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PubMed2002

Characterisation of the expression of a novel constitutive maize promoter in transgenic wheat and maize.

A novel constitutive promoter from the maize histone H2Bgene was recently identified. In this study, we characterised H2B promoter activity in both wheat and maize tissues using the gusA reporter gene and two synthetic versions of the pat (phosphinothricin acetyl transferase) selectable marker gene, namely mopat and popat. Analyses of transgenic plants showed that the H2B promoter is able to drive the expression of gusA to strong, constitutive levels in wheat and maize tissues. Using an H2B:mopat construct and phosphinothricin selection, we recovered transgenic wheat plants at efficiencies ranging from 0.3% to 7.4% (mean 1.6%), and the efficiency of selection ranged from 40% to 100% (mean 77.7%). In another application, H2B was combined with the maize Ubi-1 or the maize Adh-1 intron to drive the expression of mopat and popat. Transformation efficiencies with the Ubi-1 intron were between 1.4- to 16-fold greater than with the Adh-1 intron. However, the use of either of the introns was necessary for the recovery of transgenic plants. Mopat gave higher transformation efficiencies and induced higher levels of PAT protein in maize tissues than popat.

Acetyltransferases↗

PubMed1988

Repair of the plasmid pBR322 damaged by gamma-irradiation or by restriction endonucleases using different recombination-proficient E. coli strains.

The plasmid pBR322 was treated with BamHI, PvuII and gamma-irradiation to generate double-strand breaks (dsb) containing differently structured ends. Transformation efficiencies, mutation frequencies and clone analyses of enzymatically damaged DNA are compared with the corresponding results from radiolytically damaged DNA. In E. coli K-12 SFX, the yield of transformants produced by the action of BamHI, PvuII and gamma-irradiation (30 Gy) is 4.3%, 0.14%, and 0.10%, respectively. The survival of open circular DNA (ocDNA) produced by 30 Gy is 1.3%. The transformation efficiencies show only a slight dependence on SOS induction and on the RecA protein. Mutation frequencies to tetracycline sensitivity (tets) per surviving plasmid are 2.6% (BamHI), 11.8% (PvuII) and 0.2% (gamma-irradiated DNA with 30 Gy containing approximately 50% ocDNA and 50% linearized (lin) DNA). The mutation frequency is low at all radiation doses studied (1-50 Gy). Only 15% of the DNA of the tets mutants from gamma-irradiated plasmids contained deletions whereas with enzymatically damaged DNA, 30-50% (BamHI) or 90% (PvuII) contained deletions. In all cases, the deletions comprised 500-1700 base pairs (bp). After SOS induction of the host cells, the mutation frequency of gamma-irradiated plasmids increased by a factor of 4, whereas that of the enzymatically damaged plasmids did not change. For the repair of the enzymatically linearized DNA 2 recombinational pathways are discussed which lead to deletant (pathway I) and non-deletant transformants (pathway II). In addition, BamHI-linearized plasmids may be repaired by enzyme-induced or spontaneous circular alignment followed by ligation. The high percentage of deletions of the tets mutations for PvuII-linearized DNA with blunt ends is explained by the illegitimate or site-specific recombination pathway I (see text). The lower percentage of deletions of the tets mutations with BamHI-linearized DNA with short cohesive ends (4 bp) is proposed to be due to a greater contribution of pathway II and/or by circular alignment followed by ligation. The very small yield and the low percentage of deletant mutations of tets mutants from radiolytically damaged DNA is proposed to be due to the large overlapping ends (16-100 bp) of the linDNA which easily leads to circular alignment followed by excision repair. The repair of radiolytically produced ocDNA is predominantly due to excision repair. In agreement with this interpretation is the observation that SOS induction of the host increases the mutation incidence of radiolytically damaged DNA but not of enzymatically damaged DNA.

DNA Repair↗

PubMed1991

Optimised cDNA size selection and cloning procedure for the construction of representative plasmid cDNA libraries.

Plasmid libraries are more versatile than phage libraries since they allow expression cloning in eukaryotic systems. However, high numbers of primary clones are sometimes difficult to obtain and more efficient transformation procedures are often required. In this paper, a detailed protocol is presented, for the construction of large plasmid libraries from ng quantities of cDNA, based on a highly efficient transformation step. Drop dialysis and electroporation are optimised: complete ligase removal and yeast tRNA addition before dialysis appear critical, while exclusive use of double-distilled water as well as rapid preparation of fresh cells provide excellent electroporation yields. A novel, simple and flexible cDNA size selection procedure is also presented, based on sucrose density gradient. Two libraries were constructed using an expression vector for mammalian cells (pCDM8) and its Escherichia coli host strain (MC1061[p3]). Numbers of 2 to 10 x 10(6) primary transformants were obtained from 1 microgram of poly(A)+ RNA. Up to 85% of the clones had inserts and half of the inserts were larger than 1.5 kb.

Cloning, Molecular↗

PubMed2005

Genetic transformation of Ascochyta rabiei using Agrobacterium-mediated transformation.

In order to study pathogenic mechanisms of the plant pathogen Ascochyta rabiei, conditions for efficient transformation using Agrobacterium-mediated transformation were investigated. Hygromycin B resistance (hph) was superior to geneticin resistance (nptII) for selecting transformants, and the hph gene was more efficiently expressed by the Aspergillus nidulans trpC promoter than by the Cauliflower mosaic virus 35S promoter CaMV35S. Co-cultivation on solid media for 72 h was optimal for generating transformants, but increasing the ratio of bacterial cells to conidia did not affect transformation efficiency. All hygromycin B-resistant transformants carried transfer-DNA (T-DNA) as determined by polymerase chain reaction (PCR) and the T-DNA integrations appeared to be random and in single copy as detected by Southern hybridization. Transformants remained resistant to hygromycin B in the absence of selection. Variations in colony morphology were observed in the presence of hygromycin B under different culture conditions, and a variety of altered phenotypes including reduced virulence were observed among 550 transformants. Inverse PCR was more efficient than TAIL-PCR in identifying flanking genomic sequences from T-DNA borders, and the possible causes are discussed. This transformation technique and recovery of flanking DNA using inverse PCR will provide a useful tool for genetic studies of A. rabiei.

Ascomycota↗

PubMed1994

An efficient method for large-scale isolation of plasmid DNAs by heat-alkali co-denaturation.

A method is presented for efficient and large-scale isolation of plasmid DNAs from bacterial cells. Based on the cooperativity of heat and alkali actions, the method provides DNA preparations with high quality and yield (about 2 micrograms of DNA/ml culture), which are completely digestable by restriction enzymes and have a high transformation efficiency. Furthermore, the DNA preparations are extremely stable, and even through 4-year storage at -20 degrees C, the electrophorogram and transformation efficiency remain as high as before. The factors affecting the stability of various DNA samples are discussed.

1-Propanol↗

PubMed1993

Improvement of EBV transformation and cloning efficiency of human B cells using culture supernatants from lymphoblastoid cell lines.

Exposure of human B cells to Epstein-Barr virus (EBV) usually results in low frequencies of transformed cells. The transformed cells can be cloned poorly by limiting dilution, even when feeder cells are used. In recent years it has become clear that growth and antibody production of EBV-transformed cells are influenced by auto- and paracrine growth factors. Therefore, supernatants from the lymphoblastoid B cell lines JY and Raji were used as a source of growth factors to investigate their effect during EBV transformation of human B cells and consequently on cloning by limiting dilution of these transformed cells. Initial experiments to clone three established EBV-transformed B cell lines showed a strong increase in outgrowth of the number of cells in the presence of the supernatant (range: 1 per 2-8 of the originally plated cells) as compared to cells cultured without the supernatant (range: 1 per 17-100 of the originally plated cells). Transformation efficiencies of freshly isolated tonsil B cells were not influenced by the supernatant and were generally less than 1%. In contrast, transformation efficiency was increased up to 9.4% if B cells were both transformed and cultured in the presence of the supernatant. Cloning efficiencies increased if the cells used were transformed in the presence of the supernatant. Best results were seen when the supernatant was present during transformation and cloning of the cells. The presence of the supernatant during transformation and/or cloning of the B cells dramatically enhanced the number of B cells secreting IgG. Cloning of two established tonsil B cell lines resulted in a large number of B cell clones.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibody Formation↗

PubMed1992

Plasmid transformation of naturally competent Acinetobacter calcoaceticus in non-sterile soil extract and groundwater.

The natural transformation of Acinetobacter calcoaceticus BD413 (trpE27) was characterized with respect to features that might be important for a possible gene transfer by extracellular DNA in natural environments. Transformation of competent cells with chromosomal DNA (marker trp+) occurred in aqueous solutions of single divalent cations. Uptake of DNA into the DNase I-resistant state but not the binding of DNA to cells was strongly stimulated by divalent cations. An increase of transformation of nearly 3 orders of magnitude was obtained as a response to the presence of 0.25 mM Ca2+. With CaCl2 solutions the transformation frequencies approached the highest values obtained under standard broth conditions, followed by MnCl2 and MgCl2. It is concluded that transformation requires divalent cations. DNA competition experiments showed that A. calcoaceticus does not discriminate between homologous and heterologous DNA. Furthermore, circular plasmid DNA competed with chromosomal DNA fragments and vice versa. The equally efficient transformation with plasmid pKT210 isolated from A. calcoaceticus or Escherichia coli indicated absence of DNA restriction in transformation. High efficiency plasmid transformation was obtained in samples of non-sterile natural groundwater and in non-sterile extracts of fresh and air-dried soil. Heat-treatment (10 min, 80 degrees C) of the non-sterile liquid samples increased transformation only in the dried soil extract, probably by inactivation of DNases. The results presented suggest that competent cells of A. calcoaceticus can take up free high molecular weight DNA including plasmids of any source in natural environments such as soil, sediment or groundwater.

Acinetobacter calcoaceticus↗

PubMed1979

Donor deoxyribonucleic acid length and marker effect in pneumococcal transformation.

The efficiency of transformation of point mutations depends upon base pair mismatches during the recombination process. For low-efficiency markers, the genetic information carried on the donor deoxyribonucleic acid is preferentially lost. To understand this elimination process, we investigated the effect of the size of donor deoxyribonucleic acid on the relative efficiency of low-efficiency point mutations. The deoxyribonucleic acid was shortened either by mechanical shearing or by restriction enzyme treatments. The results indicate that transformation by low-efficiency markers was not affected by shortening the distance between them and the end of the molecule any more than was transformation by the other markers. Moreover, no lethal event could be detected for either cell or chromosomal marker survival. These data do not exclude the double-strand-break hypothesis that was proposed to explain the loss of genetic information for low-efficiency markers, but they offer no support for it.

DNA Restriction Enzymes↗

PubMed2003

[Cotransformation of rice by bar and cecropin B gene expression cassettes lacking vector backbone sequences].

Whole plasmids are used in both Agrobacterium-mediated transformation and direct DNA transfer, generally leading to the integration of vector backbone sequences into the host genome along with the transgene(s). The undesirable vector backbone sequences may not only promote transgene rearrangements and affect transgene or endogenous gene expression negatively, but have disadvantage on the safe assessment of the transformants as "desert DNA". The direct DNA transforming systems can transfer minimal gene expression cassettes (promoter, open reading frame, terminator) into plant genome and generate "safer" transformants, also it can delivery multiple genes of agronomic relevance to economically-important crop plants. But there is seldom researching reports on the topic till now. The present paper studied some factors that affecting the transforming efficiency of liner gene expression cassettes to rice varieties by particle bombardment, and the integration patterns of the gene expression cassettes in rice genome were compared with that of the whole plasmids. The results showed: (1) The transforming frequency of gene expression cassettes to rice via particle bombardment is 0.1%-0.5%, the cotransforming frequency of non-selectable gene is about 50%-60% when two separate gene expression cassettes were used for transformation. Increasing the DNA mole content can increase the transforming frequency and the beside sequences of gene constructs may play an important role on the variation of transforming efficiency between different rice varieties. (2) It's reported that the selectable and non-selectable transgene expression cassettes generated low-copy-number transgenic plants with simple integration patterns. While our results showed that the non-selectable cecropin B gene cassette generated simple integration patterns with 1-3 copies in the rice genome, but the selectable bar gene cassette which got 4-14 copies had much more complex integration patterns than that of the whole plasmids which got 1-3 copies only. As the bar gene is promoted by the CaMV35 promoter, in which there is a 19 bp palindromic sequence could act as recombination hot spot and lead to DNA rearrangement, we presumed that the transgene recombination events happened during the integration course have generated the complex Southern patterns of bar gene expression cassette. The recombination character, the heredity behavior and the expression law of gene expression cassettes in the rice genomes will be reported in our future papers.

Acetyltransferases↗

PubMed1987

DNA-mediated gene transfer as an indicator of DNA damage and its repair by recipient cells.

Preparations of plasmid containing the thymidine kinase gene (pHSV106) were treated with the alkylating agents methyl methanesulphonate or N-methyl-N-nitrosourea prior to transfection into thymidine kinase-deficient mouse L-cells using the DNA-calcium phosphate co-precipitation technique. Relative to transfection with unmodified plasmid, a reduced transformation efficiency was observed using alkylation-damaged plasmid, N-methyl-N-nitrosourea causing the greatest inhibition. Treatment of recipient cells with arabinosyl cytosine or dideoxythymidine during the expression period following transfection by the 'damaged' plasmid reduced transformation efficiency, suggesting that DNA repair 4-6 h post-transfection was required for gene expression.

Animals↗

PubMed2005

The maize Knotted1 gene is an effective positive selectable marker gene for Agrobacterium-mediated tobacco transformation.

We have assessed the use of a homeobox gene knotted1 (kn1) from maize as a selectable marker gene for plant transformation. The kn1 gene under the control of cauliflower mosaic virus 35S promoter (35S::kn1) was introduced into Nicotiana tabacum cv. Xanthi via Agrobacterium-mediated transformation. Under nonselective conditions (without antibiotic selection) on a hormone-free medium (MS), a large number of transgenic calli and shoots were obtained from explants that were infected with Agrobacterium tumefaciens LBA4404 harboring the 35S::kn1 gene. On the other hand, no calli or shoots were produced from explants that were infected with an Agrobacterium strain harboring pBI121 (nptII selection) or from uninfected controls cultured under identical conditions. Relative to kanamycin selection conferred by nptII, the use of kn1 resulted in a 3-fold increase in transformation efficiency. The transgenic status of shoots obtained was confirmed by both histochemical detection of GUS activity and molecular analysis. The results presented here suggest that kn1 gene could be used as an effective alternative selection marker with a potential to enhance plant transformation efficiency in many plant species. With kn1 gene as a selection marker gene, no antibiotic-resistance or herbicide-resistance genes are needed so that potential risks associated with the use of these traditional selection marker genes can be eliminated.

Caulimovirus↗

PubMed1995

Genetic competence in Escherichia coli requires poly-beta-hydroxybutyrate/calcium polyphosphate membrane complexes and certain divalent cations.

In earlier studies of genetic competence in Escherichia coli induced with calcium-containing buffers, a strong correlation was found between transformation efficiency and the formation of poly-beta-hydroxybutyrate/calcium polyphosphate (PHB/Ca2+/PPi) complexes in the plasma membranes. In this study, we replaced Ca2+ with one of a number of other cations--monovalent, divalent, and trivalent--and found significant numbers of transformants (transformation efficiency, > 10(5)/micrograms of pBR322 DNA) only when the cells had high levels of PHB/Ca2+/PPi and the medium contained at least one of the divalent cations Ca2+, Mn2+, Sr2+, or Mg2+. Cells with high levels of the complexes were not competent when the medium did not contain these cations, but the cations were also ineffectual when the cells had few complexes. Surprisingly, Mn, Sr, and Mg were not incorporated into the complexes in place of Ca. These results indicate that PHB/Ca2+/PPi complexes and the above-mentioned divalent cations each have essential but disparate roles in genetic competence. Moreover, the strong selectivity of PHB/PPi for Ca2+ suggests the binding sites in the complexes are ionophoretic.

Calcium↗

PubMed1995

The Mos/MAP kinase pathway stabilizes c-Fos by phosphorylation and augments its transforming activity in NIH 3T3 cells.

The c-mos proto-oncogene product, Mos, is a serine/threonine kinase that can activate ERK1 and 2 mitogen-activated protein (MAP) kinases by direct phosphorylation of MAPK/ERK kinase (MEK). ERK activation is essential for oncogenic transformation of NIH 3T3 cells by Mos. In this study, we examined how mitogenic and oncogenic signalling from the Mos/MEK/ERK pathway reaches the nucleus to activate downstream target genes. We show that c-Fos (the c-fos protooncogene product), which is an intrinsically unstable nuclear protein, is metabolically highly stabilized, and greatly enhances the transforming efficiency of NIH 3T3 cells, by Mos. This stabilization of c-Fos required Mos-induced phosphorylation of its C-terminal region on Ser362 and Ser374, and double replacements of these serines with acidic (Asp) residues markedly increased the stability and transforming efficiency of c-Fos even in the absence of Mos. Moreover, activation of the ERK pathway was necessary and sufficient for the c-Fos phosphorylation and stabilization by Mos. These results indicate that c-Fos undergoes stabilization, and mediates at least partly the oncogenic signalling, by the Mos/MEK/ERK pathway. The present findings also suggest that, in general, the ERK pathway may regulate the cell fate and function by affecting the metabolic stability of c-Fos.

3T3 Cells↗

PubMed1982

Marker rescue by a homologous recipient plasmid during transformation of gonococci by a hybrid Pcr plasmid.

A 42-kilobase hybrid Pcr plasmid (pFA14) was formed when the naturally occurring 7.2-kilobase Pcr plasmid pFA3 was introduced by transformation into a competent gonococcal recipient containing the 36-kilobase conjugative plasmid pFA2 (Sox et al., J. Bacteriol. 138:510-518). Analysis of the structure of pFA14 showed that it was a stable recombinant between pFA3 and pFA2. The transformation efficiency of pFA14 was increased 300- to 10,000-fold by the presence in isogenic recipients of the homologous plasmid pFA2. The presence of a homologous plasmid in the recipient also markedly increased the likelihood of recovery of intact donor-size Pcr plasmids in the transformants. The presence of pFA2 had no effect on the competence of piliated or nonpiliated gonococci for transformation by either linear chromosomal DNA or a nonhomologous Pcr plasmid. Increased transformation efficiency of the hybrid Pcr plasmid pFA14 may have been due to recombination between the nicked or linearized donor plasmid and the homologous recipient plasmid (marker rescue).

Chromosomes, Bacterial↗

PubMed1983

Intermolecular recombination during transformation of Bacillus subtilis competent cells by monomeric and dimeric plasmids.

Bacillus subtilis competent cells harboring plasmid pUB110 were transformed by plasmids unable to replicate in this host but carrying segments of pUB110, 260 to 4500 bp long. Recombinants between the incoming and the resident plasmids were found in the transformed cells. Transforming efficiency of the incoming plasmids depended strongly on their molecular form and the length of their region homologous with the resident plasmid. It increased with the fourth to fifth power of that length for monomers having at least 900 bp of homology. Activity of monomers having less than 900 bp homology was too low to be measured in our experiments. Transforming efficiency of dimers was much greater than that of monomers, and varied with the square of the length of the homologous region. These results indicate that dimeric and monomeric plasmid molecules are processed differently during transformation of B. subtilis competent cells.

Bacillus subtilis↗

PubMed2006

A cyclic enamine derived from 1,2-O-isopropylidene-alpha-D-xylofuranose as a novel carbohydrate intermediate to achieve skeletal diversity.

The commercially available carbohydrate 1,2-O-isopropylidene-alpha-D-xylofuranose was efficiently transformed into the high-added-value synthetic scaffold 8. The transformation requires the synthesis of the 5-O-tosyl derivative 7 and its subsequent intramolecular cyclization under basic conditions to give the cyclic enamine 8. Reaction of 8 with O-, N-, S-, and C-nucleophiles and amino acids allowed its efficient transformation (one-step, high yields, and easy purifications) into fused cyclic sugar derivatives with rather unusual molecular skeletons in a completely regio- and stereoselective manner. The characteristics of the sugar derivative 8 established here, high reactivity, synthetic accessibility, and the potential for conversion into a vast collection of products by the action of different nucleophiles, indicate that it will prove to be a useful chiral intermediate for achieving skeletal diversity. The constrained structures and dense functionalization of the polycyclic sugar derivatives generated from 8 make these compounds promising candidates for use as starting agents for the production of new analogues and as drugs.

Amines↗

PubMed2006

Cucumber (Cucumis sativus L.).

We describe two novel Agrobacterium tumefaciens-based methods of cucumber transformation. The first involves direct regeneration from leaf microexplants selected on kanamycin-containing medium. The second involves regeneration from a long-term established embryogenic suspension culture emitting green autofluorescence (GAF) and selection on medium containing hygromycin. In the latter method, GAF was used as a reporter, thereby allowing a simple and reliable identification of transgenic cells with a high regeneration capacity. (No false positives were observed.) The transformation efficiency in the leaf microexplants fluctuated from 0.8 to 6.5% of the primary explants, whereas in the embryogenic suspension-cultured cells it varied from 6.4 to 17.9% of the aggregates. In the GAF method, the step involving the elimination of the Agrobacterium cells by antibiotics could be omitted; however, this reduced the transformation efficiency to about 3%. The time required from inoculation to regenerated plant in the greenhouse was the same for both methods, but the GAF method required more preinoculation time than the leaf microexplant method.

Agrobacterium tumefaciens↗

PubMed1988

44-amino-acid E5 transforming protein of bovine papillomavirus requires a hydrophobic core and specific carboxyl-terminal amino acids.

The 44-amino-acid E5 protein of bovine papillomavirus type 1 is the shortest known protein with transforming activity. To identify the specific amino acids required for in vitro focus formation in mouse C127 cells, we used oligonucleotide-directed saturation mutagenesis to construct an extensive collection of mutants with missense mutations in the E5 gene. Characterization of mutants with amino acid substitutions in the hydrophobic middle third of the E5 protein indicated that efficient transformation requires a stretch of hydrophobic amino acids but not a specific amino acid sequence in this portion of the protein. Many amino acids in the carboxyl-terminal third of the protein can also undergo substitution without impairment of focus-forming activity, but the amino acids at seven positions, including two cysteine residues that mediate dimer formation, appear essential for efficient transforming activity. These essential amino acids are the most well conserved among related fibropapillomaviruses. The small size of the E5 protein, its lack of similarity to other transforming proteins, and its ability to tolerate many amino acid substitutions implies that it transforms cells via a novel mechanism.

Amino Acid Sequence↗