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PubMed2002

Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants.

An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.

Agrobacterium tumefaciens↗
PubMed1999

N(2)-(1-Carboxyethyl)deoxyguanosine, a nonenzymatic glycation adduct of DNA, induces single-strand breaks and increases mutation frequencies.

N(2)-(1-Carboxyethyl)deoxyguanosine (CEdG) is a major nonenzymatic glycation product of DNA. The effect of CEdG modification, which was specifically prepared by incubation with dihydroxyacetone, on plasmid DNA topology was evaluated by gel electrophoresis. A time-dependent decrease of supercoiled plasmid-DNA was observed in parallel to the increase of CEdG adducts; the half-life time of the supercoiled plasmid-DNA was estimated to be approximately 16-18 h. CEdG-modified plasmid DNA showed a 25-fold reduced transformation efficiency. When modified DNA was used to transform Escherichia coli cells, a 6-fold increase in mutation frequency was determined by measuring loss of alpha-complementation. For the mutator strain BMH71-18mutS, an 8-fold increase in mutation frequency was observed. Although the exact mechanism of DNA damage is unclear, the occurrence of spontaneous depurination is likely. These findings suggest that a defined DNA glycation reaction can lead to DNA damage in vivo.

DNA Adducts↗
PubMed1988

A point mutation in the extracellular domain of the human CSF-1 receptor (c-fms proto-oncogene product) activates its transforming potential.

A human CSF-1 receptor containing an "activating" mutation in its extracellular domain (serine for leucine 301) induced morphologic transformation, anchorage-independent growth, and tumorigenicity in mouse NIH 3T3 cells. A second regulatory mutation within the receptor's intracytoplasmic carboxy-terminal tail (phenylalanine for tyrosine 969) augmented transforming efficiency but was itself insufficient to induce transformation. Like the v-fms oncogene product, receptors bearing the activating mutation retained high-affinity binding sites for CSF-1 but were retarded in transport to the cell surface and were phosphorylated on tyrosine in the absence of ligand. Although the activating mutation does not affect the CSF-1 binding site in the receptor extracellular domain, it must induce a conformational change that mimics the effect of ligand binding, resulting in CSF-1-independent signals for cell growth.

Animals↗
PubMed1988

Participation of hup gene product in ori2-dependent replication of fertility plasmid F.

The fertility plasmid F'gal was not stably maintained in a hupA-hupB double mutant of Escherichia coli. Moreover, mini-F plasmids pFZY1, pFTC1 and pFTC2 were unable to transform the double mutant, though these plasmids efficiently transformed cells harboring a hupA or hupB single mutation. The composite plasmid pFHS1, which consists of the f5 DNA fragment of F plasmid and the whole DNA of a pSC101 derivative that carries a temperature-sensitive mutation for DNA replication, was not stably maintained in the hup double mutant at 42 degrees C. These findings strongly suggest that HU protein is required for ori2-dependent replication of the F plasmid.

Bacterial Proteins↗
PubMed2005

Generation of variability by in vivo recombination of halves of a (beta/alpha)8 barrel protein.

Similar to what has been achieved with nucleic acids, directed evolution of proteins would be greatly facilitated by the availability of large libraries and efficient selection methods. So far, host cell transformation efficiency has been a bottleneck, practically limiting libraries to sizes less than 10(9). One way to circumvent this problem has been implemented with antibody systems, where contribution to the binding site is provided by two different polypeptides (light and heavy chains). The central concept is the construction of binary systems in which the gene from the two chains are separated by a cre-lox recombinase recognition site, packaged in a phage, and subsequently introduced, by multiple infection, into a recombinase expressing cell [Sblattero D, Bradbury A. Nat Biotechnol 2000;18(1):75-80]. Here, we describe the development of a system which applies the same concept to a single-domain enzyme, the cytoplasmic (beta/alpha)8 barrel protein phosphoribosyl anthranilate isomerase (PRAI) from E. coli. For that purpose, we identified the site at which a loop containing the recognition sequence for cre-lox recombinase could be inserted yielding a functional enzyme. We evaluated the effect of this insertion on the capability of the engineered gene to complement a trp F-E. coli strain and the efficiency of the system to recover the original sequence from an abundance of non-functional mutant genes.

Aldose-Ketose Isomerases↗
PubMed2001

Electrotransformation studies in Clostridium cellulolyticum.

Electropermeabilization of Clostridium cellulolyticum was optimized using ATP leakage assays. Electrotransformation was then performed under optimized conditions (6 to 7.5 kV x cm(-1) field strength applied during 5 ms to a mixture containing methylated plasmids and late exponential phase cell suspensions (10 molecules:1 cell) in a sucrose-containing buffer). Transformants were only obtained when 7 or 7.5 kV x cm(-1) pulses were applied. Transformation efficiencies evaluated from the growth curves of transformed cells were between 10(5) and 10(7) transformants per microgram of plasmid DNA for five different replicon-based plasmids. Restriction nuclease digestion patterns of pJIR418 purified from transformed Clostridia and Escherichia coli were indistinguishable, indicating that heterologous DNA was structurally stable in the Clostridium strain. Copy numbers of 130, 70 and 10 were estimated from purification yield for pCTC1, pKNT19 and pJIR418, respectively.

Cell Membrane Permeability↗
PubMed1989

Transformation in restriction-deficient Salmonella typhimurium LT2.

Stable restriction-deficient, modification-proficient galE (JR501) and F'galE+ (JR502) strains of Salmonella typhimurium were constructed and the effects of restriction on transformation by plasmid pBR322 were tested. Several factors which affect transformation efficiency were systematically examined to determine optimum transformation conditions and a simplified method is presented.

Ampicillin Resistance↗
PubMed1994

Natural and Electroporation-Mediated Transformation of Methanococcus voltae Protoplasts.

The lack of high-efficiency transformation systems has severely impeded genetic research on methanogenic members of the kingdom Archaeobacteria. By using protoplasts of Methanococcus voltae and an integration vector, Mip1, previously shown to impart puromycin resistance, we obtained natural transformation frequencies that were about 80-fold higher (705 transformants per mug of transforming DNA) than that reported with whole cells. Electroporation-mediated transformation of M. voltae protoplasts with covalently closed circular Mip1 DNA was possible, but at lower frequencies of ca. 177 transformants per mug of vector DNA. However, a 380-fold improvement (3,417 transformants per mug of DNA) over the frequency of natural transformation with whole cells was achieved by electroporation of protoplasts with linearized DNA. This general approach, of using protoplasts, should allow the transformation of other methanogens, especially those that may be gently converted to protoplasts as a result of their tendency to lyse in hypotonic solutions.

Journal Article↗
PubMed1986

Transformation of encapsulated Streptococcus pneumoniae.

We describe the high-efficiency transformation of several virulent, encapsulated isolates of Streptococcus pneumoniae. Transformation was effected by the induction of competence with competence factor and was apparently the result both of inducing noncompetent recipients and overcoming the inhibition imposed by the capsule.

Bacterial Proteins↗
PubMed1989

Transposon mutagenesis, characterization, and cloning of transformation genes of Haemophilus influenzae Rd.

A plasmid library of PstI fragments of Haemophilus influenzae Rd genomic DNA was mutagenized in Escherichia coli with mini-Tn10kan. The mutagenized PstI fragments were introduced by transformation into the H. influenzae chromosome, and kanamycin-resistant transformants were screened for the transformation-deficient phenotype by a cyclic AMP-DNA plate method. Fifty-four mutant strains containing 24 unique insertions that mapped to 10 different PstI fragments were isolated. Strains carrying unique insertions were tested individually for DNA uptake, transformation efficiency, UV sensitivity, and growth rate. The transformation frequencies of these mutants were decreased by factors of 10(-2) to 10(-6). Five of the mutants had normal competence-induced DNA uptake, and the rest were variably deficient in competence development. Three strains were moderately UV sensitive. All strains but one had doubling times within 50% of that of the wild type. Mutated genes were cloned into an H. influenzae-E. coli shuttle vector, and wild-type loci were recovered by in vivo recombinational exchange. Hybridization of these clones to SmaI genomic fragments separated in pulsed-field gels showed that these insertions were not clustered in a particular region of the chromosome.

Alleles↗
PubMed1969

Transformation of Acinetobacter calco-aceticus (Bacterium anitratum).

A highly efficient transformation system has been demonstrated in a strain of Acinetobacter calco-aceticus (Bacterium anitratrum). During mixed growth of various stable, unencapsulated, mutant strains, deoxyribonucleic acid (DNA) is liberated and fully encapuslated transformants can be isolated. Purified DNA preparations have been used to transform suitable recipient mutant strains for ability to synthesize capsules, ability to dispense with a growth factor requirement, and resistance to streptomycin. When the wild-type strain is deprived of its capsule, either by mechanical stripping or by mutation, the unencapsulated cells tend to form large clumped masses. A nonclumping mutant of an unencapsulated strain has been isolated. When ability to synthesize capsules is transformed into this nonclumping strain, the resultant cells no longer form chains, unlike the wild-type encapsulated strain. It appears likely that the occurrence of transformation during growth of mixed cultures, with glucose or gluconate as the carbon source, may be the result of osmotic rupture resulting from the inability of unencapsulated strains to oxidize triose phosphates as fast as they are formed. The finding of transformation in Acinetobacter may provide an additional useful organism for the study of this mode of genetic transfer since this strain grows well in a simple mineral medium containing a single oxidizable source of carbon. Furthermore, no special supplementary factors seem to be required for transformation to take place.

Alcaligenes↗
PubMed1997

PLANT TRANSFORMATION: Problems and Strategies for Practical Application.

Plant transformation is now a core research tool in plant biology and a practical tool for cultivar improvement. There are verified methods for stable introduction of novel genes into the nuclear genomes of over 120 diverse plant species. This review examines the criteria to verify plant transformation; the biological and practical requirements for transformation systems; the integration of tissue culture, gene transfer, selection, and transgene expression strategies to achieve transformation in recalcitrant species; and other constraints to plant transformation including regulatory environment, public perceptions, intellectual property, and economics. Because the costs of screening populations showing diverse genetic changes can far exceed the costs of transformation, it is important to distinguish absolute and useful transformation efficiencies. The major technical challenge facing plant transformation biology is the development of methods and constructs to produce a high proportion of plants showing predictable transgene expression without collateral genetic damage. This will require answers to a series of biological and technical questions, some of which are defined.

Journal Article↗
PubMed2004

Agrobacterium-mediated transformation of European chestnut embryogenic cultures.

An innovative and efficient genetic transformation protocol for European chestnut is described in which embryogenic cultures are used as the target material. When somatic embryos at the globular or early-torpedo stages were cocultured for 4 days with Agrobacterium tumefaciens strain EHA105 harbouring the pUbiGUSINT plasmid containing marker genes, a transformation efficiency of 25% was recorded. Murashige and Skoog culture medium containing 150 mg/l of kanamycin was used as the selection medium. The addition of acetosyringone was detrimental to the transformation efficiency. Transformation was confirmed by a histochemical beta-glucuronidase (GUS ) assay, PCR and Southern blot analyses for the uidA (GUS) and nptII (neomycin phosphotransferase II) genes. At present, 93 GUS-positive chestnut embryogenic lines are being maintained in culture. Low germination rates (6.3%) were recorded for the transformed somatic embryos. The presence of the transferred genes in leaves and shoots derived from the germinated embryos was also verified by the GUS assay and PCR analysis.

Acetophenones↗
PubMed2003

Efficient and genotype-independent Agrobacterium--mediated tomato transformation.

An efficient method to transform five cultivars of tomato (Lycopersicon esculentum), Micro-Tom, Red Cherry, Rubion, Piedmont, and E6203 is reported. A comparison was made of leaf, cotyledon, and hypocotyl explants on 7 different regeneration media without Agrobacterium tumefaciens cocultivation and on 11 different media with cocultivation. Although all cultivars and explants formed callus and regenerated on the initial 7 media, cocultivation with A. tumefaciens significantly reduced the callus induction and regeneration. From these experiments, a transformation methodology using either hypocotyls or cotyledons cultured for one day on BA 1 mgL-1, NAA 0.1 mgL-1 and 3 days cocultivation with the Agrobacterium on this same medium followed by a transfer to a medium with zeatin 2 mgL-1 and IAA 0.1 mgL-1 for 4-6 weeks resulted in a greater than 20% transformation frequency for all five cultivars tested. In this transformation method, no feeder layers of tobacco, petunia or tomato suspension cultures were used, and the subculture media was minimal. Stable integration and transmission of the transgene in T1 generation plants were confirmed by Southern blot analysis. This procedure represents a simple, efficient and general means of transforming tomato.

Culture Media↗
PubMed1994

Constitutive expression of the virulence genes improves the efficiency of plant transformation by Agrobacterium.

Inducible virulence (vir) genes of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid are under control of a two-component regulatory system. In response to environmental factors (phenolic compounds, sugars, pH) VirA protein phosphorylates VirG, which in turn interacts with the promoters of other vir genes, causing induction. A mutation of virG, virGN54D (which codes for a Asn-54-->Asp amino acid change in the product), causes constitutive expression of other vir genes independent of virA. We have investigated whether providing Agrobacterium with a plasmid containing virGN54D augments the efficiency of transfer of the T-DNA (transferred DNA). For both tobacco and cotton, we observed an enhancement of transformation efficiency when the inciting Agrobacterium strain carries the virGN54D mutation. We also tested whether supplying Agrobacterium with a similar plasmid containing wild-type virG affects the efficiency of T-DNA transfer. An intermediate efficiency was observed when this plasmid was employed. Using a beta-glucuronidase (GUS) reporter gene to assess transient expression of T-DNA after transfer to tobacco and maize tissues, we observed a higher frequency of GUS-expressing foci after inoculation with Agrobacterium strains carrying virGN54D than with Agrobacterium carrying the wild-type virG. Gene-transfer efficiency to maize by an octopine strain was greatly improved upon introduction of virGN54D. Multiple copies of wild-type virG were equally effective in promoting transient expression efficiency in tobacco but were virtually ineffective in maize. We propose the use of virGN54D to improve the efficiency of Agrobacterium-mediated transformation, especially for recalcitrant plant species.

Agrobacterium tumefaciens↗
PubMed2001

Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet (Beta vulgaris L.).

Molecular approaches to sugar beet improvement will benefit from an efficient transformation procedure that does not rely upon exploitation of selectable marker genes such as those which confer antibiotic or herbicide resistance upon the transgenic plants. The expression of the green fluorescent protein (GFP) signal has been investigated during a program of research that was designed to address the need to increase the speed and efficiency of selection of sugar beet transformants. It was envisaged that the GFP reporter could be used initially as a supplement to current selection regimes in order to help eliminate "escapes" and perhaps eventually as a replacement marker in order to avoid the public disquiet associated with antibiotic/herbicide-resistance genes in field-released crops. The sgfp-S65T gene has been modified to have a plant-compatible codon usage, and a serine to threonine mutation at position 65 for enhanced fluorescence under blue light. This gene, under the control of the CaMV 35S promoter, was introduced into sugar beet via Agrobacterium-mediated transformation. Early gene expression in cocultivated sugar beet cultures was signified by green fluorescence several days after cocultivation. Stably transformed calli, which showed green fluorescence at a range of densities, were obtained at frequencies of 3-11% after transferring the inoculated cultures to selection media. Cocultivated shoot explants or embryogenic calli were regularly monitored under the microscope with blue light when they were transferred to media without selective agents. Green fluorescent shoots were obtained at frequencies of 2-5%. It was concluded that the sgfp-S65T gene can be used as a vital marker for noninvasive screening of cells and shoots for transformation, and that it has potential for the development of selectable marker-free transgenic sugar beet.

Base Sequence↗
PubMed2006

Production of soft rot resistant calla lily by expressing a ferredoxin-like protein gene (pflp) in transgenic plants.

An efficient protocol for the Agrobacterium tumefaciens-mediated transformation of calla lily (Zantedeschia elliottiana (W. Wats.) Engl. cultivar 'Florex Gold') is described. Shoot basal discs were co-cultivated with A. tumefaciens C58C1 carrying a plasmid containing neomycin phosphotransferase (nptII) and plant ferredoxin-like protein (pflp) genes. After Agrobacterium co-cultivation, the shoot basal discs were exposed to 100 mg l(-1) kanamycin for selection. Twenty-eight out of 260 discs (10.8%) were found to have survived and produced shoot clusters. Twenty-six of these were confirmed to contain the pflp transgene by PCR, ending up in 10% transformation efficiency. The disease resistance investigation revealed that 18 transgenic plants exhibited resistance to soft rot disease caused by Erwinia carotovora subsp. carotovora. The presence of pflp gene was demonstrated by PCR, and its accumulation and activity was confirmed by Western blot and disease resistance assay. This was the first report to show the successful transformation and resistance to a bacterial pathogen in Zantedeschia. The protocol is useful for the quality improvement of calla lily through genetic transformation.

Agrobacterium tumefaciens↗
PubMed2003

Stability of plasmid pA387 derivatives in Amycolatopsis mediterranei producing rifamycin.

Genetic studies on the biosynthesis of rifamycins in producer strains such as Amylcolaptopsis mediterranei U-32 are severely hampered by the availability of efficient transformation procedures and stable plasmid vectors. Using an efficient electroporation procedure we have studied the replication and stability of a pA387 derivative, pDXM32. This plasmid confers enhanced plasmid stability and copy number compared to pA387 derivatives commonly used as cloning vectors in A. mediterranei. Deletion derivatives in the region previously identified as being a minimal replication origin were also examined with respect to their ability to transform A. mediterranei and at least one locus was essential for replication. A 5.4 kbp DNA fragment was sequenced and annotated encoding the replication and plasmid stability functions. A parA homologue was identified which is likely to confer plasmid stability.

Actinomycetaceae↗