Mathias; Behringer, Richard R.; Chaboissier, Marie-Christine

2010-01-01

The sex of an individual results from the paternal transmission of the SRY gene located on the Y chromosome. In turn, SRY initiates Sox9 expression, a transcription factor required for testicular differentiation. Ectopic activation of SOX9 in XX Wt1:Sox9 transgenic mice, induces female-to-male sex reversal in adult mice. Here we show that complete sex reversal is preceded by a transient phase of ovotestis differentiation with XX Wt1:Sox9 transgenic gonads containing a testicular central region and one or both ovarian poles indicating that Wt1:Sox9 is not as efficient as Sry to induce male development. In XX Wt1:Sox9Tg/+ gonads, transgenic Sox9 is expressed earlier than Sox9 in XY gonads, and is able to induce the expression of EGFP, knocked into the 3′ UTR of Sox9 indicating that SOX9 is involved in the initiation and maintenance of its own expression. However, the delayed onset of expression of endogenous Sox9-EGFP suggests that this activation requires other factors, whose expression depends on SOX9. In the testicular regions of the XX Wt1:Sox9 ovotestes, proliferation of the XX foetal germ cells is hampered and they differentiate as pro-spermatogonia. This indicates that XX germ cells are not competent to respond to proliferative signals released from a testicular environment. In the ovarian regions, despite the continuous mRNA expression of the WT1:Sox9 transgene, the SOX9 protein does not accumulate suggesting that regulation of this gene in ovarian cells involves post-transcriptional mechanisms. Finally, ovarian cells of the XX Wt1:Sox9 ovotestis undergo apoptosis during late embryogenesis leading to complete female-to-male sex reversal of the transgenic mice at birth. PMID:20965161

Regulation of COL1A1 expression in type I collagen producing tissues: identification of a 49 base pair region which is required for transgene expression in bone of transgenic mice

NASA Technical Reports Server (NTRS)

Bedalov, A.; Salvatori, R.; Dodig, M.; Kronenberg, M. S.; Kapural, B.; Bogdanovic, Z.; Kream, B. E.; Woody, C. O.; Clark, S. H.; Mack, K.;

Arabidopsis TRANSPARENT TESTA GLABRA2 is directly regulated by R2R3 MYB transcription factors and is involved in regulation of GLABRA2 transcription in epidermal differentiation.

PubMed

Ishida, Tetsuya; Hattori, Sayoko; Sano, Ryosuke; Inoue, Kayoko; Shirano, Yumiko; Hayashi, Hiroaki; Shibata, Daisuke; Sato, Shusei; Kato, Tomohiko; Tabata, Satoshi; Okada, Kiyotaka; Wada, Takuji

2007-08-01

Arabidopsis thaliana TRANSPARENT TESTA GLABRA2 (TTG2) encodes a WRKY transcription factor and is expressed in young leaves, trichomes, seed coats, and root hairless cells. An examination of several trichome and root hair mutants indicates that MYB and bHLH genes regulate TTG2 expression. Two MYB binding sites in the TTG2 5' regulatory region act as cis regulatory elements and as direct targets of R2R3 MYB transcription factors such as WEREWOLF, GLABRA1, and TRANSPARENT TESTA2. Mutations in TTG2 cause phenotypic defects in trichome development and seed color pigmentation. Transgenic plants expressing a chimeric repressor version of the TTG2 protein (TTG2:SRDX) showed defects in trichome formation, anthocyanin accumulation, seed color pigmentation, and differentiation of root hairless cells. GLABRA2 (GL2) expression was markedly reduced in roots of ProTTG2:TTG2:SRDX transgenic plants, suggesting that TTG2 is involved in the regulation of GL2 expression, although GL2 expression in the ttg2 mutant was similar to that in the wild type. Our analysis suggests a new step in a regulatory cascade of epidermal differentiation, in which complexes containing R2R3 MYB and bHLH transcription factors regulate the expression of TTG2, which then regulates GL2 expression with complexes containing R2R3 MYB and bHLH in the differentiation of trichomes and root hairless cells.

Arabidopsis TRANSPARENT TESTA GLABRA2 Is Directly Regulated by R2R3 MYB Transcription Factors and Is Involved in Regulation of GLABRA2 Transcription in Epidermal Differentiation[W

PubMed Central

Ishida, Tetsuya; Hattori, Sayoko; Sano, Ryosuke; Inoue, Kayoko; Shirano, Yumiko; Hayashi, Hiroaki; Shibata, Daisuke; Sato, Shusei; Kato, Tomohiko; Tabata, Satoshi; Okada, Kiyotaka; Wada, Takuji

2007-01-01

Arabidopsis thaliana TRANSPARENT TESTA GLABRA2 (TTG2) encodes a WRKY transcription factor and is expressed in young leaves, trichomes, seed coats, and root hairless cells. An examination of several trichome and root hair mutants indicates that MYB and bHLH genes regulate TTG2 expression. Two MYB binding sites in the TTG2 5′ regulatory region act as cis regulatory elements and as direct targets of R2R3 MYB transcription factors such as WEREWOLF, GLABRA1, and TRANSPARENT TESTA2. Mutations in TTG2 cause phenotypic defects in trichome development and seed color pigmentation. Transgenic plants expressing a chimeric repressor version of the TTG2 protein (TTG2:SRDX) showed defects in trichome formation, anthocyanin accumulation, seed color pigmentation, and differentiation of root hairless cells. GLABRA2 (GL2) expression was markedly reduced in roots of ProTTG2:TTG2:SRDX transgenic plants, suggesting that TTG2 is involved in the regulation of GL2 expression, although GL2 expression in the ttg2 mutant was similar to that in the wild type. Our analysis suggests a new step in a regulatory cascade of epidermal differentiation, in which complexes containing R2R3 MYB and bHLH transcription factors regulate the expression of TTG2, which then regulates GL2 expression with complexes containing R2R3 MYB and bHLH in the differentiation of trichomes and root hairless cells. PMID:17766401

Ubiquitin fusion expression and tissue-dependent targeting of hG-CSF in transgenic tobacco

PubMed Central

2011-01-01

Background Human granulocyte colony-stimulating factor (hG-CSF) is an important human cytokine which has been widely used in oncology and infection protection. To satisfy clinical needs, expression of recombinant hG-CSF has been studied in several organisms, including rice cell suspension culture and transient expression in tobacco leaves, but there was no published report on its expression in stably transformed plants which can serve as a more economical expression platform with potential industrial application. Results In this study, hG-CSF expression was investigated in transgenic tobacco leaves and seeds in which the accumulation of hG-CSF could be enhanced through fusion with ubiquitin by up to 7 fold in leaves and 2 fold in seeds, leading to an accumulation level of 2.5 mg/g total soluble protein (TSP) in leaves and 1.3 mg/g TSP in seeds, relative to hG-CSF expressed without a fusion partner. Immunoblot analysis showed that ubiquitin was processed from the final protein product, and ubiquitination was up-regulated in all transgenic plants analyzed. Driven by CaMV 35S promoter and phaseolin signal peptide, hG-CSF was observed to be secreted into apoplast in leaves but deposited in protein storage vacuole (PSV) in seeds, indicating that targeting of the hG-CSF was tissue-dependent in transgenic tobacco. Bioactivity assay showed that hG-CSF expressed in both seeds and leaves was bioactive to support the proliferation of NFS-60 cells. Conclusions In this study, the expression of bioactive hG-CSF in transgenic plants was improved through ubiquitin fusion strategy, demonstrating that protein expression can be enhanced in both plant leaves and seeds through fusion with ubiquitin and providing a typical case of tissue-dependent expression of recombinant protein in transgenic plants. PMID:21985646

Transcriptome Profiling Revealed Stress-Induced and Disease Resistance Genes Up-Regulated in PRSV Resistant Transgenic Papaya

PubMed Central

Fang, Jingping; Lin, Aiting; Qiu, Weijing; Cai, Hanyang; Umar, Muhammad; Chen, Rukai; Ming, Ray

2016-01-01

Papaya is a productive and nutritious tropical fruit. Papaya Ringspot Virus (PRSV) is the most devastating pathogen threatening papaya production worldwide. Development of transgenic resistant varieties is the most effective strategy to control this disease. However, little is known about the genome-wide functional changes induced by particle bombardment transformation. We conducted transcriptome sequencing of PRSV resistant transgenic papaya SunUp and its PRSV susceptible progenitor Sunset to compare the transcriptional changes in young healthy leaves prior to infection with PRSV. In total, 20,700 transcripts were identified, and 842 differentially expressed genes (DEGs) randomly distributed among papaya chromosomes. Gene ontology (GO) category analysis revealed that microtubule-related categories were highly enriched among these DEGs. Numerous DEGs related to various transcription factors, transporters and hormone biosynthesis showed clear differences between the two cultivars, and most were up-regulated in transgenic papaya. Many known and novel stress-induced and disease-resistance genes were most highly expressed in SunUp, including MYB, WRKY, ERF, NAC, nitrate and zinc transporters, and genes involved in the abscisic acid, salicylic acid, and ethylene signaling pathways. We also identified 67,686 alternative splicing (AS) events in Sunset and 68,455 AS events in SunUp, mapping to 10,994 and 10,995 papaya annotated genes, respectively. GO enrichment for the genes displaying AS events exclusively in Sunset was significantly different from those in SunUp. Transcriptomes in Sunset and transgenic SunUp are very similar with noteworthy differences, which increased PRSV-resistance in transgenic papaya. No detrimental pathways and allergenic or toxic proteins were induced on a genome-wide scale in transgenic SunUp. Our results provide a foundation for unraveling the mechanism of PRSV resistance in transgenic papaya. PMID:27379138

Expression of Xylella fastidiosa RpfF in citrus disrupts signaling in Xanthomonas citri subsp. citri and thereby its virulence.

PubMed

Caserta, R; Picchi, S C; Takita, M A; Tomaz, J P; Pereira, W E L; Machado, M A; Ionescu, M; Lindow, S; De Souza, A A

2014-11-01

Xylella fastidiosa and Xanthomonas citri subsp. citri, that cause citrus variegated chlorosis (CVC) and citrus canker diseases, respectively, utilize diffusible signal factor (DSF) for quorum sensing. DSF, produced by RpfF, are similar fatty acids in both organisms, although a different set of genes is regulated by DSF in each species. Because of this similarity, Xylella fastidiosa DSF might be recognized and affect the biology of Xanthomonas citri. Therefore, transgenic Citrus sinensis and Carrizo citrange plants overexpressing the Xylella fastidiosa rpfF were inoculated with Xanthomonas citri and changes in symptoms of citrus canker were observed. X. citri biofilms formed only at wound sites on transgenic leaves and were thicker; however, bacteria were unable to break through the tissue and form pustules elsewhere. Although abundant growth of X. citri occurred at wound sites on inoculated transgenic leaves, little growth was observed on unwounded tissue. Genes in the DFS-responsive core in X. citri were downregulated in bacteria isolated from transgenic leaves. DSF-dependent expression of engA was suppressed in cells exposed to xylem sap from transgenic plants. Thus, altered symptom development appears to be due to reduced expression of virulence genes because of the presence of antagonists of DSF signaling in X. citri in rpfF-expressing plants.

Transgenic cotton over-producing spinach sucrose phosphate synthase showed enhanced leaf sucrose synthesis and improved fiber quality under controlled environmental conditions.

PubMed

Haigler, Candace H; Singh, Bir; Zhang, Deshui; Hwang, Sangjoon; Wu, Chunfa; Cai, Wendy X; Hozain, Mohamed; Kang, Wonhee; Kiedaisch, Brett; Strauss, Richard E; Hequet, Eric F; Wyatt, Bobby G; Jividen, Gay M; Holaday, A Scott

2007-04-01

Prior data indicated that enhanced availability of sucrose, a major product of photosynthesis in source leaves and the carbon source for secondary wall cellulose synthesis in fiber sinks, might improve fiber quality under abiotic stress conditions. To test this hypothesis, a family of transgenic cotton plants (Gossypium hirsutum cv. Coker 312 elite) was produced that over-expressed spinach sucrose-phosphate synthase (SPS) because of its role in regulation of sucrose synthesis in photosynthetic and heterotrophic tissues. A family of 12 independent transgenic lines was characterized in terms of foreign gene insertion, expression of spinach SPS, production of spinach SPS protein, and development of enhanced extractable V (max) SPS activity in leaf and fiber. Lines with the highest V (max) SPS activity were further characterized in terms of carbon partitioning and fiber quality compared to wild-type and transgenic null controls. Leaves of transgenic SPS over-expressing lines showed higher sucrose:starch ratio and partitioning of (14)C to sucrose in preference to starch. In two growth chamber experiments with cool nights, ambient CO(2) concentration, and limited light below the canopy, the transgenic line with the highest SPS activity in leaf and fiber had higher fiber micronaire and maturity ratio associated with greater thickness of the cellulosic secondary wall.

Expression of a calpastatin transgene slows muscle wasting and obviates changes in myosin isoform expression during murine muscle disuse

NASA Technical Reports Server (NTRS)

Tidball, James G.; Spencer, Melissa J.

2002-01-01

Muscle wasting is a prominent feature of several systemic diseases, neurological damage and muscle disuse. The contribution of calpain proteases to muscle wasting in any instance of muscle injury or disease has remained unknown because of the inability to specifically perturb calpain activity in vivo. We have generated a transgenic mouse with muscle-specific overexpression of calpastatin, which is the endogenous inhibitor of calpains, and induced muscle atrophy by unloading hindlimb musculature for 10 days. Expression of the transgene resulted in increases in calpastatin concentration in muscle by 30- to 50-fold, and eliminated all calpain activity that was detectable on zymograms. Muscle fibres in ambulatory, transgenic mice were smaller in diameter, but more numerous, so that muscle mass did not differ between transgenic and non-transgenic mice. This is consistent with the role of the calpain-calpastatin system in muscle cell fusion that has been observed in vitro. Overexpression of calpastatin reduced muscle atrophy by 30 % during the 10 day unloading period. In addition, calpastatin overexpression completely prevented the shift in myofibrillar myosin content from slow to fast isoforms, which normally occurs in muscle unloading. These findings indicate that therapeutics directed toward regulating the calpain-calpastatin system may be beneficial in preventing muscle mass loss in muscle injury and disease.

Expression of a calpastatin transgene slows muscle wasting and obviates changes in myosin isoform expression during murine muscle disuse.

PubMed

Tidball, James G; Spencer, Melissa J

2002-12-15

Muscle wasting is a prominent feature of several systemic diseases, neurological damage and muscle disuse. The contribution of calpain proteases to muscle wasting in any instance of muscle injury or disease has remained unknown because of the inability to specifically perturb calpain activity in vivo. We have generated a transgenic mouse with muscle-specific overexpression of calpastatin, which is the endogenous inhibitor of calpains, and induced muscle atrophy by unloading hindlimb musculature for 10 days. Expression of the transgene resulted in increases in calpastatin concentration in muscle by 30- to 50-fold, and eliminated all calpain activity that was detectable on zymograms. Muscle fibres in ambulatory, transgenic mice were smaller in diameter, but more numerous, so that muscle mass did not differ between transgenic and non-transgenic mice. This is consistent with the role of the calpain-calpastatin system in muscle cell fusion that has been observed in vitro. Overexpression of calpastatin reduced muscle atrophy by 30 % during the 10 day unloading period. In addition, calpastatin overexpression completely prevented the shift in myofibrillar myosin content from slow to fast isoforms, which normally occurs in muscle unloading. These findings indicate that therapeutics directed toward regulating the calpain-calpastatin system may be beneficial in preventing muscle mass loss in muscle injury and disease.

Improved forage digestibility of tall fescue (Festuca arundinacea) by transgenic down-regulation of cinnamyl alcohol dehydrogenase.

PubMed

Chen, Lei; Auh, Chung-Kyoon; Dowling, Paul; Bell, Jeremey; Chen, Fang; Hopkins, Andrew; Dixon, Richard A; Wang, Zeng-Yu

2003-11-01

Lignification of cell walls during plant development has been identified as the major factor limiting forage digestibility and concomitantly animal productivity. cDNA sequences encoding a key lignin biosynthetic enzyme, cinnamyl alcohol dehydrogenase (CAD), were cloned from the widely grown monocotyledonous forage species tall fescue (Festuca arundinacea Schreb.). Recombinant tall fescue CAD expressed in E. coli exhibited the highest V(max)/K(m) values when coniferaldehyde and sinapaldehyde were used as substrates. Transgenic tall fescue plants carrying either sense or antisense CAD gene constructs were obtained by microprojectile bombardment of single genotype-derived embryogenic suspension cells. Severely reduced levels of mRNA transcripts and significantly reduced CAD enzymatic activities were found in two transgenic plants carrying sense and antisense CAD transgenes, respectively. These CAD down-regulated transgenic lines had significantly decreased lignin content and altered ratios of syringyl (S) to guaiacyl (G), G to p-hydroxyphenyl (H) and S to H units. No significant changes in cellulose, hemicellulose, neutral sugar composition, p-coumaric acid and ferulic acid levels were observed in the transgenic plants. Increases of in vitro dry matter digestibility of 7.2-9.5% were achieved in the CAD down-regulated lines, thus providing a novel germplasm to be used for the development of grass cultivars with improved forage quality.

Metabolic Regulation of Carotenoid-Enriched Golden Rice Line.

PubMed

Gayen, Dipak; Ghosh, Subhrajyoti; Paul, Soumitra; Sarkar, Sailendra N; Datta, Swapan K; Datta, Karabi

2016-01-01

Vitamin A deficiency (VAD) is the leading cause of blindness among children and is associated with high risk of maternal mortality. In order to enhance the bioavailability of vitamin A, high carotenoid transgenic golden rice has been developed by manipulating enzymes, such as phytoene synthase ( psy) and phytoene desaturase ( crtI ). In this study, proteome and metabolite analyses were carried out to comprehend metabolic regulation and adaptation of transgenic golden rice after the manipulation of endosperm specific carotenoid pathways. The main alteration was observed in carbohydrate metabolism pathways of the transgenic seeds. The 2D based proteomic studies demonstrated that carbohydrate metabolism-related enzymes, such as pullulanase, UDP-glucose pyrophosphorylase, and glucose-1-phosphate adenylyltransferase, were primarily up-regulated in transgenic rice seeds. In addition, the enzyme PPDK was also elevated in transgenic seeds thus enhancing pyruvate biosynthesis, which is the precursor in the carotenoids biosynthetic pathway. GC-MS based metabolite profiling demonstrated an increase in the levels of glyceric acid, fructo-furanose, and galactose, while decrease in galactonic acid and gentiobiose in the transgenic rice compared to WT. It is noteworthy to mention that the carotenoid content, especially β-carotene level in transgenic rice (4.3 μg/g) was significantly enhanced. The present study highlights the metabolic adaptation process of a transgenic golden rice line (homozygous T4 progeny of SKBR-244) after enhancing carotenoid biosynthesis. The presented information would be helpful in the development of crops enriched in carotenoids by expressing metabolic flux of pyruvate biosynthesis.

Metabolic Regulation of Carotenoid-Enriched Golden Rice Line

PubMed Central

Gayen, Dipak; Ghosh, Subhrajyoti; Paul, Soumitra; Sarkar, Sailendra N.; Datta, Swapan K.; Datta, Karabi

2016-01-01

Vitamin A deficiency (VAD) is the leading cause of blindness among children and is associated with high risk of maternal mortality. In order to enhance the bioavailability of vitamin A, high carotenoid transgenic golden rice has been developed by manipulating enzymes, such as phytoene synthase (psy) and phytoene desaturase (crtI). In this study, proteome and metabolite analyses were carried out to comprehend metabolic regulation and adaptation of transgenic golden rice after the manipulation of endosperm specific carotenoid pathways. The main alteration was observed in carbohydrate metabolism pathways of the transgenic seeds. The 2D based proteomic studies demonstrated that carbohydrate metabolism-related enzymes, such as pullulanase, UDP-glucose pyrophosphorylase, and glucose-1-phosphate adenylyltransferase, were primarily up-regulated in transgenic rice seeds. In addition, the enzyme PPDK was also elevated in transgenic seeds thus enhancing pyruvate biosynthesis, which is the precursor in the carotenoids biosynthetic pathway. GC-MS based metabolite profiling demonstrated an increase in the levels of glyceric acid, fructo-furanose, and galactose, while decrease in galactonic acid and gentiobiose in the transgenic rice compared to WT. It is noteworthy to mention that the carotenoid content, especially β-carotene level in transgenic rice (4.3 μg/g) was significantly enhanced. The present study highlights the metabolic adaptation process of a transgenic golden rice line (homozygous T4 progeny of SKBR-244) after enhancing carotenoid biosynthesis. The presented information would be helpful in the development of crops enriched in carotenoids by expressing metabolic flux of pyruvate biosynthesis. PMID:27840631

SRC-like adaptor protein regulates B cell development and function.

PubMed

Dragone, Leonard L; Myers, Margaret D; White, Carmen; Sosinowski, Tomasz; Weiss, Arthur

2006-01-01

The avidity of BCRs and TCRs influences signal strength during processes of lymphocyte development. Avidity is determined by both the intrinsic affinity for Ag and surface levels of the Ag receptor. The Src-like adaptor protein (SLAP) is a regulator of TCR levels on thymocytes, and its deficiency alters thymocyte development. We hypothesized that SLAP, which is expressed in B cells, also is important in regulating BCR levels, signal strength, and B cell development. To test this hypothesis, we analyzed the B cell compartment in SLAP-deficient mice. We found increased splenic B cell numbers and decreased surface IgM levels on mature, splenic B cells deficient in SLAP. Immature bone marrow and splenic B cells from BCR-transgenic, SLAP-deficient mice were found to express higher surface levels of IgM. In contrast, mature splenic B cells from BCR-transgenic mice expressed decreased levels of surface BCR associated with decreased calcium flux and activation-induced markers, compared with controls. These data suggest that SLAP regulates BCR levels and signal strength during lymphocyte development.

Expression of wheat expansin driven by the RD29 promoter in tobacco confers water-stress tolerance without impacting growth and development.

PubMed

Li, Feng; Han, Yangyang; Feng, Yanan; Xing, Shichao; Zhao, Meirong; Chen, Yanhui; Wang, Wei

2013-02-10

Expansins are the key regulators of cell wall extension during plant growth. Previously, we produced transgenic tobacco plants with increased tolerance to water stress by overexpressing the wheat expansin gene TaEXPB23 driven by the constitutive 35S cauliflower mosaic virus (CaMV) promoter. However, the growth and development of 35S::TaEXPB23 transgenic tobacco plants were altered under normal growth conditions, with a faster growth rate at the seedling stage, earlier flowering and maturation, and a shorter plant height compared to WT. In the current study, we determined that cellular characteristics and carbohydrate metabolism were altered in 35S::TaEXPB23 transgenic tobacco plants. We also generated transgenic Arabidopsis plants using the same vector. The transgenic Arabidopsis plants had the same phenotype as the transgenic tobacco plants, which may have resulted from the altered expression of several flowering-related genes. We then produced TaEXPB23 transgenic tobacco plants using the stress-inducible RD29A promoter. The use of this promoter reduced the negative effects of TaEXPB23 on plant growth and development. The RD29A::TaEXPB23 transgenic tobacco plants had greater tolerance to water stress than WT, as determined by examining physiological and biochemical parameters. Therefore, the use of stress-inducible promoters, such as RD29A, may minimize the negative effects of constitutive transgene expression and improve the water-stress tolerance of plants. Copyright © 2012 Elsevier B.V. All rights reserved.

Cytokinin signaling regulates cambial development in poplar

PubMed Central

Nieminen, Kaisa; Immanen, Juha; Laxell, Marjukka; Kauppinen, Leila; Tarkowski, Petr; Dolezal, Karel; Tähtiharju, Sari; Elo, Annakaisa; Decourteix, Mélanie; Ljung, Karin; Bhalerao, Rishikesh; Keinonen, Kaija; Albert, Victor A.; Helariutta, Ykä

2008-01-01

Although a substantial proportion of plant biomass originates from the activity of vascular cambium, the molecular basis of radial plant growth is still largely unknown. To address whether cytokinins are required for cambial activity, we studied cytokinin signaling across the cambial zones of 2 tree species, poplar (Populus trichocarpa) and birch (Betula pendula). We observed an expression peak for genes encoding cytokinin receptors in the dividing cambial cells. We reduced cytokinin levels endogenously by engineering transgenic poplar trees (P. tremula × tremuloides) to express a cytokinin catabolic gene, Arabidopsis CYTOKININ OXIDASE 2, under the promoter of a birch CYTOKININ RECEPTOR 1 gene. Transgenic trees showed reduced concentration of a biologically active cytokinin, correlating with impaired cytokinin responsiveness. In these trees, both apical and radial growth was compromised. However, radial growth was more affected, as illustrated by a thinner stem diameter than in WT at same height. To dissect radial from apical growth inhibition, we performed a reciprocal grafting experiment. WT scion outgrew the diameter of transgenic stock, implicating cytokinin activity as a direct determinant of radial growth. The reduced radial growth correlated with a reduced number of cambial cell layers. Moreover, expression of a cytokinin primary response gene was dramatically reduced in the thin-stemmed transgenic trees. Thus, a reduced level of cytokinin signaling is the primary basis for the impaired cambial growth observed. Together, our results show that cytokinins are major hormonal regulators required for cambial development. PMID:19064928

Generating high temperature tolerant transgenic plants: Achievements and challenges.

PubMed

Grover, Anil; Mittal, Dheeraj; Negi, Manisha; Lavania, Dhruv

2013-05-01

Production of plants tolerant to high temperature stress is of immense significance in the light of global warming and climate change. Plant cells respond to high temperature stress by re-programming their genetic machinery for survival and reproduction. High temperature tolerance in transgenic plants has largely been achieved either by over-expressing heat shock protein genes or by altering levels of heat shock factors that regulate expression of heat shock and non-heat shock genes. Apart from heat shock factors, over-expression of other trans-acting factors like DREB2A, bZIP28 and WRKY proteins has proven useful in imparting high temperature tolerance. Besides these, elevating the genetic levels of proteins involved in osmotic adjustment, reactive oxygen species removal, saturation of membrane-associated lipids, photosynthetic reactions, production of polyamines and protein biosynthesis process have yielded positive results in equipping transgenic plants with high temperature tolerance. Cyclic nucleotide gated calcium channel proteins that regulate calcium influxes across the cell membrane have recently been shown to be the key players in induction of high temperature tolerance. The involvement of calmodulins and kinases in activation of heat shock factors has been implicated as an important event in governing high temperature tolerance. Unfilled gaps limiting the production of high temperature tolerant transgenic plants for field level cultivation are discussed. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Differential Top10 promoter regulation by six tetracycline analogues in plant cells

NASA Technical Reports Server (NTRS)

Love, John; Allen, George C.; Gatz, Christiane; Thompson, William F.; Brown, C. S. (Principal Investigator)

2002-01-01

The effects of five tetracycline analogues, anhydrotetracycline, doxycycline, minocycline, oxytetracycline, and tetracycline, on Top10 promoter activity in NT1 tobacco tissue culture cells have been analysed. The concentration that repressed Top10 promoter activity, the level of transgene repression and the kinetics of transgene de-repression were determined for each analogue, and could not be predicted from in vitro binding affinity to the tetracycline repressor or from comparison with animal cells. Doxycycline had the most potent effect on the Top10 promoter and completely inhibited transgene expression at 4 nmol l(-1). Tetracycline was the most versatile of the analogues tested; tetracycline inhibited the Top10 promoter at 10 nmol l(-1) and was easily washed out to restore Top10-driven expression in 12-24 h. A study was also made of the suitability for plant research of a novel tetracycline analogue, GR33076X. In animal cells, GR33076X de-repressed Top10 promoter activity in the presence of inhibitory concentrations of anhydrotetracycline. In NT1, it is shown that GR 33076X can antagonize repression of the Top10 promoter in the presence of tetracycline, but not of anhydrotetracycline or of doxycycline. Different tetracycline analogues can therefore be used to regulate the Top10 promoter in plant cells and this property may be exploited in planning an optimum course of transgene regulation.

Differential Top10 promoter regulation by six tetracycline analogues in plant cells.

PubMed

Love, John; Allen, George C; Gatz, Christiane; Thompson, William F

2002-09-01

The effects of five tetracycline analogues, anhydrotetracycline, doxycycline, minocycline, oxytetracycline, and tetracycline, on Top10 promoter activity in NT1 tobacco tissue culture cells have been analysed. The concentration that repressed Top10 promoter activity, the level of transgene repression and the kinetics of transgene de-repression were determined for each analogue, and could not be predicted from in vitro binding affinity to the tetracycline repressor or from comparison with animal cells. Doxycycline had the most potent effect on the Top10 promoter and completely inhibited transgene expression at 4 nmol l(-1). Tetracycline was the most versatile of the analogues tested; tetracycline inhibited the Top10 promoter at 10 nmol l(-1) and was easily washed out to restore Top10-driven expression in 12-24 h. A study was also made of the suitability for plant research of a novel tetracycline analogue, GR33076X. In animal cells, GR33076X de-repressed Top10 promoter activity in the presence of inhibitory concentrations of anhydrotetracycline. In NT1, it is shown that GR 33076X can antagonize repression of the Top10 promoter in the presence of tetracycline, but not of anhydrotetracycline or of doxycycline. Different tetracycline analogues can therefore be used to regulate the Top10 promoter in plant cells and this property may be exploited in planning an optimum course of transgene regulation.

Validation of the use of an artificial mitochondrial reporter DNA vector containing a Cytomegalovirus promoter for mitochondrial transgene expression.

PubMed

Yamada, Yuma; Ishikawa, Takuya; Harashima, Hideyoshi

2017-08-01

Mitochondria have their own gene expression system that is independent of the nuclear system, and control cellular functions in cooperation with the nucleus. While a number of useful technologies for achieving nuclear transgene expression have been reported, only a few have focused on mitochondria. In this study, we validated the utility of an artificial mitochondrial DNA vector with a virus promoter on mitochondrial transgene expression. We designed and constructed pCMV-mtLuc (CGG) that contains a CMV promotor derived from Cytomegalovirus and an artificial mitochondrial genome with a NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system. Nluc luciferase activity measurements showed that the pCMV-mtLuc (CGG) efficiently produced the Nluc luciferase protein in human HeLa cells. Moreover, we optimized the mitochondrial transfection of pCMV-mtLuc (CGG) using a MITO-Porter system, a liposome-based carrier for mitochondrial delivery via membrane fusion. As a result, we found that transfection of pCMV-mtLuc (CGG) by MITO-Porter modified with the KALA peptide (cationic amphipathic cell-penetrating peptide) showed a high mitochondrial transgene expression. The developed mitochondrial transgene expression system represents a potentially useful tool for the fields of nanoscience and nanotechnology for controlling the intracellular microenvironment via the regulation of mitochondrial function and promises to open additional innovative research fields of study. Copyright © 2017 Elsevier Ltd. All rights reserved.

Patterns of expression of position-dependent integrated transgenes in mouse embryo.

PubMed Central

Bonnerot, C; Grimber, G; Briand, P; Nicolas, J F

1990-01-01

The abilities to introduce foreign DNA into the genome of mice and to visualize gene expression at the single-cell level underlie a method for defining individual elements of a genetic program. We describe the use of an Escherichia coli lacZ reporter gene fused to the promoter of the gene for hypoxanthine phosphoribosyl transferase that is expressed in all tissues. Most transgenic mice (six of seven) obtained with this construct express the lacZ gene from the hypoxanthine phosphoribosyltransferase promoter. Unexpectedly, however, the expression is temporally and spatially regulated. Each transgenic line is characterized by a specific, highly reproducible pattern of lacZ expression. These results show that, for expression, the integrated construct must be complemented by elements of the genome. These elements exert dominant developmental control on the hypoxanthine phosphoribosyltransferase promoter. The expression patterns in some transgenic mice conform to a typological marker and in others to a subtle combination of typology and topography. These observations define discrete heterogeneities of cell types and of certain structures, particularly in the nervous system and in the mesoderm. This system opens opportunities for developmental studies by providing cellular, molecular, and genetic markers of cell types, cell states, and cells from developmental compartments. Finally this method illustrates that genes transduced or transposed to a different position in the genome acquire different spatiotemporal specificities, a result that has implications for evolution. Images PMID:1696727

Functional conservation between rodents and chicken of regulatory sequences driving skeletal muscle gene expression in transgenic chickens

PubMed Central

2010-01-01

Background Regulatory elements that control expression of specific genes during development have been shown in many cases to contain functionally-conserved modules that can be transferred between species and direct gene expression in a comparable developmental pattern. An example of such a module has been identified at the rat myosin light chain (MLC) 1/3 locus, which has been well characterised in transgenic mouse studies. This locus contains two promoters encoding two alternatively spliced isoforms of alkali myosin light chain. These promoters are differentially regulated during development through the activity of two enhancer elements. The MLC3 promoter alone has been shown to confer expression of a reporter gene in skeletal and cardiac muscle in transgenic mice and the addition of the downstream MLC enhancer increased expression levels in skeletal muscle. We asked whether this regulatory module, sufficient for striated muscle gene expression in the mouse, would drive expression in similar domains in the chicken. Results We have observed that a conserved downstream MLC enhancer is present in the chicken MLC locus. We found that the rat MLC1/3 regulatory elements were transcriptionally active in chick skeletal muscle primary cultures. We observed that a single copy lentiviral insert containing this regulatory cassette was able to drive expression of a lacZ reporter gene in the fast-fibres of skeletal muscle in chicken in three independent transgenic chicken lines in a pattern similar to the endogenous MLC locus. Reporter gene expression in cardiac muscle tissues was not observed for any of these lines. Conclusions From these results we conclude that skeletal expression from this regulatory module is conserved in a genomic context between rodents and chickens. This transgenic module will be useful in future investigations of muscle development in avian species. PMID:20184756

Transgenic expression of the Trichoderma harzianum hsp70 gene increases Arabidopsis resistance to heat and other abiotic stresses.

PubMed

Montero-Barrientos, Marta; Hermosa, Rosa; Cardoza, Rosa E; Gutiérrez, Santiago; Nicolás, Carlos; Monte, Enrique

2010-05-15

The ability of some Trichoderma strains, a biological control agent, to overcome extreme environmental conditions has previously been reported and related to heat-shock proteins (HSPs). These proteins are induced environmentally and are involved in important processes, acting as molecular chaperones in all organisms. In a previous study, we demonstrated, by overexpression, that the Trichoderma harzianum hsp70 gene conferred tolerance to heat and other abiotic stresses to this fungus. In this work, we investigate the function of the T. harzianum T34 hsp70 gene in Arabidopsis thaliana. We analyze transgenic plant responses under adverse environmental conditions and the expression levels of a set of seven stress genes, using quantitative RT-PCR. As expected, transgenic plants expressing the T. harzianum hsp70 gene exhibited enhanced tolerance to heat stress. In addition, they did not show growth inhibition and, after heat pre-treatment, transgenic seedlings were more tolerant to osmotic, salt and oxidative stresses with respect to the wild-type behavior. Transgenic lines also had increased transcript levels of the Na(+)/H(+) exchanger 1 (SOS1) and ascorbate peroxidase 1 (APX1) genes, involved in salt and oxidative stress responses, respectively. However, the heat-shock factor (HSF) and four HSP genes tested were down-regulated in 35S:hsp70 plants. Overall, our results indicate that hsp70 confers tolerance to heat and other abiotic stresses and that the fungal HSP70 protein acts as a negative regulator of the HSF transcriptional activity in Arabidopsis. (c) 2009 Elsevier GmbH. All rights reserved.

Upregulation of CREB-mediated transcription enhances both short- and long-term memory.

PubMed

Suzuki, Akinobu; Fukushima, Hotaka; Mukawa, Takuya; Toyoda, Hiroki; Wu, Long-Jun; Zhao, Ming-Gao; Xu, Hui; Shang, Yuze; Endoh, Kengo; Iwamoto, Taku; Mamiya, Nori; Okano, Emiko; Hasegawa, Shunsuke; Mercaldo, Valentina; Zhang, Yue; Maeda, Ryouta; Ohta, Miho; Josselyn, Sheena A; Zhuo, Min; Kida, Satoshi

2011-06-15

Unraveling the mechanisms by which the molecular manipulation of genes of interest enhances cognitive function is important to establish genetic therapies for cognitive disorders. Although CREB is thought to positively regulate formation of long-term memory (LTM), gain-of-function effects of CREB remain poorly understood, especially at the behavioral level. To address this, we generated four lines of transgenic mice expressing dominant active CREB mutants (CREB-Y134F or CREB-DIEDML) in the forebrain that exhibited moderate upregulation of CREB activity. These transgenic lines improved not only LTM but also long-lasting long-term potentiation in the CA1 area in the hippocampus. However, we also observed enhanced short-term memory (STM) in contextual fear-conditioning and social recognition tasks. Enhanced LTM and STM could be dissociated behaviorally in these four lines of transgenic mice, suggesting that the underlying mechanism for enhanced STM and LTM are distinct. LTM enhancement seems to be attributable to the improvement of memory consolidation by the upregulation of CREB transcriptional activity, whereas higher basal levels of BDNF, a CREB target gene, predicted enhanced shorter-term memory. The importance of BDNF in STM was verified by microinfusing BDNF or BDNF inhibitors into the hippocampus of wild-type or transgenic mice. Additionally, increasing BDNF further enhanced LTM in one of the lines of transgenic mice that displayed a normal BDNF level but enhanced LTM, suggesting that upregulation of BDNF and CREB activity cooperatively enhances LTM formation. Our findings suggest that CREB positively regulates memory consolidation and affects memory performance by regulating BDNF expression.

Molecular and phenotypic characterization of transgenic soybean expressing the Arabidopsis ferric chelate reductase gene, FRO2.

PubMed

Vasconcelos, Marta; Eckert, Helene; Arahana, Venancio; Graef, George; Grusak, Michael A; Clemente, Tom

2006-10-01

Soybean (Glycine max Merr.) production is reduced under iron-limiting calcareous soils throughout the upper Midwest regions of the US. Like other dicotyledonous plants, soybean responds to iron-limiting environments by induction of an active proton pump, a ferric iron reductase and an iron transporter. Here we demonstrate that heterologous expression of the Arabidopsis thaliana ferric chelate reductase gene, FRO2, in transgenic soybean significantly enhances Fe(+3) reduction in roots and leaves. Root ferric reductase activity was up to tenfold higher in transgenic plants and was not subjected to post-transcriptional regulation. In leaves, reductase activity was threefold higher in the transgenic plants when compared to control. The enhanced ferric reductase activity led to reduced chlorosis, increased chlorophyll concentration and a lessening in biomass loss in the transgenic events between Fe treatments as compared to control plants grown under hydroponics that mimicked Fe-sufficient and Fe-deficient soil environments. However, the data indicate that constitutive FRO2 expression under non-iron stress conditions may lead to a decrease in plant productivity as reflected by reduced biomass accumulation in the transgenic events under non-iron stress conditions. When grown at Fe(III)-EDDHA levels greater than 10 microM, iron concentration in the shoots of transgenic plants was significantly higher than control. The same observation was found in the roots in plants grown at iron levels higher than 32 microM Fe(III)-EDDHA. These results suggest that heterologous expression of an iron chelate reductase in soybean can provide a route to alleviate iron deficiency chlorosis.

Applications of transgenics in studies of bone sialoprotein.

PubMed

Zhang, Jin; Tu, Qisheng; Chen, Jake

2009-07-01

Bone sialoprotein (BSP) is a major non-collagenous protein in mineralizing connective tissues such as dentin, cementum and calcified cartilage tissues. As a member of the Small Integrin-Binding Ligand, N-linked Glycoprotein (SIBLING) gene family of glycoproteins, BSP is involved in regulating hydroxyapatite crystal formation in bones and teeth, and has long been used as a marker gene for osteogenic differentiation. In the most recent decade, new discoveries in BSP gene expression and regulation, bone remodeling, bone metastasis, and bone tissue engineering have been achieved with the help of transgenic mice. In this review, we discuss these new discoveries obtained from the literatures and from our own laboratory, which were derived from the use of transgenic mouse mutants related to BSP gene or its promoter activity.

Overexpression of a defensin enhances resistance to a fruit-specific anthracnose fungus in pepper.

PubMed

Seo, Hyo-Hyoun; Park, Sangkyu; Park, Soomin; Oh, Byung-Jun; Back, Kyoungwhan; Han, Oksoo; Kim, Jeong-Il; Kim, Young Soon

2014-01-01

Functional characterization of a defensin, J1-1, was conducted to evaluate its biotechnological potentiality in transgenic pepper plants against the causal agent of anthracnose disease, Colletotrichum gloeosporioides. To determine antifungal activity, J1-1 recombinant protein was generated and tested for the activity against C. gloeosporioides, resulting in 50% inhibition of fungal growth at a protein concentration of 0.1 mg·mL-1. To develop transgenic pepper plants resistant to anthracnose disease, J1-1 cDNA under the control of 35S promoter was introduced into pepper via Agrobacterium-mediated genetic transformation method. Southern and Northern blot analyses confirmed that a single copy of the transgene in selected transgenic plants was normally expressed and also stably transmitted to subsequent generations. The insertion of T-DNA was further analyzed in three independent homozygous lines using inverse PCR, and confirmed the integration of transgene in non-coding region of genomic DNA. Immunoblot results showed that the level of J1-1 proteins, which was not normally accumulated in unripe fruits, accumulated high in transgenic plants but appeared to differ among transgenic lines. Moreover, the expression of jasmonic acid-biosynthetic genes and pathogenesis-related genes were up-regulated in the transgenic lines, which is co-related with the resistance of J1-1 transgenic plants to anthracnose disease. Consequently, the constitutive expression of J1-1 in transgenic pepper plants provided strong resistance to the anthracnose fungus that was associated with highly reduced lesion formation and fungal colonization. These results implied the significance of the antifungal protein, J1-1, as a useful agronomic trait to control fungal disease.

Overexpression of a Defensin Enhances Resistance to a Fruit-Specific Anthracnose Fungus in Pepper

PubMed Central

Seo, Hyo-Hyoun; Park, Sangkyu; Park, Soomin; Oh, Byung-Jun; Back, Kyoungwhan; Han, Oksoo; Kim, Jeong-Il; Kim, Young Soon

2014-01-01

Functional characterization of a defensin, J1-1, was conducted to evaluate its biotechnological potentiality in transgenic pepper plants against the causal agent of anthracnose disease, Colletotrichum gloeosporioides. To determine antifungal activity, J1-1 recombinant protein was generated and tested for the activity against C. gloeosporioides, resulting in 50% inhibition of fungal growth at a protein concentration of 0.1 mg·mL−1. To develop transgenic pepper plants resistant to anthracnose disease, J1-1 cDNA under the control of 35S promoter was introduced into pepper via Agrobacterium-mediated genetic transformation method. Southern and Northern blot analyses confirmed that a single copy of the transgene in selected transgenic plants was normally expressed and also stably transmitted to subsequent generations. The insertion of T-DNA was further analyzed in three independent homozygous lines using inverse PCR, and confirmed the integration of transgene in non-coding region of genomic DNA. Immunoblot results showed that the level of J1-1 proteins, which was not normally accumulated in unripe fruits, accumulated high in transgenic plants but appeared to differ among transgenic lines. Moreover, the expression of jasmonic acid-biosynthetic genes and pathogenesis-related genes were up-regulated in the transgenic lines, which is co-related with the resistance of J1-1 transgenic plants to anthracnose disease. Consequently, the constitutive expression of J1-1 in transgenic pepper plants provided strong resistance to the anthracnose fungus that was associated with highly reduced lesion formation and fungal colonization. These results implied the significance of the antifungal protein, J1-1, as a useful agronomic trait to control fungal disease. PMID:24848280

Targeted expression of suicide gene by tissue-specific promoter and microRNA regulation for cancer gene therapy.

PubMed

Danda, Ravikanth; Krishnan, Gopinath; Ganapathy, Kalaivani; Krishnan, Uma Maheswari; Vikas, Khetan; Elchuri, Sailaja; Chatterjee, Nivedita; Krishnakumar, Subramanian

2013-01-01

In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus-thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3'UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM (+ve)/let-7b(down-regulated)), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM (-ve)/let-7b(up-regulated)), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM (+ve)/let-7b(up-regulated)) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells.

A hobo transgene that encodes the P-element transposase in Drosophila melanogaster: autoregulation and cytotype control of transposase activity.

PubMed Central

Simmons, Michael J; Haley, Kevin J; Grimes, Craig D; Raymond, John D; Niemi, Jarad B

2002-01-01

Drosophila were genetically transformed with a hobo transgene that contains a terminally truncated but otherwise complete P element fused to the promoter from the Drosophila hsp70 gene. Insertions of this H(hsp/CP) transgene on either of the major autosomes produced the P transposase in both the male and female germlines, but not in the soma. Heat-shock treatments significantly increased transposase activity in the female germline; in the male germline, these treatments had little effect. The transposase activity of two insertions of the H(hsp/CP) transgene was not significantly greater than their separate activities, and one insertion of this transgene reduced the transposase activity of P(ry(+), Delta2-3)99B, a stable P transgene, in the germline as well as in the soma. These observations suggest that, through alternate splicing, the H(hsp/CP) transgene produces a repressor that feeds back negatively to regulate transposase expression or function in both the somatic and germline tissues. The H(hsp/CP) transgenes are able to induce gonadal dysgenesis when the transposase they encode has P-element targets to attack. However, this ability and the ability to induce P-element excisions are repressed by the P cytotype, a chromosomal/cytoplasmic state that regulates P elements in the germline. PMID:12019234

The rice YABBY1 gene is involved in the feedback regulation of gibberellin metabolism.

PubMed

Dai, Mingqiu; Zhao, Yu; Ma, Qian; Hu, Yongfeng; Hedden, Peter; Zhang, Qifa; Zhou, Dao-Xiu

2007-05-01

Gibberellin (GA) biosynthesis is regulated by feedback control providing a mechanism for GA homeostasis in plants. However, regulatory elements involved in the feedback control are not known. In this report, we show that a rice (Oryza sativa) YABBY1 (YAB1) gene had a similar expression pattern as key rice GA biosynthetic genes GA3ox2 and GA20ox2. Overexpression of YAB1 in transgenic rice resulted in a semidwarf phenotype that could be fully rescued by applied GA. Quantification of the endogenous GA content revealed increases of GA(20) and decreases of GA(1) levels in the overexpression plants, in which the transcripts of the biosynthetic gene GA3ox2 were decreased. Cosuppression of YAB1 in transgenic plants induced expression of GA3ox2. The repression of GA3ox2 could be obtained upon treatment by dexamethasone of transgenic plants expressing a YAB1-glucocorticoid receptor fusion. Importantly, we show that YAB1 bound to a GA-responsive element within the GA3ox2 promoter. In addition, the expression of YAB1 was deregulated in GA biosynthesis and signaling mutants and could be either transiently induced by GA or repressed by a GA inhibitor. Finally, either overexpression or cosuppression of YAB1 impaired GA-mediated repression of GA3ox2. These data together suggest that YAB1 is involved in the feedback regulation of GA biosynthesis in rice.

Network inference analysis identifies an APRR2-like gene linked to pigment accumulation in tomato and pepper fruits.

PubMed

Pan, Yu; Bradley, Glyn; Pyke, Kevin; Ball, Graham; Lu, Chungui; Fray, Rupert; Marshall, Alexandra; Jayasuta, Subhalai; Baxter, Charles; van Wijk, Rik; Boyden, Laurie; Cade, Rebecca; Chapman, Natalie H; Fraser, Paul D; Hodgman, Charlie; Seymour, Graham B

2013-03-01

Carotenoids represent some of the most important secondary metabolites in the human diet, and tomato (Solanum lycopersicum) is a rich source of these health-promoting compounds. In this work, a novel and fruit-related regulator of pigment accumulation in tomato has been identified by artificial neural network inference analysis and its function validated in transgenic plants. A tomato fruit gene regulatory network was generated using artificial neural network inference analysis and transcription factor gene expression profiles derived from fruits sampled at various points during development and ripening. One of the transcription factor gene expression profiles with a sequence related to an Arabidopsis (Arabidopsis thaliana) ARABIDOPSIS PSEUDO RESPONSE REGULATOR2-LIKE gene (APRR2-Like) was up-regulated at the breaker stage in wild-type tomato fruits and, when overexpressed in transgenic lines, increased plastid number, area, and pigment content, enhancing the levels of chlorophyll in immature unripe fruits and carotenoids in red ripe fruits. Analysis of the transcriptome of transgenic lines overexpressing the tomato APPR2-Like gene revealed up-regulation of several ripening-related genes in the overexpression lines, providing a link between the expression of this tomato gene and the ripening process. A putative ortholog of the tomato APPR2-Like gene in sweet pepper (Capsicum annuum) was associated with pigment accumulation in fruit tissues. We conclude that the function of this gene is conserved across taxa and that it encodes a protein that has an important role in ripening.

Mammalian cytochrome CYP2E1 triggered differential gene regulation in response to trichloroethylene (TCE) in a transgenic poplar.

PubMed

Kang, Jun Won; Wilkerson, Hui-Wen; Farin, Federico M; Bammler, Theo K; Beyer, Richard P; Strand, Stuart E; Doty, Sharon L

2010-08-01

Trichloroethylene (TCE) is an important environmental contaminant of soil, groundwater, and air. Studies of the metabolism of TCE by poplar trees suggest that cytochrome P450 enzymes are involved. Using poplar genome microarrays, we report a number of putative genes that are differentially expressed in response to TCE. In a previous study, transgenic hybrid poplar plants expressing mammalian cytochrome P450 2E1 (CYP2E1) had increased metabolism of TCE. In the vector control plants for this construct, 24 h following TCE exposure, 517 genes were upregulated and 650 genes were downregulated over 2-fold when compared with the non-exposed vector control plants. However, in the transgenic CYP2E1 plant, line 78, 1,601 genes were upregulated and 1,705 genes were downregulated over 2-fold when compared with the non-exposed transgenic CYP2E1 plant. It appeared that the CYP2E1 transgenic hybrid poplar plants overexpressing mammalian CYP2E1 showed a larger number of differentially expressed transcripts, suggesting a metabolic pathway for TCE to metabolites had been initiated by activity of CYP2E1 on TCE. These results suggest that either the over-expression of the CYP2E1 gene or the abundance of TCE metabolites from CYP450 2E1 activity triggered a strong genetic response to TCE. Particularly, cytochrome p450s, glutathione S-transferases, glucosyltransferases, and ABC transporters in the CYP2E1 transgenic hybrid poplar plants were highly expressed compared with in vector controls.

Multiple Different Defense Mechanisms Are Activated in the Young Transgenic Tobacco Plants Which Express the Full Length Genome of the Tobacco Mosaic Virus, and Are Resistant against this Virus

PubMed Central

Jada, Balaji; Soitamo, Arto J.; Siddiqui, Shahid Aslam; Murukesan, Gayatri; Aro, Eva-Mari; Salakoski, Tapio; Lehto, Kirsi

2014-01-01

Previously described transgenic tobacco lines express the full length infectious Tobacco mosaic virus (TMV) genome under the 35S promoter (Siddiqui et al., 2007. Mol Plant Microbe Interact, 20: 1489–1494). Through their young stages these plants exhibit strong resistance against both the endogenously expressed and exogenously inoculated TMV, but at the age of about 7–8 weeks they break into TMV infection, with typical severe virus symptoms. Infections with some other viruses (Potato viruses Y, A, and X) induce the breaking of the TMV resistance and lead to synergistic proliferation of both viruses. To deduce the gene functions related to this early resistance, we have performed microarray analysis of the transgenic plants during the early resistant stage, and after the resistance break, and also of TMV-infected wild type tobacco plants. Comparison of these transcriptomes to those of corresponding wild type healthy plants indicated that 1362, 1150 and 550 transcripts were up-regulated in the transgenic plants before and after the resistance break, and in the TMV-infected wild type tobacco plants, respectively, and 1422, 1200 and 480 transcripts were down-regulated in these plants, respectively. These transcriptome alterations were distinctly different between the three types of plants, and it appears that several different mechanisms, such as the enhanced expression of the defense, hormone signaling and protein degradation pathways contributed to the TMV-resistance in the young transgenic plants. In addition to these alterations, we also observed a distinct and unique gene expression alteration in these plants, which was the strong suppression of the translational machinery. This may also contribute to the resistance by slowing down the synthesis of viral proteins. Viral replication potential may also be suppressed, to some extent, by the reduction of the translation initiation and elongation factors eIF-3 and eEF1A and B, which are required for the TMV replication complex. PMID:25244327

Sex-dependent regulation of hypothalamic neuropeptide Y-Y1 receptor gene expression in moderate/high fat, high-energy diet-fed mice

PubMed Central

Zammaretti, Francesca; Panzica, Giancarlo; Eva, Carola

2007-01-01

In this study we investigated whether long-term consumption of a moderate/high fat (MHF), high-energy diet can affect the gene expression of the Y1 receptor (Y1R) for neuropeptide Y (NPY) in the dorsomedial (DMH), ventromedial (VMH), arcuate (ARC) and paraventricular (PVN) hypothalamic nuclei of male and female Y1R/LacZ transgenic mice, carrying the murine Y1R promoter linked to the LacZ gene. MHF diet-fed male mice showed an increased consumption of metabolizable energy that was associated with a significant increase in body weight as compared with chow-fed controls. In parallel, consumption of a MHF diet for 8 weeks significantly decreased Y1R/LacZ transgene expression in the DMH and VMH of male mice whereas no changes were found in the ARC and PVN. Leptin treatment reduced body weight of both MHF diet- and chow-fed male mice but failed to prevent the decrease in Y1R/LacZ transgene expression apparent in the DMH and VMH of male mice after 8 weeks of MHF diet intake. Conversely, no significant changes of metabolizable energy intake, body weight or hypothalamic β-galactosidase expression were found in MHF diet-fed female Y1R/LacZ transgenic mice. A gender-related difference of Y1R/LacZ transgenic mice was also observed in response to leptin treatment that failed to decrease body weight of both MHF diet- and chow-fed female mice. Results herein demonstrate that Y1R/LacZ FVB mice show a sexual dimorphism both on energy intake and on nucleus-specific regulation of the NPY Y1R system in the hypothalamus. Overall, these results provide new insights into the mechanism by which diet composition affects the hypothalamic circuit that controls energy homeostasis. PMID:17584829

Compensation of the AKT signaling by ERK signaling in transgenic mice hearts overexpressing TRIM72

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ham, Young-Mi, E-mail: youngmi_ham@hms.harvard.edu; Department of Cell Biology, Harvard Medical School, Boston, MA 02115; Mahoney, Sarah Jane

The AKT and ERK signaling pathways are known to be involved in cell hypertrophy, proliferation, survival and differentiation. Although there is evidence for crosstalk between these two signaling pathways in cellulo, there is less evidence for cross talk in vivo. Here, we show that crosstalk between AKT and ERK signaling in the hearts of TRIM72-overexpressing transgenic mice (TRIM72-Tg) with alpha-MHC promoter regulates and maintains their heart size. TRIM72, a heart- and skeletal muscle-specific protein, downregulates AKT-mTOR signaling via IRS-1 degradation and reduces the size of rat cardiomyocytes and the size of postnatal TRIM72-Tg hearts. TRIM72 expression was upregulated by hypertrophicmore » inducers in cardiomyocytes, while IRS-1 was downregulated by IGF-1. TRIM72 specifically regulated IGF-1-dependent AKT-mTOR signaling, resulting in a reduction of the size of cardiomyocytes. Postnatal TRIM72-Tg hearts were smaller than control-treated hearts with inhibition of AKT-mTOR signaling. However, adult TRIM72-Tg hearts were larger than of control despite the suppression of AKT-mTOR signaling. Activation of ERK, PKC-α, and JNK were observed to be elevated in adult TRIM72-Tg, and these signals were mediated by ET-1 via the ET receptors A and B. Altogether, these results suggest that AKT signaling regulates cardiac hypertrophy in physiological conditions, and ERK signaling compensates for the absence of AKT signaling during TRIM72 overexpression, leading to pathological hypertrophy. -- Highlights: • TRIM72 inhibits AKT signaling through ubiquitination of IRS-1 in cardiac cells. • TRIM72 regulates the size of cardiac cells. • TRIM72 regulates size of postnatal TRIM72-overexpressing transgenic mice hearts. • Adult TRIM72-overexpressing transgenic mice hearts showed cardiac dysfunction. • Adult TRIM72 transgenic mice hearts showed higher expression of endothelin receptors.« less

Magnetogenetics: Remote Control of Cellular Signaling with Magnetic Fields

NASA Astrophysics Data System (ADS)

Sauer, Jeremy P.

Means for temporally regulating gene expression and cellular activity are invaluable for elucidating the underlying physiological processes and have therapeutic implications. Here we report the development of a system for remote regulation of gene expression by low frequency radiowaves (RF) or by a static magnetic field. We accomplished this by first adding iron oxide nanoparticles - either exogenously or as genetically encoded ferritin/ferric oxyhydroxide particle. These particles have been designed with affinity to the plasma membrane ion channel Transient Receptor Potential Vanilloid 1 (TRPV1) by a conjugated antibody. Application of a magnetic field stimulates the particle to gate the ion channel and this, in turn, initiates calcium-dependent transgene expression. We first demonstrated in vitro that TRPV1 can be actuated to cause calcium flux into the cell by directly applying a localized magnetic field. In mice expressing these genetically encoded components, application of external magnetic field caused remote stimulation of insulin transgene expression and significantly lowered blood glucose. In addition, we are investigating mechanisms by which iron oxide nanoparticles can absorb RF, and transduce this energy to cause channel opening. This robust, repeatable method for remote cellular regulation in vivo may ultimately have applications in basic science, as well as in technology and therapeutics.

Transgenic Mice Expressing Yeast CUP1 Exhibit Increased Copper Utilization from Feeds

PubMed Central

Chen, Zhenliang; Liao, Rongrong; Zhang, Xiangzhe; Wang, Qishan; Pan, Yuchun

2014-01-01

Copper is required for structural and catalytic properties of a variety of enzymes participating in many vital biological processes for growth and development. Feeds provide most of the copper as an essential micronutrient consumed by animals, but inorganic copper could not be utilized effectively. In the present study, we aimed to develop transgenic mouse models to test if copper utilization will be increased by providing the animals with an exogenous gene for generation of copper chelatin in saliva. Considering that the S. cerevisiae CUP1 gene encodes a Cys-rich protein that can bind copper as specifically as copper chelatin in yeast, we therefore constructed a transgene plasmid containing the CUP1 gene regulated for specific expression in the salivary glands by a promoter of gene coding pig parotid secretory protein. Transgenic CUP1 was highly expressed in the parotid and submandibular salivary glands and secreted in saliva as a 9-kDa copper-chelating protein. Expression of salivary copper-chelating proteins reduced fecal copper contents by 21.61% and increased body-weight by 12.97%, suggesting that chelating proteins improve the utilization and absorbed efficacy of copper. No negative effects on the health of the transgenic mice were found by blood biochemistry and histology analysis. These results demonstrate that the introduction of the salivary CUP1 transgene into animals offers a possible approach to increase the utilization efficiency of copper and decrease the fecal copper contents. PMID:25265503

Multiple effects of genetic background on variegated transgene expression in mice.

PubMed Central

Opsahl, Margaret L; McClenaghan, Margaret; Springbett, Anthea; Reid, Sarah; Lathe, Richard; Colman, Alan; Whitelaw, C Bruce A

2002-01-01

BLG/7 transgenic mice express an ovine beta-lactoglobulin transgene during lactation. Unusually, transgene expression levels in milk differ between siblings. This variable expression is due to variegated transgene expression in the mammary gland and is reminiscent of position-effect variegation. The BLG/7 line was created and maintained on a mixed CBA x C57BL/6 background. We have investigated the effect on transgene expression of backcrossing for 13 generations into these backgrounds. Variable transgene expression was observed in all populations examined, confirming that it is an inherent property of the transgene array at its site of integration. There were also strain-specific effects on transgene expression that appear to be independent of the inherent variegation. The transgene, compared to endogenous milk protein genes, is specifically susceptible to inbreeding depression. Outcrossing restored transgene expression levels to that of the parental population; thus suppression was not inherited. Finally, no generation-dependent decrease in mean expression levels was observed in the parental population. Thus, although the BLG/7 transgene is expressed in a variegated manner, there was no generation-associated accumulated silencing of transgene expression. PMID:11901126

Multiple effects of genetic background on variegated transgene expression in mice.

PubMed

Opsahl, Margaret L; McClenaghan, Margaret; Springbett, Anthea; Reid, Sarah; Lathe, Richard; Colman, Alan; Whitelaw, C Bruce A

2002-03-01

BLG/7 transgenic mice express an ovine beta-lactoglobulin transgene during lactation. Unusually, transgene expression levels in milk differ between siblings. This variable expression is due to variegated transgene expression in the mammary gland and is reminiscent of position-effect variegation. The BLG/7 line was created and maintained on a mixed CBA x C57BL/6 background. We have investigated the effect on transgene expression of backcrossing for 13 generations into these backgrounds. Variable transgene expression was observed in all populations examined, confirming that it is an inherent property of the transgene array at its site of integration. There were also strain-specific effects on transgene expression that appear to be independent of the inherent variegation. The transgene, compared to endogenous milk protein genes, is specifically susceptible to inbreeding depression. Outcrossing restored transgene expression levels to that of the parental population; thus suppression was not inherited. Finally, no generation-dependent decrease in mean expression levels was observed in the parental population. Thus, although the BLG/7 transgene is expressed in a variegated manner, there was no generation-associated accumulated silencing of transgene expression.

Comparative functional analysis of wheat (Triticum aestivum) zinc finger-containing glycine-rich RNA-binding proteins in response to abiotic stresses.

PubMed

Xu, Tao; Gu, Lili; Choi, Min Ji; Kim, Ryeo Jin; Suh, Mi Chung; Kang, Hunseung

2014-01-01

Although the functional roles of zinc finger-containing glycine-rich RNA-binding proteins (RZs) have been characterized in several plant species, including Arabidopsis thaliana and rice (Oryza sativa), the physiological functions of RZs in wheat (Triticum aestivum) remain largely unknown. Here, the functional roles of the three wheat RZ family members, named TaRZ1, TaRZ2, and TaRZ3, were investigated using transgenic Arabidopsis plants under various abiotic stress conditions. Expression of TaRZs was markedly regulated by salt, dehydration, or cold stress. The TaRZ1 and TaRZ3 proteins were localized to the nucleus, whereas the TaRZ2 protein was localized to the nucleus, endoplasmic reticulum, and cytoplasm. Germination of all three TaRZ-expressing transgenic Arabidopsis seeds was retarded compared with that of wild-type seeds under salt stress conditions, whereas germination of TaRZ2- or TaRZ3-expressing transgenic Arabidopsis seeds was retarded under dehydration stress conditions. Seedling growth of TaRZ1-expressing transgenic plants was severely inhibited under cold or salt stress conditions, and seedling growth of TaRZ2-expressing plants was inhibited under salt stress conditions. By contrast, expression of TaRZ3 did not affect seedling growth of transgenic plants under any of the stress conditions. In addition, expression of TaRZ2 conferred freeze tolerance in Arabidopsis. Taken together, these results suggest that different TaRZ family members play various roles in seed germination, seedling growth, and freeze tolerance in plants under abiotic stress.

Inducible transgenics. New lessons on events governing the induction and commitment in mammary tumorigenesis.

PubMed

Hulit, J; Di Vizio, D; Pestell, R G

2001-01-01

Breast cancer arises from multiple genetic events that together contribute to the established, irreversible malignant phenotype. The development of inducible tissue-specific transgenics has allowed a careful dissection of the events required for induction and subsequent maintenance of tumorigenesis. Mammary gland targeted expression of oncogenic Ras or c-Myc is sufficient for the induction of mammary gland tumorigenesis in the rodent, and when overexpressed together the rate of tumor onset is substantially enhanced. In an exciting recent finding, D'Cruz et al discovered tetracycline-regulated c-Myc overexpression in the mammary gland induced invasive mammary tumors that regressed upon withdrawal of c-Myc expression. Almost one-half of the c-Myc-induced tumors harbored K-ras or N-ras gene point mutations, correlating with tumor persistence on withdrawal of c-Myc transgene expression. These findings suggest maintenance of tumorigenesis may involve a second mutation within the Ras pathway.

Expression of biologically active human interferon alpha 2 in aloe vera

USDA-ARS?s Scientific Manuscript database

We have developed a system for transgenic expression of proteins in Aloe Vera. Using this approach we have generated plants expressing the human gene interferon alpha 2, IFNa2. IFNa2 is a small secreted cytokine that plays a vital role in regulating the body’s immune response to viral infections a...

Ectopic expression of pMADS3 in transgenic petunia phenocopies the petunia blind mutant.

PubMed Central

Tsuchimoto, S; van der Krol, A R; Chua, N H

1993-01-01

We cloned a MADS-box gene, pMADS3, from Petunia hybrida, which shows high sequence homology to the Arabidopsis AGAMOUS and Antirrhinum PLENA. pMADS3 is expressed exclusively in stamens and carpels of wild-type petunia plants. In the petunia mutant blind, which shows homeotic conversions of corolla limbs into antheroid structures with pollen grains and small parts of sepals into carpelloid tissue, pMADS3 is expressed in all floral organs as well as in leaves. Ectopic expression of pMADS3 in transgenic petunia leads to phenocopies of the blind mutant, i.e., the formation of antheroid structures on limbs and carpelloid tissue on sepals. Transgenic tobacco plants that overexpress pMADS3 exhibit an even more severe phenotype, with the sepals forming a carpel-like structure encasing the interior floral organs. Our results identify BLIND as a negative regulator of pMADS3, which specifies stamens and carpels during petunia flower development. PMID:8104573

PU.1 regulates TCR expression by modulating GATA-3 activity

PubMed Central

Chang, Hua-Chen; Han, Ling; Jabeen, Rukhsana; Carotta, Sebastian; Nutt, Stephen L.; Kaplan, Mark H.

2009-01-01

The Ets transcription factor PU.1 is a master regulator for the development of multiple lineages during hematopoiesis. The expression pattern of PU.1 is dynamically regulated during early T lineage development in the thymus. We previously revealed that PU.1 delineates heterogeneity of effector Th2 populations. In this study, we further define the function of PU.1 on the Th2 phenotype using mice that specifically lack PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpi1lck-/-). While deletion of PU.1 by the lck-Cre transgene does not affect T cell development, Sfpi1lck-/- T cells have a lower activation threshold than wild type T cells. When TCR engagement is limiting, Sfpi1lck-/- T cells cultured in Th2 polarizing conditions secrete higher levels of Th2 cytokines and have greater cytokine homogeneity than wild type cells. We show that PU.1 modulates the levels of TCR expression in CD4+ T cells by regulating the DNA-binding activity of GATA-3 and limiting GATA-3 regulation of TCR gene expression. GATA-3 dependent regulation of TCR expression is also observed in Th1 and Th2 cells. In CD4+ T cells, PU.1 expression segregates into subpopulations of cells that have lower levels of surface TCR, suggesting that PU.1 contributes to the heterogeneity of TCR expression. Thus, we have identified a mechanism whereby increased GATA-3 function in the absence of the antagonizing activity of PU.1 leads to increased TCR expression, a reduced activation threshold and increased homogeneity in Th2 populations. PMID:19801513

Liver BCATm transgenic mouse model reveals the important role of the liver in maintaining BCAA homeostasis.

PubMed

Ananieva, Elitsa A; Van Horn, Cynthia G; Jones, Meghan R; Hutson, Susan M

2017-02-01

Unlike other amino acids, the branched-chain amino acids (BCAAs) largely bypass first-pass liver degradation due to a lack of hepatocyte expression of the mitochondrial branched-chain aminotransferase (BCATm). This sets up interorgan shuttling of BCAAs and liver-skeletal muscle cooperation in BCAA catabolism. To explore whether complete liver catabolism of BCAAs may impact BCAA shuttling in peripheral tissues, the BCATm gene was stably introduced into mouse liver. Two transgenic mouse lines with low and high hepatocyte expression of the BCATm transgene (LivTg-LE and LivTg-HE) were created and used to measure liver and plasma amino acid concentrations and determine whether the first two BCAA enzymatic steps in liver, skeletal muscle, heart and kidney were impacted. Expression of the hepatic BCATm transgene lowered the concentrations of hepatic BCAAs while enhancing the concentrations of some nonessential amino acids. Extrahepatic BCAA metabolic enzymes and plasma amino acids were largely unaffected, and no growth rate or body composition differences were observed in the transgenic animals as compared to wild-type mice. Feeding the transgenic animals a high-fat diet did not reverse the effect of the BCATm transgene on the hepatic BCAA catabolism, nor did the high-fat diet cause elevation in plasma BCAAs. However, the high-fat-diet-fed BCATm transgenic animals experienced attenuation in the mammalian target of rapamycin (mTOR) pathway in the liver and had impaired blood glucose tolerance. These results suggest that complete liver BCAA metabolism influences the regulation of glucose utilization during diet-induced obesity. Copyright © 2016 Elsevier Inc. All rights reserved.

Liver BCATm transgenic mouse model reveals the important role of the liver in maintaining BCAA homeostasis

PubMed Central

Ananieva, Elitsa A.; Van Horn, Cynthia G.; Jones, Meghan R.; Hutson, Susan M.

2016-01-01

Unlike other amino acids, the branched chain amino acids (BCAAs) largely bypass first pass liver degradation due to a lack of hepatocyte expression of the mitochondrial branched chain aminotransferase (BCATm). This sets up interorgan shuttling of BCAAs and liver-skeletal muscle cooperation in BCAA catabolism. To explore whether complete liver catabolism of BCAAs may impact BCAA shuttling in peripheral tissues, the BCATm gene was stably introduced into mouse liver. Two transgenic mouse lines with low and high hepatocyte expression of the BCATm transgene (LivTg-LE and LivTg-HE) were created and used to measure liver and plasma amino acid concentrations and determine whether the first two BCAA enzymatic steps in liver, skeletal muscle, heart, and kidney were impacted. Expression of the hepatic BCATm transgene lowered the concentrations of hepatic BCAAs while enhancing the concentrations of some nonessential amino acids. Extrahepatic BCAA metabolic enzymes and plasma amino acids were largely unaffected and no growth rate or body composition differences were observed in the transgenic animals as compared to wild type (WT) mice. Feeding the transgenic animals a high fat diet did not reverse the effect of the BCATm transgene on the hepatic BCAA catabolism nor did the high fat diet cause elevation in plasma BCAAs. However, the high fat diet fed BCATm transgenic animals experienced attenuation in the mammalian target of rapamycin (mTOR) pathway in the liver and had impaired blood glucose tolerance. These results suggest that complete liver BCAA metabolism influences the regulation of glucose utilization during diet-induced obesity. PMID:27886623

Tumor formation initiated by nondividing epidermal cells via an inflammatory infiltrate.

PubMed

Arwert, Esther N; Lal, Rohit; Quist, Sven; Rosewell, Ian; van Rooijen, Nico; Watt, Fiona M

2010-11-16

In mammalian epidermis, integrin expression is normally confined to the basal proliferative layer that contains stem cells. However, in epidermal hyperproliferative disorders and tumors, integrins are also expressed by suprabasal cells, with concomitant up-regulation of Erk mitogen-activated protein kinase (MAPK) signaling. In transgenic mice, expression of activated MAPK kinase 1 (MEK1) in the suprabasal, nondividing, differentiated cell layers (InvEE transgenics) results in epidermal hyperproliferation and skin inflammation. We now demonstrate that wounding induces benign tumors (papillomas and keratoacanthomas) in InvEE mice. By generating chimeras between InvEE mice and mice that lack the MEK1 transgene, we demonstrate that differentiating, nondividing cells that express MEK1 stimulate adjacent transgene-negative cells to divide and become incorporated into the tumor mass. Dexamethasone treatment inhibits tumor formation, suggesting that inflammation is involved. InvEE skin and tumors express high levels of IL1α; treatment with an IL1 receptor antagonist delays tumor onset and reduces incidence. Depletion of γδ T cells and macrophages also reduces tumor incidence. Because a hallmark of cancer is uncontrolled proliferation, it is widely assumed that tumors arise only from dividing cells. In contrast, our studies show that differentiated epidermal cells can initiate tumor formation without reacquiring the ability to divide and that they do so by triggering an inflammatory infiltrate.

Doxycycline modulates VEGF-A expression: Failure of doxycycline-inducible lentivirus shRNA vector to knockdown VEGF-A expression in transgenic mice.

PubMed

Merentie, Mari; Rissanen, Riina; Lottonen-Raikaslehto, Line; Huusko, Jenni; Gurzeler, Erika; Turunen, Mikko P; Holappa, Lari; Mäkinen, Petri; Ylä-Herttuala, Seppo

2018-01-01

Vascular endothelial growth factor-A (VEGF-A) is the master regulator of angiogenesis, vascular permeability and growth. However, its role in mature blood vessels is still not well understood. To better understand the role of VEGF-A in the adult vasculature, we generated a VEGF-A knockdown mouse model carrying a doxycycline (dox)-regulatable short hairpin RNA (shRNA) transgene, which silences VEGF-A. The aim was to find the critical level of VEGF-A reduction for vascular well-being in vivo. In vitro, the dox-inducible lentiviral shRNA vector decreased VEGF-A expression efficiently and dose-dependently in mouse endothelial cells and cardiomyocytes. In the generated transgenic mice plasma VEGF-A levels decreased shortly after the dox treatment but returned back to normal after two weeks. VEGF-A expression decreased shortly after the dox treatment only in some tissues. Surprisingly, increasing the dox exposure time and dose led to elevated VEGF-A expression in some tissues of both wildtype and knockdown mice, suggesting that dox itself has an effect on VEGF-A expression. When the effect of dox on VEGF-A levels was further tested in naïve/non-transduced cells, the dox administration led to a decreased VEGF-A expression in endothelial cells but to an increased expression in cardiomyocytes. In conclusion, the VEGF-A knockdown was achieved in a dox-regulatable fashion with a VEGF-A shRNA vector in vitro, but not in the knockdown mouse model in vivo. Dox itself was found to regulate VEGF-A expression explaining the unexpected results in mice. The effect of dox on VEGF-A levels might at least partly explain its previously reported beneficial effects on myocardial and brain ischemia. Also, this effect on VEGF-A should be taken into account in all studies using dox-regulated vectors.

Development of marker-free transgenic Jatropha curcas producing curcin-deficient seeds through endosperm-specific RNAi-mediated gene silencing.

PubMed

Gu, Keyu; Tian, Dongsheng; Mao, Huizhu; Wu, Lifang; Yin, Zhongchao

2015-10-08

Jatropha curcas L. is a potential biofuel plant and its seed oil is suitable for biodiesel production. Despite this promising application, jatropha seeds contain two major toxic components, namely phorbol esters and curcins. These compounds would reduce commercial value of seed cake and raise safety and environment concerns on jatropha plantation and processing. Curcins are Type I ribosome inactivating proteins. Several curcin genes have been identified in the jatropha genome. Among which, the Curcin 1 (C1) gene is identified to be specifically expressed in endosperm, whereas the Curcin 2A (C2A) is mainly expressed in young leaves. A marker-free RNAi construct carrying a β-estradiol-regulated Cre/loxP system and a C1 promoter-driven RNAi cassette for C1 gene was made and used to generate marker-free transgenic RNAi plants to specifically silence the C1 gene in the endosperm of J. curcas. Plants of transgenic line L1, derived from T0-1, carry two copies of marker-free RNAi cassette, whereas plants of L35, derived from T0-35, harbored one copy of marker-free RNAi cassette and three copies of closely linked and yet truncated Hpt genes. The C1 protein content in endosperm of L1 and L35 seeds was greatly reduced or undetectable, while the C2A proteins in young leaves of T0-1 and T0-35 plants were unaffected. In addition, the C1 mRNA transcripts were undetectable in the endosperm of T3 seeds of L1 and L35. The results demonstrated that the expression of the C1 gene was specifically down-regulated or silenced by the double-stranded RNA-mediated RNA interference generated from the RNAi cassette. The C1 promoter-driven RNAi cassette for the C1 gene in transgenic plants was functional and heritable. Both C1 transcripts and C1 proteins were greatly down-regulated or silenced in the endosperm of transgenic J. curcas. The marker-free transgenic plants and curcin-deficient seeds developed in this study provided a solution for the toxicity of curcins in jatropha seeds and addressed the safety concerns of the marker genes in transgenic plants on the environments.

Arabidopsis ABI5 plays a role in regulating ROS homeostasis by activating CATALASE 1 transcription in seed germination.

PubMed

Bi, Chao; Ma, Yu; Wu, Zhen; Yu, Yong-Tao; Liang, Shan; Lu, Kai; Wang, Xiao-Fang

2017-05-01

It has been known that ABA INSENSITIVE 5 (ABI5) plays a vital role in regulating seed germination. In the present study, we showed that inhibition of the catalase activity with 3-amino-1,2,4-triazole (3-AT) inhibits seed germination of Col-0, abi5 mutants and ABI5-overexpression transgenic lines. Compared with Col-0, the seeds of abi5 mutants showed more sensitive to 3-AT during seed germination, while the seeds of ABI5-overexpression transgenic lines showed more insensitive. H 2 O 2 showed the same effect on seed germination of Col-0, abi5 mutants and ABI5-overexpression transgenic lines as 3-AT. These results suggest that ROS is involved in the seed germination mediated by ABI5. Further, we observed that T-DNA insertion mutants of the three catalase members in Arabidopsis displayed 3-AT-insensitive or -hypersensitive phenotypes during seed germination, suggesting that these catalase members regulate ROS homeostasis in a highly complex way. ABI5 affects reactive oxygen species (ROS) homeostasis by affecting CATALASE expression and catalase activity. Furthermore, we showed that ABI5 directly binds to the CAT1 promoter and activates CAT1 expression. Genetic evidence supports the idea that CAT1 functions downstream of ABI5 in ROS signaling during seed germination. RNA-sequencing analysis indicates that the transcription of the genes involved in ROS metabolic process or genes responsive to ROS stress is impaired in abi5-1 seeds. Additionally, expression changes in some genes correlative to seed germination were showed due to the change in ABI5 expression under 3-AT treatment. Together, all the findings suggest that ABI5 regulates seed germination at least partly by affecting ROS homeostasis.

Inhibiting effects of rhynchophylline on zebrafish methamphetamine dependence are associated with amelioration of neurotransmitters content and down-regulation of TH and NR2B expression.

PubMed

Jiang, Mingjin; Chen, Yifei; Li, Chan; Peng, Qiuxian; Fang, Miao; Liu, Wei; Kang, Qunzhao; Lin, Yingbo; Yung, Ken Kin Lam; Mo, Zhixian

2016-07-04

Others and we have reported that rhynchophylline reverses amphetamine-induced conditioned place preference (CPP) effect which may be partly mediated by amelioration of central neurotransmitters and N-methyl-d-aspartate receptor 2B (NR2B) levels in the rat brains. The current study investigated the inhibiting effects of rhynchophylline on methamphetamine-induced (METH-induced) CPP in adult zebrafish and METH-induced locomotor activity in tyrosine hydroxylase-green fluorescent protein (TH-GFP) transgenic zebrafish larvae and attempted to confirm the hypothesis that these effects were mediated via regulation of neurotransmitters and dopaminergic and glutamatergic systems. After baseline preference test (on days 1-3), zebrafish were injected intraperitoneally METH (on days 4, 6 and 8) or the same volume of fish physiological saline (on days 5 and 7) and were immediately conditioned. Rhynchophylline was administered at 12h after injection of METH. On day 9, zebrafish were tested for METH-induced CPP. Results revealed that rhynchophylline (100mg/kg) significantly inhibited the acquisition of METH-induced CPP, reduced the content of dopamine and glutamate and down-regulated the expression of TH and NR2B in the CPP zebrafish brains. Furthermore, the influence of rhynchophylline on METH-induced locomotor activity was also observed in TH-GFP transgenic zebrafish larvae. Results showed that rhynchophylline (50mg/L) treatment led to a significant reduction on the locomotor activity and TH expression in TH-GFP transgenic zebrafish larvae. Taken together, these data indicate that the inhibition of the formation of METH dependence by rhynchophylline in zebrafish is associated with amelioration of the neurotransmitters dopamine and glutamate content and down-regulation of TH and NR2B expression. Copyright © 2016 Elsevier Inc. All rights reserved.

The Drosophila melanogaster Eip74EF-PA transcription factor directly binds the sciarid BhC4-1 promoter.

PubMed

Frank, Henrique Oliveira; Sanchez, Danilo Garcia; de Freitas Oliveira, Lucas; Kobarg, Jörg; Monesi, Nadia

2017-11-01

The DNA puff BhC4-1 gene of Bradysia hygida (Diptera, Sciaridae) is amplified and expressed in the salivary glands at the end of the last larval instar. Even though there are no BhC4-1 orthologs in Drosophila melanogaster, the mechanisms that regulate BhC4-1 gene expression in B. hygida are for the most part conserved in D. melanogaster. The BhC4-1 promoter contains a 129bp (-186/-58) cis-regulatory module (CRM) that drives developmentally regulated expression in transgenic salivary glands at the onset of metamorphosis. Both in the sciarid and in transgenic D. melanogaster, BhC4-1 gene expression is induced by the increase in ecdysone titers that triggers metamorphosis. Genetic interaction experiments revealed that in the absence of the Eip74EF-PA early gene isoform BhC4-1-lacZ levels of expression in the salivary gland are severely reduced. Here we show that the overexpression of the Eip74EF-PA transcription factor is sufficient to anticipate BhC4-1-lacZ expression in transgenic D. melanogaster. Through yeast one-hybrid assays we confirm that the Eip74EF-PA transcription factor directly binds to the 129 bp sciarid CRM. Together, these results contribute to the characterization of an insect CRM and indicate that the ecdysone gene regulatory network that promotes metamorphosis is conserved between D. melanogaster and the sciarid B. hygida. © 2017 Wiley Periodicals, Inc.

Functional analysis of rice HOMEOBOX4 (Oshox4) gene reveals a negative function in gibberellin responses.

PubMed

Dai, Mingqiu; Hu, Yongfeng; Ma, Qian; Zhao, Yu; Zhou, Dao-Xiu

2008-02-01

The homeodomain-leucine zipper (HD-Zip) putative transcription factor genes are divided into 4 families. In this work, we studied the function of a rice HD-Zip I gene, H OME O BO X4 (Oshox4). Oshox4 transcripts were detected in leaf and floral organ primordia but excluded from the shoot apical meristem and the protein was nuclear localized. Over-expression of Oshox4 in rice induced a semi-dwarf phenotype that could not be complemented by applied GA3. The over-expression plants accumulated elevated levels of bioactive GA, while the GA catabolic gene GA2ox3 was upregulated in the transgenic plants. In addition, over-expression of Oshox4 blocked GA-dependent alpha-amylase production. However, down-regulation of Oshox4 in RNAi transgenic plants induced no phenotypic alteration. Interestingly, the expression of YAB1 that is involved in the negative feedback regulation of the GA biosynthesis was upregulated in the Oshox4 over-expressing plants. One-hybrid assays showed that Oshox4 could interact with YAB1 promoter in yeast. In addition, Oshox4 expression was upregulated by GA. These data together suggest that Oshox4 may be involved in the negative regulation of GA signalling and may play a role to fine tune GA responses in rice.

Human HLA-Ev (147) Expression in Transgenic Animals.

PubMed

Matsuura, R; Maeda, A; Sakai, R; Eguchi, H; Lo, P-C; Hasuwa, H; Ikawa, M; Nakahata, K; Zenitani, M; Yamamichi, T; Umeda, S; Deguchi, K; Okuyama, H; Miyagawa, S

2016-05-01

In our previous study, we reported on the development of substituting S147C for HLA-E as a useful gene tool for xenotransplantation. In this study we exchanged the codon of HLA-Ev (147), checked its function, and established a line of transgenic mice. A new construct, a codon exchanging human HLA-Ev (147) + IRES + human beta 2-microgloblin, was established. The construct was subcloned into pCXN2 (the chick beta-actin promoter and cytomegalovirus enhancer) vector. Natural killer cell- and macrophage-mediated cytotoxicities were performed using the established the pig endothelial cell (PEC) line with the new gene. Transgenic mice with it were next produced using a micro-injection method. The expression of the molecule on PECs was confirmed by the transfection of the plasmid. The established molecules on PECs functioned well in regulating natural killer cell-mediated cytotoxicity and macrophage-mediated cytotoxicity. We have also successfully generated several lines of transgenic mice with this plasmid. The expression of HLA-Ev (147) in each mouse organ was confirmed by assessing the mRNA. The chick beta-actin promoter and cytomegalovirus enhancer resulted in a relatively broad expression of the gene in each organ, and a strong expression in the cases of the heart and lung. A synthetic HLA-Ev (147) gene with a codon usage optimized to a mammalian system represents a critical factor in the development of transgenic animals for xenotransplantation. Copyright © 2016 Elsevier Inc. All rights reserved.

Over-expression of bacterial gamma-glutamylcysteine synthetase (GSH1) in plastids affects photosynthesis, growth and sulphur metabolism in poplar (Populus tremula x Populus alba) dependent on the resulting gamma-glutamylcysteine and glutathione levels.

PubMed

Herschbach, Cornelia; Rizzini, Luca; Mult, Susanne; Hartmann, Tanja; Busch, Florian; Peuke, Andreas D; Kopriva, Stanislav; Ensminger, Ingo

2010-07-01

We compared three transgenic poplar lines over-expressing the bacterial gamma-glutamylcysteine synthetase (GSH1) targeted to plastids. Lines Lggs6 and Lggs12 have two copies, while line Lggs20 has three copies of the transgene. The three lines differ in their expression levels of the transgene and in the accumulation of gamma-glutamylcysteine (gamma-EC) and glutathione (GSH) in leaves, roots and phloem exudates. The lowest transgene expression level was observed in line Lggs6 which showed an increased growth, an enhanced rate of photosynthesis and a decreased excitation pressure (1-qP). The latter typically represents a lower reduction state of the plastoquinone pool, and thereby facilitates electron flow along the electron transport chain. Line Lggs12 showed the highest transgene expression level, highest gamma-EC accumulation in leaves and highest GSH enrichment in phloem exudates and roots. This line also exhibited a reduced growth, and after a prolonged growth of 4.5 months, symptoms of leaf injury. Decreased maximum quantum yield (F(v)/F(m)) indicated down-regulation of photosystem II reaction centre (PSII RC), which correlates with decreased PSII RC protein D1 (PsbA) and diminished light-harvesting complex (Lhcb1). Potential effects of changes in chloroplastic and cytosolic GSH contents on photosynthesis, growth and the whole-plant sulphur nutrition are discussed for each line.

Expression of a calpastatin transgene slows muscle wasting and obviates changes in myosin isoform expression during murine muscle disuse

PubMed Central

Tidball, James G; Spencer, Melissa J

2002-01-01

Muscle wasting is a prominent feature of several systemic diseases, neurological damage and muscle disuse. The contribution of calpain proteases to muscle wasting in any instance of muscle injury or disease has remained unknown because of the inability to specifically perturb calpain activity in vivo. We have generated a transgenic mouse with muscle-specific overexpression of calpastatin, which is the endogenous inhibitor of calpains, and induced muscle atrophy by unloading hindlimb musculature for 10 days. Expression of the transgene resulted in increases in calpastatin concentration in muscle by 30- to 50-fold, and eliminated all calpain activity that was detectable on zymograms. Muscle fibres in ambulatory, transgenic mice were smaller in diameter, but more numerous, so that muscle mass did not differ between transgenic and non-transgenic mice. This is consistent with the role of the calpain-calpastatin system in muscle cell fusion that has been observed in vitro. Overexpression of calpastatin reduced muscle atrophy by 30 % during the 10 day unloading period. In addition, calpastatin overexpression completely prevented the shift in myofibrillar myosin content from slow to fast isoforms, which normally occurs in muscle unloading. These findings indicate that therapeutics directed toward regulating the calpain-calpastatin system may be beneficial in preventing muscle mass loss in muscle injury and disease. PMID:12482888

Generation of transgenic chickens expressing the human erythropoietin (hEPO) gene in an oviduct-specific manner: Production of transgenic chicken eggs containing human erythropoietin in egg whites

PubMed Central

Kim, Dohyang; Nam, Yu Hwa; Cui, Xiang-Shun; Kim, Nam-Hyung

2018-01-01

The transgenic chicken has been considered as a prospective bioreactor for large-scale production of costly pharmaceutical proteins. In the present study, we report successful generation of transgenic hens that lay eggs containing a high concentration of human erythropoietin (hEPO) in the ovalbumin. Using a feline immunodeficiency virus (FIV)-based pseudotyped lentivirus vector enveloped with G glycoproteins of the vesicular stomatitis virus, the replication-defective vector virus carrying the hEPO gene under the control of the chicken ovalbumin promoter was microinjected to the subgerminal cavity of freshly laid chicken eggs (stage X). Stable germline transmission of the hEPO transgene to the G1 progeny, which were non-mosaic and hemizygous for the hEPO gene under the ovalbumin promoter, was confirmed by mating of a G0 rooster with non-transgenic hens. Quantitative analysis of hEPO in the egg whites and in the blood samples taken from G1 transgenic chickens showed 4,810 ~ 6,600 IU/ml (40.1 ~ 55.0 μg/ml) and almost no detectable concentration, respectively, indicating tightly regulated oviduct-specific expression of the hEPO transgene. In terms of biological activity, there was no difference between the recombinant hEPO contained in the transgenic egg white and the commercially available counterpart, in vitro. We suggest that these results imply an important step toward efficient production of human cytokines from a transgenic animal bioreactor. PMID:29847554

A CBL-Interacting Protein Kinase TaCIPK2 Confers Drought Tolerance in Transgenic Tobacco Plants through Regulating the Stomatal Movement.

PubMed

Wang, Yan; Sun, Tao; Li, Tingting; Wang, Meng; Yang, Guangxiao; He, Guangyuan

2016-01-01

In plants, the CBL-CIPK signaling pathways play key roles in the response to abiotic stresses. However, functional studies of CIPKs in the important staple crop wheat are very rare. In this study, we identified a CIPK gene from wheat, designated TaCIPK2. Expression analysis results showed that TaCIPK2 could be up-regulated in wheat leaves by polyethylene glycol, abscisic acid and H2O2 treatments. Subcellular localization analyses revealed that TaCIPK2 was present in whole wheat epidermal cells. A yeast two-hybrid assay indicated that TaCIPK2 interacted with TaCBL1, 2, 3 and 4 in vitro. Transgenic tobacco plants over-expressing TaCIPK2 exhibited increased drought tolerance, indicated by a larger proportion of green cotyledons and higher survival rates under the osmotic and drought stress conditions compared with control plants. Additionally, physiological index analyses revealed that the transgenic tobacco plants had lower water loss rates and ion leakage, accumulated less malondialdehyde and H2O2, and had higher catalase and superoxide dismutase activities than the control plants. The transgenic plants also exhibited faster stomatal closure following exposure to osmotic stress conditions. The seed germination rates and stomatal aperture of TaCIPK2-overexpressing tobacco plants decreased after exogenous abscisic acid treatment was applied, implying that the transgenic tobacco plants were more sensitive to exogenous abscisic acid than the control plants. Our results indicate that TaCIPK2 plays a positive regulatory role in drought stress responses in transgenic tobacco plants.

Harnessing endogenous miR-181a to segregate transgenic antigen receptor expression in developing versus post-thymic T cells in murine hematopoietic chimeras.

PubMed

Papapetrou, Eirini P; Kovalovsky, Damian; Beloeil, Laurent; Sant'angelo, Derek; Sadelain, Michel

2009-01-01

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by targeting complementary sequences, referred to as miRNA recognition elements (MREs), typically located in the 3' untranslated region of mRNAs. miR-181a is highly expressed in developing thymocytes and markedly downregulated in post-thymic T cells. We investigated whether endogenous miR-181a can be harnessed to segregate expression of chimeric antigen receptors (CARs) and TCRs between developing and mature T cells. Lentiviral-encoded antigen receptors were tagged with a miR-181a-specific MRE and transduced into mouse BM cells that were used to generate hematopoietic chimeras. Expression of a CAR specific for human CD19 (hCD19) was selectively suppressed in late double-negative and double-positive thymocytes, coinciding with the peak in endogenous miR-181a expression. Receptor expression was fully restored in post-thymic resting and activated T cells, affording protection against a subsequent challenge with hCD19+ tumors. Hematopoietic mouse chimeras engrafted with a conalbumin-specific TCR prone to thymic clonal deletion acquired peptide-specific T cell responsiveness only when the vector-encoded TCR transcript was similarly engineered to be subject to regulation by miR-181a. These results demonstrate the potential of miRNA-regulated transgene expression in stem cell-based therapies, including cancer immunotherapy.

Ectopically expressing MdPIP1;3, an aquaporin gene, increased fruit size and enhanced drought tolerance of transgenic tomatoes.

PubMed

Wang, Lin; Li, Qing-Tian; Lei, Qiong; Feng, Chao; Zheng, Xiaodong; Zhou, Fangfang; Li, Lingzi; Liu, Xuan; Wang, Zhi; Kong, Jin

2017-12-19

Water deficit severely reduces apple growth and production, is detrimental to fruit quality and size. This problem is exacerbated as global warming is implicated in producing more severe drought stress. Thus water-efficiency has becomes the major target for apple breeding. A desired apple tree can absorb and transport water efficiently, which not only confers improved drought tolerance, but also guarantees fruit size for higher income returns. Aquaporins, as water channels, control water transportation across membranes and can regulate water flow by changing their amount and activity. The exploration of molecular mechanism of water efficiency and the gene wealth will pave a way for molecular breeding of drought tolerant apple tree. In the current study, we screened out a drought inducible aquaporin gene MdPIP1;3, which specifically enhanced its expression during fruit expansion in 'Fuji' apple (Malus domestica Borkh. cv. Red Fuji). It localized on plasma membranes and belonged to PIP1 subfamily. The tolerance to drought stress enhanced in transgenic tomato plants ectopically expressing MdPIP1;3, showing that the rate of losing water in isolated transgenic leaves was slower than wild type, and stomata of transgenic plants closed sensitively to respond to drought compared with wild type. Besides, length and diameter of transgenic tomato fruits increased faster than wild type, and in final, fruit sizes and fresh weights of transgenic tomatoes were bigger than wild type. Specially, in cell levels, fruit cell size from transgenic tomatoes was larger than wild type, showing that cell number per mm 2 in transgenic fruits was less than wild type. Altogether, ectopically expressing MdPIP1;3 enhanced drought tolerance of transgenic tomatoes partially via reduced water loss controlled by stomata closure in leaves. In addition, the transgenic tomato fruits are larger and heavier with larger cells via more efficient water transportation across membranes. Our research will contribute to apple production, by engineering apples with big fruits via efficient water transportation when well watered and enhanced drought tolerance in transgenic apples under water deficit.

Applications of Transgenics in Studies of Bone Sialoprotein

PubMed Central

Zhang, Jin; Tu, Qisheng; Chen, Jake

2010-01-01

Bone sialoprotein (BSP) is a major non-collagenous protein in mineralizing connective tissues such as dentin, cementum and calcified cartilage tissues. As a member of the SIBLING (Small Integrin-Binding Ligand, N-linked Glycoprotein) gene family of glycoproteins, BSP is involved in regulating hydroxyapatite crystal formation in bones and teeth, and has long been used as a marker gene for osteogenic differentiation. In the most recent decade, new discoveries in BSP gene expression and regulation, bone remodeling, bone metastasis, and bone tissue engineering have been achieved with the help of transgenic mice. In this review, we discuss these new discoveries obtained from the literatures and from our own laboratory, which were derived from the use of transgenic mouse mutants related to BSP gene or its promoter activity. PMID:19326395

GH indirectly enhances the regeneration of transgenic zebrafish fins through IGF2a and IGF2b.

PubMed

Nornberg, Bruna Félix; Almeida, Daniela Volcan; Figueiredo, Márcio Azevedo; Marins, Luis Fernando

2016-10-01

The somatotropic axis, composed essentially of the growth hormone (GH) and insulin-like growth factors (IGFs), is the main regulator of somatic growth in vertebrates. However, these protein hormones are also involved in various other major physiological processes. Although the importance of IGFs in mechanisms involving tissue regeneration has already been established, little is known regarding the direct effects of GH in these processes. In this study, we used a transgenic zebrafish (Danio rerio) model, which overexpresses GH from the beta-actin constitutive promoter. The regenerative ability of the caudal fin was assessed after repeated amputations, as well as the expression of genes related to the GH/IGF axis. The results revealed that GH overexpression increased the regenerated area of the caudal fin in transgenic fish after the second amputation. Transgenic fish also presented a decrease in gene expression of the GH receptor (ghrb), in opposition to the increased expression of the IGF1 receptors (igf1ra and igf1rb). These results suggest that transgenic fish have a higher sensitivity to IGFs than to GH during fin regeneration. With respect to the different IGFs produced locally, a decrease in igf1a expression and a significant increase in both igf2a and igf2b expression was observed, suggesting that igf1a is not directly involved in fin regeneration. Overall, the results revealed that excess GH enhances fin regeneration in zebrafish through igf2a and igf2b expression, acting indirectly on this major physiological process.

Targeted Enhancement of Glutamate-to-γ-Aminobutyrate Conversion in Arabidopsis Seeds Affects Carbon-Nitrogen Balance and Storage Reserves in a Development-Dependent Manner1[W][OA

PubMed Central

Fait, Aaron; Nesi, Adriano Nunes; Angelovici, Ruthie; Lehmann, Martin; Pham, Phuong Anh; Song, Luhua; Haslam, Richard P.; Napier, Johnathan A.; Galili, Gad; Fernie, Alisdair R.

2011-01-01

In seeds, glutamate decarboxylase (GAD) operates at the metabolic nexus between carbon and nitrogen metabolism by catalyzing the unidirectional decarboxylation of glutamate to form γ-aminobutyric acid (GABA). To elucidate the regulatory role of GAD in seed development, we generated Arabidopsis (Arabidopsis thaliana) transgenic plants expressing a truncated GAD from Petunia hybrida missing the carboxyl-terminal regulatory Ca2+-calmodulin-binding domain under the transcriptional regulation of the seed maturation-specific phaseolin promoter. Dry seeds of the transgenic plants accumulated considerable amounts of GABA, and during desiccation the content of several amino acids increased, although not glutamate or proline. Dry transgenic seeds had higher protein content than wild-type seeds but lower amounts of the intermediates of glycolysis, glycerol and malate. The total fatty acid content of the transgenic seeds was 50% lower than in the wild type, while acyl-coenzyme A accumulated in the transgenic seeds. Labeling experiments revealed altered levels of respiration in the transgenic seeds, and fractionation studies indicated reduced incorporation of label in the sugar and lipid fractions extracted from transgenic seeds. Comparative transcript profiling of the dry seeds supported the metabolic data. Cellular processes up-regulated at the transcript level included the tricarboxylic acid cycle, fatty acid elongation, the shikimate pathway, tryptophan metabolism, nitrogen-carbon remobilization, and programmed cell death. Genes involved in the regulation of germination were similarly up-regulated. Taken together, these results indicate that the GAD-mediated conversion of glutamate to GABA during seed development plays an important role in balancing carbon and nitrogen metabolism and in storage reserve accumulation. PMID:21921115

Targeted enhancement of glutamate-to-γ-aminobutyrate conversion in Arabidopsis seeds affects carbon-nitrogen balance and storage reserves in a development-dependent manner.

PubMed

Fait, Aaron; Nesi, Adriano Nunes; Angelovici, Ruthie; Lehmann, Martin; Pham, Phuong Anh; Song, Luhua; Haslam, Richard P; Napier, Johnathan A; Galili, Gad; Fernie, Alisdair R

2011-11-01

In seeds, glutamate decarboxylase (GAD) operates at the metabolic nexus between carbon and nitrogen metabolism by catalyzing the unidirectional decarboxylation of glutamate to form γ-aminobutyric acid (GABA). To elucidate the regulatory role of GAD in seed development, we generated Arabidopsis (Arabidopsis thaliana) transgenic plants expressing a truncated GAD from Petunia hybrida missing the carboxyl-terminal regulatory Ca(2+)-calmodulin-binding domain under the transcriptional regulation of the seed maturation-specific phaseolin promoter. Dry seeds of the transgenic plants accumulated considerable amounts of GABA, and during desiccation the content of several amino acids increased, although not glutamate or proline. Dry transgenic seeds had higher protein content than wild-type seeds but lower amounts of the intermediates of glycolysis, glycerol and malate. The total fatty acid content of the transgenic seeds was 50% lower than in the wild type, while acyl-coenzyme A accumulated in the transgenic seeds. Labeling experiments revealed altered levels of respiration in the transgenic seeds, and fractionation studies indicated reduced incorporation of label in the sugar and lipid fractions extracted from transgenic seeds. Comparative transcript profiling of the dry seeds supported the metabolic data. Cellular processes up-regulated at the transcript level included the tricarboxylic acid cycle, fatty acid elongation, the shikimate pathway, tryptophan metabolism, nitrogen-carbon remobilization, and programmed cell death. Genes involved in the regulation of germination were similarly up-regulated. Taken together, these results indicate that the GAD-mediated conversion of glutamate to GABA during seed development plays an important role in balancing carbon and nitrogen metabolism and in storage reserve accumulation.

Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes

PubMed Central

Ivanov, Sergey V.; Kuzmin, Igor; Wei, Ming-Hui; Pack, Svetlana; Geil, Laura; Johnson, Bruce E.; Stanbridge, Eric J.; Lerman, Michael I.

1998-01-01

To discover genes involved in von Hippel-Lindau (VHL)-mediated carcinogenesis, we used renal cell carcinoma cell lines stably transfected with wild-type VHL-expressing transgenes. Large-scale RNA differential display technology applied to these cell lines identified several differentially expressed genes, including an alpha carbonic anhydrase gene, termed CA12. The deduced protein sequence was classified as a one-pass transmembrane CA possessing an apparently intact catalytic domain in the extracellular CA module. Reintroduced wild-type VHL strongly inhibited the overexpression of the CA12 gene in the parental renal cell carcinoma cell lines. Similar results were obtained with CA9, encoding another transmembrane CA with an intact catalytic domain. Although both domains of the VHL protein contribute to regulation of CA12 expression, the elongin binding domain alone could effectively regulate CA9 expression. We mapped CA12 and CA9 loci to chromosome bands 15q22 and 17q21.2 respectively, regions prone to amplification in some human cancers. Additional experiments are needed to define the role of CA IX and CA XII enzymes in the regulation of pH in the extracellular microenvironment and its potential impact on cancer cell growth. PMID:9770531

Characterization and isolation of a T-DNA tagged banana promoter active during in vitro culture and low temperature stress.

PubMed

Santos, Efrén; Remy, Serge; Thiry, Els; Windelinckx, Saskia; Swennen, Rony; Sági, László

2009-06-24

Next-generation transgenic plants will require a more precise regulation of transgene expression, preferably under the control of native promoters. A genome-wide T-DNA tagging strategy was therefore performed for the identification and characterization of novel banana promoters. Embryogenic cell suspensions of a plantain-type banana were transformed with a promoterless, codon-optimized luciferase (luc+) gene and low temperature-responsive luciferase activation was monitored in real time. Around 16,000 transgenic cell colonies were screened for baseline luciferase activity at room temperature 2 months after transformation. After discarding positive colonies, cultures were re-screened in real-time at 26 degrees C followed by a gradual decrease to 8 degrees C. The baseline activation frequency was 0.98%, while the frequency of low temperature-responsive luciferase activity was 0.61% in the same population of cell cultures. Transgenic colonies with luciferase activity responsive to low temperature were regenerated to plantlets and luciferase expression patterns monitored during different regeneration stages. Twenty four banana DNA sequences flanking the right T-DNA borders in seven independent lines were cloned via PCR walking. RT-PCR analysis in one line containing five inserts allowed the identification of the sequence that had activated luciferase expression under low temperature stress in a developmentally regulated manner. This activating sequence was fused to the uidA reporter gene and back-transformed into a commercial dessert banana cultivar, in which its original expression pattern was confirmed. This promoter tagging and real-time screening platform proved valuable for the identification of novel promoters and genes in banana and for monitoring expression patterns throughout in vitro development and low temperature treatment. Combination of PCR walking techniques was efficient for the isolation of candidate promoters even in a multicopy T-DNA line. Qualitative and quantitative GUS expression analyses of one tagged promoter in a commercial cultivar demonstrated a reproducible promoter activity pattern during in vitro culture. Thus, this promoter could be used during in vitro selection and generation of commercial transgenic plants.

Global Screening of Antiviral Genes that Suppress Baculovirus Transgene Expression in Mammalian Cells.

PubMed

Wang, Chia-Hung; Naik, Nenavath Gopal; Liao, Lin-Li; Wei, Sung-Chan; Chao, Yu-Chan

2017-09-15

Although baculovirus has been used as a safe and convenient gene delivery vector in mammalian cells, baculovirus-mediated transgene expression is less effective in various mammalian cell lines. Identification of the negative regulators in host cells is necessary to improve baculovirus-based expression systems. Here, we performed high-throughput shRNA library screening, targeting 176 antiviral innate immune genes, and identified 43 host restriction factor genes in a human A549 lung carcinoma cell line. Among them, suppression of receptor interaction protein kinase 1 (RIP1, also known as RIPK1) significantly increased baculoviral transgene expression without resulting in significant cell death. Silencing of RIP1 did not affect viral entry or cell viability, but it did inhibit nuclear translocation of the IRF3 and NF-κB transcription factors. Also, activation of downstream signaling mediators (such as TBK1 and IRF7) was affected, and subsequent interferon and cytokine gene expression levels were abolished. Further, Necrostatin-1 (Nec-1)-an inhibitor of RIP1 kinase activity-dramatically increased baculoviral transgene expression in RIP1-silenced cells. Using baculovirus as a model system, this study presents an initial investigation of large numbers of human cell antiviral innate immune response factors against a "nonadaptive virus." In addition, our study has made baculovirus a more efficient gene transfer vector for some of the most frequently used mammalian cell systems.

In Vivo Regulation of Hepatitis B Virus Replication by Peroxisome Proliferators†

PubMed Central

Guidotti, Luca G.; Eggers, Carrie M.; Raney, Anneke K.; Chi, Susan Y.; Peters, Jeffrey M.; Gonzalez, Frank J.; McLachlan, Alan

1999-01-01

The role of the peroxisome proliferator-activated receptor α (PPARα) in regulating hepatitis B virus (HBV) transcription and replication in vivo was investigated in an HBV transgenic mouse model. Treatment of HBV transgenic mice with the peroxisome proliferators Wy-14,643 and clofibric acid resulted in a less than twofold increase in HBV transcription rates and steady-state levels of HBV RNAs in the livers of these mice. In male mice, this increase in transcription was associated with a 2- to 3-fold increase in replication intermediates, whereas in female mice it was associated with a 7- to 14-fold increase in replication intermediates. The observed increases in transcription and replication were dependent on PPARα. HBV transgenic mice lacking this nuclear hormone receptor showed similar levels of HBV transcripts and replication intermediates as untreated HBV transgenic mice expressing PPARα but failed to demonstrate alterations in either RNA or DNA synthesis in response to peroxisome proliferators. Therefore, it appears that very modest alterations in transcription can, under certain circumstances, result in relatively large increases in HBV replication in HBV transgenic mice. PMID:10559356

Soybean GmMYB73 promotes lipid accumulation in transgenic plants

PubMed Central

2014-01-01

Background Soybean is one of the most important oil crops. The regulatory genes involved in oil accumulation are largely unclear. We initiated studies to identify genes that regulate this process. Results One MYB-type gene GmMYB73 was found to display differential expression in soybean seeds of different developing stages by microarray analysis and was further investigated for its functions in lipid accumulation. GmMYB73 is a small protein with single MYB repeat and has similarity to CPC-like MYB proteins from Arabidopsis. GmMYB73 interacted with GL3 and EGL3, and then suppressed GL2, a negative regulator of oil accumulation. GmMYB73 overexpression enhanced lipid contents in both seeds and leaves of transgenic Arabidopsis plants. Seed length and thousand-seed weight were also promoted. GmMYB73 introduction into the Arabidopsis try cpc double mutant rescued the total lipids, seed size and thousand-seed weight. GmMYB73 also elevated lipid levels in seeds and leaves of transgenic Lotus, and in transgenic hairy roots of soybean plants. GmMYB73 promoted PLDα1 expression, whose promoter can be bound and inhibited by GL2. PLDα1 mutation reduced triacylglycerol levels mildly in seeds but significantly in leaves of Arabidopsis plants. Conclusions GmMYB73 may reduce GL2, and then release GL2-inhibited PLDα1 expression for lipid accumulation. Manipulation of GmMYB73 may potentially improve oil production in legume crop plants. PMID:24655684

Overexpression of MusaMYB31, a R2R3 type MYB transcription factor gene indicate its role as a negative regulator of lignin biosynthesis in banana

PubMed Central

Ganapathi, T. R.

2017-01-01

Lignin and polyphenols are important cellular components biosynthesized through phenylpropanoid pathway. Phenylpropanoid pathway in plants is regulated by some important transcription factors including R2R3 MYB transcription factors. In this study, we report the cloning and functional characterization of a banana R2R3-MYB transcription factor (MusaMYB31) by overexpression in transgenic banana plants and evaluated its potential role in regulating biosynthesis of lignin and polyphenols. Sequence analysis of MusaMYB31 indicated its clustering with members of subgroup 4 (Sg4) of R2R3MYB family which are well known for their role as repressors of lignin biosynthesis. Expression analysis indicated higher expression of MusaMYB31 in corm and root tissue, known for presence of highly lignified tissue than other organs of banana. Overexpression of MusaMYB31 in banana cultivar Rasthali was carried out and four transgenic lines were confirmed by GUS histochemical staining, PCR analysis and Southern blot. Histological and biochemical analysis suggested reduction of cell wall lignin in vascular elements of banana. Transgenic lines showed alteration in transcript levels of general phenylpropanoid pathway genes including lignin biosynthesis pathway genes. Reduction of total polyphenols content in transgenic lines was in line with the observation related to repression of general phenylpropanoid pathway genes. This study suggested the potential role of MusaMYB31 as repressor of lignin and polyphenols biosynthesis in banana. PMID:28234982

Firewalls Prevent Systemic Dissemination of Vectors Derived from Human Adenovirus Type 5 and Suppress Production of Transgene-Encoded Antigen in a Murine Model of Oral Vaccination

PubMed Central

Revaud, Julien; Unterfinger, Yves; Rol, Nicolas; Suleman, Muhammad; Shaw, Julia; Galea, Sandra; Gavard, Françoise; Lacour, Sandrine A.; Coulpier, Muriel; Versillé, Nicolas; Havenga, Menzo; Klonjkowski, Bernard; Zanella, Gina; Biacchesi, Stéphane; Cordonnier, Nathalie; Corthésy, Blaise; Ben Arous, Juliette; Richardson, Jennifer P.

2018-01-01

To define the bottlenecks that restrict antigen expression after oral administration of viral-vectored vaccines, we tracked vectors derived from the human adenovirus type 5 at whole body, tissue, and cellular scales throughout the digestive tract in a murine model of oral delivery. After intragastric administration of vectors encoding firefly luciferase or a model antigen, detectable levels of transgene-encoded protein or mRNA were confined to the intestine, and restricted to delimited anatomical zones. Expression of luciferase in the form of multiple small bioluminescent foci in the distal ileum, cecum, and proximal colon suggested multiple crossing points. Many foci were unassociated with visible Peyer's patches, implying that transduced cells lay in proximity to villous rather than follicle-associated epithelium, as supported by detection of transgene-encoded antigen in villous epithelial cells. Transgene-encoded mRNA but not protein was readily detected in Peyer's patches, suggesting that post-transcriptional regulation of viral gene expression might limit expression of transgene-encoded antigen in this tissue. To characterize the pathways by which the vector crossed the intestinal epithelium and encountered sentinel cells, a fluorescent-labeled vector was administered to mice by the intragastric route or inoculated into ligated intestinal loops comprising a Peyer's patch. The vector adhered selectively to microfold cells in the follicle-associated epithelium, and, after translocation to the subepithelial dome region, was captured by phagocytes that expressed CD11c and lysozyme. In conclusion, although a large number of crossing events took place throughout the intestine within and without Peyer's patches, multiple firewalls prevented systemic dissemination of vector and suppressed production of transgene-encoded protein in Peyer's patches. PMID:29423380

BnLATE, a Cys2/His2-Type Zinc-Finger Protein, Enhances Silique Shattering Resistance by Negatively Regulating Lignin Accumulation in the Silique Walls of Brassica napus

PubMed Central

Tao, Zhangsheng; Huang, Yi; Zhang, Lida; Wang, Xinfa; Liu, Guihua; Wang, Hanzhong

2017-01-01

Silique shattering resistance is one of the most important agricultural traits in oil crop breeding. Seed shedding from siliques prior to and during harvest causes devastating losses in oilseed yield. Lignin biosynthesis in the silique walls is thought to affect silique-shattering resistance in oil crops. Here, we identified and characterized B. napus LATE FLOWERING (BnLATE), which encodes a Cys2/His2-type zinc-finger protein. Heterologous expression of BnLATE under the double enhanced CaMV 35S promoter (D35S) in wild-type Arabidopsis plants resulted in a marked decrease in lignification in the replum, valve layer (carpel) and dehiscence zone. pBnLATE::GUS activity was strong in the yellowing silique walls of transgenic lines. Furthermore, the expression pattern of BnLATE and the lignin content gradient in the silique walls at 48 days after pollination (DAP) of 73290, a B. napus silique shattering-resistant line, are similar to those in transgenic Arabidopsis lines expressing BnLATE. Transcriptome sequencing of the silique walls revealed that genes encoding peroxidases, which polymerize monolignols and lignin in the phenylpropanoid pathway, were down-regulated at least two-fold change in the D35S::BnLATE transgenic lines. pBnLATE::BnLATE transgenic lines were further used to identify the function of BnLATE, and the results showed that lignification in the carpel and dehiscence zone of yellowing silique also remarkably decreased compared with the wild-type control, the silique shattering-resistance and expression pattern of peroxidase genes are very similar to results with D35S::BnLATE. These results suggest that BnLATE is a negative regulator of lignin biosynthesis in the yellowing silique walls, and promotes silique-shattering resistance in B. napus through restraining the polymerization of monolignols and lignin. PMID:28081140

MicroRNA166 Modulates Cadmium Tolerance and Accumulation in Rice.

PubMed

Ding, Yanfei; Gong, Shaohua; Wang, Yi; Wang, Feijuan; Bao, Hexigeduleng; Sun, Junwei; Cai, Chong; Yi, Keke; Chen, Zhixiang; Zhu, Cheng

2018-06-20

MicroRNAs (miRNAs) are 20- to 24-nucleotide small non-coding RNAs that regulate gene expression in eukaryotic organisms. Several plant miRNAs, such as miR166, have vital roles in plant growth, development and responses to environmental stresses. One such environmental stress encountered by crop plants is exposure to cadmium (Cd), an element highly toxic to most organisms, including humans and plants. In this study, we analyzed the role of miR166 in Cd accumulation and tolerance in rice (Oryza sativa). The expression levels of miR166 in both root and leaf tissues were significantly higher in the reproductive stage than in the seedling stage in rice. The expression of miR166 in the roots of rice seedlings was reduced after Cd treatment. Overexpression of miR166 in rice improved Cd tolerance, a result associated with the reduction of Cd-induced oxidative stress in transgenic rice plants. Furthermore, overexpression of miR166 reduced both Cd translocation from roots to shoots and Cd accumulation in the grains. miR166 targets genes encoding the class-III homeodomain-leucine zipper (HD-Zip) family proteins in plants. In rice, HOMEODOMAIN CONTAINING PROTEIN 4 (OsHB4) gene (Os03g43930), which encodes an HD-Zip protein, was up-regulated by Cd treatment but down-regulated by overexpression of miR166 in transgenic rice plants. Overexpression of OsHB4 increased Cd sensitivity and Cd accumulation in the leaves and grains of transgenic rice plants. By contrast, silencing OsHB4 by RNA interference enhanced Cd tolerance in transgenic rice plants. These results indicate a critical role for miR166 in Cd accumulation and tolerance through regulation of its target gene, OsHB4, in rice. {copyright, serif} 2018 American Society of Plant Biologists. All rights reserved.

Cross-regulation in the mouse HoxB complex: the expression of Hoxb2 in rhombomere 4 is regulated by Hoxb1.

PubMed

Maconochie, M K; Nonchev, S; Studer, M; Chan, S K; Pöpperl, H; Sham, M H; Mann, R S; Krumlauf, R

1997-07-15

Correct regulation of the segment-restricted patterns of Hox gene expression is essential for proper patterning of the vertebrate hindbrain. We have examined the molecular basis of restricted expression of Hoxb2 in rhombomere 4 (r4), by using deletion analysis in transgenic mice to identify an r4 enhancer from the mouse gene. A bipartite Hox/Pbx binding motif is located within this enhancer, and in vitro DNA binding experiments showed that the vertebrate labial-related protein Hoxb1 will cooperatively bind to this site in a Pbx/Exd-dependent manner. The Hoxb2 r4 enhancer can be transactivated in vivo by the ectopic expression of Hoxb1, Hoxa1, and Drosophila labial in transgenic mice. In contrast, ectopic Hoxb2 and Hoxb4 are unable to induce expression, indicating that in vivo this enhancer preferentially responds to labial family members. Mutational analysis demonstrated that the bipartite Hox/Pbx motif is required for r4 enhancer activity and the responses to retinoids and ectopic Hox expression. Furthermore, three copies of the Hoxb2 motif are sufficient to mediate r4 expression in transgenic mouse embryos and a labial pattern in Drosophila embryos. This reporter expression in Drosophila embryos is dependent upon endogenous labial and exd, suggesting that the ability of this Hox/Pbx site to interact with labial-related proteins has been evolutionarily conserved. The endogenous Hoxb2 gene is no longer upregulated in r4 in Hoxb1 homozygous mutant embryos. On the basis of these experiments we conclude that the r4-restricted domain of Hoxb2 in the hindbrain is the result of a direct cross-regulatory interaction by Hoxb1 involving vertebrate Pbx proteins as cofactors. This suggests that part of the functional role of Hoxb1 in maintaining r4 identity may be mediated by the Hoxb2 gene.

Molecular cloning and characterization of a gene regulating flowering time from Alfalfa (Medicago sativa L.).

PubMed

Zhang, Tiejun; Chao, Yuehui; Kang, Junmei; Ding, Wang; Yang, Qingchuan

2013-07-01

Genes that regulate flowering time play crucial roles in plant development and biomass formation. Based on the cDNA sequence of Medicago truncatula (accession no. AY690425), the LFY gene of alfalfa was cloned. Sequence similarity analysis revealed high homology with FLO/LFY family genes of other plants. When fused to the green fluorescent protein, MsLFY protein was localized in the nucleus of onion (Allium cepa L.) epidermal cells. The RT-qPCR analysis of MsLFY expression patterns showed that the expression of MsLFY gene was at a low level in roots, stems, leaves and pods, and the expression level in floral buds was the highest. The expression of MsLFY was induced by GA3 and long photoperiod. Plant expression vector was constructed and transformed into Arabidopsis by the agrobacterium-mediated methods. PCR amplification with the transgenic Arabidopsis genome DNA indicated that MsLFY gene had integrated in Arabidopsis genome. Overexpression of MsLFY specifically caused early flowering under long day conditions compared with non-transgenic plants. These results indicated MsLFY played roles in promoting flowering time.

The Wheat NAC Transcription Factor TaNAC2L Is Regulated at the Transcriptional and Post-Translational Levels and Promotes Heat Stress Tolerance in Transgenic Arabidopsis.

PubMed

Guo, Weiwei; Zhang, Jinxia; Zhang, Ning; Xin, Mingming; Peng, Huiru; Hu, Zhaorong; Ni, Zhongfu; Du, Jinkun

2015-01-01

Heat stress poses a serious threat to global crop production. In efforts that aim to mitigate the adverse effects of heat stress on crops, a variety of genetic tools are being used to develop plants with improved thermotolerance. The characterization of important regulators of heat stress tolerance provides essential information for this aim. In this study, we examine the wheat (Triticum aestivum) NAC transcription factor gene TaNAC2L. High temperature induced TaNAC2L expression in wheat and overexpression of TaNAC2L in Arabidopsis thaliana enhanced acquired heat tolerance without causing obvious alterations in phenotype compared with wild type under normal conditions. TaNAC2L overexpression also activated the expression of heat-related genes in the transgenic Arabidopsis plants, suggesting that TaNAC2L may improve heat tolerance by regulating the expression of stress-responsive genes. Notably, TaNAC2L is also regulated at the post-translational level and might be degraded via a proteasome-mediated pathway. Thus, this wheat transcription factor may have potential uses in enhancing thermotolerance in crops.

Senescence-inducible LEC2 enhances triacylglycerol accumulation in leaves without negatively affecting plant growth.

PubMed

Kim, Hyun Uk; Lee, Kyeong-Ryeol; Jung, Su-Jin; Shin, Hyun A; Go, Young Sam; Suh, Mi-Chung; Kim, Jong Bum

2015-12-01

The synthesis of fatty acids and glycerolipids in wild-type Arabidopsis leaves does not typically lead to strong triacylglycerol (TAG) accumulation. LEAFY COTYLEDON2 (LEC2) is a master regulator of seed maturation and oil accumulation in seeds. Constitutive ectopic LEC2 expression causes somatic embryogenesis and defects in seedling growth. Here, we report that senescence-inducible LEC2 expression caused a threefold increase in TAG levels in transgenic leaves compared with that in the leaves of wild-type plants. Plant growth was not severely affected by the accumulation the TAG in response to LEC2 expression. The levels of plastid-synthesized lipids, mono- and di-galactosyldiacylglycerol and phosphatidylglycerol were reduced more in senescence-induced LEC2 than in endoplasmic reticulum-synthesized lipids, including phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol. Senescence-induced LEC2 up-regulated the expression of many genes involved in fatty acid and TAG biosynthesis at precise times in senescent leaves, including WRINKLED1 (WRI1), which encodes a fatty acid transcription factor. The expressions of glycerol-3-phosphate dehydrogenase 1 and phospholipid:diacylglycerol 2 were increased in the transgenic leaves. Five seed-type oleosin-encoding genes, expressed during oil-body formation, and the seed-specific FAE1 gene, which encodes the enzyme responsible for the synthesis of C20:1 and C22:1 fatty acids, were also expressed at higher levels in senescing transgenic leaves than in wild-type leaves. Senescence-inducible LEC2 triggers the key metabolic steps that increase TAG accumulation in vegetative tissues. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

CsWRKY46, a WRKY transcription factor from cucumber, confers cold resistance in transgenic-plant by regulating a set of cold-stress responsive genes in an ABA-dependent manner.

PubMed

Zhang, Ying; Yu, Hongjun; Yang, Xueyong; Li, Qiang; Ling, Jian; Wang, Hong; Gu, Xingfang; Huang, Sanwen; Jiang, Weijie

2016-11-01

Plant WRKY transcription factors are trans-regulatory proteins that are involved in plant immune responses, development and senescence; however, their roles in abiotic stress are still not well understood, especially in the horticultural crop cucumber. In this study, a novel cucumber WRKY gene, CsWRKY46 was cloned and identified, which was up-regulated in response to cold stress and exogenous abscisic acid (ABA) treatment. CsWRKY46 is belonging to group II of the WRKY family, CsWRKY46 was found exclusively in the nucleus, as indicated by a transient expression assay. Yeast one-hybrid assay shown that CsWRKY46 interact with the W-box in the promoter of ABI5. Transgenic Arabidopsis lines over-expressing CsWRKY46, WRK46-OE1 and WRK46-OE5 had higher seedling survival rates upon freezing treatment compared with that of the wild-type. The above over-expression lines also showed much a higher proline accumulation, less electrolyte leakage and lower malondialdehyde (MDA) levels. Furthermore, the CsWRKY46 overexpression lines were hypersensitive to ABA during seed germination, but the seedlings were not. Quantitative RT-PCR analyses revealed that the expression levels of the ABA-responsive transcription factor ABI5 were higher in the WRKY46-OE lines than in wild-type and that the overexpression of CsWRKY46 increased the expression of stress-inducible genes, including RD29A and COR47. Taken together, our results demonstrated that CsWRKY46 from cucumber conferred cold tolerance to transgenic plants and positively regulated the cold signaling pathway in an ABA-dependent manner. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Phaseolin expression in tobacco chloroplast reveals an autoregulatory mechanism in heterologous protein translation.

PubMed

De Marchis, Francesca; Bellucci, Michele; Pompa, Andrea

2016-02-01

Plastid DNA engineering is a well-established research area of plant biotechnology, and plastid transgenes often give high expression levels. However, it is still almost impossible to predict the accumulation rate of heterologous protein in transplastomic plants, and there are many cases of unsuccessful transgene expression. Chloroplasts regulate their proteome at the post-transcriptional level, mainly through translation control. One of the mechanisms to modulate the translation has been described in plant chloroplasts for the chloroplast-encoded subunits of multiprotein complexes, and the autoregulation of the translation initiation of these subunits depends on the availability of their assembly partners [control by epistasy of synthesis (CES)]. In Chlamydomonas reinhardtii, autoregulation of endogenous proteins recruited in the assembly of functional complexes has also been reported. In this study, we revealed a self-regulation mechanism triggered by the accumulation of a soluble recombinant protein, phaseolin, in the stroma of chloroplast-transformed tobacco plants. Immunoblotting experiments showed that phaseolin could avoid this self-regulation mechanism when targeted to the thylakoids in transplastomic plants. To inhibit the thylakoid-targeted phaseolin translation as well, this protein was expressed in the presence of a nuclear version of the phaseolin gene with a transit peptide. Pulse-chase and polysome analysis revealed that phaseolin mRNA translation on plastid ribosomes was repressed due to the accumulation in the stroma of the same soluble polypeptide imported from the cytosol. We suggest that translation autoregulation in chloroplast is not limited to heteromeric protein subunits but also involves at least some of the foreign soluble recombinant proteins, leading to the inhibition of plastome-encoded transgene expression in chloroplast. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

CCN3 Protein Participates in Bone Regeneration as an Inhibitory Factor*

PubMed Central

Matsushita, Yuki; Sakamoto, Kei; Tamamura, Yoshihiro; Shibata, Yasuaki; Minamizato, Tokutaro; Kihara, Tasuku; Ito, Masako; Katsube, Ken-ichi; Hiraoka, Shuichi; Koseki, Haruhiko; Harada, Kiyoshi; Yamaguchi, Akira

2013-01-01

CCN3, a member of the CCN protein family, inhibits osteoblast differentiation in vitro. However, the role of CCN3 in bone regeneration has not been well elucidated. In this study, we investigated the role of CCN3 in bone regeneration. We identified the Ccn3 gene by microarray analysis as a highly expressed gene at the early phase of bone regeneration in a mouse bone regeneration model. We confirmed the up-regulation of Ccn3 at the early phase of bone regeneration by RT-PCR, Western blot, and immunofluorescence analyses. Ccn3 transgenic mice, in which Ccn3 expression was driven by 2.3-kb Col1a1 promoter, showed osteopenia compared with wild-type mice, but Ccn3 knock-out mice showed no skeletal changes compared with wild-type mice. We analyzed the bone regeneration process in Ccn3 transgenic mice and Ccn3 knock-out mice by microcomputed tomography and histological analyses. Bone regeneration in Ccn3 knock-out mice was accelerated compared with that in wild-type mice. The mRNA expression levels of osteoblast-related genes (Runx2, Sp7, Col1a1, Alpl, and Bglap) in Ccn3 knock-out mice were up-regulated earlier than those in wild-type mice, as demonstrated by RT-PCR. Bone regeneration in Ccn3 transgenic mice showed no significant changes compared with that in wild-type mice. Phosphorylation of Smad1/5 was highly up-regulated at bone regeneration sites in Ccn3 KO mice compared with wild-type mice. These results indicate that CCN3 is up-regulated in the early phase of bone regeneration and acts as a negative regulator for bone regeneration. This study may contribute to the development of new strategies for bone regeneration therapy. PMID:23653360

Regulating the ethylene response of a plant by modulation of F-box proteins

DOEpatents

Guo, Hongwei [Beijing, CN; Ecker, Joseph R [Carlsbad, CA

2014-01-07

The relationship between F-box proteins and proteins invovled in the ethylene response in plants is described. In particular, F-box proteins may bind to proteins involved in the ethylene response and target them for degradation by the ubiquitin/proteasome pathway. The transcription factor EIN3 is a key transcription factor mediating ethylne-regulated gene expression and morphological responses. EIN3 is degraded through a ubiquitin/proteasome pathway mediated by F-box proteins EBF1 and EBF2. The link between F-box proteins and the ethylene response is a key step in modulating or regulating the response of a plant to ethylene. Described herein are transgenic plants having an altered sensitivity to ethylene, and methods for making transgenic plant haing an althered sensitivity to ethylene by modulating the level of activity of F-box proteins. Methods of altering the ethylene response in a plant by modulating the activity or expression of an F-box protein are described. Also described are methods of identifying compounds that modulate the ethylene response in plants by modulating the level of F-box protein expression or activity.

IrrE, a Global Regulator of Extreme Radiation Resistance in Deinococcus radiodurans, Enhances Salt Tolerance in Escherichia coli and Brassica napus

PubMed Central

Zhou, Zhengfu; Yan, Yongliang; Zhang, Wei; Lu, Wei; Ping, Shuzhen; Dai, Qilin; Yuan, Menglong; Feng, Bin; Hou, Xiaoguang; Zhang, Ying; Ruiqiang; Liu, Tingting; Feng, Lu; Wang, Lei; Chen, Ming; Lin, Min

2009-01-01

Background Globally, about 20% of cultivated land is now affected by salinity. Salt tolerance is a trait of importance to all crops in saline soils. Previous efforts to improve salt tolerance in crop plants have met with only limited success. Bacteria of the genus Deinococcus are known for their ability to survive highly stressful conditions, and therefore possess a unique pool of genes conferring extreme resistance. In Deinococcus radiodurans, the irrE gene encodes a global regulator responsible for extreme radioresistance. Methodology/Principal Findings Using plate assays, we showed that IrrE protected E. coli cells against salt shock and other abiotic stresses such as oxidative, osmotic and thermal shocks. Comparative proteomic analysis revealed that IrrE functions as a switch to regulate different sets of proteins such as stress responsive proteins, protein kinases, glycerol-degrading enzymes, detoxification proteins, and growth-related proteins in E. coli. We also used quantitative RT-PCR to investigate expression of nine selected stress-responsive genes in transgenic and wild-type Brassica napus plants. Transgenic B. napus plants expressing the IrrE protein can tolerate 350 mM NaCl, a concentration that inhibits the growth of almost all crop plants. Conclusions Expression of IrrE, a global regulator for extreme radiation resistance in D. radiodurans, confers significantly enhanced salt tolerance in both E. coli and B. napus. We thus propose that the irrE gene might be used as a potentially promising transgene to improve abiotic stress tolerances in crop plants. PMID:19204796

Network Inference Analysis Identifies an APRR2-Like Gene Linked to Pigment Accumulation in Tomato and Pepper Fruits1[W][OA

PubMed Central

Pan, Yu; Bradley, Glyn; Pyke, Kevin; Ball, Graham; Lu, Chungui; Fray, Rupert; Marshall, Alexandra; Jayasuta, Subhalai; Baxter, Charles; van Wijk, Rik; Boyden, Laurie; Cade, Rebecca; Chapman, Natalie H.; Fraser, Paul D.; Hodgman, Charlie; Seymour, Graham B.

2013-01-01

Carotenoids represent some of the most important secondary metabolites in the human diet, and tomato (Solanum lycopersicum) is a rich source of these health-promoting compounds. In this work, a novel and fruit-related regulator of pigment accumulation in tomato has been identified by artificial neural network inference analysis and its function validated in transgenic plants. A tomato fruit gene regulatory network was generated using artificial neural network inference analysis and transcription factor gene expression profiles derived from fruits sampled at various points during development and ripening. One of the transcription factor gene expression profiles with a sequence related to an Arabidopsis (Arabidopsis thaliana) ARABIDOPSIS PSEUDO RESPONSE REGULATOR2-LIKE gene (APRR2-Like) was up-regulated at the breaker stage in wild-type tomato fruits and, when overexpressed in transgenic lines, increased plastid number, area, and pigment content, enhancing the levels of chlorophyll in immature unripe fruits and carotenoids in red ripe fruits. Analysis of the transcriptome of transgenic lines overexpressing the tomato APPR2-Like gene revealed up-regulation of several ripening-related genes in the overexpression lines, providing a link between the expression of this tomato gene and the ripening process. A putative ortholog of the tomato APPR2-Like gene in sweet pepper (Capsicum annuum) was associated with pigment accumulation in fruit tissues. We conclude that the function of this gene is conserved across taxa and that it encodes a protein that has an important role in ripening. PMID:23292788

Involvement of miR528 in the Regulation of Arsenite Tolerance in Rice (Oryza sativa L.).

PubMed

Liu, Qingpo; Hu, Haichao; Zhu, Leyi; Li, Ruochen; Feng, Ying; Zhang, Liqing; Yang, Yuyan; Liu, Xingquan; Zhang, Hengmu

2015-10-14

Tens of miRNAs were previously established as being arsenic (As) stress responsive in rice. However, their functional role in As tolerance remains unclear. This study demonstrates that transgenic plants overexpressing miR528 (Ubi::MIR528) were more sensitive to arsenite [As(III)] compared with wild-type (WT) rice. Under normal and stress conditions, miR528-5p and -3p were highly up-regulated in both the roots and leaves of transgenic plants, which exhibited a negative correlation with the expression of seven target genes. Compared with WT plants, Ubi::MIR528 plants showed excessive oxidative stress generation and remarkable amino acid content changes in the roots and leaves upon As(III) exposure. Notably, the expression profiles of diverse functional genes were clearly different between WT and transgenic plants. Thus, the observed As(III) sensitivity of Ubi::MIR528 plants was likely due to the strong alteration of antioxidant enzyme activity and amino acid profiles and the impairment of the As(III) uptake, translocation, and tolerance systems of rice.

Transient development of ovotestes in XX Sox9 transgenic mice.

PubMed

Gregoire, Elodie P; Lavery, Rowena; Chassot, Anne-Amandine; Akiyama, Haruhiko; Treier, Mathias; Behringer, Richard R; Chaboissier, Marie-Christine

2011-01-01

The sex of an individual results from the paternal transmission of the SRY gene located on the Y chromosome. In turn, SRY initiates Sox9 expression, a transcription factor required for testicular differentiation. Ectopic activation of SOX9 in XX Wt1:Sox9 transgenic mice induces female-to-male sex reversal in adult mice. Here we show that complete sex reversal is preceded by a transient phase of ovotestis differentiation with XX Wt1:Sox9 transgenic gonads containing a testicular central region and one or both ovarian poles indicating that Wt1:Sox9 is not as efficient as Sry to induce male development. In XX Wt1:Sox9(Tg/+) gonads, transgenic Sox9 is expressed earlier than Sox9 in XY gonads and is able to induce the expression of EGFP, knocked into the 3' UTR of Sox9 indicating that SOX9 is involved in the initiation and maintenance of its own expression. However, the delayed onset of expression of endogenous Sox9-EGFP suggests that this activation requires other factors, whose expression depends on SOX9. In the testicular regions of the XX Wt1:Sox9 ovotestes, proliferation of the XX fetal germ cells is hampered and they differentiate as pro-spermatogonia. This indicates that XX germ cells are not competent to respond to proliferative signals released from a testicular environment. In the ovarian regions, despite the continuous mRNA expression of the WT1:Sox9 transgene, the SOX9 protein does not accumulate suggesting that regulation of this gene in ovarian cells involves post-transcriptional mechanisms. Finally, ovarian cells of the XX Wt1:Sox9 ovotestis undergo apoptosis during late embryogenesis leading to complete female-to-male sex reversal of the transgenic mice at birth. Copyright © 2010 Elsevier Inc. All rights reserved.

Human CD22 Inhibits Murine B Cell Receptor Activation in a Human CD22 Transgenic Mouse Model.

PubMed

Bednar, Kyle J; Shanina, Elena; Ballet, Romain; Connors, Edward P; Duan, Shiteng; Juan, Joana; Arlian, Britni M; Kulis, Michael D; Butcher, Eugene C; Fung-Leung, Wai-Ping; Rao, Tadimeti S; Paulson, James C; Macauley, Matthew S

2017-11-01

CD22, a sialic acid-binding Ig-type lectin (Siglec) family member, is an inhibitory coreceptor of the BCR with established roles in health and disease. The restricted expression pattern of CD22 on B cells and most B cell lymphomas has made CD22 a therapeutic target for B cell-mediated diseases. Models to better understand how in vivo targeting of CD22 translates to human disease are needed. In this article, we report the development of a transgenic mouse expressing human CD22 (hCD22) in B cells and assess its ability to functionally substitute for murine CD22 (mCD22) for regulation of BCR signaling, Ab responses, homing, and tolerance. Expression of hCD22 on transgenic murine B cells is comparable to expression on human primary B cells, and it colocalizes with mCD22 on the cell surface. Murine B cells expressing only hCD22 have identical calcium (Ca 2+ ) flux responses to anti-IgM as mCD22-expressing wild-type B cells. Furthermore, hCD22 transgenic mice on an mCD22 -/- background have restored levels of marginal zone B cells and Ab responses compared with deficiencies observed in CD22 -/- mice. Consistent with these observations, hCD22 transgenic mice develop normal humoral responses in a peanut allergy oral sensitization model. Homing of B cells to Peyer's patches was partially rescued by expression of hCD22 compared with CD22 -/- B cells, although not to wild-type levels. Notably, Siglec-engaging antigenic liposomes formulated with an hCD22 ligand were shown to prevent B cell activation, increase cell death, and induce tolerance in vivo. This hCD22 transgenic mouse will be a valuable model for investigating the function of hCD22 and preclinical studies targeting hCD22. Copyright © 2017 by The American Association of Immunologists, Inc.

Highly conserved proximal promoter element harbouring paired Sox9-binding sites contributes to the tissue- and developmental stage-specific activity of the matrilin-1 gene.

PubMed

Rentsendorj, Otgonchimeg; Nagy, Andrea; Sinkó, Ildikó; Daraba, Andreea; Barta, Endre; Kiss, Ibolya

2005-08-01

The matrilin-1 gene has the unique feature that it is expressed in chondrocytes in a developmental stage-specific manner. Previously, we found that the chicken matrilin-1 long promoter with or without the intronic enhancer and the short promoter with the intronic enhancer restricted the transgene expression to the columnar proliferative chondroblasts and prehypertrophic chondrocytes of growth-plate cartilage in transgenic mice. To study whether the short promoter shared by these transgenes harbours cartilage-specific control elements, we generated transgenic mice expressing the LacZ reporter gene under the control of the matrilin-1 promoter between -338 and +67. Histological analysis of the founder embryos demonstrated relatively weak transgene activity in the developing chondrocranium, axial and appendicular skeleton with highest level of expression in the columnar proliferating chondroblasts and prehypertrophic chondrocytes. Computer analysis of the matrilin-1 genes of amniotes revealed a highly conserved Pe1 (proximal promoter element 1) and two less-conserved sequence blocks in the distal promoter region. The inverted Sox motifs of the Pe1 element interacted with chondrogenic transcription factors Sox9, L-Sox5 and Sox6 in vitro and another factor bound to the spacer region. Point mutations in the Sox motifs or in the spacer region interfered with or altered the formation of nucleoprotein complexes in vitro and significantly decreased the reporter gene activity in transient expression assays in chondrocytes. In vivo occupancy of the Sox motifs in genomic footprinting in the expressing cell type, but not in fibroblasts, also supported the involvement of Pe1 in the tissue-specific regulation of the gene. Our results indicate that interaction of Pe1 with distal DNA elements is required for the high level, cartilage- and developmental stage-specific transgene expression.

Expression of toxin co-regulated pilus subunit A (TCPA) of Vibrio cholerae and its immunogenic epitopes fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum).

PubMed

Sharma, Manoj Kumar; Singh, Nirmal Kumar; Jani, Dewal; Sisodia, Rama; Thungapathra, M; Gautam, J K; Meena, L S; Singh, Yogendra; Ghosh, Amit; Tyagi, Akhilesh Kumar; Sharma, Arun Kumar

2008-02-01

For protection against cholera, it is important to develop efficient vaccine capable of inducing anti-toxin as well as anti-colonizing immunity against Vibrio cholerae infections. Earlier, expression of cholera toxin B subunit (CTB) in tomato was reported by us. In the present investigation, toxin co-regulated pilus subunit A (TCPA), earlier reported to be an antigen capable of providing anti-colonization immunity, has been expressed in tomato. Further, to generate more potent combinatorial antigens, nucleotides encoding P4 or P6 epitope of TCPA were fused to cholera toxin B subunit gene (ctxB) and expressed in tomato. Presence of transgenes in the tomato genome was confirmed by PCR and expression of genes was confirmed at transcript and protein level. TCPA, chimeric CTB-P4 and CTB-P6 proteins were also expressed in E. coli. TCPA protein expressed in E. coli was purified to generate anti-TCPA antibodies in rabbit. Immunoblot and G(M1)-ELISA verified the synthesis and assembly of pentameric chimeric proteins in fruit tissue of transgenic tomato plants. The chimeric protein CTB-P4 and CTB-P6 accumulated up to 0.17 and 0.096% of total soluble protein (TSP), respectively, in tomato fruits. Whereas expression of TCPA, CTB-P4 and CTB-P6 in E. coli can be utilized for development of conventional vaccine, expression of these antigens which can provide both anti-toxin as well as anti-colonization immunity, has been demonstrated in plants, in a form which is potentially capable of inducing immune response against cholera infection.

Multiple Renal Cyst Development but Not Situs Abnormalities in Transgenic RNAi Mice against Inv::GFP Rescue Gene

PubMed Central

Kamijho, Yuki; Shiozaki, Yayoi; Sakurai, Eiki; Hanaoka, Kazunori; Watanabe, Daisuke

2014-01-01

In this study we generated RNA interference (RNAi)-mediated gene knockdown transgenic mice (transgenic RNAi mice) against the functional Inv gene. Inv mutant mice show consistently reversed internal organs (situs inversus), multiple renal cysts and neonatal lethality. The Inv::GFP-rescue mice, which introduced the Inv::GFP fusion gene, can rescue inv mutant mice phenotypes. This indicates that the Inv::GFP gene is functional in vivo. To analyze the physiological functions of the Inv gene, and to demonstrate the availability of transgenic RNAi mice, we introduced a short hairpin RNA expression vector against GFP mRNA into Inv::GFP-rescue mice and analyzed the gene silencing effects and Inv functions by examining phenotypes. Transgenic RNAi mice with the Inv::GFP-rescue gene (Inv-KD mice) down-regulated Inv::GFP fusion protein and showed hypomorphic phenotypes of inv mutant mice, such as renal cyst development, but not situs abnormalities or postnatal lethality. This indicates that shRNAi-mediated gene silencing systems that target the tag sequence of the fusion gene work properly in vivo, and suggests that a relatively high level of Inv protein is required for kidney development in contrast to left/right axis determination. Inv::GFP protein was significantly down-regulated in the germ cells of Inv-KD mice testis compared with somatic cells, suggesting the existence of a testicular germ cell-specific enhanced RNAi system that regulates germ cell development. The Inv-KD mouse is useful for studying Inv gene functions in adult tissue that are unable to be analyzed in inv mutant mice showing postnatal lethality. In addition, the shRNA-based gene silencing system against the tag sequence of the fusion gene can be utilized as a new technique to regulate gene expression in either in vitro or in vivo experiments. PMID:24586938

Comparison of growth and metabolic regulation between wild, domesticated and transgenic salmonids.

USDA-ARS?s Scientific Manuscript database

To gain a better understanding of the aspects underlying normal and growth hormone enhanced growth in salmonids, quantitative expression analysis was performed for a number of genes related to muscle growth, metabolism, immunology and energy regulation. This analysis was performed in liver and musc...

[Effect of ectopic expression of NtEXPA5 gene on cell size and growth of organs of transgenic tobacco plants].

PubMed

Kuluev, B R; Safiullina, M G; Kniazev, A V; Chemeris, A V

2013-01-01

We obtained transgenic tobacco plants demonstrating overexpression of NtEXPA5 gene that encodes alpha-expansin of Nicotiana tabacum. The transgenic plants were characterized by increased size of leaves and stems. However, size of flowers remained almost unchanged. The increase of organ sizes was induced by cell stretching only. Moreover, the number of cell divisions was even decreased. The obtained data suggest tight interaction between cell stretching regulation and cell division, which together provide the basic mechanism aimed at the controlling of plant organ sizes.

Polycomb-group protein SlMSI1 represses the expression of fruit-ripening genes to prolong shelf life in tomato.

PubMed

Liu, Dan-Dan; Zhou, Li-Jie; Fang, Mou-Jing; Dong, Qing-Long; An, Xiu-Hong; You, Chun-Xiang; Hao, Yu-Jin

2016-08-25

Polycomb-group (PcG) protein MULTICOPY SUPPRESSOR OF IRA1 (MSI1) protein is an evolutionarily conserved developmental suppressor and plays a crucial role in regulating epigenetic modulations. However, the potential role and function of MSI1 in fleshy fruits remain unknown. In this study, SlMSI1 was cloned and transformed into tomato to explore its function. The quantitative real-time PCR results showed that SlMSI1 was highly expressed in flowers and fruits and that its transcript and protein levels were significantly decreased in fruits after the breaker stage. Additionally, SlMSI1-overexpressing transgenic tomatoes displayed abnormal non-ripening fruit formation, whereas its suppression promoted fruit ripening in transgenic tomatoes. Quantitative real-time PCR assays also showed that RIN and its regulons were decreased in SlMSI1 overexpression transgenic tomato fruits. Furthermore, RNA-seq analysis demonstrated that SlMSI1 inhibits fruit ripening by negatively regulating a large set of fruit-ripening genes in addition to RIN and its regulons. Finally, genetic manipulation of SlMSI1 and RIN successfully prolonged the fruit shelf life by regulating the fruit-ripening genes in tomato. Our findings reveal a novel regulatory function of SlMSI1 in fruit ripening and provide a new regulator that may be useful for genetic engineering and modification of fruit shelf life.

Polycomb-group protein SlMSI1 represses the expression of fruit-ripening genes to prolong shelf life in tomato

PubMed Central

Liu, Dan-Dan; Zhou, Li-Jie; Fang, Mou-Jing; Dong, Qing-Long; An, Xiu-Hong; You, Chun-Xiang; Hao, Yu-Jin

2016-01-01

Polycomb-group (PcG) protein MULTICOPY SUPPRESSOR OF IRA1 (MSI1) protein is an evolutionarily conserved developmental suppressor and plays a crucial role in regulating epigenetic modulations. However, the potential role and function of MSI1 in fleshy fruits remain unknown. In this study, SlMSI1 was cloned and transformed into tomato to explore its function. The quantitative real-time PCR results showed that SlMSI1 was highly expressed in flowers and fruits and that its transcript and protein levels were significantly decreased in fruits after the breaker stage. Additionally, SlMSI1-overexpressing transgenic tomatoes displayed abnormal non-ripening fruit formation, whereas its suppression promoted fruit ripening in transgenic tomatoes. Quantitative real-time PCR assays also showed that RIN and its regulons were decreased in SlMSI1 overexpression transgenic tomato fruits. Furthermore, RNA-seq analysis demonstrated that SlMSI1 inhibits fruit ripening by negatively regulating a large set of fruit-ripening genes in addition to RIN and its regulons. Finally, genetic manipulation of SlMSI1 and RIN successfully prolonged the fruit shelf life by regulating the fruit-ripening genes in tomato. Our findings reveal a novel regulatory function of SlMSI1 in fruit ripening and provide a new regulator that may be useful for genetic engineering and modification of fruit shelf life. PMID:27558543

Petunia × hybrida floral scent production is negatively affected by high-temperature growth conditions.

PubMed

Cna'ani, Alon; Mühlemann, Joelle K; Ravid, Jasmin; Masci, Tania; Klempien, Antje; Nguyen, Thuong T H; Dudareva, Natalia; Pichersky, Eran; Vainstein, Alexander

2015-07-01

Increasing temperatures due to changing global climate are interfering with plant-pollinator mutualism, an interaction facilitated mainly by floral colour and scent. Gas chromatography-mass spectroscopy analyses revealed that increasing ambient temperature leads to a decrease in phenylpropanoid-based floral scent production in two Petunia × hybrida varieties, P720 and Blue Spark, acclimated at 22/16 or 28/22 °C (day/night). This decrease could be attributed to down-regulation of scent-related structural gene expression from both phenylpropanoid and shikimate pathways, and up-regulation of a negative regulator of scent production, emission of benzenoids V (EOBV). To test whether the negative effect of increased temperature on scent production can be reduced in flowers with enhanced metabolic flow in the phenylpropanoid pathway, we analysed floral volatile production by transgenic 'Blue Spark' plants overexpressing CaMV 35S-driven Arabidopsis thaliana production of anthocyanin pigments 1 (PAP1) under elevated versus standard temperature conditions. Flowers of 35S:PAP1 transgenic plants produced the same or even higher levels of volatiles when exposed to a long-term high-temperature regime. This phenotype was also evident when analysing relevant gene expression as inferred from sequencing the transcriptome of 35S:PAP1 transgenic flowers under the two temperature regimes. Thus, up-regulation of transcription might negate the adverse effects of temperature on scent production. © 2014 John Wiley & Sons Ltd.

Transcriptome changes associated with delayed flower senescence on transgenic petunia by inducing expression of etr1-1, a mutant ethylene receptor.

PubMed

Wang, Hong; Stier, Genevieve; Lin, Jing; Liu, Gang; Zhang, Zhen; Chang, Youhong; Reid, Michael S; Jiang, Cai-Zhong

2013-01-01

Flowers of ethylene-sensitive ornamental plants transformed with ethylene-insensitive 1-1(etr1-1), a mutant ethylene receptor first isolated from Arabidopsis, are known to have longer shelf lives. We have generated petunia plants in which the etr1-1 gene was over-expressed under the control of a chemically-inducible promoter, which would allow expression of etr1-1 to be initiated at the desired time and stage of development. Here, we showed that transgenic plants grew and developed normally without a chemical inducer. Semi-quantitative RT-PCR demonstrated that the abundance of transcripts of Arabidopsis etr1-1 gene was substantially induced in flowers with 30 μM dexamethasone (DEX). Consequently, t he life of the flowers was almost doubled and the peak of ethylene production was delayed. We compared gene expression changes of petals with DEX to those without DEX at 24 h and 48 h by microarray. Our results indicated that transcripts of many putative genes encoding transcription factors were down-regulated by etr1-1 induced expression at the early stage. In addition, putative genes involved in gibberellin biosynthesis, response to jasmonic acid/gibberellins stimulus, cell wall modification, ethylene biosynthesis, and cell death were down-regulated associating with etr1-1 induced expression. We investigated time-course gene expression profiles and found two profiles which displayed totally opposite expression patterns under these two treatments. In these profiles, 'the regulation of transcription' was predominant in GO categories. Taking all results together, we concluded those transcription factors down-regulated at early stage might exert a major role in regulating the senescence process which were consequently characterized by cell wall modification and cell death.

Transcriptome Changes Associated with Delayed Flower Senescence on Transgenic Petunia by Inducing Expression of etr1-1, a Mutant Ethylene Receptor

PubMed Central

Lin, Jing; Liu, Gang; Zhang, Zhen; Chang, Youhong; Reid, Michael S.; Jiang, Cai-Zhong

2013-01-01

Flowers of ethylene-sensitive ornamental plants transformed with ethylene-insensitive 1-1(etr1-1), a mutant ethylene receptor first isolated from Arabidopsis, are known to have longer shelf lives. We have generated petunia plants in which the etr1-1 gene was over-expressed under the control of a chemically-inducible promoter, which would allow expression of etr1-1 to be initiated at the desired time and stage of development. Here, we showed that transgenic plants grew and developed normally without a chemical inducer. Semi-quantitative RT-PCR demonstrated that the abundance of transcripts of Arabidopsis etr1-1 gene was substantially induced in flowers with 30 μM dexamethasone (DEX). Consequently, t he life of the flowers was almost doubled and the peak of ethylene production was delayed. We compared gene expression changes of petals with DEX to those without DEX at 24 h and 48 h by microarray. Our results indicated that transcripts of many putative genes encoding transcription factors were down-regulated by etr1-1 induced expression at the early stage. In addition, putative genes involved in gibberellin biosynthesis, response to jasmonic acid/gibberellins stimulus, cell wall modification, ethylene biosynthesis, and cell death were down-regulated associating with etr1-1 induced expression. We investigated time-course gene expression profiles and found two profiles which displayed totally opposite expression patterns under these two treatments. In these profiles, ‘the regulation of transcription’ was predominant in GO categories. Taking all results together, we concluded those transcription factors down-regulated at early stage might exert a major role in regulating the senescence process which were consequently characterized by cell wall modification and cell death. PMID:23874385

Arabidopsis DREB2C modulates ABA biosynthesis during germination.

PubMed

Je, Jihyun; Chen, Huan; Song, Chieun; Lim, Chae Oh

2014-09-12

Plant dehydration-responsive element binding factors (DREBs) are transcriptional regulators of the APETELA2/Ethylene Responsive element-binding Factor (AP2/ERF) family that control expression of abiotic stress-related genes. We show here that under conditions of mild heat stress, constitutive overexpression seeds of transgenic DREB2C overexpression Arabidopsis exhibit delayed germination and increased abscisic acid (ABA) content compared to untransformed wild-type (WT). Treatment with fluridone, an inhibitor of the ABA biosynthesis abrogated these effects. Expression of an ABA biosynthesis-related gene, 9-cis-epoxycarotenoid dioxygenase 9 (NCED9) was up-regulated in the DREB2C overexpression lines compared to WT. DREB2C was able to trans-activate expression of NCED9 in Arabidopsis leaf protoplasts in vitro. Direct and specific binding of DREB2C to a complete DRE on the NCED9 promoter was observed in electrophoretic mobility shift assays. Exogenous ABA treatment induced DREB2C expression in germinating seeds of WT. Vegetative growth of transgenic DREB2C overexpression lines was more strongly inhibited by exogenous ABA compared to WT. These results suggest that DREB2C is a stress- and ABA-inducible gene that acts as a positive regulator of ABA biosynthesis in germinating seeds through activating NCED9 expression. Copyright © 2014 Elsevier Inc. All rights reserved.

Generation of Rab-based transgenic lines for in vivo studies of endosome biology in zebrafish

PubMed Central

Clark, Brian S.; Winter, Mark; Cohen, Andrew R.; Link, Brian A.

2011-01-01

The Rab family of small GTPases function as molecular switches regulating membrane and protein trafficking. Individual Rab isoforms define and are required for specific endosomal compartments. To facilitate in vivo investigation of specific Rab proteins, and endosome biology in general, we have generated transgenic zebrafish lines to mark and manipulate Rab proteins. We also developed software to track and quantify endosome dynamics within time-lapse movies. The established transgenic lines ubiquitously express EGFP fusions of Rab5c (early endosomes), Rab11a (recycling endosomes), and Rab7 (late endosomes) to study localization and dynamics during development. Additionally, we generated UAS-based transgenic lines expressing constitutive active (CA) and dominant negative (DN) versions for each of these Rab proteins. Predicted localization and functional consequences for each line were verified through a variety of assays, including lipophilic dye uptake and Crumbs2a localization. In summary, we have established a toolset for in vivo analyses of endosome dynamics and functions. PMID:21976318

A laser pointer driven microheater for precise local heating and conditional gene regulation in vivo. Microheater driven gene regulation in zebrafish.

PubMed

Placinta, Mike; Shen, Meng-Chieh; Achermann, Marc; Karlstrom, Rolf O

2009-12-30

Tissue heating has been employed to study a variety of biological processes, including the study of genes that control embryonic development. Conditional regulation of gene expression is a particularly powerful approach for understanding gene function. One popular method for mis-expressing a gene of interest employs heat-inducible heat shock protein (hsp) promoters. Global heat shock of hsp-promoter-containing transgenic animals induces gene expression throughout all tissues, but does not allow for spatial control. Local heating allows for spatial control of hsp-promoter-driven transgenes, but methods for local heating are cumbersome and variably effective. We describe a simple, highly controllable, and versatile apparatus for heating biological tissue and other materials on the micron-scale. This microheater employs micron-scale fiber optics and uses an inexpensive laser-pointer as a power source. Optical fibers can be pulled on a standard electrode puller to produce tips of varying sizes that can then be used to reliably heat 20-100 mum targets. We demonstrate precise spatiotemporal control of hsp70l:GFP transgene expression in a variety of tissue types in zebrafish embryos and larvae. We also show how this system can be employed as part of a new method for lineage tracing that would greatly facilitate the study of organogenesis and tissue regulation at any time in the life cycle. This versatile and simple local heater has broad utility for the study of gene function and for lineage tracing. This system could be used to control hsp-driven gene expression in any organism simply by bringing the fiber optic tip in contact with the tissue of interest. Beyond these uses for the study of gene function, this device has wide-ranging utility in materials science and could easily be adapted for therapeutic purposes in humans.

Characterization of growth and reproduction performance, transgene integration, expression and transmission patterns in transgenic pigs produced by piggyBac transposition-mediated gene transfer

PubMed Central

Zeng, Fang; Li, Zicong; Cai, Gengyuan; Gao, Wenchao; Jiang, Gelong; Liu, Dewu; Urschitz, Johann; Moisyadi, Stefan; Wu, Zhenfang

2016-01-01

Previously we successfully produced a group of EGFP-expressing founder transgenic pigs by a newly developed efficient and simple pig transgenesis method based on cytoplasmic injection of piggyBac plasmids. In this study, we investigated the growth and reproduction performance, and characterized the transgene insertion, transmission and expression patterns in transgenic pigs generated by piggyBac transposition. Results showed that transgene has no injurious effect on the growth and reproduction of transgenic pigs. Multiple copies of monogenic EGFP transgene were inserted at noncoding sequences of host genome, and passed from founder transgenic pigs to their transgenic offspring in segregation or linkage manner. The EGFP transgene was ubiquitously expressed in transgenic pigs, and its expression intensity was associated with transgene copy number but not related to its promoter DNA methylation level. To the best of our knowledge, this is first study that fully described the growth and reproduction performance, transgene insertion, expression and transmission profiles in transgenic pigs produced by piggyBac system. It not only demonstrates that piggyBac transposition-mediated gene transfer is an effective and favourable approach for pig transgenesis, but also provides scientific information for understanding the transgene insertion, expression and transmission patterns in transgenic animals produced by piggyBac transposition. PMID:27565868

Novel marker for the onset of frontotemporal dementia: early increase in activity-dependent neuroprotective protein (ADNP) in the face of Tau mutation.

PubMed

Schirer, Yulie; Malishkevich, Anna; Ophir, Yotam; Lewis, Jada; Giladi, Eliezer; Gozes, Illana

2014-01-01

Tauopathy, a major pathology in Alzheimer's disease, is also found in ~50% of frontotemporal dementias (FTDs). Tau transcript, a product of a single gene, undergoes alternative splicing to yield 6 protein species, each with either 3 or 4 microtubule binding repeat domains (tau 3R or 4R, associated with dynamic and stable microtubules, respectively). While the healthy human brain shows a 1/1 ratio of tau 3R/4R, this ratio may be dramatically changed in the FTD brain. We have previously discovered that activity-dependent neuroprotective protein (ADNP) is essential for brain formation in the mouse, with ADNP+/- mice exhibiting tauopathy, age-driven neurodegeneration and behavioral deficits. Here, in transgenic mice overexpressing a mutated tau 4R species, in the cerebral cortex but not in the cerebellum, we showed significantly increased ADNP expression (~3-fold transcripts) in the cerebral cortex of young transgenic mice (~disease onset), but not in the cerebellum, as compared to control littermates. The transgene-age-related increased ADNP expression paralleled augmented dynamic tau 3R transcript level compared to control littermates. Blocking mutated tau 4R transgene expression resulted in normalization of ADNP and tau 3R expression. ADNP was previously shown to be a member of the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex. Here, Brahma (Brm), a component of the SWI/SNF complex regulating alternative splicing, showed a similar developmental expression pattern to ADNP. Immunoprecipitations further suggested Brm-ADNP interaction coupled to ADNP - polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF)-binding, with PSF being a direct regulator of tau transcript splicing. It should be noted that although we have shown a correlation between levels of ADNP and tau isoform expression three months of age, we are not presenting evidence of a direct link between the two. Future research into ADNP/tau relations is warranted.

Muscle-specific transgenic expression of porcine myostatin propeptide enhances muscle growth in mice.

PubMed

Wang, Kaiyun; Li, Zicong; Li, Yang; Zeng, Jinyong; He, Chang; Yang, Jinzeng; Liu, Dewu; Wu, Zhenfang

2013-10-01

Myostatin is a well-known negative regulator of skeletal muscle growth. Inhibition of myostatin activity results in increased muscle mass. Myostatin propeptide, as a myostatin antagonist, could be applied to promote meat production in livestock such as pigs. In this study, we generated a transgenic mouse model expressing porcine myostatin propeptide under the control of muscle-specific regulatory elements. The mean body weight of transgenic mice from a line expressing the highest level of porcine myostatin propeptide was increased by 5.4 % (P = 0.023) and 3.2 % (P = 0.031) in males and females, respectively, at 8 weeks of age. Weight of carcass, fore limb and hind limb was respectively increased by 6.0 % (P = 0.038), 9.0 % (P = 0.014), 8.7 % (P = 0.036) in transgenic male mice, compared to wild-type male controls at the age of 9 weeks. Similarly, carcass, fore limb and hind limb of transgenic female mice was 11.4 % (P = 0.002), 14.5 % (P = 0.006) and 14.5 % (P = 0.03) respectively heavier than that of wild-type female mice. The mean cross-section area of muscle fiber was increased by 17 % (P = 0.002) in transgenic mice, in comparison with wild-type controls. These results demonstrated that porcine myostatin propeptide is effective in enhancement of muscle growth. The present study provided useful information for future study on generation of transgenic pigs overexpressing porcine myostatin propeptide for improvement of muscle mass.

Improved photobio-H2 production regulated by artificial miRNA targeting psbA in green microalga Chlamydomonas reinhardtii.

PubMed

Li, Hui; Liu, Yanmei; Wang, Yuting; Chen, Meirong; Zhuang, Xiaoshan; Wang, Chaogang; Wang, Jiangxin; Hu, Zhangli

2018-01-01

Sulfur-deprived cultivation of Chlamydomonas reinhardtii , referred as "two-stage culture" transferring the cells from regular algal medium to sulfur-deplete one, has been extensively studied to improve photobio-H 2 production in this green microalga. During sulfur-deprivation treatment, the synthesis of a key component of photosystem II complex, D1 protein, was inhibited and improved photobio-H 2 production could be established in C. reinhardtii . However, separation of algal cells from a regular liquid culture medium to a sulfur-deprived one is not only a discontinuous process, but also a cost- and time-consuming operation. More applicable and economic alternatives for sustained H 2 production by C. reinhardtii are still highly required. In the present study, a significant improvement in photobio-H 2 production was observed in the transgenic green microalga C. reinhardtii , which employed a newly designed strategy based on a heat-inducible artificial miRNA (amiRNA) expression system targeting D1-encoded gene, psbA . A transgenic algal strain referred as "amiRNA-D1" has been successfully obtained by transforming the expression vector containing a heat-inducible promoter. After heat shock conducted in the same algal cultures, the expression of amiRNA-D1 was detected increased 15-fold accompanied with a 73% decrease of target gene psbA . More interestingly, this transgenic alga accumulated about 60% more H 2 content than the wild-type strain CC-849 at the end of 7-day cultivation. The photobio-H 2 production in the engineered transgenic alga was significantly improved. Without imposing any nutrient-deprived stress, this novel strategy provided a convenient and efficient way for regulation of photobio-H 2 production in green microalga by simply "turn on" the expression of a designed amiRNA.

A Novel WRKY Transcription Factor, MuWRKY3 (Macrotyloma uniflorum Lam. Verdc.) Enhances Drought Stress Tolerance in Transgenic Groundnut (Arachis hypogaea L.) Plants.

PubMed

Kiranmai, Kurnool; Lokanadha Rao, Gunupuru; Pandurangaiah, Merum; Nareshkumar, Ambekar; Amaranatha Reddy, Vennapusa; Lokesh, Uppala; Venkatesh, Boya; Anthony Johnson, A M; Sudhakar, Chinta

2018-01-01

Drought stress has adverse effects on growth, water relations, photosynthesis and yield of groundnut. WRKY transcription factors (TFs) are the plant-specific TFs which regulate several down-stream stress-responsive genes and play an essential role in plant biotic and abiotic stress responses. We found that WRKY3 gene is highly up-regulated under drought stress conditions and therefore isolated a new WRKY3TF gene from a drought-adapted horsegram ( Macrotyloma uniflorum Lam. Verdc.). Conserved domain studies revealed that protein encoded by this gene contains highly conserved regions of two WRKY domains and two C2H2 zinc-finger motifs. The fusion protein localization studies of transient MuWRKY 3-YFP revealed its nuclear localization. Overexpression of MuWRKY3 TF gene in groundnut ( Arachis hypogaea L.) showed increased tolerance to drought stress compared to wild-type (WT) plants. MuWRKY3 groundnut transgenics displayed lesser and delayed wilting symptoms than WT plants after 10-days of drought stress imposition. The transgenic groundnut plants expressing MuWRKY3 showed less accumulation of malondialdehyde, hydrogen peroxide (H 2 O 2 ), and superoxide anion (O 2 ∙- ), accompanied by more free proline, total soluble sugar content, and activities of antioxidant enzymes than WT plants under drought stress. Moreover, a series of stress-related LEA, HSP, MIPS, APX, SOD , and CAT genes found up-regulated in the transgenic groundnut plants. The study demonstrates that nuclear-localized MuWRKY3 TF regulates the expression of stress-responsive genes and the activity of ROS scavenging enzymes which results in improved drought tolerance in groundnut. We conclude that MuWRKY3 may serve as a new putative candidate gene for the improvement of stress resistance in plants.

A Novel WRKY Transcription Factor, MuWRKY3 (Macrotyloma uniflorum Lam. Verdc.) Enhances Drought Stress Tolerance in Transgenic Groundnut (Arachis hypogaea L.) Plants

PubMed Central

Kiranmai, Kurnool; Lokanadha Rao, Gunupuru; Pandurangaiah, Merum; Nareshkumar, Ambekar; Amaranatha Reddy, Vennapusa; Lokesh, Uppala; Venkatesh, Boya; Anthony Johnson, A. M.; Sudhakar, Chinta

2018-01-01

Drought stress has adverse effects on growth, water relations, photosynthesis and yield of groundnut. WRKY transcription factors (TFs) are the plant-specific TFs which regulate several down-stream stress-responsive genes and play an essential role in plant biotic and abiotic stress responses. We found that WRKY3 gene is highly up-regulated under drought stress conditions and therefore isolated a new WRKY3TF gene from a drought-adapted horsegram (Macrotyloma uniflorum Lam. Verdc.). Conserved domain studies revealed that protein encoded by this gene contains highly conserved regions of two WRKY domains and two C2H2 zinc-finger motifs. The fusion protein localization studies of transient MuWRKY3-YFP revealed its nuclear localization. Overexpression of MuWRKY3 TF gene in groundnut (Arachis hypogaea L.) showed increased tolerance to drought stress compared to wild-type (WT) plants. MuWRKY3 groundnut transgenics displayed lesser and delayed wilting symptoms than WT plants after 10-days of drought stress imposition. The transgenic groundnut plants expressing MuWRKY3 showed less accumulation of malondialdehyde, hydrogen peroxide (H2O2), and superoxide anion (O2∙-), accompanied by more free proline, total soluble sugar content, and activities of antioxidant enzymes than WT plants under drought stress. Moreover, a series of stress-related LEA, HSP, MIPS, APX, SOD, and CAT genes found up-regulated in the transgenic groundnut plants. The study demonstrates that nuclear-localized MuWRKY3 TF regulates the expression of stress-responsive genes and the activity of ROS scavenging enzymes which results in improved drought tolerance in groundnut. We conclude that MuWRKY3 may serve as a new putative candidate gene for the improvement of stress resistance in plants. PMID:29616059

Twist functions in vertebral column formation in medaka, Oryzias latipes.

PubMed

Yasutake, Junichi; Inohaya, Keiji; Kudo, Akira

2004-07-01

Medaka twist, a basic helix-loop-helix (bHLH) transcription factor, is expressed in the sclerotome during embryogenesis. We previously established a line of twist-EGFP transgenic medaka, whose EGFP expression is regulated by the twist promoter; therefore, we could observe the behavior of sclerotomal cells in vivo. In the transgenic medaka embryos, EGFP-positive sclerotomal cells migrated dorsally around the notochord and the neural tube, where at a later stage the vertebral column would be formed. This finding strongly suggests that twist-expressing sclerotomal cells participate in vertebral column formation in medaka. To clarify the function of twist gene in the sclerotome, we performed knockdown analysis of twist by using two kinds of morpholino antisense oligonucleotides targeted against twist (MO1 and MO2). Both the MO1 and MO2 morphants exhibited absence of neural arches, which are bilaterally paired, dorsomedially oriented bones on the dorsal aspect of the centrum. In addition, MO2, which blocks translation of only endogenous twist mRNA in the twist-EGFP transgenic medaka, did not affect the migration pattern of EGFP-positive cells, revealing that the migration of sclerotome-derived cells were normal in the absence of twist gene function. These results demonstrate that medaka twist functions in vertebral column formation by regulating the sclerotomal cell differentiation.

Ectopic expression of Triticum aestivum SERK genes (TaSERKs) control plant growth and development in Arabidopsis.

PubMed

Singh, Akanksha; Khurana, Paramjit

2017-09-28

Somatic embryogenesis receptor kinases (SERKs) belong to a small gene family of receptor-like kinases involved in signal transduction. A total of 54 genes were shortlisted from the wheat genome survey sequence of which 5 were classified as SERKs and 49 were identified as SERK-like (SERLs). Tissue- specific expression of TaSERKs at major developmental stages of wheat corroborates their indispensable role during somatic and zygotic embryogenesis. TaSERK transcripts show inherent differences in their hormonal sensitivities, i.e. TaSERK2 and TaSERK3 elicits auxin- specific responses while TaSERK1, 4 and 5 were more specific towards BR-mediated regulation. The ectopic expression of TaSERK1, 2, 3, 4 and 5 in Arabidopsis led to enhanced plant height, larger silique size and increased seed yield. Zygotic embryogenesis specific genes showed a differential pattern in TaSERK Arabidopsis transgenics specifically in the silique tissues. Elongated hypocotyls and enhanced root growth were observed in the overexpression transgenic lines of all five TaSERKs. The inhibitory action of auxin and brassinosteroid in all the TaSERK transgenic lines indicates their role in regulating root development. The results obtained imply redundant functions of TaSERKs in maintaining plant growth and development.

Divergent Regulation of CBF Regulon on Cold Tolerance and Plant Phenotype in Cassava Overexpressing Arabidopsis CBF3 Gene.

PubMed

An, Dong; Ma, Qiuxiang; Yan, Wei; Zhou, Wenzhi; Liu, Guanghua; Zhang, Peng

2016-01-01

Cassava is a tropical origin plant that is sensitive to chilling stress. In order to understand the CBF cold response pathway, a well-recognized regulatory mechanism in temperate plants, in cassava, overexpression of an Arabidopsis CBF3 gene is studied. This gene renders cassava increasingly tolerant to cold and drought stresses but is associated with retarded plant growth, leaf curling, reduced storage root yield, and reduced anthocyanin accumulation in a transcript abundance-dependent manner. Physiological analysis revealed that the transgenic cassava increased proline accumulation, reduced malondialdehyde production, and electrolyte leakage under cold stress. These transgenic lines also showed high relative water content when faced with drought. The expression of partial CBF-targeted genes in response to cold displayed temporal and spatial variations in the wild-type and transgenic plants: highly inducible in leaves and less altered in apical buds. In addition, anthocyanin accumulation was inhibited by downregulating the expression of genes involved in its biosynthesis and by interplaying between the CBF3 and the endogenous transcription factors. Thus, the heterologous CBF3 modulates the expression of stress-related genes and carries out a series of physiological adjustments under stressful conditions, showing a varied regulation pattern of CBF regulon from that of cassava CBFs.

Genome-wide analysis of Glycine soja ubiquitin (UBQ) genes and functional analysis of GsUBQ10 in response to alkaline stress.

PubMed

Chen, Chao; Chen, Ranran; Wu, Shengyang; Zhu, Dan; Sun, Xiaoli; Liu, Beidong; Li, Qiang; Zhu, Yanming

2018-03-26

Ubiquitin is a highly conserved protein with multiple essential regulation functions through the ubiquitin-proteasome system. Even though its functions in the ubiquitin-mediated protein degradation pathway were very well characterized. The functions of ubiquitin genes in regulating alkaline stress response are not fully established. In this study, we identified 12 potential UBQ genes in Glycine soja genome, and analyzed their evolutionary relationship, conserved domains and promoter cis-elements. We also explored the expression profiles of G. soja UBQ genes under alkaline stress, based on the transcriptome sequencing. We found that the expression of GsUBQ10 was significantly induced by alkaline stress, and function of GsUBQ10 was characterized using overexpression transgenic alfalfa (Medicago sativa). Our results suggested that GsUBQ10 transgenic lines significantly improved the alkaline tolerance in alfalfa. The GsUBQ10 transgenic lines showed lower relative membrane permeability, lower malon dialdehyde content and higher catalase activity than in the wild-type plants. This indicates that GsUBQ10 is involved in regulating the reactive oxygen species accumulation under alkaline stress. Taken together, we identified an ubiquitin gene GsUBQ10 from G. soja, which plays a positive role in responses to alkaline stress in alfalfa. This article is protected by copyright. All rights reserved.

Employing epigenetics to augment the expression of therapeutic proteins in mammalian cells.

PubMed

Kwaks, Ted H J; Otte, Arie P

2006-03-01

Recombinant proteins form an increasingly large part of the portfolio of biopharmaceutical companies. Production of these often complex transgenic proteins is achieved predominantly in mammalian cell lines but the process is hampered by low yields and unstable expression. Some of these problems are caused by gene silencing at the level of chromatin - so-called epigenetic gene silencing. Here, we describe approaches, which have emerged during the past few years, designed to interfere with epigenetic gene silencing with the aim of enhancing and stabilizing transgene expression. These include targeting histones, the inclusion of specific DNA elements and targeting sites of high gene-expression. We conclude that employing epigenetic gene regulation tools, in combination with further process optimization, might represent the next step forward in the production of therapeutic proteins.

Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Wen-Ta; Li, Hui-Chun; Lee, Shen-Kao

Highlights: •HCV core and F proteins could induce hepatocyte proliferation in the transgenic mice. •β-Catenin signaling pathway was activated by core protein in the transgenic mice. •β-Catenin signaling pathway was activated by myc-F protein in the transgenic mice. •Expression of SMA protein was enhanced by core but not myc-F protein. -- Abstract: The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made itmore » more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway.« less

Targeted taste cell-specific overexpression of brain-derived neurotrophic factor in adult taste buds elevates phosphorylated TrkB protein levels in taste cells, increases taste bud size, and promotes gustatory innervation.

PubMed

Nosrat, Irina V; Margolskee, Robert F; Nosrat, Christopher A

2012-05-11

Brain-derived neurotrophic factor (BDNF) is the most potent neurotrophic factor in the peripheral taste system during embryonic development. It is also expressed in adult taste buds. There is a lack of understanding of the role of BDNF in the adult taste system. To address this, we generated novel transgenic mice in which transgene expression was driven by an α-gustducin promoter coupling BDNF expression to the postnatal expression of gustducin in taste cells. Immunohistochemistry revealed significantly stronger BDNF labeling in taste cells of high BDNF-expressing mouse lines compared with controls. We show that taste buds in these mice are significantly larger and have a larger number of taste cells compared with controls. To examine whether innervation was affected in Gust-BDNF mice, we used antibodies to neural cell adhesion molecule (NCAM) and ATP receptor P2X3. The total density of general innervation and specifically the gustatory innervation was markedly increased in high BDNF-expressing mice compared with controls. TrkB and NCAM gene expression in laser capture microdissected taste epithelia were significantly up-regulated in these mice. Up-regulation of TrkB transcripts in taste buds and elevated taste cell-specific TrkB phosphorylation in response to increased BDNF levels indicate that BDNF controls the expression and activation of its high affinity receptor in taste cells. This demonstrates a direct taste cell function for BDNF. BDNF also orchestrates and maintains taste bud innervation. We propose that the Gust-BDNF transgenic mouse models can be employed to further dissect the specific roles of BDNF in the adult taste system.

Targeted Taste Cell-specific Overexpression of Brain-derived Neurotrophic Factor in Adult Taste Buds Elevates Phosphorylated TrkB Protein Levels in Taste Cells, Increases Taste Bud Size, and Promotes Gustatory Innervation*

PubMed Central

Nosrat, Irina V.; Margolskee, Robert F.; Nosrat, Christopher A.

2012-01-01

Brain-derived neurotrophic factor (BDNF) is the most potent neurotrophic factor in the peripheral taste system during embryonic development. It is also expressed in adult taste buds. There is a lack of understanding of the role of BDNF in the adult taste system. To address this, we generated novel transgenic mice in which transgene expression was driven by an α-gustducin promoter coupling BDNF expression to the postnatal expression of gustducin in taste cells. Immunohistochemistry revealed significantly stronger BDNF labeling in taste cells of high BDNF-expressing mouse lines compared with controls. We show that taste buds in these mice are significantly larger and have a larger number of taste cells compared with controls. To examine whether innervation was affected in Gust-BDNF mice, we used antibodies to neural cell adhesion molecule (NCAM) and ATP receptor P2X3. The total density of general innervation and specifically the gustatory innervation was markedly increased in high BDNF-expressing mice compared with controls. TrkB and NCAM gene expression in laser capture microdissected taste epithelia were significantly up-regulated in these mice. Up-regulation of TrkB transcripts in taste buds and elevated taste cell-specific TrkB phosphorylation in response to increased BDNF levels indicate that BDNF controls the expression and activation of its high affinity receptor in taste cells. This demonstrates a direct taste cell function for BDNF. BDNF also orchestrates and maintains taste bud innervation. We propose that the Gust-BDNF transgenic mouse models can be employed to further dissect the specific roles of BDNF in the adult taste system. PMID:22442142

OsRACK1 Is Involved in Abscisic Acid- and H2O2-Mediated Signaling to Regulate Seed Germination in Rice (Oryza sativa, L.)

PubMed Central

Zhang, Dongping; Chen, Li; Li, Dahong; Lv, Bing; Chen, Yun; Chen, Jingui; XuejiaoYan; Liang, Jiansheng

2014-01-01

The receptor for activated C kinase 1 (RACK1) is one member of the most important WD repeat–containing family of proteins found in all eukaryotes and is involved in multiple signaling pathways. However, compared with the progress in the area of mammalian RACK1, our understanding of the functions and molecular mechanisms of RACK1 in the regulation of plant growth and development is still in its infancy. In the present study, we investigated the roles of rice RACK1A gene (OsRACK1A) in controlling seed germination and its molecular mechanisms by generating a series of transgenic rice lines, of which OsRACK1A was either over-expressed or under-expressed. Our results showed that OsRACK1A positively regulated seed germination and negatively regulated the responses of seed germination to both exogenous ABA and H2O2. Inhibition of ABA biosynthesis had no enhancing effect on germination, whereas inhibition of ABA catabolism significantly suppressed germination. ABA inhibition on seed germination was almost fully recovered by exogenous H2O2 treatment. Quantitative analyses showed that endogenous ABA levels were significantly higher and H2O2 levels significantly lower in OsRACK1A-down regulated transgenic lines as compared with those in wildtype or OsRACK1A-up regulated lines. Quantitative real-time PCR analyses showed that the transcript levels of OsRbohs and amylase genes, RAmy1A and RAmy3D, were significantly lower in OsRACK1A-down regulated transgenic lines. It is concluded that OsRACK1A positively regulates seed germination by controlling endogenous levels of ABA and H2O2 and their interaction. PMID:24865690

Negative Regulation of Abscisic Acid Signaling by the Fagus sylvatica FsPP2C1 Plays A Role in Seed Dormancy Regulation and Promotion of Seed Germination1

PubMed Central

González-García, Mary Paz; Rodríguez, Dolores; Nicolás, Carlos; Rodríguez, Pedro Luis; Nicolás, Gregorio; Lorenzo, Oscar

2003-01-01

FsPP2C1 was previously isolated from beech (Fagus sylvatica) seeds as a functional protein phosphatase type-2C (PP2C) with all the conserved features of these enzymes and high homology to ABI1, ABI2, and PP2CA, PP2Cs identified as negative regulators of ABA signaling. The expression of FsPP2C1 was induced upon abscisic acid (ABA) treatment and was also up-regulated during early weeks of stratification. Furthermore, this gene was specifically expressed in ABA-treated seeds and was hardly detectable in vegetative tissues. In this report, to provide genetic evidence on FsPP2C1 function in seed dormancy and germination, we used an overexpression approach in Arabidopsis because transgenic work is not feasible in beech. Constitutive expression of FsPP2C1 under the cauliflower mosaic virus 35S promoter confers ABA insensitivity in Arabidopsis seeds and, consequently, a reduced degree of seed dormancy. Additionally, transgenic 35S:FsPP2C1 plants are able to germinate under unfavorable conditions, as inhibitory concentrations of mannitol, NaCl, or paclobutrazol. In vegetative tissues, Arabidopsis FsPP2C1 transgenic plants show ABA-resistant early root growth and diminished induction of the ABA-response genes RAB18 and KIN2, but no effect on stomatal closure regulation. Seed and vegetative phenotypes of Arabidopsis 35S:FsPP2C1 plants suggest that FsPP2C1 negatively regulates ABA signaling. The ABA inducibility of FsPP2C1 expression, together with the transcript accumulation mainly in seeds, suggest that it could play an important role modulating ABA signaling in beechnuts through a negative feedback loop. Finally, we suggest that negative regulation of ABA signaling by FsPP2C1 is a factor contributing to promote the transition from seed dormancy to germination during early weeks of stratification. PMID:12970481

Post-transcriptional regulation mediated by specific neurofilament introns in vivo.

PubMed

Wang, Chen; Szaro, Ben G

2016-04-01

Neurons regulate genes post-transcriptionally to coordinate the supply of cytoskeletal proteins, such as the medium neurofilament (NEFM), with demand for structural materials in response to extracellular cues encountered by developing axons. By using a method for evaluating functionality of cis-regulatory gene elements in vivo through plasmid injection into Xenopus embryos, we discovered that splicing of a specific nefm intron was required for robust transgene expression, regardless of promoter or cell type. Transgenes utilizing the nefm 3'-UTR but substituting other nefm introns expressed little or no protein owing to defects in handling of the messenger (m)RNA as opposed to transcription or splicing. Post-transcriptional events at multiple steps, but mainly during nucleocytoplasmic export, contributed to these varied levels of protein expression. An intron of the β-globin gene was also able to promote expression in a manner identical to that of the nefm intron, implying a more general preference for certain introns in controlling nefm expression. These results expand our knowledge of intron-mediated gene expression to encompass neurofilaments, indicating an additional layer of complexity in the control of a cytoskeletal gene needed for developing and maintaining healthy axons. © 2016. Published by The Company of Biologists Ltd.

Constitutive expression of a putative high-affinity nitrate transporter in Nicotiana plumbaginifolia: evidence for post-transcriptional regulation by a reduced nitrogen source.

PubMed

Fraisier, V; Gojon, A; Tillard, P; Daniel-Vedele, F

2000-08-01

The NpNRT2.1 gene encodes a putative inducible component of the high-affinity nitrate (NO3-) uptake system in Nicotiana plumbaginifolia. Here we report functional and physiological analyses of transgenic plants expressing the NpNRT2.1 coding sequence fused to the CaMV 35S or rolD promoters. Irrespective of the level of NO3- supplied, NO3- contents were found to be remarkably similar in wild-type and transgenic plants. Under specific conditions (growth on 10 mM NO3-), the steady-state NpNRT2. 1 mRNA level resulting from the deregulated transgene expression was accompanied by an increase in 15NO3- influx measured in the low concentration range. This demonstrates for the first time that the NRT2.1 sequence codes a limiting element of the inducible high-affinity transport system. Both 15NO3- influx and mRNA levels decreased in the wild type after exposure to ammonium, in agreement with previous results from many species. Surprisingly, however, influx was also markedly decreased in transgenic plants, despite stable levels of transgene expression in independent transformants after ammonium addition. We conclude that the conditions associated with the supply of a reduced nitrogen source such as ammonium, or with the generation of a further downstream metabolite, probably exert a repressive effect on NO3- influx at both transcriptional and post-transcriptional levels.

V(D)J recombination and allelic exclusion of a TCR beta-chain minilocus occurs in the absence of a functional promoter.

PubMed

Alvarez, J D; Anderson, S J; Loh, D Y

1995-08-01

Transcriptional activation of rearranging Ag receptor gene segments has been hypothesized to regulate their accessibility to V(D)J recombination. We analyzed the role of a functional promoter in the rearrangement of the murine TCR beta-chain locus using two transgenic minilocus constructs. These miniloci each contain an unrearranged V beta 8.3 gene. One has a wild-type V beta 8.3 gene, but the other has a V beta 8.3 gene with a promoter mutation that was previously shown to abrogate transcription in tissue culture. FACS analysis of thymus and lymph node cells from transgenic mouse lines showed that only the lines with the wild-type V beta 8.3 gene promoter express an 8.3 TCR beta-chain. Consistent with the protein expression data, V beta 8.3 gene transcripts were found only in the transgenic lines with the wild-type promoter. Using a quantitative PCR-based assay, it was shown that both types of transgenic lines recombine the V beta 8.3 gene at similar levels. Rearrangement of the transgenes was normal with respect to thymic development and junctional reading frame. Interestingly, both types of miniloci also underwent allelic exclusion in that recombination was blocked by the expression of a rearranged TCR beta-chain transgene. We conclude that a functional V beta gene promoter is not necessary for proper V(D)J recombination to occur.

Pathogen Phytosensing: Plants to Report Plant Pathogens.

PubMed

Mazarei, Mitra; Teplova, Irina; Hajimorad, M Reza; Stewart, C Neal

2008-04-14

Real-time systems that provide evidence of pathogen contamination in crops can be an important new line of early defense in agricultural centers. Plants possess defense mechanisms to protect against pathogen attack. Inducible plant defense is controlled by signal transduction pathways, inducible promoters and cis-regulatory elements corresponding to key genes involved in defense, and pathogen-specific responses. Identified inducible promoters and cis-acting elements could be utilized in plant sentinels, or 'phytosensors', by fusing these to reporter genes to produce plants with altered phenotypes in response to the presence of pathogens. Here, we have employed cis-acting elements from promoter regions of pathogen inducible genes as well as those responsive to the plant defense signal molecules salicylic acid, jasmonic acid, and ethylene. Synthetic promoters were constructed by combining various regulatory elements supplemented with the enhancer elements from the Cauliflower mosaic virus (CaMV) 35S promoter to increase basal level of the GUS expression. The inducibility of each synthetic promoter was first assessed in transient expression assays using Arabidopsis thaliana protoplasts and then examined for efficacy in stably transgenic Arabidopsis and tobacco plants. Histochemical and fluorometric GUS expression analyses showed that both transgenic Arabidopsis and tobacco plants responded to elicitor and phytohormone treatments with increased GUS expression when compared to untreated plants. Pathogen-inducible phytosensor studies were initiated by analyzing the sensitivity of the synthetic promoters against virus infection. Transgenic tobacco plants infected with Alfalfa mosaic virus showed an increase in GUS expression when compared to mock-inoculated control plants, whereas Tobacco mosaic virus infection caused no changes in GUS expression. Further research, using these transgenic plants against a range of different pathogens with the regulation of detectable reporter gene could provide biological evidence to define the functional differences between pathogens, and provide new technology and applications for transgenic plants as phytosensors.

Effect of CRC::etr1-1 transgene expression on ethylene production, sex expression, fruit set and fruit ripening in transgenic melon (Cucumis melo L.).

PubMed

Switzenberg, Jessica A; Beaudry, Randy M; Grumet, Rebecca

2015-06-01

Ethylene is a key factor regulating sex expression in cucurbits. Commercial melons (Cucumis melo L.) are typically andromonoecious, producing male and bisexual flowers. Our prior greenhouse studies of transgenic melon plants expressing the dominant negative ethylene perception mutant gene, etr1-1, under control of the carpel- and nectary-primordia targeted CRAB'S CLAW (CRC) promoter showed increased number and earlier appearance of carpel-bearing flowers. To further investigate this phenomenon which could be potentially useful for earlier fruit production, we observed CRC::etr1-1 plants in the field for sex expression, fruit set, fruit development, and ripening. CRC::etr1-1 melon plants showed increased number of carpel-bearing open flowers on the main stem and earlier onset by 7-10 nodes. Additional phenotypes observed in the greenhouse and field were conversion of approximately 50% of bisexual buds to female, and elongated ovaries and fruits. Earlier and greater fruit set occurred on the transgenic plants. However, CRC::etr1-1 plants had greater abscission of young fruit, and smaller fruit, so that final yield (kg/plot) was equivalent to wild type. Earlier fruit set in line M5 was accompanied by earlier appearance of ripe fruit. Fruit from line M15 frequently did not exhibit external ripening processes of rind color change and abscission, but when cut open, the majority showed a ripe or overripe interior accompanied by elevated internal ethylene. The non-ripening external phenotype in M15 fruit corresponded with elevated etr1-1 transgene expression in the exocarp. These results provide insight into the role of ethylene perception in carpel-bearing flower production, fruit set, and ripening.

Vascular endothelium-specific overexpression of human catalase in cloned pigs

PubMed Central

Samuel, M.; Mahan, E.; Padilla, J.; Simmons, G. H.; Arce-Esquivel, A. A.; Bender, S. B.; Whitworth, K. M.; Hao, Y. H.; Murphy, C. N.; Walters, E. M.; Prather, R. S.; Laughlin, M. H.

2012-01-01

The objective of this study was to develop transgenic Yucatan minipigs that overexpress human catalase (hCat) in an endothelial-specific manner. Catalase metabolizes hydrogen peroxide (H2O2), an important regulator of vascular tone that contributes to diseases such as atherosclerosis and preeclampsia. A large animal model to study reduced endothelium-derived H2O2 would therefore generate valuable translational data on vascular regulation in health and disease. Yucatan minipig fetal fibroblasts stably co-transfected with human catalase (Tie2-hCat) and eGFP expression constructs were isolated into single-cell populations. The presence of the Tie2-hCat transgene in individual colonies of fibroblasts was determined by PCR. Transgenic fibroblasts were used for nuclear transfer into enucleated oocytes by electrofusion. A minimum of 140 cloned embryos were transferred per surrogate sow (n = 4). All four surrogates maintained pregnancies and piglets were delivered by cesarean section. Nine male piglets from three of the four litters carried the Tie2-hCat transgene. Expression of human catalase mRNA and overall elevated catalase protein in isolated umbilical endothelial cells from transgenic piglets were verified by RT–PCR and western blot, respectively, and endothelial localization was confirmed by immunohistochemistry. Increased enzymatic activity of catalase in transgenic versus wild-type endothelial cells was inferred based on significantly reduced levels of H2O2 in culture. The similarities in swine and human cardiovascular anatomy and physiology will make this pig model a valuable source of information on the putative role of endothelium-derived H2O2 in vasodilation and in the mechanisms underlying vascular health and disease. PMID:21170678

Arabidopsis and Brachypodium distachyon Transgenic Plants Expressing Aspergillus nidulans Acetylesterases Have Decreased Degree of Polysaccharide Acetylation and Increased Resistance to Pathogens1[C][W][OA

PubMed Central

Pogorelko, Gennady; Lionetti, Vincenzo; Fursova, Oksana; Sundaram, Raman M.; Qi, Mingsheng; Whitham, Steven A.; Bogdanove, Adam J.; Bellincampi, Daniela; Zabotina, Olga A.

2013-01-01

The plant cell wall has many significant structural and physiological roles, but the contributions of the various components to these roles remain unclear. Modification of cell wall properties can affect key agronomic traits such as disease resistance and plant growth. The plant cell wall is composed of diverse polysaccharides often decorated with methyl, acetyl, and feruloyl groups linked to the sugar subunits. In this study, we examined the effect of perturbing cell wall acetylation by making transgenic Arabidopsis (Arabidopsis thaliana) and Brachypodium (Brachypodium distachyon) plants expressing hemicellulose- and pectin-specific fungal acetylesterases. All transgenic plants carried highly expressed active Aspergillus nidulans acetylesterases localized to the apoplast and had significant reduction of cell wall acetylation compared with wild-type plants. Partial deacetylation of polysaccharides caused compensatory up-regulation of three known acetyltransferases and increased polysaccharide accessibility to glycosyl hydrolases. Transgenic plants showed increased resistance to the fungal pathogens Botrytis cinerea and Bipolaris sorokiniana but not to the bacterial pathogens Pseudomonas syringae and Xanthomonas oryzae. These results demonstrate a role, in both monocot and dicot plants, of hemicellulose and pectin acetylation in plant defense against fungal pathogens. PMID:23463782

A chloroplast lipoxygenase is required for wound-induced jasmonic acid accumulation in Arabidopsis.

PubMed

Bell, E; Creelman, R A; Mullet, J E

1995-09-12

Plant lipoxygenases are thought to be involved in the biosynthesis of lipid-derived signaling molecules. The potential involvement of a specific Arabidopsis thaliana lipoxygenase isozyme, LOX2, in the biosynthesis of the plant growth regulators jasmonic acid (JA) and abscisic acid was investigated. Our characterization of LOX2 indicates that the protein is targeted to chloroplasts. The physiological role of this chloroplast lipoxygenase was analyzed in transgenic plants where cosuppression reduced LOX2 accumulation. The reduction in LOX2 levels caused no obvious changes in plant growth or in the accumulation of abscisic acid. However, the wound-induced accumulation of JA observed in control plants was absent in leaves of transgenic plants that lacked LOX2. Thus, LOX2 is required for the wound-induced synthesis of the plant growth regulator JA in leaves. We also examined the expression of a wound- and JA-inducible Arabidopsis gene, vsp, in transgenic and control plants. Leaves of transgenic plants lacking LOX2 accumulated less vsp mRNA than did control leaves in response to wounding. This result suggests that wound-induced JA (or some other LOX2-requiring component of the wound response pathway) is involved in the wound-induced regulation of this gene.

A unique virulence factor for proliferation and dwarfism in plants identified from a phytopathogenic bacterium

PubMed Central

Hoshi, Ayaka; Oshima, Kenro; Kakizawa, Shigeyuki; Ishii, Yoshiko; Ozeki, Johji; Hashimoto, Masayoshi; Komatsu, Ken; Kagiwada, Satoshi; Yamaji, Yasuyuki; Namba, Shigetou

2009-01-01

One of the most important themes in agricultural science is the identification of virulence factors involved in plant disease. Here, we show that a single virulence factor, tengu-su inducer (TENGU), induces witches' broom and dwarfism and is a small secreted protein of the plant-pathogenic bacterium, phytoplasma. When tengu was expressed in Nicotiana benthamiana plants, these plants showed symptoms of witches' broom and dwarfism, which are typical of phytoplasma infection. Transgenic Arabidopsis thaliana lines expressing tengu exhibited similar symptoms, confirming the effects of tengu expression on plants. Although the localization of phytoplasma was restricted to the phloem, TENGU protein was detected in apical buds by immunohistochemical analysis, suggesting that TENGU was transported from the phloem to other cells. Microarray analyses showed that auxin-responsive genes were significantly down-regulated in the tengu-transgenic plants compared with GUS-transgenic control plants. These results suggest that TENGU inhibits auxin-related pathways, thereby affecting plant development. PMID:19329488

A comparative cDNA microarray analysis reveals a spectrum of genes regulated by Pax6 in mouse lens

PubMed Central

Chauhan, Bharesh K.; Reed, Nathan A.; Yang, Ying; Čermák, Lukáš; Reneker, Lixing; Duncan, Melinda K.; Cvekl, Aleš

2007-01-01

Background Pax6 is a transcription factor that is required for induction, growth, and maintenance of the lens; however, few direct target genes of Pax6 are known. Results In this report, we describe the results of a cDNA microarray analysis of lens transcripts from transgenic mice over-expressing Pax6 in lens fibre cells in order to narrow the field of potential direct Pax6 target genes. This study revealed that the transcript levels were significantly altered for 508 of the 9700 genes analysed, including five genes encoding the cell adhesion molecules β1-integrin, JAM1, L1 CAM, NCAM-140 and neogenin. Notably, comparisons between the genes differentially expressed in Pax6 heterozygous and Pax6 over-expressing lenses identified 13 common genes, including paralemmin, GDIβ, ATF1, Hrp12 and Brg1. Immunohistochemistry and Western blotting demonstrated that Brg1 is expressed in the embryonic and neonatal (2-week-old) but not in 14-week adult lenses, and confirmed altered expression in transgenic lenses over-expressing Pax6. Furthermore, EMSA demonstrated that the BRG1 promoter contains Pax6 binding sites, further supporting the proposition that it is directly regulated by Pax6. Conclusions These results provide a list of genes with possible roles in lens biology and cataracts that are directly or indirectly regulated by Pax6. PMID:12485166

Sex chromosome-specific regulation in the Drosophila male germline but little evidence for chromosomal dosage compensation or meiotic inactivation.

PubMed

Meiklejohn, Colin D; Landeen, Emily L; Cook, Jodi M; Kingan, Sarah B; Presgraves, Daven C

2011-08-01

The evolution of heteromorphic sex chromosomes (e.g., XY in males or ZW in females) has repeatedly elicited the evolution of two kinds of chromosome-specific regulation: dosage compensation--the equalization of X chromosome gene expression in males and females--and meiotic sex chromosome inactivation (MSCI)--the transcriptional silencing and heterochromatinization of the X during meiosis in the male (or Z in the female) germline. How the X chromosome is regulated in the Drosophila melanogaster male germline is unclear. Here we report three new findings concerning gene expression from the X in Drosophila testes. First, X chromosome-wide dosage compensation appears to be absent from most of the Drosophila male germline. Second, microarray analysis provides no evidence for X chromosome-specific inactivation during meiosis. Third, we confirm the previous discovery that the expression of transgene reporters driven by autosomal spermatogenesis-specific promoters is strongly reduced when inserted on the X chromosome versus the autosomes; but we show that this chromosomal difference in expression is established in premeiotic cells and persists in meiotic cells. The magnitude of the X-autosome difference in transgene expression cannot be explained by the absence of dosage compensation, suggesting that a previously unrecognized mechanism limits expression from the X during spermatogenesis in Drosophila. These findings help to resolve several previously conflicting reports and have implications for patterns of genome evolution and speciation in Drosophila.

Why is golden rice golden (yellow) instead of red?

PubMed

Schaub, Patrick; Al-Babili, Salim; Drake, Rachel; Beyer, Peter

2005-05-01

The endosperm of Golden Rice (Oryza sativa) is yellow due to the accumulation of beta-carotene (provitamin A) and xanthophylls. The product of the two carotenoid biosynthesis transgenes used in Golden Rice, phytoene synthase (PSY) and the bacterial carotene desaturase (CRTI), is lycopene, which has a red color. The absence of lycopene in Golden Rice shows that the pathway proceeds beyond the transgenic end point and thus that the endogenous pathway must also be acting. By using TaqMan real-time PCR, we show in wild-type rice endosperm the mRNA expression of the relevant carotenoid biosynthetic enzymes encoding phytoene desaturase, zeta-carotene desaturase, carotene cis-trans-isomerase, beta-lycopene cyclase, and beta-carotene hydroxylase; only PSY mRNA was virtually absent. We show that the transgenic phenotype is not due to up-regulation of expression of the endogenous rice pathway in response to the transgenes, as was suggested to be the case in tomato (Lycopersicon esculentum) fruit, where CRTI expression resulted in a similar carotenoid phenomenon. This means that beta-carotene and xanthophyll formation in Golden Rice relies on the activity of constitutively expressed intrinsic rice genes (carotene cis-trans-isomerase, alpha/beta-lycopene cyclase, beta-carotene hydroxylase). PSY needs to be supplemented and the need for the CrtI transgene in Golden Rice is presumably due to insufficient activity of the phytoene desaturase and/or zeta-carotene desaturase enzyme in endosperm. The effect of CRTI expression was also investigated in leaves of transgenic rice and Arabidopsis (Arabidopsis thaliana). Here, again, the mRNA levels of intrinsic carotenogenic enzymes remained unaffected; nevertheless, the carotenoid pattern changed, showing a decrease in lutein, while the beta-carotene-derived xanthophylls increased. This shift correlated with CRTI-expression and is most likely governed at the enzyme level by lycopene-cis-trans-isomerism. Possible implications are discussed.

Why Is Golden Rice Golden (Yellow) Instead of Red?1[w

PubMed Central

Schaub, Patrick; Al-Babili, Salim; Drake, Rachel; Beyer, Peter

2005-01-01

The endosperm of Golden Rice (Oryza sativa) is yellow due to the accumulation of β-carotene (provitamin A) and xanthophylls. The product of the two carotenoid biosynthesis transgenes used in Golden Rice, phytoene synthase (PSY) and the bacterial carotene desaturase (CRTI), is lycopene, which has a red color. The absence of lycopene in Golden Rice shows that the pathway proceeds beyond the transgenic end point and thus that the endogenous pathway must also be acting. By using TaqMan real-time PCR, we show in wild-type rice endosperm the mRNA expression of the relevant carotenoid biosynthetic enzymes encoding phytoene desaturase, ζ-carotene desaturase, carotene cis-trans-isomerase, β-lycopene cyclase, and β-carotene hydroxylase; only PSY mRNA was virtually absent. We show that the transgenic phenotype is not due to up-regulation of expression of the endogenous rice pathway in response to the transgenes, as was suggested to be the case in tomato (Lycopersicon esculentum) fruit, where CRTI expression resulted in a similar carotenoid phenomenon. This means that β-carotene and xanthophyll formation in Golden Rice relies on the activity of constitutively expressed intrinsic rice genes (carotene cis-trans-isomerase, α/β-lycopene cyclase, β-carotene hydroxylase). PSY needs to be supplemented and the need for the CrtI transgene in Golden Rice is presumably due to insufficient activity of the phytoene desaturase and/or ζ-carotene desaturase enzyme in endosperm. The effect of CRTI expression was also investigated in leaves of transgenic rice and Arabidopsis (Arabidopsis thaliana). Here, again, the mRNA levels of intrinsic carotenogenic enzymes remained unaffected; nevertheless, the carotenoid pattern changed, showing a decrease in lutein, while the β-carotene-derived xanthophylls increased. This shift correlated with CRTI-expression and is most likely governed at the enzyme level by lycopene-cis-trans-isomerism. Possible implications are discussed. PMID:15821145

Tissue-specifically regulated site-specific excision of selectable marker genes in bivalent insecticidal, genetically-modified rice.

PubMed

Hu, Zhan; Ding, Xuezhi; Hu, Shengbiao; Sun, Yunjun; Xia, Liqiu

2013-12-01

Marker-free, genetically-modified rice was created by the tissue-specifically regulated Cre/loxP system, in which the Cre recombinase gene and hygromycin phosphotransferase gene (hpt) were flanked by two directly oriented loxP sites. Cre expression was activated by the tissue-specific promoter OsMADS45 in flower or napin in seed, resulting in simultaneous excision of the recombinase and marker genes. Segregation of T1 progeny was performed to select recombined plants. The excision was confirmed by PCR, Southern blot and sequence analyses indicating that efficiency varied from 10 to 53 % for OsMADS45 and from 12 to 36 % for napin. The expression of cry1Ac and vip3A was detected by RT-PCR analysis in marker-free transgenic rice. These results suggested that our tissue-specifically regulated Cre/loxP system could auto-excise marker genes from transgenic rice and alleviate public concerns about the security of GM crops.

Regulation of zebrafish heart regeneration by miR-133

PubMed Central

Yin, Viravuth P.; Lepilina, Alexandra; Smith, Ashley; Poss, Kenneth D.

2012-01-01

Zebrafish regenerate cardiac muscle after severe injuries through the activation and proliferation of spared cardiomyocytes. Little is known about factors that control these events. Here we investigated the extent to which miRNAs regulate zebrafish heart regeneration. Microarray analysis identified many miRNAs with increased or reduced levels during regeneration. miR-133, a miRNA with known roles in cardiac development and disease, showed diminished expression during regeneration. Induced transgenic elevation of miR-133 levels after injury inhibited myocardial regeneration, while transgenic miR-133 depletion enhanced cardiomyocyte proliferation. Expression analyses indicated that cell cycle factors mps1, cdc37, and PA2G4, and cell junction components cx43 and cldn5, are miR-133 targets during regeneration. With pharmacological inhibition and EGFP sensor interaction studies, we demonstrated that cx43 is a new miR-133 target and regeneration gene. Our results reveal dynamic regulation of miRNAs during heart regeneration, and indicate that miR-133 restricts injury-induced cardiomyocyte proliferation. PMID:22374218

Distal-less regulates eyespot patterns and melanization in Bicyclus butterflies.

PubMed

Monteiro, Antónia; Chen, Bin; Ramos, Diane M; Oliver, Jeffrey C; Tong, Xiaoling; Guo, Min; Wang, Wen-Kai; Fazzino, Lisa; Kamal, Firdous

2013-07-01

Butterfly eyespots represent novel complex traits that display substantial diversity in number and size within and across species. Correlative gene expression studies have implicated a large suite of transcription factors, including Distal-less (Dll), Engrailed (En), and Spalt (Sal), in eyespot development in butterflies, but direct evidence testing the function of any of these proteins is still missing. Here we show that the characteristic two-eyespot pattern of wildtype Bicyclus anynana forewings is correlated with dynamic progression of Dll, En, and Sal expression in larval wings from four spots to two spots, whereas no such decline in gene expression ensues in a four-eyespot mutant. We then conduct transgenic experiments testing whether over-expression of any of these genes in a wild-type genetic background is sufficient to induce eyespot differentiation in these pre-patterned wing compartments. We also produce a Dll-RNAi transgenic line to test how Dll down-regulation affects eyespot development. Finally we test how ectopic expression of these genes during the pupal stages of development alters adults color patters. We show that over-expressing Dll in larvae is sufficient to induce the differentiation of additional eyespots and increase the size of eyespots, whereas down-regulating Dll leads to a decrease in eyespot size. Furthermore, ectopic expression of Dll in the early pupal wing led to the appearance of ectopic patches of black scales. We conclude that Dll is a positive regulator of focal differentiation and eyespot signaling and that this gene is also a possible selector gene for scale melanization in butterflies. Copyright © 2013 Wiley Periodicals, Inc.

Overexpression of Mafb in Podocytes Protects against Diabetic Nephropathy

PubMed Central

Yoh, Keigyou; Ojima, Masami; Okamura, Midori; Nakamura, Megumi; Hamada, Michito; Shimohata, Homare; Moriguchi, Takashi; Yamagata, Kunihiro; Takahashi, Satoru

2014-01-01

We previously showed that the transcription factor Mafb is essential for podocyte differentiation and foot process formation. Podocytes are susceptible to injury in diabetes, and this injury leads to progression of diabetic nephropathy. In this study, we generated transgenic mice that overexpress Mafb in podocytes using the nephrin promoter/enhancer. To examine a potential pathogenetic role for Mafb in diabetic nephropathy, Mafb transgenic mice were treated with either streptozotocin or saline solution. Diabetic nephropathy was assessed by renal histology and biochemical analyses of urine and serum. Podocyte-specific overexpression of Mafb had no effect on body weight or blood glucose levels in either diabetic or control mice. Notably, albuminuria and changes in BUN levels and renal histology observed in diabetic wild-type animals were ameliorated in diabetic Mafb transgenic mice. Moreover, hyperglycemia-induced downregulation of Nephrin was mitigated in diabetic Mafb transgenic mice, and reporter assay results suggested that Mafb regulates Nephrin directly. Mafb transgenic glomeruli also overexpressed glutathione peroxidase, an antioxidative stress enzyme, and levels of the oxidative stress marker 8-hydroxydeoxyguanosine decreased in the urine of diabetic Mafb transgenic mice. Finally, Notch2 expression increased in diabetic glomeruli, and this effect was enhanced in diabetic Mafb transgenic glomeruli. These data indicate Mafb has a protective role in diabetic nephropathy through regulation of slit diaphragm proteins, antioxidative enzymes, and Notch pathways in podocytes and suggest that Mafb could be a therapeutic target. PMID:24722438

Transgenic Mice Expressing an Inhibitory Truncated Form of p300 Exhibit Long-Term Memory Deficits

ERIC Educational Resources Information Center

Oliveira, Ana M. M.; Wood, Marcelo A.; McDonough, Conor B.; Abel, Ted

2007-01-01

The formation of many forms of long-term memory requires several molecular mechanisms including regulation of gene expression. The mechanisms directing transcription require not only activation of individual transcription factors but also recruitment of transcriptional coactivators. CBP and p300 are transcriptional coactivators that interact with…

Expression pattern conferred by a glutamic acid-rich protein gene promoter in field-grown transgenic cassava (Manihot esculenta Crantz).

PubMed

Beltrán, J; Prías, M; Al-Babili, S; Ladino, Y; López, D; Beyer, P; Chavarriaga, P; Tohme, J

2010-05-01

A major constraint for incorporating new traits into cassava using biotechnology is the limited list of known/tested promoters that encourage the expression of transgenes in the cassava's starchy roots. Based on a previous report on the glutamic-acid-rich protein Pt2L4, indicating a preferential expression in roots, we cloned the corresponding gene including promoter sequence. A promoter fragment (CP2; 731 bp) was evaluated for its potential to regulate the expression of the reporter gene GUSPlus in transgenic cassava plants grown in the field. Intense GUS staining was observed in storage roots and vascular stem tissues; less intense staining in leaves; and none in the pith. Consistent with determined mRNA levels of the GUSPlus gene, fluorometric analyses revealed equal activities in root pulp and stems, but 3.5 times less in leaves. In a second approach, the activity of a longer promoter fragment (CP1) including an intrinsic intron was evaluated in carrot plants. CP1 exhibited a pronounced tissue preference, conferring high expression in the secondary phloem and vascular cambium of roots, but six times lower expression levels in leaf vascular tissues. Thus, CP1 and CP2 may be useful tools to improve nutritional and agronomical traits of cassava by genetic engineering. To date, this is the first study presenting field data on the specificity and potential of promoters for transgenic cassava.

OsDOG, a gibberellin-induced A20/AN1 zinc-finger protein, negatively regulates gibberellin-mediated cell elongation in rice.

PubMed

Liu, Yaju; Xu, Yunyuan; Xiao, Jun; Ma, Qibin; Li, Dan; Xue, Zhen; Chong, Kang

2011-07-01

The A20/AN1 zinc-finger proteins (ZFPs) play pivotal roles in animal immune responses and plant stress responses. From previous gibberellin (GA) microarray data and A20/AN1 ZFP family member association, we chose Oryza sativa dwarf rice with overexpression of gibberellin-induced gene (OsDOG) to examine its function in the GA pathway. OsDOG was induced by gibberellic acid (GA(3)) and repressed by the GA-synthesis inhibitor paclobutrazol. Different transgenic lines with constitutive expression of OsDOG showed dwarf phenotypes due to deficiency of cell elongation. Additional GA(1) and real-time PCR quantitative assay analyses confirmed that the decrease of GA(1) in the overexpression lines resulted from reduced expression of GA3ox2 and enhanced expression of GA2ox1 and GA2ox3. Adding exogenous GA rescued the constitutive expression phenotypes of the transgenic lines. OsDOG has a novel function in regulating GA homeostasis and in negative maintenance of plant cell elongation in rice. Copyright © 2011 Elsevier GmbH. All rights reserved.

Betacellulin transgenic mice develop urothelial hyperplasia and show sex-dependent reduction in urinary major urinary protein content.

PubMed

Schulz, Helene; Dahlhoff, Maik; Glogowska, Aleksandra; Zhang, Lin; Arnold, Georg J; Fröhlich, Thomas; Schneider, Marlon R; Klonisch, Thomas

2015-08-01

The epidermal growth factor (EGF)-like ligands and their cognate ERBB1-4 receptors represent important signaling pathways that regulate tissue and cell proliferation, differentiation and regeneration in a wide variety of tissues, including the urogenital tract. Betacellulin (BTC) can activate all four ERBB tyrosine kinase receptors and is a multifunctional EGF-like ligand with diverse roles in β cell differentiation, bone maturation, formation of functional epithelial linings and vascular permeability in different organs. Using transgenic BTC mice, we have studied the effect of constitutive systemic BTC over-expression on the urinary bladder. BTC was detected in microvascular structures of the stromal bladder compartment and in umbrella cells representing the protective apical lining of the uroepithelium. ERBB1 and ERBB4 receptors were co-localized in the urothelium. Mice transgenic for BTC and double transgenic for both BTC and the dominant kinase-dead mutant of EGFR (Waved 5) developed hyperplasia of the uroepithelium at 5months of age, suggesting that urothelial hyperplasia was not exclusively dependent on ERBB1/EGFR. Mass spectrometric analysis of urine revealed a significant down-regulation of major urinary proteins in female BTC transgenic mice, suggesting a novel role for systemic BTC in odor-based signaling in female transgenic BTC mice. Copyright © 2015 Elsevier Inc. All rights reserved.

GENETIC MODIFICATION OF GIBBERELLIC ACID SIGNALING TO PROMOTE CARBON SEQUESTRATION IN TREE ROOTS AND STEMS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Busov, Victor

Semidwarfism has been used extensively in row crops and horticulture to promote yield, reduce lodging, and improve harvest index, and it might have similar benefits for trees for short-rotation forestry or energy plantations, reclamation, phytoremediation, or other applications. We studied the effects of the dominant semidwarfism transgenes GA Insensitive (GAI) and Repressor of GAI-Like, which affect gibberellin (GA) action, and the GA catabolic gene, GA 2-oxidase, in nursery beds and in 2-year-old high-density stands of hybrid poplar (Populus tremula - Populus alba). Twenty-nine traits were analyzed, including measures of growth, morphology, and physiology. Endogenous GA levels were modified in mostmore » transgenic events; GA(20) and GA(8), in particular, had strong inverse associations with tree height. Nearly all measured traits varied significantly among genotypes, and several traits interacted with planting density, including aboveground biomass, root-shoot ratio, root fraction, branch angle, and crown depth. Semidwarfism promoted biomass allocation to roots over shoots and substantially increased rooting efficiency with most genes tested. The increased root proportion and increased leaf chlorophyll levels were associated with changes in leaf carbon isotope discrimination, indicating altered water use efficiency. Semidwarf trees had dramatically reduced growth when in direct competition with wild-type trees, supporting the hypothesis that semidwarfism genes could be effective tools to mitigate the spread of exotic, hybrid, and transgenic plants in wild and feral populations. We modified gibberellin (GA) metabolism and signaling in transgenic poplars using dominant transgenes and studied their effects for 3 years under field conditions. The transgenes that we employed either reduced the bioactive GAs, or attenuated their signaling. The majority of transgenic trees had significant and in many cases dramatic changes in height, crown architecture, foliage morphology, flowering onset, floral structure, and vegetative phenology. Most transgenes elicited various levels of height reduction consistent with the roles of GA in elongation growth. Several other growth traits were proportionally reduced, including branch length, internode distance, and leaf length. In contrast to elongation growth, stem diameter growth was much less affected, suggesting that semi-dwarf trees in dense stands might provide high levels of biomass production and carbon sequestration. The severity of phenotypic effects was strongly correlated with transgene expression among independent transgenic events, but often in a non-linear manner, the form of which varied widely among constructs. The majority of semi-dwarfed, transgenic plants showed delayed bud flush and early bud set, and expression of a native GAI transgene accelerated first time flowering in the field. All of the phenotypic changes observed in multiple years were stable over the 3 years of field study. Our results suggest that transgenic modification of GA action may be useful for producing semi-dwarf trees with modified growth and morphology for horticulture and other uses. We studied the poplar C(19) gibberellin 2-oxidase (GA2ox) gene subfamily. We show that a set of paralogous gene pairs differentially regulate shoot and root development. ? PtGA2ox4 and its paralogous gene PtGA2ox5 are primarily expressed in aerial organs, and overexpression of PtGA2ox5 produced a strong dwarfing phenotype characteristic of GA deficiency. Suppression of PtGA2ox4 and PtGA2ox5 led to increased biomass growth, but had no effect on root development. By contrast, the PtGA2ox2 and PtGA2ox7 paralogous pair was predominantly expressed in roots, and when these two genes were RNAi-suppressed it led to a decrease of root biomass. ? The morphological changes in the transgenic plants were underpinned by tissue-specific increases in bioactive GAs that corresponded to the predominant native expression of the targeted paralogous gene pair. Although RNAi suppression of both paralogous pairs led to changes in wood development, they were much greater in the transgenics with suppressed PtGA2ox4 and PtGA2ox5. The degree of gene suppression in independent events was strongly associated with phenotypes, demonstrating dose-dependent control of growth by GA2ox RNA concentrations. ? The expression and transgenic modifications reported here show that shoot- and leaf-expressed PtGA2ox4 and PtGA2ox5 specifically restrain aerial shoot growth, while root-expressed PtGA2ox2 and PtGA2ox7 promote root development. Genes controlling plant growth and form are of considerable interest, because they affect survival and productivity traits, and are largely unknown or poorly characterized. The SHORT INTERNODES(SHI) gene is one of a 10-member SHI-RELATED SEQUENCE (SRS) gene family in Arabidopsis that includes important developmental regulators. ? Using comparative sequence analysis of the SRS gene families in poplar and Arabidopsis, we identified two poplar proteins that are most similar to SHI and its closely related gene STYLISH1 (STY1). The two poplar genes are very similar in sequence and expression and are therefore probably paralogs with redundant functions. ? RNAi suppression of the two Populus genes enhanced shoot and root growth, whereas the overexpression of Arabidopsis SHI in poplar reduced internode and petiole length. The suppression of the two genes increased fiber length and the proportion of xylem tissue, mainly through increased xylem cell proliferation. The transgenic modifications were also associated with significant changes in the concentrations of gibberellins and cytokinin. ? We conclude that Populus SHI-RELATED SEQUENCE (SRS) genes play an important role in the regulation of vegetative growth, including wood formation, and thus could be useful tools for the modification of biomass productivity, wood quality or plant form. We studied the effects on plant growth from insertion of five cisgenes that encode proteins involved in gibberellin metabolism or signalling. Intact genomic copies of PtGA20ox7, PtGA2ox2,Pt RGL1_1, PtRGL1_2 and PtGAI1 genes from the genome-sequenced Populus trichocarpa clone Nisqually-1 were transformed into Populus tremula - alba (clone INRA 717-1B4), and growth, morphology and xylem cell size characterized in the greenhouse. Each cisgene encompassed 1-2?kb of 5' and 1?kb of 3' flanking DNA, as well as all native exons and introns. Large numbers of independent insertion events per cisgene (19-38), including empty vector controls, were studied. Three of the cisgenic modifications had significant effects on plant growth rate, morphology or wood properties. The PtGA20ox7 cisgene increased rate of shoot regeneration in vitro, accelerated early growth, and variation in growth rate was correlated with PtGA20ox7 gene expression. PtRGL1_1 and PtGA2ox2 caused reduced growth, while PtRGL1_2 gave rise to plants that grew normally but had significantly longer xylem fibres. RT-PCR studies suggested that the lack of growth inhibition observed in PtRGL1_2 cisgenic plants was a result of co-suppression. PtGAI1 slowed regeneration rate and both PtGAI1 and PtGA20ox7 gave rise to increased variance among events for early diameter and volume index, respectively. Our work suggests that cisgenic insertion of additional copies of native genes involved in growth regulation may provide tools to help modify plant architecture, expand the genetic variance in plant architecture available to breeders and accelerate transfer of alleles between difficult-to-cross species. The role of gibberellins (GAs) in regulation of lateral root development is poorly understood. We show that GA-deficient (35S:PcGA2ox1) and GA-insensitive (35S:rgl1) transgenic Populus exhibited increased lateral root proliferation and elongation under in vitro and greenhouse conditions, and these effects were reversed by exogenous GA treatment. In addition, RNA interference suppression of two poplar GA 2-oxidases predominantly expressed in roots also decreased lateral root formation. GAs negatively affected lateral root formation by inhibiting lateral root primordium initiation. A whole-genome microarray analysis of root development in GA-modified transgenic plants revealed 2069 genes with significantly altered expression. The expression of 1178 genes, including genes that promote cell proliferation, growth, and cell wall loosening, corresponded to the phenotypic severity of the root traits when transgenic events with differential phenotypic expression were compared. The array data and direct hormone measurements suggested crosstalk of GA signaling with other hormone pathways, including auxin and abscisic acid. Transgenic modification of a differentially expressed gene encoding an auxin efflux carrier suggests that GA modulation of lateral root development is at least partly imparted by polar auxin transport modification. These results suggest a mechanism for GA-regulated modulation of lateral root proliferation associated with regulation of plant allometry during the stress response. Here we summarize progress in identification of three classes of genes useful for control of plant architecture: those affecting hormone metabolism and signaling; transcription and other regulatory factors; and the cell cycle. We focus on strong modifiers of stature and form that may be useful for directed modification of plant architecture, rather than the detailed mechanisms of gene action. Gibberellin (GA) metabolic and response genes are particularly attractive targets for manipulation because many act in a dose-dependent manner; similar phenotypic effects can be readily achieved in heterologous species; and induced pleiotropic effects--such as on nitrogen assimilation, photosynthesis, and lateral root production--are usually positive with respect to crop performance. Genes encoding transcription factors represent strong candidates for manipulation of plant architecture. For example, AINTEGUMENTA, ARGOS (auxin-regulated gene controlling organ size), and growth-regulating factors (GRFs) are strong modifiers of leaf and/or flower size. Plants overexpressing these genes had increased organ size and did not display negative pleiotropic effects in glasshouse environments. TCP-domain genes such as CINCINNATA, and the associated regulatory miRNAs such as miRJAW, may provide useful means to modulate leaf curvature and other foliage properties. There are considerable opportunities for comparative and translational genomics in nonmodel plant systems.« less

Increased gibberellin contents contribute to accelerated growth and development of transgenic tobacco overexpressing a wheat ubiquitin gene.

PubMed

Wang, Guo-Kun; Zhang, Meng; Gong, Jiang-Feng; Guo, Qi-Fang; Feng, Ya-Nan; Wang, Wei

2012-12-01

Overexpressing TaUb2 promoted stem growth and resulted in early flowering in transgenic tobacco plants. Ubiquitin are involved in the production, metabolism and proper function of gibberellin. The ubiquitin-26S proteasome system (UPS), in which ubiquitin (Ub) functions as a marker, is a post-translational regulatory system that plays a prominent role in various biological processes. To investigate the impact of different Ub levels on plant growth and development, transgenic tobacco (Nicotiana tabacum L.) plants were engineered to express an Ub gene (TaUb2) from wheat (Triticum aestivum L.) under the control of cauliflower mosaic virus 35S promoter. Transgenic tobacco plants overexpressing TaUb2 demonstrated an accelerated growth rate at early stage and an early flowering phenotype in development. The preceding expression of MADS-box genes also corresponded to the accelerated developmental phenotypes of the transgenic tobacco plants compared to that of wild-type (WT). Total gibberellin (GA) and active GA contents in transgenic tobacco plants were higher than those in WT at the corresponding developmental stages, and some GA metabolism genes were upregulated. Treatment with GA(3) conferred a similarly accelerated grown rate in WT plants to that of transgenic tobacco plants, while growth was inhibited when transgenic tobacco plants were treated with a GA biosynthesis inhibitor. Thus, the results suggest that Ub are involved in the production, metabolism and proper function of GA, which is important in the regulation of plant growth and development.

Enrichment of provitamin A content in wheat (Triticum aestivum L.) by introduction of the bacterial carotenoid biosynthetic genes CrtB and CrtI

PubMed Central

Wang, Cheng; Zeng, Jian; Li, Yin; Yang, Guangxiao; He, Guangyuan

2014-01-01

Carotenoid content is a primary determinant of wheat nutritional value and affects its end-use quality. Wheat grains contain very low carotenoid levels and trace amounts of provitamin A content. In order to enrich the carotenoid content in wheat grains, the bacterial phytoene synthase gene (CrtB) and carotene desaturase gene (CrtI) were transformed into the common wheat cultivar Bobwhite. Expression of CrtB or CrtI alone slightly increased the carotenoid content in the grains of transgenic wheat, while co-expression of both genes resulted in a darker red/yellow grain phenotype, accompanied by a total carotenoid content increase of approximately 8-fold achieving 4.76 μg g–1 of seed dry weight, a β-carotene increase of 65-fold to 3.21 μg g–1 of seed dry weight, and a provitamin A content (sum of α-carotene, β-carotene, and β-cryptoxanthin) increase of 76-fold to 3.82 μg g–1 of seed dry weight. The high provitamin A content in the transgenic wheat was stably inherited over four generations. Quantitative PCR analysis revealed that enhancement of provitamin A content in transgenic wheat was also a result of the highly coordinated regulation of endogenous carotenoid biosynthetic genes, suggesting a metabolic feedback regulation in the wheat carotenoid biosynthetic pathway. These transgenic wheat lines are not only valuable for breeding wheat varieties with nutritional benefits for human health but also for understanding the mechanism regulating carotenoid biosynthesis in wheat endosperm. PMID:24692648

Sepsis reveals compartment-specific responses in intestinal proliferation and apoptosis in transgenic mice whose enterocytes re-enter the cell cycle.

PubMed

Lyons, John D; Klingensmith, Nathan J; Otani, Shunsuke; Mittal, Rohit; Liang, Zhe; Ford, Mandy L; Coopersmith, Craig M

2017-12-01

Cell production and death are tightly regulated in the rapidly renewing gut epithelium, with proliferation confined to crypts and apoptosis occurring in villi and crypts. This study sought to determine how stress alters these compartmentalized processes. Wild-type mice made septic via cecal ligation and puncture had decreased crypt proliferation and increased crypt and villus apoptosis. Fabpi -TAg mice expressing large T-antigen solely in villi had ectopic enterocyte proliferation with increased villus apoptosis in unmanipulated animals. Septic fabpi -TAg mice had an unexpected increase in villus proliferation compared with unmanipulated littermates, whereas crypt proliferation was decreased. Cell cycle regulators cyclin D1 and cyclin D2 were decreased in jejunal tissue in septic transgenic mice. In contrast, villus and crypt apoptosis were increased in septic fabpi -TAg mice. To examine the relationship between apoptosis and proliferation in a compartment-specific manner, fabpi -TAg mice were crossed with fabpl -Bcl-2 mice, resulting in expression of both genes in the villus but Bcl-2 alone in the crypt. Septic bi-transgenic animals had decreased crypt apoptosis but had a paradoxical increase in villus apoptosis compared with septic fabpi -TAg mice, associated with decreased proliferation in both compartments. Thus, sepsis unmasks compartment-specific proliferative and apoptotic regulation that is not present under homeostatic conditions.-Lyons, J. D., Klingensmith, N. J., Otani, S., Mittal, R., Liang, Z., Ford, M. L., Coopersmith, C. M. Sepsis reveals compartment-specific responses in intestinal proliferation and apoptosis in transgenic mice whose enterocytes re-enter the cell cycle. © FASEB.

Increased expression of native cytosolic Cu/Zn superoxide dismutase and ascorbate peroxidase improves tolerance to oxidative and chilling stresses in cassava (Manihot esculenta Crantz)

PubMed Central

2014-01-01

Background Cassava (Manihot esculenta Crantz) is a tropical root crop, and is therefore, extremely sensitive to low temperature; its antioxidative response is pivotal for its survival under stress. Timely turnover of reactive oxygen species (ROS) in plant cells generated by chilling-induced oxidative damages, and scavenging can be achieved by non-enzymatic and enzymatic reactions in order to maintain ROS homeostasis. Results Transgenic cassava plants that co-express cytosolic superoxide dismutase (SOD), MeCu/ZnSOD, and ascorbate peroxidase (APX), MeAPX2, were produced and tested for tolerance against oxidative and chilling stresses. The up-regulation of MeCu/ZnSOD and MeAPX2 expression was confirmed by the quantitative reverse transcriptase-polymerase chain reaction, and enzymatic activity analyses in the leaves of transgenic cassava plant lines with a single-transgene integration site. Upon exposure to ROS-generating agents, 100 μM ROS-generating reagent methyl viologen and 0.5 M H2O2, higher levels of enzymatic activities of SOD and APX were detected in transgenic plants than the wild type. Consequently, the oxidative stress parameters, such as lipid peroxidation, chlorophyll degradation and H2O2 synthesis, were lower in the transgenic lines than the wild type. Tolerance to chilling stress at 4°C for 2 d was greater in transgenic cassava, as observed by the higher levels of SOD, catalase, and ascorbate-glutathione cycle enzymes (e.g., APX, monodehydroascorbate reductase, dehydroascorbate reducatase and glutathione reductase) and lower levels of malondialdehyde content. Conclusions These results suggest that the expression of native cytosolic SOD and APX simultaneously activated the antioxidative defense mechanisms via cyclic ROS scavenging, thereby improving its tolerance to cold stress. PMID:25091029

Increased expression of native cytosolic Cu/Zn superoxide dismutase and ascorbate peroxidase improves tolerance to oxidative and chilling stresses in cassava (Manihot esculenta Crantz).

PubMed

Xu, Jia; Yang, Jun; Duan, Xiaoguang; Jiang, Yueming; Zhang, Peng

2014-08-05

Cassava (Manihot esculenta Crantz) is a tropical root crop, and is therefore, extremely sensitive to low temperature; its antioxidative response is pivotal for its survival under stress. Timely turnover of reactive oxygen species (ROS) in plant cells generated by chilling-induced oxidative damages, and scavenging can be achieved by non-enzymatic and enzymatic reactions in order to maintain ROS homeostasis. Transgenic cassava plants that co-express cytosolic superoxide dismutase (SOD), MeCu/ZnSOD, and ascorbate peroxidase (APX), MeAPX2, were produced and tested for tolerance against oxidative and chilling stresses. The up-regulation of MeCu/ZnSOD and MeAPX2 expression was confirmed by the quantitative reverse transcriptase-polymerase chain reaction, and enzymatic activity analyses in the leaves of transgenic cassava plant lines with a single-transgene integration site. Upon exposure to ROS-generating agents, 100 μM ROS-generating reagent methyl viologen and 0.5 M H₂O₂, higher levels of enzymatic activities of SOD and APX were detected in transgenic plants than the wild type. Consequently, the oxidative stress parameters, such as lipid peroxidation, chlorophyll degradation and H₂O₂ synthesis, were lower in the transgenic lines than the wild type. Tolerance to chilling stress at 4°C for 2 d was greater in transgenic cassava, as observed by the higher levels of SOD, catalase, and ascorbate-glutathione cycle enzymes (e.g., APX, monodehydroascorbate reductase, dehydroascorbate reducatase and glutathione reductase) and lower levels of malondialdehyde content. These results suggest that the expression of native cytosolic SOD and APX simultaneously activated the antioxidative defense mechanisms via cyclic ROS scavenging, thereby improving its tolerance to cold stress.

B-Bolivia, an Allele of the Maize b1 Gene with Variable Expression, Contains a High Copy Retrotransposon-Related Sequence Immediately Upstream1

PubMed Central

Selinger, David A.; Chandler, Vicki L.

2001-01-01

The maize (Zea mays) b1 gene encodes a transcription factor that regulates the anthocyanin pigment pathway. Of the b1 alleles with distinct tissue-specific expression, B-Peru and B-Bolivia are the only alleles that confer seed pigmentation. B-Bolivia produces variable and weaker seed expression but darker, more regular plant expression relative to B-Peru. Our experiments demonstrated that B-Bolivia is not expressed in the seed when transmitted through the male. When transmitted through the female the proportion of kernels pigmented and the intensity of pigment varied. Molecular characterization of B-Bolivia demonstrated that it shares the first 530 bp of the upstream region with B-Peru, a region sufficient for seed expression. Immediately upstream of 530 bp, B-Bolivia is completely divergent from B-Peru. These sequences share sequence similarity to retrotransposons. Transient expression assays of various promoter constructs identified a 33-bp region in B-Bolivia that can account for the reduced aleurone pigment amounts (40%) observed with B-Bolivia relative to B-Peru. Transgenic plants carrying the B-Bolivia promoter proximal region produced pigmented seeds. Similar to native B-Bolivia, some transgene loci are variably expressed in seeds. In contrast to native B-Bolivia, the transgene loci are expressed in seeds when transmitted through both the male and female. Some transgenic lines produced pigment in vegetative tissues, but the tissue-specificity was different from B-Bolivia, suggesting the introduced sequences do not contain the B-Bolivia plant-specific regulatory sequences. We hypothesize that the chromatin context of the B-Bolivia allele controls its epigenetic seed expression properties, which could be influenced by the adjacent highly repeated retrotransposon sequence. PMID:11244116

Osteoblasts are target cells for transformation in c-fos transgenic mice

PubMed Central

1993-01-01

We have generated transgenic mice expressing the proto-oncogene c-fos from an H-2Kb class I MHC promoter as a tool to identify and isolate cell populations which are sensitive to altered levels of Fos protein. All homozygous H2-c-fosLTR mice develop osteosarcomas with a short latency period. This phenotype is specific for c-fos as transgenic mice expressing the fos- and jun-related genes, fosB and c-jun, from the same regulatory elements do not develop any pathology despite high expression in bone tissues. The c-fos transgene is not expressed during embryogenesis but is expressed after birth in bone tissues before the onset of tumor formation, specifically in putative preosteoblasts, bone- forming osteoblasts, osteocytes, as well as in osteoblastic cells present within the tumors. Primary and clonal cell lines established from c-fos-induced tumors expressed high levels of exogenous c-fos as well as the bone cell marker genes, type I collagen, alkaline phosphatase, and osteopontin/2ar. In contrast, osteocalcin/BGP expression was either low or absent. All cell lines were tumorigenic in vivo, some of which gave rise to osteosarcomas, expressing exogenous c- fos mRNA, and Fos protein in osteoblastic cells. Detailed analysis of one osteogenic cell line, P1, and several P1-derived clonal cell lines indicated that bone-forming osteoblastic cells were transformed by Fos. The regulation of osteocalcin/BGP and alkaline phosphatase gene expression by 1,25-dihydroxyvitamin D3 was abrogated in P1-derived clonal cells, whereas glucocorticoid responsiveness was unaltered. These results suggest that high levels of Fos perturb the normal growth control of osteoblastic cells and exert specific effects on the expression of the osteoblast phenotype. PMID:8335693

Developmental Activation of the Proteolipid Protein Promoter Transgene in Neuronal and Oligodendroglial Cells of Neostriatum in Mice

PubMed Central

Fulton, Daniel; Paez, Pablo; Spreur, Vilma; Handley, Vance; Colwell, Christopher S.; Campagnoni, Anthony; Fisher, Robin

2011-01-01

Prior studies suggest that non-canonical proteolipid protein (PLP) gene expression occurs during development in non-myelinating neurons as well as myelinating oligodendroglia in mammalian brain. To assess this possibility in neostriatum, a region of uncertain PLP gene expression in neurons, morphological and electrophysiological tools were used to determine phenotypes of cells with activation of a PLP promoter transgene during the early postnatal period in mice. PLP gene expression is evident in both neuronal and oligodendroglial phenotypes in developing neostriatum, a conclusion based on three novel observations: (1) An enhanced green fluorescent protein (EGFP) reporter of PLP promoter activation was localized in two distinct populations of cells, which exhibit collective, developmental differences of morphological and electrophysiological characteristics in accord with neuronal and oligodendroglial phenotypes of neostriatal cells found during the early postnatal period in both transgenic and wild-type mice. (2) The EGFP reporter of PLP promoter activation was appropriately positioned to serve as a regulator of PLP gene expression. It colocalized with native PLP proteins in both neuronal and oligodendroglial phenotypes; however, only soma-restricted PLP protein isoforms were found in the neuronal phenotype, while classic and soma-restricted PLP protein isoforms were found in the oligodendroglial phenotype. (3) As shown by EGFP reporter, PLP promoter activation was placed to regulate PLP gene expression in only one neuronal phenotype among the several that constitute neostriatum. It was localized in medium spiny neurons, but not large aspiny neurons. These outcomes have significant implications for the non-canonical functional roles of PLP gene expression in addition to myelinogenesis in mammalian brain, and are consistent with potentially independent pathologic loci in neurons during the course of human mutational disorders of PLP gene expression. PMID:21912090

A Rice Immunophilin Gene, OsFKBP16-3, Confers Tolerance to Environmental Stress in Arabidopsis and Rice

PubMed Central

Park, Hyun Ji; Lee, Sang Sook; You, Young Nim; Yoon, Dae Hwa; Kim, Beom-Gi; Ahn, Jun Cheul; Cho, Hye Sun

2013-01-01

The putative thylakoid lumen immunophilin, FKBP16-3, has not yet been characterized, although this protein is known to be regulated by thioredoxin and possesses a well-conserved CxxxC motif in photosynthetic organisms. Here, we characterized rice OsFKBP16-3 and examined the role of this gene in the regulation of abiotic stress in plants. FKBP16-3s are well conserved in eukaryotic photosynthetic organisms, including the presence of a unique disulfide-forming CxxxC motif in their N-terminal regions. OsFKBP16-3 was mainly expressed in rice leaf tissues and was upregulated by various abiotic stresses, including salt, drought, high light, hydrogen peroxide, heat and methyl viologen. The chloroplast localization of OsFKBP16-3-GFP was confirmed through the transient expression of OsFKBP16-3 in Nicotiana benthamiana leaves. Transgenic Arabidopsis and transgenic rice plants that constitutively expressed OsFKBP16-3 exhibited increased tolerance to salinity, drought and oxidative stresses, but showed no change in growth or phenotype, compared with vector control plants, when grown under non-stressed conditions. This is the first report to demonstrate the potential role of FKBP16-3 in the environmental stress response, which may be regulated by a redox relay process in the thylakoid lumen, suggesting that artificial regulation of FKBP16-3 expression is a candidate for stress-tolerant crop breeding. PMID:23485991

Mild expression differences of MECP2 influencing aggressive social behavior

PubMed Central

Tantra, Martesa; Hammer, Christian; Kästner, Anne; Dahm, Liane; Begemann, Martin; Bodda, Chiranjeevi; Hammerschmidt, Kurt; Giegling, Ina; Stepniak, Beata; Castillo Venzor, Aracely; Konte, Bettina; Erbaba, Begun; Hartmann, Annette; Tarami, Asieh; Schulz-Schaeffer, Walter; Rujescu, Dan; Mannan, Ashraf U; Ehrenreich, Hannelore

2014-01-01

The X-chromosomal MECP2/Mecp2 gene encodes methyl-CpG-binding protein 2, a transcriptional activator and repressor regulating many other genes. We discovered in male FVB/N mice that mild (∼50%) transgenic overexpression of Mecp2 enhances aggression. Surprisingly, when the same transgene was expressed in C57BL/6N mice, transgenics showed reduced aggression and social interaction. This suggests that Mecp2 modulates aggressive social behavior. To test this hypothesis in humans, we performed a phenotype-based genetic association study (PGAS) in >1000 schizophrenic individuals. We found MECP2 SNPs rs2239464 (G/A) and rs2734647 (C/T; 3′UTR) associated with aggression, with the G and C carriers, respectively, being more aggressive. This finding was replicated in an independent schizophrenia cohort. Allele-specific MECP2mRNA expression differs in peripheral blood mononuclear cells by ∼50% (rs2734647: C > T). Notably, the brain-expressed, species-conserved miR-511 binds to MECP2 3′UTR only in T carriers, thereby suppressing gene expression. To conclude, subtle MECP2/Mecp2 expression alterations impact aggression. While the mouse data provides evidence of an interaction between genetic background and mild Mecp2 overexpression, the human data convey means by which genetic variation affects MECP2 expression and behavior. PMID:24648499

Mild expression differences of MECP2 influencing aggressive social behavior.

PubMed

Tantra, Martesa; Hammer, Christian; Kästner, Anne; Dahm, Liane; Begemann, Martin; Bodda, Chiranjeevi; Hammerschmidt, Kurt; Giegling, Ina; Stepniak, Beata; Castillo Venzor, Aracely; Konte, Bettina; Erbaba, Begun; Hartmann, Annette; Tarami, Asieh; Schulz-Schaeffer, Walter; Rujescu, Dan; Mannan, Ashraf U; Ehrenreich, Hannelore

2014-05-01

The X-chromosomal MECP2/Mecp2 gene encodes methyl-CpG-binding protein 2, a transcriptional activator and repressor regulating many other genes. We discovered in male FVB/N mice that mild (~50%) transgenic overexpression of Mecp2 enhances aggression. Surprisingly, when the same transgene was expressed in C57BL/6N mice, transgenics showed reduced aggression and social interaction. This suggests that Mecp2 modulates aggressive social behavior. To test this hypothesis in humans, we performed a phenotype-based genetic association study (PGAS) in >1000 schizophrenic individuals. We found MECP2 SNPs rs2239464 (G/A) and rs2734647 (C/T; 3'UTR) associated with aggression, with the G and C carriers, respectively, being more aggressive. This finding was replicated in an independent schizophrenia cohort. Allele-specific MECP2 mRNA expression differs in peripheral blood mononuclear cells by ~50% (rs2734647: C > T). Notably, the brain-expressed, species-conserved miR-511 binds to MECP2 3'UTR only in T carriers, thereby suppressing gene expression. To conclude, subtle MECP2/Mecp2 expression alterations impact aggression. While the mouse data provides evidence of an interaction between genetic background and mild Mecp2 overexpression, the human data convey means by which genetic variation affects MECP2 expression and behavior.

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.

Embryo-specific expression of soybean oleosin altered oil body morphogenesis and increased lipid content in transgenic rice seeds.

PubMed

Liu, Wen Xian; Liu, Hua Liang; Qu, Le Qing

2013-09-01

Oleosin is the most abundant protein in the oil bodies of plant seeds, playing an important role in regulating oil body formation and lipid accumulation. To investigate whether lipid accumulation in transgenic rice seeds depends on the expression level of oleosin, we introduced two soybean oleosin genes encoding 24 kDa proteins into rice under the control of an embryo-specific rice promoter REG-2. Overexpression of soybean oleosin in transgenic rice leads to an increase of seed lipid content up to 36.93 and 46.06 % higher than that of the non-transgenic control, respectively, while the overall fatty acid profiles of triacylglycerols remained unchanged. The overexpression of soybean oleosin in transgenic rice seeds resulted in more numerous and smaller oil bodies compared with wild type, suggesting that an inverse relationship exists between oil body size and the total oleosin level. The increase in lipid content is accompanied by a reduction in the accumulation of total seed protein. Our results suggest that it is possible to increase rice seed oil content for food use and for use as a low-cost feedstock for biodiesel by overexpressing oleosin in rice seeds.

Stimulation of the cell cycle and maize transformation by disruption of the plant retinoblastoma pathway

PubMed Central

Gordon-Kamm, William; Dilkes, Brian P.; Lowe, Keith; Hoerster, George; Sun, Xifan; Ross, Margit; Church, Laura; Bunde, Chris; Farrell, Jeff; Hill, Patrea; Maddock, Sheila; Snyder, Jane; Sykes, Louisa; Li, Zhongsen; Woo, Young-min; Bidney, Dennis; Larkins, Brian A.

2002-01-01

The genome of the Mastreviruses encodes a replication-associated protein (RepA) that interacts with members of the plant retinoblastoma-related protein family, which are putative cell cycle regulators. Expression of ZmRb1, a maize retinoblastoma-related gene, and RepA inhibited and stimulated, respectively, cell division in tobacco cell cultures. The effect of RepA was mitigated by over-expression of ZmRb1. RepA increased transformation frequency and callus growth rate of high type II maize germplasm. RepA-containing transgenic maize calli remained embryogenic, were readily regenerable, and produced fertile plants that transmitted transgene expression in a Mendelian fashion. In high type II, transformation frequency increased with the strength of the promoter driving RepA expression. When a construct in which RepA was expressed behind its native LIR promoter was used, primary transformation frequencies did not improve for two elite Pioneer maize inbreds. However, when LIR:RepA-containing transgenic embryos were used in subsequent rounds of transformation, frequencies were higher in the RepA+ embryos. These data demonstrate that RepA can stimulate cell division and callus growth in culture, and improve maize transformation. PMID:12185243

Human amyloid β peptide and tau co-expression impairs behavior and causes specific gene expression changes in Caenorhabditis elegans.

PubMed

Wang, Chenyin; Saar, Valeria; Leung, Ka Lai; Chen, Liang; Wong, Garry

2018-01-01

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the presence of extracellular amyloid plaques consisting of Amyloid-β peptide (Aβ) aggregates and neurofibrillary tangles formed by aggregation of hyperphosphorylated microtubule-associated protein tau. We generated a novel invertebrate model of AD by crossing Aβ1-42 (strain CL2355) with either pro-aggregating tau (strain BR5270) or anti-aggregating tau (strain BR5271) pan-neuronal expressing transgenic Caenorhabditis elegans. The lifespan and progeny viability of the double transgenic strains were significantly decreased compared with wild type N2 (P<0.0001). In addition, co-expression of these transgenes interfered with neurotransmitter signaling pathways, caused deficits in chemotaxis associative learning, increased protein aggregation visualized by Congo red staining, and increased neuronal loss. Global transcriptomic RNA-seq analysis revealed 248 up- and 805 down-regulated genes in N2 wild type versus Aβ1-42+pro-aggregating tau animals, compared to 293 up- and 295 down-regulated genes in N2 wild type versus Aβ1-42+anti-aggregating tau animals. Gene set enrichment analysis of Aβ1-42+pro-aggregating tau animals uncovered up-regulated annotation clusters UDP-glucuronosyltransferase (5 genes, P<4.2E-4), protein phosphorylation (5 genes, P<2.60E-02), and aging (5 genes, P<8.1E-2) while the down-regulated clusters included nematode cuticle collagen (36 genes, P<1.5E-21). RNA interference of 13 available top up-regulated genes in Aβ1-42+pro-aggregating tau animals revealed that F-box family genes and nep-4 could enhance life span deficits and chemotaxis deficits while Y39G8C.2 (TTBK2) could suppress these behaviors. Comparing the list of regulated genes from C. elegans to the top 60 genes related to human AD confirmed an overlap of 8 genes: patched homolog 1, PTCH1 (ptc-3), the Rab GTPase activating protein, TBC1D16 (tbc-16), the WD repeat and FYVE domain-containing protein 3, WDFY3 (wdfy-3), ADP-ribosylation factor guanine nucleotide exchange factor 2, ARFGEF2 (agef-1), Early B-cell Factor, EBF1 (unc-3), d-amino-acid oxidase, DAO (daao-1), glutamate receptor, metabotropic 1, GRM1 (mgl-2), prolyl 4-hydroxylase subunit alpha 2, P4HA2 (dpy-18 and phy-2). Taken together, our C. elegans double transgenic model provides insight on the fundamental neurobiologic processes underlying human AD and recapitulates selected transcriptomic changes observed in human AD brains. Copyright © 2017 Elsevier Inc. All rights reserved.

HC-Pro silencing suppressor significantly alters the gene expression profile in tobacco leaves and flowers

PubMed Central

2011-01-01

Background RNA silencing is used in plants as a major defence mechanism against invasive nucleic acids, such as viruses. Accordingly, plant viruses have evolved to produce counter defensive RNA-silencing suppressors (RSSs). These factors interfere in various ways with the RNA silencing machinery in cells, and thereby disturb the microRNA (miRNA) mediated endogene regulation and induce developmental and morphological changes in plants. In this study we have explored these effects using previously characterized transgenic tobacco plants which constitutively express (under CaMV 35S promoter) the helper component-proteinase (HC-Pro) derived from a potyviral genome. The transcript levels of leaves and flowers of these plants were analysed using microarray techniques (Tobacco 4 × 44 k, Agilent). Results Over expression of HC-Pro RSS induced clear phenotypic changes both in growth rate and in leaf and flower morphology of the tobacco plants. The expression of 748 and 332 genes was significantly changed in the leaves and flowers, respectively, in the HC-Pro expressing transgenic plants. Interestingly, these transcriptome alterations in the HC-Pro expressing tobacco plants were similar as those previously detected in plants infected with ssRNA-viruses. Particularly, many defense-related and hormone-responsive genes (e.g. ethylene responsive transcription factor 1, ERF1) were differentially regulated in these plants. Also the expression of several stress-related genes, and genes related to cell wall modifications, protein processing, transcriptional regulation and photosynthesis were strongly altered. Moreover, genes regulating circadian cycle and flowering time were significantly altered, which may have induced a late flowering phenotype in HC-Pro expressing plants. The results also suggest that photosynthetic oxygen evolution, sugar metabolism and energy levels were significantly changed in these transgenic plants. Transcript levels of S-adenosyl-L-methionine (SAM) were also decreased in these plants, apparently leading to decreased transmethylation capacity. The proteome analysis using 2D-PAGE indicated significantly altered proteome profile, which may have been both due to altered transcript levels, decreased translation, and increased proteosomal/protease activity. Conclusion Expression of the HC-Pro RSS mimics transcriptional changes previously shown to occur in plants infected with intact viruses (e.g. Tobacco etch virus, TEV). The results indicate that the HC-Pro RSS contributes a significant part of virus-plant interactions by changing the levels of multiple cellular RNAs and proteins. PMID:21507209

Over-expression of a NAC 67 transcription factor from finger millet (Eleusine coracana L.) confers tolerance against salinity and drought stress in rice.

PubMed

Rahman, Hifzur; Ramanathan, Valarmathi; Nallathambi, Jagedeeshselvam; Duraialagaraja, Sudhakar; Muthurajan, Raveendran

2016-05-11

NAC proteins (NAM (No apical meristem), ATAF (Arabidopsis transcription activation factor) and CUC (cup-shaped cotyledon)) are plant-specific transcription factors reported to be involved in regulating growth, development and stress responses. Salinity responsive transcriptome profiling in a set of contrasting finger millet genotypes through RNA-sequencing resulted in the identification of a NAC homolog (EcNAC 67) exhibiting differential salinity responsive expression pattern. Full length cDNA of EcNAC67 was isolated, characterized and validated for its role in abiotic stress tolerance through agrobacterium mediated genetic transformation in a rice cultivar ASD16. Bioinformatics analysis of putative NAC transcription factor (TF) isolated from a salinity tolerant finger millet showed its genetic relatedness to NAC67 family TFs in related cereals. Putative transgenic lines of rice over-expressing EcNAC67 were generated through Agrobacterium mediated transformation and presence/integration of transgene was confirmed through PCR and southern hybridization analysis. Transgenic rice plants harboring EcNAC67 showed enhanced tolerance against drought and salinity under greenhouse conditions. Transgenic rice plants were found to possess higher root and shoot biomass during stress and showed better revival ability upon relief from salinity stress. Upon drought stress, transgenic lines were found to maintain higher relative water content and lesser reduction in grain yield when compared to non-transgenic ASD16 plants. Drought induced spikelet sterility was found to be much lower in the transgenic lines than the non-transgenic ASD16. Results revealed the significant role of EcNAC67 in modulating responses against dehydration stress in rice. No detectable abnormalities in the phenotypic traits were observed in the transgenic plants under normal growth conditions. Results indicate that EcNAC67 can be used as a novel source for engineering tolerance against drought and salinity stress in rice and other crop plants.

Inducible Transgenic Models of BRCA1 Function

DTIC Science & Technology

1999-10-01

inducible expression vectors were created to conditionally express four different hammerhead ribozymes designed to specifically cleave the Brca...transcript. Hammerhead ribozymes are catalytic RNAs that efficiently cleave RNA and thereby down- regulate gene expression. Hammerhead ribozymes can cleave...any RNA containing its 5’-UH-3’ consensus sequence where U can be replaced by a C, and H=C, U or A. Hammerhead ribozymes effectively and selectively

Ectopic Expression of Nolz-1 in Neural Progenitors Promotes Cell Cycle Exit/Premature Neuronal Differentiation Accompanying with Abnormal Apoptosis in the Developing Mouse Telencephalon

PubMed Central

Chang, Sunny Li-Yun; Chen, Shih-Yun; Huang, Huai-Huei; Ko, Hsin-An; Liu, Pei-Tsen; Liu, Ya-Chi; Chen, Ping-Hau; Liu, Fu-Chin

2013-01-01

Nolz-1, as a murine member of the NET zinc-finger protein family, is expressed in post-mitotic differentiating neurons of striatum during development. To explore the function of Nolz-1 in regulating the neurogenesis of forebrain, we studied the effects of ectopic expression of Nolz-1 in neural progenitors. We generated the Cre-loxP dependent conditional transgenic mice in which Nolz-1 was ectopically expressed in proliferative neural progenitors. Ectopic expression of Nolz-1 in neural progenitors by intercrossing the Nolz-1 conditional transgenic mice with the nestin-Cre mice resulted in hypoplasia of telencephalon in double transgenic mice. Decreased proliferation of neural progenitor cells were found in the telencephalon, as evidenced by the reduction of BrdU−, Ki67− and phospho-histone 3-positive cells in E11.5–12.5 germinal zone of telencephalon. Transgenic Nolz-1 also promoted cell cycle exit and as a consequence might facilitate premature differentiation of progenitors, because TuJ1-positive neurons were ectopically found in the ventricular zone and there was a general increase of TuJ1 immunoreactivity in the telencephalon. Moreover, clusters of strong TuJ1-expressing neurons were present in E12.5 germinal zone. Some of these strong TuJ1-positive clusters, however, contained apoptotic condensed DNA, suggesting that inappropriate premature differentiation may lead to abnormal apoptosis in some progenitor cells. Consistent with the transgenic mouse analysis in vivo, similar effects of Nozl-1 over-expression in induction of apoptosis, inhibition of cell proliferation and promotion of neuronal differentiation were also observed in three different N18, ST14A and N2A neural cell lines in vitro. Taken together, our study indicates that ectopic expression of Nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation and induces abnormal apoptosis in the developing telencephalon. PMID:24073229

Ectopic expression of nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation accompanying with abnormal apoptosis in the developing mouse telencephalon.

PubMed

Chang, Sunny Li-Yun; Chen, Shih-Yun; Huang, Huai-Huei; Ko, Hsin-An; Liu, Pei-Tsen; Liu, Ya-Chi; Chen, Ping-Hau; Liu, Fu-Chin

2013-01-01

Nolz-1, as a murine member of the NET zinc-finger protein family, is expressed in post-mitotic differentiating neurons of striatum during development. To explore the function of Nolz-1 in regulating the neurogenesis of forebrain, we studied the effects of ectopic expression of Nolz-1 in neural progenitors. We generated the Cre-loxP dependent conditional transgenic mice in which Nolz-1 was ectopically expressed in proliferative neural progenitors. Ectopic expression of Nolz-1 in neural progenitors by intercrossing the Nolz-1 conditional transgenic mice with the nestin-Cre mice resulted in hypoplasia of telencephalon in double transgenic mice. Decreased proliferation of neural progenitor cells were found in the telencephalon, as evidenced by the reduction of BrdU-, Ki67- and phospho-histone 3-positive cells in E11.5-12.5 germinal zone of telencephalon. Transgenic Nolz-1 also promoted cell cycle exit and as a consequence might facilitate premature differentiation of progenitors, because TuJ1-positive neurons were ectopically found in the ventricular zone and there was a general increase of TuJ1 immunoreactivity in the telencephalon. Moreover, clusters of strong TuJ1-expressing neurons were present in E12.5 germinal zone. Some of these strong TuJ1-positive clusters, however, contained apoptotic condensed DNA, suggesting that inappropriate premature differentiation may lead to abnormal apoptosis in some progenitor cells. Consistent with the transgenic mouse analysis in vivo, similar effects of Nozl-1 over-expression in induction of apoptosis, inhibition of cell proliferation and promotion of neuronal differentiation were also observed in three different N18, ST14A and N2A neural cell lines in vitro. Taken together, our study indicates that ectopic expression of Nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation and induces abnormal apoptosis in the developing telencephalon.

Characterization of a wheat (Triticum aestivum L.) expansin gene, TaEXPB23, involved in the abiotic stress response and phytohormone regulation.

PubMed

Han, Yang yang; Li, Ai xiu; Li, Feng; Zhao, Mei rong; Wang, Wei

2012-05-01

Expansins are proteins that are generally accepted to be key regulators of cell wall extension and plant growth. We examined the expression pattern of TaEXPB23, a wheat (Triticum aestivum L.) expansin gene, under exogenous phytohormone and abiotic stress treatments. In addition, we evaluated its function in the tolerance to salt stress and high temperature (HT) by overexpressing it in transgenic tobacco plants. In subcellular localization assays, TaEXPB23 localized to the cell wall. Expression analysis demonstrated that the transcription pattern of TaEXPB23 corresponded to wheat coleoptile growth. Real-time RT-PCR analysis revealed that TaEXPB23 transcript expression was upregulated by exogenous methyl jasmonate (MeJA) and salt stress, but downregulated by exogenous gibberellins (GA₃), ethylene (ET), indole-3-acetic acid (IAA) and α-naphthlcetic acid (NAA). Overexpression of TaEXPB23 in tobacco (tabacum) conferred tolerance to salt stress by enhancing water retention ability (WRA) and decreasing osmotic potential (OP). However, transgenic plants overexpressing TaEXPB23 did not show any improvement in the tolerance to HT stress. These results suggested that TaEXPB23 is regulated by phytohormones and is involved in the regulation of salt stress tolerance. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

The bZIP transcription factor MdHY5 regulates anthocyanin accumulation and nitrate assimilation in apple.

PubMed

An, Jian-Ping; Qu, Feng-Jia; Yao, Ji-Fang; Wang, Xiao-Na; You, Chun-Xiang; Wang, Xiao-Fei; Hao, Yu-Jin

2017-01-01

The basic leucine zipper (bZIP) transcription factor HY5 plays a multifaceted role in plant growth and development. Here the apple MdHY5 gene was cloned based on its homology with Arabidopsis HY5 . Expression analysis demonstrated that MdHY5 transcription was induced by light and abscisic acid treatments. Electrophoretic mobility shift assays and transient expression assays subsequently showed that MdHY5 positively regulated both its own transcription and that of MdMYB10 by binding to E-box and G-box motifs, respectively. Furthermore, we obtained transgenic apple calli that overexpressed the MdHY5 gene, and apple calli coloration assays showed that MdHY5 promoted anthocyanin accumulation by regulating expression of the MdMYB10 gene and downstream anthocyanin biosynthesis genes. In addition, the transcript levels of a series of nitrate reductase genes and nitrate uptake genes in both wild-type and transgenic apple calli were detected. In association with increased nitrate reductase activities and nitrate contents, the results indicated that MdHY5 might be an important regulator in nutrient assimilation. Taken together, these results indicate that MdHY5 plays a vital role in anthocyanin accumulation and nitrate assimilation in apple.

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.

Up-Regulation of Phosphoinositide Metabolism in Tobacco Cells Constitutively Expressing the Human Type I Inositol Polyphosphate 5-Phosphatase1

PubMed Central

Perera, Imara Y.; Love, John; Heilmann, Ingo; Thompson, William F.; Boss, Wendy F.

2002-01-01

To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP3) 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 InsP3. 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 InsP3 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 InsP3 in both wild-type and transgenic cells. However, even with stimulation, InsP3 levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP3 signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP2), the lipid precursor of InsP3, was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP2 metabolism showed that the activity of the PtdInsP2-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 32P into PtdInsP2 in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP2 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 PtdInsP2 synthesis as a regulatory step in this system. PMID:12177493

Characterization of the yeast copper-inducible promoter system in Arabidopsis thaliana

NASA Technical Reports Server (NTRS)

Granger, C. L.; Cyr, R. J.

2001-01-01

Inducible promoters or gene-switches are used to both spatially and temporally regulate gene expression. Such regulation can provide information concerning the function of a gene in a developmental context as well as avoid potential harmful effects due to overexpression. A gfp construct under the control of a copper-inducible promoter was introduced into Arabidopsis thaliana (L.) Heynh. and the regulatory parameters of this inducible promoter were determined. Here, we describe the time-course of up- and down-regulation of GFP expression in response to copper level, the optimal regulatory levels of copper, and the tissue specificity of expression in three transgenic lines. We conclude that the copper-inducible promoter system may be useful in regulating the time and location of gene expression in A. thaliana.

Identification and Characterization of CYC-Like Genes in Regulation of Ray Floret Development in Chrysanthemum morifolium.

PubMed

Huang, Di; Li, Xiaowei; Sun, Ming; Zhang, Tengxun; Pan, Huitang; Cheng, Tangren; Wang, Jia; Zhang, Qixiang

2016-01-01

Chrysanthemum morifolium , one of the most economically important ornamental crops worldwide, is well-known for the elaborate and complex inflorescence which is composed of both bilaterally symmetrical ray florets and radially symmetrical disc florets. Despite continuing efforts, the molecular mechanisms underlying regulation of the two flower types are still unclear so far. CYC -like proteins have been shown to control flower symmetry or regulate flower-type identity in several angiosperm plant lineages. In this study, we conducted comparative analysis of the CmCYC2 genes in two chrysanthemum cultivars and their F1 progenies with various whorls of ray florets. Six CmCYC genes were identified and sequenced, all of which were grouped into the CYC2 subclade. All the six CmCYC2 genes were predominantly expressed in reproductive organs, and in particular in the petal of ray florets. Of these genes, the transcription level of CmCYC2c was highly up-regulated in ray florets of the double-ray flowered heads. In addition, the result that CmCYC2c was highly expressed at key developing stages indicates its role in regulating petal development. Furthermore, overexpression of CmCYC2c in C. lavandulifolium , one of the original species of C. morifolium , led to significant increase in flower numbers and petal ligule length of ray florets. Besides CmCYC2c , the expression of CmCYC2f was also significantly up-regulated in transgenic lines, implying a possible role in regulating development of ray florets. Both results of expression patterns and transgenic phenotypes suggest that CmCYC2c is involved in regulating ray floret identity in the chrysanthemum. This study will be useful for genetic manipulation of flower shape in chrysanthemum and hence promote the process of molecular breeding.

Manipulation of MKS1 gene expression affects Kalanchoë blossfeldiana and Petunia hybrida phenotypes.

PubMed

Gargul, Joanna Maria; Mibus, Heiko; Serek, Margrethe

2015-01-01

The establishment of alternative methods to chemical treatments for growth retardation and pathogen protection in ornamental plant production has become a major goal in recent breeding programmes. This study evaluates the effect of manipulating MAP kinase 4 nuclear substrate 1 (MKS1) expression in Kalanchoë blossfeldiana and Petunia hybrida. The Arabidopsis thaliana MKS1 gene was overexpressed in both species via Agrobacterium-mediated transformation, resulting in dwarfed phenotypes and delayed flowering in both species and increased tolerance to Pseudomonas syringae pv. tomato in transgenic Petunia plants. The lengths of the stems and internodes were decreased, while the number of nodes in the transgenic plants was similar to that of the control plants in both species. The transgenic Kalanchoë flowers had an increased anthocyanin concentration, and the length of the inflorescence stem was decreased. The morphology of transgenic Petunia flowers was not altered. The results of the Pseudomonas syringae tolerance test showed that Petunia plants with one copy of the transgene reacted similarly to the nontransgenic control plants; however, plants with four copies of the transgene exhibited considerably higher tolerance to bacterial attack. Transgene integration and expression was determined by Southern blot hybridization and RT-PCR analyses. MKS1 in wild-type Petunia plants was down-regulated through a virus-induced gene silencing (VIGS) method using tobacco rattle virus vectors. There were no significant phenotypic differences between the plants with silenced MKS1 genes and the controls. The relative concentration of the MKS1 transcript in VIGS-treated plants was estimated by quantitative RT-PCR. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Insulated hsp70B' promoter: stringent heat-inducible activity in replication-deficient, but not replication-competent adenoviruses.

PubMed

Rohmer, Stanimira; Mainka, Astrid; Knippertz, Ilka; Hesse, Andrea; Nettelbeck, Dirk M

2008-04-01

Key to the realization of gene therapy is the development of efficient and targeted gene transfer vectors. Therapeutic gene transfer by replication-deficient or more recently by conditionally replication-competent/oncolytic adenoviruses has shown much promise. For specific applications, however, it will be advantageous to provide vectors that allow for external control of gene expression. The efficient cellular heat shock system in combination with available technology for focused and controlled hyperthermia suggests heat-regulated transcription control as a promising tool for this purpose. We investigated the feasibility of a short fragment of the human hsp70B' promoter, with and without upstream insulator elements, for the regulation of transgene expression by replication-deficient or oncolytic adenoviruses. Two novel adenoviral vectors with an insulated hsp70B' promoter were developed and showed stringent heat-inducible gene expression with induction ratios up to 8000-fold. In contrast, regulation of gene expression from the hsp70B' promoter without insulation was suboptimal. In replication-competent/oncolytic adenoviruses regulation of the hsp70B' promoter was lost specifically during late replication in permissive cells and could not be restored by the insulators. We developed novel adenovirus gene transfer vectors that feature improved and stringent regulation of transgene expression from the hsp70B' promoter using promoter insulation. These vectors have potential for gene therapy applications that benefit from external modulation of therapeutic gene expression or for combination therapy with hyperthermia. Furthermore, our study reveals that vector replication can deregulate inserted cellular promoters, an observation which is of relevance for the development of replication-competent/oncolytic gene transfer vectors. (c) 2008 John Wiley & Sons, Ltd.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ikeda, Kazuhiro; Tsukui, Tohru; Imazawa, Yukiko

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 activemore » 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.« less

Antisense Down-Regulation of 4CL Expression Alters Lignification, Tree Growth, and Saccharification Potential of Field-Grown Poplar1[W][OA

PubMed Central

Voelker, Steven L.; Lachenbruch, Barbara; Meinzer, Frederick C.; Jourdes, Michael; Ki, Chanyoung; Patten, Ann M.; Davin, Laurence B.; Lewis, Norman G.; Tuskan, Gerald A.; Gunter, Lee; Decker, Stephen R.; Selig, Michael J.; Sykes, Robert; Himmel, Michael E.; Kitin, Peter; Shevchenko, Olga; Strauss, Steven H.

2010-01-01

Transgenic down-regulation of the Pt4CL1 gene family encoding 4-coumarate:coenzyme A ligase (4CL) has been reported as a means for reducing lignin content in cell walls and increasing overall growth rates, thereby improving feedstock quality for paper and bioethanol production. Using hybrid poplar (Populus tremula × Populus alba), we applied this strategy and examined field-grown transformants for both effects on wood biochemistry and tree productivity. The reductions in lignin contents obtained correlated well with 4CL RNA expression, with a sharp decrease in lignin amount being observed for RNA expression below approximately 50% of the nontransgenic control. Relatively small lignin reductions of approximately 10% were associated with reduced productivity, decreased wood syringyl/guaiacyl lignin monomer ratios, and a small increase in the level of incorporation of H-monomers (p-hydroxyphenyl) into cell walls. Transgenic events with less than approximately 50% 4CL RNA expression were characterized by patches of reddish-brown discolored wood that had approximately twice the extractive content of controls (largely complex polyphenolics). There was no evidence that substantially reduced lignin contents increased growth rates or saccharification potential. Our results suggest that the capacity for lignin reduction is limited; below a threshold, large changes in wood chemistry and plant metabolism were observed that adversely affected productivity and potential ethanol yield. They also underline the importance of field studies to obtain physiologically meaningful results and to support technology development with transgenic trees. PMID:20729393

Overexpression of Transcription Factor Sp2 Inhibits Epidermal Differentiation and Increases Susceptibility to Wound and Carcinogen-Induced Tumorigenesis

PubMed Central

Kim, Tae-Hyung; Chiera, Shannon L.; Linder, Keith E.; Trempus, Carol S.; Smart, Robert C.; Horowitz, Jonathan M.

2010-01-01

Sp proteins are evolutionarily-conserved transcription factors required for the expression of a wide variety of genes that are critical for development and cell-cycle progression. De-regulated expression of certain Sp proteins is associated with the formation of a variety of human tumors, however direct evidence that any given Sp protein is oncogenic has been lacking. Here we report that Sp2 protein abundance in mice increases in concert with the progression of carcinogen-induced murine squamous cell carcinomas. Transgenic mice specifically overexpressing murine Sp2 in epidermal basal keratinocytes were highly susceptible to wound- and carcinogen-induced papillomagenesis. Transgenic animals that were homozygous rather than hemizygous for the Sp2 transgene exhibited a striking arrest in the epidermal differentiation program, perishing within two weeks of birth. Our results directly support the likelihood that Sp2 overexpression occurring in various human cancers has significant functional impact. PMID:20959487

CTRP3 attenuates diet-induced hepatic steatosis by regulating triglyceride metabolism

PubMed Central

Peterson, Jonathan M.; Seldin, Marcus M.; Wei, Zhikui; Aja, Susan

2013-01-01

CTRP3 is a secreted plasma protein of the C1q family that helps regulate hepatic gluconeogenesis and is downregulated in a diet-induced obese state. However, the role of CTRP3 in regulating lipid metabolism has not been established. Here, we used a transgenic mouse model to address the potential function of CTRP3 in ameliorating high-fat diet-induced metabolic stress. Both transgenic and wild-type mice fed a high-fat diet showed similar body weight gain, food intake, and energy expenditure. Despite similar adiposity to wild-type mice upon diet-induced obesity (DIO), CTRP3 transgenic mice were strikingly resistant to the development of hepatic steatosis, had reduced serum TNF-α levels, and demonstrated a modest improvement in systemic insulin sensitivity. Additionally, reduced hepatic triglyceride levels were due to decreased expression of enzymes (GPAT, AGPAT, and DGAT) involved in triglyceride synthesis. Importantly, short-term daily administration of recombinant CTRP3 to DIO mice for 5 days was sufficient to improve the fatty liver phenotype, evident as reduced hepatic triglyceride content and expression of triglyceride synthesis genes. Consistent with a direct effect on liver cells, recombinant CTRP3 treatment reduced fatty acid synthesis and neutral lipid accumulation in cultured rat H4IIE hepatocytes. Together, these results establish a novel role for CTRP3 hormone in regulating hepatic lipid metabolism and highlight its protective function and therapeutic potential in attenuating hepatic steatosis. PMID:23744740

Transcriptome Profiling Reveals the Negative Regulation of Multiple Plant Hormone Signaling Pathways Elicited by Overexpression of C-Repeat Binding Factors.

PubMed

Li, Aixin; Zhou, Mingqi; Wei, Donghui; Chen, Hu; You, Chenjiang; Lin, Juan

2017-01-01

C-repeat binding factors (CBF) are a subfamily of AP2 transcription factors that play critical roles in the regulation of plant cold tolerance and growth in low temperature. In the present work, we sought to perform a detailed investigation into global transcriptional regulation of plant hormone signaling associated genes in transgenic plants engineered with CBF genes. RNA samples from Arabidopsis thaliana plants overexpressing two CBF genes, CBF2 and CBF3 , were subjected to Illumina HiSeq 2000 RNA sequencing (RNA-Seq). Our results showed that more than half of the hormone associated genes that were differentially expressed in CBF2 or CBF3 transgenic plants were related to auxin signal transduction and metabolism. Most of these alterations in gene expression could lead to repression of auxin signaling. Accordingly, the IAA content was significantly decreased in young tissues of plants overexpressing CBF2 and CBF3 compared with wild type. In addition, genes associated with the biosynthesis of Jasmonate (JA) and Salicylic acid (SA), as well as the signal sensing of Brassinolide (BR) and SA, were down-regulated, while genes associated with Gibberellin (GA) deactivation were up-regulated. In general, overexpression of CBF2 and CBF3 negatively affects multiple plant hormone signaling pathways in Arabidopsis . The transcriptome analysis using CBF2 and CBF3 transgenic plants provides novel and integrated insights into the interaction between CBFs and plant hormones, particularly the modulation of auxin signaling, which may contribute to the improvement of crop yields under abiotic stress via molecular engineering using CBF genes.

Transcriptome Profiling Reveals the Negative Regulation of Multiple Plant Hormone Signaling Pathways Elicited by Overexpression of C-Repeat Binding Factors

PubMed Central

Li, Aixin; Zhou, Mingqi; Wei, Donghui; Chen, Hu; You, Chenjiang; Lin, Juan

2017-01-01

C-repeat binding factors (CBF) are a subfamily of AP2 transcription factors that play critical roles in the regulation of plant cold tolerance and growth in low temperature. In the present work, we sought to perform a detailed investigation into global transcriptional regulation of plant hormone signaling associated genes in transgenic plants engineered with CBF genes. RNA samples from Arabidopsis thaliana plants overexpressing two CBF genes, CBF2 and CBF3, were subjected to Illumina HiSeq 2000 RNA sequencing (RNA-Seq). Our results showed that more than half of the hormone associated genes that were differentially expressed in CBF2 or CBF3 transgenic plants were related to auxin signal transduction and metabolism. Most of these alterations in gene expression could lead to repression of auxin signaling. Accordingly, the IAA content was significantly decreased in young tissues of plants overexpressing CBF2 and CBF3 compared with wild type. In addition, genes associated with the biosynthesis of Jasmonate (JA) and Salicylic acid (SA), as well as the signal sensing of Brassinolide (BR) and SA, were down-regulated, while genes associated with Gibberellin (GA) deactivation were up-regulated. In general, overexpression of CBF2 and CBF3 negatively affects multiple plant hormone signaling pathways in Arabidopsis. The transcriptome analysis using CBF2 and CBF3 transgenic plants provides novel and integrated insights into the interaction between CBFs and plant hormones, particularly the modulation of auxin signaling, which may contribute to the improvement of crop yields under abiotic stress via molecular engineering using CBF genes. PMID:28983312

Over-Expression of GmGIa-Regulated Soybean miR172a Confers Early Flowering in Transgenic Arabidopsis thaliana.

PubMed

Wang, Tao; Sun, Ming-Yang; Wang, Xue-Song; Li, Wen-Bin; Li, Yong-Guang

2016-04-29

Flowering is a pivotal event in the life cycle of plants. miR172 has been widely confirmed to play critical roles in flowering time control by regulating its target gene expression in Arabidopsis. However, the role of its counterpart in soybean remains largely unclear. In the present study, we found that the gma-miR172a was regulated by a GIGANTEA ortholog, GmGIa, in soybean through miRNA metabolism. The expression analysis revealed that gma-miR172a has a pattern of diurnal rhythm expression and its abundance increased rapidly as plants grew until the initiation of flowering phase in soybean. One target gene of gma-miR172a, Glyma03g33470, was predicted and verified using a modified RLM 5'-RACE (RNA ligase-mediated rapid amplification of 5' cDNA ends) assay. Overexpression of gma-miR172a exhibited an early flowering phenotype and the expression of FT, AP1 and LFY were simultaneously increased in gma-miR172a-transgenic Arabidopsis plants, suggesting that the early flowering phenotype was associated with up-regulation of these genes. The overexpression of the gma-miR172a-resistant version of Glyma03g33470 weakened early flowering phenotype in the toe1 mutant of Arabidopsis. Taken together, our results suggested that gma-miR172a played an important role in GmGIa-mediated flowering by repressing Glyma03g33470, which in turn increased the expression of FT, AP1 and LFY to promote flowering in soybean.

The phosphatidylinositol synthase gene (GhPIS) contributes to longer, stronger, and finer fibers in cotton.

PubMed

Long, Qin; Yue, Fang; Liu, Ruochen; Song, Shuiqing; Li, Xianbi; Ding, Bo; Yan, Xingying; Pei, Yan

2018-05-11

Cotton fibers are the most important natural raw material used in textile industries world-wide. Fiber length, strength, and fineness are the three major traits which determine the quality and economic value of cotton. It is known that exogenous application of phosphatidylinositols (PtdIns), important structural phospholipids, can promote cotton fiber elongation. Here, we sought to increase the in planta production of PtdIns to improve fiber traits. Transgenic cotton plants were generated in which the expression of a cotton phosphatidylinositol synthase gene (i.e., GhPIS) was controlled by the fiber-specific SCFP promoter element, resulting in the specific up-regulation of GhPIS during cotton fiber development. We demonstrate that PtdIns content was significantly enhanced in transgenic cotton fibers and the elevated level of PtdIns stimulated the expression of genes involved in PtdIns phosphorylation as well as promoting lignin/lignin-like phenolic biosynthesis. Fiber length, strength and fineness were also improved in the transgenic plants as compared to the wild-type cotton, with no loss in overall fiber yield. Our data indicate that fiber-specific up-regulation of PtdIns synthesis is a promising strategy for cotton fiber quality improvement.

Tissue-specific and pathogen-inducible expression of a fusion protein containing a Fusarium-specific antibody and a fungal chitinase protects wheat against Fusarium pathogens and mycotoxins.

PubMed

Cheng, Wei; Li, He-Ping; Zhang, Jing-Bo; Du, Hong-Jie; Wei, Qi-Yong; Huang, Tao; Yang, Peng; Kong, Xian-Wei; Liao, Yu-Cai

2015-06-01

Fusarium head blight (FHB) in wheat and other small grain cereals is a globally devastating disease caused by toxigenic Fusarium pathogens. Controlling FHB is a challenge because germplasm that is naturally resistant against these pathogens is inadequate. Current control measures rely on fungicides. Here, an antibody fusion comprised of the Fusarium spp.-specific recombinant antibody gene CWP2 derived from chicken, and the endochitinase gene Ech42 from the biocontrol fungus Trichoderma atroviride was introduced into the elite wheat cultivar Zhengmai9023 by particle bombardment. Expression of this fusion gene was regulated by the lemma/palea-specific promoter Lem2 derived from barley; its expression was confirmed as lemma/palea-specific in transgenic wheat. Single-floret inoculation of independent transgenic wheat lines of the T3 to T6 generations revealed significant resistance (type II) to fungal spreading, and natural infection assays in the field showed significant resistance (type I) to initial infection. Gas chromatography-mass spectrometry analysis revealed marked reduction of mycotoxins in the grains of the transgenic wheat lines. Progenies of crosses between the transgenic lines and the FHB-susceptible cultivar Huamai13 also showed significantly enhanced FHB resistance. Quantitative real-time PCR analysis revealed that the tissue-specific expression of the antibody fusion was induced by salicylic acid drenching and induced to a greater extent by F. graminearum infection. Histochemical analysis showed substantial restriction of mycelial growth in the lemma tissues of the transgenic plants. Thus, the combined tissue-specific and pathogen-inducible expression of this Fusarium-specific antibody fusion can effectively protect wheat against Fusarium pathogens and reduce mycotoxin content in grain. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Transgenic medaka fish as models to analyze bone homeostasis under micro-gravity conditions in vivo

NASA Astrophysics Data System (ADS)

Winkler, C.; Wagner, T.; Renn, J.; Goerlich, R.; Schartl, M.

Long-term space flight and microgravity results in bone loss that can be explained by reduced activity of bone-forming osteoblast cells and/or an increase in activity of bone resorbing osteoclast cells. Osteoprotegerin (OPG), a secreted protein of 401 amino acids, has been shown to regulate the balance between osteoblast and osteoclast formation and thereby warrants constant bone mass under normal gravitational conditions. Consistent with this, earlier reports using transgenic mice have shown that increased activation of OPG leads to exc essive bone formation (osteopetrosis), while inactivation of OPG leads to bone loss (osteoporosis). Importantly, it has recently been reported that expression of murine OPG is regulated by vector averaged gravity (Kanematsu et al., 2002, Bone 30, p553). The small bony fish medaka (Oryzias latipes ) has attracted increasing attention as genetic model system to study developmental and pathological processes. To analyze the molecular mechanisms of bone formation in this small vertebrate, we have isolated two related genes, opr-1 and opr -2, from medaka. Our phylogenetic analysis revealed that both genes originated from a common ancestor by fish-specific gene duplication and represent the orthologs of the mammalian OPG gene. Both opr genes are differentially expressed during embryonic and larval development, in adult tissues and in cultured primary osteoblast cells. We have characterized their promoter regions and identified consensus binding sites for transcription factors of the bone-morphogenetic-protein (BMP) p thway and for core-binding-factor-1Aa (cbfa1). Cbfa1 has been shown to be the key regulator of OPG expression during several steps of osteoblast differentiation in mammals. This opens the possibility that the mechanisms controlling bone formation in teleost fish and higher vertebrates are regulated by related mechanisms. We are currently generating transgenic medakafish expressing a GFP reporter gene under control of the teleost OPG promoter in order to visualize osteoblast activity in a living organism under different gravity conditions. This work is supported by the German Aerospace Center, DLR.

The BnGRF2 gene (GRF2-like gene from Brassica napus) enhances seed oil production through regulating cell number and plant photosynthesis

PubMed Central

Liu, Jing; Hua, Wei; Yang, Hong-Li; Zhan, Gao-Miao; Deng, Lin-Bin; Wang, Xin-Fa; Liu, Gui-Hua; Wang, Han-Zhong

2012-01-01

Seed yield and oil content are two important agricultural characteristics in oil crop breeding, and a lot of functional gene research is being concentrated on increasing these factors. In this study, by differential gene expression analyses between rapeseed lines (zy036 and 51070) which exhibit different levels of seed oil production, BnGRF2 (Brassica napus growth-regulating factor 2-like gene) was identified in the high oil-producing line zy036. To elucidate the possible roles of BnGRF2 in seed oil production, the cDNA sequences of the rapeseed GRF2 gene were isolated. The Blastn result showed that rapeseed contained BnGRF2a/2b which were located in the A genome (A1 and A3) and C genome (C1 and C6), respectively, and the dominantly expressed gene BnGRF2a was chosen for transgenic research. Analysis of 35S-BnGRF2a transgenic Arabidopsis showed that overexpressed BnGRF2a resulted in an increase in seed oil production of >50%. Moreover, BnGRF2a also induced a >20% enlargement in extended leaves and >40% improvement in photosynthetic efficiency because of an increase in the chlorophyll content. Furthermore, transcriptome analyses indicated that some genes associated with cell proliferation, photosynthesis, and oil synthesis were up-regulated, which revealed that cell number and plant photosynthesis contributed to the increased seed weight and oil content. Because of less efficient self-fertilization induced by the longer pistil in the 35S-BnGRF2a transgenic line, Napin-BnGRF2a transgenic lines were further used to identify the function of BnGRF2, and the results showed that seed oil production also could increase >40% compared with the wild-type control. The results suggest that improvement to economically important characteristics in oil crops may be achieved by manipulation of the GRF2 expression level. PMID:22442419

Structural characterization of a novel full-length transcript promoter from Horseradish Latent Virus (HRLV) and its transcriptional regulation by multiple stress responsive transcription factors.

PubMed

Khan, Ahamed; Shrestha, Ankita; Bhuyan, Kashyap; Maiti, Indu B; Dey, Nrisingha

2018-01-01

The promoter fragment described in this study can be employed for strong transgene expression under both biotic and abiotic stress conditions. Plant-infecting Caulimoviruses have evolved multiple regulatory mechanisms to address various environmental stimuli during the course of evolution. One such mechanism involves the retention of discrete stress responsive cis-elements which are required for their survival and host-specificity. Here we describe the characterization of a novel Caulimoviral promoter isolated from Horseradish Latent Virus (HRLV) and its regulation by multiple stress responsive Transcription factors (TFs) namely DREB1, AREB1 and TGA1a. The activity of full length transcript (Flt-) promoter from HRLV (- 677 to + 283) was investigated in both transient and transgenic assays where we identified H12 (- 427 to + 73) as the highest expressing fragment having ~ 2.5-fold stronger activity than the CaMV35S promoter. The H12 promoter was highly active and near-constitutive in the vegetative and reproductive parts of both Tobacco and Arabidopsis transgenic plants. Interestingly, H12 contains a distinct cluster of cis-elements like dehydration-responsive element (DRE-core; GCCGAC), an ABA-responsive element (ABRE; ACGTGTC) and as-1 element (TGACG) which are known to be induced by cold, drought and pathogen/SA respectively. The specific binding of DREB1, AREB1 and TGA1a to DRE, ABRE and as-1 elements respectively were confirmed by the gel-binding assays using H12 promoter-specific probes. Detailed mutational analysis of the H12 promoter suggested that the presence of DRE-core and as-1 element was indispensable for its activity which was further confirmed by the transactivation assays. Our studies imply that H12 could be a valuable genetic tool for regulated transgene expression under diverse environmental conditions.

The BnGRF2 gene (GRF2-like gene from Brassica napus) enhances seed oil production through regulating cell number and plant photosynthesis.

PubMed

Liu, Jing; Hua, Wei; Yang, Hong-Li; Zhan, Gao-Miao; Li, Rong-Jun; Deng, Lin-Bin; Wang, Xin-Fa; Liu, Gui-Hua; Wang, Han-Zhong

2012-06-01

Seed yield and oil content are two important agricultural characteristics in oil crop breeding, and a lot of functional gene research is being concentrated on increasing these factors. In this study, by differential gene expression analyses between rapeseed lines (zy036 and 51070) which exhibit different levels of seed oil production, BnGRF2 (Brassica napus growth-regulating factor 2-like gene) was identified in the high oil-producing line zy036. To elucidate the possible roles of BnGRF2 in seed oil production, the cDNA sequences of the rapeseed GRF2 gene were isolated. The Blastn result showed that rapeseed contained BnGRF2a/2b which were located in the A genome (A1 and A3) and C genome (C1 and C6), respectively, and the dominantly expressed gene BnGRF2a was chosen for transgenic research. Analysis of 35S-BnGRF2a transgenic Arabidopsis showed that overexpressed BnGRF2a resulted in an increase in seed oil production of >50%. Moreover, BnGRF2a also induced a >20% enlargement in extended leaves and >40% improvement in photosynthetic efficiency because of an increase in the chlorophyll content. Furthermore, transcriptome analyses indicated that some genes associated with cell proliferation, photosynthesis, and oil synthesis were up-regulated, which revealed that cell number and plant photosynthesis contributed to the increased seed weight and oil content. Because of less efficient self-fertilization induced by the longer pistil in the 35S-BnGRF2a transgenic line, Napin-BnGRF2a transgenic lines were further used to identify the function of BnGRF2, and the results showed that seed oil production also could increase >40% compared with the wild-type control. The results suggest that improvement to economically important characteristics in oil crops may be achieved by manipulation of the GRF2 expression level.

Adapting in vitro embryonic stem cell differentiation to the study of locus control regions.

PubMed

Lahiji, Armin; Kučerová-Levisohn, Martina; Holmes, Roxanne; Zúñiga-Pflücker, Juan Carlos; Ortiz, Benjamin D

2014-05-01

Numerous locus control region (LCR) activities have been discovered in gene loci important to immune cell development and function. LCRs are a distinct class of cis-acting gene regulatory elements that appear to contain all the DNA sequence information required to establish an independently and predictably regulated gene expression program at any genomic site in native chromatin of a whole animal. As such, LCR-regulated transgenic reporter systems provide invaluable opportunities to investigate the mechanisms of gene regulatory DNA action during development. Furthermore the qualities of LCR-driven gene expression, including spatiotemporal specificity and "integration site-independence" would be highly desirable to incorporate into vectors used in therapeutic genetic engineering. Thus, advancement in the methods used to investigate LCRs is of considerable basic and translational significance. We study the LCR present in the mouse T cell receptor (TCR)-α gene locus. Until recently, transgenic mice provided the only experimental model capable of supporting the entire spectrum of LCR activities. We have recently reported complete manifestation of TCRα LCR function in T cells derived in vitro from mouse embryonic stem cells (ESC), thus validating a complete cell culture model for the full range of LCR activities seen in transgenic mice. Here we discuss the critical parameters involved in studying LCR-regulated gene expression during in vitro hematopoietic differentiation from ESCs. This advance provides an approach to speed progress in the LCR field, and facilitate the clinical application of its findings, particularly to the genetic engineering of T cells. Copyright © 2014 Elsevier B.V. All rights reserved.

GFP reporter mice for the retinoblastoma-related cell cycle regulator p107

PubMed Central

Burkhart, Deborah L.; Viatour, Patrick; Ho, Victoria M.; Sage, Julien

2009-01-01

The RB tumor suppressor gene is mutated in a broad range of human cancers, including pediatric retinoblastoma. Strikingly, however, Rb mutant mice develop tumors of the pituitary and thyroid glands, but not retinoblastoma. Mouse genetics experiments have demonstrated that p107, a protein related to pRB, is capable of preventing retinoblastoma, but not pituitary tumors, in Rb-deficient mice. Evidence suggests that the basis for this compensatory function of p107 is increased transcription of the p107 gene in response to Rb inactivation. To begin to address the context-dependency of this compensatory role of p107 and to follow p107 expression in vivo, we have generated transgenic mice carrying an enhanced GFP (eGFP) reporter inserted into a bacterial artificial chromosome (BAC) containing the mouse p107 gene. Expression of the eGFP transgene parallels that of p107 in these transgenic mice and identifies cells with a broad range of expression level for p107, even within particular organs or tissues. We also show that loss of Rb results in the upregulation of p107 transcription in specific cell populations in vivo, including subpopulations of hematopoietic cells. Thus, p107 BAC-eGFP transgenic mice serve as a useful tool to identify distinct cell types in which p107 is expressed and may have key functions in vivo, and to characterize changes in cellular networks accompanying Rb deficiency. PMID:18719374

VEGF-C and VEGF-D blockade inhibits inflammatory skin carcinogenesis.

PubMed

Alitalo, Annamari K; Proulx, Steven T; Karaman, Sinem; Aebischer, David; Martino, Stefania; Jost, Manuela; Schneider, Nicole; Bry, Maija; Detmar, Michael

2013-07-15

VEGF-C and VEGF-D were identified as lymphangiogenic growth factors and later shown to promote tumor metastasis, but their effects on carcinogenesis are poorly understood. Here, we have studied the effects of VEGF-C and VEGF-D on tumor development in the murine multistep chemical carcinogenesis model of squamous cell carcinoma by using a soluble VEGF-C/VEGF-D inhibitor. After topical treatment with a tumor initiator and repeated tumor promoter applications, transgenic mice expressing a soluble VEGF-C/VEGF-D receptor (sVEGFR-3) in the skin developed significantly fewer squamous cell tumors with a delayed onset when compared with wild-type mice or mice expressing sVEGFR-3 lacking the ligand-binding site. Epidermal proliferation was reduced in the carcinogen-treated transgenic skin, whereas epidermal keratinocyte proliferation in vitro was not affected by VEGF-C or VEGF-D, indicating indirect effects of sVEGFR-3 expression. Importantly, transgenic mouse skin was less sensitive to tumor promoter-induced inflammation, with reduced angiogenesis and blood vessel leakage. Cutaneous leukocytes, especially macrophages, were reduced in transgenic skin without major changes in macrophage polarization or blood monocyte numbers. Several macrophage-associated cytokines were also reduced in transgenic papillomas, although the dermal macrophages themselves did not express VEGFR-3. These findings indicate that VEGF-C/VEGF-D are involved in shaping the inflammatory tumor microenvironment that regulates early tumor progression. Our results support the use of VEGF-C/VEGF-D-blocking agents not only to inhibit metastatic progression, but also during the early stages of tumor growth. ©2013 AACR.

Transgenic mouse lines for non-invasive ratiometric monitoring of intracellular chloride

PubMed Central

Batti, Laura; Mukhtarov, Marat; Audero, Enrica; Ivanov, Anton; Paolicelli, Rosa Chiara; Zurborg, Sandra; Gross, Cornelius; Bregestovski, Piotr; Heppenstall, Paul A.

2013-01-01

Chloride is the most abundant physiological anion and participates in a variety of cellular processes including trans-epithelial transport, cell volume regulation, and regulation of electrical excitability. The development of tools to monitor intracellular chloride concentration ([Cli]) is therefore important for the evaluation of cellular function in normal and pathological conditions. Recently, several Cl-sensitive genetically encoded probes have been described which allow for non-invasive monitoring of [Cli]. Here we describe two mouse lines expressing a CFP-YFP-based Cl probe called Cl-Sensor. First, we generated transgenic mice expressing Cl-Sensor under the control of the mouse Thy1 mini promoter. Cl-Sensor exhibited good expression from postnatal day two (P2) in neurons of the hippocampus and cortex, and its level increased strongly during development. Using simultaneous whole-cell monitoring of ionic currents and Cl-dependent fluorescence, we determined that the apparent EC50 for Cli was 46 mM, indicating that this line is appropriate for measuring neuronal [Cli] in postnatal mice. We also describe a transgenic mouse reporter line for Cre-dependent conditional expression of Cl-Sensor, which was targeted to the Rosa26 locus and by incorporating a strong exogenous promoter induced robust expression upon Cre-mediated recombination. We demonstrate high levels of tissue-specific expression in two different Cre-driver lines targeting cells of the myeloid lineage and peripheral sensory neurons. Using these mice the apparent EC50 for Cli was estimated to be 61 and 54 mM in macrophages and DRG, respectively. Our data suggest that these mouse lines will be useful models for ratiometric monitoring of Cli in specific cell types in vivo. PMID:23734096

The Central Role of PhEIN2 in Ethylene Responses throughout Plant Development in Petunia1

PubMed Central

Shibuya, Kenichi; Barry, Kristin G.; Ciardi, Joseph A.; Loucas, Holly M.; Underwood, Beverly A.; Nourizadeh, Saeid; Ecker, Joseph R.; Klee, Harry J.; Clark, David G.

2004-01-01

The plant hormone ethylene regulates many aspects of growth and development. Loss-of-function mutations in ETHYLENE INSENSITIVE2 (EIN2) result in ethylene insensitivity in Arabidopsis, indicating an essential role of EIN2 in ethylene signaling. However, little is known about the role of EIN2 in species other than Arabidopsis. To gain a better understanding of EIN2, a petunia (Petunia × hybrida cv Mitchell Diploid [MD]) homolog of the Arabidopsis EIN2 gene (PhEIN2) was isolated, and the role of PhEIN2 was analyzed in a wide range of plant responses to ethylene, many that do not occur in Arabidopsis. PhEIN2 mRNA was present at varying levels in tissues examined, and the PhEIN2 expression decreased after ethylene treatment in petals. These results indicate that expression of PhEIN2 mRNA is spatially and temporally regulated in petunia during plant development. Transgenic petunia plants with reduced PhEIN2 expression were compared to wild-type MD and ethylene-insensitive petunia plants expressing the Arabidopsis etr1-1 gene for several physiological processes. Both PhEIN2 and etr1-1 transgenic plants exhibited significant delays in flower senescence and fruit ripening, inhibited adventitious root and seedling root hair formation, premature death, and increased hypocotyl length in seedling ethylene response assays compared to MD. Moderate or strong levels of reduction in ethylene sensitivity were achieved with expression of both etr1-1 and PhEIN2 transgenes, as measured by downstream expression of PhEIL1. These results demonstrate that PhEIN2 mediates ethylene signals in a wide range of physiological processes and also indicate the central role of EIN2 in ethylene signal transduction. PMID:15466231

A role for small RNA in regulating innate immunity during plant growth

PubMed Central

Deng, Yingtian; Wang, Jubin; Tung, Jeffrey; Liu, Dan; Zhou, Yingjia; He, Shuang; Baker, Barbara

2018-01-01

Plant genomes encode large numbers of nucleotide-binding (NB) leucine-rich repeat (LRR) immune receptors (NLR) that mediate effector triggered immunity (ETI) and play key roles in protecting crops from diseases caused by devastating pathogens. Fitness costs are associated with plant NLR genes and regulation of NLR genes by micro(mi)RNAs and phased small interfering RNAs (phasiRNA) is proposed as a mechanism for reducing these fitness costs. However, whether NLR expression and NLR-mediated immunity are regulated during plant growth is unclear. We conducted genome-wide transcriptome analysis and showed that NLR expression gradually increased while expression of their regulatory small RNAs (sRNA) gradually decreased as plants matured, indicating that sRNAs could play a role in regulating NLR expression during plant growth. We further tested the role of miRNA in the growth regulation of NLRs using the tobacco mosaic virus (TMV) resistance gene N, which was targeted by miR6019 and miR6020. We showed that N-mediated resistance to TMV effectively restricted this virus to the infected leaves of 6-week old plants, whereas TMV infection was lethal in 1- and 3-week old seedlings due to virus-induced systemic necrosis. We further found that N transcript levels gradually increased while miR6019 levels gradually decreased during seedling maturation that occurs in the weeks after germination. Analyses of reporter genes in transgenic plants showed that growth regulation of N expression was post-transcriptionally mediated by MIR6019/6020 whereas MIR6019/6020 was regulated at the transcriptional level during plant growth. TMV infection of MIR6019/6020 transgenic plants indicated a key role for miR6019-triggered phasiRNA production for regulation of N-mediated immunity. Together our results demonstrate a mechanistic role for miRNAs in regulating innate immunity during plant growth. PMID:29293695

Heterotrimeric G-Protein γ Subunit CsGG3.2 Positively Regulates the Expression of CBF Genes and Chilling Tolerance in Cucumber

PubMed Central

Bai, Longqiang; Liu, Yumei; Mu, Ying; Anwar, Ali; He, Chaoxing; Yan, Yan; Li, Yansu; Yu, Xianchang

2018-01-01

Heterotrimeric guanine nucleotide-binding proteins (G proteins) composed of alpha (Gα), beta (Gβ), and gamma (Gγ) subunits are central signal transducers mediating the cellular response to multiple stimuli, such as cold, in eukaryotes. Plant Gγ subunits, divided into A, B, and C three structurally distinct types, provide proper cellular localization and functional specificity to the heterotrimer complex. Here, we demonstrate that a type C Gγ subunit CsGG3.2 is involved in the regulation of the CBF regulon and plant tolerance to cold stresses in cucumber (Cucumis sativus L.). We showed that CsGG3.2 transcript abundance was positively induced by cold treatments. Transgenic cucumber plants (T1) constitutively over-expressing CsGG3.2 exhibits tolerance to chilling conditions and increased expression of CBF genes and their regulon. Antioxidative enzymes, i.e., superoxide dismutase, catalase, peroxidase, and glutathione reductase activities increased in cold-stressed transgenic plants. The reactive oxygen species, oxygen free radical and H2O2, production, as well as membrane lipid peroxidation (MDA) production decreased in transgenic plants, suggesting a better antioxidant system to cope the oxidative-damages caused by cold stress. These findings provide evidence for a critical role of CsGG3.2 in mediating cold signal transduction in plant cells. PMID:29719547

β-Catenin Dosage Is a Critical Determinant of Tracheal Basal Cell Fate Determination

PubMed Central

Brechbuhl, Heather M.; Ghosh, Moumita; Smith, Mary Kathryn; Smith, Russell W.; Li, Bilan; Hicks, Douglas A.; Cole, Brook B.; Reynolds, Paul R.; Reynolds, Susan D.

2011-01-01

The purpose of this study was to determine whether β-catenin regulates basal cell fate determination in the mouse trachea. Analysis of TOPGal transgene reporter activity and Wnt/β-catenin pathway gene expression suggested a role for β-catenin in basal cell proliferation and differentiation after naphthalene-mediated Clara-like and ciliated cell depletion. However, these basal cell activities occurred simultaneously, limiting precise determination of the role(s) played by β-catenin. This issue was overcome by analysis of β-catenin signaling in tracheal air-liquid interface cultures. The cultures could be divided into two phases: basal cell proliferation and basal cell differentiation. A role for β-catenin in basal cell proliferation was indicated by activation of the TOPGal transgene on proliferation days 3 to 5 and by transient expression of Myc (alias c-myc). Another peak of TOPGal transgene activity was detected on differentiation days 2 to 10 and was associated with the expression of Axin 2. These results suggest a role for β-catenin in basal to ciliated and basal to Clara-like cell differentiation. Genetic stabilization of β-catenin in basal cells shortened the period of basal cell proliferation but had a minor effect on this process. Persistent β-catenin signaling regulated basal cell fate by driving the generation of ciliated cells and preventing the production of Clara-like cells. PMID:21703416

Ectopic Activation of Wnt/β-Catenin Signaling in Lens Fiber Cells Results in Cataract Formation and Aberrant Fiber Cell Differentiation

PubMed Central

Antosova, Barbora; Smolikova, Jana; Borkovcova, Romana; Strnad, Hynek; Lachova, Jitka; Machon, Ondrej; Kozmik, Zbynek

2013-01-01

The Wnt/β-catenin signaling pathway controls many processes during development, including cell proliferation, cell differentiation and tissue homeostasis, and its aberrant regulation has been linked to various pathologies. In this study we investigated the effect of ectopic activation of Wnt/β-catenin signaling during lens fiber cell differentiation. To activate Wnt/β-catenin signaling in lens fiber cells, the transgenic mouse referred to as αA-CLEF was generated, in which the transactivation domain of β-catenin was fused to the DNA-binding protein LEF1, and expression of the transgene was controlled by αA-crystallin promoter. Constitutive activation of Wnt/β-catenin signaling in lens fiber cells of αA-CLEF mice resulted in abnormal and delayed fiber cell differentiation. Moreover, adult αA-CLEF mice developed cataract, microphthalmia and manifested downregulated levels of γ-crystallins in lenses. We provide evidence of aberrant expression of cell cycle regulators in embryonic lenses of αA-CLEF transgenic mice resulting in the delay in cell cycle exit and in the shift of fiber cell differentiation to the central fiber cell compartment. Our results indicate that precise regulation of the Wnt/β-catenin signaling activity during later stages of lens development is essential for proper lens fiber cell differentiation and lens transparency. PMID:24205179

Cross-talk between miR-471-5p and autophagy component proteins regulates LC3-associated phagocytosis (LAP) of apoptotic germ cells.

PubMed

Panneerdoss, Subbarayalu; Viswanadhapalli, Suryavathi; Abdelfattah, Nourhan; Onyeagucha, Benjamin C; Timilsina, Santosh; Mohammad, Tabrez A; Chen, Yidong; Drake, Michael; Vuori, Kristiina; Kumar, T Rajendra; Rao, Manjeet K

2017-09-19

Phagocytic clearance of apoptotic germ cells by Sertoli cells is vital for germ cell development and differentiation. Here, using a tissue-specific miRNA transgenic mouse model, we show that interaction between miR-471-5p and autophagy member proteins regulates clearance of apoptotic germ cells via LC3-associated phagocytosis (LAP). Transgenic mice expressing miR-471-5p in Sertoli cells show increased germ cell apoptosis and compromised male fertility. Those effects are due to defective engulfment and impaired LAP-mediated clearance of apoptotic germ cells as miR-471-5p transgenic mice show lower levels of Dock180, LC3, Atg12, Becn1, Rab5 and Rubicon in Sertoli cells. Our results reveal that Dock180 interacts with autophagy member proteins to constitute a functional LC3-dependent phagocytic complex. We find that androgen regulates Sertoli cell phagocytosis by controlling expression of miR-471-5p and its target proteins. These findings suggest that recruitment of autophagy machinery is essential for efficient clearance of apoptotic germ cells by Sertoli cells using LAP.Although phagocytic clearance of apoptotic germ cells by Sertoli cells is essential for spermatogenesis, little of the mechanism is known. Here the authors show that Sertoli cells employ LC3-associated phagocytosis (LAP) by recruiting autophagy member proteins to clear apoptotic germ cells.

Liver tumor formation by a mutant retinoblastoma protein in the transgenic mice is caused by an upregulation of c-Myc target genes.

PubMed

Wang, Bo; Hikosaka, Keisuke; Sultana, Nishat; Sharkar, Mohammad Tofael Kabir; Noritake, Hidenao; Kimura, Wataru; Wu, Yi-Xin; Kobayashi, Yoshimasa; Uezato, Tadayoshi; Miura, Naoyuki

2012-01-06

The retinoblastoma (Rb) tumor suppressor encodes a nuclear phosphoprotein that regulates cellular proliferation, apoptosis and differentiation. In order to adapt itself to these biological functions, Rb is subjected to modification cycle, phosphorylation and dephosphorylation. To directly determine the effect of phosphorylation-resistant Rb on liver development and function, we generated transgenic mice expressing phosphorylation-resistant human mutant Rb (mt-Rb) under the control of the rat hepatocyte nuclear factor-1 gene promoter/enhancer. Expression of mt-Rb in the liver resulted in macroscopic neoplastic nodules (adenomas) with ∼50% incidence within 15 months old. Interestingly, quantitative reverse transcriptase-PCR analysis showed that c-Myc was up-regulated in the liver of mt-Rb transgenic mice irrespective of having tumor tissues or no tumor. In tumor tissues, several c-Myc target genes, Foxm1, c-Jun, c-Fos, Bmi1 and Skp2, were also up-regulated dramatically. We determined whether mt-Rb activated the Myc promoter in the HTP9 cells and demonstrated that mt-Rb acted as an inhibitor of wild-type Rb-induced repression on the Myc promoter. Our results suggest that continued upregulation of c-Myc target genes promotes the liver tumor formation after about 1 year of age. Copyright © 2011 Elsevier Inc. All rights reserved.

Molecular Mechanisms Regulating Muscle Fiber Composition Under Microgravity

NASA Technical Reports Server (NTRS)

Rosenthal, Nadia A.

1999-01-01

The overall goal of this project is to reveal the molecular mechanisms underlying the selective and debilitating atrophy of specific skeletal muscle fiber types that accompanies sustained conditions of microgravity. Since little is currently known about the regulation of fiber-specific gene expression programs in mammalian muscle, elucidation of the basic mechanisms of fiber diversification is a necessary prerequisite to the generation of therapeutic strategies for attenuation of muscle atrophy on earth or in space. Vertebrate skeletal muscle development involves the fusion of undifferentiated mononucleated myoblasts to form multinucleated myofibers, with a concomitant activation of muscle-specific genes encoding proteins that form the force-generating contractile apparatus. The regulatory circuitry controlling skeletal muscle gene expression has been well studied in a number of vertebrate animal systems. The goal of this project has been to achieve a similar level of understanding of the mechanisms underlying the further specification of muscles into different fiber types, and the role played by innervation and physical activity in the maintenance and adaptation of different fiber phenotypes into adulthood. Our recent research on the genetic basis of fiber specificity has focused on the emergence of mature fiber types and have implicated a group of transcriptional regulatory proteins, known as E proteins, in the control of fiber specificity. The restriction of E proteins to selected muscle fiber types is an attractive hypothetical mechanism for the generation of muscle fiber-specific patterns of gene expression. To date our results support a model wherein different E proteins are selectively expressed in muscle cells to determine fiber-restricted gene expression. These studies are a first step to define the molecular mechanisms responsible for the shifts in fiber type under conditions of microgravity, and to determine the potential importance of E proteins as upstream targets for the effects of weightlessness. In the past year we have determined that the expression of E Proteins is restricted to specific fiber types by post-transcriptional mechanisms. By far, the most prevalent mechanism of cellular control for achieving post-transcriptional regulation of gene expression is selective proteolysis -through the ubiquitin -proteasome pathway. Steady-state levels of HEB message are similar in all fast and slow skeletal muscle fiber types, yet the protein is restricted to Type IIX fibers. HEB appears to be a nodal point for regulating fiber-specific transcription, as expression of the transcription factor is regulated at the post-transcriptional level. It is not clear at present whether the regulation is at the level of protein synthesis or degradation. We are now poised to evaluate the biological role of ubiquitination in fiber specific-gene expression by controlling the post-transcriptional expression of E Proteins. The use of metabolic labelling and pharmacological inhibitors of the ubiquitin pathway will be used to identify the mode of regulation of the Type IIX expression pattern. The potential role of specific kinases in effecting the restriction of HEB expression will be examined by using both inhibitors and activators. The results of these studies will provide the necessary information to evaluate the biological role of E proteins in controlling fiber type transitions, and in potentially attenuating the atrophic effects of microgravity conditions. We have also recently shown that ectopic expression of the HEB protein transactivates the Type IIX-specific skeletal a-actin reporter. The 218 bp skeletal a-actin promoter drives transgene expression solely in mature Type IIX fibers. A mouse also carrying the transgene MLCI/HEB (which ectopically expresses the E Protein HEB in Type IIB fibers) forces expression of the skeletal a-actin reporter gene in Type IIB fibers. We can now dissect the composition of this fiber-specific cis-element. The skeletal a-actin promoter is quite compact and has been extensively characterized in vitro for activity and binding factors. The single E box may act as a binding target of myogenic factor/HEB heterodimer to allow for IIX expression. The HEB transcription factor may recognize either the precise flanking sequences of the E Box, or perhaps interacting with other proteins bound nearby, and activating expression in Type IIX fibers. This E box will be both ablated, and alternatively, as ablation may well destroy any muscle-specific transcriptional activity, flanking sequences substituted with those surrounding the E box (El) of the myogenin promoter. Modification of fiber-specific transgene expression will be tested in transgenic mice. The results of these studies will provide basic information on the regulatory circuitry underlying fiber specificity, and will form the basis for building appropriate transgenic regulatory cassettes to effect fiber transitions in subsequent experimental manipulations on unweighted muscles.

[MYB-like transcription factor SiMYB42 from foxtail millet (Setaria italica L.) enhances Arabidopsis tolerance to low-nitrogen stress].

PubMed

Ding, Qing Qian; Wang, Xiao Ting; Hu, Li Qin; Qi, Xin; Ge, Lin Hao; Xu, Wei Ya; Xu, Zhao Shi; Zhou, Yong Bin; Jia, Guan Qing; Diao, Xian Min; Min, Dong Hong; Ma, You Zhi; Chen, Ming

2018-04-20

Myeloblastosis (MYB) transcription factors are one of the largest families of transcription factors in higher plants. They play an important role in plant development, defense response processes, and non-biological stresses, i.e., drought stress. Foxtail millet (Setaria italica L.), originated in China, is resistant to drought and low nutrition stresses and has been regarded as an ideal material for studying abiotic stress resistance in monocotyledon. In this study, we ran a transcription profile analysis of zheng 204 under low-nitrogen conditions and identified a MYB-like transcription factor SiMYB42, which was up-regulated under low-nitrogen stress. Phylogenetic tree analysis showed that SiMYB42 belongs to R2R3-MYB subfamily and has two MYB conserved domains. Expression pattern analysis showed that SiMYB42 was significantly up-regulated under various stress conditions, including low-nitrogen stress, high salt, drought and ABA conditions. The results of subcellular localization, quantitative real-time PCR and transcriptional activation analysis indicated that SiMYB42 protein localizes to the nucleus and cell membrane of plant cells, mainly expressed in the leaf or root of foxtail millet, and has transcription activation activity. Functional analysis showed that there was no significant difference between transgenic SiMYB42 Arabidopsis and wild-type (WT) Arabidopsis under normal conditions; however, under low-nitrogen condition, the root length, surface area and seedling fresh weight in transgenic SiMYB42 Arabidopsis, were significantly higher than their counterparts in WT. These results suggest that SiMYB42 transgenic plants exhibit higher tolerance to low-nitrogen stress. Expression levels of nitrate transporters genes NRT2.1, NRT2.4 and NRT2.5, which are the transcriptional targets of SiMYB42, were higher in transgenic SiMYB42 Arabidopsis plants than those in WT; the promoter regions of NRT2.1, NRT2.4 and NRT2.5 all have MYB binding sites. These results indicate that SiMYB42 might enhance foxtail millet tolerance to low-nitrogen condition through regulating the expression of nitrate transporter genes. This study reveals the possible functions of SiMYB42 in a low-nitrogen stress response pathway, and provides a foundation for further understanding the entire regulation network of foxtail millet in response to low-nitrogen stress.

Combined exposure to Maneb and Paraquat alters transcriptional regulation of neurogenesis-related genes in mice models of Parkinson's disease.

PubMed

Desplats, Paula; Patel, Pruthul; Kosberg, Kori; Mante, Michael; Patrick, Christina; Rockenstein, Edward; Fujita, Masayo; Hashimoto, Makoto; Masliah, Eliezer

2012-09-28

Parkinson's disease (PD) is a multifactorial disease where environmental factors act on genetically predisposed individuals. Although only 5% of PD manifestations are associated with specific mutations, majority of PD cases are of idiopathic origin, where environment plays a prominent role. Concurrent exposure to Paraquat (PQ) and Maneb (MB) in rural workers increases the risk for PD and exposure of adult mice to MB/PQ results in dopamine fiber loss and decreased locomotor activity. While PD is characterized by neuronal loss in the substantia nigra, we previously showed that accumulation of α-synuclein in the limbic system contributes to neurodegeneration by interfering with adult neurogenesis. We investigated the effect of pesticides on adult hippocampal neurogenesis in two transgenic models: Line 61, expressing the human wild type SNCA gene and Line LRRK2(G2019S), expressing the human LRRK2 gene with the mutation G2019S. Combined exposure to MB/PQ resulted in significant reduction of neuronal precursors and proliferating cells in non-transgenic animals, and this effect was increased in transgenic mice, in particular for Line 61, suggesting that α-synuclein accumulation and environmental toxins have a synergistic effect. We further investigated the transcription of 84 genes with direct function on neurogenesis. Overexpresion of α-synuclein resulted in the downregulation of 12% of target genes, most of which were functionally related to cell differentiation, while LRRK2 mutation had a minor impact on gene expression. MB/PQ also affected transcription in non-transgenic backgrounds, but when transgenic mice were exposed to the pesticides, profound alterations in gene expression affecting 27% of the studied targets were observed in both transgenic lines. Gene enrichment analysis showed that 1:3 of those genes were under the regulation of FoxF2 and FoxO3A, suggesting a primary role of these proteins in the response to genetic and environmental cues. We report that adult neurogenesis is highly susceptible to multiple "risk factors" for PD, including α-synuclein accumulation, LRRK2 G2019 mutation and exposure to environmental toxins. We identified specific groups of genes that are responsive to each stressor, while uncovering a novel function for Fox transcription factors in PD.

A R2R3-MYB transcription factor gene in common wheat (namely TaMYBsm1) involved in enhancement of drought tolerance in transgenic Arabidopsis.

PubMed

Li, Meng-Jun; Qiao, Yu; Li, Ya-Qing; Shi, Zhan-Liang; Zhang, Nan; Bi, Cai-Li; Guo, Jin-Kao

2016-11-01

We isolated the TaMYBsm1 genes, encoding R2R3-type MYB proteins in common wheat, aimed to uncover the possible molecular mechanisms related to drought response. The TaMYBsm1 genes, TaMYBsm1-A, TaMYBsm1-B and TaMYBsm1-D, were isolated and analyzed from the common wheat cultivar Shimai 15. Their expression patterns under PEG 6000 and mannitol were monitored by semi-quantitative RT-PCR and β-glucuronidase (Gus) assay. The function of TaMYBsm1-D under drought stress in transgenic Arabidopsis plants was investigated, and the germination rate, water loss rate, as well as the proline and malondialdehyde (MDA) content were compared with that in wild type (WT) plants. The expression of three downstream genes (DREB2A, P5CS1 and RD29A) in TaMYBsm1-D transgenic plants was analyzed. The R2R3-MYB domains of the MYBsm1 proteins were highly conserved in plants. In addition, the TaMYBsm1 proteins were targeted to the nucleus and contained transcriptional activation domains (TADs). Gus assay and semi-quantitative RT-PCR analysis demonstrated that the TaMYBsm1 genes were up-regulated when the wheat was treated by PEG and mannitol. Compared with WT plants, the germination rates were much higher, but the water loss rates were much lower in TaMYBsm1-D overexpression plants. TaMYBsm1-D transgenic plants showed distinct higher proline contents but a lower MDA content than the WT plants. The three downstream genes were highly expressed in TaMYBsm1-D transgenic plants. We concluded from these results that TaMYBsm1 genes play an important role in plant drought stress tolerance through up-regulation of DREB2A, P5CS1 and RD29A. The increase of proline content and decrease of MDA content may also be involved in the drought response.

Switchgrass (Panicum virgatum L.) promoters for green tissue-specific expression of the MYB4 transcription factor for reduced-recalcitrance transgenic switchgrass

DOE PAGES

Liu, Wusheng; Mazarei, Mitra; Ye, Rongjian; ...

2018-04-24

Genetic engineering of switchgrass (Panicum virgatum L.) for reduced cell wall recalcitrance and improved biofuel production has been a long pursued goal. Up to now, constitutive promoters have been used to direct the expression of cell wall biosynthesis genes toward attaining that goal. While generally sufficient to gauge a transgene's effects in the heterologous host, constitutive overexpression often leads to undesirable plant phenotypic effects. Green tissue-specific promoters from switchgrass are potentially valuable to directly alter cell wall traits exclusively in harvestable aboveground biomass while not changing root phenotypes. We identified and functionally characterized three switchgrass green tissue-specific promoters and assessedmore » marker gene expression patterns and intensity in stably transformed rice (Oryza sativa L.), and then used them to direct the expression of the switchgrass MYB4 (PvMYB4) transcription factor gene in transgenic switchgrass to endow reduced recalcitrance in aboveground biomass. These promoters correspond to photosynthesis-related light-harvesting complex II chlorophyll-a/b binding gene (PvLhcb), phosphoenolpyruvate carboxylase (PvPEPC), and the photosystem II 10 kDa R subunit (PvPsbR). Real-time RT-PCR analysis detected their strong expression in the aboveground tissues including leaf blades, leaf sheaths, internodes, inflorescences, and nodes of switchgrass, which was tightly up-regulated by light. Stable transgenic rice expressing the GUS reporter under the control of each promoter (756-2005 bp in length) further confirmed their strong expression patterns in leaves and stems. With the exception of the serial promoter deletions of PvLhcb, all GUS marker patterns under the control of each 5'-end serial promoter deletion were not different from that conveyed by their respective promoters. All of the shortest promoter fragments (199-275 bp in length) conveyed strong green tissue-specific GUS expression in transgenic rice. PvMYB4 is a master repressor of lignin biosynthesis. The green tissue-specific expression of PvMYB4 via each promoter in transgenic switchgrass led to significant gains in saccharification efficiency, decreased lignin, and decreased S/G lignin ratios. In contrast to constitutive overexpression of PvMYB4, which negatively impacts switchgrass root growth, plant growth was not compromised in green tissue-expressed PvMYB4 switchgrass plants in the current study. Each of the newly described green tissue-specific promoters from switchgrass has utility to change cell wall biosynthesis exclusively in aboveground harvestable biomass without altering root systems. The truncated green tissue promoters are very short and should be useful for targeted expression in a number of monocots to improve shoot traits while restricting gene expression from roots. Green tissue-specific expression of PvMYB4 is an effective strategy for improvement of transgenic feedstocks.« less

Switchgrass (Panicum virgatum L.) promoters for green tissue-specific expression of the MYB4 transcription factor for reduced-recalcitrance transgenic switchgrass

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Wusheng; Mazarei, Mitra; Ye, Rongjian

Genetic engineering of switchgrass (Panicum virgatum L.) for reduced cell wall recalcitrance and improved biofuel production has been a long pursued goal. Up to now, constitutive promoters have been used to direct the expression of cell wall biosynthesis genes toward attaining that goal. While generally sufficient to gauge a transgene's effects in the heterologous host, constitutive overexpression often leads to undesirable plant phenotypic effects. Green tissue-specific promoters from switchgrass are potentially valuable to directly alter cell wall traits exclusively in harvestable aboveground biomass while not changing root phenotypes. We identified and functionally characterized three switchgrass green tissue-specific promoters and assessedmore » marker gene expression patterns and intensity in stably transformed rice (Oryza sativa L.), and then used them to direct the expression of the switchgrass MYB4 (PvMYB4) transcription factor gene in transgenic switchgrass to endow reduced recalcitrance in aboveground biomass. These promoters correspond to photosynthesis-related light-harvesting complex II chlorophyll-a/b binding gene (PvLhcb), phosphoenolpyruvate carboxylase (PvPEPC), and the photosystem II 10 kDa R subunit (PvPsbR). Real-time RT-PCR analysis detected their strong expression in the aboveground tissues including leaf blades, leaf sheaths, internodes, inflorescences, and nodes of switchgrass, which was tightly up-regulated by light. Stable transgenic rice expressing the GUS reporter under the control of each promoter (756-2005 bp in length) further confirmed their strong expression patterns in leaves and stems. With the exception of the serial promoter deletions of PvLhcb, all GUS marker patterns under the control of each 5'-end serial promoter deletion were not different from that conveyed by their respective promoters. All of the shortest promoter fragments (199-275 bp in length) conveyed strong green tissue-specific GUS expression in transgenic rice. PvMYB4 is a master repressor of lignin biosynthesis. The green tissue-specific expression of PvMYB4 via each promoter in transgenic switchgrass led to significant gains in saccharification efficiency, decreased lignin, and decreased S/G lignin ratios. In contrast to constitutive overexpression of PvMYB4, which negatively impacts switchgrass root growth, plant growth was not compromised in green tissue-expressed PvMYB4 switchgrass plants in the current study. Each of the newly described green tissue-specific promoters from switchgrass has utility to change cell wall biosynthesis exclusively in aboveground harvestable biomass without altering root systems. The truncated green tissue promoters are very short and should be useful for targeted expression in a number of monocots to improve shoot traits while restricting gene expression from roots. Green tissue-specific expression of PvMYB4 is an effective strategy for improvement of transgenic feedstocks.« less

[Progress in application of targeting viral vector regulated by microRNA in gene therapy: a review].

PubMed

Zhang, Guohai; Wang, Qizhao; Zhang, Jinghong; Xu, Ruian

2010-06-01

A safe and effective targeting viral vector is the key factor for successful clinical gene therapy. microRNA, a class of small, single-stranded endogenous RNAs, act as post-transcriptional regulators of gene expression. The discovery of these kind regulatory elements provides a new approach to regulate gene expression more accurately. In this review, we elucidated the principle of microRNA in regulation of targeting viral vector. The applications of microRNA in the fields of elimination contamination from replication competent virus, reduction of transgene-specific immunity, promotion of cancer-targeted gene therapy and development of live attenuated vaccines were also discussed.

Transgenic increases in seed oil content are associated with the differential expression of novel Brassica-specific transcripts.

PubMed

Sharma, Nirmala; Anderson, Maureen; Kumar, Arvind; Zhang, Yan; Giblin, E Michael; Abrams, Suzanne R; Zaharia, L Irina; Taylor, David C; Fobert, Pierre R

2008-12-19

Seed oil accumulates primarily as triacylglycerol (TAG). While the biochemical pathway for TAG biosynthesis is known, its regulation remains unclear. Previous research identified microsomal diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) as controlling a rate-limiting step in the TAG biosynthesis pathway. Of note, overexpression of DGAT1 results in substantial increases in oil content and seed size. To further analyze the global consequences of manipulating DGAT1 levels during seed development, a concerted transcriptome and metabolome analysis of transgenic B. napus prototypes was performed. Using a targeted Brassica cDNA microarray, about 200 genes were differentially expressed in two independent transgenic lines analyzed. Interestingly, 24-33% of the targets showing significant changes have no matching gene in Arabidopsis although these represent only 5% of the targets on the microarray. Further analysis of some of these novel transcripts indicated that several are inducible by ABA in microspore-derived embryos. Of the 200 Arabidopsis genes implicated in lipid biology present on the microarray, 36 were found to be differentially regulated in DGAT transgenic lines. Furthermore, kinetic reverse transcriptase Polymerase Chain Reaction (k-PCR) analysis revealed up-regulation of genes encoding enzymes of the Kennedy pathway involved in assembly of TAGs. Hormone profiling indicated that levels of auxins and cytokinins varied between transgenic lines and untransformed controls, while differences in the pool sizes of ABA and catabolites were only observed at later stages of development. Our results indicate that the increased TAG accumulation observed in transgenic DGAT1 plants is associated with modest transcriptional and hormonal changes during seed development that are not limited to the TAG biosynthesis pathway. These might be associated with feedback or feed-forward effects due to altered levels of DGAT1 activity. The fact that a large fraction of significant amplicons have no matching genes in Arabidopsis compromised our ability to draw concrete inferences from the data at this stage, but has led to the identification of novel genes of potential interest.

Morphological Analysis of the Axonal Projections of EGFP-Labeled Esr1-Expressing Neurons in Transgenic Female Medaka.

PubMed

Zempo, Buntaro; Karigo, Tomomi; Kanda, Shinji; Akazome, Yasuhisa; Oka, Yoshitaka

2018-02-01

Some hypothalamic neurons expressing estrogen receptor α (Esr1) are thought to transmit a gonadal estrogen feedback signal to gonadotropin-releasing hormone 1 (GnRH1) neurons, which is the final common pathway for feedback regulation of reproductive functions. Moreover, estrogen-sensitive neurons are suggested to control sexual behaviors in coordination with reproduction. In mammals, hypothalamic estrogen-sensitive neurons release the peptide kisspeptin and regulate GnRH1 neurons. However, a growing body of evidence in nonmammalian species casts doubt on the regulation of GnRH1 neurons by kisspeptin neurons. As a step toward understanding how estrogen regulates neuronal circuits for reproduction and sex behavior in vertebrates in general, we generated a transgenic (Tg) medaka that expresses enhanced green fluorescent protein (EGFP) specifically in esr1-expressing neurons (esr1 neurons) and analyzed their axonal projections. We found that esr1 neurons in the preoptic area (POA) project to the gnrh1 neurons. We also demonstrated by transcriptome and histological analyses that these esr1 neurons are glutamatergic or γ-aminobutyric acidergic (GABAergic) but not kisspeptinergic. We therefore suggest that glutamatergic and GABAergic esr1 neurons in the POA regulate gnrh1 neurons. This hypothesis is consistent with previous studies in mice that found that glutamatergic and GABAergic transmission is critical for estrogen-dependent changes in GnRH1 neuron firing. Thus, we propose that this neuronal circuit may provide an evolutionarily conserved mechanism for regulation of reproduction. In addition, we showed that telencephalic esr1 neurons project to medulla, which may control sexual behavior. Moreover, we found that some POA-esr1 neurons coexpress progesterone receptors. These neurons may form the neuronal circuits that regulate reproduction and sex behavior in response to the serum estrogen/progesterone. Copyright © 2018 Endocrine Society.

On risk and plant-based biopharmaceuticals.

PubMed

Peterson, Robert K D; Arntzen, Charles J

2004-02-01

Research into plant-based expression of pharmaceutical proteins is proceeding at a blistering pace. Indeed, plants expressing pharmaceutical proteins are currently being grown in field environments throughout the USA. But how are these plants and proteins being assessed for environmental risk and how are they being regulated? Here, we examine the applicability of the risk assessment paradigm for assessing human and ecological risks from field-grown transgenic plants that express pharmaceutical proteins.

Transcription factor ERG and joint and articular cartilage formation during mouse limb and spine skeletogenesis.

PubMed

Iwamoto, Masahiro; Tamamura, Yoshihiro; Koyama, Eiki; Komori, Toshihisa; Takeshita, Nobuo; Williams, Julie A; Nakamura, Takashi; Enomoto-Iwamoto, Motomi; Pacifici, Maurizio

2007-05-01

Articular cartilage and synovial joints are critical for skeletal function, but the mechanisms regulating their development are largely unknown. In previous studies we found that the ets transcription factor ERG and its alternatively-spliced variant C-1-1 have roles in joint formation in chick. Here, we extended our studies to mouse. We found that ERG is also expressed in developing mouse limb joints. To test regulation of ERG expression, beads coated with the joint master regulator protein GDF-5 were implanted close to incipient joints in mouse limb explants; this led to rapid and strong ectopic ERG expression. We cloned and characterized several mammalian ERG variants and expressed a human C-1-1 counterpart (hERG3Delta81) throughout the cartilaginous skeleton of transgenic mice, using Col2a1 gene promoter/enhancer sequences. The skeletal phenotype was severe and neonatal lethal, and the transgenic mice were smaller than wild type littermates and their skeletons were largely cartilaginous. Limb long bone anlagen were entirely composed of chondrocytes actively expressing collagen IX and aggrecan as well as articular markers such as tenascin-C. Typical growth plates were absent and there was very low expression of maturation and hypertrophy markers, including Indian hedgehog, collagen X and MMP-13. The results suggest that ERG is part of molecular mechanisms leading chondrocytes into a permanent developmental path and become joint forming cells, and may do so by acting downstream of GDF-5.

Overexpression of Mafb in podocytes protects against diabetic nephropathy.

PubMed

Morito, Naoki; Yoh, Keigyou; Ojima, Masami; Okamura, Midori; Nakamura, Megumi; Hamada, Michito; Shimohata, Homare; Moriguchi, Takashi; Yamagata, Kunihiro; Takahashi, Satoru

2014-11-01

We previously showed that the transcription factor Mafb is essential for podocyte differentiation and foot process formation. Podocytes are susceptible to injury in diabetes, and this injury leads to progression of diabetic nephropathy. In this study, we generated transgenic mice that overexpress Mafb in podocytes using the nephrin promoter/enhancer. To examine a potential pathogenetic role for Mafb in diabetic nephropathy, Mafb transgenic mice were treated with either streptozotocin or saline solution. Diabetic nephropathy was assessed by renal histology and biochemical analyses of urine and serum. Podocyte-specific overexpression of Mafb had no effect on body weight or blood glucose levels in either diabetic or control mice. Notably, albuminuria and changes in BUN levels and renal histology observed in diabetic wild-type animals were ameliorated in diabetic Mafb transgenic mice. Moreover, hyperglycemia-induced downregulation of Nephrin was mitigated in diabetic Mafb transgenic mice, and reporter assay results suggested that Mafb regulates Nephrin directly. Mafb transgenic glomeruli also overexpressed glutathione peroxidase, an antioxidative stress enzyme, and levels of the oxidative stress marker 8-hydroxydeoxyguanosine decreased in the urine of diabetic Mafb transgenic mice. Finally, Notch2 expression increased in diabetic glomeruli, and this effect was enhanced in diabetic Mafb transgenic glomeruli. These data indicate Mafb has a protective role in diabetic nephropathy through regulation of slit diaphragm proteins, antioxidative enzymes, and Notch pathways in podocytes and suggest that Mafb could be a therapeutic target. Copyright © 2014 by the American Society of Nephrology.

On the Occurrence of Hypomyelination in a Transgenic Mouse Model: A Consequence of the Myelin Basic Protein Promoter?

PubMed Central

Gaupp, Stefanie; Arezzo, Joseph; Dutta, Dipankar J.; John, Gareth R.; Raine, Cedric S.

2013-01-01

Central nervous system hypomyelination is a feature common to a number of transgenic (Tg) mouse lines that express a variety of unrelated exogenous (i.e. non-CNS) transgenes. In this report we document hypomyelination structurally by immunocytochemistry and functionally in the Tg line MBP-JE, which overexpresses the chemokine CCL2 (MCP-1) within oligodendrocytes targeted by a myelin basic protein (MBP) promoter. Analysis of hypomyelinated optic nerves of Tg mice revealed progressive decrease in oligodendrocyte numbers with age (p < 0.01). Although molecular mechanisms underlying hypomyelination in this and other Tg models remain largely unknown, we present preliminary findings on oligodendrocyte progenitor cell (OPC) cultures in which, although OPC expressed CCR2, the receptor for CCL2, treatment with CCL2 had no significant effect on OPC proliferation, differentiation or apoptosis. We suggest that hypomyelination in the MBP-JE model might not be due to CCL2 expression but rather the result of transcriptional dysfunction related to random insertion of the MBP promoter that disrupts myelinogenesis and leads to oligodendrocytes demise. Because an MBP promoter is a common denominator in most Tg lines displaying hypomyelination, we hypothesize that use of myelin gene sequences in the regulator region of transgenic constructs might underlie this perturbation of myelination in such models. PMID:22082665

A chloroplast lipoxygenase is required for wound-induced jasmonic acid accumulation in Arabidopsis.

PubMed Central

Bell, E; Creelman, R A; Mullet, J E

1995-01-01

Plant lipoxygenases are thought to be involved in the biosynthesis of lipid-derived signaling molecules. The potential involvement of a specific Arabidopsis thaliana lipoxygenase isozyme, LOX2, in the biosynthesis of the plant growth regulators jasmonic acid (JA) and abscisic acid was investigated. Our characterization of LOX2 indicates that the protein is targeted to chloroplasts. The physiological role of this chloroplast lipoxygenase was analyzed in transgenic plants where cosuppression reduced LOX2 accumulation. The reduction in LOX2 levels caused no obvious changes in plant growth or in the accumulation of abscisic acid. However, the wound-induced accumulation of JA observed in control plants was absent in leaves of transgenic plants that lacked LOX2. Thus, LOX2 is required for the wound-induced synthesis of the plant growth regulator JA in leaves. We also examined the expression of a wound- and JA-inducible Arabidopsis gene, vsp, in transgenic and control plants. Leaves of transgenic plants lacking LOX2 accumulated less vsp mRNA than did control leaves in response to wounding. This result suggests that wound-induced JA (or some other LOX2-requiring component of the wound response pathway) is involved in the wound-induced regulation of this gene. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7567995

A 90-day safety study in Sprague-Dawley rats fed milk powder containing recombinant human lactoferrin (rhLF) derived from transgenic cloned cattle.

PubMed

Zhou, Cui; Wang, Jian Wu; Huang, Kun Lun; He, XiaoYun; Chen, Xiu Ping; Sun, Hong; Yu, Tian; Che, Hui Lian

2011-10-01

Transgenic cloned animals expressing beneficial human nutritional traits offer a new strategy for large-scale production of some kinds of functional substances. In some cases, the required safety testing for genetically modified (GM) foods do not seem appropriate for human food safety, though regulations do not seem to provide alternatives. A 90-day rat feeding study is the core study for the safety assessment of GM foods. The test material in this 90-day study was prepared nonfat milk powder containing recombinant human lactoferrin (rhLF), which was expressed in transgenic cloned cattle. Groups of 10 male and female Sprague-Dawley rats were given a nutritionally balanced purified diet containing 7.5, 15, or 30% transgenic or conventional milk powder for 90 days. A commercial AIN93G diet was used as an additional control group. Clinical, biological, and pathological parameters were compared between groups. The only significant effect of treatment was higher mean ferritin and Fe(+) concentrations for both male and female rats fed the transgenic milk powder diets, as compared to rats fed nontransgenic milk diets or the commercial diet. The results of the present study are consistent with previous research, which indicates that milk powder containing rhLF derived from healthy transgenic cloned cattle is as safe as conventional milk powder.

Molecular and Functional Characterization of GR2-R1 Event Based Backcross Derived Lines of Golden Rice in the Genetic Background of a Mega Rice Variety Swarna

PubMed Central

Bollinedi, Haritha; S., Gopala Krishnan; Prabhu, Kumble Vinod; Singh, Nagendra Kumar; Mishra, Sushma; Khurana, Jitendra P.; Singh, Ashok Kumar

2017-01-01

Homozygous Golden Rice lines developed in the background of Swarna through marker assisted backcross breeding (MABB) using transgenic GR2-R1 event as a donor for the provitamin A trait have high levels of provitamin A (up to 20 ppm) but are dwarf with pale green leaves and drastically reduced panicle size, grain number and yield as compared to the recurrent parent, Swarna. In this study, we carried out detailed morphological, biochemical and molecular characterization of these lines in a quest to identify the probable reasons for their abnormal phenotype. Nucleotide blast analysis with the primer sequences used to amplify the transgene revealed that the integration of transgene disrupted the native OsAux1 gene, which codes for an auxin transmembrane transporter protein. Real time expression analysis of the transgenes (ZmPsy and CrtI) driven by endosperm-specific promoter revealed the leaky expression of the transgene in the vegetative tissues. We propose that the disruption of OsAux1 disturbed the fine balance of plant growth regulators viz., auxins, gibberellic acid and abscisic acid, leading to the abnormalities in the growth and development of the lines homozygous for the transgene. The study demonstrates the conserved roles of OsAux1 gene in rice and Arabidopsis. PMID:28068433

Molecular and Functional Characterization of GR2-R1 Event Based Backcross Derived Lines of Golden Rice in the Genetic Background of a Mega Rice Variety Swarna.

PubMed

Bollinedi, Haritha; S, Gopala Krishnan; Prabhu, Kumble Vinod; Singh, Nagendra Kumar; Mishra, Sushma; Khurana, Jitendra P; Singh, Ashok Kumar

2017-01-01

Homozygous Golden Rice lines developed in the background of Swarna through marker assisted backcross breeding (MABB) using transgenic GR2-R1 event as a donor for the provitamin A trait have high levels of provitamin A (up to 20 ppm) but are dwarf with pale green leaves and drastically reduced panicle size, grain number and yield as compared to the recurrent parent, Swarna. In this study, we carried out detailed morphological, biochemical and molecular characterization of these lines in a quest to identify the probable reasons for their abnormal phenotype. Nucleotide blast analysis with the primer sequences used to amplify the transgene revealed that the integration of transgene disrupted the native OsAux1 gene, which codes for an auxin transmembrane transporter protein. Real time expression analysis of the transgenes (ZmPsy and CrtI) driven by endosperm-specific promoter revealed the leaky expression of the transgene in the vegetative tissues. We propose that the disruption of OsAux1 disturbed the fine balance of plant growth regulators viz., auxins, gibberellic acid and abscisic acid, leading to the abnormalities in the growth and development of the lines homozygous for the transgene. The study demonstrates the conserved roles of OsAux1 gene in rice and Arabidopsis.

A distal ABA responsive element in AtNCED3 promoter is required for positive feedback regulation of ABA biosynthesis in Arabidopsis.

PubMed

Yang, Yan-Zhuo; Tan, Bao-Cai

2014-01-01

The plant hormone abscisic acid (ABA) plays a crucial role in plant development and responses to abiotic stresses. Recent studies indicate that a positive feedback regulation by ABA exists in ABA biosynthesis in plants under dehydration stress. To understand the molecular basis of this regulation, we analyzed the cis-elements of the AtNCED3 promoter in Arabidopsis. AtNCED3 encodes the first committed and highly regulated dioxygenase in the ABA biosynthetic pathway. Through delineated and mutagenesis analyses in stable-transformed Arabidopsis, we revealed that a distal ABA responsive element (ABRE: GGCACGTG, -2372 to -2364 bp) is required for ABA-induced AtNCED3 expression. By analyzing the AtNCED3 expression in ABRE binding protein ABF3 over-expression transgenic plants and knock-out mutants, we provide evidence that the ABA feedback regulation of AtNCED3 expression is not mediated by ABF3.

OsBIRH1, a DEAD-box RNA helicase with functions in modulating defence responses against pathogen infection and oxidative stress

PubMed Central

Li, Dayong; Liu, Huizhi; Zhang, Huijuan; Wang, Xiaoe; Song, Fengming

2008-01-01

DEAD-box proteins comprise a large protein family with members from all kingdoms and play important roles in all types of processes in RNA metabolism. In this study, a rice gene OsBIRH1, which encodes a DEAD-box RNA helicase protein, was cloned and characterized. The predicted OsBIRH1 protein contains a DEAD domain and all conserved motifs that are common characteristics of DEAD-box RNA helicases. Recombinant OsBIRH1 protein purified from Escherichia coli was shown to have both RNA-dependent ATPase and ATP-dependent RNA helicase activities in vitro. Expression of OsBIRH1 was activated in rice seedling leaves after treatment with defence-related signal chemicals, for example benzothiadiazole, salicylic acid, l-aminocyclopropane-1-carboxylic acid, and jasmonic acid, and was also up-regulated in an incompatible interaction between a resistant rice genotype and the blast fungus, Magnaporthe grisea. Transgenic Arabidopsis plants that overexpress the OsBIRH1 gene were generated. Disease resistance phenotype assays revealed that the OsBIRH1-overexpressing transgenic plants showed an enhanced disease resistance against Alternaria brassicicola and Pseudomonas syringae pv. tomato DC3000. Meanwhile, defence-related genes, for example PR-1, PR-2, PR-5, and PDF1.2, showed an up-regulated expression in the transgenic plants. Moreover, the OsBIRH1 transgenic Arabidopsis plants also showed increased tolerance to oxidative stress and elevated expression levels of oxidative defence genes, AtApx1, AtApx2, and AtFSD1. The results suggest that OsBIRH1 encodes a functional DEAD-box RNA helicase and plays important roles in defence responses against biotic and abiotic stresses. PMID:18441339

Flowering-Related RING Protein 1 (FRRP1) Regulates Flowering Time and Yield Potential by Affecting Histone H2B Monoubiquitination in Rice (Oryza Sativa).

PubMed

Du, Yiwei; He, Wei; Deng, Changwang; Chen, Xi; Gou, Lanming; Zhu, Fugui; Guo, Wei; Zhang, Jianfu; Wang, Tao

2016-01-01

Flowering time is a critical trait for crops cultivated under various temperature/photoperiod conditions around the world. To understand better the flowering time of rice, we used the vector pTCK303 to produce several lines of RNAi knockdown transgenic rice and investigated their flowering times and other agronomic traits. Among them, the heading date of FRRP1-RNAi knockdown transgenic rice was 23-26 days earlier than that of wild-type plants. FRRP1 is a novel rice gene that encodes a C3HC4-type Really Interesting Novel Gene (RING) finger domain protein. In addition to the early flowering time, FRRP1-RNAi knockdown transgenic rice caused changes on an array of agronomic traits, including plant height, panicle length and grain length. We analyzed the expression of some key genes associated with the flowering time and other agronomic traits in the FRRP1-RNAi knockdown lines and compared with that in wild-type lines. The expression of Hd3a increased significantly, which was the key factor in the early flowering time. Further experiments showed that the level of histone H2B monoubiquitination (H2Bub1) was noticeably reduced in the FRRP1-RNAi knockdown transgenic rice lines compared with wild-type plants and MBP-FRRP1-F1 was capable of self-ubiquitination. The results indicate that Flowering Related RING Protein 1 (FRRP1) is involved in histone H2B monoubiquitination and suggest that FRRP1 functions as an E3 ligase in vivo and in vitro. In conclusion, FRRP1 probably regulates flowering time and yield potential in rice by affecting histone H2B monoubiquitination, which leads to changes in gene expression in multiple processes.

Flowering-Related RING Protein 1 (FRRP1) Regulates Flowering Time and Yield Potential by Affecting Histone H2B Monoubiquitination in Rice (Oryza Sativa)

PubMed Central

Deng, Changwang; Chen, Xi; Gou, Lanming; Zhu, Fugui; Guo, Wei; Zhang, Jianfu; Wang, Tao

2016-01-01

Flowering time is a critical trait for crops cultivated under various temperature/photoperiod conditions around the world. To understand better the flowering time of rice, we used the vector pTCK303 to produce several lines of RNAi knockdown transgenic rice and investigated their flowering times and other agronomic traits. Among them, the heading date of FRRP1-RNAi knockdown transgenic rice was 23–26 days earlier than that of wild-type plants. FRRP1 is a novel rice gene that encodes a C3HC4-type Really Interesting Novel Gene (RING) finger domain protein. In addition to the early flowering time, FRRP1-RNAi knockdown transgenic rice caused changes on an array of agronomic traits, including plant height, panicle length and grain length. We analyzed the expression of some key genes associated with the flowering time and other agronomic traits in the FRRP1-RNAi knockdown lines and compared with that in wild-type lines. The expression of Hd3a increased significantly, which was the key factor in the early flowering time. Further experiments showed that the level of histone H2B monoubiquitination (H2Bub1) was noticeably reduced in the FRRP1-RNAi knockdown transgenic rice lines compared with wild-type plants and MBP-FRRP1-F1 was capable of self-ubiquitination. The results indicate that Flowering Related RING Protein 1 (FRRP1) is involved in histone H2B monoubiquitination and suggest that FRRP1 functions as an E3 ligase in vivo and in vitro. In conclusion, FRRP1 probably regulates flowering time and yield potential in rice by affecting histone H2B monoubiquitination, which leads to changes in gene expression in multiple processes. PMID:26934377

Novel R2R3-MYB transcription factors from Prunus americana regulate differential patterns of anthocyanin accumulation in tobacco and citrus

PubMed Central

Dasgupta, Kasturi; Stover, Ed; Oliveira, Maria Luiza; Thomson, James

2017-01-01

ABSTRACT The level of anthocyanins in plants vary widely among cultivars, developmental stages and environmental stimuli. Previous studies have reported that the expression of various MYBs regulate anthocyanin pigmentation during growth and development. Here we examine the activity of 3 novel R2R3-MYB transcription factor (TF) genes, PamMybA.1, PamMybA.3 and PamMybA.5 from Prunus americana. The anthocyanin accumulation patterns mediated by CaMV double35S promoter (db35Sp) controlled expression of the TFs in transgenic tobacco were compared with citrus-MoroMybA, Arabidopsis-AtMybA1 and grapevine-VvMybA1 transgenics during their entire growth cycles. The db35Sp-PamMybA.1 and db35Sp-PamMybA.5 constructs induced high levels of anthocyanin accumulation in both transformed tobacco calli and the regenerated plants. The red/purple color pigmentation induced in the PamMybA.1 and PamMybA.5 lines was not uniformly distributed, but appeared as patches in the leaves, whereas the flowers showed intense uniform pigmentation similar to the VvMybA1 expressing lines. MoroMybA and AtMybA1 showed more uniform pink coloration in both vegetative and reproductive tissues. Plant morphology, anthocyanin content, seed viability, and transgene inheritance were examined for the PamMybA.5 transgenic plants and compared with the controls. We conclude that these TFs alone are sufficient for activating anthocyanin production in plants and may be used as visible reporter genes for plant transformation. Evaluating these TFs in a heterologous crop species such as citrus further validated that these genes can be useful for the metabolic engineering of anthocyanin production and cultivar enhancement. PMID:28051907

Enrichment of provitamin A content in wheat (Triticum aestivum L.) by introduction of the bacterial carotenoid biosynthetic genes CrtB and CrtI.

PubMed

Wang, Cheng; Zeng, Jian; Li, Yin; Hu, Wei; Chen, Ling; Miao, Yingjie; Deng, Pengyi; Yuan, Cuihong; Ma, Cheng; Chen, Xi; Zang, Mingli; Wang, Qiong; Li, Kexiu; Chang, Junli; Wang, Yuesheng; Yang, Guangxiao; He, Guangyuan

2014-06-01

Carotenoid content is a primary determinant of wheat nutritional value and affects its end-use quality. Wheat grains contain very low carotenoid levels and trace amounts of provitamin A content. In order to enrich the carotenoid content in wheat grains, the bacterial phytoene synthase gene (CrtB) and carotene desaturase gene (CrtI) were transformed into the common wheat cultivar Bobwhite. Expression of CrtB or CrtI alone slightly increased the carotenoid content in the grains of transgenic wheat, while co-expression of both genes resulted in a darker red/yellow grain phenotype, accompanied by a total carotenoid content increase of approximately 8-fold achieving 4.76 μg g(-1) of seed dry weight, a β-carotene increase of 65-fold to 3.21 μg g(-1) of seed dry weight, and a provitamin A content (sum of α-carotene, β-carotene, and β-cryptoxanthin) increase of 76-fold to 3.82 μg g(-1) of seed dry weight. The high provitamin A content in the transgenic wheat was stably inherited over four generations. Quantitative PCR analysis revealed that enhancement of provitamin A content in transgenic wheat was also a result of the highly coordinated regulation of endogenous carotenoid biosynthetic genes, suggesting a metabolic feedback regulation in the wheat carotenoid biosynthetic pathway. These transgenic wheat lines are not only valuable for breeding wheat varieties with nutritional benefits for human health but also for understanding the mechanism regulating carotenoid biosynthesis in wheat endosperm. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

H+ -pyrophosphatase IbVP1 promotes efficient iron use in sweet potato [Ipomoea batatas (L.) Lam.].

PubMed

Fan, Weijuan; Wang, Hongxia; Wu, Yinliang; Yang, Nan; Yang, Jun; Zhang, Peng

2017-06-01

Iron (Fe) deficiency is one of the most common micronutrient deficiencies limiting crop production globally, especially in arid regions because of decreased availability of iron in alkaline soils. Sweet potato [Ipomoea batatas (L.) Lam.] grows well in arid regions and is tolerant to Fe deficiency. Here, we report that the transcription of type I H + -pyrophosphatase (H + -PPase) gene IbVP1 in sweet potato plants was strongly induced by Fe deficiency and auxin in hydroponics, improving Fe acquisition via increased rhizosphere acidification and auxin regulation. When overexpressed, transgenic plants show higher pyrophosphate hydrolysis and plasma membrane H + -ATPase activity compared with the wild type, leading to increased rhizosphere acidification. The IbVP1-overexpressing plants showed better growth, including enlarged root systems, under Fe-sufficient or Fe-deficient conditions. Increased ferric precipitation and ferric chelate reductase activity in the roots of transgenic lines indicate improved iron uptake, which is also confirmed by increased Fe content and up-regulation of Fe uptake genes, e.g. FRO2, IRT1 and FIT. Carbohydrate metabolism is significantly affected in the transgenic lines, showing increased sugar and starch content associated with the increased expression of AGPase and SUT1 genes and the decrease in β-amylase gene expression. Improved antioxidant capacities were also detected in the transgenic plants, which showed reduced H 2 O 2 accumulation associated with up-regulated ROS-scavenging activity. Therefore, H + -PPase plays a key role in the response to Fe deficiency by sweet potato and effectively improves the Fe acquisition by overexpressing IbVP1 in crops cultivated in micronutrient-deficient soils. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

A Cyclin Dependent Kinase Regulatory Subunit (CKS) Gene of Pigeonpea Imparts Abiotic Stress Tolerance and Regulates Plant Growth and Development in Arabidopsis

PubMed Central

Tamirisa, Srinath; Vudem, Dashavantha R.; Khareedu, Venkateswara R.

2017-01-01

Frequent climatic changes in conjunction with other extreme environmental factors are known to affect growth, development and productivity of diverse crop plants. Pigeonpea, a major grain legume of the semiarid tropics, endowed with an excellent deep-root system, is known as one of the important drought tolerant crop plants. Cyclin dependent kinases (CDKs) are core cell cycle regulators and play important role in different aspects of plant growth and development. The cyclin-dependent kinase regulatory subunit gene (CKS) was isolated from the cDNA library of pigeonpea plants subjected to drought stress. Pigeonpea CKS (CcCKS) gene expression was detected in both the root and leaf tissues of pigeonpea and was upregulated by polyethylene glycol (PEG), mannitol, NaCl and abscisic acid (ABA) treatments. The overexpression of CcCKS gene in Arabidopsis significantly enhanced tolerance of transgenics to drought and salt stresses as evidenced by different physiological parameters. Under stress conditions, transgenics showed higher biomass, decreased rate of water loss, decreased MDA levels, higher free proline contents, and glutathione levels. Moreover, under stress conditions transgenics exhibited lower stomatal conductance, lower transpiration, and higher photosynthetic rates. However, under normal conditions, CcCKS-transgenics displayed decreased plant growth rate, increased cell size and decreased stomatal number compared to those of wild-type plants. Real-time polymerase chain reaction revealed that CcCKS could regulate the expression of both ABA-dependent and ABA-independent genes associated with abiotic stress tolerance as well as plant growth and development. As such, the CcCKS seems promising and might serve as a potential candidate gene for enhancing the abiotic stress tolerance of crop plants. PMID:28239388

A Cyclin Dependent Kinase Regulatory Subunit (CKS) Gene of Pigeonpea Imparts Abiotic Stress Tolerance and Regulates Plant Growth and Development in Arabidopsis.

PubMed

Tamirisa, Srinath; Vudem, Dashavantha R; Khareedu, Venkateswara R

2017-01-01

Frequent climatic changes in conjunction with other extreme environmental factors are known to affect growth, development and productivity of diverse crop plants. Pigeonpea, a major grain legume of the semiarid tropics, endowed with an excellent deep-root system, is known as one of the important drought tolerant crop plants. Cyclin dependent kinases (CDKs) are core cell cycle regulators and play important role in different aspects of plant growth and development. The cyclin-dependent kinase regulatory subunit gene ( CKS ) was isolated from the cDNA library of pigeonpea plants subjected to drought stress. Pigeonpea CKS ( CcCKS ) gene expression was detected in both the root and leaf tissues of pigeonpea and was upregulated by polyethylene glycol (PEG), mannitol, NaCl and abscisic acid (ABA) treatments. The overexpression of CcCKS gene in Arabidopsis significantly enhanced tolerance of transgenics to drought and salt stresses as evidenced by different physiological parameters. Under stress conditions, transgenics showed higher biomass, decreased rate of water loss, decreased MDA levels, higher free proline contents, and glutathione levels. Moreover, under stress conditions transgenics exhibited lower stomatal conductance, lower transpiration, and higher photosynthetic rates. However, under normal conditions, CcCKS -transgenics displayed decreased plant growth rate, increased cell size and decreased stomatal number compared to those of wild-type plants. Real-time polymerase chain reaction revealed that Cc CKS could regulate the expression of both ABA-dependent and ABA-independent genes associated with abiotic stress tolerance as well as plant growth and development. As such, the CcCKS seems promising and might serve as a potential candidate gene for enhancing the abiotic stress tolerance of crop plants.

A complex interaction of imprinted and maternal-effect genes modifies sex determination in Odd Sex (Ods) mice.

PubMed

Poirier, Christophe; Qin, Yangjun; Adams, Carolyn P; Anaya, Yanett; Singer, Jonathan B; Hill, Annie E; Lander, Eric S; Nadeau, Joseph H; Bishop, Colin E

2004-11-01

The transgenic insertional mouse mutation Odd Sex (Ods) represents a model for the long-range regulation of Sox9. The mutation causes complete female-to-male sex reversal by inducing a male-specific expression pattern of Sox9 in XX Ods/+ embryonic gonads. We previously described an A/J strain-specific suppressor of Ods termed Odsm1(A). Here we show that phenotypic sex depends on a complex interaction between the suppressor and the transgene. Suppression can be achieved only if the transgene is transmitted paternally. In addition, the suppressor itself exhibits a maternal effect, suggesting that it may act on chromatin in the early embryo.

A Complex Interaction of Imprinted and Maternal-Effect Genes Modifies Sex Determination in Odd Sex (Ods) Mice

PubMed Central

Poirier, Christophe; Qin, Yangjun; Adams, Carolyn P.; Anaya, Yanett; Singer, Jonathan B.; Hill, Annie E.; Lander, Eric S.; Nadeau, Joseph H.; Bishop, Colin E.

2004-01-01

The transgenic insertional mouse mutation Odd Sex (Ods) represents a model for the long-range regulation of Sox9. The mutation causes complete female-to-male sex reversal by inducing a male-specific expression pattern of Sox9 in XX Ods/+ embryonic gonads. We previously described an A/J strain-specific suppressor of Ods termed Odsm1A. Here we show that phenotypic sex depends on a complex interaction between the suppressor and the transgene. Suppression can be achieved only if the transgene is transmitted paternally. In addition, the suppressor itself exhibits a maternal effect, suggesting that it may act on chromatin in the early embryo. PMID:15579706

Hibernoma formation in transgenic mice and isolation of a brown adipocyte cell line expressing the uncoupling protein gene.

PubMed Central

Ross, S R; Choy, L; Graves, R A; Fox, N; Solevjeva, V; Klaus, S; Ricquier, D; Spiegelman, B M

1992-01-01

Transgenic mice were produced containing the adipocyte-specific regulatory region from the adipocyte P2 (aP2) gene linked to the simian virus 40 transforming genes. Most of the transgenic mice developed brown fat tumors (hibernomas) in their interscapular brown adipose tissue. Hibernoma formation was noticeable in some of the mice as early as 1 day after birth and most of the mice developed very large tumors by 1 month of age. All of the tumor tissue expressed the brown fat-specific uncoupling protein (UCP) gene as well as the aP2 gene. Several of the tumors have been used to establish cultured cell lines and at least one of these lines can be induced to differentiate into brown adipocytes. The cultured adipocytes express mRNA for UCP upon stimulation with N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, norepinephrine, isoproterenol or D7114, a beta 3 adrenergic agonist. Thus, regulation of the key thermogenic gene UCP can now be studied in an established cell line. Images PMID:1323843

Tunable Protein Stabilization In Vivo Mediated by Shield-1 in Transgenic Medaka

PubMed Central

Froschauer, Alexander; Kube, Lisa; Kegler, Alexandra; Rieger, Christiane; Gutzeit, Herwig O.

2015-01-01

Techniques for conditional gene or protein expression are important tools in developmental biology and in the analysis of physiology and disease. On the protein level, the tunable and reversible expression of proteins can be achieved by the fusion of the protein of interest to a destabilizing domain (DD). In the absence of its specific ligand (Shield-1), the protein is degraded by the proteasome. The DD-Shield system has proven to be an excellent tool to regulate the expression of proteins of interests in mammalian systems but has not been applied in teleosts like the medaka. We present the application of the DD-Shield technique in transgenic medaka and show the ubiquitous conditional expression throughout life. Shield-1 administration to the water leads to concentration-dependent induction of a YFP reporter gene in various organs and in spermatogonia at the cellular level. PMID:26148066

Stress inducible overexpression of AtHDG11 leads to improved drought and salt stress tolerance in peanut (Arachis hypogaea L.)

NASA Astrophysics Data System (ADS)

Banavath, Jayanna N.; Chakradhar, Thammineni; Pandit, Varakumar; Konduru, Sravani; Guduru, Krishna K.; Akila, Chandra S.; Podha, Sudhakar; Puli, Chandra O. R.

2018-03-01

Peanut is an important oilseed and food legume cultivated as a rain-fed crop in semi-arid tropics. Drought and high salinity are the major abiotic stresses limiting the peanut productivity in this region. Development of drought and salt tolerant peanut varieties with improved yield potential using biotechnological approach is highly desirable to improve the peanut productivity in marginal geographies. As abiotic stress tolerance and yield represent complex traits, engineering of regulatory genes to produce abiotic stress-resilient transgenic crops appears to be a viable approach. In the present study, we developed transgenic peanut plants expressing an Arabidopsis homeodomain-leucine zipper transcription factor (AtHDG11) under stress inducible rd29Apromoter. A stress-inducible expression of AtHDG11 in three independent homozygous transgenic peanut lines resulted in improved drought and salt tolerance through up-regulation of known stress responsive genes(LEA, HSP70, Cu/Zn SOD, APX, P5CS, NCED1, RRS5, ERF1, NAC4, MIPS, Aquaporin, TIP, ELIP ) in the stress gene network , antioxidative enzymes, free proline along with improved water use efficiency traits such as longer root system, reduced stomatal density, higher chlorophyll content, increased specific leaf area, improved photosynthetic rates and increased intrinsic instantaneous WUE. Transgenic peanut plants displayed high yield compared to non-transgenic plants under both drought and salt stress conditions. Holistically, our study demonstrates the potentiality of stress-induced expression of AtHDG11 to improve the drought, salt tolerance in peanut.

A DNA segment spanning the mouse Tnfsf11 transcription unit and its upstream regulatory domain rescues the pleiotropic biologic phenotype of the RANKL null mouse.

PubMed

Onal, Melda; Bishop, Kathleen A; St John, Hillary C; Danielson, Allison L; Riley, Erin M; Piemontese, Marilina; Xiong, Jinhu; Goellner, Joseph J; O'Brien, Charles A; Pike, J Wesley

2015-05-01

Receptor activator of NF-κB ligand (RANKL) is a TNFα-like cytokine that is produced by a diverse set of lineage-specific cells and is involved in a wide variety of physiological processes that include skeletal remodeling, lymph node organogenesis, mammary gland development, and thermal regulation. Consistent with these diverse functions, control of RANKL expression is accomplished in a cell-specific fashion via a set of at least 10 regulatory enhancers that are located up to 170 kb upstream of the gene's transcriptional start site. Here we examined the in vivo consequence of introducing a contiguous DNA segment containing these components into a genetically deleted RANKL null mouse strain. In contrast to RANKL null littermates, null mice containing the transgene exhibited normalized body size, skeletal development, and bone mass as well as normal bone marrow cavities, normalized spleen weights, and the presence of developed lymph nodes. These mice also manifested normalized reproductive capacity, including the ability to lactate and to produce normal healthy litters. Consistent with this, the transgene restored endogenous-like RANKL transcript levels in several RANKL-expressing tissues. Most importantly, restoration of RANKL expression from this segment of DNA was fully capable of rescuing the complex aberrant skeletal and immune phenotype of the RANKL null mouse. RANKL also restored appropriate levels of B220+ IgM+ and B220+ IgD+ B cells in spleen. Finally, we found that RANKL expression from this transgene was regulated by exogenously administered 1,25(OH)2 D3 , parathyroid hormone (PTH), and lipopolysaccharide (LPS), thus recapitulating the ability of these same factors to regulate the endogenous gene. These findings fully highlight the properties of the Tnfsf11 gene locus predicted through previous in vitro dissection. We conclude that the mouse Tnfsf11 gene locus identified originally through unbiased chromatin immunoprecipitation with DNA microarray (ChIP-chip) analysis contains the necessary genetic information to direct appropriate tissue-specific and factor-regulated RANKL expression in vivo. © 2014 American Society for Bone and Mineral Research.

An auxin responsive CLE gene regulates shoot apical meristem development in Arabidopsis

PubMed Central

Guo, Hongyan; Zhang, Wei; Tian, Hainan; Zheng, Kaijie; Dai, Xuemei; Liu, Shanda; Hu, Qingnan; Wang, Xianling; Liu, Bao; Wang, Shucai

2015-01-01

Plant hormone auxin regulates most, if not all aspects of plant growth and development, including lateral root formation, organ pattering, apical dominance, and tropisms. Peptide hormones are peptides with hormone activities. Some of the functions of peptide hormones in regulating plant growth and development are similar to that of auxin, however, the relationship between auxin and peptide hormones remains largely unknown. Here we report the identification of OsCLE48, a rice (Oryza sativa) CLE (CLAVATA3/ENDOSPERM SURROUNDING REGION) gene, as an auxin response gene, and the functional characterization of OsCLE48 in Arabidopsis and rice. OsCLE48 encodes a CLE peptide hormone that is similar to Arabidopsis CLEs. RT-PCR analysis showed that OsCLE48 was induced by exogenously application of IAA (indole-3-acetic acid), a naturally occurred auxin. Expression of integrated OsCLE48p:GUS reporter gene in transgenic Arabidopsis plants was also induced by exogenously IAA treatment. These results indicate that OsCLE48 is an auxin responsive gene. Histochemical staining showed that GUS activity was detected in all the tissue and organs of the OsCLE48p:GUS transgenic Arabidopsis plants. Expression of OsCLE48 under the control of the 35S promoter in Arabidopsis inhibited shoot apical meristem development. Expression of OsCLE48 under the control of the CLV3 native regulatory elements almost completely complemented clv3-2 mutant phenotypes, suggesting that OsCLE48 is functionally similar to CLV3. On the other hand, expression of OsCLE48 under the control of the 35S promoter in Arabidopsis has little, if any effects on root apical meristem development, and transgenic rice plants overexpressing OsCLE48 are morphologically indistinguishable from wild type plants, suggesting that the functions of some CLE peptides may not be fully conserved in Arabidopsis and rice. Taken together, our results showed that OsCLE48 is an auxin responsive peptide hormone gene, and it regulates shoot apical meristem development when expressed in Arabidopsis. PMID:25983737

Silencing C19-GA 2-oxidases induces parthenocarpic development and inhibits lateral branching in tomato plants

PubMed Central

Martínez-Bello, Liliam; Moritz, Thomas; López-Díaz, Isabel

2015-01-01

Gibberellins (GAs) are phytohormones that regulate a wide range of developmental processes in plants. Levels of active GAs are regulated by biosynthetic and catabolic enzymes like the GA 2-oxidases (GA2oxs). In tomato (Solanum lycopersicum L.) C19 GA2oxs are encoded by a small multigenic family of five members with some degree of redundancy. In order to investigate their roles in tomato, the silencing of all five genes in transgenic plants was induced. A significant increase in active GA4 content was found in the ovaries of transgenic plants. In addition, the transgenic unfertilized ovaries were much bigger than wild-type ovaries (about 30 times) and a certain proportion (5–37%) were able to develop parthenocarpically. Among the GA2ox family, genes GA2ox1 and -2 seem to be the most relevant for this phenotype since their expression was induced in unfertilized ovaries and repressed in developing fruits, inversely correlating with ovary growth. Interestingly, transgenic lines exhibited a significant inhibition of branching and a higher content of active GA4 in axillary buds. This phenotype was reverted, in transgenic plants, by the application of paclobutrazol, a GA biosynthesis inhibitor, suggesting a role for GAs as repressors of branching. In summary, this work demonstrates that GA 2-oxidases regulate gibberellin levels in ovaries and axillary buds of tomato plants and their silencing is responsible for parthenocarpic fruit growth and branching inhibition. PMID:26093022

MicroR828 regulates lignin and H2O2 accumulation in sweet potato on wounding.

PubMed

Lin, Jeng-Shane; Lin, Chih-Ching; Lin, Hsin-Hung; Chen, Yu-Chi; Jeng, Shih-Tong

2012-10-01

MicroRNAs (miRNAs) are small noncoding RNAs which post-transcriptionally regulate gene expression by directing mRNA cleavage or translational inhibition. miRNAs play multiple roles in the growth, development and stress responses in plants. However, little is known of the wounding-responsive miRNAs and their regulation. Here, we investigated the expression patterns of microR828 (miR828) on wounding in sweet potato (Ipomoea batatas cv Tainung 57). The expression of miR828 was only detected in leaves, and was induced by wounding rather than by ethylene, hydrogen peroxide (H2O2), methyl jasmonate or nitric oxide (NO). Moreover, cyclic guanosine monophosphate (cGMP) was necessary for miR828 accumulation in leaves on wounding. Two miR828 target candidates, named IbMYB and IbTLD, were obtained by cDNA cloning, and their mRNA cleavage caused by miR828 was confirmed by cleavage site mapping, agro-infiltration and transgenics studies. The reduction in IbMYB and IbTLD expression coincided with the induction of miR828, demonstrating that IbMYB and IbTLD might be miR828 targets. Furthermore, transgenic sweet potato overexpressing miR828 precursor affected lignin and H2O2 contents. These results showed that cGMP could regulate wounding-responsive miR828, which repressed the expression of IbMYB and IbTLD. Subsequently, lignin and H2O2 were accumulated to participate in defense mechanisms. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

Generation of transgenic mice expressing EGFP protein fused to NP68 MHC class I epitope using lentivirus vectors.

PubMed

Tomkowiak, Martine; Ghittoni, Raffaella; Teixeira, Marie; Blanquier, Bariza; Szécsi, Judit; Nègre, Didier; Aubert, Denise; Coupet, Charles-Antoine; Brunner, Molly; Verhoeyen, Els; Thoumas, Jean-Louis; Cosset, François-Loïc; Leverrier, Yann; Marvel, Jacqueline

2013-03-01

Immune tolerance to self-antigens is a complex process that utilizes multiple mechanisms working in concert to maintain homeostasis and prevent autoimmunity. Considerable progress in deciphering the mechanisms controlling the activation or deletion of T cells has been made by using T cell receptor (TCR) transgenic mice. One such model is the F5 model in which CD8 T cells express a TCR specific for an epitope derived from the influenza NP68 protein. Our aim was to create transgenic mouse models expressing constitutively the NP68 epitope fused to enhanced green fluorescent protein (EGFP) in order to assess unambiguously the relative levels of NP68 epitope expressed by single cells. We used a lentiviral-based approach to generate two independent transgenic mouse strains expressing the fusion protein EGFP-NP68 under the control of CAG (CMV immediate early enhancer and the chicken β-actin promoter) or spleen focus-forming virus (SFFV) promoters. Analysis of the pattern of EGFP expression in the hematopoietic compartment showed that CAG and SFFV promoters are differentially regulated during T cell development. However, both promoters drove high EGFP-NP68 expression in dendritic cells (pDCs, CD8α(+) cDCs, and CD8α(-) cDCs) from spleen or generated in vitro following differentiation from bone-marrow progenitors. NP68 epitope was properly processed and successfully presented by dendritic cells (DCs) by direct presentation and cross-presentation to F5 CD8 T cells. The models presented here are valuable tools to investigate the priming of F5 CD8 T cells by different subsets of DCs. Copyright © 2013 Wiley Periodicals, Inc.

A putative positive feedback regulation mechanism in CsACS2 expression suggests a modified model for sex determination in cucumber (Cucumis sativus L.)

PubMed Central

Wang, Shu; Tao, Qianyi; Pan, Junsong; Si, Longting; Gong, Zhenhui; Cai, Run

2012-01-01

It is well established that the plant hormone ethylene plays a key role in cucumber sex determination. Since the unisexual control gene M was cloned and shown to encode an ethylene synthase, instead of an ethylene receptor, the ‘one-hormone hypothesis’, which was used to explain the cucumber sex phenotype, has been challenged. Here, the physiological function of CsACS2 (the gene encoded by the M locus) was studied using the transgenic tobacco system. The results indicated that overexpression of CsACS2 increased ethylene production in the tobacco plant, and the native cucumber promoter had no activity in transgenic tobacco (PM). However, when PM plants were treated with exogenous ethylene, CsACS2 expression could be detected. In cucumber, ethylene treatment could also induce transcription of CsACS2, while inhibition of ethylene action reduced the expression level. These findings suggest a positive feedback regulation mechanism for CsACS2, and a modified ‘one-hormone hypothesis’ for sex determination in cucumber is proposed. PMID:22577183

A putative positive feedback regulation mechanism in CsACS2 expression suggests a modified model for sex determination in cucumber (Cucumis sativus L.).

PubMed

Li, Zheng; Wang, Shu; Tao, Qianyi; Pan, Junsong; Si, Longting; Gong, Zhenhui; Cai, Run

2012-07-01

It is well established that the plant hormone ethylene plays a key role in cucumber sex determination. Since the unisexual control gene M was cloned and shown to encode an ethylene synthase, instead of an ethylene receptor, the 'one-hormone hypothesis', which was used to explain the cucumber sex phenotype, has been challenged. Here, the physiological function of CsACS2 (the gene encoded by the M locus) was studied using the transgenic tobacco system. The results indicated that overexpression of CsACS2 increased ethylene production in the tobacco plant, and the native cucumber promoter had no activity in transgenic tobacco (PM). However, when PM plants were treated with exogenous ethylene, CsACS2 expression could be detected. In cucumber, ethylene treatment could also induce transcription of CsACS2, while inhibition of ethylene action reduced the expression level. These findings suggest a positive feedback regulation mechanism for CsACS2, and a modified 'one-hormone hypothesis' for sex determination in cucumber is proposed.

Coordinate cytokine regulatory sequences

DOEpatents

Frazer, Kelly A.; Rubin, Edward M.; Loots, Gabriela G.

2005-05-10

The present invention provides CNS sequences that regulate the cytokine gene expression, expression cassettes and vectors comprising or lacking the CNS sequences, host cells and non-human transgenic animals comprising the CNS sequences or lacking the CNS sequences. The present invention also provides methods for identifying compounds that modulate the functions of CNS sequences as well as methods for diagnosing defects in the CNS sequences of patients.

Regulation of zebrafish heart regeneration by miR-133.

PubMed

Yin, Viravuth P; Lepilina, Alexandra; Smith, Ashley; Poss, Kenneth D

2012-05-15

Zebrafish regenerate cardiac muscle after severe injuries through the activation and proliferation of spared cardiomyocytes. Little is known about factors that control these events. Here we investigated the extent to which miRNAs regulate zebrafish heart regeneration. Microarray analysis identified many miRNAs with increased or reduced levels during regeneration. miR-133, a miRNA with known roles in cardiac development and disease, showed diminished expression during regeneration. Induced transgenic elevation of miR-133 levels after injury inhibited myocardial regeneration, while transgenic miR-133 depletion enhanced cardiomyocyte proliferation. Expression analyses indicated that cell cycle factors mps1, cdc37, and PA2G4, and cell junction components cx43 and cldn5, are miR-133 targets during regeneration. Using pharmacological inhibition and EGFP sensor interaction studies, we found that cx43 is a new miR-133 target and regeneration gene. Our results reveal dynamic regulation of miRNAs during heart regeneration, and indicate that miR-133 restricts injury-induced cardiomyocyte proliferation. Copyright © 2012. Published by Elsevier Inc.

Antisense down-regulation of 4CL expression alters lignification, tree growth, and saccharification potential of field-grown poplar

Treesearch

Steven L. Voelker; Barbara Lachenbruch; Frederick C. Meinzer; Michael Jourdes; Chanyoung Ki; Ann M. Patten; Laurence B. Davin; Norman G. Lewis; Gerald A. Tuskan; Lee Gunter; Stephen R. Decker; Michael J. Selig; Robert Sykes; Michael E. Himmel; Peter Kitin; Olga Shevchenko; Steven H. Strauss

2010-01-01

Transgenic down-regulation of the Pt4CL1 gene family encoding 4-coumarate:coenzyme A ligase (4CL) has been reported as a means for reducing lignin content in cell walls and increasing overall growth rates, thereby improving feedstock quality for paper and bioethanol production. Using hybrid poplar (Populus tremula...

GmWRKY31 and GmHDL56 Enhances Resistance to Phytophthora sojae by Regulating Defense-Related Gene Expression in Soybean.

PubMed

Fan, Sujie; Dong, Lidong; Han, Dan; Zhang, Feng; Wu, Junjiang; Jiang, Liangyu; Cheng, Qun; Li, Rongpeng; Lu, Wencheng; Meng, Fanshan; Zhang, Shuzhen; Xu, Pengfei

2017-01-01

Phytophthora root and stem rot of soybean [ Glycine max (L.) Merr.] caused by the oomycete Phytophthora sojae , is a destructive disease worldwide. The molecular mechanism of the soybean response to P. sojae is largely unclear. We report a novel WRKY transcription factor (TF) in soybean, GmWRKY31, in the host response to P. sojae . Overexpression and RNA interference analysis demonstrated that GmWRKY31 enhanced resistance to P. sojae in transgenic soybean plants. GmWRKY31 was targeted to the nucleus, where it bound to the W-box and acted as an activator of gene transcription. Moreover, we determined that GmWRKY31 physically interacted with GmHDL56, which improved resistance to P. sojae in transgenic soybean roots. GmWRKY31 and GmHDL56 shared a common target GmNPR1 which was induced by P. sojae . Overexpression and RNA interference analysis demonstrated that GmNPR1 enhanced resistance to P. sojae in transgenic soybean plants. Several pathogenesis-related ( PR ) genes were constitutively activated, including GmPR1a , GmPR2 , GmPR3 , GmPR4 , GmPR5a , and GmPR10 , in soybean plants overexpressing GmNPR1 transcripts. By contrast, the induction of PR genes was compromised in transgenic GmNPR1 -RNAi lines. Taken together, these findings suggested that the interaction between GmWRKY31 and GmHDL56 enhances resistance to P. sojae by regulating defense-related gene expression in soybean.

Enhanced disease resistance and drought tolerance in transgenic rice plants overexpressing protein elicitors from Magnaporthe oryzae.

PubMed

Wang, Zhenzhen; Han, Qiang; Zi, Qian; Lv, Shun; Qiu, Dewen; Zeng, Hongmei

2017-01-01

Exogenous application of the protein elicitors MoHrip1 and MoHrip2, which were isolated from the pathogenic fungus Magnaporthe oryzae (M. oryzae), was previously shown to induce a hypersensitive response in tobacco and to enhance resistance to rice blast. In this work, we successfully transformed rice with the mohrip1 and mohrip2 genes separately. The MoHrip1 and MoHrip2 transgenic rice plants displayed higher resistance to rice blast and stronger tolerance to drought stress than wild-type (WT) rice and the vector-control pCXUN rice. The expression of salicylic acid (SA)- and abscisic acid (ABA)-related genes was also increased, suggesting that these two elicitors may trigger SA signaling to protect the rice from damage during pathogen infection and regulate the ABA content to increase drought tolerance in transgenic rice. Trypan blue staining indicated that expressing MoHrip1 and MoHrip2 in rice plants inhibited hyphal growth of the rice blast fungus. Relative water content (RWC), water usage efficiency (WUE) and water loss rate (WLR) were measured to confirm the high capacity for water retention in transgenic rice. The MoHrip1 and MoHrip2 transgenic rice also exhibited enhanced agronomic traits such as increased plant height and tiller number.

Transgenic plants with increased calcium stores

NASA Technical Reports Server (NTRS)

Robertson, Dominique (Inventor); Wyatt, Sarah (Inventor); Tsou, Pei-Lan (Inventor); Boss, Wendy (Inventor)

2004-01-01

The present invention provides transgenic plants over-expressing a transgene encoding a calcium-binding protein or peptide (CaBP). Preferably, the CaBP is a calcium storage protein and over-expression thereof does not have undue adverse effects on calcium homeostasis or biochemical pathways that are regulated by calcium. In preferred embodiments, the CaBP is calreticulin (CRT) or calsequestrin. In more preferred embodiments, the CaBP is the C-domain of CRT, a fragment of the C-domain, or multimers of the foregoing. In other preferred embodiments, the CaBP is localized to the endoplasmic reticulum by operatively associating the transgene encoding the CaBP with an endoplasmic reticulum localization peptide. Alternatively, the CaBP is targeted to any other sub-cellular compartment that permits the calcium to be stored in a form that is biologically available to the plant. Also provided are methods of producing plants with desirable phenotypic traits by transformation of the plant with a transgene encoding a CaBP. Such phenotypic traits include increased calcium storage, enhanced resistance to calcium-limiting conditions, enhanced growth and viability, increased disease and stress resistance, enhanced flower and fruit production, reduced senescence, and a decreased need for fertilizer production. Further provided are plants with enhanced nutritional value as human food or animal feed.

Identification of a Retroelement from the Resurrection Plant Boea hygrometrica That Confers Osmotic and Alkaline Tolerance in Arabidopsis thaliana

PubMed Central

Shen, Chun-Ying; Xu, Guang-Hui; Chen, Shi-Xuan; Song, Li-Zhen; Li, Mei-Jing; Wang, Li-Li; Zhu, Yan; Lv, Wei-Tao; Gong, Zhi-Zhong; Liu, Chun-Ming; Deng, Xin

2014-01-01

Functional genomic elements, including transposable elements, small RNAs and non-coding RNAs, are involved in regulation of gene expression in response to plant stress. To identify genomic elements that regulate dehydration and alkaline tolerance in Boea hygrometrica, a resurrection plant that inhabits drought and alkaline Karst areas, a genomic DNA library from B. hygrometrica was constructed and subsequently transformed into Arabidopsis using binary bacterial artificial chromosome (BIBAC) vectors. Transgenic lines were screened under osmotic and alkaline conditions, leading to the identification of Clone L1-4 that conferred osmotic and alkaline tolerance. Sequence analyses revealed that L1-4 contained a 49-kb retroelement fragment from B. hygrometrica, of which only a truncated sequence was present in L1-4 transgenic Arabidopsis plants. Additional subcloning revealed that activity resided in a 2-kb sequence, designated Osmotic and Alkaline Resistance 1 (OAR1). In addition, transgenic Arabidopsis lines carrying an OAR1-homologue also showed similar stress tolerance phenotypes. Physiological and molecular analyses demonstrated that OAR1-transgenic plants exhibited improved photochemical efficiency and membrane integrity and biomarker gene expression under both osmotic and alkaline stresses. Short transcripts that originated from OAR1 were increased under stress conditions in both B. hygrometrica and Arabidopsis carrying OAR1. The relative copy number of OAR1 was stable in transgenic Arabidopsis under stress but increased in B. hygrometrica. Taken together, our results indicated a potential role of OAR1 element in plant tolerance to osmotic and alkaline stresses, and verified the feasibility of the BIBAC transformation technique to identify functional genomic elements from physiological model species. PMID:24851859

Competitive Expression of Endogenous Wheat CENH3 May Lead to Suppression of Alien ZmCENH3 in Transgenic Wheat × Maize Hybrids.

PubMed

Chen, Wei; Zhu, Qilin; Wang, Haiyan; Xiao, Jin; Xing, Liping; Chen, Peidu; Jin, Weiwei; Wang, Xiu-E

2015-11-20

Uniparental chromosome elimination in wheat × maize hybrid embryos is widely used in double haploid production of wheat. Several explanations have been proposed for this phenomenon, one of which is that the lack of cross-species CENH3 incorporation may act as a barrier to interspecies hybridization. However, it is unknown if this mechanism applies universally. To study the role of CENH3 in maize chromosome elimination of wheat × maize hybrid embryos, maize ZmCENH3 and wheat αTaCENH3-B driven by the constitutive CaMV35S promoter were transformed into wheat variety Yangmai 158. Five transgenic lines for ZmCENH3 and six transgenic lines for αTaCENH3-B were identified. RT-PCR analysis showed that the transgene could be transcribed at a low level in all ZmCENH3 transgenic lines, whereas transcription of endogenous wheat CENH3 was significantly up-regulated. Interestingly, the expression levels of both wheat CENH3 and ZmCENH3 in the ZmCENH3 transgenic wheat × maize hybrid embryos were higher than those in the non-transformed Yangmai 158 × maize hybrid embryos. This indicates that the alien ZmCENH3 in wheat may induce competitive expression of endogenous wheat CENH3, leading to suppression of ZmCENH3 over-expression. Eliminations of maize chromosomes in hybrid embryos of ZmCENH3 transgenic wheat × maize and Yangmai 158 × maize were compared by observations on micronuclei presence, by marker analysis using maize SSRs (simple sequence repeats), and by FISH (fluorescence in situ hybridization) using 45S rDNA as a probe. The results indicate that maize chromosome elimination events in the two crosses are not significantly different. Fusion protein ZmCENH3-YFP could not be detected in ZmCENH3 transgenic wheat by either Western blotting or immnunostaining, whereas accumulation and loading of the αTaCENH3-B-GFP fusion protein was normal in αTaCENH3-B transgenic lines. As ZmCENH3-YFP did not accumulate after AM114 treatment, we speculate that low levels of ZmCENH3 protein in transgenic wheat may be one of the factors that lead to failure of suppression of maize chromatin elimination in ZmCENH3 transgenic wheat × maize hybrids. Copyright © 2015 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Effects of sulfate concentrations on the expression of a soybean seed storage protein gene and its reversibility in transgenic Arabidopsis thaliana.

PubMed

Hirai, M Y; Fujiwara, T; Chino, M; Naito, S

1995-10-01

Transgenic expression of genes encoding the alpha' and beta subunits of beta-conglycinin, one of the major seed storage proteins of soybean (Glycine max [L.] Merr.), was analyzed in Arabidopsis thaliana (L.) Heynh. under conditions of sulfate deficiency. Temporal patterns of expression of both the intact beta subunit gene and the beta subunit gene promoter fused to the beta-glucuronidase (GUS) gene are similar in soil-less cultures using rockwool, suggesting that the response to sulfate deficiency is regulated mainly at the level of transcription. In hydroponic cultures with various concentrations of sulfate, expression of both the intact beta subunit gene and the beta subunit gene promoter-GUS fusion gene were negatively correlated to increased sulfate concentrations in the culture medium. Transfer of transgenic A. thaliana plants carrying the beta subunit gene promoter-GUS fusion from sulfate-deficient to sulfate-sufficient control medium caused GUS activity in developing siliques to be repressed within two days. A reverse shift, where the plants were transferred from the control to sulfate-deficient medium, caused GUS activity to become higher than that in seeds of the control plants within two days. These results indicate that the expression of the beta subunit gene promoter responds rapidly to changes of sulfate availability.

Pineal Photoreceptor Cells Are Required for Maintaining the Circadian Rhythms of Behavioral Visual Sensitivity in Zebrafish

PubMed Central

Li, Xinle; Montgomery, Jake; Cheng, Wesley; Noh, Jung Hyun; Hyde, David R.; Li, Lei

2012-01-01

In non-mammalian vertebrates, the pineal gland functions as the central pacemaker that regulates the circadian rhythms of animal behavior and physiology. We generated a transgenic zebrafish line [Tg(Gnat2:gal4-VP16/UAS:nfsB-mCherry)] in which the E. coli nitroreductase is expressed in pineal photoreceptor cells. In developing embryos and young adults, the transgene is expressed in both retinal and pineal photoreceptor cells. During aging, the expression of the transgene in retinal photoreceptor cells gradually diminishes. By 8 months of age, the Gnat2 promoter-driven nitroreductase is no longer expressed in retinal photoreceptor cells, but its expression in pineal photoreceptor cells persists. This provides a tool for selective ablation of pineal photoreceptor cells, i.e., by treatments with metronidazole. In the absence of pineal photoreceptor cells, the behavioral visual sensitivity of the fish remains unchanged; however, the circadian rhythms of rod and cone sensitivity are diminished. Brief light exposures restore the circadian rhythms of behavioral visual sensitivity. Together, the data suggest that retinal photoreceptor cells respond to environmental cues and are capable of entraining the circadian rhythms of visual sensitivity; however, they are insufficient for maintaining the rhythms. Cellular signals from the pineal photoreceptor cells may be required for maintaining the circadian rhythms of visual sensitivity. PMID:22815753

Hoxa5 overexpression correlates with IGFBP1 upregulation and postnatal dwarfism: evidence for an interaction between Hoxa5 and Forkhead box transcription factors.

PubMed

Foucher, Isabelle; Volovitch, Michel; Frain, Monique; Kim, J Julie; Souberbielle, Jean-Claude; Gan, Lixia; Unterman, Terry G; Prochiantz, Alain; Trembleau, Alain

2002-09-01

Transgenic mice expressing the homeobox gene Hoxa5 under the control of Hoxb2 regulatory elements present a growth arrest during weeks two and three of postnatal development, resulting in proportionate dwarfism. These mice present a liver phenotype illustrated by a 12-fold increase in liver insulin-like growth factor binding protein 1 (IGFBP1) mRNA and a 50% decrease in liver insulin-like growth factor 1 (IGF1) mRNA correlated with a 50% decrease in circulating IGF1. We show that the Hoxa5 transgene is expressed in the liver of these mice, leading to an overexpression of total (endogenous plus transgene) Hoxa5 mRNA in this tissue. We have used several cell lines to investigate a possible physiological interaction of Hoxa5 with the main regulator of IGFBP1 promoter activity, the Forkhead box transcription factor FKHR. In HepG2 cells, Hoxa5 has little effect by itself but inhibits the FKHR-dependent activation of the IGFBP1 promoter. In HuF cells, Hoxa5 cooperates with FKHR to dramatically enhance IGFBP1 promoter activity. This context-dependent physiological interaction probably corresponds to the existence of a direct interaction between Hoxa5 and FKHR and FoxA2/HNF3beta, as demonstrated by pull-down experiments achieved either in vitro or after cellular co-expression. In conclusion, we propose that the impaired growth observed in this transgenic line relates to a liver phenotype best explained by a direct interaction between Hoxa5 and liver-specific Forkhead box transcription factors, in particular FKHR but also Foxa2/HNF3beta. Because Hoxa5 and homeogenes of the same paralog group are normally expressed in the liver, the present results raise the possibility that homeoproteins, in addition to their established role during early development, regulate systemic physiological functions.

Altering Trehalose-6-Phosphate Content in Transgenic Potato Tubers Affects Tuber Growth and Alters Responsiveness to Hormones during Sprouting1[C][W

PubMed Central

Debast, Stefan; Nunes-Nesi, Adriano; Hajirezaei, Mohammad R.; Hofmann, Jörg; Sonnewald, Uwe; Fernie, Alisdair R.; Börnke, Frederik

2011-01-01

Trehalose-6-phosphate (T6P) is a signaling metabolite that regulates carbon metabolism, developmental processes, and growth in plants. In Arabidopsis (Arabidopsis thaliana), T6P signaling is, at least in part, mediated through inhibition of the SNF1-related protein kinase SnRK1. To investigate the role of T6P signaling in a heterotrophic, starch-accumulating storage organ, transgenic potato (Solanum tuberosum) plants with altered T6P levels specifically in their tubers were generated. Transgenic lines with elevated T6P levels (B33-TPS, expressing Escherichia coli osmoregulatory trehalose synthesis A [OtsA], which encodes a T6P synthase) displayed reduced starch content, decreased ATP contents, and increased respiration rate diagnostic for high metabolic activity. On the other hand, lines with significantly reduced T6P (B33-TPP, expressing E. coli OtsB, which encodes a T6P phosphatase) showed accumulation of soluble carbohydrates, hexose phosphates, and ATP, no change in starch when calculated on a fresh weight basis, and a strongly reduced tuber yield. [14C]Glucose feeding to transgenic tubers indicated that carbon partitioning between starch and soluble carbohydrates was not altered. Transcriptional profiling of B33-TPP tubers revealed that target genes of SnRK1 were strongly up-regulated and that T6P inhibited potato tuber SnRK1 activity in vitro. Among the SnRK1 target genes in B33-TPP tubers, those involved in the promotion of cell proliferation and growth were down-regulated, while an inhibitor of cell cycle progression was up-regulated. T6P-accumulating tubers were strongly delayed in sprouting, while those with reduced T6P sprouted earlier than the wild type. Early sprouting of B33-TPP tubers correlated with a reduced abscisic acid content. Collectively, our data indicate that T6P plays an important role for potato tuber growth. PMID:21670224

Pathogen Phytosensing: Plants to Report Plant Pathogens

PubMed Central

Mazarei, Mitra; Teplova, Irina; Hajimorad, M. Reza; Stewart, C. Neal

2008-01-01

Real-time systems that provide evidence of pathogen contamination in crops can be an important new line of early defense in agricultural centers. Plants possess defense mechanisms to protect against pathogen attack. Inducible plant defense is controlled by signal transduction pathways, inducible promoters and cis-regulatory elements corresponding to key genes involved in defense, and pathogen-specific responses. Identified inducible promoters and cis-acting elements could be utilized in plant sentinels, or ‘phytosensors’, by fusing these to reporter genes to produce plants with altered phenotypes in response to the presence of pathogens. Here, we have employed cis-acting elements from promoter regions of pathogen inducible genes as well as those responsive to the plant defense signal molecules salicylic acid, jasmonic acid, and ethylene. Synthetic promoters were constructed by combining various regulatory elements supplemented with the enhancer elements from the Cauliflower mosaic virus (CaMV) 35S promoter to increase basal level of the GUS expression. The inducibility of each synthetic promoter was first assessed in transient expression assays using Arabidopsis thaliana protoplasts and then examined for efficacy in stably transgenic Arabidopsis and tobacco plants. Histochemical and fluorometric GUS expression analyses showed that both transgenic Arabidopsis and tobacco plants responded to elicitor and phytohormone treatments with increased GUS expression when compared to untreated plants. Pathogen-inducible phytosensor studies were initiated by analyzing the sensitivity of the synthetic promoters against virus infection. Transgenic tobacco plants infected with Alfalfa mosaic virus showed an increase in GUS expression when compared to mock-inoculated control plants, whereas Tobacco mosaic virus infection caused no changes in GUS expression. Further research, using these transgenic plants against a range of different pathogens with the regulation of detectable reporter gene could provide biological evidence to define the functional differences between pathogens, and provide new technology and applications for transgenic plants as phytosensors. PMID:27879840

Expression of the Arabidopsis thaliana BBX32 gene in soybean increases grain yield.

PubMed

Preuss, Sasha B; Meister, Robert; Xu, Qingzhang; Urwin, Carl P; Tripodi, Federico A; Screen, Steven E; Anil, Veena S; Zhu, Shuquan; Morrell, James A; Liu, Grace; Ratcliffe, Oliver J; Reuber, T Lynne; Khanna, Rajnish; Goldman, Barry S; Bell, Erin; Ziegler, Todd E; McClerren, Amanda L; Ruff, Thomas G; Petracek, Marie E

2012-01-01

Crop yield is a highly complex quantitative trait. Historically, successful breeding for improved grain yield has led to crop plants with improved source capacity, altered plant architecture, and increased resistance to abiotic and biotic stresses. To date, transgenic approaches towards improving crop grain yield have primarily focused on protecting plants from herbicide, insects, or disease. In contrast, we have focused on identifying genes that, when expressed in soybean, improve the intrinsic ability of the plant to yield more. Through the large scale screening of candidate genes in transgenic soybean, we identified an Arabidopsis thaliana B-box domain gene (AtBBX32) that significantly increases soybean grain yield year after year in multiple transgenic events in multi-location field trials. In order to understand the underlying physiological changes that are associated with increased yield in transgenic soybean, we examined phenotypic differences in two AtBBX32-expressing lines and found increases in plant height and node, flower, pod, and seed number. We propose that these phenotypic changes are likely the result of changes in the timing of reproductive development in transgenic soybean that lead to the increased duration of the pod and seed development period. Consistent with the role of BBX32 in A. thaliana in regulating light signaling, we show that the constitutive expression of AtBBX32 in soybean alters the abundance of a subset of gene transcripts in the early morning hours. In particular, AtBBX32 alters transcript levels of the soybean clock genes GmTOC1 and LHY-CCA1-like2 (GmLCL2). We propose that through the expression of AtBBX32 and modulation of the abundance of circadian clock genes during the transition from dark to light, the timing of critical phases of reproductive development are altered. These findings demonstrate a specific role for AtBBX32 in modulating soybean development, and demonstrate the validity of expressing single genes in crops to deliver increased agricultural productivity.

The grapevine root-specific aquaporin VvPIP2;4N controls root hydraulic conductance and leaf gas exchange under well-watered conditions but not under water stress.

PubMed

Perrone, Irene; Gambino, Giorgio; Chitarra, Walter; Vitali, Marco; Pagliarani, Chiara; Riccomagno, Nadia; Balestrini, Raffaella; Kaldenhoff, Ralf; Uehlein, Norbert; Gribaudo, Ivana; Schubert, Andrea; Lovisolo, Claudio

2012-10-01

We functionally characterized the grape (Vitis vinifera) VvPIP2;4N (for Plasma membrane Intrinsic Protein) aquaporin gene. Expression of VvPIP2;4N in Xenopus laevis oocytes increased their swelling rate 54-fold. Northern blot and quantitative reverse transcription-polymerase chain reaction analyses showed that VvPIP2;4N is the most expressed PIP2 gene in root. In situ hybridization confirmed root localization in the cortical parenchyma and close to the endodermis. We then constitutively overexpressed VvPIP2;4N in grape 'Brachetto', and in the resulting transgenic plants we analyzed (1) the expression of endogenous and transgenic VvPIP2;4N and of four other aquaporins, (2) whole-plant, root, and leaf ecophysiological parameters, and (3) leaf abscisic acid content. Expression of transgenic VvPIP2;4N inhibited neither the expression of the endogenous gene nor that of other PIP aquaporins in both root and leaf. Under well-watered conditions, transgenic plants showed higher stomatal conductance, gas exchange, and shoot growth. The expression level of VvPIP2;4N (endogenous + transgene) was inversely correlated to root hydraulic resistance. The leaf component of total plant hydraulic resistance was low and unaffected by overexpression of VvPIP2;4N. Upon water stress, the overexpression of VvPIP2;4N induced a surge in leaf abscisic acid content and a decrease in stomatal conductance and leaf gas exchange. Our results show that aquaporin-mediated modifications of root hydraulics play a substantial role in the regulation of water flow in well-watered grapevine plants, while they have a minor role upon drought, probably because other signals, such as abscisic acid, take over the control of water flow.

Proteomic identification of OsCYP2, a rice cyclophilin that confers salt tolerance in rice (Oryza sativa L.) seedlings when overexpressed

PubMed Central

2011-01-01

Background High Salinity is a major environmental stress influencing growth and development of rice. Comparative proteomic analysis of hybrid rice shoot proteins from Shanyou 10 seedlings, a salt-tolerant hybrid variety, and Liangyoupeijiu seedlings, a salt-sensitive hybrid variety, was performed to identify new components involved in salt-stress signaling. Results Phenotypic analysis of one protein that was upregulated during salt-induced stress, cyclophilin 2 (OsCYP2), indicated that OsCYP2 transgenic rice seedlings had better tolerance to salt stress than did wild-type seedlings. Interestingly, wild-type seedlings exhibited a marked reduction in maximal photochemical efficiency under salt stress, whereas no such change was observed for OsCYP2-transgenic seedlings. OsCYP2-transgenic seedlings had lower levels of lipid peroxidation products and higher activities of antioxidant enzymes than wild-type seedlings. Spatiotemporal expression analysis of OsCYP2 showed that it could be induced by salt stress in both Shanyou 10 and Liangyoupeijiu seedlings, but Shanyou 10 seedlings showed higher OsCYP2 expression levels. Moreover, circadian rhythm expression of OsCYP2 in Shanyou 10 seedlings occurred earlier than in Liangyoupeijiu seedlings. Treatment with PEG, heat, or ABA induced OsCYP2 expression in Shanyou 10 seedlings but inhibited its expression in Liangyoupeijiu seedlings. Cold stress inhibited OsCYP2 expression in Shanyou 10 and Liangyoupeijiu seedlings. In addition, OsCYP2 was strongly expressed in shoots but rarely in roots in two rice hybrid varieties. Conclusions Together, these data suggest that OsCYP2 may act as a key regulator that controls ROS level by modulating activities of antioxidant enzymes at translation level. OsCYP2 expression is not only induced by salt stress, but also regulated by circadian rhythm. Moreover, OsCYP2 is also likely to act as a key component that is involved in signal pathways of other types of stresses-PEG, heat, cold, or ABA. PMID:21324151

Increasing Sucrose Uptake Capacity of Wheat Grains Stimulates Storage Protein Synthesis1[W

PubMed Central

Weichert, Nicola; Saalbach, Isolde; Weichert, Heiko; Kohl, Stefan; Erban, Alexander; Kopka, Joachim; Hause, Bettina; Varshney, Alok; Sreenivasulu, Nese; Strickert, Marc; Kumlehn, Jochen; Weschke, Winfriede; Weber, Hans

2010-01-01

Increasing grain sink strength by improving assimilate uptake capacity could be a promising approach toward getting higher yield. The barley (Hordeum vulgare) sucrose transporter HvSUT1 (SUT) was expressed under control of the endosperm-specific Hordein B1 promoter (HO). Compared with the wild type, transgenic HOSUT grains take up more sucrose (Suc) in vitro, showing that the transgene is functional. Grain Suc levels are not altered, indicating that Suc fluxes are influenced rather than steady-state levels. HOSUT grains have increased percentages of total nitrogen and prolamins, which is reflected in increased levels of phenylalanine, tyrosine, tryptophan, isoleucine, and leucine at late grain development. Transcript profiling indicates specific stimulation of prolamin gene expression at the onset of storage phase. Changes in gene expression and metabolite levels related to carbon metabolism and amino acid biosynthesis suggest deregulated carbon-nitrogen balance, which together indicate carbon sufficiency and relative depletion of nitrogen. Genes, deregulated together with prolamin genes, might represent candidates, which respond positively to assimilate supply and are related to sugar-starch metabolism, cytokinin and brassinosteroid functions, cell proliferation, and sugar/abscisic acid signaling. Genes showing inverse expression patterns represent potential negative regulators. It is concluded that HvSUT1 overexpression increases grain protein content but also deregulates the metabolic status of wheat (Triticum aestivum) grains, accompanied by up-regulated gene expression of positive and negative regulators related to sugar signaling and assimilate supply. In HOSUT grains, alternating stimulation of positive and negative regulators causes oscillatory patterns of gene expression and highlights the capacity and great flexibility to adjust wheat grain storage metabolism in response to metabolic alterations. PMID:20018590

Alterations of Clock Gene RNA Expression in Brain Regions of a Triple Transgenic Model of Alzheimer’s Disease

PubMed Central

Bellanti, Francesco; Iannelli, Giuseppina; Blonda, Maria; Tamborra, Rosanna; Villani, Rosanna; Romano, Adele; Calcagnini, Silvio; Mazzoccoli, Gianluigi; Vinciguerra, Manlio; Gaetani, Silvana; Giudetti, Anna Maria; Vendemiale, Gianluigi; Cassano, Tommaso; Serviddio, Gaetano

2017-01-01

A disruption to circadian rhythmicity and the sleep/wake cycle constitutes a major feature of Alzheimer’s disease (AD). The maintenance of circadian rhythmicity is regulated by endogenous clock genes and a number of external Zeitgebers, including light. This study investigated the light induced changes in the expression of clock genes in a triple transgenic model of AD (3×Tg-AD) and their wild type littermates (Non-Tg). Changes in gene expression were evaluated in four brain areas¾suprachiasmatic nucleus (SCN), hippocampus, frontal cortex and brainstem¾of 6- and 18-month-old Non-Tg and 3×Tg-AD mice after 12 h exposure to light or darkness. Light exposure exerted significant effects on clock gene expression in the SCN, the site of the major circadian pacemaker. These patterns of expression were disrupted in 3×Tg-AD and in 18-month-old compared with 6-month-old Non-Tg mice. In other brain areas, age rather than genotype affected gene expression; the effect of genotype was observed on hippocampal Sirt1 expression, while it modified the expression of genes regulating the negative feedback loop as well as Rorα, Csnk1ɛ and Sirt1 in the brainstem. In conclusion, during the early development of AD, there is a disruption to the normal expression of genes regulating circadian function after exposure to light, particularly in the SCN but also in extra-hypothalamic brain areas supporting circadian regulation, suggesting a severe impairment of functioning of the clock gene pathway. Even though this study did not demonstrate a direct association between these alterations in clock gene expression among brain areas with the cognitive impairments and chrono-disruption that characterize the early onset of AD, our novel results encourage further investigation aimed at testing this hypothesis. PMID:28671110

Expression Analysis of CB2-GFP BAC Transgenic Mice.

PubMed

Schmöle, Anne-Caroline; Lundt, Ramona; Gennequin, Benjamin; Schrage, Hanna; Beins, Eva; Krämer, Alexandra; Zimmer, Till; Limmer, Andreas; Zimmer, Andreas; Otte, David-Marian

2015-01-01

The endocannabinoid system (ECS) is a retrograde messenger system, consisting of lipid signaling molecules that bind to at least two G-protein-coupled receptors, Cannabinoid receptor 1 and 2 (CB1 and 2). As CB2 is primarily expressed on immune cells such as B cells, T cells, macrophages, dendritic cells, and microglia, it is of great interest how CB2 contributes to immune cell development and function in health and disease. Here, understanding the mechanisms of CB2 involvement in immune-cell function as well as the trafficking and regulation of CB2 expressing cells are crucial issues. Up to now, CB2 antibodies produce unclear results, especially those targeting the murine protein. Therefore, we have generated BAC transgenic GFP reporter mice (CB2-GFPTg) to trace CB2 expression in vitro and in situ. Those mice express GFP under the CB2 promoter and display GFP expression paralleling CB2 expression on the transcript level in spleen, thymus and brain tissue. Furthermore, by using fluorescence techniques we show that the major sources for GFP-CB2 expression are B cells in spleen and blood and microglia in the brain. This novel CB2-GFP transgenic reporter mouse line represents a powerful resource to study CB2 expression in different cell types. Furthermore, it could be used for analyzing CB2-mediated mobilization and trafficking of immune cells as well as studying the fate of recruited immune cells in models of acute and chronic inflammation.

GNC and CGA1 Modulate Chlorophyll Biosynthesis and Glutamate Synthase (GLU1/Fd-GOGAT) Expression in Arabidopsis

PubMed Central

Hudson, Darryl; Guevara, David; Yaish, Mahmoud W.; Hannam, Carol; Long, Nykoll; Clarke, Joseph D.; Bi, Yong-Mei; Rothstein, Steven J.

2011-01-01

Chloroplast development is an important determinant of plant productivity and is controlled by environmental factors including amounts of light and nitrogen as well as internal phytohormones including cytokinins and gibberellins (GA). The paralog GATA transcription factors GNC and CGA1/GNL up-regulated by light, nitrogen and cytokinin while also being repressed by GA signaling. Modifying the expression of these genes has previously been shown to influence chlorophyll content in Arabidopsis while also altering aspects of germination, elongation growth and flowering time. In this work, we also use transgenic lines to demonstrate that GNC and CGA1 exhibit a partially redundant control over chlorophyll biosynthesis. We provide novel evidence that GNC and CGA1 influence both chloroplast number and leaf starch in proportion to their transcript level. GNC and CGA1 were found to modify the expression of chloroplast localized GLUTAMATE SYNTHASE (GLU1/Fd-GOGAT), which is the primary factor controlling nitrogen assimilation in green tissue. Altering GNC and CGA1 expression was also found to modulate the expression of important chlorophyll biosynthesis genes (GUN4, HEMA1, PORB, and PORC). As previously demonstrated, the CGA1 transgenic plants demonstrated significantly altered timing to a number of developmental events including germination, leaf production, flowering time and senescence. In contrast, the GNC transgenic lines we analyzed maintain relatively normal growth phenotypes outside of differences in chloroplast development. Despite some evidence for partial divergence, results indicate that regulation of both GNC and CGA1 by light, nitrogen, cytokinin, and GA acts to modulate nitrogen assimilation, chloroplast development and starch production. Understanding the mechanisms controlling these processes is important for agricultural biotechnology. PMID:22102866

RcRR1, a Rosa canina type-A response regulator gene, is involved in cytokinin-modulated rhizoid organogenesis.

PubMed

Gao, Bin; Fan, Lusheng; Li, Xingxing; Yang, Huifang; Liu, Fengluan; Wang, Ling; Xi, Lin; Ma, Nan; Zhao, Liangjun

2013-01-01

In vitro, a new protocol of plant regeneration in rose was achieved via protocorm-like bodies (PLBs) induced from the root-like organs named rhizoids that developed from leaf explants. The development of rhizoids is a critical stage for efficient regeneration, which is triggered by exogenous auxin. However, the role of cytokinin in the control of organogenesis in rose is as yet uncharacterized. The aim of this study was to elucidate the molecular mechanism of cytokinin-modulated rhizoid formation in Rosa canina. Here, we found that cytokinin is a key regulator in the formation of rhizoids. Treatment with cytokinin reduced callus activity and significantly inhibited rhizoid formation in Rosa canina. We further isolated the full-length cDNA of a type-A response regulator gene of cytokinin signaling, RcRR1, from which the deduced amino acid sequence contained the conserved DDK motif. Gene expression analysis revealed that RcRR1 was differentially expressed during rhizoid formation and its expression level was rapidly up-regulated by cytokinin. In addition, the functionality of RcRR1 was tested in Arabidopsis. RcRR1 was found to be localized to the nucleus in GFP-RcRR1 transgenic plants and overexpression of RcRR1 resulted in increased primary root length and lateral root density. More importantly, RcRR1 overexpression transgenic plants also showed reduced sensitivity to cytokinin during root growth; auxin distribution and the expression of auxin efflux carriers PIN genes were altered in RcRR1 overexpression plants. Taken together, these results demonstrate that RcRR1 is a functional type-A response regulator which is involved in cytokinin-regulated rhizoid formation in Rosa canina.

RcRR1, a Rosa canina Type-A Response Regulator Gene, Is Involved in Cytokinin-Modulated Rhizoid Organogenesis

PubMed Central

Li, Xingxing; Yang, Huifang; Liu, Fengluan; Wang, Ling; Xi, Lin; Ma, Nan; Zhao, Liangjun

2013-01-01

In vitro, a new protocol of plant regeneration in rose was achieved via protocorm-like bodies (PLBs) induced from the root-like organs named rhizoids that developed from leaf explants. The development of rhizoids is a critical stage for efficient regeneration, which is triggered by exogenous auxin. However, the role of cytokinin in the control of organogenesis in rose is as yet uncharacterized. The aim of this study was to elucidate the molecular mechanism of cytokinin-modulated rhizoid formation in Rosa canina. Here, we found that cytokinin is a key regulator in the formation of rhizoids. Treatment with cytokinin reduced callus activity and significantly inhibited rhizoid formation in Rosa canina. We further isolated the full-length cDNA of a type-A response regulator gene of cytokinin signaling, RcRR1, from which the deduced amino acid sequence contained the conserved DDK motif. Gene expression analysis revealed that RcRR1 was differentially expressed during rhizoid formation and its expression level was rapidly up-regulated by cytokinin. In addition, the functionality of RcRR1 was tested in Arabidopsis. RcRR1 was found to be localized to the nucleus in GFP-RcRR1 transgenic plants and overexpression of RcRR1 resulted in increased primary root length and lateral root density. More importantly, RcRR1 overexpression transgenic plants also showed reduced sensitivity to cytokinin during root growth; auxin distribution and the expression of auxin efflux carriers PIN genes were altered in RcRR1 overexpression plants. Taken together, these results demonstrate that RcRR1 is a functional type-A response regulator which is involved in cytokinin-regulated rhizoid formation in Rosa canina. PMID:24009713

Both alpha(1A)- and alpha(1B)-adrenergic receptors crosstalk to down regulate beta(1)-ARs in mouse heart: coupling to differential PTX-sensitive pathways.

PubMed

Rorabaugh, Boyd R; Gaivin, Robert J; Papay, Robert S; Shi, Ting; Simpson, Paul C; Perez, Dianne M

2005-11-01

Adrenergic receptors (ARs) play an important role in the regulation of cardiac function. Cardiac inotropy is primarily regulated by beta(1)-ARs. However, alpha(1)-ARs may play an important role in inotropy during heart failure. Previous work has suggested that the alpha(1B)-AR modulates beta(1)-AR function in the heart. The potential role of the alpha(1A)-AR has not been previously studied. We used transgenic mice that express constitutively active mutant (CAM) forms of the alpha(1A)-AR or alpha(1B)-AR regulated by their endogenous promoters. Expression of the CAM alpha(1A)-AR or CAM alpha(1B)-AR had no effect on basal cardiac function (developed pressure, +dP/dT, -dP/dT, heart rate, flow rate). However, both alpha(1)-AR subtypes significantly decreased isoproterenol-stimulated +dP/dT. Pertussis toxin had no effect on +dP/dT in CAM alpha(1A)-AR hearts but restored +dP/dT to non-transgenic values in CAM alpha(1B)-AR hearts. Radioligand binding indicated a selective decrease in the density of beta(1)-ARs in both CAM mice. However, G-proteins, cAMP, or the percentage of high and low affinity states were unchanged in either transgenic compared with control. These data demonstrate that CAM alpha(1A)- and alpha(1B)-ARs both down regulate beta(1)-AR-mediated inotropy in the mouse heart. However, alpha(1)-AR subtypes are coupled to different beta-AR mediated signaling pathways with the alpha(1B)-AR being pertussis toxin sensitive.

PGC-1α-mediated branched-chain amino acid metabolism in the skeletal muscle.

PubMed

Hatazawa, Yukino; Tadaishi, Miki; Nagaike, Yuta; Morita, Akihito; Ogawa, Yoshihiro; Ezaki, Osamu; Takai-Igarashi, Takako; Kitaura, Yasuyuki; Shimomura, Yoshiharu; Kamei, Yasutomi; Miura, Shinji

2014-01-01

Peroxisome proliferator-activated receptor (PPAR) γ coactivator 1α (PGC-1α) is a coactivator of various nuclear receptors and other transcription factors, which is involved in the regulation of energy metabolism, thermogenesis, and other biological processes that control phenotypic characteristics of various organ systems including skeletal muscle. PGC-1α in skeletal muscle is considered to be involved in contractile protein function, mitochondrial function, metabolic regulation, intracellular signaling, and transcriptional responses. Branched-chain amino acid (BCAA) metabolism mainly occurs in skeletal muscle mitochondria, and enzymes related to BCAA metabolism are increased by exercise. Using murine skeletal muscle overexpressing PGC-1α and cultured cells, we investigated whether PGC-1α stimulates BCAA metabolism by increasing the expression of enzymes involved in BCAA metabolism. Transgenic mice overexpressing PGC-1α specifically in the skeletal muscle had increased the expression of branched-chain aminotransferase (BCAT) 2, branched-chain α-keto acid dehydrogenase (BCKDH), which catabolize BCAA. The expression of BCKDH kinase (BCKDK), which phosphorylates BCKDH and suppresses its enzymatic activity, was unchanged. The amount of BCAA in the skeletal muscle was significantly decreased in the transgenic mice compared with that in the wild-type mice. The amount of glutamic acid, a metabolite of BCAA catabolism, was increased in the transgenic mice, suggesting the activation of muscle BCAA metabolism by PGC-1α. In C2C12 cells, the overexpression of PGC-1α significantly increased the expression of BCAT2 and BCKDH but not BCKDK. Thus, PGC-1α in the skeletal muscle is considered to significantly contribute to BCAA metabolism.

A novel GhBEE1-Like gene of cotton causes anther indehiscence in transgenic Arabidopsis under uncontrolled transcription level.

PubMed

Chen, Eryong; Wang, Xiaoqian; Gong, Qian; Butt, Hamama Islam; Chen, Yanli; Zhang, Chaojun; Yang, Zuoren; Wu, Zhixia; Ge, Xiaoyang; Zhang, Xianlong; Li, Fuguang; Zhang, Xueyan

2017-09-05

Male-sterile lines are very important for selective breeding, and anther dehiscence defect is an effective way to generate male-sterile lines. Although several bHLH-family proteins in Arabidopsis have been characterized, little is known about the role of bHLH-family proteins in cotton. Here, we isolated a novel bHLH protein from cotton (Gossypium hirsutum), named GhBEE1-Like. Protein domain analysis showed that GhBEE1-Like contained a basic domain and an HLH domain. Subcellular localization analysis revealed that GhBEE1-Like was a nuclear-localized protein. Expression pattern analysis showed GhBEE1-Like was highly expressed in floral organs, and its expression was induced by the active brassinosteroid (BR) substance 24-epi-BL. GhBEE1-Like overexpression in Arabidopsis resulted in two types of transgenic lines, one with normal anther dehiscence and the other with defective anther dehiscence. Semi-qRT-PCR and qRT-PCR analyses revealed that GhBEE1-Like transcript levels acted as a check-point determining how anther dehiscence proceeds in these transgenic lines; regulated transcript levels result in normal anther dehiscence, whereas uncontrolled transcript levels lead to anther indehiscence. These results suggest that GhBEE1-Like plays an important role via its accumulation in regulating anther dehiscence. Therefore, controlling the level of GhBEE1-Like expression in cotton could be a convenient tool for generating male-sterile lines to use in selective breeding. Copyright © 2017. Published by Elsevier B.V.

PGC-1α-Mediated Branched-Chain Amino Acid Metabolism in the Skeletal Muscle

PubMed Central

Nagaike, Yuta; Morita, Akihito; Ogawa, Yoshihiro; Ezaki, Osamu; Takai-Igarashi, Takako; Kitaura, Yasuyuki; Shimomura, Yoshiharu; Kamei, Yasutomi; Miura, Shinji

2014-01-01

Peroxisome proliferator-activated receptor (PPAR) γ coactivator 1α (PGC-1α) is a coactivator of various nuclear receptors and other transcription factors, which is involved in the regulation of energy metabolism, thermogenesis, and other biological processes that control phenotypic characteristics of various organ systems including skeletal muscle. PGC-1α in skeletal muscle is considered to be involved in contractile protein function, mitochondrial function, metabolic regulation, intracellular signaling, and transcriptional responses. Branched-chain amino acid (BCAA) metabolism mainly occurs in skeletal muscle mitochondria, and enzymes related to BCAA metabolism are increased by exercise. Using murine skeletal muscle overexpressing PGC-1α and cultured cells, we investigated whether PGC-1α stimulates BCAA metabolism by increasing the expression of enzymes involved in BCAA metabolism. Transgenic mice overexpressing PGC-1α specifically in the skeletal muscle had increased the expression of branched-chain aminotransferase (BCAT) 2, branched-chain α-keto acid dehydrogenase (BCKDH), which catabolize BCAA. The expression of BCKDH kinase (BCKDK), which phosphorylates BCKDH and suppresses its enzymatic activity, was unchanged. The amount of BCAA in the skeletal muscle was significantly decreased in the transgenic mice compared with that in the wild-type mice. The amount of glutamic acid, a metabolite of BCAA catabolism, was increased in the transgenic mice, suggesting the activation of muscle BCAA metabolism by PGC-1α. In C2C12 cells, the overexpression of PGC-1α significantly increased the expression of BCAT2 and BCKDH but not BCKDK. Thus, PGC-1α in the skeletal muscle is considered to significantly contribute to BCAA metabolism. PMID:24638054

Highly Efficient Generation of Transgenic Sheep by Lentivirus Accompanying the Alteration of Methylation Status

PubMed Central

Liu, Chenxi; Wang, Liqin; Li, Wenrong; Zhang, Xuemei; Tian, Yongzhi; Zhang, Ning; He, Sangang; Chen, Tong; Huang, Juncheng; Liu, Mingjun

2013-01-01

Background Low efficiency of gene transfer and silence of transgene expression are the critical factors hampering the development of transgenic livestock. Recently, transfer of recombinant lentivirus has been demonstrated to be an efficient transgene delivery method in various animals. However, the lentiviral transgenesis and the methylation status of transgene in sheep have not been well addressed. Methodology/Principle Findings EGFP transgenic sheep were generated by injecting recombinant lentivirus into zygotes. Of the 13 lambs born, 8 carried the EGFP transgene, and its chromosomal integration was identified in all tested tissues. Western blotting showed that GFP was expressed in all transgenic founders and their various tissues. Analysis of CpG methylation status of CMV promoter by bisulfate sequencing unraveled remarkable variation of methylation levels in transgenic sheep. The average methylation levels ranged from 37.6% to 79.1% in the transgenic individuals and 34.7% to 83% in the tested tissues. Correlative analysis of methylation status with GFP expression revealed that the GFP expression level was inversely correlated with methylation density. The similar phenomenon was also observed in tested tissues. Transgene integration determined by Southern blotting presented multiple integrants ranging from 2 to 6 copies in the genome of transgenic sheep. Conclusions/Significance Injection of lentiviral transgene into zygotes could be a promising efficient gene delivery system to generate transgenic sheep and achieved widespread transgene expression. The promoter of integrants transferred by lentiviral vector was subjected to dramatic alteration of methylation status and the transgene expression level was inversely correlative with promoter methylation density. Our work illustrated for the first time that generation of transgenic sheep by injecting recombinant lentivirus into zygote could be an efficient tool to improve sheep performance by genetic modification. PMID:23382924

Nell-1-Induced Bone Regeneration in Calvarial Defects

PubMed Central

Aghaloo, Tara; Cowan, Catherine M.; Chou, Yu-Fen; Zhang, Xinli; Lee, Haofu; Miao, Steve; Hong, Nichole; Kuroda, Shun’ichi; Wu, Benjamin; Ting, Kang; Soo, Chia

2006-01-01

Many craniofacial birth defects contain skeletal components requiring bone grafting. We previously identified the novel secreted osteogenic molecule NELL-1, first noted to be overexpressed during premature bone formation in calvarial sutures of craniosynostosis patients. Nell-1 overexpression significantly increases differentiation and mineralization selectively in osteoblasts, while newborn Nell-1 transgenic mice significantly increase premature bone formation in calvarial sutures. In the current study, cultured calvarial explants isolated from Nell-1 transgenic newborn mice (with mild sagittal synostosis) demonstrated continuous bone growth and overlapping sagittal sutures. Further investigation into gene expression cascades revealed that fibroblast growth factor-2 and transforming growth factor-β1 stimulated Nell-1 expression, whereas bone morphogenetic protein (BMP)-2 had no direct effect. Additionally, Nell-1-induced osteogenesis in MC3T3-E1 osteoblasts through reduction in the expression of early up-regulated osteogenic regulators (OSX and ALP) but induction of later markers (OPN and OCN). Grafting Nell-1 protein-coated PLGA scaffolds into rat calvarial defects revealed the osteogenic potential of Nell-1 to induce bone regeneration equivalent to BMP-2, whereas immunohistochemistry indicated that Nell-1 reduced osterix-producing cells and increased bone sialoprotein, osteocalcin, and BMP-7 expression. Insights into Nell-1-regulated osteogenesis coupled with its ability to stimulate bone regeneration revealed a potential therapeutic role and an alternative to the currently accepted techniques for bone regeneration. PMID:16936265

Sp8 and COUP-TF1 reciprocally regulate patterning and Fgf signaling in cortical progenitors.

PubMed

Borello, Ugo; Madhavan, Mayur; Vilinsky, Ilya; Faedo, Andrea; Pierani, Alessandra; Rubenstein, John; Campbell, Kenneth

2014-06-01

To gain new insights into the transcriptional regulation of cortical development, we examined the role of the transcription factor Sp8, which is downstream of Fgf8 signaling and known to promote rostral cortical development. We have used a binary transgenic system to express Sp8 throughout the mouse telencephalon in a temporally restricted manner. Our results show that misexpression of Sp8 throughout the telencephalon, at early but not late embryonic stages, results in cortical hypoplasia, which is accompanied by increased cell death, reduced proliferation, and precocious neuronal differentiation. Misexpression of Sp8 at early developmental stages represses COUP-TF1 expression, a negative effector of Fgf signaling and a key promoter of posterior cortical identity, while ablation of Sp8 has the opposite effect. In addition, transgenic misexpression of COUP-TF1 resulted in downregulation of Sp8, indicating a reciprocal cross-regulation between these 2 transcription factors. Although Sp8 has been suggested to induce and/or maintain Fgf8 expression in the embryonic telencephalon, neither Fgf8 nor Fgf15 was upregulated using our gain-of-function approach. However, misexpression of Sp8 greatly increased the expression of Fgf target molecules, suggesting enhanced Fgf signaling. Thus, we propose that Sp8 promotes rostral and dorsomedial cortical development by repressing COUP-TF1 and promoting Fgf signaling in pallial progenitors.

Sp8 and COUP-TF1 Reciprocally Regulate Patterning and Fgf Signaling in Cortical Progenitors

PubMed Central

Borello, Ugo; Madhavan, Mayur; Vilinsky, Ilya; Faedo, Andrea; Pierani, Alessandra; Rubenstein, John; Campbell, Kenneth

2014-01-01

To gain new insights into the transcriptional regulation of cortical development, we examined the role of the transcription factor Sp8, which is downstream of Fgf8 signaling and known to promote rostral cortical development. We have used a binary transgenic system to express Sp8 throughout the mouse telencephalon in a temporally restricted manner. Our results show that misexpression of Sp8 throughout the telencephalon, at early but not late embryonic stages, results in cortical hypoplasia, which is accompanied by increased cell death, reduced proliferation, and precocious neuronal differentiation. Misexpression of Sp8 at early developmental stages represses COUP-TF1 expression, a negative effector of Fgf signaling and a key promoter of posterior cortical identity, while ablation of Sp8 has the opposite effect. In addition, transgenic misexpression of COUP-TF1 resulted in downregulation of Sp8, indicating a reciprocal cross-regulation between these 2 transcription factors. Although Sp8 has been suggested to induce and/or maintain Fgf8 expression in the embryonic telencephalon, neither Fgf8 nor Fgf15 was upregulated using our gain-of-function approach. However, misexpression of Sp8 greatly increased the expression of Fgf target molecules, suggesting enhanced Fgf signaling. Thus, we propose that Sp8 promotes rostral and dorsomedial cortical development by repressing COUP-TF1 and promoting Fgf signaling in pallial progenitors. PMID:23307639

lin-4 and the NRDE pathway are required to activate a transgenic lin-4 reporter but not the endogenous lin-4 locus in C. elegans.

PubMed

Jiao, Alan L; Foster, Daniel J; Dixon, Julia; Slack, Frank J

2018-01-01

As the founding member of the microRNA (miRNA) gene family, insights into lin-4 regulation and function have laid a conceptual foundation for countless miRNA-related studies that followed. We previously showed that a transcriptional lin-4 reporter in C. elegans was positively regulated by a lin-4-complementary element (LCE), and by lin-4 itself. In this study, we sought to (1) identify additional factors required for lin-4 reporter expression, and (2) validate the endogenous relevance of a potential positive autoregulatory mechanism of lin-4 expression. We report that all four core nuclear RNAi factors (nrde-1, nrde-2, nrde-3 and nrde-4), positively regulate lin-4 reporter expression. In contrast, endogenous lin-4 levels were largely unaffected in nrde-2;nrde-3 mutants. Further, an endogenous LCE deletion generated by CRISPR-Cas9 revealed that the LCE was also not necessary for the activity of the endogenous lin-4 promoter. Finally, mutations in mature lin-4 did not reduce primary lin-4 transcript levels. Taken together, these data indicate that under growth conditions that reveal effects at the transgenic locus, a direct, positive autoregulatory mechanism of lin-4 expression does not occur in the context of the endogenous lin-4 locus.

Mono-allelic expression of variegating transgene locus in the mouse.

PubMed

Opsahl, Margaret L; Springbett, Anthea; Lathe, Richard; Colman, Alan; McClenaghan, Margaret; Whitelaw, C Bruce A

2003-12-01

We have generated transgenic mice which express an ovine beta-lactoglobulin transgene during lactation. In two transgenic lines, BLG/7 and BLG/45, beta-lactoglobulin protein levels vary between siblings, reflected at the cellular level by a mosaic transgene expression pattern in the mammary tissue that is reminiscent of position effect variegation. To investigate whether this variegating expression profile can be affected by the introduction of an identical variegating locus on the homologous chromosome, we compared the beta-lactoglobulin expression profiles in mice hemizygous or homozygous for the transgene locus. In BLG/45 mice, milk protein analysis revealed that transgene expression was effectively doubled in homozygous compared to hemizygous mice. In contrast, beta-lactoglobulin protein in hemizygous and homozygous BLG/7 mice displayed a similar range; although minimum expression levels were doubled in the homozygous population, the maximum level of expression was indistinguishable between the two populations. Fluorescent in situ hybridisation (FISH) for transgene mRNA indicated that for a given protein level, the extent of cellular expression is similar in both BLG/7 populations. In homozygous mice genomic DNA and nuclear RNA FISH demonstrated that only one of the two BLG/7 loci is active in expressing cells, while two transcription foci were present in BLG/45 homozygous mice. This mono-allelic transgene expression pattern is not inherited through the germline, as hemizygous mice bred from homozygous parents expressed at the expected hemizygous population level. We discuss these observations in the context of known epigenetic events such as imprinting and trans-inactivation.

Homologous expression of cytosolic dehydroascorbate reductase increases grain yield and biomass under paddy field conditions in transgenic rice (Oryza sativa L. japonica).

PubMed

Kim, Young-Saeng; Kim, Il-Sup; Bae, Mi-Jung; Choe, Yong-Hoe; Kim, Yul-Ho; Park, Hyang-Mi; Kang, Hong-Gyu; Yoon, Ho-Sung

2013-06-01

Dehydroascorbate reductase (DHAR, EC 1.8.5.1) maintains redox pools of ascorbate (AsA) by recycling oxidized AsA to reduced AsA. To investigate whether DHAR affects rice yield under normal environmental conditions, cDNA-encoding DHAR (OsDHAR1) was isolated from rice and used to develop OsDHAR1-overexpressing transgenic rice plants, under the regulation of a maize ubiquitin promoter. Incorporation and expression of the transgene in transgenic rice plants was confirmed by genomic polymerase chain reaction (PCR), semi-quantitative reverse transcription PCR (RT-PCR), western blot, and enzyme activity. The expression levels were at least twofold higher in transgenic (TG) rice plants than in control wild-type (WT) rice plants. In addition, OsDHAR1-overexpression in seven-independent homologous transgenic plants, as compared to WT plants, increased photosynthetic capacity and antioxidant enzyme activities under paddy field conditions, which led to an improved AsA pool and redox homeostasis. Furthermore, OsDHAR1 overexpression significantly improved grain yield and biomass due to the increase of culm and root weights and to enhance panicle and spikelet numbers in the same seven independent TG rice plants during the farming season (2010 and 2011) in South Korea. The OsDHAR protein contained the redox-active site (Cys20), as well as the conserved GSH-binding region, GSH-binding motif, glutathione-S-transferase (GST) N-terminal domain, C-terminal domain interface, and GST C-terminal domain. Therefore, our results indicate that OsDHAR1 overexpression, capable of functioning in AsA recycling, and protein folding increases environmental adaptation to paddy field conditions by the improving AsA pool and redox homeostasis, which enhances rice grain yield and biomass.

Antisense down-regulation of the strawberry β-galactosidase gene FaβGal4 increases cell wall galactose levels and reduces fruit softening

PubMed Central

Paniagua, Candelas; Blanco-Portales, Rosario; Barceló-Muñoz, Marta; García-Gago, Juan A.; Waldron, Keith W.; Quesada, Miguel A.; Muñoz-Blanco, Juan; Mercado, José A.

2016-01-01

Strawberry softening is characterized by an increase in the solubilization and depolymerization of pectins from cell walls. Galactose release from pectin side chains by β-galactosidase enzymes has been proposed as one reason for the increase in soluble pectins. A putative β-galactosidase gene, FaβGal4, has been identified using a custom-made oligonucleotide-based strawberry microarray platform. FaβGal4 was expressed mainly in the receptacle during fruit ripening, and was positively regulated by abscisic acid and negatively regulated by auxins. To ascertain the role of FaβGal4 in strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the CaMV35S promoter were generated. Phenotypic analyses were carried out in transgenic plants during three consecutive growing seasons, using non-transformed plants as control. Two out of nine independent transgenic lines yielded fruits that were 30% firmer than control at the ripe stage. FaβGal4 mRNA levels were reduced by 70% in ripe fruits from these selected transgenic lines, but they also showed significant silencing of FaβGal1, although the genes did not share significant similarity. These two transgenic lines also showed an increase in pectin covalently bound to the cell wall, extracted using Na2CO3. The amount of galactose in cell walls from transgenic fruits was 30% higher than in control; notably, the galactose increase was larger in the 1 M KOH fraction, which is enriched in hemicellulose. These results suggest that FaβGal4 participates in the solubilization of covalently bound pectins during ripening, reducing strawberry fruit firmness. PMID:26585222

Antisense down-regulation of the strawberry β-galactosidase gene FaβGal4 increases cell wall galactose levels and reduces fruit softening.

PubMed

Paniagua, Candelas; Blanco-Portales, Rosario; Barceló-Muñoz, Marta; García-Gago, Juan A; Waldron, Keith W; Quesada, Miguel A; Muñoz-Blanco, Juan; Mercado, José A

2016-02-01

Strawberry softening is characterized by an increase in the solubilization and depolymerization of pectins from cell walls. Galactose release from pectin side chains by β-galactosidase enzymes has been proposed as one reason for the increase in soluble pectins. A putative β-galactosidase gene, FaβGal4, has been identified using a custom-made oligonucleotide-based strawberry microarray platform. FaβGal4 was expressed mainly in the receptacle during fruit ripening, and was positively regulated by abscisic acid and negatively regulated by auxins. To ascertain the role of FaβGal4 in strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the CaMV35S promoter were generated. Phenotypic analyses were carried out in transgenic plants during three consecutive growing seasons, using non-transformed plants as control. Two out of nine independent transgenic lines yielded fruits that were 30% firmer than control at the ripe stage. FaβGal4 mRNA levels were reduced by 70% in ripe fruits from these selected transgenic lines, but they also showed significant silencing of FaβGal1, although the genes did not share significant similarity. These two transgenic lines also showed an increase in pectin covalently bound to the cell wall, extracted using Na2CO3. The amount of galactose in cell walls from transgenic fruits was 30% higher than in control; notably, the galactose increase was larger in the 1 M KOH fraction, which is enriched in hemicellulose. These results suggest that FaβGal4 participates in the solubilization of covalently bound pectins during ripening, reducing strawberry fruit firmness. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Insulin-secreting non-islet cells are resistant to autoimmune destruction.

PubMed Central

Lipes, M A; Cooper, E M; Skelly, R; Rhodes, C J; Boschetti, E; Weir, G C; Davalli, A M

1996-01-01

Transgenic nonobese diabetic mice were created in which insulin expression was targeted to proopiomelanocortin-expressing pituitary cells. Proopiomelanocortin-expressing intermediate lobe pituitary cells efficiently secrete fully processed, mature insulin via a regulated secretory pathway, similar to islet beta cells. However, in contrast to the insulin-producing islet beta cells, the insulin-producing intermediate lobe pituitaries are not targeted or destroyed by cells of the immune system. Transplantation of the transgenic intermediate lobe tissues into diabetic nonobese diabetic mice resulted in the restoration of near-normoglycemia and the reversal of diabetic symptoms. The absence of autoimmunity in intermediate lobe pituitary cells engineered to secrete bona fide insulin raises the potential of these cell types for beta-cell replacement therapy for the treatment of insulin-dependent diabetes mellitus. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8710916

PgLOX6 encoding a lipoxygenase contributes to jasmonic acid biosynthesis and ginsenoside production in Panax ginseng

PubMed Central

Rahimi, Shadi; Kim, Yu-Jin; Sukweenadhi, Johan; Zhang, Dabing; Yang, Deok-Chun

2016-01-01

Ginsenosides, the valuable pharmaceutical compounds in Panax ginseng, are triterpene saponins that occur mainly in ginseng plants. It was shown that in vitro treatment with the phytohormone jasmonic acid (JA) is able to increase ginsenoside production in ginseng plants. To understand the molecular link between JA biosynthesis and ginsenoside biosynthesis, we identified a JA biosynthetic 13-lipoxygenase gene (PgLOX6) in P. ginseng that promotes ginsenoside production. The expression of PgLOX6 was high in vascular bundles, which corresponds with expression of ginsenoside biosynthetic genes. Consistent with the role of PgLOX6 in synthesizing JA and promoting ginsenoside synthesis, transgenic plants overexpressing PgLOX6 in Arabidopsis had increased amounts of JA and methyl jasmonate (MJ), increased expression of triterpene biosynthetic genes such as squalene synthase (AtSS1) and squalene epoxidase (AtSE1), and increased squalene content. Moreover, transgenic ginseng roots overexpressing PgLOX6 had around 1.4-fold increased ginsenoside content and upregulation of ginsenoside biosynthesis-related genes including PgSS1, PgSE1, and dammarenediol synthase (PgDDS), which is similar to that of treatment with MJ. However, MJ treatment of transgenic ginseng significantly enhanced JA and MJ, associated with a 2.8-fold increase of ginsenoside content compared with the non-treated, non-transgenic control plant, which was 1.4 times higher than the MJ treatment effect on non-transgenic plants. These results demonstrate that PgLOX6 is responsible for the biosynthesis of JA and promotion of the production of triterpenoid saponin through up-regulating the expression of ginsenoside biosynthetic genes. This work provides insight into the role of JA in biosynthesizing secondary metabolites and provides a molecular tool for increasing ginsenoside production. PMID:27811076

The Regulation of Sox9 Gene Expression by the GATA4/FOG2 Transcriptional Complex in Dominant XX Sex Reversal Mouse Models.

PubMed Central

Manuylov, Nikolay L.; Fujiwara, Yuko; Adameyko, Igor I.; Poulat, Francis

2007-01-01

We have previously established an in vivo requirement for GATA4 and FOG2 transcription factors in sexual differentiation. Fog2 null mouse fetuses or fetuses homozygous for a targeted mutation in Gata4 (Gata4ki), which cripples the GATA4-FOG2 interaction, exhibit a profound and early block in testis differentiation in both sexes. Others have shown that XX mice with the Ods transgenic insertion or the Wt1-Sox9 YAC transgene overexpress the testis differentiation gene, Sox9. Thus, these XX animals undergo dominant sex-reversal by developing into phenotypically normal, but sterile, males. Now we have determined that Fog2 haploinsufficiency prevents (suppresses) this dominant sex-reversal and Fog2+/− Wt1-Sox9 or Ods XX animals develop normally - as fertile females. The suppression of sex-reversal in Fog2 heterozygous females results from approximately 50% downregulation of the expression from the transgene-associated allele of Sox9. The GATA4/FOG2-dependent sex reversal observed in the transgenic XX gonads has to rely on gene targets other than the Y chromosome-linked Sry gene. Importantly, Fog2 null or Gata4ki/ki embryos (either XX or XY) fail to express detectable levels of Sox9 despite carrying the Ods mutation or Wt1-Sox9 transgene. Fog2 haploinsufficiency leads to a decreased amount of SOX9-positive cells in XY gonads. We conclude that FOG2 is a limiting factor in the formation of a functional GATA4/FOG2 transcription complex that is required for Sox9 expression during gonadogenesis. PMID:17540364

Human Oncoprotein MDM2 Up-regulates Expression of NF-κB2 Precursor p100 Conferring a Survival Advantage to Lung Cells

PubMed Central

Vaughan, Catherine; Mohanraj, Lathika; Singh, Shilpa; Dumur, Catherine I.; Ramamoorthy, Mahesh; Garrett, Carleton T.; Windle, Brad; Yeudall, W. Andrew; Deb, Sumitra

2011-01-01

The current model predicts that MDM2 is primarily overexpressed in cancers with wild-type (WT) p53 and contributes to oncogenesis by degrading p53. Following a correlated expression of MDM2 and NF-κB2 transcripts in human lung tumors, we have identified a novel transactivation function of MDM2. Here, we report that in human lung tumors, overexpression of MDM2 was found in approximately 30% of cases irrespective of their p53 status, and expression of MDM2 and NF-κB2 transcripts showed a highly significant statistical correlation in tumors with WT p53. We investigated the significance of this correlated expression in terms of mechanism and biological function. Increase in MDM2 expression from its own promoter in transgenic mice remarkably enhanced expression of NF-κB2 compared with its non-transgenic littermates. Knockdown or elimination of endogenous MDM2 expression in cultured non-transformed or lung tumor cells drastically reduced expression of NF-κB2 transcripts, suggesting a normal physiological role of MDM2 in regulating NF-κB2 transcription. MDM2 could up-regulate expression of NF-κB2 transcripts when its p53-interaction domain was blocked with Nutlin-3, indicating that the MDM2-p53 interaction is dispensable for up-regulation of NF-κB2 expression. Consistently, analysis of functional domains of MDM2 indicated that although the p53-interaction domain of MDM2 contributes to the up-regulation of the NFκB2 promoter, MDM2 does not require direct interactions with p53 for this function. Accordingly, MDM2 overexpression in non-transformed or lung cancer cells devoid of p53 also generated a significant increase in the expression of NF-κB2 transcript and its targets CXCL-1 and CXCL-10, whereas elimination of MDM2 expression had the opposite effects. MDM2-mediated increase in p100/NF-κB2 expression reduced cell death mediated by paclitaxel. Furthermore, knockdown of NF-κB2 expression retarded cell proliferation. Based on these data, we propose that MDM2-mediated NF-κB2 up-regulation is a combined effect of p53-dependent and independent mechanisms and that it confers a survival advantage to lung cancer cells. PMID:22701761

Fibre elongation requires normal redox homeostasis modulated by cytosolic ascorbate peroxidase in cotton (Gossypium hirsutum).

PubMed

Guo, Kai; Du, Xueqiong; Tu, Lili; Tang, Wenxin; Wang, Pengcheng; Wang, Maojun; Liu, Zhen; Zhang, Xianlong

2016-05-01

High-quality cotton fibre equates to a more comfortable textile. Fibre length is an important index of fibre quality. Hydrogen peroxide (H2O2) acts as a signalling molecule in the regulation of fibre elongation. Results from in vitro ovule culture suggest that the alteration of fibre cell H2O2 levels affects fibre development. Ascorbate peroxidase (APX) is an important reactive oxygen species (ROS) scavenging enzyme, and we found that GhAPX1AT/DT encoded one member of the previously unrealized group of cytosolic APXs (cAPXs) that were preferentially expressed during the fibre elongation stage. Transgenic cottons with up- and down-regulation of GhAPX1AT/DT were generated to control fibre endogenous levels of H2O2 Suppression of all cAPX (IAO) resulted in a 3.5-fold increase in H2O2 level in fibres and oxidative stress, which significantly suppressed fibre elongation. The fibre length of transgenic lines with over-expression or specific down-regulation of GhAPX1AT/DT did not show any obvious change. However, the fibres in the over-expression lines exhibited higher tolerance to oxidative stress. Differentially expressed genes (DEGs) in fibres at 10 days post-anthesis (DPA) of IAO lines identified by RNA-seq were related to redox homeostasis, signalling pathways, stress responses and cell wall synthesis, and the DEGs that were up-regulated in IAO lines were also up-regulated in the 10 DPA and 20 DPA fibres of wild cotton compared with domesticated cotton. These results suggest that optimal H2O2 levels and redox state regulated by cytosolic APX are key mechanisms regulating fibre elongation, and dysregulation of the increase in H2O2 induces oxidative stress and results in shorter fibres by initiating secondary cell wall-related gene expression. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Altered expression of CmNRRa changes flowering time of Chrysanthemum morifolium.

PubMed

Zhang, Yuman; Lian, Lijuan; Liu, Qing; Xiao, Na; Fang, Rongxiang; Liu, Qinglin; Chen, Xiaoying

2013-04-01

Flowering time is an important ornamental trait for chrysanthemum (Chrysanthemum morifolium, Dendranthema x grandiflorum) floricultural production. In this study, CmNRRa, an orthologous gene of OsNRRa that regulates root growth in response to nutrient stress in rice, was identified from Chrysanthemum and its role in flowering time was studied. The entire CmNRRa cDNA sequence was determined using a combinatorial PCR approach along with 5' and 3' RACE methods. CmNRRa expression levels in various tissues were monitored by real-time RT-PCR. CmNRRa was strongly expressed in flower buds and peduncles, suggesting that CmNRRa plays a regulatory role in floral development. To investigate the biological function of CmNRRa in chrysanthemums, overexpression and knockdown of CmNRRa were carried out using transgenic Chrysanthemum plants generated through Agrobacterium-mediated transformation. CmNRRa expression levels in the transgenic plants were assayed by real-time RT-PCR and Northern blot analysis. The transgenic plants showed altered flowering times compared with nontransgenic plants. CmNRRa-RNAi transgenic plants flowered 40-64 days earlier, while CmNRRa-overexpressing plants exhibited a delayed flowering phenotype. These results revealed a negative effect of CmNRRa on flowering time modulation. Alteration of CmNRRa expression levels might be an effective means of controlling flowering time in Chrysanthemum. These results possess potential application in molecular breeding of chrysanthemums that production year-round, and may improve commercial chrysanthemum production in the flower industry. © 2012 The Authors Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Functional and comparative genomics analyses of pmp22 in medaka fish

PubMed Central

Itou, Junji; Suyama, Mikita; Imamura, Yukio; Deguchi, Tomonori; Fujimori, Kazuhiro; Yuba, Shunsuke; Kawarabayasi, Yutaka; Kawasaki, Takashi

2009-01-01

Background Pmp22, a member of the junction protein family Claudin/EMP/PMP22, plays an important role in myelin formation. Increase of pmp22 transcription causes peripheral neuropathy, Charcot-Marie-Tooth disease type1A (CMT1A). The pathophysiological phenotype of CMT1A is aberrant axonal myelination which induces a reduction in nerve conduction velocity (NCV). Several CMT1A model rodents have been established by overexpressing pmp22. Thus, it is thought that pmp22 expression must be tightly regulated for correct myelin formation in mammals. Interestingly, the myelin sheath is also present in other jawed vertebrates. The purpose of this study is to analyze the evolutionary conservation of the association between pmp22 transcription level and vertebrate myelin formation, and to find the conserved non-coding sequences for pmp22 regulation by comparative genomics analyses between jawed fishes and mammals. Results A transgenic pmp22 over-expression medaka fish line was established. The transgenic fish had approximately one fifth the peripheral NCV values of controls, and aberrant myelination of transgenic fish in the peripheral nerve system (PNS) was observed. We successfully confirmed that medaka fish pmp22 has the same exon-intron structure as mammals, and identified some known conserved regulatory motifs. Furthermore, we found novel conserved sequences in the first intron and 3'UTR. Conclusion Medaka fish undergo abnormalities in the PNS when pmp22 transcription increases. This result indicates that an adequate pmp22 transcription level is necessary for correct myelination of jawed vertebrates. Comparison of pmp22 orthologs between distantly related species identifies evolutionary conserved sequences that contribute to precise regulation of pmp22 expression. PMID:19534778

A cotton Raf-like MAP3K gene, GhMAP3K40, mediates reduced tolerance to biotic and abiotic stress in Nicotiana benthamiana by negatively regulating growth and development.

PubMed

Chen, Xiaobo; Wang, Ji; Zhu, Ming; Jia, Haihong; Liu, Dongdong; Hao, Lili; Guo, Xingqi

2015-11-01

Mitogen-activated protein kinase (MAPK) cascades mediate various responses in plants. As the top component, MAP3Ks deserve more attention; however, little is known about the role of MAP3Ks, especially in cotton, a worldwide economic crop. In this study, a gene encoding a putative Raf-like MAP3K, GhMAP3K40, was isolated. GhMAP3K40 expression was induced by stress and multiple signal molecules. The plants overexpressing GhMAP3K40 had an enhanced tolerance to drought and salt stress at the germination stage. However, at the seedling stage, the transgenic plants suffered more severe damage after drought, exposure to pathogens and oxidative stress. The defence-related genes and the antioxidant system were activated in transgenic palnts, suggesting that GhMAP3K40 positively regulate the defence response. The transgenic plants were less able to prevent pathogenic invasion, which was due to defects in the cell structure of the leaves. The root system of the control plants were stronger compared with the transgenic plants. These results indicated a negative role of GhMAP3K40 in growth and development and GhMAP3K40 possibly caused the defects by down-regulating the lignin biosynthesis. Overall, these results suggest that GhMAP3K40 may positively regulate defence response but cause reduced tolerance to biotic and abiotic stress by negatively regulating growth and development. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Transgenic over-expression of YY1 induces pathologic cardiac hypertrophy in a sex-specific manner

PubMed Central

Stauffer, Brian L.; Dockstader, Karen; Russell, Gloria; Hijmans, Jamie; Walker, Lisa; Cecil, Mackenzie; Demos-Davies, Kimberly; Medway, Allen; McKinsey, Timothy A.; Sucharov, Carmen C.

2015-01-01

YY1 can activate or repress transcription of various genes. In cardiac myocytes in culture YY1 has been shown to regulate expression of several genes involved in myocyte pathology. YY1 can also acutely protect the heart against detrimental changes in gene expression. In this study we show that cardiac over-expression of YY1 induces pathologic cardiac hypertrophy in male mice, measured by changes in gene expression and lower ejection fraction/fractional shortening. In contrast, female animals are protected against pathologic gene expression changes and cardiac dysfunction. Furthermore, we show that YY1 regulates, in a sex-specific manner, the expression of mammalian enable (Mena), a factor that regulates cytoskeletal actin dynamics and whose expression is increased in several models of cardiac pathology, and that Mena expression in humans with heart failure is sex-dependent. Finally, we show that sex differences in YY1 expression are also observed in human heart failure. In summary, this is the first work to show that YY1 has a sex-specific effect in the regulation of cardiac pathology. PMID:25935483

A transgenic zebrafish model for monitoring xbp1 splicing and endoplasmic reticulum stress in vivo.

PubMed

Li, Junling; Chen, Zhiliang; Gao, Lian-Yong; Colorni, Angelo; Ucko, Michal; Fang, Shengyun; Du, Shao Jun

2015-08-01

Accumulation of misfolded or unfolded proteins in the endoplasmic reticulum (ER) triggers ER stress that initiates unfolded protein response (UPR). XBP1 is a transcription factor that mediates one of the key signaling pathways of UPR to cope with ER stress through regulating gene expression. Activation of XBP1 involves an unconventional mRNA splicing catalyzed by IRE1 endonuclease that removes an internal 26 nucleotides from xbp1 mRNA transcripts in the cytoplasm. Researchers have taken advantage of this unique activation mechanism to monitor XBP1 activation, thereby UPR, in cell culture and transgenic models. Here we report a Tg(ef1α:xbp1δ-gfp) transgenic zebrafish line to monitor XBP1 activation using GFP as a reporter especially in zebrafish oocytes and developing embryos. The Tg(ef1α:xbp1δ-gfp) transgene was constructed using part of the zebrafish xbp1 cDNA containing the splicing element. ER stress induced splicing results in the cDNA encoding a GFP-tagged partial XBP1 without the transactivation activation domain (XBP1Δ-GFP). The results showed that xbp1 transcripts mainly exist as the spliced active isoform in unfertilized oocytes and zebrafish embryos prior to zygotic gene activation at 3 hours post fertilization. A strong GFP expression was observed in unfertilized oocytes, eyes, brain and skeletal muscle in addition to a weak expression in the hatching gland. Incubation of transgenic zebrafish embryos with (dithiothreitol) DTT significantly induced XBP1Δ-GFP expression. Collectively, these studies unveil the presence of maternal xbp1 splicing in zebrafish oocytes, fertilized eggs and early stage embryos. The Tg(ef1α:xbp1δ-gfp) transgenic zebrafish provides a useful model for in vivo monitoring xbp1 splicing during development and under ER stress conditions. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Relationship between Hexokinase and the Aquaporin PIP1 in the Regulation of Photosynthesis and Plant Growth

PubMed Central

Kelly, Gilor; Sade, Nir; Attia, Ziv; Secchi, Francesca; Zwieniecki, Maciej; Holbrook, N. Michele; Levi, Asher; Alchanatis, Victor; Moshelion, Menachem; Granot, David

2014-01-01

Increased expression of the aquaporin NtAQP1, which is known to function as a plasmalemma channel for CO2 and water, increases the rate of both photosynthesis and transpiration. In contrast, increased expression of Arabidopsis hexokinase1 (AtHXK1), a dual-function enzyme that mediates sugar sensing, decreases the expression of photosynthetic genes and the rate of transpiration and inhibits growth. Here, we show that AtHXK1 also decreases root and stem hydraulic conductivity and leaf mesophyll CO2 conductance (g m). Due to their opposite effects on plant development and physiology, we examined the relationship between NtAQP1 and AtHXK1 at the whole-plant level using transgenic tomato plants expressing both genes simultaneously. NtAQP1 significantly improved growth and increased the transpiration rates of AtHXK1-expressing plants. Reciprocal grafting experiments indicated that this complementation occurs when both genes are expressed simultaneously in the shoot. Yet, NtAQP1 had only a marginal effect on the hydraulic conductivity of the double-transgenic plants, suggesting that the complementary effect of NtAQP1 is unrelated to shoot water transport. Rather, NtAQP1 significantly increased leaf mesophyll CO2 conductance and enhanced the rate of photosynthesis, suggesting that NtAQP1 facilitated the growth of the double-transgenic plants by enhancing mesophyll conductance of CO2. PMID:24498392

MicroRNA-mediated gene regulation: potential applications for plant genetic engineering.

PubMed

Zhou, Man; Luo, Hong

2013-09-01

Food security is one of the most important issues challenging the world today. Any strategies to solve this problem must include increasing crop yields and quality. MicroRNA-based genetic modification technology (miRNA-based GM tech) can be one of the most promising solutions that contribute to agricultural productivity directly by developing superior crop cultivars with enhanced biotic and abiotic stress tolerance and increased biomass yields. Indirectly, the technology may increase usage of marginal soils and decrease pesticide use, among other benefits. This review highlights the most recent progress of transgenic studies utilizing various miRNAs and their targets for plant trait modifications, and analyzes the potential of miRNA-mediated gene regulation for use in crop improvement. Strategies for manipulating miRNAs and their targets in transgenic plants including constitutive, stress-induced, or tissue-specific expression of miRNAs or their targets, RNA interference, expressing miRNA-resistant target genes, artificial target mimic and artificial miRNAs were discussed. We also discussed potential risks of utilizing miRNA-based GM tech. In general, miRNAs and their targets not only provide an invaluable source of novel transgenes, but also inspire the development of several new GM strategies, allowing advances in breeding novel crop cultivars with agronomically useful characteristics.

Constitutive expression of nitrate reductase allows normal growth and development of Nicotiana plumbaginifolia plants.

PubMed Central

Vincentz, M; Caboche, M

1991-01-01

A nitrate reductase (NR) deficient mutant of Nicotiana plumbaginifolia totally impaired in the production of NR transcript and protein was restored for NR activity by transformation with a chimaeric NR gene. This gene was composed of a full-length tobacco NR cDNA fused to the CaMV 35S promoter and to termination signals from the tobacco NR gene. The transgenic plants we obtained were viable and fertile and expressed from one-fifth to three times the wild-type NR activity in their leaves. The analysis of chimeric NR gene expression in these plants showed, by comparison with wild-type plants, that the regulation of NR gene expression by light, nitrate and circadian rhythm takes place at the transcriptional level. However, unlike nitrate, light was required for the accumulation of NR protein in transgenic plants, suggesting that NR expression is also controlled at the translational and/or post-translational level. Images PMID:2022181

Cadmium resistance in tobacco plants expressing the MuSI gene.

PubMed

Kim, Young-Nam; Kim, Ji-Seoung; Seo, Sang-Gyu; Lee, Youngwoo; Baek, Seung-Woo; Kim, Il-Sup; Yoon, Ho-Sung; Kim, Kwon-Rae; Kim, Sun-Hyung; Kim, Kye-Hoon

2011-10-01

MuSI, a gene that corresponds to a domain that contains the rubber elongation factor (REF), is highly homologous to many stress-related proteins in plants. Since MuSI is up-regulated in the roots of plants treated with cadmium or copper, the involvement of MuSI in cadmium tolerance was investigated in this study. Escherichia coli cells overexpressing MuSI were more resistant to Cd than wild-type cells transfected with vector alone. MuSI transgenic plants were also more resistant to Cd. MuSI transgenic tobacco plants absorbed less Cd than wild-type plants. Cd translocation from roots to shoots was reduced in the transgenic plants, thereby avoiding Cd toxicity. The number of short trichomes in the leaves of wild-type tobacco plants was increased by Cd treatment, while this was unchanged in MuSI transgenic tobacco. These results suggest that MuSI transgenic tobacco plants have enhanced tolerance to Cd via reduced Cd uptake and/or increased Cd immobilization in the roots, resulting in less Cd translocation to the shoots.

Regulating the ethylene response of a plant by modulation of F-box proteins

DOEpatents

Guo, Hongwei [Beijing, CN; Ecker, Joseph R [Carlsbad, CA

2011-03-08

The invention relates to transgenic plants having reduced sensitivity to ethylene as a result of having a recombinant nucleic acid encoding an F-box protein that interacts with a EIN3 involved in an ethylene response of plants, and a method of producing a transgenic plant with reduced ethylene sensitivity by transforming the plant with a nucleic acid sequence encoding an F-box protein. The inventions also relates to methods of altering the ethylene response in a plant by modulating the activity or expression of an F-box protein.

Dynamic and differential expression of the gonadal aromatase during the process of sexual differentiation in a novel transgenic cyp19a1a-eGFP zebrafish line.

PubMed

Hinfray, Nathalie; Sohm, Frédéric; Caulier, Morgane; Chadili, Edith; Piccini, Benjamin; Torchy, Camille; Porcher, Jean-Marc; Guiguen, Yann; Brion, François

2018-05-15

In zebrafish, there exists a clear need for new tools to study sex differentiation dynamic and its perturbation by endocrine disrupting chemicals. In this context, we developed and characterized a novel transgenic zebrafish line expressing green fluorescent protein (GFP) under the control of the zebrafish cyp19a1a (gonadal aromatase) promoter. In most gonochoristic fish species including zebrafish, cyp19a1a, the enzyme responsible for the synthesis of estrogens, has been shown to play a critical role in the processes of reproduction and sexual differentiation. This novel cyp19a1a-eGFP transgenic line allowed a deeper characterization of expression and localization of cyp19a1a gene in zebrafish gonads both at the adult stage and during development. At the adult stage, GFP expression was higher in ovaries than in testis. We showed a perfect co-expression of GFP and endogenous Cyp19a1a protein in gonads that was mainly localized in the cytoplasm of peri-follicular cells in the ovary and of Leydig and germ cells in the testis. During development, GFP was expressed in all immature gonads of 20 dpf-old zebrafish. Then, GFP expression increased in early differentiated female at 30 and 35dpf to reach a high GFP intensity in well-differentiated ovaries at 40dpf. On the contrary, males consistently displayed low GFP expression as compared to female whatever their stage of development, resulting in a clear dimorphic expression between both sexes. Interestingly, fish that undergoes ovary-to-testis transition (35 and 40dpf) presented GFP levels similar to males or intermediate between females and males. In this transgenic line our results confirm that cyp19a1a is expressed early during development, before the histological differentiation of the gonads, and that the down-regulation of cyp19a1a expression is likely responsible for the testicular differentiation. Moreover, we show that although cyp19a1a expression exhibits a clear dimorphic expression pattern in gonads during sexual differentiation, its expression persists whatever the sex suggesting that estradiol synthesis is important for gonadal development of both sexes. Monitoring the expression of GFP in control and exposed-fish will help determine the sensitivity of this transgenic line to EDCs and to refine mechanistic based-assays for the study of EDCs. In fine, this transgenic zebrafish line will be a useful tool to study physiological processes such as reproduction and sexual differentiation, and their perturbations by EDCs. Copyright © 2017 Elsevier Inc. All rights reserved.

Cloning and Functional Analysis of Phosphoethanolamine Methyltransferase Promoter from Maize (Zea mays L.)

PubMed Central

Niu, Gai-Li; Gou, Wei; Han, Xiang-Long; Qin, Cheng; Zhang, Li-Xin; Ashraf, Muhammad

2018-01-01

Betaine, a non-toxic osmoprotectant, is believed to accumulate considerably in plants under stress conditions to maintain the osmotic pressure and promote a variety of processes involved in growth and development. Phosphoethanolamine N-methyltransferase (PEAMT), a key enzyme for betaine synthesis, is reported to be regulated by its upstream promoter. In the present investigation, by using the transgenic approach, a 1048 bp long promoter region of ZmPEAMT gene from Zea mays was cloned and functionally characterized in tobacco. Computational analysis affirmed the existence of abiotic stress responsive cis-elements like ABRE, MYC, HST, LST etc., as well as pathogen, wound and phytohormone responsive motifs. For transformation in tobacco, four 5′-deletion constructs of 826 bp (P2), 642 bp (P3), 428 bp (P4) and 245 bp (P5) were constructed from the 1048 bp (P1) promoter fragment. The transgenic plants generated through a single event exhibited a promising expression of GUS reporter protein in the leaf tissues of treated with salt, drought, oxidative and cold stress as well as control plants. The GUS expression level progressively reduced from P1 to P5 in the leaf tissues, whereas a maximal expression was observed with the P3 construct in the leaves of control plants. The expression of GUS was noted to be higher in the leaves of osmotically- or salt-treated transgenic plants than that in the untreated (control) plants. An effective expression of GUS in the transgenic plants manifests that this promoter can be employed for both stress-inducible and constitutive expression of gene(s). Due to this characteristic, this potential promoter can be effectively used for genetic engineering of several crops. PMID:29316727

GC-rich coding sequences reduce transposon-like, small RNA-mediated transgene silencing.

PubMed

Sidorenko, Lyudmila V; Lee, Tzuu-Fen; Woosley, Aaron; Moskal, William A; Bevan, Scott A; Merlo, P Ann Owens; Walsh, Terence A; Wang, Xiujuan; Weaver, Staci; Glancy, Todd P; Wang, PoHao; Yang, Xiaozeng; Sriram, Shreedharan; Meyers, Blake C

2017-11-01

The molecular basis of transgene susceptibility to silencing is poorly characterized in plants; thus, we evaluated several transgene design parameters as means to reduce heritable transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposon-like silencing of canola-biased PUFA synthase transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in a remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three crystal (Cry) proteins from Bacillus thuringiensis (Bt) tested the impact of CDS recoding using different codon bias tables. Transgenes with higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing, providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.

Key gene regulating cell wall biosynthesis and recalcitrance in Populus, gene Y

DOEpatents

Chen, Jay; Engle, Nancy; Gunter, Lee E.; Jawdy, Sara; Tschaplinski, Timothy J.; Tuskan, Gerald A.

2015-12-08

This disclosure provides methods and transgenic plants for improved production of renewable biofuels and other plant-derived biomaterials by altering the expression and/or activity of Gene Y, an O-acetyltransferase. This disclosure also provides expression vectors containing a nucleic acid (Gene Y) which encodes the polypeptide of SEQ ID NO: 1 and is operably linked to a heterologous promoter.

Two tobacco AP1-like gene promoters with highly specific, tightly regulated and uniquely expressed activity during floral transition, initiation and development

USDA-ARS?s Scientific Manuscript database

Biotech engineering of agronomic traits requires an array of highly specific and tightly regulated promoters in flower or other tissues. In this study, we isolated and characterized two tobacco AP1-like promoters (termed NtAP1La and NtAP1Lb1) in transgenic plants using GUS reporter and tissue-speci...

MLF1IP promotes normal erythroid proliferation and is involved in the pathogenesis of polycythemia vera.

PubMed

Feng, Gege; Zhang, Tianjiao; Liu, Jinqin; Ma, Xiaotang; Li, Bing; Yang, Lin; Zhang, Yue; Xu, Zefeng; Qin, Tiejun; Zhou, Jiaxi; Huang, Gang; Shi, Lihong; Xiao, Zhijian

2017-03-01

Myelodysplasia/myeloid leukemia factor 1-interacting protein (MLF1IP) appears to be an erythroid lineage-specific gene in mice; however, its role in normal erythropoiesis and erythropoietic disorders have not yet been elucidated. Here, we found that MLF1IP is abundantly expressed in human erythroid progenitor cells and that MLF1IP-deficiency reduces cell proliferation resulting from cell cycle arrest. Moreover, MLF1IP expression is exclusively elevated in CFU-E cells from polycythemia vera (PV) patients, and MLF1IP transgenic mice develop a PV-like disorder. Further analyses revealed that the erythroid progenitors and early-stage erythroblasts from these transgenic mice expand by up-regulating cyclin D2 and down-regulating p27 and p21. Thus, our data demonstrate that MLF1IP promotes erythroid proliferation and is involved in the pathogenesis of PV, suggesting that it might be a novel molecular target for erythropoietic disorders. © 2017 Federation of European Biochemical Societies.

An oilseed rape WRKY-type transcription factor regulates ROS accumulation and leaf senescence in Nicotiana benthamiana and Arabidopsis through modulating transcription of RbohD and RbohF.

PubMed

Yang, Liu; Ye, Chaofei; Zhao, Yuting; Cheng, Xiaolin; Wang, Yiqiao; Jiang, Yuan-Qing; Yang, Bo

2018-06-01

Overexpression of BnaWGR1 causes ROS accumulation and promotes leaf senescence. BnaWGR1 binds to promoters of RbohD and RbohF and regulates their expression. Manipulation of leaf senescence process affects agricultural traits of crop plants, including biomass, seed yield and stress resistance. Since delayed leaf senescence usually enhances tolerance to multiple stresses, we analyzed the function of specific MAPK-WRKY cascades in abiotic and biotic stress tolerance as well as leaf senescence in oilseed rape (Brassica napus L.), one of the important oil crops. In the present study, we showed that expression of one WRKY gene from oilseed rape, BnaWGR1, induced an accumulation of reactive oxygen species (ROS), cell death and precocious leaf senescence both in Nicotiana benthamiana and transgenic Arabidopsis (Arabidopsis thaliana). BnaWGR1 regulates the transcription of two genes encoding key enzymes implicated in production of ROS, that is, respiratory burst oxidase homolog (Rboh) D and RbohF. A dual-luciferase reporter assay confirmed the transcriptional regulation of RbohD and RbohF by BnaWGR1. In vitro electrophoresis mobility shift assay (EMSA) showed that BnaWGR1 could bind to W-box cis-elements within promoters of RbohD and RbohF. Moreover, RbohD and RbohF were significantly upregulated in transgenic Arabidopsis overexpressing BnaWGR1. In summary, these results suggest that BnaWGR1 could positively regulate leaf senescence through regulating the expression of RbohD and RbohF genes.

Regulations on growth and development in tomato cotyledon, flower and fruit via destruction of miR396 with short tandem target mimic.

PubMed

Cao, Dongyan; Wang, Jiao; Ju, Zheng; Liu, Qingqing; Li, Shan; Tian, Huiqin; Fu, Daqi; Zhu, Hongliang; Luo, Yunbo; Zhu, Benzhong

2016-06-01

Despite many studies about functions of miR396 were concentrated on cotyledon and leaf growth and development, only few researches were focused on flower and fruit, especially for fleshy fruit, for example, tomato fruit. Here, the roles of miR396 throughout the growth and development of tomato plant were explored with combining bioinformatics and transgene-mediated methods. In tomato, miR396 had two mature types (miR396a and miR396b), and miR396a expressed significantly higher than miR396b in cotyledon, flower, sepal and fruit. Generally, plant growth and development were regulated by miR396 via growth-regulating factors (GRFs). In tomato, all 13 SlGRFs were analyzed comprehensively, including phylogeny, domain and expression patterns. To investigate the roles of miR396 further, STTM396a/396a-88 was over-expressed in tomato, which induced miR396a and miR396b both dramatical down-regulation, and the target GRFs general up-regulation. As a result, the flowers, sepals and fruits all obviously became bigger. Most significantly, the sepal length of transgenic lines #3 and #4 at 39 days post-anthesis was separately increased 75% and 81%, and the fruit weight was added 45% and 39%, respectively. Overall, these results revealed novel roles of miR396 in regulating flower and fruit development, and provided a new potential way for improving tomato fruit yield. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Reduced expression of the SHORT-ROOT gene increases the rates of growth and development in hybrid poplar and Arabidopsis.

PubMed

Wang, Jiehua; Andersson-Gunnerås, Sara; Gaboreanu, Ioana; Hertzberg, Magnus; Tucker, Matthew R; Zheng, Bo; Leśniewska, Joanna; Mellerowicz, Ewa J; Laux, Thomas; Sandberg, Göran; Jones, Brian

2011-01-01

SHORT-ROOT (SHR) is a well characterized regulator of cell division and cell fate determination in the Arabidopsis primary root. However, much less is known about the functions of SHR in the aerial parts of the plant. In this work, we cloned SHR gene from Populus trichocarpa (PtSHR1) as an AtSHR ortholog and down-regulated its expression in hybrid poplar (Populus tremula×P. tremuloides Michx-clone T89) in order to determine its physiological functions in shoot development. Sharing a 90% similarity to AtSHR at amino acid level, PtSHR1 was able to complement the Arabidopsis shr mutant. Down regulation of PtSHR1 led to a strong enhancement of primary (height) and secondary (girth) growth rates in the transgenic poplars. A similar approach in Arabidopsis showed a comparable accelerated growth and development phenotype. Our results suggest that the response to SHR could be dose-dependent and that a partial down-regulation of SHR could lead to enhanced meristem activity and a coordinated acceleration of plant growth in woody species. Therefore, SHR functions in plant growth and development as a regulator of cell division and meristem activity not only in the roots but also in the shoots. Reducing SHR expression in transgenic poplar was shown to lead to significant increases in primary and secondary growth rates. Given the current interest in bioenergy crops, SHR has a broader role as a key regulator of whole plant growth and development and SHR suppression has considerable potential for accelerating biomass accumulation in a variety of species.

Ectopic Expression of JcWRKY Transcription Factor Confers Salinity Tolerance via Salicylic Acid Signaling.

PubMed

Agarwal, Parinita; Dabi, Mitali; Sapara, Komal K; Joshi, Priyanka S; Agarwal, Pradeep K

2016-01-01

Plants, being sessile, have developed intricate signaling network to specifically respond to the diverse environmental stress. The plant-specific WRKY TFs form one of the largest TF family and are involved in diverse plant processes, involving growth, development and stress signaling through auto and cross regulation with different genes and TFs. Here, we report the functional characterization of a salicylic acid -inducible JcWRKY TF. The JcWRKY overexpression confers salinity tolerance in transgenic tobacco, as was evident by increased chlorophyll content and seed germination potential. The transgenic plants showed increased soluble sugar, membrane stability, reduced electrolyte leakage and generation of reactive oxygen species (H 2 O 2 and [Formula: see text]) as compared to the wild type. Furthermore, the low SA treatment along with salinity improved the tolerance potential of the transgenics by maintaining ROS homeostasis and high K + /Na + ratio. The transcript expression of SA biosynthetic gene ICS1 and antioxidative enzymes ( CAT and SOD ) showed upregulation during stress. Thus, the present study reflects that JcWRKY is working in co-ordination with SA signaling to orchestrate the different biochemical and molecular pathways to maneuvre salt stress tolerance of the transgenic plants.

Chromogranin A deficiency in transgenic mice leads to aberrant chromaffin granule biogenesis.

PubMed

Kim, Taeyoon; Zhang, Chun-fa; Sun, Ziqing; Wu, Heling; Loh, Y Peng

2005-07-27

The biogenesis of dense-core secretory granules (DCGs), organelles responsible for the storage and secretion of neurotransmitters and neuropeptides in chromaffin cells, is poorly understood. Chromogranin A (CgA), which binds catecholamines for storage in the lumen of chromaffin granules, has been shown to be involved in DCG biogenesis in neuroendocrine PC12 cells. Here, we report that downregulation of CgA expression in vivo by expressing antisense RNA against CgA in transgenic mice led to a significant reduction in DCG formation in adrenal chromaffin cells. The number of DCGs formed in CgA antisense transgenic mice was directly correlated with the amount of CgA present in adrenal medulla. In addition, DCGs showed an increase in size, with enlargement in the volume around the dense core, a phenomenon that occurs to maintain constant "free" catecholamine concentration in the lumen of these granules. The extent of DCG swelling was inversely correlated with the number of DCGs formed, as well as the amount of CgA present in the adrenal glands of CgA antisense transgenic mice. These data indicate an essential role of CgA in regulating chromaffin DCG biogenesis and catecholamine storage in vivo.

Caudal dysgenesis in islet-1 transgenic mice

PubMed Central

Muller, Yunhua Li; Yueh, Yir Gloria; Yaworsky, Paul J.; Salbaum, J. Michael; Kappen, Claudia

2014-01-01

Maternal diabetes during pregnancy is responsible for the occurrence of diabetic embryopathy, a spectrum of birth defects that includes heart abnormalities, neural tube defects, and caudal dysgenesis syndromes. Here, we report that mice transgenic for the homeodomain transcription factor Isl-1 develop profound caudal growth defects that resemble human sacral/caudal agenesis. Isl-1 is normally expressed in the pancreas and is required for pancreas development and endocrine cell differentiation. Aberrant regulation of this pancreatic transcription factor causes increased mesodermal cell death, and the severity of defects is dependent on transgene dosage. Together with the finding that mutation of the pancreatic transcription factor HLXB9 causes sacral agenesis, our results implicate pancreatic transcription factors in the pathogenesis of birth defects associated with diabetes. PMID:12738808

The sunflower transcription factor HaHB11 confers tolerance to water deficit and salinity to transgenic Arabidopsis and alfalfa plants.

PubMed

Cabello, Julieta V; Giacomelli, Jorge I; Gómez, María C; Chan, Raquel L

2017-09-10

Homeodomain-leucine zipper (HD-Zip) transcription factors are unique to the plant kingdom; members of subfamily I are known to be involved in abiotic stress responses. HaHB11 belongs to this subfamily and it was previously shown that it is able to confer improved yield and tolerance to flooding via a quiescent strategy. Here we show that HaHB11 expression is induced by ABA, NaCl and water deficit in sunflower seedlings and leaves. Arabidopsis transgenic plants expressing HaHB11, controlled either by its own promoter or by the constitutive 35S CaMV, presented rolled leaves and longer roots than WT when grown under standard conditions. In addition, these plants showed wider stems and more vascular bundles. To deal with drought, HaHB11 transgenic plants closed their stomata faster and lost less water than controls, triggering an enhanced tolerance to such stress condition and also to salinity stress. Concomitantly, ABA-synthesis and sensing related genes were differentially regulated in HaHB11 transgenic plants. Either under long-term salinity stress or mild drought stress, HaHB11 transgenic plants did not exhibit yield penalties. Moreover, alfalfa transgenic plants were generated which also showed enhanced drought tolerance. Altogether, the results indicated that HaHB11 was able to confer drought and salinity tolerance via a complex mechanism which involves morphological, physiological and molecular changes. Copyright © 2016 Elsevier B.V. All rights reserved.

Coat color determination by miR-137 mediated down-regulation of microphthalmia-associated transcription factor in a mouse model

PubMed Central

Dong, Changsheng; Wang, Haidong; Xue, Linli; Dong, Yanjun; Yang, Lei; Fan, Ruiwen; Yu, Xiuju; Tian, Xue; Ma, Shuhui; Smith, George W.

2012-01-01

Coat color is a key economic trait in wool-producing species. Color development and pigmentation are controlled by complex mechanisms in animals. Here, we report the first production of an altered coat color by overexpression of miR-137 in transgenic mice. Transgenic mice overexpressing miR-137 developed a range of coat color changes from dark black to light color. Molecular analyses of the transgenic mice showed decreased expression of the major target gene termed MITF and its downstream genes, including TYR, TYRP1, and TYRP2. We also showed that melanogenesis altered by miR-137 is distinct from that affected by UV radiation in transgenic mice. Our study provides the first mouse model for the study of coat color controlled by miRNAs in animals and may have important applications in wool production. PMID:22847819

Genetic Evidence for a Role for Protein Kinase A in the Maintenance of Sleep and Thalamocortical Oscillations

PubMed Central

Hellman, Kevin; Hernandez, Pepe; Park, Alice; Abel, Ted

2010-01-01

Study Objectives: Genetic manipulation of cAMP-dependent protein kinase A (PKA) in Drosophila has implicated an important role for PKA in sleep/wake state regulation. Here, we characterize the role of this signaling pathway in the regulation of sleep using electroencephalographic (EEG) and electromyographic (EMG) recordings in R(AB) transgenic mice that express a dominant negative form of the regulatory subunit of PKA in neurons within cortex and hippocampus. Previous studies have revealed that these mutant mice have reduced PKA activity that results in the impairment of hippocampus-dependent long-term memory and long-lasting forms of hippocampal synaptic plasticity. Design: PKA assays, in situ hybridization, immunoblots, and sleep studies were performed in R(AB) transgenic mice and wild-type control mice. Measurements and Results: We have found that R(AB) transgenic mice have reduced PKA activity within cortex and reduced Ser845 phosphorylation of the glutamate receptor subunit GluR1. R(AB) transgenic mice exhibit non-rapid eye movement (NREM) sleep fragmentation and increased amounts of rapid eye movement (REM) sleep relative to wild-type mice. Further, R(AB) transgenic mice have more delta power but less sigma power during NREM sleep relative to wild-type mice. After sleep deprivation, the amounts of NREM and REM sleep were comparable between wild-type and R(AB) transgenic mice. However, the homeostatic rebound of sigma power in R(AB) transgenic mice was reduced. Conclusions: Alterations in cortical synaptic receptors, impairments in sleep continuity, and alterations in sleep oscillations in R(AB) mice imply that PKA is involved not only in synaptic plasticity and memory storage but also in the regulation of sleep/wake states. Citation: Hellman K; Hernandez P; Park A; Abel T. Genetic evidence for a role for protein kinase a in the maintenance of sleep and thalamocortical oscillations. SLEEP 2010;33(1):19-28. PMID:20120617

ABI-like transcription factor gene TaABL1 from wheat improves multiple abiotic stress tolerances in transgenic plants.

PubMed

Xu, Dong-Bei; Gao, Shi-Qing; Ma, You-Zhi; Xu, Zhao-Shi; Zhao, Chang-Ping; Tang, Yi-Miao; Li, Xue-Yin; Li, Lian-Cheng; Chen, Yao-Feng; Chen, Ming

2014-12-01

The phytohormone abscisic acid (ABA) plays crucial roles in adaptive responses of plants to abiotic stresses. ABA-responsive element binding proteins (AREBs) are basic leucine zipper transcription factors that regulate the expression of downstream genes containing ABA-responsive elements (ABREs) in promoter regions. A novel ABI-like (ABA-insensitive) transcription factor gene, named TaABL1, containing a conserved basic leucine zipper (bZIP) domain was cloned from wheat. Southern blotting showed that three copies were present in the wheat genome. Phylogenetic analyses indicated that TaABL1 belonged to the AREB subfamily of the bZIP transcription factor family and was most closely related to ZmABI5 in maize and OsAREB2 in rice. Expression of TaABL1 was highly induced in wheat roots, stems, and leaves by ABA, drought, high salt, and low temperature stresses. TaABL1 was localized inside the nuclei of transformed wheat mesophyll protoplast. Overexpression of TaABL1 enhanced responses of transgenic plants to ABA and hastened stomatal closure under stress, thereby improving tolerance to multiple abiotic stresses. Furthermore, overexpression of TaABL1 upregulated or downregulated the expression of some stress-related genes controlling stomatal closure in transgenic plants under ABA and drought stress conditions, suggesting that TaABL1 might be a valuable genetic resource for transgenic molecular breeding.

Next-generation transcriptome analysis in transgenic birch overexpressing and suppressing APETALA1 sheds lights in reproduction development and diterpenoid biosynthesis.

PubMed

Huang, Haijiao; Chen, Su; Li, Huiyu; Jiang, Jing

2015-09-01

Overexpression of BpAP1 could cause early flowering in birch. BpAP1 affected the expression of many flowering-related unigenes and diterpenoid biosynthesis in transgenic birch, and BpPI was a putative target gene of BpAP1. APETALA1 (AP1) is an MADS-box transcription factor that is involved in the flowering process in plants and has been a focus of genetic studies examining flower development. Here, we carried out transcriptome analysis of birch (Betula platyphylla Suk.), including BpAP1 overexpression lines, BpAP1 suppression lines, and non-transgenic line (NT). Compared with NT, we detected 8302 and 7813 differentially expressed unigenes in 35S::BpAP1 and 35S::BpAP1RNAi transgenic lines, respectively. Overexpression and suppression of BpAP1 in birch affected diterpenoid biosynthesis and altered expression of many flowering-related unigenes. Moreover, combining information from the RNA-seq database and the birch genome, we predicted downstream target genes of BpAP1. Among the 166 putative target genes of BpAP1, there was a positive correlation between BpAP1 and BpPI. These results provide references for further examining the relationship between BpAP1 and its target genes, and reveal that BpAP1 functions as a transcription regulator in birch.

Novel hypophysiotropic AgRP2 neurons and pineal cells revealed by BAC transgenesis in zebrafish.

PubMed

Shainer, Inbal; Buchshtab, Adi; Hawkins, Thomas A; Wilson, Stephen W; Cone, Roger D; Gothilf, Yoav

2017-03-20

The neuropeptide agouti-related protein (AgRP) is expressed in the arcuate nucleus of the mammalian hypothalamus and plays a key role in regulating food consumption and energy homeostasis. Fish express two agrp genes in the brain: agrp1, considered functionally homologous with the mammalian AgRP, and agrp2. The role of agrp2 and its relationship to agrp1 are not fully understood. Utilizing BAC transgenesis, we generated transgenic zebrafish in which agrp1- and agrp2-expressing cells can be visualized and manipulated. By characterizing these transgenic lines, we showed that agrp1-expressing neurons are located in the ventral periventricular hypothalamus (the equivalent of the mammalian arcuate nucleus), projecting throughout the hypothalamus and towards the preoptic area. The agrp2 gene was expressed in the pineal gland in a previously uncharacterized subgroup of cells. Additionally, agrp2 was expressed in a small group of neurons in the preoptic area that project directly towards the pituitary and form an interface with the pituitary vasculature, suggesting that preoptic AgRP2 neurons are hypophysiotropic. We showed that direct synaptic connection can exist between AgRP1 and AgRP2 neurons in the hypothalamus, suggesting communication and coordination between AgRP1 and AgRP2 neurons and, therefore, probably also between the processes they regulate.

Matrix Metalloproteinase-20 Over-Expression Is Detrimental to Enamel Development: A Mus musculus Model

PubMed Central

Shin, Masashi; Hu, Yuanyuan; Tye, Coralee E.; Guan, Xiaomu; Deagle, Craig C.; Antone, Jerry V.; Smith, Charles E.; Simmer, James P.; Bartlett, John D.

2014-01-01

Background Matrix metalloproteinase-20 (Mmp20) ablated mice have enamel that is thin and soft with an abnormal rod pattern that abrades from the underlying dentin. We asked if introduction of transgenes expressing Mmp20 would revert this Mmp20 null phenotype back to normal. Unexpectedly, for transgenes expressing medium or high levels of Mmp20, we found opposite enamel phenotypes depending on the genetic background (Mmp20−/− or Mmp20+/+) in which the transgenes were expressed. Methodology/Principal Findings Amelx-promoter-Mmp20 transgenic founder mouse lines were assessed for transgene expression and those expressing low, medium or high levels of Mmp20 were selected for breeding into the Mmp20 null background. Regardless of expression level, each transgene brought the null enamel back to full thickness. However, the high and medium expressing Mmp20 transgenes in the Mmp20 null background had significantly harder more mineralized enamel than did the low transgene expresser. Strikingly, when the high and medium expressing Mmp20 transgenes were present in the wild-type background, the enamel was significantly less well mineralized than normal. Protein gel analysis of enamel matrix proteins from the high and medium expressing transgenes present in the wild-type background demonstrated that greater than normal amounts of cleavage products and smaller quantities of higher molecular weight proteins were present within their enamel matrices. Conclusions/Significance Mmp20 expression levels must be within a specific range for normal enamel development to occur. Creation of a normally thick enamel layer may occur over a wider range of Mmp20 expression levels, but acquisition of normal enamel hardness has a narrower range. Since over-expression of Mmp20 results in decreased enamel hardness, this suggests that a balance exists between cleaved and full-length enamel matrix proteins that are essential for formation of a properly hardened enamel layer. It also suggests that few feedback controls are present in the enamel matrix to prevent excessive MMP20 activity. PMID:24466234

Discoidin domain receptor 2 (DDR2) regulates proliferation of endochondral cells in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawai, Ikuma; Hisaki, Tomoka; Sugiura, Koji

2012-10-26

Highlights: Black-Right-Pointing-Pointer Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase. Black-Right-Pointing-Pointer DDR2 regulates cell proliferation, cell adhesion, migration, and extracellular matrix remodeling. Black-Right-Pointing-Pointer We produced in vitro and in vivo model to better understand the role of DDR2. Black-Right-Pointing-Pointer DDR2 might play an inhibitory role in the proliferation of chondrocyte. -- Abstract: Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase that is activated by fibrillar collagens. DDR2 regulates cell proliferation, cell adhesion, migration, and extracellular matrix remodeling. The decrement of endogenous DDR2 represses osteoblastic marker gene expression and osteogenic differentiation in murine preosteoblastic cells, but themore » functions of DDR2 in chondrogenic cellular proliferation remain unclear. To better understand the role of DDR2 signaling in cellular proliferation in endochondral ossification, we inhibited Ddr2 expression via the inhibitory effect of miRNA on Ddr2 mRNA (miDdr2) and analyzed the cellular proliferation and differentiation in the prechondrocyte ATDC5 cell lines. To investigate DDR2's molecular role in endochondral cellular proliferation in vivo, we also produced transgenic mice in which the expression of truncated, kinase dead (KD) DDR2 protein is induced, and evaluated the DDR2 function in cellular proliferation in chondrocytes. Although the miDdr2-transfected ATDC5 cell lines retained normal differentiation ability, DDR2 reduction finally promoted cellular proliferation in proportion to the decreasing ratio of Ddr2 expression, and it also promoted earlier differentiation to cartilage cells by insulin induction. The layer of hypertrophic chondrocytes in KD Ddr2 transgenic mice was not significantly thicker than that of normal littermates, but the layer of proliferative chondrocytes in KD-Ddr2 transgenic mice was significantly thicker than that of normal littermates. Taken together, our data demonstrated that DDR2 might play a local and essential role in the proliferation of chondrocytes.« less

Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase gene are resistant to diphenyl ether herbicide oxyfluorfen.

PubMed

Lee, H J; Lee, S B; Chung, J S; Han, S U; Han, O; Guh, J O; Jeon, J S; An, G; Back, K

2000-06-01

Protoporphyrinogen oxidase (Protox), the penultimate step enzyme of the branch point for the biosynthetic pathway of Chl and hemes, is the target site of action of diphenyl ether (DPE) herbicides. However, Bacillus subtilis Protox is known to be resistant to the herbicides. In order to develop the herbicide-resistant plants, the transgenic rice plants were generated via expression of B. subtilis Protox gene under ubiquitin promoter targeted to the cytoplasm or to the plastid using Agrobacterium-mediated gene transformation. The integration and expression of the transgene were investigated at T0 generation by DNA and RNA blots. Most transgenic rice plants revealed one copy transgene insertion into the rice genome, but some with 3 copies. The expression levels of B. subtilis Protox mRNA appeared to correlate with the copy number. Furthermore, the plastidal transgenic lines exhibited much higher expression of the Protox mRNA than the cytoplasmic transgenic lines. The transgenic plants expressing the B. subtilis Protox gene at T0 generation were found to be resistant to oxyfluorfen when judged by cellular damage with respect to cellular leakage, Chl loss, and lipid peroxidation. The transgenic rice plants targeted to the plastid exhibited higher resistance to the herbicide than the transgenic plants targeted to the cytoplasm. In addition, possible resistance mechanisms in the transgenic plants to DPE herbicides are discussed.

Transgenic plants expressing the AaIT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests.

PubMed

Liu, Shu-Min; Li, Jie; Zhu, Jin-Qi; Wang, Xiao-Wei; Wang, Cheng-Shu; Liu, Shu-Sheng; Chen, Xue-Xin; Li, Sheng

2016-04-01

The adoption of pest-resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops. © 2015 Institute of Zoology, Chinese Academy of Sciences.

OsNucleolin1-L Expression in Arabidopsis Enhances Photosynthesis via Transcriptome Modification under Salt Stress Conditions.

PubMed

Udomchalothorn, Thanikarn; Plaimas, Kitiporn; Sripinyowanich, Siriporn; Boonchai, Chutamas; Kojonna, Thammaporn; Chutimanukul, Panita; Comai, Luca; Buaboocha, Teerapong; Chadchawan, Supachitra

2017-04-01

OsNUC1 encodes rice nucleolin, which has been shown to be involved in salt stress responses. Expression of the full-length OsNUC1 gene in Arabidopsis resulted in hypersensitivity to ABA during germination. Transcriptome analysis of the transgenic lines, in comparison with the wild type, revealed that the RNA abundance of >1,900 genes was significantly changed under normal growth conditions, while under salt stress conditions the RNAs of 999 genes were found to be significantly regulated. Gene enrichment analysis showed that under normal conditions OsNUC1 resulted in repression of genes involved in photosynthesis, while in salt stress conditions OsNUC1 increased expression of the genes involved in the light-harvesting complex. Correspondingly, the net rate of photosynthesis of the transgenic lines was increased under salt stress. Transgenic rice lines with overexpression of the OsNUC1-L gene were generated and tested for photosynthetic performance under salt stress conditions. The transgenic rice lines treated with salt stress at the booting stage had a higher photosynthetic rate and stomatal conductance in flag leaves and second leaves than the wild type. Moreover, higher contents of Chl a and carotenoids were found in flag leaves of the transgenic rice. These results suggest a role for OsNUC1 in the modification of the transcriptome, especially the gene transcripts responsible for photosynthesis, leading to stabilization of photosynthesis under salt stress conditions. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Transcription factors SOHLH1 and SOHLH2 coordinate oocyte differentiation without affecting meiosis I.

PubMed

Shin, Yong-Hyun; Ren, Yu; Suzuki, Hitomi; Golnoski, Kayla J; Ahn, Hyo Won; Mico, Vasil; Rajkovic, Aleksandar

2017-06-01

Following migration of primordial germ cells to the genital ridge, oogonia undergo several rounds of mitotic division and enter meiosis at approximately E13.5. Most oocytes arrest in the dictyate (diplotene) stage of meiosis circa E18.5. The genes necessary to drive oocyte differentiation in parallel with meiosis are unknown. Here, we have investigated whether expression of spermatogenesis and oogenesis bHLH transcription factor 1 (Sohlh1) and Sohlh2 coordinates oocyte differentiation within the embryonic ovary. We found that SOHLH2 protein was expressed in the mouse germline as early as E12.5 and preceded SOHLH1 protein expression, which occurred circa E15.5. SOHLH1 protein appearance at E15.5 correlated with SOHLH2 translocation from the cytoplasm into the nucleus and was dependent on SOHLH1 expression. NOBOX oogenesis homeobox (NOBOX) and LIM homeobox protein 8 (LHX8), two important regulators of postnatal oogenesis, were coexpressed with SOHLH1. Single deficiency of Sohlh1 or Sohlh2 disrupted the expression of LHX8 and NOBOX in the embryonic gonad without affecting meiosis. Sohlh1-KO infertility was rescued by conditional expression of the Sohlh1 transgene after the onset of meiosis. However, Sohlh1 or Sohlh2 transgene expression could not rescue Sohlh2-KO infertility due to a lack of Sohlh1 or Sohlh2 expression in rescued mice. Our results indicate that Sohlh1 and Sohlh2 are essential regulators of oocyte differentiation but do not affect meiosis I.

Improved production of genetically modified fetuses with homogeneous transgene expression after transgene integration site analysis and recloning in cattle.

PubMed

Bressan, Fabiana Fernandes; Dos Santos Miranda, Moyses; Perecin, Felipe; De Bem, Tiago Henrique; Pereira, Flavia Thomaz Verechia; Russo-Carbolante, Elisa Maria; Alves, Daiani; Strauss, Bryan; Bajgelman, Marcio; Krieger, José Eduardo; Binelli, Mario; Meirelles, Flavio Vieira

2011-02-01

Animal cloning by nuclear transfer (NT) has made the production of transgenic animals using genetically modified donor cells possible and ensures the presence of the gene construct in the offspring. The identification of transgene insertion sites in donor cells before cloning may avoid the production of animals that carry undesirable characteristics due to positional effects. This article compares blastocyst development and competence to establish pregnancies of bovine cloned embryos reconstructed with lentivirus-mediated transgenic fibroblasts containing either random integration of a transgene (random integration group) or nuclear transfer derived transgenic fibroblasts with known transgene insertion sites submitted to recloning (recloned group). In the random integration group, eGFP-expressing bovine fetal fibroblasts were selected by fluorescence activated cell sorting (FACS) and used as nuclei donor cells for NT. In the recloned group, a fibroblast cell line derived from a transgenic cloned fetus was characterized regarding transgene insertion and submitted to recloning. The recloned group had higher blastocyst production (25.38 vs. 14.42%) and higher percentage of 30-day pregnancies (14.29 vs. 2.56%) when compared to the random integration group. Relative eGFP expression analysis in fibroblasts derived from each cloned embryo revealed more homogeneous expression in the recloned group. In conclusion, the use of cell lines recovered from transgenic fetuses after identification of the transgene integration site allowed for the production of cells and fetuses with stable transgene expression, and recloning may improve transgenic animal yields.

Stability of Lentiviral Vector-Mediated Transgene Expression in the Brain in the Presence of Systemic Antivector Immune Responses

PubMed Central

ABORDO-ADESIDA, EVELYN; FOLLENZI, ANTONIA; BARCIA, CARLOS; SCIASCIA, SANDRA; CASTRO, MARIA G.; NALDINI, LUIGI; LOWENSTEIN, PEDRO R.

2009-01-01

Lentiviral vectors are promising tools for gene therapy in the CNS. It is therefore important to characterize their interactions with the immune system in the CNS. This work characterizes transgene expression and brain inflammation in the presence or absence of immune responses generated after systemic immunization with lentiviral vectors. We characterized transduction with SIN-LV vectors in the CNS. A dose—response curve using SIN-LV-GFP demonstrated detectable transgene expression in the striatum at a dose of 102, and maximum expression at 106, transducing units of lentiviral vector, with minimal increase in inflammatory markers between the lowest and highest dose of vector injected. Our studies demonstrate that injection of a lentiviral vector into the CNS did not cause a measurable inflammatory response. Systemic immunization after CNS injection, with the lentiviral vector expressing the same transgene as a vector injected into the CNS, caused a decrease in transgene expression in the CNS, concomitantly with an infiltration of inflammatory cells into the CNS parenchyma at the injection site. However, peripheral immunization with a lentiviral vector carrying a different transgene did not diminish transgene expression, or cause CNS inflammation. Systemic immunization preceding injection of lentiviral vectors into the CNS determined that preexisting antilentiviral immunity, regardless of the transgene, did not affect transgene expression. Furthermore, we showed that the transgene, but not the virion or vector components, is responsible for providing antigenic epitopes to the activated immune system, on systemic immunization with lentivirus. Low immunogenicity and prolonged transgene expression in the presence of preexisting lentiviral immunity are encouraging data for the future use of lentiviral vectors in CNS gene therapy. In summary, the lentiviral vectors tested induced undetectable activation of innate immune responses, and stimulation of adaptive immune responses against lentiviral vectors was effective in causing a decrease in transgene expression only if the immune response was directed against the transgene. A systemic immune response against vector components alone did not cause brain inflammation, possibly because vector-derived epitopes were not being presented in the CNS. PMID:15960605

Isolation, characterization, and evaluation of three Citrus sinensis-derived constitutive gene promoters.

PubMed

Erpen, L; Tavano, E C R; Harakava, R; Dutt, M; Grosser, J W; Piedade, S M S; Mendes, B M J; Mourão Filho, F A A

2018-05-23

Regulatory sequences from the citrus constitutive genes cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1) were isolated, fused to the uidA gene, and qualitatively and quantitatively evaluated in transgenic sweet orange plants. The 5' upstream region of a gene (the promoter) is the most important component for the initiation and regulation of gene transcription of both native genes and transgenes in plants. The isolation and characterization of gene regulatory sequences are essential to the development of intragenic or cisgenic genetic manipulation strategies, which imply the use of genetic material from the same species or from closely related species. We describe herein the isolation and evaluation of the promoter sequence from three constitutively expressed citrus genes: cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1). The functionality of the promoters was confirmed by a histochemical GUS assay in leaves, stems, and roots of stably transformed citrus plants expressing the promoter-uidA construct. Lower uidA mRNA levels were detected when the transgene was under the control of citrus promoters as compared to the expression under the control of the CaMV35S promoter. The association of the uidA gene with the citrus-derived promoters resulted in mRNA levels of up to 60-41.8% of the value obtained with the construct containing CaMV35S driving the uidA gene. Moreover, a lower inter-individual variability in transgene expression was observed amongst the different transgenic lines, where gene constructs containing citrus-derived promoters were used. In silico analysis of the citrus-derived promoter sequences revealed that their activity may be controlled by several putative cis-regulatory elements. These citrus promoters will expand the availability of regulatory sequences for driving gene expression in citrus gene-modification programs.

Exon 2-mediated c-myc mRNA decay in vivo is independent of its translation.

PubMed Central

Pistoi, S; Roland, J; Babinet, C; Morello, D

1996-01-01

We have previously shown that the steady-state level of c-myc mRNA in vivo is primarily controlled by posttranscriptional regulatory mechanisms. To identify the sequences involved in this process, we constructed a series of H-2/myc transgenic lines in which various regions of the human c-MYC gene were placed under the control of the quasi-ubiquitous H-2K class I regulatory sequences. We demonstrated that the presence of one of the two coding exons, exon 2 or exon 3, is sufficient to confer a level of expression of transgene mRNA similar to that of endogenous c-myc in various adult tissues as well as after partial hepatectomy or after protein synthesis inhibition. We now focus on the molecular mechanisms involved in modulation of expression of mRNAs containing c-myc exon 2 sequences, with special emphasis on the coupling between translation and c-myc mRNA turnover. We have undertaken an analysis of expression, both at the mRNA level and at the protein level, of new transgenic constructs in which the translation is impaired either by disruption of the initiation codon or by addition of stop codons upstream of exon 2. Our results show that the translation of c-myc exon 2 is not required for regulated expression of the transgene in the different situations analyzed, and therefore they indicate that the mRNA destabilizing function of exon 2 is independent of translation by ribosomes. Our investigations also reveal that, in the thymus, some H-2/myc transgenes express high levels of mRNA but low levels of protein. Besides the fact that these results suggest the existence of tissue-specific mechanisms that control c-myc translatability in vivo, they also bring another indication of the uncoupling of c-myc mRNA translation and degradation. PMID:8756668

Expression of the SIN3 homologue from banana, MaSIN3, suppresses ABA responses globally during plant growth in Arabidopsis.

PubMed

Luxmi, Raj; Garg, Rashmi; Srivastava, Sudhakar; Sane, Aniruddha P

2017-11-01

The SIN3 family of co-repressors is a family of highly conserved eukaryotic repressor proteins that regulates diverse functions in yeasts and animals but remains largely uncharacterized functionally even in plants like Arabidopsis. The sole SIN3 homologue in banana, MaSIN3, was identified as a 1408 amino acids, nuclear localized protein conserved to other SIN3s in the PAH, HID and HCR domains. Interestingly, MaSIN3 over-expression in Arabidopsis mimics a state of reduced ABA responses throughout plant development affecting growth processes such as germination, root growth, stomatal closure and water loss, flowering and senescence. The reduction in ABA responses is not due to reduced ABA levels but due to suppression of expression of several transcription factors mediating ABA responses. Transcript levels of negative regulators of germination (ABI3, ABI5, PIL5, RGL2 and RGL3) are reduced post-imbibition while those responsible for GA biosynthesis are up-regulated in transgenic MaSIN3 over-expressers. ABA-associated transcription factors are also down-regulated in response to ABA treatment. The HDAC inhibitors, SAHA and sodium butyrate, in combination with ABA differentially suppress germination in control and transgenic lines suggesting the recruitment by MaSIN3 of HDACs involved in suppression of ABA responses in different processes. The studies provide an insight into the ability of MaSIN3 to specifically affect a subset of developmental processes governed largely by ABA. Copyright © 2017 Elsevier B.V. All rights reserved.

Human umbilical cord mesenchymal stromal cells suppress MHC class II expression on rat vascular endothelium and prolong survival time of cardiac allograft

PubMed Central

Qiu, Ying; Yun, Mark M; Han, Xia; Zhao, Ruidong; Zhou, Erxia; Yun, Sheng

2014-01-01

Background: Human umbilical cord mesenchymal stromal cells (UC-MSCs) have low immunogenicity and immune regulation. To investigate immunomodulatory effects of human UC-MSCs on MHC class II expression and allograft, we transplanted heart of transgenic rats with MHC class II expression on vascular endothelium. Methods: UC-MSCs were obtained from human umbilical cords and confirmed with flow cytometry analysis. Transgenic rat line was established using the construct of human MHC class II transactivator gene (CIITA) under mouse ICAM-2 promoter control. The induced MHC class II expression on transgenic rat vascular endothelial cells (VECs) was assessed with immunohistological staining. And the survival time of cardiac allograft was compared between the recipients with and without UC-MSC transfusion. Results: Flow cytometry confirmed that the human UC-MSCs were positive for CD29, CD44, CD73, CD90, CD105, CD271, and negative for CD34 and HLA-DR. Repeated infusion of human UC-MSCs reduced MHC class II expression on vascular endothelia of transplanted hearts, and increased survival time of allograft. The UC-MSCs increased regulatory cytokines IL10, transforming growth factor (TGF)-β1 and suppressed proinflammatory cytokines IL2 and IFN-γ in vivo. The UC-MSC culture supernatant had similar effects on cytokine expression, and decreased lymphocyte proliferation in vitro. Conclusions: Repeated transfusion of the human UC-MSCs reduced MHC class II expression on vascular endothelia and prolonged the survival time of rat cardiac allograft. PMID:25126177

Induced over-expression of AtDREB2A CA improves drought tolerance in sugarcane.

PubMed

Reis, Rafaela Ribeiro; da Cunha, Bárbara Andrade Dias Brito; Martins, Polyana Kelly; Martins, Maria Thereza Bazzo; Alekcevetch, Jean Carlos; Chalfun, Antônio; Andrade, Alan Carvalho; Ribeiro, Ana Paula; Qin, Feng; Mizoi, Junya; Yamaguchi-Shinozaki, Kazuko; Nakashima, Kazuo; Carvalho, Josirley de Fátima Corrêa; de Sousa, Carlos Antônio Ferreira; Nepomuceno, Alexandre Lima; Kobayashi, Adilson Kenji; Molinari, Hugo Bruno Correa

2014-05-01

Drought is one of the most challenging agricultural issues limiting sustainable sugarcane production and, in some cases, yield losses caused by drought are nearly 50%. DREB proteins play vital regulatory roles in abiotic stress responses in plants. The transcription factor DREB2A interacts with a cis-acting DRE sequence to activate the expression of downstream genes that are involved in drought-, salt- and heat-stress response in Arabidopsis thaliana. In the present study, we evaluated the effects of stress-inducible over-expression of AtDREB2A CA on gene expression, leaf water potential (ΨL), relative water content (RWC), sucrose content and gas exchanges of sugarcane plants submitted to a four-days water deficit treatment in a rhizotron-grown root system. The plants were also phenotyped by scanning the roots and measuring morphological parameters of the shoot. The stress-inducible expression of AtDREB2A CA in transgenic sugarcane led to the up-regulation of genes involved in plant response to drought stress. The transgenic plants maintained higher RWC and ΨL over 4 days after withholding water and had higher photosynthetic rates until the 3rd day of water-deficit. Induced expression of AtDREB2A CA in sugarcane increased sucrose levels and improved bud sprouting of the transgenic plants. Our results indicate that induced expression of AtDREB2A CA in sugarcane enhanced its drought tolerance without biomass penalty. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Cyclin Polynucleotides, Polypeptides And Uses Thereof.

DOEpatents

Lowe, Keith S.; Tao, Yumin; Gordon-Kamm, William J.; Gregory, Carolyn A.; Hoerster, George J.; McElver, John A.

2003-02-11

The invention provides isolated polynucleotides and their encoded proteins that are involved in cell cycle regulation. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions. The present invention provides methods and compositions relating to altering cell cycle protein content and/or composition of plants.

Transgenic mouse models in the study of reproduction: insights into GATA protein function.

PubMed

Tevosian, Sergei G

2014-07-01

For the past 2 decades, transgenic technology in mice has allowed for an unprecedented insight into the transcriptional control of reproductive development and function. The key factor among the mouse genetic tools that made this rapid advance possible is a conditional transgenic approach, a particularly versatile method of creating gene deletions and substitutions in the mouse genome. A centerpiece of this strategy is an enzyme, Cre recombinase, which is expressed from defined DNA regulatory elements that are active in the tissue of choice. The regulatory DNA element (either genetically engineered or natural) assures Cre expression only in predetermined cell types, leading to the guided deletion of genetically modified (flanked by loxP or 'floxed' by loxP) gene loci. This review summarizes and compares the studies in which genes encoding GATA family transcription factors were targeted either globally or by Cre recombinases active in the somatic cells of ovaries and testes. The conditional gene loss experiments require detailed knowledge of the spatial and temporal expression of Cre activity, and the challenges in interpreting the outcomes are highlighted. These studies also expose the complexity of GATA-dependent regulation of gonadal gene expression and suggest that gene function is highly context dependent. © 2014 Society for Reproduction and Fertility.

[TSA improve transgenic porcine cloned embryo development and transgene expression].

PubMed

Kong, Qing-Ran; Zhu, Jiang; Huang, Bo; Huan, Yan-Jun; Wang, Feng; Shi, Yong-Qian; Liu, Zhong-Feng; Wu, Mei-Ling; Liu, Zhong-Hua

2011-07-01

Uncompleted epigenetic reprogramming is attributed to the low efficiency of producing transgenic cloned animals. Histone modification associated with epigenetics can directly influence the embryo development and transgene expression. Trichostatin A (TSA), as an inhibitor of histone deacetylase, can change the status of histone acetylation, improve somatic cell reprogramming, and enhance cloning efficiency. TSA prevents the chromatin structure from being condensed, so that transcription factor could binds to DNA sequence easily and enhance transgene expression. Our study established the optimal TSA treatment on porcine donor cells and cloned embryos, 250 nmol/L, 24 h and 40 nmol/L, 24 h, respectively. Furthermore, we found that both the cloned embryo and the donor cell treated by TSA resulted in the highest development efficiency. Meanwhile, TSA can improve transgene expression in donor cell and cloned embryo. In summary, TSA can significantly improve porcine reconstructed embryo development and transgene expression.

CASK and CaMKII function in Drosophila memory

PubMed Central

Malik, Bilal R.; Hodge, James J. L.

2014-01-01

Calcium (Ca2+) and Calmodulin (CaM)-dependent serine/threonine kinase II (CaMKII) plays a central role in synaptic plasticity and memory due to its ability to phosphorylate itself and regulate its own kinase activity. Autophosphorylation at threonine 287 (T287) switches CaMKII to a Ca2+ independent and constitutively active state replicated by overexpression of a phosphomimetic CaMKII-T287D transgene or blocked by expression of a T287A transgene. A second pair of sites, T306 T307 in the CaM binding region once autophosphorylated, prevents CaM binding and inactivates the kinase during synaptic plasticity and memory, and can be blocked by a TT306/7AA transgene. Recently the synaptic scaffolding molecule called CASK (Ca2+/CaM-associated serine kinase) has been shown to control both sets of CaMKII autophosphorylation events during neuronal growth, Ca2+ signaling and memory in Drosophila. Deletion of either full length CASK or just its CaMK-like and L27 domains removed middle-term memory (MTM) and long-term memory (LTM), with CASK function in the α′/ß′ mushroom body neurons being required for memory. In a similar manner directly changing the levels of CaMKII autophosphorylation (T287D, T287A, or TT306/7AA) in the α′/ß′ neurons also removed MTM and LTM. In the CASK null mutant expression of either the Drosophila or human CASK transgene in the α′/ß′ neurons was found to completely rescue memory, confirming that CASK signaling in α′/β′ neurons is necessary and sufficient for Drosophila memory formation and that the neuronal function of CASK is conserved between Drosophila and human. Expression of human CASK in Drosophila also rescued the effect of CASK deletion on the activity state of CaMKII, suggesting that human CASK may also regulate CaMKII autophosphorylation. Mutations in human CASK have recently been shown to result in intellectual disability and neurological defects suggesting a role in plasticity and learning possibly via regulation of CaMKII autophosphorylation. PMID:25009461

GmWRKY31 and GmHDL56 Enhances Resistance to Phytophthora sojae by Regulating Defense-Related Gene Expression in Soybean

PubMed Central

Fan, Sujie; Dong, Lidong; Han, Dan; Zhang, Feng; Wu, Junjiang; Jiang, Liangyu; Cheng, Qun; Li, Rongpeng; Lu, Wencheng; Meng, Fanshan; Zhang, Shuzhen; Xu, Pengfei

2017-01-01

Phytophthora root and stem rot of soybean [Glycine max (L.) Merr.] caused by the oomycete Phytophthora sojae, is a destructive disease worldwide. The molecular mechanism of the soybean response to P. sojae is largely unclear. We report a novel WRKY transcription factor (TF) in soybean, GmWRKY31, in the host response to P. sojae. Overexpression and RNA interference analysis demonstrated that GmWRKY31 enhanced resistance to P. sojae in transgenic soybean plants. GmWRKY31 was targeted to the nucleus, where it bound to the W-box and acted as an activator of gene transcription. Moreover, we determined that GmWRKY31 physically interacted with GmHDL56, which improved resistance to P. sojae in transgenic soybean roots. GmWRKY31 and GmHDL56 shared a common target GmNPR1 which was induced by P. sojae. Overexpression and RNA interference analysis demonstrated that GmNPR1 enhanced resistance to P. sojae in transgenic soybean plants. Several pathogenesis-related (PR) genes were constitutively activated, including GmPR1a, GmPR2, GmPR3, GmPR4, GmPR5a, and GmPR10, in soybean plants overexpressing GmNPR1 transcripts. By contrast, the induction of PR genes was compromised in transgenic GmNPR1-RNAi lines. Taken together, these findings suggested that the interaction between GmWRKY31 and GmHDL56 enhances resistance to P. sojae by regulating defense-related gene expression in soybean. PMID:28553307

GARP-TGF-β complexes negatively regulate regulatory T cell development and maintenance of peripheral CD4+ T cells in vivo.

PubMed

Zhou, Angela X; Kozhaya, Lina; Fujii, Hodaka; Unutmaz, Derya

2013-05-15

The role of surface-bound TGF-β on regulatory T cells (Tregs) and the mechanisms that mediate its functions are not well defined. We recently identified a cell-surface molecule called Glycoprotein A Repetitions Predominant (GARP), which is expressed specifically on activated Tregs and was found to bind latent TGF-β and mediate a portion of Treg suppressive activity in vitro. In this article, we address the role of GARP in regulating Treg and conventional T cell development and immune suppression in vivo using a transgenic mouse expressing GARP on all T cells. We found that, despite forced expression of GARP on all T cells, stimulation through the TCR was required for efficient localization of GARP to the cell surface. In addition, IL-2 signals enhanced GARP cell surface expression specifically on Tregs. GARP-transgenic CD4(+) T cells and Tregs, especially those expressing higher levels of GARP, were significantly reduced in the periphery. Mature Tregs, but not conventional CD4(+) T cells, were also reduced in the thymus. CD4(+) T cell reduction was more pronounced within the effector/memory subset, especially as the mouse aged. In addition, GARP-overexpressing CD4(+) T cells stimulated through the TCR displayed reduced proliferative capacity, which was restored by inhibiting TGF-β signaling. Furthermore, inhibiting TGF-β signals greatly enhanced surface expression of GARP on Tregs and blocked the induction of Foxp3 in activated CD4(+) T cells overexpressing GARP. These findings suggest a role for GARP in natural and induced Treg development through activation of bound latent TGF-β and signaling, which negatively regulates GARP expression on Tregs.

Clk post-transcriptional control denoises circadian transcription both temporally and spatially.

PubMed

Lerner, Immanuel; Bartok, Osnat; Wolfson, Victoria; Menet, Jerome S; Weissbein, Uri; Afik, Shaked; Haimovich, Daniel; Gafni, Chen; Friedman, Nir; Rosbash, Michael; Kadener, Sebastian

2015-05-08

The transcription factor CLOCK (CLK) is essential for the development and maintenance of circadian rhythms in Drosophila. However, little is known about how CLK levels are controlled. Here we show that Clk mRNA is strongly regulated post-transcriptionally through its 3' UTR. Flies expressing Clk transgenes without normal 3' UTR exhibit variable CLK-driven transcription and circadian behaviour as well as ectopic expression of CLK-target genes in the brain. In these flies, the number of the key circadian neurons differs stochastically between individuals and within the two hemispheres of the same brain. Moreover, flies carrying Clk transgenes with deletions in the binding sites for the miRNA bantam have stochastic number of pacemaker neurons, suggesting that this miRNA mediates the deterministic expression of CLK. Overall our results demonstrate a key role of Clk post-transcriptional control in stabilizing circadian transcription, which is essential for proper development and maintenance of circadian rhythms in Drosophila.

BMP signaling is required for development of the ciliary body.

PubMed

Zhao, Shulei; Chen, Qin; Hung, Fang-Cheng; Overbeek, Paul A

2002-10-01

The ciliary body in the eye secretes aqueous humor and glycoproteins of the vitreous body and maintains the intraocular pressure. The ciliary muscle controls the shape of the lens through the ciliary zonules to focus the image onto the retina. During embryonic development, the ciliary epithelium is derived from the optic vesicle, but the molecular signals that control morphogenesis of the ciliary body are unknown. We report that lens-specific expression of a transgenic protein, Noggin, can block BMP signaling in the mouse eye and result in failure in formation of the ciliary processes. Co-expression of transgenic BMP7 restores normal development of the ciliary epithelium. Ectopic expression of Noggin also promotes differentiation of retinal ganglion cells. These results indicate that BMP signaling is required for development of the ciliary body and may also play a role in regulation of neuronal differentiation in the developing eye.

Prototypic chromatin insulator cHS4 protects retroviral transgene from silencing in Schistosoma mansoni

PubMed Central

Suttiprapa, Sutas; Rinaldi, Gabriel; Brindley, Paul J.

2011-01-01

Vesicular stomatitis virus glycoprotein (VSVG) pseudotyped murine leukemia virus (MLV) virions can transduce schistosomes, leading to chromosomal integration of reporter transgenes. To develop VSVG-MLV for functional genomics in schistosomes, the influence of the chicken β-globin cHS4 element, a prototypic chromatin insulator, on transgene expression was examined. Plasmid pLNHX encoding the MLV 5′- and 3′-Long Terminal Repeats (LTRs) flanking the neomycin phosphotransferase gene (neo) was modified to include, within the U3 region of the 3′-LTR, active components of cHS4 insulator, the 250 bp core fused to the 400 bp 3′-region. Cultured larvae of Schistosoma mansoni were transduced with virions from producer cells transfected with control or cHS4-bearing plasmids. Schistosomules transduced with cHS4 virions expressed two to 20 times higher levels of neo than controls, while carrying comparable numbers of integrated proviral transgenes. The findings not only demonstrated that cHS4 was active in schistosomes but also they represent the first report of activity of cHS4 in any Lophotrochozoan species, which has significant implications for evolutionary conservation of heterochromatin regulation. The findings advance prospects for transgenesis in functional genomics of the schistosome genome to discover intervention targets because they provide the means to enhance and extend transgene activity including for vector based RNA interference. PMID:21918820

The RING finger/B-box factor TAM-1 and a retinoblastoma-like protein LIN-35 modulate context-dependent gene silencing in Caenorhabditis elegans.

PubMed

Hsieh, J; Liu, J; Kostas, S A; Chang, C; Sternberg, P W; Fire, A

1999-11-15

Context-dependent gene silencing is used by many organisms to stably modulate gene activity for large chromosomal regions. We have used tandem array transgenes as a model substrate in a screen for Caenorhabditis elegans mutants that affect context-dependent gene silencing in somatic tissues. This screen yielded multiple alleles of a previously uncharacterized gene, designated tam-1 (for tandem-array-modifier). Loss-of-function mutations in tam-1 led to a dramatic reduction in the activity of numerous highly repeated transgenes. These effects were apparently context dependent, as nonrepetitive transgenes retained activity in a tam-1 mutant background. In addition to the dramatic alterations in transgene activity, tam-1 mutants showed modest alterations in expression of a subset of endogenous cellular genes. These effects include genetic interactions that place tam-1 into a group called the class B synMuv genes (for a Synthetic Multivulva phenotype); this family plays a negative role in the regulation of RAS pathway activity in C. elegans. Loss-of-function mutants in other members of the class-B synMuv family, including lin-35, which encodes a protein similar to the tumor suppressor Rb, exhibit a hypersilencing in somatic transgenes similar to that of tam-1 mutants. Molecular analysis reveals that tam-1 encodes a broadly expressed nuclear protein with RING finger and B-box motifs.

GhMAP3K65, a Cotton Raf-Like MAP3K Gene, Enhances Susceptibility to Pathogen Infection and Heat Stress by Negatively Modulating Growth and Development in Transgenic Nicotiana benthamiana.

PubMed

Zhai, Na; Jia, Haihong; Liu, Dongdong; Liu, Shuchang; Ma, Manli; Guo, Xingqi; Li, Han

2017-11-21

Mitogen-activated protein kinase kinase kinases (MAP3Ks), the top components of MAPK cascades, modulate many biological processes, such as growth, development and various environmental stresses. Nevertheless, the roles of MAP3Ks remain poorly understood in cotton. In this study, GhMAP3K65 was identified in cotton, and its transcription was inducible by pathogen infection, heat stress, and multiple signalling molecules. Silencing of GhMAP3K65 enhanced resistance to pathogen infection and heat stress in cotton. In contrast, overexpression of GhMAP3K65 enhanced susceptibility to pathogen infection and heat stress in transgenic Nicotiana benthamiana . The expression of defence-associated genes was activated in transgenic N. benthamiana plants after pathogen infection and heat stress, indicating that GhMAP3K65 positively regulates plant defence responses. Nevertheless, transgenic N. benthamiana plants impaired lignin biosynthesis and stomatal immunity in their leaves and repressed vitality of their root systems. In addition, the expression of lignin biosynthesis genes and lignin content were inhibited after pathogen infection and heat stress. Collectively, these results demonstrate that GhMAP3K65 enhances susceptibility to pathogen infection and heat stress by negatively modulating growth and development in transgenic N. benthamiana plants.

SlCOR413IM1: A novel cold-regulation gene from tomato, enhances drought stress tolerance in tobacco.

PubMed

Ma, Xiaocui; Wang, Guodong; Zhao, Weiyang; Yang, Minmin; Ma, Nana; Kong, Fanying; Dong, Xinchun; Meng, Qingwei

2017-09-01

Drought stress adversely affects plant growth, development, and productivity. Genes functioning in plant response to drought stress are essential for drought tolerance. In this study, SlCOR413IM1, a cold-regulated gene isolated from Solanum lycopersium, was transferred to Nicotiana tabacum to investigate its function under drought stress. The subcellular localisation of SlCOR413IM1-GFP fusion protein in Arabidopsis protoplasts suggested that SlCOR413IM1 is a chloroplast protein. Expression analyses revealed that SlCOR413IM1 responded to drought and cold stresses. Under drought stress, transgenic plants maintained the high maximum photochemical efficiency, net photosynthetic rate (Pn) and D1 protein content of photosystem II (PSII). Compared with wild-type (WT) plants, transgenic plants showed higher superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities and proline and soluble sugar content, which reduced reactive oxygen species (ROS) generation. However, the high SOD and APX activities in transgenic plants were independent of their transcription levels. Moreover, the transgenic plants exhibited better seed germination, water status and survival, as well as lower malondialdehyde (MDA) content and relative electrical conductivity (REC) than WT plants under drought stress. Taken together, these data demonstrated that overexpression of SlCOR413IM1 enhanced drought stress tolerance in transgenic tobacco. Copyright © 2017. Published by Elsevier GmbH.

Characterization of fructose-bisphosphate aldolase regulated by gibberellin in roots of rice seedling.

PubMed

Konishi, Hirosato; Yamane, Hisakazu; Maeshima, Masayoshi; Komatsu, Setsuko

2004-12-01

Fructose-bisphosphate aldolase is a glycolytic enzyme whose activity increases in rice roots treated with gibberellin (GA). To investigate the relationship between aldolase and root growth, GA-induced root aldolase was characterized. GA3 promoted an increase in aldolase accumulation when 0.1 microM GA3 was added exogenously to rice roots. Aldolase accumulated abundantly in roots, especially in the apical region. To examine the effect of aldolase function on root growth, transgenic rice plants expressing antisense aldolase were constructed. Root growth of aldolase-antisense transgenic rice was repressed compared with that of the vector control transgenic rice. Although aldolase activity increased by 25% in vector control rice roots treated with 0.1 microM GA3, FBPA activity increased very little by 0.1 microM GA3 treatment in the root of aldolase-antisense transgenic rice. Furthermore, aldolase co-immunoprecipitated with antibodies against vacuolar H+ -ATPase in rice roots. In the root of OsCDPK13-antisense transgenic rice, aldolase did not accumulate even after treatment with GA3. These results suggest that the activation of glycolytic pathway function accelerates root growth and that GA3-induced root aldolase may be modulated through OsCDPK13. Aldolase physically associates with vacuolar H-ATPase in roots and may regulate the vacuolar H-ATPase mediated control of cell elongation that determines root length.

Cloning of B cell-specific membrane tetraspanning molecule BTS possessing B cell proliferation-inhibitory function.

PubMed

Suenaga, Tadahiro; Arase, Hisashi; Yamasaki, Sho; Kohno, Masayuki; Yokosuka, Tadashi; Takeuchi, Arata; Hattori, Takamichi; Saito, Takashi

2007-11-01

Lymphocyte proliferation is regulated by signals through antigen receptors, co-stimulatory receptors, and other positive and negative modulators. Several membrane tetraspanning molecules are also involved in the regulation of lymphocyte growth and death. We cloned a new B cell-specific tetraspanning (BTS) membrane molecule, which is similar to CD20 in terms of expression, structure and function. BTS is specifically expressed in the B cell line and its expression is increased after the pre-B cell stage. BTS is expressed in intracellular granules and on the cell surface. Overexpression of BTS in immature B cell lines induces growth retardation through inhibition of cell cycle progression and cell size increase without inducing apoptosis. This inhibitory function is mediated predominantly by the N terminus of BTS. The development of mature B cells is inhibited in transgenic mice expressing BTS, suggesting that BTS is involved in the in vivo regulation of B cells. These results indicate that BTS plays a role in the regulation of cell division and B cell growth.