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PubMed1996

Bioproduction of human enzymes in transgenic tobacco.

Transgenic plants have significant potential in the bioproduction of complex human therapeutic proteins due to ease of genetic manipulation, lack of potential contamination with human pathogens, conservation of eukaryotic cell machinery mediating protein modification, and low cost of biomass production. Tobacco has been used as our initial transgenic system because Agrobacterium-mediated transformation is highly efficient, prolific seed production greatly facilitates biomass scale-up, and development of new "health-positive" uses for tobacco has significant regional support. We have targeted bioproduction of complex recombinant human proteins with commercial potential as human pharmaceuticals. Human protein C (hPC), a highly processed serum protease of the coagulation/anticoagulation cascade, was produced at low levels in transgenic tobacco leaves. Analogous to its processing in mammalian systems, tobacco-synthesized hPC appears to undergo multiple proteolytic cleavages, disulfide bond formation, and N-linked glycosylation. Although tobacco-derived hPC has not yet been tested for all posttranslational modifications or for enzymatic (anticlotting) activity, these results are promising and suggest considerable conservation of protein processing machinery between plants and animals. CropTech researchers have also produced the human lysosomal enzyme glucocerebrosidase (hGC) in transgenic tobacco. This glycoprotein has significant commercial potential as replacement therapy in patients with Gaucher's disease. Regular intravenous administration of modified glucocerebrosidase, derived from human placentae or CHO cells, has proven highly effective in reducing disease manifestations in patients with Gaucher's disease. However, the enzyme is expensive (dubbed the "world's most expensive drug" by the media), making it a dramatic model for evaluating the potential of plants to provide a safe, low-cost source of bioactive human enzymes. Transgenic tobacco plants were generated that contained the human glucocerebrosidase cDNA under the control of an inducible plant promoter. hGC expression was demonstrated in plant extracts by enzyme activity assay and immunologic cross-reactivity with anti-hGC antibodies. Tobacco-synthesized hGC comigrates with human placental-derived hGC during electrophoretic separations, is glycosylated, and, most significantly, is enzymatically active. Although expression levels vary depending on transformant and induction protocol, hGC production of > 1 mg/g fresh weight of leaf tissue has been attained in crude extracts. Our studies provide strong support for the utilization of tobacco for high-level production of active hGC for purification and eventual therapeutic use at potentially much reduced costs. Furthermore, this technology should be directly adaptable to the production of a variety of other complex human proteins of biologic and pharmaceutical interest.

Enzymes↗

PubMed1996

The 5' terminal sequence of alfalfa mosaic virus RNA 3 is dispensable for replication and contains a determinant for symptom formation.

Transgenic P12 tobacco plants, transformed with the replicase genes P1 and P2 of alfalfa mosaic virus (AIMV), can be infected with RNA 3 of the tripartitite AIMV genome or with a DNA copy of RNA 3 fused to the CaMV 35S promoter and nos terminator. The effect of various modifications on the infectivity of the 35S/cDNA 3 construct to P12 plants was studied. When nonviral sequences ranging from 11 to 200 bp were inserted between the 35S promoter and cDNA 3, the infection became dependent on addition of coat protein (CP) to the inoculum. About 80% of the progeny RNAs resulting from these infections were full-length and had lost the nonviral sequence, whereas 20% were truncated by a deletion of the 5' terminal 79 nucleotides (nt). When the sequence corresponding to the 5' terminal 22 nt of RNA 3 was deleted from the 35S/cDNA 3 construct, the clone was as infectious as the wild type (wt), provided that CP was added to the inoculum, but only progeny RNA with a 5' terminal deletion of 79 nt was produced. The 5' truncated RNA 3 molecules induced necrotic ringspot-like symptoms on P12 tobacco plants, whereas wt RNA 3 did not induce detectable symptoms on these plants. It is proposed that in the infections with the modified 35S/cDNA 3 clones, CP is required in the inoculum to permit internal initiation of plus-strand RNA 3 synthesis on 3'-extended or 3'-truncated minus-strand RNA templates. Evidence was obtained that minus-strand RNA 3 synthesized under the control of the 35S promoter was not infectious to P12 plants.

Alfalfa mosaic virus↗

PubMed1989

Endoplasmic reticulum targeting and glycosylation of hybrid proteins in transgenic tobacco.

The correct compartmentation of proteins to the endomembrane system, mitochondria, or chloroplasts requires an amino-terminal signal peptide. The major tuber protein of potato, patatin, has a signal peptide in common with many other plant storage proteins. When the putative signal peptide of patatin was fused to the bacterial reporter protein beta-glucuronidase, the fusion proteins were translocated to the endoplasmic reticulum in planta and in vitro. In addition, translocated beta-glucuronidase was modified by glycosylation, and the signal peptide was correctly processed. In the presence of an inhibitor of glycosylation, tunicamycin, the enzymatically active form of beta-glucuronidase was assembled in the endoplasmic reticulum. This is the first report of targeting a cytoplasmic protein to the endoplasmic reticulum of plants using a signal peptide.

Amino Acid Sequence↗

PubMed2000

Modulation of a lectin insecticidal activity by carbohydrates.

Lectins from plants present an insecticidal activity most probably through their carbohydrate binding properties; as a consequence, their toxicity should vary with the presence of a competitive sugar in the ingested food. In order to test this hypothesis, we performed competition experiments between insecticidal activity and carbohydrate binding. For this purpose, we used a lectin from Lathyrus ochrus and the specific carbohydrate for this protein, glucose. In toxicological tests with Drosophila melanogaster, we observed a decrease of lectin toxicity when glucose was added to the larva-rearing medium. This result suggests that the toxicity of the lectin is correlated to its ability to bind sugar in the insect digestive tract and stresses the importance of sugar composition of the nutriment used for toxicological testing of lectins or in genetically modified plants.

Animals↗

PubMed2000

Metabolic engineering of astaxanthin production in tobacco flowers.

Using metabolic engineering, we have modified the carotenoid biosynthesis pathway in tobacco (Nicotiana tabacum) to produce astaxanthin, a red pigment of considerable economic value. To alter the carotenoid pathway in chromoplasts of higher plants, the cDNA of the gene CrtO from the alga Haematococcus pluvialis, encoding beta-carotene ketolase, was transferred to tobacco under the regulation of the tomato Pds (phytoene desaturase) promoter. The transit peptide of PDS from tomato was used to target the CRTO polypeptide to the plastids. Chromoplasts in the nectary tissue of transgenic plants accumulated (3S,3'S) astaxanthin and other ketocarotenoids, changing the color of the nectary from yellow to red. This accomplishment demonstrates that plants can be used as a source of novel carotenoid pigments such as astaxanthin. The procedures described in this work can serve as a platform technology for future genetic manipulations of pigmentation of fruits and flowers of horticultural and floricultural importance.

Bacterial Proteins↗

PubMed1997

Transactivation of a target gene using a suppressor tRNA in transgenic tobacco plants.

The abundance of tRNAs, together with their central role in translation, has generated considerable interest in the use of tRNA genes for biotechnological applications. One such application is the use of suppressor tRNAs to transactive target genes containing premature stop codons. Previous work has shown that such systems can work in transient expression experiments in plant protoplasts; here these experiments are extended to show that suppression of stop codons can occur in whole plants. Transgenic tobacco plants homozygous for a modified tRNA(Leu) gene expressing a strong amber suppressor tRNA, and plants carrying a beta-glucuronidase (gus) gene inactivated by a premature amber stop codon have been obtained. When the two types of plants are crossed, many of the F1 hybrids show significant GUS activity. The GUS activity is dependent on the presence of both the suppressor tRNA gene and the gus gene. Tobacco plants carrying the suppressor tRNA gene are phenotypically normal, fertile and the gene shows normal Menedelian inheritance. The potential applications of such a system are discussed.

Genes, Plant↗

PubMed2001

Manipulation of the napin primary structure alters its packaging and deposition in transgenic tobacco (Nicotiana tabacum L.) seeds.

Napin is a 2S storage protein found in the seeds of oilseed rape (Brassica napus L.) and related species. Using protein structural prediction programs we have identified a region in the napin protein sequence which forms a 'hydrophilic loop' composed of amino acid residues located at the protein surface. Targeting this region, we have constructed two napin chimeric genes containing the coding sequence for the peptide hormone leucine-enkephalin as a topological marker. One version has a single enkephalin sequence of 11 amino acids including linkers and the second contains a tandem repeat of this peptide comprising 22 amino acids, inserted into the napin large subunit. The inserted peptide sequences alter the balance of hydrophilic to hydrophobic amino acids and introduce flexibility into this region of the polypeptide chain. The chimeric genes have been expressed in tobacco plants under the control of the seed-specific napA gene promoter. Analyses indicate that the engineered napin proteins are expressed, transported, post-translationally modified and deposited inside the protein bodies of the transgenic seeds demonstrating that the altered napin proteins behave in a similar fashion to the authentic napin protein. Detailed immunolocalisation studies indicate that the insertion of the peptide sequences has a significant effect on the distribution of the napin proteins within the tobacco seed protein bodies.

2S Albumins, Plant↗

PubMed2005

Traceability of biotech-derived animals: application of DNA technology.

Traceability is increasingly becoming standard across the agri-food industry, largely driven by recent food crises and the consequent demands for transparency within the food chain. This is leading to the development of a range of traceability concepts and technologies adapted to different industry needs. Experience with genetically modified plants has shown that traceability can play a role in increasing public confidence in biotechnology, and might similarly help allay concerns relating to the development of animal biotechnology. Traceability also forms an essential component of any risk management strategy and is a key requirement for post-marketing surveillance. Given the diversity of traceability concepts and technologies available, consideration needs to be given to the scope and precision of traceability systems for animal biotechnology. Experience to date has shown that conventional tagging and labelling systems can incorporate levels of error and may not have sufficient precision for biotech-derived animals. Deoxyribonucleic acid (DNA) technology can overcome these difficulties by tracing animals and animal by-products through their DNA code rather than an associated label. This offers the possibility of tracing some by-products of animal biotechnology through the supply chain back to source animals, offering unprecedented levels of traceability. Developments in both DNA sampling and analysis technology are making large-scale applications of DNA traceability increasingly cost effective and feasible, and are likely to lead to a broader uptake of DNA traceability concepts.

Animal Identification Systems↗

PubMed1994

Accumulation of fructose polymers in transgenic tobacco.

Fructan, a polyfructose molecule, is a storage compound in a limited number of plant species. Usually these species accumulate fructan with a low degree of polymerization (DP) and most of these plants have properties which preclude their use as a fructan source. With the eventual aim of allowing the accumulation of high DP fructans in non-fructan storing plants, we have investigated whether carbohydrate flow in the plant cell can be directed to produce this polymer. For this purpose the SacB gene from Bacillus subtilis, which encodes levansucrase, was modified and introduced into tobacco plants. Transgenic plants containing the sacB gene accumulate fructans. The size and properties of this fructan are similar to fructan produced by Bacillus subtilis, and is stable in plants. Although the level of fructan accumulation in the transgenic tobacco plants ranged from 3-8 percent of the dry weight, no levansucrase mRNA or protein could be detected in these plants. Extension of this work should permit the production of this high molecular weight biopolymer in crop plants for applications in food and non-food products.

Bacillus subtilis↗

PubMed2000

Immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants.

Transgenic plants expressing recombinant proteins from pathogenic microorganisms provide an inexpensive edible vaccine for induction of local immunity. Three transgenic plant lines were generated expressing the spike (S) protein of transmissible gastroenteritis virus (TGEV), a protein crucial for establishing mucosal immunity. All three of them were driven by a strong plant promoter. One construct contained the 3.7 kb 5' end of the native S gene sequence. In the second construct part of the S gene, from nucleotide 49 to 1785, was modified for optimal plant recognition and was fused to a plant signal peptide coding sequence. The third construct contained the D epitope-coding region of the S gene, from nucleotide 1201 to 1591, which was fused to the alfalfa beta-amylase gene. The S gene products were detected by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Antigens from all three transgenic plant lines induced TGEV-specific immune responses in pigs as determined by virus neutralization and ELISA, and the resultant antibody titers for all three constructs were similar.

Animals↗

PubMed2006

Transformation of Acinetobacter sp. BD413 with DNA from commercially available genetically modified potato and papaya.

AIM: To estimate the likelihood of transfer of kanamycin-resistance gene (nptII) from commercially available genetically modified (GM) plants. METHODS AND RESULTS: Acinetobacter sp. BD413 carrying a plasmid containing an inactivated nptII gene was treated with DNA derived from GM potato and GM papaya. Kanamycin-resistant transformants were obtained at a frequency of 10-30 microg(-1) DNA. Calculation of the results suggested that 6-9 x 10(4) molecules of genomic DNA from GM plants were needed to obtain one transformant. However, such transformation events were not detectable in the absence of the plasmid in the host strain. CONCLUSIONS: Acinetobacter sp. BD413 was transformed with DNA derived from GM potato and GM papaya, in the presence of an inactivated nptII gene on a plasmid. However, the frequency of such events in the natural environment on wild-type strains, while evidently low, remains unknown. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results may help to evaluate potential risks associated with the use of antibiotic-resistance determinants as genetic markers in GM plants. Complete risk assessment must consider factors other than transformation frequency alone, including the natural background of antibiotic resistance present in bacterial populations, and the spectrum and clinical use of the antimicrobial agents in question.

Acinetobacter↗

PubMed1995

Studies of coat protein-mediated resistance to tobacco mosaic virus (TMV). II. Challenge by a mutant with altered virion surface does not overcome resistance conferred by TMV coat protein.

Transgenic tobacco plants expressing the coat protein (CP) gene of the U1 strain of tobacco mosaic virus (TMV) exhibit CP-mediated resistance (CP-MR) against some, but not all, tobamoviruses. To investigate the role of the amino acid sequences on the surface of the challenge virus in CP-MR, mutant strains of U1 TMV were constructed to contain the amino or carboxy termini of the CP of Sunn hemp mosaic tobamovirus (SHMV). The modified virus was unable to overcome CP-MR in transgenic plants that contained the TMV CP. In contrast, TMV in which the CP was replaced by the SHMV CP overcame CP-MR to the same extent as did SHMV. We conclude that CP-MR conferred by TMV CP involves interactions between amino acid sequences of the challenge viruses and the transgene protein other than those on the surface of the challenge virus.

Amino Acid Sequence↗

PubMed1994

The 3a protein from cucumber mosaic virus increases the gating capacity of plasmodesmata in transgenic tobacco plants.

The 3a protein, encoded by RNA 3 of cucumber mosaic virus (CMV), is the putative movement protein of viral progeny in infected plants. An analysis of transgenic tobacco plants constitutively expressing the CMV 3a protein showed that the protein is accumulated in leaves at every stage of development. In fully expanded leaves the protein is immunodetectable mostly in a cell-wall-enriched fraction. Dye-coupling experiments using fluorescent-dextran probes were performed on fully expanded leaves to study the modifying effect of CMV 3a protein on the gating capacity of plasmodesmata. Movement of fluorescein-isothiocyanate-labelled dextran with a mean molecular mass of 10,000 Da, and an approximate Stokes' radius of 2.3 nm, was detected between cells of the 3a protein transgenic plants, but not in the control plants. These results are consistent with the idea that the CMV 3a protein is involved in the modification of plasmodesmata and, therefore, in the cell-to-cell spread of the virus.

Blotting, Western↗

PubMed2001

A cDNA from grapevine (Vitis vinifera L.), which shows homology to AGAMOUS and SHATTERPROOF, is not only expressed in flowers but also throughout berry development.

An AGAMOUS/SHATTERPROOF homologue (Vvmads1) was isolated from grapevine by differential display between berry and leaf mRNA. The predicted protein sequence of the full-length clone shows a high degree of homology to PLENA (77% identity) and to SHP1 and SHP2 (75% and 74% identity respectively), and is grouped with AGAMOUS/PLENA homologues when the conserved MADS and K domains are compared. Vvmads1 is expressed only in the later stages of flower development and throughout berry development, although expression is reduced after ripening commenced. When Vvmads1 was over-expressed in tobacco, the resulting plants display altered morphologies in the outer two floral whorls. In the most extreme cases, the inner whorls were surrounded by a carpelloid structure created by the modified sepals. Within these sepals were petals which had been split into sections and which were attached at the base of the flower by structures with the appearance of filaments. The results of this study suggest that Vvmads1 has a regulatory role in flower development before fertilisation and a role in fruit development after fertilisation.

AGAMOUS Protein, Arabidopsis↗

PubMed2001

Expression of bar in the plastid genome confers herbicide resistance.

Phosphinothricin (PPT) is the active component of a family of environmentally safe, nonselective herbicides. Resistance to PPT in transgenic crops has been reported by nuclear expression of a bar transgene encoding phosphinothricin acetyltransferase, a detoxifying enzyme. We report here expression of a bacterial bar gene (b-bar1) in tobacco (Nicotiana tabacum cv Petit Havana) plastids that confers field-level tolerance to Liberty, an herbicide containing PPT. We also describe a second bacterial bar gene (b-bar2) and a codon-optimized synthetic bar (s-bar) gene with significantly elevated levels of expression in plastids (>7% of total soluble cellular protein). Although these genes are expressed at a high level, direct selection thus far did not yield transplastomic clones, indicating that subcellular localization rather than the absolute amount of the enzyme is critical for direct selection of transgenic clones. The codon-modified s-bar gene is poorly expressed in Escherichia coli, a common enteric bacterium, due to differences in codon use. We propose to use codon usage differences as a precautionary measure to prevent expression of marker genes in the unlikely event of horizontal gene transfer from plastids to bacteria. Localization of the bar gene in the plastid genome is an attractive alternative to incorporation in the nuclear genome since there is no transmission of plastid-encoded genes via pollen.

Acetyltransferases↗

PubMed2001

Galactose-extended glycans of antibodies produced by transgenic plants.

Plant-specific N-glycosylation can represent an important limitation for the use of recombinant glycoproteins of mammalian origin produced by transgenic plants. Comparison of plant and mammalian N-glycan biosynthesis indicates that beta1,4-galactosyltransferase is the most important enzyme that is missing for conversion of typical plant N-glycans into mammalian-like N-glycans. Here, the stable expression of human beta1,4-galactosyltransferase in tobacco plants is described. Proteins isolated from transgenic tobacco plants expressing the mammalian enzyme bear N-glycans, of which about 15% exhibit terminal beta1,4-galactose residues in addition to the specific plant N-glycan epitopes. The results indicate that the human enzyme is fully functional and localizes correctly in the Golgi apparatus. Despite the fact that through the modified glycosylation machinery numerous proteins have acquired unusual N-glycans with terminal beta1,4-galactose residues, no obvious changes in the physiology of the transgenic plants are observed, and the feature is inheritable. The crossing of a tobacco plant expressing human beta1,4-galactosyltransferase with a plant expressing the heavy and light chains of a mouse antibody results in the expression of a plantibody that exhibits partially galactosylated N-glycans (30%), which is approximately as abundant as when the same antibody is produced by hybridoma cells. These results are a major step in the in planta engineering of the N-glycosylation of recombinant antibodies.

Antibodies↗

PubMed2000

Induced expression of sarcotoxin IA enhanced host resistance against both bacterial and fungal pathogens in transgenic tobacco.

We demonstrate here that induced expression of sarcotoxin IA, a bactericidal peptide from Sarcophaga peregrina, enhanced the resistance of transgenic tobacco plants to both bacterial and fungal pathogens. The peptide was produced with a modified PR1a promoter, which is further activated by salicylic acid treatment and necrotic lesion formation by pathogen infection. Host resistance to infection of bacteria Erwinia carotovora subsp. carotovora and Pseudomonas syringae pv. tabaci was shown to be dependent on the amounts of sarcotoxin IA expressed. Since we found antifungal activity of the peptide in vitro, transgenic seedlings were also inoculated with fungal pathogens Rhizoctonia solani and Pythium aphanidermatum. Transgenic plants expressing higher levels of sarcotoxin were able to withstand fungal infection and remained healthy even after 4 weeks, while control plants were dead by fungal infection after 2 weeks.

Anti-Bacterial Agents↗

PubMed2006

Letting the gene out of the bottle: the population genetics of genetically modified crops.

Genetically modified (GM) plants are rapidly becoming a common feature of modern agriculture. This transition to engineered crops has been driven by a variety of potential benefits, both economic and ecological. The increase in the use of GM crops has, however, been accompanied by growing concerns regarding their potential impact on the environment. Here, we focus on the escape of transgenes from cultivation via crop x wild hybridization. We begin by reviewing the literature on natural hybridization, with particular reference to gene flow between crop plants and their wild relatives. We further show that natural selection, and not the overall rate of gene flow, is the most important factor governing the spread of favorable alleles. Hence, much of this review focuses on the likely effects of transgenes once they escape. Finally, we consider strategies for transgene containment.

Crops, Agricultural↗