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PubMed1989

One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution.

We have developed a simple, one-step procedure for the preparation of competent Escherichia coli that uses a transformation and storage solution [TSS; 1 x TSS is LB broth containing 10% (wt/vol) polyethylene glycol, 5% (vol/vol) dimethyl sulfoxide, and 50 mM Mg2+ at pH 6.5]. Cells are mixed with an equal volume of ice-cold 2 x TSS and are immediately ready for use. Genetic transformation is equally simple: plasmid DNA is added and the cells are incubated for 5-60 min at 4 degrees C. A heat pulse is not necessary and the incubation time at 4 degrees C is not crucial, so there are no critical timing steps in the transformation procedure. Transformed bacteria are grown and selected by standard methods. Thus, this procedure eliminates the centrifugation, washing, and long-term incubation steps of current methods. Although cells taken early in the growth cycle (OD600 0.3-0.4) yield the highest transformation efficiencies (10(7)-10(8) transformants per micrograms of plasmid DNA), cells harvested at other stages in the growth cycle (including stationary phase) are capable of undergoing transformation (10(5)-10(7) transformants per micrograms of DNA). For long-term storage of competent cells, bacteria can be frozen in TSS without addition of other components. Our procedure represents a simple and convenient method for the preparation, transformation, and storage of competent bacterial cells.

Cations, Divalent↗
PubMed2001

A new dominant selectable marker for use in Cryptococcus neoformans.

Cryptococcus neoformans is an excellent model system for studies on the molecular pathogenesis of fungal infections. There is only one dominant selectable market that can be used in the transformation of this organism, and we wanted to develop another. We found that various strains of C. neoformans are very sensitive to the aminoglycoside antibiotic nourseothricin, and that spontaneous resistance to this drug must be an extremely rare event. Resistance to nourseothricin is conferred by the product of the nourseothricin acetyltransferase gene (nat1) from Streptomyces noursei. In order to express this gene in C. neoformans, we created a fusion construct by driving expression of natl with the promoter sequence from a C. neoformans actin gene. Biolistic transformation of the serotype A C. neoformans strain H99 and the serotype D strain JEC21 with this construct resulted in transformation efficiencies of approximately 1,000 transformants microg(-1) of DNA and 20 transformants microg(-1) of DNA, respectively. Southern blots were performed using DNA from some of the H99 transformants, and this confirmed that all of the resistant isolates had the construct integrated in a random fashion within the genome. There was no cross-resistance of the nourseothricin-resistant transformants to hygromycin B, which is the other antibiotic used as a dominant selection marker in C. neoformans. The development of nourseothricin resistance as a second dominant selectable market will be helpful in future molecular studies on this important pathogenic fungus.

Acetyltransferases↗
PubMed2003

Nitrogen-regulated hypermutator strain of Synechococcus sp. for use in in vivo artificial evolution.

Artificially evolved variants of proteins with roles in photosynthesis may be selected most conveniently by using a photosynthetic organism, such as a cyanobacterium, whose growth depends on the function of the target protein. However, the limited transformation efficiency of even the most transformable cyanobacteria wastes much of the diversity of mutant libraries of genes produced in vitro, impairing the coverage of sequence space. This highlights the advantages of an in vivo approach for generating diversity in the selection organism itself. We constructed two different hypermutator strains of Synechococcus sp. strain PCC 7942 by insertionally inactivating or nutritionally repressing the DNA mismatch repair gene, mutS. Inactivation of mutS greatly increases the mutation rate of the cyanobacterium's genes, leading to an up-to-300-fold increase in the frequency of resistance to the antibiotics rifampin and spectinomycin. In order to control the rate of mutation and to limit cellular damage resulting from prolonged hypermutation, we placed the uninterrupted mutS gene in the cyanobacterial chromosome under the transcriptional control of the cyanobacterial nirA promoter, which is repressed in the presence of NH(4)(+) as an N source and derepressed in its absence. By removing or adding this substrate, hypermutation was activated or repressed as required. As expected, hypermutation caused by repression in PnirA-mutS transformants led to an accumulation of spectinomycin resistance mutations during growth.

Adenosine Triphosphatases↗
PubMed1993

Genetic definition of a new bovine papillomavirus type 1 open reading frame, E5B, that encodes a hydrophobic protein involved in altering host-cell protein processing.

We have previously observed that bovine papillomavirus type 1 (BPV-1) induces the appearance of five cellular proteins in C127 mouse fibroblasts, four of which appear to arise by altered processing of resident endoplasmic reticulum proteins. Studies of various cell lines revealed that expression of the 3' end of the BPV early region was sufficient for induction of these changes. To identify the BPV gene responsible, we have utilized the simian virus 40 (SV40)/BPV-1 recombinant virus Pava-1, which expresses the 3' end of the BPV early region behind an SV40 early promoter. C127 cells infected with Pava-1 for 48 h show the expected BPV-associated alterations, as do cells infected with Pava constructs mutated in the E5 or E2 genes. However, a mutation in the start codon of a previously ignored open reading frame extending from nucleotides 4013 to 4170 (E5B) eliminated the BPV-associated changes. Similar results were obtained with COS cells infected with the Pava mutants and C127 cells transformed by full-length mutated BPV. Despite its influence on the processing of cellular endoplasmic reticulum proteins, this mutation in E5B did not alter BPV-transforming efficiency or the ability of transformants to form colonies in soft agar. The E5B open reading frame encodes a hydrophobic 52-amino-acid polypeptide that shares structural similarities with HPV6 E5A and HPV16 E5. Speculations on a role for E5B in the viral life cycle are discussed.

Amino Acid Sequence↗
PubMed2006

Pine (Pinus radiata).

This chapter describes the transformation of Pinus radiata using organogenic cotyledon explants rather than the more common somatic embryogenesis methods for conifers. The advantages of our method are the year round availability of seed and that over 80% of genotypes can be easily regenerated from the mature cotyledon explants. The transformation efficiency (i.e., the number of transformed shoots regenerated from excised cotyledons) is 1.7% and, as with other Agrobacterium tumefaciens transformation methods, the majority of transgene integrations are single copy. Critical factors for success are survival of the cotyledons, Agrobacterium strain, and selection pressure after cocultivation.

Agrobacterium tumefaciens↗
PubMed1999

Transformation of a flocculating Saccharomyces cerevisiae using lithium acetate and pYAC4.

A flocculating yeast Saccharomyces cerevisiae ura3 was transformed by the method based on treatment of intact cells with lithium acetate plus single-stranded carrier DNA using the shuttle vector pYAC4. The transformation efficiency was above 10(3) transformants per microgram of plasmid DNA which is similar to other described yeast transformation systems. Under selective pressure, the transformed cells were stable and maintained the flocculation ability. Thus, this simple transformation system can be used for gene expression studies in flocculating yeasts, overcoming disadvantages of conventional methods such as the spheroplast one.

Acetates↗
PubMed1990

A procedure for cloning restriction fragments of DNA as single inserts in yeast artificial chromosomes.

A novel procedure is described for the cloning of partial EcoRI fragments of bovine DNA: it reduces the chance of sequence rearrangements due to multiple insertions (co-cloning) of restriction fragments in the resulting YAC. The DNA to be inserted has been dephosphorylated, whereas the matching ends of the vector, pYAC4, have not. The ligation was essentially complete, the transformation efficiency was close to 19 transformants per ng of vector and the frequency of clones carrying YAC, 60-100 kb in size, was close to 70%. The YACs show segregative and replicative stability.

Animals↗
PubMed1987

Inhibition of H-ras oncogene transformation of NIH3T3 cells by protease inhibitors.

The protease inhibitors antipain, leupeptin, alpha 1-antitrypsin, and epsilon-aminocaproic acid were found to inhibit transformation of NIH3T3 cells after transfection with an activated H-ras oncogene. Inhibition of focus formation by protease inhibitors was concentration dependent and maximal at 50% of control values. Transfection of a gene for neomycin resistance was not affected by protease inhibitors. Antipain was inactive if present only during the first 2 days of the gene transfer protocol or only during the final 10 days of the experiment. However, the full effect was observed when antipain was added at the subculture step on day 3 and during the subsequent cell proliferation. If cells were not subcultured, the yield of the foci per microgram of DNA was sharply reduced and addition of antipain did not further suppress the transformation rate. Subculture of NIH3T3 cells 3 days after transfection at lower cell densities resulted in higher transformation efficiency. The results suggest that transformation of NIH3T3 cells by a single mutated oncogene may involve multiple stages including cell proliferation and that part of this process is susceptible to inhibition by protease inhibitors.

Aminocaproic Acid↗
PubMed1994

Transformation of Solanum dulcamara protoplasts and regeneration of transgenic plants.

We have achieved successful transformation of Solanum dulcamara protoplasts by direct DNA uptake and regeneration of transgenic plants. The plasmids pDW2 carrying CAT gene and pCaMVNEO carrying NPTII gene were used. The electroporation voltage was 1500 V, which gave a field strength of 1500 V/cm with a time decay constant of 59.4 sec. The concentration of plasmid was 20 micrograms/2 x 10(6) protoplasts. Under these conditions, a very high transformation efficiency was obtained, with relative transformation frequency being up to 12.4% and absolute transformation frequency 2.4 x 10(-4). The activity of NPTII was detected in 75% of the kanamycin resistant calli and all of the plants regenerated from resistant calli. Southern blot analysis showed that the DNA sequence of NPTII gene derived from pCaMVNEO plasmid existed in the transformed plants of S. dulcamara.

Blotting, Southern↗
PubMed1981

Integration and excision of a plasmid in Bacillus subtilis.

We have studied the behaviour in Bacillus subtilis of a plasmid (pPV21) carrying the thymidylate synthetase gene of phage phi3T (thyP3). The plasmid can transform efficiently the competent cells of all the strains tested. Polyethylene glycol (PEG)-mediated protoplast transformation is efficient only for recE, recD or recF mutants. When present in recombination proficient strains, the plasmid can be integrated into the chromosome, primarily at the thyA locus. This has been shown by genetic mapping and by blot-hybridization. A second less efficient site is at (or near to) the attachment site of phage phi3T. Excision of the plasmid restores the EcoRI restriction pattern of the parental DNA, although with the loss of the defective thyA endogenotic allele and the retention of the thyP exogenotic gene.

Bacillus subtilis↗
PubMed1989

High efficiency electroporation of intact Corynebacterium glutamicum cells.

High-frequency electroporation of whole Corynebacterium glutamicum cells without enzymatic pretreatment was achieved. Under optimized conditions concerning growth stage, washing of cells, cell concentration and pulse parameter transformation efficiencies of far more than 10(7) transformants per microgram pWST4B plasmid DNA were reached. Using electroporation, linearised and subsequently religated plasmid as well as chimeric ligase reaction products were directly introduced into C. glutamicum with reasonable efficiencies. Electrotransformation efficiency was reduced about 10(5)-fold for plasmid DNA cycled through E. coli JM83. Restriction deficient mutants of C. glutamicum were isolated which could be efficiently transformed with foreign DNA.

Corynebacterium↗
PubMed2004

Improvement of transformation and electroduction in avermectin high-producer, Streptomyces avermitilis.

Factors affecting the PEG-mediated transformation and electrotransformation of Streptomyces avermitilis protoplasts, an industrial avermectin high-producer, were evaluated. The maximum protoplast transformation efficiency under optimum conditions with PEG was 3 x 106 transformants per microg plasmid pIJ702 DNA. The efficiency of electrotransformation with the same plasmid the intact cells grown in medium with 0.5 mmol/L CaCl2, suspended in buffer with 0.5 mol/L sucrose +1 mmol/L MgCl2, and pulsed at an electric field strength of 10 kV/cm, 800 ohms, 25 microF, was of 2 x 10(3) transformants per microg DNA. When the cells were electroporated after mild lysozyme-treatment, the efficiency was up to 10(4) transformants per microg DNA. Electroporation of protoplasts and germlings had a lower efficiency (10(2) transformants per microg DNA). We report that electroporation under optimum conditions can be used for direct transfer of nonconjugative plasmid pIJ699 between two different Streptomyces species, S. avermitilis and S. lividans.

Electroporation↗
PubMed1986

Inhibition of protein synthesis enhances transformation of primary cells by viral DNA.

Inhibition of protein synthesis by cycloheximide after transfection and subsequent removal of the drug increased the transformation efficiency of primary cells by plasmids containing the left 4.5, 6.7, or 16% of the adenovirus (Ad) genome. The enhancement factor ranged from 2 to as much as 70 depending on the size of the viral DNA fragments used. Addition of cycloheximide before or at the time of transfection inhibited transformation, suggesting that viral protein synthesis is important during the early phase of transformation. Transient expression assays showed that cells treated with cycloheximide post-transfection contained as much as three times the amount of viral RNA transcribed from regions E1A and E1B. Conversion of a rat cell line lacking thymidine kinase activity (TK-) to the TK+ phenotype by a plasmid containing the herpes TK gene was severely inhibited by the drug treatment, suggesting that the enhancement effects of cycloheximide on transformation may be specific for Ad DNA. Cycloheximide treatment also increased the number of transformants induced by a transformation defective E1B mutant of Ad12 (cyt mutant). Plasmid containing only the E1A region of Ad12 transformed primary rat kidney cells with very low efficiency. The inclusion of E1B in the transfecting DNA fragments increased the transformation frequency by more than 400-fold, much higher than that achieved by cycloheximide. Thus, cycloheximide cannot replace E1B functions in transformation efficiency.

Adenoviruses, Human↗
PubMed1991

Genetic transformation of Chainia and heat attenuation of its restriction system.

A PEG-mediated transformation system for Chainia (NCL 82-5-1) was developed using a broad host range Streptomyces vector, pIJ702. Protoplasts prepared from Chainia (NCL 82-5-1) were regenerated with 5% efficiency. Transformation of the protoplasts with pIJ702 gave 10-20 transformants/micrograms DNA. The low efficiency of transformation is attributed to a restriction system in Chainia; this could be inhibited by treating the protoplasts at 42 degrees C for 10 min just before transformation. The yield of transformants increased 100-fold when pIJ702 was modified by passage in Chainia. Because the plasmid replicon was functional in Chainia and the modified plasmid was stably maintained, the transformation system should be useful for self-cloning in Chainia NCL 82-5-1 of the many commercially important enzymes this strain is known to produce.

Cloning, Molecular↗
PubMed2002

[Factors affecting Agrobacterium tumefaciens-mediated transformation of wheat (Triticum aestivum L.)].

Immature embryos and embryo-derived calli from two cultivars of winter wheat (Triticum aestivum L.), BAU146 and BAU170, were transformed with three strains of Agrobacterium tumefaciens, AGL-1, EHA105 and LBA4404 harboring expression vector p3301 or pBTAaB. Both vectors contained bar gene and p3301 contained also gus gene with an intron. The highest explant survival rate and transformation efficiency was obtained when the bacterial cell density was OD600 1.0 with 1 h of infection incubation. Higher osmotic treatment of the explants before inoculation had a positive effect on transformation, while addition of acetosyringone showed ambiguous one, depending on the explant types and bacterium strains. The efficiencies of transformation and transgenic plant regeneration were varied greatly with the bacterium strain, receptor genotype, explant type and its age and physiological state. After optimizing these factors, a large number of PPT-resistant calli and some of PPT-resistant plants were obtained. The resistant plantlet tested and 50% to 60% of the resistant calli were GUS-positive. The integration of foreign DNA into the genome of transgenic plants (3 out 6) was further confirmed by PCR and Southern Blot analysis.

Agrobacterium tumefaciens↗
PubMed2003

An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens.

The moss Physcomitrella patens is the only land plant known to date with highly efficient homologous recombination in its nuclear DNA, making it a unique model for plant functional genomics approaches. For high-throughput production of knockout plants, a robust transformation system based on polyethylene glycol-mediated transfection of protoplasts was developed and optimised. Both the DNA conformation and pre-culture of plants used for protoplast isolation significantly affected transformation efficiencies. Employing a newly developed PCR high-throughput method, the gene-targeting efficiency in more than 1000 plants transformed with different cDNA-based knockout constructs was determined and analysed with regard to the length and intron/exon structure of the homologous gene locus. Different targeting constructs, each containing an identical selectable marker gene, were applied as batch DNA in a single transformation experiment and resulted in double-knockout plants. Thus, the fast and efficient generation of multiple targeted gene-knockouts is now feasible in Physcomitrella.

Amino Acid Sequence↗
PubMed1986

Abelson transformed fibroblasts lacking the EGF receptor are not tumourigenic in nude mice.

Cells transformed by the v-abl-oncogene produce large amounts of the tumour growth factor alpha TGF. alpha TGF is homologous to the epidermal growth factor (EGF) and stimulates cell growth via the EGF receptor pathway. To separate metabolic events in the v-abl-transformed cells mediated by alpha TGF as opposed to the v-abl-encoded protein-tyrosine kinase, we have employed the Swiss 3T3 variant cell line NR6 which lacks a functional EGF receptor. v-abl was found to transform efficiently NR6 cells in vitro. These transformed NR6 cells displayed a variety of in vitro properties which were indistinguishable from transformed wild-type fibroblast lines. However, in contrast to the wild-type lines, v-abl-transformed NR6 cells failed to form tumours when injected into athymic nude mice. These results imply an important function for alpha TGF and the EGF receptor in the establishment of the v-abl-induced fibrosarcomas.

Abelson murine leukemia virus↗
PubMed1987

Development of a plasmid-cloning system for Streptomyces viridochromogenes Tü494.

A plasmid-cloning system was developed for Streptomyces viridochromogenes Tü494, a producer of the tripeptide antibiotic phosphinothricyl-alanyl-alanine (PTT). Parameters affecting protoplast formation and transformability of S. viridochromogenes were investigated in detail. A procedure giving rise to transformation efficiencies of 10(4)-10(5) transformants per microgram DNA was worked out. Several Streptomyces plasmid vectors such as pIJ350, pIJ61, pEB2, pGM4 and pSW2 were tested in S. viridochromogenes. Some of these vectors (pGM4, pEB2) showed a high copy number, whereas the copy number of others (pIJ350, pIJ61 and pSW2) was markedly lower. Under non-selective conditions some of the vectors (pIJ350, pEB2) were not stably maintained, in contrast to the vectors pGM4 and pSW2 which did not require any selection pressure for stable maintenance. Therefore, the plasmid vectors pGM4 and pSW2 derived from endogenous replicons of Streptomyces ghanaensis strains represent appropriate plasmid vehicles for cloning experiments in S. viridochromogenes.

Cloning, Molecular↗