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PubMed2000

Tobacco ribosomal DNA spacer element stimulates amplification and expression of heterologous genes.

Here we show that the cis-acting genetic element aps (amplification-promoting sequence), isolated from the nontranscribed spacer region of tobacco ribosomal DNA (rDNA), increases the level of expression of recombinant proteins. Transgenic tobacco plants, transformed with expression cassettes containing the herbicide-resistant acetolactate synthase (hr-ALS) gene or the green fluorescent protein (GFP) gene fused to the aps sequence, had greater levels of corresponding messenger RNAs (mRNAs) and proteins compared to transformants lacking aps. Analysis of transgenic plants showed that aps increased the copy number and transcription of the adjacent heterologous genes and, in the case of hr-ALS, enhanced the herbicide resistance phenotype. Both the increased transgene copy number and enhanced expression were stably inherited. These data provide the first evidence that the aps sequence can be used for gene amplification in transgenic plants and possibly other multicellular organisms.

Acetolactate Synthase↗

PubMed1993

The pea plastocyanin promoter directs cell-specific but not full light-regulated expression in transgenic tobacco plants.

A series of 5' deletions of the pea plastocyanin gene (petE) promoter fused to the beta-glucuronidase (GUS) reporter gene has been examined for expression in transgenic tobacco plants. Strong positive and negative cis-elements which modulate quantitative expression of the transgene in the light and the dark have been detected within the petE promoter. Disruption of a negative regulatory element at -784 bp produced the strongest photosynthesis-gene promoter so far described. Histochemical analysis demonstrated that all petE-GUS constructs directed expression in chloroplast-containing cells, and that a region from -176 bp to +4 bp from the translation start site was sufficient for such cell-specific expression. The petE-promoter fusions were expressed at high levels in etiolated transgenic tobacco seedlings but there was no marked induction of GUS activity in the light. The endogenous tobacco plastocyanin genes and the complete pea plastocyanin gene in transgenic tobacco plants were also expressed in the dark, but showed a three- to sevenfold increase in the light. This indicates a requirement for sequences 3' to the promoter for the full light response of the petE gene.

Base Sequence↗

PubMed1995

Targeted recombination in plants using Agrobacterium coincides with additional rearrangements at the target locus.

The use of Agrobacterium for gene targeting in plants has been investigated. Leaf protoplasts of five transgenic tobacco lines, containing a T-DNA insertion with a defective npt-II gene at different positions in the plant genome, were transformed via Agrobacterium with a T-DNA containing a npt-II repair gene. After selection for kanamycin resistance and PCR analysis six recombinants were derived from four of the target lines. The recombination frequencies were similar for the different target lines with one recombinant from approximately 3 x 10(5) transformants. Apparently gene targeting is more or less independent of the location of the target construct in the plant genome. Molecular analysis revealed that gene targeting had occurred in five of the six recombinant lines. However precise recombination had occurred in only one line, while in the other four lines restoration of the npt-II gene was accompanied by a deletion of part of the target locus. The sixth recombinant line showed restoration of the npt-II gene of the incoming T-DNA construct which was inserted in the plant genome at a position closely linked to the target locus. The different recombination products favour a model in which recombination is via gene conversion followed by reintegration of the synthesized DNA via homologous or illegitimate recombination rather than a reciprocal exchange of DNA between two cross-overs.

Agrobacterium tumefaciens↗

PubMed1995

Expression of a chimeric uidA gene indicates that polycistronic mRNAs are efficiently translated in tobacco plastids.

Polycistronic mRNAs are the predominant form of plastid primary transcripts. To determine if there is internal initiation of translation of promoter-distal open reading frames, a promoterless uidA reporter gene was integrated into the tobacco plastid genome downstream of the rbcL gene. Monocistronic uidA mRNA does not accumulate from the promoterless uidA construct. However, due to inefficient rbcL transcription termination, a polycistronic transcription unit is created that contains the uidA gene as the second cistron. Numerous stop codons in all three reading frames between the rbcL and uidA coding regions ensure that translation of uidA initiates only from the correct start codon. The encoded reporter gene product, beta-glucuronidase (GUS) accumulates to high levels in the transplastomic plants indicating that promoter-distal cistrons can be efficiently translated in plastids.

Base Sequence↗

PubMed1997

Gene targeting and instability of Agrobacterium T-DNA loci in the plant genome.

To develop a model system for studies of homologous recombination in plants, transgenic Nicotiana tabacum and Nicotiana plumbaginifolia lines were generated harbouring a single target T-DNA containing the negative selective codA gene encoding cytosine deaminase (CD) and the beta-glucuronidase (GUS) gene. Subsequently, the target lines were transformed with a replacement-type T-DNA vector in which the CD gene and the GUS promoter had been replaced with a kanamycin-resistance gene. For both Nicotiana species kanamycin-resistant lines were selected which had lost the CD gene and the GUS activity. One tobacco line was the result of a precise gene targeting event. However, most other lines were selected due to a chromosomal deletion of the target locus. The deletion frequency of the target locus varied between target lines, and could be present in up to 20% of the calli which were grown from leaf protoplasts. T-DNA transfer was not required for induction of the deletions, indicating that the target loci were unstable. A few lines were obtained in which the target locus had been deleted partially. Sequence analysis of the junctions revealed deletion of DNA sequences between microhomologies. We conclude that T-DNAs, which are stable during plant development as well as in transmission to the offspring, may become unstable during propagation in callus tissue. The relationships between callus culture, genetic instability and the process of T-DNA integration and deletion in the plant genome are discussed.

Base Sequence↗

PubMed1997

Integration of T-DNA binary vector 'backbone' sequences into the tobacco genome: evidence for multiple complex patterns of integration.

During the process of crown gall tumorigenesis, Agrobacterium tumefaciens transfers part of the tumor-inducing (Ti) plasmid, the T-DNA, to a plant cell where it eventually becomes stably integrated into the plant genome. Directly repeated DNA sequences, called T-DNA borders, define the left and the right ends of the T-DNA. The T-DNA can be physically separated from the remainder of the Ti-plasmid, creating a 'binary vector' system; this system is frequently used to generate transgenic plants. Scientists initially thought that only those sequences located between T-DNA left and right borders transferred to the plant. More recently, however, several reports have appeared describing the integration of the non-T-DNA binary vector 'backbone' sequences into the genome of transgenic plants. In order to investigate this phenomenon, we constructed T-DNA binary vectors containing a nos-nptll gene within the T-DNA and a mas2'-gusA (beta-glucuronidase) gene outside the T-DNA borders. We regenerated kanamycin-resistant transgenic tobacco plants and analyzed these plants for the expression of the vector-localized gusA gene and for the presence of binary vector backbone sequences. Approximately one-fifth of the plants expressed detectable GUS activity. PCR analysis indicated that approximately 75% of the plants contained the gusA gene. Southern blot analysis indicated that the vector backbone sequences could integrate into the tobacco genome linked either to the left or to the right T-DNA border. The vector backbone sequences could also integrate into the plant genome independently of (unlinked to) the T-DNA. Although we could readily detect T-strands containing the T-DNA within the bacterium, we could not detect T-strands containing only the vector backbone sequences or these vector sequences linked to the T-DNA.

Agrobacterium tumefaciens↗

PubMed1998

Dual regulation of a heat shock promoter during embryogenesis: stage-dependent role of heat shock elements.

Transgenic tobacco expression was analysed of chimeric genes with point mutations in the heat shock element (HSE) arrays of a small heat shock protein (sHSP) gene from sunflower: Ha hsp17.7 G4. The promoter was developmentally regulated during zygotic embryogenesis and responded to heat stress in vegetative tissues. Mutations in the HSE affected nucleotides crucial for human heat shock transcription factor 1 (HSF1) binding. They abolished the heat shock response of Ha hsp17.7 G4 and produced expression changes that demonstrated dual regulation of this promoter during embryogenesis. Thus, whereas activation of the chimeric genes during early maturation stages did not require intact HSE, expression at later desiccation stages was reduced by mutations in both the proximal (-57 to -89) and distal (-99 to -121) HSE. In contrast, two point mutations in the proximal HSE that did not severely affect gene expression during zygotic embryogenesis, eliminated the heat shock response of the same chimeric gene in vegetative organs. Therefore, by site-directed mutagenesis, it was possible to separate the heat shock response of Ha hsp17.7 G4 from its developmental regulation. The results indicate the co-existence, in a single promoter, of HSF-dependent and -independent regulation mechanisms that would control sHSP gene expression at different stages during plant embryogenesis.

Base Sequence↗

PubMed2000

Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase.

During the course of a search for cDNAs encoding plant sterol acyltransferases, an expressed sequence tag clone presenting substantial identity with yeast and animal acyl CoA:cholesterol acyltransferases was used to screen cDNA libraries from Arabidopsis and tobacco. This resulted in the isolation of two full-length cDNAs encoding proteins of 520 and 532 amino acids, respectively. Attempts to complement the yeast double-mutant are1 are2 defective in acyl CoA:cholesterol acyltransferase were unsuccessful, showing that neither gene encodes acyl CoA:cholesterol acyltransferase. Their deduced amino acid sequences were then shown to have 40 and 38% identity, respectively, with a murine acyl CoA:diacylglycerol acyltransferase and their expression in are1 are2 or wild-type yeast resulted in a strong increase in the incorporation of oleyl CoA into triacylglycerols. Incorporation was 2-3 times higher in microsomes from yeast transformed with these plant cDNAs than in yeast transformed with the void vector, clearly showing that these cDNAs encode acyl CoA:diacylglycerol acyltransferases. Moreover, during the preparation of microsomes from the Arabidopsis DGAT-transformed yeast, a floating layer was observed on top of the 100 000 g supernatant. This fraction was enriched in triacylglycerols and exhibited strong acyl CoA:diacylglycerol acyltransferase activity, whereas almost no activity was detected in the corresponding clear fraction from the control yeast. Thanks to the use of this active fraction and dihexanoylglycerol as a substrate, the de novo synthesis of 1,2-dihexanoyl 3-oleyl glycerol by AtDGAT could be demonstrated. Transformation of tobacco with AtDGAT was also performed. Analysis of 19 primary transformants allowed detection, in several individuals, of a marked increase (up to seven times) of triacylglycerol content which correlated with the AtDGAT mRNA expression. Furthermore, light-microscopy observations of leaf epidermis cells, stained with a lipid-specific dye, showed the presence of lipid droplets in the cells of triacylglycerol-overproducer plants, thus illustrating the potential application of acyl CoA:diacylglycerol acyltransferase-transformed plants.

Acyl Coenzyme A↗

PubMed1996

Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination.

Genomic double-strand breaks (DSBs) are key intermediates in recombination reactions of living organisms. We studied the repair of genomic DSBs by homologous sequences in plants. Tobacco plants containing a site for the highly specific restriction enzyme I-Sce I were cotransformed with Agrobacterium strains carrying sequences homologous to the transgene locus and, separately, containing the gene coding for the enzyme. We show that the induction of a DSB can increase the frequency of homologous recombination at a specific locus by up to two orders of magnitude. Analysis of the recombination products demonstrates that a DSB can be repaired via homologous recombination by at least two different but related pathways. In the major pathway, homologies on both sides of the DSB are used, analogous to the conservative DSB repair model originally proposed for meiotic recombination in yeast. Homologous recombination of the minor pathway is restricted to one side of the DSB as described by the nonconservative one-sided invasion model. The sequence of the recombination partners was absolutely conserved in two cases, whereas in a third case, a deletion of 14 bp had occurred, probably due to DNA polymerase slippage during the copy process. The induction of DSB breaks to enhance homologous recombination can be applied for a variety of approaches of plant genome manipulation.

Base Sequence↗

PubMed1993

TnpA trans-activates methylated maize Suppressor-mutator transposable elements in transgenic tobacco.

The maize Suppressor-mutator (Spm) transposable element is subject to epigenetic inactivation in transgenic tobacco, as it is in maize. Spm inactivation in tobacco is correlated with increased methylation of sequences near the element's transcription start site. To determine whether element-encoded gene products can promote the reactivation of an inactive element, we investigated the effects of introducing individual CaMV 35S promoter-driven cDNAs for tnpA, tnpB, tnpC and tnpD, the element's four known protein-coding sequences. Introduction of the tnpA cDNA promoted the reactivation of the inactive resident Spm element, as judged by the appearance of regenerants with very early excision events and transposed elements. By contrast, the tnpB, tnpC and tnpD cDNAs had no affect on the activity of the resident Spm element. Similar results were obtained when the element-encoded cDNAs were introduced either by Agrobacterium-mediated retransformation or by a genetic cross. Reactivation of an inactive Spm by the tnpA cDNA is accompanied by reduced methylation of several methylation-sensitive restriction sites near the element's transcription start site. Maintenance of the reactivated Spm element in an active state requires the continued presence of the tnpA cDNA. Elimination of the tnpA cDNA locus by genetic segregation generally results in decreased element activity, as judged by a low frequency of excision events, and is accompanied by increased methylation of the element's 5'-end. Exceptions resembling the phenomenon of "presetting" are also observed in which progeny plants that did not receive the tnpA cDNA locus after meiotic segregation maintain high excision activity and exhibit low methylation levels.

Crosses, Genetic↗

PubMed1998

The phage-type PclpP-53 plastid promoter comprises sequences downstream of the transcription initiation site.

The existence of a phage-type plastid transcription machinery (NEP), related to the mitochondrial RNA polymerase, has been recognized only recently. Here we report the cis sequences required for transcription initiation by the phage-type enzyme. The promoter chosen for the study, PclpP-53, is well expressed in tobacco leaves, unlike most NEP promoters. Promoter definition was carried out in vivo , in transplastomic tobacco plants expressing a uidA reporter gene from PclpP-53 promoter derivatives. We report here that sequences from -5 to +25 (relative to the transcription initiation site) are sufficient to support specific transcription initiation. Requirement of sequences downstream of the transcription initiation site contrasts with mitochondrial promoters, which have conserved sequences predominantly upstream. The promoter defined here is conserved in liverworts and conifers, indicating that the phage-type transcription machinery appeared in plastids early on during the evolution of land plants. The PclpP-53 promoter sequences are present in rice but do not function, suggesting that PclpP-53 recognition specificity is absent in some monocots.

Bacteriophages↗

PubMed1998

Expression of human alpha-lactalbumin in transgenic tobacco.

alphaLA-Lactalbumin (alphaLA), a major milk protein, is the regulatory subunit of lactose synthase. To assess the production of recombinant alphaLA in plants, the cDNAs for human alphaLA with or without its own signal sequence were introduced into tobacco plants under the control of the cauliflower mosaic virus 35S promoter. The gene integration and expression at the mRNA level were confirmed in several regenerated plants, while the expression at the protein level could be confirmed only in a transgenic tobacco transformed with the gene containing the signal sequence. The tobacco-expressed alphaLA migrated in SDS-PAGE with identical mobility to alphaLA prepared from human milk, indicating that the signal peptide of human alphaLA was correctly processed to yield a mature protein in tobacco plants. The expressed alphaLA (ca. 5 microg/g of fresh leaves) was found in the soluble fraction and eluted from a DEAE-Sepharose column in the same salt concentration range as the milk alphaLA. The partially purified tobacco-alphaLA was fully active in the synthesis of lactose when combined with galactosyltransferase. Thus, the transgenic tobacco produces a fully active mature alphaLA in a soluble form.

Cloning, Molecular↗

PubMedDate not supplied

Expression of a viral avirulence gene in transgenic plants is sufficient to induce the hypersensitive defense reaction.

Tobacco plants containing the N' resistance gene exhibit a hypersensitive defense reaction when infected with tomato mosaic virus (ToMV); infection results in necrotic lesions at the primary infection sites. In an attempt to investigate the molecular mechanism(s) underlying this plant-pathogen interaction, the ToMV coat protein gene was joined by a transcriptional fusion to the strong constitutive 35S RNA promoter from cauliflower mosaic virus. This chimeric gene was introduced via Agrobacterium-mediated transformation into isogenic tobacco cultivars differing only with respect to the N' gene. Strong necrotic reactions were observed on most emerging calli of the N' genotype, but never on calli lacking the N' resistance gene. These data indicate that the coat protein of ToMV is, on its own, sufficient to induce a hypersensitive reaction in tobacco. Thus, recognition of a single viral gene product may be the only prerequisite for the induction of a specific defense reaction in higher plants.

Base Sequence↗

PubMedDate not supplied

Broad resistance to tospoviruses in transgenic tobacco plants expressing three tospoviral nucleoprotein gene sequences.

Transgenic tobacco plants have been obtained expressing nucleoprotein (N) gene sequences of three different tospoviruses known to affect vegetable crops: tomato spotted wilt virus (TSWV), tomato chlorotic spot virus (TCSV), and groundnut ringspot virus (GRSV). The chimeric plant transformation vector used comprised the three viral N gene sequences, each with a copy of the CaMV 35S promoter and the nos terminator. Despite the high levels of homology between the different N gene sequences (74-82%) and the presence of repeated promoter and terminator sequences in this construct, unrearranged copies of this triple N gene construct were stably maintained in both Escherichia coli and Agrobacterium tumefaciens plasmids used during the cloning process, as well as in several generations of transgenic tobacco plants. A transgenic tobacco line was obtained that exhibited high levels of resistance to all three tospoviruses, showing the possibility of producing transgenic plants with a broad resistance to tospoviruses by introducing tandemly cloned viral N gene sequences. DNA analysis of this transgenic plant line shows that the multivirus resistance trait is confined to a single genetic locus, which is very convenient for further breeding purposes.

Agrobacterium tumefaciens↗

PubMedDate not supplied

RNA-mediated resistance with nonstructural genes from the tobacco etch virus genome.

Sense RNA-mediated virus resistance has been described for transgenic plants expressing potyviral capsid protein sequences. This study was undertaken to determine if expression of other viral sequences could induce this type of virus resistance. Plants showing highly resistant or 'recovery' phenotypes were generated by expressing the tobacco etch virus (TEV) 6 kDa/21 kDa reading frames. Expression of translatable or untranslatable versions of this TEV sequence produced resistant lines. Highly resistant and recovery phenotype plants expressing TEV coat protein sequences. High transcription rates with low steady-state levels of the transgene transcript generally correlated with resistance. During recovery and induction of the resistance, RNA and protein steady-state levels decreased 5- to 20-fold, while transcription of the transgene continued at a similar level. A posttranscriptional, cellular system eliminating sequences contained in the transgene transcript and viral genome would be consistent with the results.

Base Sequence↗

PubMed2000

Heterologous rhizobial lipochitin oligosaccharides and chitin oligomers induce cortical cell divisions in red clover roots, transformed with the pea lectin gene.

Division of cortical cells in roots of leguminous plants is triggered by lipochitin oligosaccharides (LCOs) secreted by the rhizobial microsymbiont. Previously, we have shown that presence of pea lectin in transgenic white clover hairy roots renders these roots susceptible to induction of root nodule formation by pea-specific rhizobia (C. L. Díaz, L. S. Melchers, P. J. J. Hooykaas, B. J. J. Lugtenberg, and J. W. Kijne, Nature 338:579-581, 1989). Here, we report that pea lectin-transformed red clover hairy roots form nodule primordium-like structures after inoculation with pea-, alfalfa-, and Lotus-specific rhizobia, which normally do not nodulate red clover. External application of a broad range of purified LCOs showed all of them to be active in induction of cortical cell divisions and cell expansion in a radial direction, resulting in formation of structures that resemble nodule primordia induced by clover-specific rhizobia. This activity was obvious in about 50% of the red clover plants carrying hairy roots transformed with the pea lectin gene. Also, chitopentaose, chitotetraose, chitotriose, and chitobiose were able to induce cortical cell divisions and cell expansion in a radial direction in transgenic roots, but not in control roots. Sugar-binding activity of pea lectin was essential for its effect. These results show that transformation of red clover roots with pea lectin results in a broadened response of legume root cortical cells to externally applied potentially mitogenic oligochitin signals.

Cell Division↗

PubMed1993

Expression of a 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Camptotheca acuminata is differentially regulated by wounding and methyl jasmonate.

We have isolated a gene, hmg1, for 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) from Camptotheca acuminata, a Chinese tree that produces the anti-cancer monoterpenoid indole alkaloid camptothecin (CPT). HMGR supplies mevalonate for the synthesis of the terpenoid component of CPT as well as for the formation of many other primary and secondary metabolites. In Camptotheca, hmg1 transcripts were detected only in young seedlings and not in vegetative organs of older plants. Regulation of the hmg1 promoter was studied in transgenic tobacco using three translational fusions (-1678, -1107, -165) with the beta-glucuronidase (GUS) reporter gene. Histochemical analysis of plants containing each of the three promoter fusions showed similar developmental and spatial expression patterns. In vegetative tissues, GUS staining was localized to the epidermis of young leaves and stems, particularly in glandular trichomes. Roots showed intense staining in the cortical tissues in the elongation zone and light staining in the cortex of mature roots. hmg1::GUS expression was also observed in sepals, petals, pistils, and stamens of developing flowers, with darkest staining in the ovary wall, ovules, stigmas, and pollen. Leaf discs from plants containing each of the translational fusions showed a 15- to 20-fold wound induction of hmg1::GUS expression over 72 h; however, this increase in GUS activity was completely suppressed by treatment with methyl jasmonate. Taken together, these data show that a 165-bp fragment of Camptotheca hmg1 promoter is sufficient to confer developmental regulation as well as wound induction and methyl jasmonate suppression of GUS expression in transgenic tobacco.

Acetates↗

PubMed1995

Light-dependent and tissue-specific expression of the H-protein of the glycine decarboxylase complex.

Glycine decarboxylase is a mitochondrial enzyme complex, which is the site of photorespiratory CO2 and NH3 release. Although the proteins that constitute the complex are located within the mitochondria, because of their intimate association with photosynthesis their expression is controlled by light. Comparisons of the kinetics of mRNA accumulation between the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and the H-protein of glycine decarboxylase during the greening of etiolated Arabidopsis thaliana suggest that their expression is controlled in parallel. A genomic clone for the H-protein (gdcH) was isolated from Arabidopsis and sequenced. The upstream region from -856 to +62 was fused to the beta-glucuronidase (GUS) reporter gene, and this construct was transformed into tobacco. This 5' upstream regulatory region appears to control GUS expression in a manner very similar to that of the endogenous H-protein gene. Constructs with deletions in the 5' upstream region were transformed into tobacco. These deletions revealed that light-dependent and tissue-specific expression was largely controlled by a 259-bp region between -376 and -117 bp. This region contains several putative GT boxes with the GGTTAA consensus core sequence. Once these strong light-dependent elements were removed, a second level of control was revealed. In constructs in which the gdcH 5' regulatory region was shortened to -117 bp or less, there was more GUS activity in the roots than in the leaves, and in dark-grown plants than in light-grown plants. This suggests that more proximal control elements may be responsible for the constitutive low levels of gene expression noted in all nonphotosynthetic tissues.

Amino Acid Oxidoreductases↗