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PubMed1992

Regulation of the activities of African cassava mosaic virus promoters by the AC1, AC2, and AC3 gene products.

DNA fragments comprising each of the promoter regions from the geminivirus African cassava mosaic virus (ACMV) were cloned into the pUC18-based vector, pG1, producing transcriptional fusions with the beta-glucuronidase gene (GUS) and nopaline synthase terminator sequence. The relative activity of each promoter construct was analyzed by a GUS expression assay of extracts from Nicotiana clevelandii protoplasts coelectroporated with the GUS reporter constructs and constructs in which individual ACMV open reading frames (ORFs) were placed under control of a cauliflower mosaic virus 35 S promoter. Results suggest repression of the AC1 gene by its gene product, which is required for ACMV DNA synthesis. The promoter activity observed for the single promoter for the DNA A genes encoding functions of spread and the regulation of replication (AC2 and AC3 ORFs) was unaffected by coelectroporation with any of the ACMV ORF constructs. Promoters for the AV1 (coat protein) gene and the two DNA B genes (BV1 and BC1) were activated by electroporation of the AC2 ORF construct. To a lesser extent promoters for the AV1 and BV1 genes were activated with the AC3 ORF construct. The same pattern of promoter repression and activation was observed when transgenic N. benthamiana plants expressing the GUS reporter constructions were inoculated with ACMV DNA A.

DNA-Directed DNA Polymerase↗
PubMed1993

The barley alpha-thionin promoter is rich in negative regulatory motifs and directs tissue-specific expression of a reporter gene in tobacco.

The promoter of the barley alpha-thionin gene (1.6 kb) fused to the beta-glucuronidase (GUS) gene directs temporally-controlled, tissue-specific expression in the endosperm of transgenic tobacco. The nucleotide sequence of this promoter shows negative regulatory motifs which have been functionally analyzed in other genes.

Antimicrobial Cationic Peptides↗
PubMed2006

Harnessing the potential of hairy roots: dawn of a new era.

In the past two decades, hairy root research for the production of important secondary metabolites has received a lot of attention. The addition of knowledge to overcome the limiting culture parameters of the regulation of the metabolic pathway by specific molecules and the development of novel tools for metabolic engineering now offer new possibilities to improve the hairy root technique for the production of metabolites. Furthermore, engineering hairy roots for the production of animal proteins of therapeutic interest in confined and controlled in vitro conditions is seen as one of the exciting spin-offs of the technology. Recent progress made in the scale-up of the hairy root cultures has paved the way for industrial exploitation of this system. This review highlights some of the significant progress made in the past three years and discusses the potential implications of that research.

Biotechnology↗
PubMed2000

Drug-induced hypomethylation of a posttranscriptionally silenced transgene locus of tobacco leads to partial release of silencing.

The effect of DNA methylation upon posttranscriptional gene silencing (PTGS) has been investigated in transgenic tobacco lines showing PTGS and methylation of the neomycin phosphotransferase II (nptII) reporter genes. Application of the hypomethylation drugs dihydroxypropyladenine or 5-azacytidine resulted in approximately 30% reduced methylation of cytosines located in a non-symmetrical context in the 3' untranslated region of the nptII transgenes. The hypomethylation was accompanied by up to 12-fold increase in NPTII protein levels, suggesting that methylation of non-symmetrical motifs may account for an increased degree of PTGS. Models for the possible role of DNA methylation in PTGS are discussed.

3' Untranslated Regions↗
PubMed1997

Fructan synthesis in transgenic tobacco and chicory plants expressing barley sucrose: fructan 6-fructosyltransferase.

We have recently cloned a cDNA encoding sucrose:fructan 6-fructosyltransferase (6-SFT), a key enzyme of fructan synthesis forming the beta-2,6 linkages typical of the grass fructans, graminans and phleins [Sprenger et al. (1995) Proc. Natl. Acad. Sci. USA 92, 11652-11656]. Here we report functional expression of 6-SFT from barley in transgenic tobacco and chicory. Transformants of tobacco, a plant naturally unable to form fructans, synthesized the trisaccharide kestose and a series of unbranched fructans of the phlein type (beta-2,6 linkages). Transformants of chicory, a plant naturally producing only unbranched fructans of the inulin type (beta-2,1 linkages), synthesized in addition branched fructans of the graminan type, particularly the tetrasaccharide bifurcose which is also a main fructan in barley leaves.

Fructans↗
PubMed1998

Promoter activity of the 5' flanking region of a tobacco glycolate oxidase gene in transgenic tobacco plants.

A clone harboring the 5' flanking region of a tobacco glycolate oxidase (GLO) gene was isolated from a lambda EMBL3-tobacco genomic DNA library. Primer extension analysis indicated two major transcripts with 76 and 81 bp 5' UTRs. An RT-PCR assay mapped the major mRNA transcription initiation site to thymine at position 81 upstream of the translation initiation codon. A putative TATA box spanning positions -56 to -50 upstream of the transcription initiation site was found. Promoter activity of the 5' flanking region (-3.0 kb to +82 bp) was demonstrated in tobacco plants transformed with a GLO-beta-glucuronidase (GUS) chimeric gene. Furthermore, in these transgenic plants, GUS expression patterns mimicked the expression patterns of the endogenous GLO.

Alcohol Oxidoreductases↗
PubMed2000

Characterization of the structure and expression of a highly conserved ribosomal protein gene, L9, from pea.

The eukaryotic ribosomal protein (RP) L9 is highly conserved in nature, and its gene is expressed to high levels in the actively growing tissues of pea. The transcriptional activity of the gene is highest in root, cambial and shoot meristems and immature tissues of the plant. Promoter deletion analysis using constructs employing the reporter gene gus were stably transferred into tobacco and revealed that the fully functional promoter is found in the first 316bp upstream from the start codon. Transgenic pea plants carrying one of these constructs show that translational efficiency mirrors gene transcription; gene expression appears to be developmentally regulated at the level of transcription. The coding region of the gene shares 80% amino acid homology with Arabidopsis and 76% homology with rice. Comparisons of the gene structure to that of the human, fruit fly, yeast, and Arabidopsis homologues reveal a close relationship in both promoter structure and intron insertion sites with the Arabidopsis gene. A nucleotide sequence alignment of the pea gene with other plant RP genes revealed that a sequence, -TTAGGGTTTT-, was commonly found in the forward and/or the inverted orientation at or near the TATA boxes of the promoters of these genes and may have a role in regulating the coordinate production of the RP genes in plants.

Amino Acid Sequence↗
PubMed1997

Distinct cis-acting sequences are required for the germination and sugar responses of the cucumber isocitrate lyase gene.

Recent data have shown that distinct DNA sequence elements direct the germination and sugar responses of the cucumber (Cucumis sativus, L.) malate synthase (Ms) gene (Sarah et al. (1996) Mol. Gen. Genet., 250, 153-161). Such information is, however, lacking for the isocitrate lyase (Icl) gene which is coordinately regulated with Ms. Deletions from the 5' end of the Icl promoter were therefore created specifically to address this question. Analysis of expression in seeds of transgenic Nicotiana plumbaginifolia plants showed that whereas a promoter sequence of 2.9 kilobases (kb) produced a normal germination response, deletion to -1568 base pairs (bp) dramatically reduced this response. Examination of the sugar response employed a transgenic cucumber root system. In this case, the 2900 bp and 1568 bp promoters both gave a strong sugar response, but further deletion to -1367 bp eliminated the response. Therefore, the germination and sugar responses of the Icl gene require distinct cis-acting elements, located respectively upstream and downstream of -1568 bp. This observation is consistent with distinct signal transduction systems regulating gene expression in each case.

Cucumis sativus↗
PubMed1997

Optimization of scFv antibody production in transgenic plants.

BACKGROUND: Plants offer various advantages for the production of pharmaceutical proteins over conventional production systems such as bacterial or mammalian cell culture. In order to explore transgenic plants for large-scale production and storage of recombinant antibodies we tried to optimize the accumulation and stability of functionally active single chain Fv (scFv) antibodies in transgenic tobacco plants. OBJECTIVES: Two different scFv antibodies which were expressed in different plant organs and plant cell compartments have been used for the study. Accumulation levels and antibody properties such as stability and antigen-binding activity were investigated. STUDY DESIGN: For ubiquitous expression in tobacco plants, transcription of the scFv genes was controlled by the strong cauliflower mosaic virus (CaMV) 35S promoter. We used seed specific legumin B4 (LeB4) and the unknown seed protein (USP) promoters from Vicia faba for storage organ specific expression. RESULTS: High accumulation of the two different scFv proteins in transgenic tobacco plants was only achieved by retention of the recombinant antibodies in the lumen of the endoplasmic reticulum (ER). Expression levels of scFv antibodies reached up to 4-6.8% of total soluble proteins (TSP) in leaves and up to 3-4% in ripe tobacco seeds. Transgenic tobacco seeds as well as tobacco leaves facilitated stable storage of ER-accumulated scFvs over an extended (seeds) or a short (leaves) period of time. Functionally active scFv proteins could be extracted after harvesting of the leaf material--drying and storage for 1 week at room temperature. Both the amount and the binding activity of the scFv proteins remained unchanged. CONCLUSION: A plant expression system where the scFv-proteins are targeted in the ER provides not only the highest accumulation level of active single chain Fv antibodies ever reported but also a short- or long-term storage of the foreign protein in the harvested plant material.

Chimera↗
PubMed2001

Replicating satellite RNA induces sequence-specific DNA methylation and truncated transcripts in plants.

Tobacco plants were transformed with a chimeric transgene comprising sequences encoding beta-glucuronidase (GUS) and the satellite RNA (satRNA) of cereal yellow dwarf luteovirus. When transgenic plants were infected with potato leafroll luteovirus (PLRV), which replicated the transgene-derived satRNA to a high level, the satellite sequence of the GUS:Sat transgene became densely methylated. Within the satellite region, all 86 cytosines in the upper strand and 73 of the 75 cytosines in the lower strand were either partially or fully methylated. In contrast, very low levels of DNA methylation were detected in the satellite sequence of the transgene in uninfected plants and in the flanking nonsatellite sequences in both infected and uninfected plants. Substantial amounts of truncated GUS:Sat RNA accumulated in the satRNA-replicating plants, and most of the molecules terminated at nucleotides within the first 60 bp of the satellite sequence. Whereas this RNA truncation was associated with high levels of satRNA replication, it appeared to be independent of the levels of DNA methylation in the satellite sequence, suggesting that it is not caused by methylation. All the sequenced GUS:Sat DNA molecules were hypermethylated in plants with replicating satRNA despite the phloem restriction of the helper PLRV. Also, small, sense and antisense approximately 22 nt RNAs, derived from the satRNA, were associated with the replicating satellite. These results suggest that the sequence-specific DNA methylation spread into cells in which no satRNA replication occurred and that this was mediated by the spread of unamplified satRNA and/or its associated 22 nt RNA molecules.

Base Sequence↗
PubMed1997

The promoter of the tobacco Tnt1 retrotransposon is induced by wounding and by abiotic stress.

The transcription of the tobacco Tnt1 retrotransposon was previously shown to be induced, in tobacco and in heterologous species, by microbial elicitors and by pathogen infections. We report here that the expression of the Tnt1 promoter is also activated in heterologous species such as tomato and Arabidopsis by wounding, freezing and by other abiotic factors known to induce the plant defence response, such as salicylic acid, CuCl2, or oxidative stress. A similar regulation is observed in tobacco for most treatments. The induction of the Tnt1 promoter expression by wounding remains localized around injury points. In CuCl2-treated Arabidopsis plants, the transcription of Tnt1 is correlated with accumulation of the phytoalexin camalexin and with the expression of the EL13 defence gene. The interest of the Tnt1 promoter as a sensitive indicator of the plant defence responses is discussed.

Arabidopsis↗
PubMed1997

Regulated expression of a wheat germin gene in tobacco: oxalate oxidase activity and apoplastic localization of the heterologous protein.

Wheat (Triticum aestivum) germin is a homopentameric glycoprotein whose synthesis is allied with seed germination. Germin pentamers show an unusual resistance to dissociation and possess an oxalate oxidase (OxO) activity. In order to increase our knowledge of germin gene expression, the function(s) of germin during development and possible uses in plant genetic engineering, an in vivo expression system is required. To this end, a gene for germin, named gf-2.8, was studied by expressing either promoter-GUS fusions or the intact gene in transgenic tobacco (Nicotiana tabacum) plants. Heterologous gene transcription was monitored in vitro and in vivo by GUS or OxO activity and was found to occur in developing seeds and in seedlings. This transcription was stimulated by auxins, as would be expected because of the presence of putative auxin-responsive elements in the promoter of the gf-2.8 gene. Auxin stimulation also extended to young leaves since OxO activity could be detected in treated but not in untreated leaves. The biochemical characteristics of wheat germin were also conserved in a transgenic host: the OxO activity was present under the form of a doublet co-migrating with germin G and G' isoforms. Also, germin distributed between a soluble and an apoplastic fractions despite the fact that wheat cell wall substantially differs from tobacco cell wall. Therefore, tobacco constitutes a suitable host for in vivo studies of this monocotyledon gene.

Amino Acid Sequence↗
PubMed1997

Structural characteristics of two wheat histone H2A genes encoding distinct types of variants and functional differences in their promoter activity.

To investigate the regulation of plant histone H2A gene expression, we isolated two H2A genes (TH254 and TH274) from wheat, which encode two variants of H2A. Both genes had an intron in the coding region. In the promoters, some characteristic sequences, such as Oct and Nona motifs, which are conserved among plant histone genes, were located in a short region (about 120 bp) upstream from the putative TATA box. Transient expression analyses of promoter activity with H2A-GUS fusion genes using tobacco protoplasts revealed novel types of positive cis-acting sequences in the TH254 promoter: a direct repeat of a 13 bp sequence (AGTTACATTATTG) and a stretch composed of an AT-rich sequence (ATATAGAAAATTAAAA) and a G-box (CACGTG). Quantitative S1 assay of the mRNA amounts from the TH254/GUS and TH274/GUS chimeric genes in stably transformed and cell cycle-synchronized tobacco cell lines showed that the promoters of both genes contained at least one cis-acting element responsible for S phase-specific expression. Histochemical analysis of transgenic tobacco plants carrying the chimeric genes showed that the promoters of the two H2A genes were active in developing seedlings and flower organs but were regulated in a different manner.

Amino Acid Sequence↗
PubMed1997

A promoter directing epidermal expression in transgenic alfalfa.

We describe the isolation, cloning and expression of a 2.8 kb promoter fragment of the Blec4 gene from pea (Pisum sativum cv. Alaska) and demonstrate that it is capable of directing the expression of the beta-glucuronidase coding region to the developing epidermal tissue of vegetative and floral shoot apices of transgenic alfalfa (Medicago sativa cv. RegenSY). The Blec4 promoter represents a useful tool with which to target the expression of foreign genes to the epidermal layer of actively growing shoots. The activity of the Blec4 promoter in the epidermis of the shoot apex makes it particularly suitable for genetically engineering defense against insects and diseases that attack the growing shoot apex.

Fabaceae↗
PubMed1997

Characterization of a barley gene coding for an alpha-amylase inhibitor subunit (CMd protein) and analysis of its promoter in transgenic tobacco plants and in maize kernels by microprojectile bombardment.

A gene coding for a barley CMd protein was isolated from a genomic library using a cDNA probe encoding the wheat CM3 protein. Promoter sequence analysis reveals motifs found in genes specifically expressed in endosperm and aleurone cells, as well as TATA and other putative functional boxes. 720 bp of the Hv85.1 CMd protein gene promoter, when fused to a gus coding region, were unable to direct GUS activity in the seeds of transgenic tobacco plants. In contrast, the same construction delivered into immature maize kernels by microprojectile bombardment was able to direct expression of GUS in the outermost cell layers of maize endosperm in both a tissue-specific and a developmentally determined manner.

Amino Acid Sequence↗
PubMed1997

Tissue-specific activation of the osmotin gene by ABA, C2H4 and NaCl involves the same promoter region.

The gene encoding the antifungal protein osmotin is induced by several hormonal and environmental signals. In this study, tissue-specific and inducer-mediated expression of the reporter gene beta-glucuronidase (uidA) fused to different fragment lengths of the osmotin promoter was evaluated in transgenic tobacco (Nicotiana tabacum). The region of the promoter between -248 to -108 (Fragment A) was found to be essential and sufficient for inducer (abscisic acid (ABA), C2H4 and NaCl)-mediated expression of the reporter gene. Expression of the reporter gene was developmentally regulated and increased with maturity of leaves, stem and flowers. Expression also was tissue-specific being most highly expressed in epidermis and vascular parenchyma of the stem. The regulators ABA, C2H4 and NaCl exhibited tissue-specific induction of this promoter. The promoter was specifically responsive to C2H4 in flowers at virtually all stages of development, but not responsive in these tissues to ABA or NaCl. Conversely, ABA and NaCl were able to induce reporter gene activity using promoter Fragment A in specific tissues of root where C2H4 was unable to induce activity. Further dissection of the promoter Fragment A into fragments containing either the conserved GCC element (PR); PR/AT; or G/AT sequences, and subsequent testing of these fragments fused to GUS in transgenic plants was performed. These experiments revealed that the promoter fragment containing PR element alone, although required, was barely able to allow responsiveness to C2H4. However, significant C2H4-induced activity was obtained with a promoter fragment containing the AT and PR elements together.

Abscisic Acid↗
PubMed1997

An ozone-responsive region of the grapevine resveratrol synthase promoter differs from the basal pathogen-responsive sequence.

Stilbene synthase (STS) is an enzyme involved in the biosynthesis of stilbenes, which are synthesized in various plants in response to pathogen attack, UV irradiation or exposure to ozone. We describe analysis of an ozone inducible STS transcript and its corresponding promoter (Vst1), combined with the beta-glucuronidase (GUS) reporter gene. A single ozone pulse (0.1 microliter/l, 10 h) resulted in 11-fold GUS expression. Histochemical localization of GUS activity revealed small spots distributed over the whole leaf. Cross-sections of leaf tissue showed that the Vst1 promoter was induced in palisade and spongy parenchyma cells and to a lesser extent in epidermal cells. Deletions at the 5' end showed that a partial promoter sequence between position -430 and -280 constituted the ozone-responsive region, whereas for effective pathogen-inducibility sequences from -280 to -140 have been shown to be necessary.

Acyltransferases↗