Showing papers in "Nature Biotechnology in 1992"
TL;DR: The ability to simultaneously amplify specific DNA sequences and detect the product of the amplification both simplifies and improves PCR and may facilitate its automation and more widespread use in the clinic or in other situations requiring high sample through–put.
Abstract: We have enhanced the polymerase chain reaction (PCR) such that specific DNA sequences can be detected without opening the reaction tube. This enhancement requires the addition of ethidium bromide (EtBr) to a PCR. Since the fluorescence of EtBr increases in the presence of double-stranded (ds) DNA an increase in fluorescence in such a PCR indicates a positive amplification, which can be easily monitored externally. In fact, amplification can be continuously monitored in order to follow its progress. The ability to simultaneously amplify specific DNA sequences and detect the product of the amplification both simplifies and improves PCR and may facilitate its automation and more widespread use in the clinic or in other situations requiring high sample throughput.
1,201 citations
TL;DR: Improved the affinity of one such “primary” antibody is improved by sequentially replacing the heavy and light chain variable (V) region genes with repertoires of V–genes (chain shuffling) obtained from unimmunized donors.
Abstract: Diverse antibody libraries can be displayed on the surface of filamentous bacteriophage, and selected by panning of the phage with antigen. This allows human antibodies to be made directly in vitro without prior immunization, thus mimicking the primary immune response. Here we have improved the affinity of one such "primary" antibody by sequentially replacing the heavy and light chain variable (V) region genes with repertoires of V-genes (chain shuffling) obtained from unimmunized donors. For a human phage antibody for the hapten 2-phenyloxazol-5-one (phOx) (Kd = 3.2 x 10(-7) M), we shuffled the light chains and isolated an antibody with a 20 fold improved affinity. By shuffling the first two hypervariable loops of the heavy chain, we isolated an antibody with a further 15-fold improved affinity. The reshuffled antibody differed in five of the six hypervariable loops from the original antibody and the affinity for phOx (Kd = 1.1 x 10(-9) M) was comparable to that of mouse hybridomas from the tertiary immune response. Reshuffling offers an alternative to random point mutation for affinity maturation of human antibodies in vitro.
1,171 citations
TL;DR: Herbicide Resistant Fertile Transgenic Wheat Plants Obtained by Microprojectile Bombardment of Regenerable Embryogenic Callus as mentioned in this paper, which can be used to produce wheat.
Abstract: Herbicide Resistant Fertile Transgenic Wheat Plants Obtained by Microprojectile Bombardment of Regenerable Embryogenic Callus
687 citations
TL;DR: A protocol is developed to directly and efficiently recover Fab′ with the single hinge cysteine in the free thiol state, allowing F(ab′)2 formation by chemically-directed coupling in vitro, and makes E. coli expression of bivalent antibody fragments for human therapy (or other uses) practical.
Abstract: Many clinical uses of antibodies will require large quantities of fragments which are bivalent and humanized. We therefore attempted to generate humanized F(ab')2 fragments by secretion from E. coli. Titers of 1-2 g l-1 of soluble and functional Fab' fragments have been routinely achieved as judged by antigen-binding ELISA. Surprisingly, this high expression level of Fab' in the periplasmic space of E. coli does not drive dimerization. However, we have developed a protocol to directly and efficiently recover Fab' with the single hinge cysteine in the free thiol state, allowing F(ab')2 formation by chemically-directed coupling in vitro. The E. coli derived humanized F(ab')2 fragment is indistinguishable from F(ab')2 derived from limited proteolysis of intact antibody in its binding affinity for the antigen, p185HER2, and anti-proliferative activity against the human breast tumor cell line, SK-BR-3, which over-expresses p185HER2. This system makes E. coli expression of bivalent antibody fragments for human therapy (or other uses) practical.
601 citations
568 citations
TL;DR: A method for introducing a glutamine synthetase (GS) selectable marker into myeloma cells in which transfectants are selected by growth in a glutamines-free medium is reported.
Abstract: We report a method for introducing a glutamine synthetase (GS) selectable marker into myeloma cells in which transfectants are selected by growth in a glutamine-free medium. Vector amplification can subsequently be selected using the specific inhibitor of GS, methionine sulphoximine (MSX). Using this system, DNA sequences encoding a chimeric B72.3 IgG4 antibody were expressed from hCMV-MIE promoters in NSO myeloma cells. A cell line was isolated after a single round of selection for vector amplification which contains approximately 4 copies of the vector, secretes 10-15 pg/cell/day cB72.3 antibody during exponential growth and can accumulate 560 mg/l antibody in a fed-batch air-lift fermentation system. Productivity is stable in the absence of MSX selection.
544 citations
TL;DR: RIPs have antiviral and abortifacient activities, and, in a widespread application, can also be linked to antibodies or ligands to form immunotixins or conjugates specifically toxic to a given type of cell.
Abstract: Plant ribosome-inactivating proteins (RIPs) are N-glycosidases which cleave the N-glycosidic bond of adenine in a specific ribosomal RNA sequence. Most commonly RIPs are single-chain proteins (type 1 RIPs), but some (type 2 RIPs) possess a galactose-specific lectin domain that binds to cell surfaces. The latter RIPs are potent toxins, the best known of which is ricin. RIPs have antiviral and abortifacient activities, and, in a widespread application, can also be linked to antibodies or ligands to form immunotoxins or conjugates specifically toxic to a given type of cell.
413 citations
TL;DR: Recent progress in the genetic and biochemical analysis of biosurfactant synthesis as well as the current status of fermentation technologies are discussed.
Abstract: Microbial surfactants are a structurally diverse group of compounds consisting of hydrophilic and hydrophobic domains and which partition preferentially at interfaces. Biosurfactants are of increasing interest commercially as substitutes for synthetic surfactants particularly for environmental applications. This article discusses recent progress in the genetic and biochemical analysis of biosurfactant synthesis as well as the current status of fermentation technologies.
406 citations
TL;DR: An “all fish” growth hormone (GH) chimeric gene construct is developed by using an antifreeze protein gene (AFP) promoter from ocean pout linked to a chinook salmon GH cDNA clone to generate transgenic Atlantic salmon.
Abstract: We have developed an “all fish” growth hormone (GH) chimeric gene construct by using an antifreeze protein gene (AFP) promoter from ocean pout linked to a chinook salmon GH cDNA clone. After microinjection into fertilized, nonactivated Atlantic salmon eggs via the micropyle, transgenic Atlantic salmon were generated. The presence of the transgene was detected by polymerase chain reaction (PCR) using specific oligonucleotide primers. A number of these transgenic fish showed dramatic increases in their growth rate. At one year old, the average increase of the transgenic fish was 2 to 6 fold and the largest transgenic fish was 13 times that of the average non-transgenic control.
379 citations
TL;DR: It is shown that extremely fragile biomolecules such as DNA restriction and modifying enzymes can be dried in vitro in the presence of trehalose with no loss of activity, even after prolonged storage, and their ability to withstand prolonged exposure to temperatures as high as +70°C is remarkable.
Abstract: We show that extremely fragile biomolecules such as DNA restriction and modifying enzymes can be dried in vitro in the presence of trehalose with no loss of activity, even after prolonged storage A remarkable and unexpected property of the dried enzyme preparations is their ability to withstand prolonged exposure to temperatures as high as +70 degrees C This stability is unique to trehalose and is not found with other sugars irrespective of their physical or chemical properties The immediate significance of these observations is the ability to convert enzymes used in molecular biology into stable reagents The indefinite stability and high temperature tolerance of these dried enzymes should permit the design of convenient formats that may be of particular significance in the automation of genome mapping and sequencing projects The stabilization of a wide range of biomolecules by trehalose also has practical implications for a number of areas ranging from basic science, through healthcare and agriculture, to bio-electronics
347 citations
TL;DR: An efficient gene transfer system utilizing microprojectile-mediated transformation of 2,4-D-treated immature zygotic embryos with a plasmid construction that contains the neomycin phosphotransferase II and β-glucuronidase genes flanking a PRV cp gene expression cassette is reported.
Abstract: Papaya ringspot virus (PRV) is a serious disease of papaya (Carica papaya L.) that has only been partially controlled by conventional methods. An alternative control method is coat protein-mediated protection (CPMP) through the transfer and expression of the PRV coat protein (cp) gene in papaya. We report an efficient gene transfer system utilizing microprojectile-mediated transformation of 2,4-D-treated immature zygotic embryos with a plasmid construction that contains the neomycin phosphotransferase II (NPTII) and β-glucuronidase (GUS) genes flanking a PRV cp gene expression cassette. Putative transgenic RO papaya plants, regenerated on kanamycin-containing medium, were assayed for GUS and PRV coat protein expression, for the presence of NPTII and PRV cp genes [with the polymerase chain reaction (PCR) and genomic blot hybridization analysis], and for PRV cp gene transcripts by Northern analysis. Four RO transgenic plant lines that contained the PRV cp gene showed varying degrees of resistance to PRV, and one line appeared to be completely resistant. These results represent the first demonstration that CPMP can be extended to a tree species such as papaya.
TL;DR: The use of an in vivo inducible promoter such as nirB may be a generally applicable approach to obtaining the stable in vivo expression of heterologous antigens in Salmonella vaccine strains.
Abstract: Plasmid pTETnir15, which directs the expression of the non-toxic immunogenic fragment C of tetanus toxin from the anaerobically inducible nirB promoter, was introduced into the Salmonella typhimurium aroA aroD live oral vaccine strain BRD509 The resulting strain, designated BRD847, was used to vaccinate orally BALB/c mice and was tested for plasmid stability and its ability to protect against a lethal tetanus toxin challenge pTETnir15 was stably inherited by bacteria growing or persisting in the tissues of immunized mice whereas another BRD509 derivative, designated BRD753, harboring plasmid pTET85 which directs fragment C expression from the tac promoter, was highly unstable Mice immunized with a single oral dose of BRD847 developed high levels of circulating anti-fragment C antibodies and were solidly protected against tetanus toxin challenge Mice immunized with a single oral dose of BRD753 developed no detectable anti-fragment C antibodies After boosting, antibodies were detected, but the mice were only partially protected against tetanus toxin challenge Thus the use of an in vivo inducible promoter such as nirB may be a generally applicable approach to obtaining the stable in vivo expression of heterologous antigens in Salmonella vaccine strains
TL;DR: This review focuses on the use of fed-batch techniques to produce recombinant products in Escherichia coli with high cell concentrations as well as high specific yields of recombinant product.
Abstract: Whereas cell concentrations of 5-10 grams dry cell weight per liter (gDCW/l) are typical of batch cultures, fed-batch techniques can be used to achieve concentrations greater than 50 gDCW/l. Feeding strategies for fed-batch cultures include feed-back control as well as pre-determined feeding profiles. The volumetric yield of recombinant products can be improved by controlling the specific growth rate and the substrate concentration. Furthermore, inhibitory by-product formation can be minimized in fed-batch cultures. This review focuses on the use of fed-batch techniques to produce recombinant products in Escherichia coli. The modes of nutrient feeding that have been employed are discussed, and the factors important in attaining high cell concentrations as well as high specific yields of recombinant product are described.
TL;DR: Transgenic tobacco plants that express a barley ribosome inactivating protein (RIP) under control of the wound-inducible promoter of the potato wun1 gene were obtained and showed increased protection against fungal inoculation.
Abstract: Expression of a Barley Ribosome-Inactivating Protein Leads to Increased Fungal Protection in Transgenic Tobacco Plants
TL;DR: The engineered strain of the yeast, Candida tropicalis, is engineered for the efficient production of long–chain dicarboxylic acids and is effective for the selective terminal oxidation of both saturated and unsaturated linear aliphatic substrates with chain–lengths ranging from 12 carbons to 22 carbons.
Abstract: We have engineered an industrial strain of the yeast, Candida tropicalis, for the efficient production of long-chain dicarboxylic acids, which are important raw materials for the chemical industry. By sequential disruption of the four genes encoding both isozymes of the acyl-CoA oxidase which catalyzes the first reaction in the beta-oxidation pathway, alkane and fatty acid substrates have been successfully redirected to the omega-oxidation pathway. Consequently, the conversion efficiency and chemical selectivity of their terminal oxidation to the corresponding dicarboxylic acids has been improved to 100 percent. The specific productivity of the bioconversion has been increased further by amplification of the cytochrome P450 monooxygenase and NADPH-cytochrome reductase genes encoding the rate-limiting omega-hydroxylase in the omega-oxidation pathway. The amplified strains demonstrated increased omega-hydroxylase activity and a 30% increase in productivity compared to the beta-oxidation-blocked strain in fermentations. The bioconversion is effective for the selective terminal oxidation of both saturated and unsaturated linear aliphatic substrates with chain-lengths ranging from 12 carbons to 22 carbons and also avoids the undesirable chain modifications associated with passage through the beta-oxidation pathway, such as unsaturation, hydroxylation, or chain shortening. It is now possible to efficiently produce a wide range of previously unavailable saturated and unsaturated dicarboxylic acids with a high degree of purity.
TL;DR: Subcloning in Bacillus subtilis and transfer to strain ATCC 824 via electrotransformation, where the corresponding enzyme activities were expressed at elevated levels, using pFNK1, a new B. subtilIS/C.
Abstract: We have previously cloned the acetone-formation pathway gene, encoding acetoacetate decarboxylase (adc), and butyrate-formation pathway gene, encoding phosphotransbutyrylase (ptb), of Clostridium acetobutylicum ATCC 824 in Escherichia coli. Here we report their subcloning in Bacillus subtilis and transfer to strain ATCC 824 via electrotransformation, where the corresponding enzyme activities were expressed at elevated levels, using pFNK1, a new B. subtilis/C. acetobutylicum shuttle vector. Plasmid pFNK1 was used because shuttle vectors that function in E. coli were unable to electrotransform ATCC 824 unless they became deleted in the E. coli-plasmid regions. The difficulties with shuttle vectors that function in E. coli are probably due to the presence of a restriction endonuclease in ATCC 824. This endonuclease recognizes the sequence 5'-GCNGC-3', which is prevalent in E. coli plasmids but occurs infrequently in pFNK1 and C. acetobutylicum genes. Cloning of genes in C. acetobutylicum is critical for redirecting the cellular metabolism (metabolic engineering) as well as for genetic studies of this industrial organism.
TL;DR: Advances in particle bombardment have extended the range of gene transfer by particle bombardment to animal and bacterial cells, and one noteworthy newer application is the direct insertion of genes into the organs of living animals.
Abstract: Over the past several years, particle bombardment has evolved into a useful tool for molecular biologists, allowing direct gene transfer to a broad range of cell and tissue types. Some of the important applications of the process include the production of transgenic crop species including maize and soybean and the introduction of DNA into plastids and mitochondria. Recent results have extended the range of gene transfer by particle bombardment to animal and bacterial cells. One noteworthy newer application is the direct insertion of genes into the organs of living animals. Here we discuss these advances and the instrument developments that contributed to them.
TL;DR: In this article, a synthetic gene that encodes an antigen-binding single-chain Fv protein (scFv) in transgenic tobacco plants was created by polymerase chain reaction (PCR) amplification of the variable domain coding regions from a mouse monoclonal hybridoma cell line.
Abstract: We have expressed a synthetic gene that encodes an antigen–binding single–chain Fv protein (scFv) in transgenic tobacco plants. The scFv gene was created by polymerase chain reaction (PCR) amplification of the variable domain coding regions from a mouse monoclonal hybridoma cell line. The monoclonal cell line secretes an IgG1 antibody that binds to the plant regulatory photoreceptor protein, phytochrome. The cloned scFv gene was expressed initially in Escherichia coli and shown to produce a 28 kD, phytochrome–binding scFv protein. Transgenic tobacco plants expressing the scFv gene were also found to produce a functional scFv protein, and seeds from transgenic R1 progeny displayed aberrant phytochrome–dependent germination. The scFv from transgenic tobacco could be isolated, to near homogeneity, by a single phytochrome–Sepharose affinity chromatography step.
TL;DR: As a first step to exploit the potential of Trichoderma reesei to produce hemicellulases, two endo-β-1,4-xylanases are purified and cloned and the enzyme encoded by xyn2 appears to more closely resemble several other bacterial and fungal xylanases than does that of xyn1.
Abstract: As a first step to exploit the potential of Trichoderma reesei to produce hemicellulases, we have purified two endo-β-1,4-xylanases (1,4-β-D-xylan xylanohydrolase, EC 3.2.1.8) and cloned their genes. The enzymes were isolated from culture filtrates of T. reesei C 30 grown on xylan as a carbon source, using two steps of cation exchange chromatography. They exhibited molecular weights of 19 (XYN I) and 21 (XYN II) kD, and isoelectric points of 5.2 and 9.0, respectively. These enzymes differed in their pH optimum for activity and affinity for xylan, and accounted for more than 90% of the total xylanolytic activity of the fungus. The purified enzymes were subjected to N-terminal sequence analysis, and after cleavage with trypsin and endoproteinase Glu-C the resulting peptides were sequenced. Oligonucleotides based on these sequences were used to clone gene fragments via PCR, and these were used as probes to isolate full-length copies of xyn1 and xyn2 from a lambda gene bank of T. reesei. The products of xyn1 and xyn2 share considerable homology, but the enzyme encoded by xyn2 appears to more closely resemble several other bacterial and fungal xylanases than does that of xyn1.
TL;DR: The imminent cloning of disease resistance genes, further molecular dissection of stress signal perception and transduction mechanisms, and identification of genes that affect symptom development will provide attractive new opportunities for enhancing crop protection, andbinatorial integration of these novel strategies should make an important contribution to effective, durable field resistance.
Abstract: There are marked differences in the pattern of host gene expression in incompatible plant: microbial pathogen interactions compared with compatible interactions, associated with the elaboration of inducible defenses Constitutive expression of genes encoding a chitinase or a ribosome-inactivating protein in transgenic plants confers partial protection against fungal attack, and a large repertoire of such antimicrobial genes has been identified for further manipulation In addition, strategies are emerging for the manipulation of multigenic defenses such as lignin deposition and synthesis of phytoalexin antibiotics by overexpression of genes encoding rate determining steps, modification of transcription factors or other regulatory genes, and engineering production of novel phytoalexins by interspecies transfer of biosynthetic genes The imminent cloning of disease resistance genes, futher molecular dissection of stress signal perception and transduction mechanisms, and identification of genes that affect symptom development will provide attractive new opportunities for enhancing crop protection Combinatorial integration of these novel strategies into ongoing breeding programs should make an important contribution to effective, durable field resistance
TL;DR: Some recent advances in the ability to construct mice with a variety of genetic modifications are addressed, including an increased understanding of the basic cell biology and in vitro growth characteristics of ES cells, which has facilitated germ line transmission of manipulated clones on a routine basis.
Abstract: Genetic modification of endogenous genes in mice has become possible by applying gene targeting techniques to embryonic stem (ES) cells and using specific clones of cells to generate mice. Despite the experimental opportunities offered by the creation of organisms with specific genetic changes, there are considerable technical obstacles which can confound the routine implementation of this technology. This review addresses some recent advances in the ability to construct mice with a variety of genetic modifications. These include an increased understanding of the basic cell biology and in vitro growth characteristics of ES cells, which has facilitated germ line transmission of manipulated clones on a routine basis. The techniques that are used to isolate "targeted" clones of ES cells have been summarized, and the current status of strategies which have been successfully used to make very specific modifications of the genome are discussed.
TL;DR: It is shown that cynomolgus macaques are phylogenetically distant enough to respond against conserved human antigens, and a human/monkey chimeric anti-CD4 antibody with an apparent affinity of 3.2 × 10−11 M and exhibits potent immunosuppressive properties in vitro.
Abstract: Immunoglobulin variable region genes from non-human primates, cynomolgus macaques, were shown to have 85%-98% homology with human immunoglobulin sequences and yet macaques are phylogenetically distant enough to respond against conserved human antigens. Immunoglobulin genes were isolated from monkeys immunized with human CD4 antigen and a human/monkey chimeric anti-CD4 antibody with 91-92% homology to human immunoglobulin framework regions was cloned and expressed. The antibody has an apparent affinity of 3.2 x 10(-11) M and exhibits potent immunosuppressive properties in vitro.
TL;DR: Recently high levels of protection against tomato spotted wilt virus (TSWV), a negative–strand RNA virus infecting plants, have been obtained by transforming tobacco with viral nucleoprotein (N) gene sequences, and hence appears to be RNA–mediated.
Abstract: Tobacco plants were transformed with different TSWV N gene constructs to obtain a generally applicable source of resistance to TSWV
TL;DR: Refolding studies reveal that PEG has significant potential for enhancing the recovery of active proteins produced in heterologous hosts and a good correlation was observed between the protein hydrophobicity and the stoichiome–tric concentration of PEG required to achieve a maximal increase in recovery ofactive protein.
Abstract: Previous studies on the refolding of recombinant bovine carbonic anhydrase B (CAB) indicated that polyethylene glycol (PEG) significantly enhanced the recovery of active protein by reducing aggregation. To further test the ability of PEG to enhance refolding, three recombinant human proteins, deoxyribonuclease (rhDNAse), tissue plasminogen activator (rhtPA), and interferon-gamma (rhIFN-gamma) were refolded in the presence of PEG (3350 MW). rhDNAse produced from CHO cells was denatured in 7.2 M urea and refolded by rapid dilution to 4.0 M urea and 0.20 mg/ml protein. When a final PEG to rhDNAse molar ratio of 5 to 1 (0.1 milligram PEG, 3350 MW) was used in the dilution buffer, refolding was improved by 30% to yield complete recovery of active protein. Impure E. coli derived inclusion body preparations of rhDNAse were solubilized in 8 M urea and refolded by dilution to 4 M urea and 0.10 mg/ml protein. Refolding with a dilution buffer which yielded a final PEG to rhDNAse molar ratio of 10 to 1 (0.1 milligram PEG, 3350 MW) resulted in a three-fold increase in the recovery of active protein. When PEG was used in the dilution buffer, aggregation of rhDNAse did not occur during refolding in either case. rhtPA produced from CHO cells was denatured in 5 M guanidine hydrochloride (GuHCl) and refolded by rapid dilution to 0.10 M GuHCl and 0.20 mg/ml protein.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: A unique application of the Random Amplified Polymorphic DNA (RAPD) technique for genetic linkage mapping of a single spruce tree using haploid DNA from megagametophyte tissue of individual seeds is reported.
Abstract: We report a unique application of the Random Amplified Polymorphic DNA (RAPD) technique for genetic linkage mapping of a single spruce tree using haploid DNA from megagametophyte tissue of individual seeds. Sixty–one segregating loci were analysed for reproducibility, inheritance and linkage. Forty–seven of the 61 markers were distributed into 12 linkage groups and covered 873.8 cM. The 14 markers not associated with any linkage group should be assigned to linkage groups as more markers are added to the map. This new approach quickly provides molecular genetic markers that are simple to evaluate for constructing genetic linkage maps and for other related genetic studies in forest tree species.
TL;DR: The bound scFv(FRP5)–PhoA protein can be detected directly on tumor cells using a substrate for alkaline phosphatase, showing that the chimeric protein retains both binding and enzymatic activity.
Abstract: We have constructed genes expressing single-chain antigen binding proteins (scFv) which recognize the human erbB-2 receptor. These genes encode the heavy and light chain variable domains of an erbB-2 receptor specific monoclonal antibody, MAb FRP5, connected by a peptide linker. In order to express a bifunctional molecule, a bacterial alkaline phosphatase gene was fused 3' to the scFv gene. The scFv(FRP5) and scFv(FRP5)-alkaline phosphatase fusion protein (scFv(FRP5)-PhoA) expressed in E. coli specifically recognize the human erbB-2 protein and compete with MAb FRP5 for binding to the receptor. The bound scFv(FRP5)-PhoA protein can be detected directly on tumor cells using a substrate for alkaline phosphatase, showing that the chimeric protein retains both binding and enzymatic activity.
TL;DR: The resulting hydrolysis products were virtually identical with those obtained from degradation with α-amylase from Bacillus licheniformis, and the difference, compared to 55.2 kD for the bacterial enzyme, was found to result from complex-type carbohydrate chains attached to the protein.
Abstract: As a first example of the feasibility of producing industrial bulk enzymes in plants, we have expressed Bacillus licheniformis alpha-amylase in transgenic tobacco, and applied the seeds directly in starch liquification. The enzyme was properly secreted into the intercellular space, and maximum expression levels of about 0.3% of total soluble protein were obtained. No apparent effect of the presence of the enzyme on plant phenotype was observed. The molecular weight of the enzyme produced in tobacco was around 64 kD. The difference, compared to 55.2 kD for the bacterial enzyme, was found to result from complex-type carbohydrate chains attached to the protein. Application studies on the liquefaction of starch were done with transgenic seeds containing the recombinant alpha-amylase. The resulting hydrolysis products were virtually identical with those obtained from degradation with alpha-amylase from Bacillus licheniformis.
TL;DR: Stable integration of the transgenes in the genomes of plants regenerated from resistant callus clones was shown by Southern hybridization analysis and in situ hybridization of a labeled transgene–probe to metaphase chromosomes is shown for one transgenic primary regenerant.
Abstract: Chimeric hygromycin phosphotransferase (hph) and phosphinothricin acetyltransferase (bar) genes were introduced, using polyethylene glycol treatment, into protoplasts isolated from embryogenic cell suspension cultures of tall fescue (Festuca arundinacea Schreb), a graminaceous plant that is an important forage crop in temperate pastures Colonies resistant to either 200 mg/l hygromycin or 100 mg/l phosphinothricin, respectively, were recovered upon selection using bead-type culture systems Stable integration of the transgenes in the genomes of plants regenerated from resistant callus clones was shown by Southern hybridization analysis In situ hybridization of a labeled transgene-probe to metaphase chromosomes is shown for one transgenic primary regenerant Expression of the transgenes in mature plants was demonstrated by HPH enzyme assay or by phosphinothricin-herbicide spraying
TL;DR: Dendrograms based on oligonucleotide fingerprint band sharing data proved to be consistent with most of the known features of the history of banana and plantain cultivation and evolution, respectively and has potential application in several areas of Musa improvement.
Abstract: Oligonucleotide and Amplification Fingerprinting of Wild Species and Cultivars of Banana (Musa spp)