Showing papers in "Nature Biotechnology in 1988"
TL;DR: The current status and potential use of baculovirus vectors for the expression of foreign genes in insect cells are described and are contributing to understanding the molecular biology of gene and protein function and regulation in both vertebrate and insect systems.
Abstract: We describe the current status and potential use of baculovirus vectors for the expression of foreign genes in insect cells. Trends in the development of transfer vectors for the expression of foreign genes under the control of the strong polyhedrin promoter of Autographa californica nuclear polyhedrosis virus and strategies for maximizing levels of expression are discussed. Baculovirus vectors have achieved widespread acceptance for their ability to express proteins of agricultural and medical importance. A baculovirus vector was used to express the first recombinant HIV envelope proteins to receive F.D.A. approval for clinical evaluation as a candidate vaccine for AIDS. These insect DNA virus vectors are contributing to understanding the molecular biology of gene and protein function and regulation in both vertebrate and insect systems.
1,144 citations
TL;DR: A general purification method for recombinant proteins based upon the selective interaction between a poly-histidine peptide, which is fused to the protein of interest, and a novel metal chelate adsorbent is described.
Abstract: We describe a general purification method for recombinant proteins based upon the selective interaction between a poly-histidine peptide, which is fused to the protein of interest, and a novel metal chelate adsorbent. The principle of the technique is illustrated with mouse dihydrofolate reductase. DNA elements coding for adjacent histidines were fused to the mouse dihydrofolate reductase gene. Subsequent expression in E. coli resulted in the production of hybrid proteins that could be purified by immobilized metal ion affinity chromatography, followed by removal of the histidine affinity peptide with carboxypeptidase A.
1,112 citations
TL;DR: A small hydrophilic peptide of eight amino acids was engineered onto the N-terminus of a variety of recombinant lymphokines for the purpose of aiding in their detection and purification from yeast supernatants or E. coli extracts.
Abstract: A small hydrophilic peptide of eight amino acids (AspTyrLysAspAspAspAspLys) was engineered onto the N-terminus of a variety of recombinant lymphokines for the purpose of aiding in their detection and purification from yeast supernatants or E. coli extracts. An antibody specific for the first four amino acids of this sequence was used as a detection reagent and for immunoaffinity purification of products under mild conditions. Because of the small size of the peptide moiety and its hydrophilic nature, the proteins were unaffected by its presence and retained a high level of biological activity. In addition, it was possible to remove the peptide via an enzymatic cleavage procedure using enterokinase.
1,075 citations
TL;DR: Experiments using this protocol have resulted in mature RO and R1 plants expressing the introduced gene(s), regarded as a major step in the practical application of genetic engineering to the useful modification of food-crop species.
Abstract: We have used particle acceleration by electric discharge to introduce DNA-coated gold particles into meristems of immature soybean seeds. Approximately 2 percent of shoots derived from these meristems via organogenesis were chimeric for expression of the introduced gene. Experiments using this protocol have resulted in mature RO and R1 plants expressing the introduced gene(s). We regard this work as a major step in the practical application of genetic engineering to the useful modification of food-crop species.
742 citations
TL;DR: Progeny from two transgenic soybean plants demonstrated co-segregation of kanamycin resistance and either GUS expression or glyphosate tolerance in a 3:1 ratio indicating a single insert inherited in a Mendelian fashion.
Abstract: Transgenic soybean plants have been produced using an Agrobacterium-mediated gene transfer system. This procedure relied on a regeneration protocol in which shoot organogenesis was induced on cotyledons of soybean genotypes selected for susceptibility to Agrobacterium. Cotyledon explants were inoculated with Agrobacterium tumefaciens pTiT37-SE harboring pMON9749 (conferring kanamycin resistance and β-glucuronidase “GUS” activity) or pTiT37-SE∷pMON894 (conferring kanamycin resistance and glyphosate tolerance) and cultured on shoot induction medium containing kanamycin. Plantlets were tested for gene insertion 3–4 months post-inoculation. Approximately 6% of the shoots (8 plants to date) produced on the kanamycin-selected cotyledons were transgenic based on assays for GUS expression, kanamycin resistance or glyphosate tolerance. Progeny from two of these plants demonstrated co-segregation of kanamycin resistance and either GUS expression or glyphosate tolerance in a 3:1 ratio indicating a single insert inherited in a Mendelian fashion.
573 citations
TL;DR: Lysates of non–transformed E. coli grown at either temperature rendered initially soluble human recombinant IFN–α2 insoluble at 37° but not at 0° or 30°C, and insolubilization was not abolished by nuclease treatment.
Abstract: Human interferon–α2 (IFN–α2), an 18 kD, acidic protein, human interferon–γ (IFN–γ), a 17 kD, basic protein, and the interferon–induced murine protein Mx (72 kD) were all found in aggregates when produced in genetically engineered strains of E. coli grown at 37°C. However, at a growth temperature of 23–30°C, 30–90% of the recombinant protein was soluble. The temperature effect was not directly dependent on the concentration of the protein and was observed for several E. coli strains and for different plasmid constructions. Lysates of non–transformed E. coli grown at either temperature rendered initially soluble human recombinant IFN–α2 insoluble at 37° but not at 0° or 30°C. Insolubilization was not abolished by nuclease treatment, and may involve sulfhydryl group shuffling, as sulfhydryl reducing agents added to a mock lysate gave a similar temperature dependent precipitation.
468 citations
TL;DR: Delivery by high–velocity microprojectiles may be useful for the stable transformation of monocot species as well as for gene delivery into suspension culture cells of Zea mays embryos.
Abstract: Factors influencing the efficiency of DNA delivery into suspension culture cells of Zea mays by the particle bombardment process were studied using a chimeric gene coding for the production of β–glucuronidase. Two days following bombardment with plasmid–coated microprojectiles, expression of the β–glucuronidase gene was detected with the synthetic substrate 5–bromo–4–chloro–3–indoyl–β–D–glucuronic acid, which, upon cleavage, forms a blue precipitate visually detectable within affected cells. The number of cells expressing β–glucuronidase was about 30 times greater when the cells were bombarded on a filter paper support than when they were bombarded while covered by liquid media without such a support. The efficiency of gene delivery was also significantly affected by the velocity of the microprojectile and the number of micro–projectiles used for bombardment, and the concentration of CaCl2 and spermidine used to adsorb DNA to the microprojectiles. In addition to cell cultures, the particle bombardment process was also used to deliver the β–glucuronidase gene to intact cells on the surface of excised Z. mays embryos. The results indicate that delivery by high–velocity microprojectiles may be useful for the stable transformation of monocot species.
425 citations
TL;DR: An Asd+ expression-cloning vector was constructed for the purpose of high-level stable expression of foreign antigen genes in a Salmonella typhimurium vaccine strain and represents a balanced lethal combination that eliminates the need for vector drug resistance markers, an essential attribute for live vaccines.
Abstract: An Asd+ expression-cloning vector was constructed for the purpose of high-level stable expression of foreign antigen genes in a Salmonella typhimurium vaccine strain. It possesses the asd+ gene of Streptococcus mutans as a unique plasmid marker and is stably maintained in Δasd mutants of S. typhimurium when using media lacking diaminopimelic acid. The recombinant Asd+ plasmid possessing the spaA gene of Streptococcus sobrinus, which is regulated by the trc promoter, produced the fused SpaA protein at high level in the S. typhimurium vaccine strain. The Asd+ vector/Δasd mutant host constructs represent a balanced lethal combination that eliminates the need for vector drug resistance markers, an essential attribute for live vaccines. This strategy also has applications whenever stable high-level expression by genetically modified bacteria, in the absence of external selection pressures, is desirable.
378 citations
TL;DR: In this article, the authors have regenerated whole plants of rice (Oryza sativa L) derived from protoplasts, which had been electroporated with plasmid DNA possessing a chimeric gene encoding aminoglycoside phosphotransferase II (APH(3′)II).
Abstract: We have regenerated whole plants of rice (Oryza sativa L.) derived from protoplasts, which had been electroporated with plasmid DNA possessing a chimeric gene encoding aminoglycoside phosphotransferase II (APH(3′)II). Transformed calli were selected on the basis of tolerance to the antibiotic, G418. Several plants were regenerated possessing functional APH(3′)II activity due to the integration of intact foreign DNA into their genome.
344 citations
TL;DR: A low cost protein-free medium is devised that supports insect cell growth, virus propagation, and product expression similar to that obtained in traditional 10% serum-supplemented media and facilitates recovery and purification of the recombinant protein product.
Abstract: We have developed a process for the large-scale suspension culture of insect (Spodoptera frugiperda) cells and the production of recombinant proteins using the baculovirus expression system. Cell densities greater than 5 × 106 cells/ml are achieved in 21 L airlift culture. We have also devised a low cost protein-free medium that supports insect cell growth, virus propagation, and product expression similar to that obtained in traditional 10% serum-supplemented media. The protein-free medium facilitates recovery and purification of the recombinant protein product. We demonstrate the utility of this system in the production of human macrophage colony stimulating factor (M-CSF) at 40 mg/L.
326 citations
TL;DR: The synthesized recombinant RNA molecules that function both as hybridization probes and as templates for exponential amplification by Qβ replicase demonstrate the feasibility of employing exponentially replicatable RNAs in bioassays, where they serve the dual role of specific probe and amplifiable reporter.
Abstract: We have synthesized recombinant RNA molecules that function both as hybridization probes and as templates for exponential amplification by Qβ replicase. Each recombinant consists of a sequence specific for the protozoan parasite, Plasmodium falciparum, embedded within the sequence of MDV-1 RNA, which is a natural template for Qβ replicase. The probe sequence was inserted within a hairpin loop that occurs on the exterior of MDV-1 RNA. The recombinant RNAs hybridize specifically to complementary DNA, despite topological constraints on the probe domain, are replicated at the same rate as MDV-1 RNA, despite their additional length, and are able to serve as templates for the synthesis of a large number of RNA copies. A Qβ replicase reaction initiated with only 0.14 femtograms of recombinant RNA (1,000 molecules) can produce 129 nanograms of recombinant RNA product in 30 minutes. This represents a one-billion fold amplification. Our results demonstrate the feasibility of employing exponentially replicatable RNAs in bioassays, where they would serve the dual role of specific probe and amplifiable reporter.
TL;DR: It is demonstrated that although it is possible to obtain protection in transgenic plants using antisense–CP message, this protection is not as effective as the CP–mediated protection.
Abstract: Progeny of transgenic tobacco plants expressing the cucumber mosaic virus (CMV) coat protein (CP +) or its antisense transcript (antisense–CP) were protected from CMV infection. Symptom development and virus accumulation were reduced or absent in CP+ plants, independent of the strength of the inoculum. Antisense–CP plants were protected only at low inoculum concentrations. These results demonstrate that although it is possible to obtain protection in transgenic plants using antisense–CP message, this protection is not as effective as the CP–mediated protection.
TL;DR: The amounts of heterologous protein obtained make this system attractive for even moderately priced industrial enzymes, as an example, the Rhizomucor miehei derived aspartic proteinase was secreted with yields in excess of three grams per liter.
Abstract: We have modified a method for transformation of Aspergillus nidulans1 to work for Aspergillus oryzae using selection for the amdS and argB genes from Aspergillus nidulans. To direct the expression of recombinant genes in A. oryzae we have used an alpha-amylase promoter cloned from a high yielding strain of A. oryzae. The amounts of heterologous protein obtained make this system attractive for even moderately priced industrial enzymes. As an example, the Rhizomucor miehei derived aspartic proteinase was secreted with yields in excess of three grams per liter. The proteinase was slightly overglycosylated, which did not, however, alter the specific activity of the enzyme.
TL;DR: There is considerable experimental support for the idea that plant growth promoting bacteria may be used as bio–fertilizers or biological disease control agents to increase agricultural yields.
Abstract: Bacteria associated with the plant rhizosphere may have beneficial effects on plant growth by providing nutrients and growth factors, or by producing antibiotics and siderophores, which antagonize phytopathogenic fungi and bacteria. There is considerable experimental support for the idea that plant growth promoting bacteria may be used as bio–fertilizers or biological disease control agents to increase agricultural yields. Recent advances in our understanding of the molecular biology of the systems responsible for plant growth stimulation are opening the way to strain improvement by genetic engineering.
TL;DR: It is demonstrated that insect cells can be grown in an agitated, spargedBioreactor and an airlift bioreactor with a growth rate and maximum cell density comparable to those obtained in spinner flasks oxygenated by surface diffusion.
Abstract: We demonstrate that insect cells can be grown in an agitated, sparged bioreactor and an airlift bioreactor with a growth rate and maximum cell density comparable to those obtained in spinner flasks oxygenated by surface diffusion. Recombinant protein synthesis by cells infected with a genetically-modified baculovirus in the sparged bioreactor is also demonstrated. The nonionic surfactant Pluronic® F-68 was responsible for the protection of insect cells from the adverse effects of sparging. The mechanism of Pluronic® F-68 protection in a sparged environment is discussed.
TL;DR: It is argued that a far better understanding of growth, development, development (including morphogenesis in vitro), physiology and molecular biology/genetics of plants—and continuous dialogue and interaction with plant breeders and geneticists—are required for the effective and useful application of the modern tools of biotechnology to major crop species.
Abstract: The economically important cereal and grass crops have generally proved to be notoriously recalcitrant to manipulation in vitro. Regeneration of plants from single cells, a prerequisite for cellular and molecular manipulation, has proven to be especially difficult. Consequently, this group of plants has until recently remained outside the main stream of plant biotechnology. The discovery and exploitation of embryogenic tissue cultures, in which plant regeneration takes place by the formation of embryos from single somatic cells, has led to the development of efficient procedures for plant regeneration in almost all of the important species of grasses, and recovery of mature plants from protoplasts in crops such as maize, rice and sugarcane. These results, along with the success in somatic hybridization and the demonstration of transient as well as stable expression of introduced genes in grass cells and plants, provide challenging opportunities for the genetic manipulation and improvement of this group of food crops. It is argued, however, that a far better understanding of growth, development (including morphogenesis in vitro), physiology and molecular biology/genetics of plants—and continuous dialogue and interaction with plant breeders and geneticists—are required for the effective and useful application of the modern tools of biotechnology to major crop species.
TL;DR: The first report of the successful transformation of a woody tree species from somatic embryos using Agrobacterium is reported, and four rooted plantlets have been transplanted to soil and acclimatized.
Abstract: Genetically transformed walnut plants were regenerated from somatic embryos inoculated with a disarmed Agrobacterium tumefaciens strain containing a gene encoding kanamycin resistance. Embryo subclones derived from the inoculated embryos were selected for continued embryo proliferation on kanamycin-containing medium. Embryos and shoots were shown to contain and express the foreign gene and have been subjected to rooting procedures. Four rooted plantlets have been transplanted to soil and acclimatized. This is the first report of the successful transformation of a woody tree species from somatic embryos using Agrobacterium.
TL;DR: A system of protoplast culture that results in high plating efficiency from embryogenic protoplasts and can be followed by plant regeneration is developed and has been successfully used with protoplast of two maize inbreds.
Abstract: Genetic engineering of maize via protoplast technology has been limited due to lack of plant regeneration from maize protoplasts. We have developed a system of protoplast culture that results in high plating efficiency from embryogenic protoplasts and can be followed by plant regeneration. Maize protoplasts were grown on filters directly over a feeder layer of nurse cells in liquid medium. Initial condition and subsequent growth of feeder cells were critical in obtaining a plating efficiency of 10% from protoplasts. Protoplasts were digested from embryogenic cell suspension cultures, and recovered callus retained the morphogenic potential of initial donor cultures. The system has been successfully used with protoplasts of two maize inbreds, one of which is an important commercial line.
TL;DR: Yields from one CP–expressing line were equal to that of the uninoculated VF36 plants suggesting that expression of the CP gene does not intrinsically cause yield depression, and the potential for use of genetically engineered protection in agriculture is indicated.
Abstract: Two transgenic tomato plants that express the coat protein (CP) of the common (U1) strain of tobacco mosaic virus (TMV) were produced from cultivar VF36 using gene transfer techniques. CP–expressing plants were partially resistant to infection and symptom development caused by TMV and tomato mosaic virus (ToMV) strains L, 2, or 22. Strains 2 and 22 normally overcome the natural resistance genes present in many commercial tomato cultivars. In the field, no more than 5% of the CP–expressing plants inoculated with TMV exhibited visual systemic disease symptoms by fruit harvest compared with 99% of the VF36 plants. Lack of visual symptoms was associated with lack of virus accumulation in the CP–expressing plants. In terms of agronomic traits, leaf and stem dry weight accumulation in greenhouse–grown uninoculated CP–expressing (line 306) and nonexpressing plants were essentially equal. In field analyses, tomato fruit yields of the VF36 plants decreased 26–35% due to virus infection, whereas yields of the CP–expressing plants were unaffected. Yields from one CP–expressing line were equal to that of the uninoculated VF36 plants suggesting that expression of the CP gene does not intrinsically cause yield depression. The results from these growth chamber, greenhouse and field experiments indicate the potential for use of genetically engineered protection in agriculture.
TL;DR: Five founder transgenic sheep described carry genes designed to direct the production of human clotting factor IX or human αl–antitrypsin in milk and may ultimately provide a new source of these and other therapeutic proteins.
Abstract: Gene transfer into animals has considerable potential for livestock improvement. If this potential is to be realized, the ease of generation of transgenic livestock will be of major importance. We report here the production of six transgenic sheep by microinjection of DNA into early embryos (1.2% of embryos transferred). Three different gene constructs were injected and transgenic sheep were obtained with each. The transgenic animals have all incorporated the DNA without detectable rearrangement, and where multiple copies were integrated, they are present in arrays of tandem repeats. Transmission of transferred genes to progeny of three of the sheep has been demonstrated. Five founder transgenic sheep described carry genes designed to direct the production of human clotting factor IX or human αl–antitrypsin in milk. Transgenic animals carrying such genes may ultimately provide a new source of these and other therapeutic proteins.
TL;DR: Glycolyse aerobic and glutaminolyse, enzymes ―Production d'ATP, strategies possibles pour l'amelioration de the productivite des bioreacteurs.
Abstract: Glycolyse aerobic et glutaminolyse. Enzymes ―Production d'ATP. Strategies possibles pour l'amelioration de la productivite des bioreacteurs
TL;DR: Atrial natriuretic peptide shows promise as an agent to treat heart failure, renal failure and fluid excess states and is commonly elevated in situations of excessive fluid volume or hypertension.
Abstract: Atrial natriuretic peptide (ANP) stimulates vasodilation, fluid egress, increased glomerular filtration and salt and water excretion, and blocks the release and/or actions of several hormones, including angiotensin II, aldosterone and vasopressin. ANP levels are commonly elevated in situations of excessive fluid volume or hypertension, and the hormone may be important in combatting these states. ANP is derived from a precursor protein produced by the cardiac atrium and at lower levels by other tissues. Two classes of ANP receptors present in vascular, renal and other tissues are linked respectively, to either guanylate cyclase and possibly other second messengers, or to ANP clearance. ANP shows promise as an agent to treat heart failure, renal failure and fluid excess states.
TL;DR: The results indicated that the carbohydrates attached to the EPO peptide are responsible for the different biological activities of these proteins.
Abstract: Erythropoietin (EPO), a heavily glycosylated protein, is a major stimulatory factor in erythropoiesis. The human EPO gene was engineered for expression in animal cells. Recombinant EPOs produced in two kinds of cells were isolated and their properties compared with those of human urinary EPO. The results indicated that the carbohydrates attached to the EPO peptide are responsible for the different biological activities of these proteins. The biological activities of recombinant glycoproteins produced in heterologous systems may vary depending on the host cells in which the proteins are modified.
TL;DR: The parB locus constitutes a convenient and efficient plasmid stabilization casette, useful in many gram negative species, and was confirmed by testing a variety of different replicons.
Abstract: The parB locus of plasmid R1, comprising at most 580 base pairs of DNA, mediates efficient plasmid stabilization via postsegregational killing of plasmid free cells. The locus encodes two small genes, hok and sok. The hok gene product is a potent cell killing protein, the expression of which is regulated by the sok product, an anti-sense RNA complementary to the hok mRNA. The hok mRNA is extraordinarily stable, while the sok-RNA is rapidly degraded. The mechanism of postsegregational killing is explained by the differential decay of the hok and sok-RNA's: In newborn plasmid free cells the prolonged persistence of the hok mRNA leads to synthesis of the Hok protein, thus ensuring a rapid and selective killing of these cells. As predicted from this simple model, any unstably inherited plasmid should become stabilized by carrying the parB locus. This important prediction was confirmed by testing a variety of different replicons. Even plasmids replicating in as distantly related organisms as Eschericia coli, Serratia marsescense, and Pseudomonas putida were efficiently stabilized. The parB locus therefore constitutes a convenient and efficient plasmid stabilization casette, useful in many gram negative species.
TL;DR: Systeme Vitromatic developpe a «Plant Biotech Industries» en Israel integrant un bioreacteur and un bioprocesseur permettant the separation, le calibrage and the repartition des propagules.
Abstract: Historique du developpement des methodes de propagation en masse des plantes in vitro. Aspects economiques de la culture de tissus vegetaux. Culture de tissus en milieu liquide. Systeme Vitromatic developpe a «Plant Biotech Industries» en Israel integrant un bioreacteur et un bioprocesseur permettant la separation, le calibrage et la repartition des propagules
TL;DR: The gene encoding the coleopteran–specific insect control protein from Bacillus thuringiensis var.
Abstract: The gene encoding the coleopteran–specific insect control protein from Bacillus thuringiensis var. tenebrionis was isolated and characterized. When expressed in either E. coli or P. fluorescens the gene directs the production of biologically active insect control protein that is lethal to Colorado potato beetle. The gene contains two functional translational initiation codons in the same reading frame leading to the production of both the full length protein and an N–terminal truncated form. The gene has been engineered to produce each of these two proteins separately, and each is active against Colorado potato beetle. Native crystals from B.t. var. tenebrionis were found to contain five distinct proteins derived from this single gene either as primary translation products or the result of proteolysis. Comparison of this gene to a lepidopteran–specific B.t. gene revealed only limited regions of homology.
TL;DR: In this article, an automated DNA sequenator using a real-time fluorescence detection method, achieved excellent sensitivity and rapid analysis by optimizing operating conditions, using unicolor labeling and four-track electrophoresis.
Abstract: We have developed an automated DNA sequenator using a real-time fluorescence detection method, and achieved excellent sensitivity and rapid analysis by optimizing operating conditions. The sequenator uses unicolor labeling and four-track electrophoresis. Fluoresceine isothiocyanate(FITC)-labeled primers are used to produce labeled DNA families. The fluorescent bands are excited by an Ar laser (488 nm, 10 mW) introduced from the side of a thin gel during electrophoresis. Analysis, with an accuracy greater than 99%, consumes 0.01–0.04 picomole sample for each DNA family, which is almost comparable to the amount used in conventional autoradiography. With the optimized electrophoresis conditions reported here, four hundred bases of DNA can be analyzed within two and a half hours using a four percent polyacrylamide gel.