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PubMed1975

Isolation and characterization of mutants of Haemophilus influenzae deficient in an adenosine 5'-triphosphate-dependent deoxyribonuclease activity.

By a direct assay approach, mutants of Haemophilus influenzae Rd that are deficient in adenosine 5'-triphosphate-dependent deoxyribonuclease activity (add-) were isolated and characterized. A large proportion (50 to 90%) of the cells in cultures of these mutants failed to produce visible colonies when plated. An extensive analysis of the recombination proficiency of these strains revealed that the transformation frequency (transformants per competent cell) in the mutants was similar to that found in the wild type, but that the transformation efficiency (transformants per microgram of irreversibly bound deoxyribonucleic acid [DNA]) was reduced approximately fourfold. Sensitivities of the mutants to gamma rays, ultraviolet radiation, and methyl methane sulfonate were only slightly greater than wild-type levels. The rate of degradation of host DNA after ultraviolet irradiation was significantly reduced in the mutants. It is suggested that the adenosine 5'-triphosphate-dependent deoxyribonuclease in H. influenzae plays a nonessential role in DNA recombination and repair.

Adenosine Triphosphate↗
PubMed1989

JC virus-simian virus 40 genomes containing heterologous regulatory signals and chimeric early regions: identification of regions restricting transformation by JC virus.

The papovavirus JC virus (JCV) is highly oncogenic in experimental animals but, unlike simian virus 40 (SV40), is severely restricted in its ability to transform cells in culture. We exploited the close genetic relatedness of these two viruses to delimit region(s) of the T protein which can restrict transforming activity. Novel chimeric genomes were produced by exchanging various segments of the JCV and SV40 T-protein-coding regions. These DNA constructs specified early proteins with in-frame substitutions of analogous amino acid sequences. A second set of genomes was prepared which, in addition to chimeric early proteins, contained substituted regulatory regions. The transformation efficiencies of these chimeric genomes were intermediate between those of SV40 and JCV, with the source of T protein exerting a greater effect than that of the regulatory region. The ability of certain constructs to induce efficient transformation required the presence of an SV40 regulatory region or specific sequences within the SV40 early coding region. Cloned cell lines prepared from representative transformants were characterized; the ability to form colonies in soft agarose was investigated, and the presence of viral T and cellular p53 proteins was determined. The various T proteins differed in amount, stability, and the ability to form stable complexes with p53.

Animals↗
PubMed2006

Brassica oleracea.

A better understanding of the genetic basis underlying the genotype dependence of Brassica oleracea transformation is enabling researchers to distinguish between recalcitrant and successful candidate genotypes for routine transformation. In this chapter we outline an A. tumefaciens-mediated transformation method for B. oleracea using 4-d-old cotyledonary explants and a model B. oleracea doubled haploid genotype, AG DH1012. After only 3 wk on kanamycin selection, the first transgenic shoots can be isolated. Transformation efficiencies in the region of 10 to 25% (based on 50-125 PCR-positive independent shoots from 500 infected explants) are typically achieved. For researchers wishing to use their own plant genotype, we highlight the tissue culture phenotypes that are conducive to efficient transformation.

Agrobacterium tumefaciens↗
PubMed2025

Ultra-high field strength electroporation enables efficient DNA transformation and genome editing in nontuberculous mycobacteria.

Efficient DNA delivery is essential for genetic manipulation of mycobacteria and for dissecting their physiology, pathogenesis, and drug resistance. Although electroporation enables transformation efficiencies exceeding 10⁵ CFU per µg DNA in Mycobacterium smegmatis and Mycobacterium tuberculosis, it remains highly inefficient in many nontuberculous mycobacteria (NTM), including Mycobacterium abscessus. Here, we discovered that NTM such as M. abscessus exhibit exceptional tolerance to ultra-high electric field strengths and that hypertonic preconditioning partially protects cells from electroporation-induced damage. Using ultra-high electric field strength (3 kV/mm) electroporation, we achieved dramatic improvements in plasmid transformation efficiency-up to 106-fold in M. abscessus, 83-fold in Mycobacterium marinum, and 37-fold in Mycobacterium kansasii-compared to standard conditions (1.25  kV/mm). Transformation efficiency was further influenced by the choice of selectable marker. Ultra-high field strength electroporation also markedly enhanced allelic exchange in M. abscessus expressing Che9c RecET recombinases, increasing the recovery of gene deletion mutants by over 1,000-fold relative to conventional electroporation. In parallel, oligonucleotide-mediated recombineering for targeted point mutations produced nearly 10,000-fold more mutants under ultra-high field conditions. Together, these findings establish ultra-high field electroporation as a robust, broadly applicable platform for genetic engineering of NTMs. This method substantially enhances transformation efficiency and enables construction of advanced genetic tools-including expression libraries and CRISPRi knockdown libraries-in species that have historically resisted genetic manipulation.IMPORTANCEInfections caused by nontuberculous mycobacteria (NTM), including Mycobacterium abscessus, are increasing globally, yet genetic manipulation of these pathogens remains technically challenging due to inefficient DNA delivery and low gene editing success. The ultra-high electric field strength electroporation strategy described here overcomes these barriers, enabling dramatic improvements in both transformation and genome editing efficiency. This advance paves the way for high-throughput functional genomics in NTMs, including the construction of genome-wide knockout, CRISPRi knockdown, and expression libraries. Broad adoption of this approach will accelerate discovery of genetic determinants of virulence and drug resistance, facilitating the development of antimicrobials and vaccines.

Electroporation↗
PubMed2006

Development of simple and efficient in Planta transformation method for wheat (Triticum aestivum L.) using Agrobacterium tumefaciens.

Wheat (Triticum aestivum L. var. Shiranekomugi) seeds were soaked in water at 22 degrees C for 1 d. Thereafter, the embryo of the soaked seeds was inoculated with Agrobacterium tumefaciens by piercing a region of the embryonic apical meristem with a needle that had been dipped in an A. tumefaciens inoculum. The inoculated seeds were incubated at 22 degrees C for 2 d and sterilized by cefotaxime (Claforan) (1000 ppm water solution) treatment and then vernalized at 5 degrees C for 25 d. Finally, the seedlings were grown to maturation (T(0) plants) and allowed to pollinate naturally for seed setting (T(1) plants) in pots under nonsterile condition. To examine the transformation by various means, four different strains of A. tumefaciens were used for transformation. The following five lines of evidence proved the transformation: altered phenotype and its transmittance to the next generation, resistance of T(1) seed germination to geneticin or hygromycin B, the detection of a transgene in T(1) plants by PCR analysis and Southern hybridization and the rescue of the plasmid consisting of the integrated T-DNA and flanking wheat genome DNA from T(1) plants. The transformation efficiency of T(1) plants, which were transformed using different A. tumefaciens strains, was estimated to be 33% by PCR analysis, 75% by Southern hybridization and 40% by plasmid rescue.

Agrobacterium tumefaciens↗
PubMed1993

A study of integrative transformation in Schizosaccharomyces pombe.

Using the one-step gene disruption technique, we studied the effect of various parameters on the disruption frequency (percentage of homologous integrants) and transformation efficiency (number of transformants per microgram of input DNA) of integrative transformation in Schizosaccharomyces pombe. We used suc1 as the target gene for disruption and ura4 as the selectable marker. Our results are as follows. 1) Use of the strong adh1 promoter to drive the expression of ura4 did not affect the disruption frequency but modestly increased the transformation efficiency. 2) The transformation method had a profound effect, with the lithium acetate method yielding both a 10-fold higher disruption frequency compared to the protoplast method and a 5- to 10-fold higher transformation efficiency. 3) The presence of increasing amounts of non-homologous sequences at the ends of the transforming DNA decreased the disruption frequency by up to 5-fold but had no effect on the transformation efficiency. We also describe the use of the sup3-5 allele in an ade6-704 genetic background to discriminate between the products of homologous versus non-homologous integration, thereby promoting the identification of rare homologous integrants.

Alleles↗
PubMed2003

Efficiency of transient transformation in tobacco protoplasts is independent of plasmid amount.

We describe an optimized protocol for the transient transformation of tobacco protoplasts mediated by polyethylene-glycol (PEG). As expected, the quantitative beta-glucuronidase (Gus) activity driven by pCaMVGus was dependent on the amount of plasmid used. Nevertheless, we demonstrate by an immunodetection method that transformation efficiency did not depend on the amount of plasmid used but on the limitation imposed by cell competence. In fact, we obtained the same percentage of transformed cells (about 60%) using a wide range of plasmid concentrations (0.1-10 microg per test). Finally, we show that, when we used two plasmid types in a mixture at a concentration ranging from 0.1 to 10 microg for each, all transformed cells expressed proteins encoded by both plasmids. Transient expression and co-transformation experiments are routinely used methods and, probably, the major results from this work were assumed by many researchers in this field, but our data experimentally support this assumption.

Plasmids↗
PubMed2000

Development of a simple and efficient method for transformation of buckwheat plants (Fagopyrum esculentum) using Agrobacterium tumefaciens.

Apical meristems of seedlings of buckwheat (Fagopyrum esculentum var. Shinano No. 1) were pricked with a needle and inoculated with Agrobacterium tumefaciens (LBA4404, pBI121). The inoculated seedlings were grown to maturation and allowed to pollinate randomly to set the seeds (T1 plants). The transformation efficiency of the T1 plants was estimated by germination in the presence of geneticin (20 microg/ml) and by detection of beta-glucuronidase (GUS) gene with PCR, indicating that 36% and 70% of the T1 plants were transformed, respectively. Four plants taking on a mutated morphology were selected from T1 plants which were transformed with the method using A. tumefaciens harboring a modified pBI121 for plasmid rescue. Southern blot analysis of plasmids rescued from the 4 T1 plants demonstrated that each plasmid contained a different flanking DNA of the buckwheat genome, an evidence that T-DNA was integrated in different sites of the genomic DNA among the 4 T1 plants.

Agrobacterium tumefaciens↗
PubMed1984

Optimal conditions for genetic transformation of the cyanobacterium Anacystis nidulans R2.

Under optimal conditions, the cyanobacterium Anacystis nidulans R2 was transformed to ampicillin resistance at frequencies of greater than 10(7) transformants per microgram of plasmid (pCH1) donor DNA. No stringent period of competency was detected, and high frequencies of transformation were achieved with cultures at various growth stages. Transformation increased with time after addition of donor DNA up to 15 to 18 h. The peak of transformation efficiency (transformants/donor molecule) occurred at plasmid concentrations of 125 to 325 ng/ml with an ampicillin resistance donor plasmid (pCH1) and 300 to 625 ng/ml for chloramphenicol resistance conferred by plasmid pSG111. The efficiency of transformation was enhanced by excluding light during the incubation or by blocking photosynthesis with the electron transport inhibitor 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU) or the uncoupler carbonyl cyanide-m-chlorophenyl hydrazone. Preincubation of cells in darkness for 15 to 18 h before addition of donor DNA significantly decreased transformation efficiency. Growth of cells in iron-deficient medium before transformation enhanced efficiency fourfold. These results were obtained with selection for ampicillin (pCH1 donor plasmid)- or chloramphenicol (pSG111 donor plasmid)-resistant transformants. Approximately 1,000 transformants per microgram were obtained when chromosomal DNA from an herbicide (DCMU)-resistant mutant was used as donor DNA. DCMU resistance was also transferred to recipient cells by using restriction fragments of chromosomal DNA from DCMU-resistant mutants. This procedure allowed size classes of fragments to be assayed for the presence of the DCMU resistance gene.

Ampicillin↗
PubMed2005

Development of simple and efficient in planta transformation method for rice (Oryza sativa L.) using Agrobacterium tumefaciens.

Seeds of rice (Oryza sativa L. var. Koshihikari) were soaked in water for 2 d. Thereafter, the embryo containing an apical meristem was inoculated with Agrobacterium tumefaciens by piercing a site of the husk overlying the embryonic apical meristem with a needle that had been dipped in an A. tumefaciens inoculum. The inoculated seeds were then grown to maturation (T0 plants) and allowed to pollinate naturally to set seeds (T1 plants) in pots under nonsterile conditions. To examine the transformation by various means, three different strains of A. tumefaciens were used for transformation: an M-21 mutant, which is an avirulent mutant with a Tn5 insertion in the iaaM gene, and two LBA4404 strains each with a different binary vector. Five different lines of evidence were demonstrated the transformation: the altered phenotype and its inheritance by the next generation, histochemical detection of beta-glucuronidase, resistance to hygromycin B, detection of the transgene by PCR and rescue of a plasmid consisting of the integrated T-DNA and the flanking rice genome DNA. Transformation efficiency of T1 plants was estimated to be 40% and 43% by PCR and a histochemical assay of beta-glucuronidase, respectively.

Agrobacterium tumefaciens↗
PubMed2006

An improved protocol for electrotransformation of Corynebacterium pseudotuberculosis.

We developed an improved protocol for the electrotransformation of Corynebacterium pseudotuberculosis, testing variations of parameters in the procedures that are routinely used for the preparation of electrocompetent cells of this species, including (i) culture conditions, (ii) cell growth phase, (iii) electroporation solutions and (iv) quantity of plasmid DNA. We obtained the greatest efficiency of transformation when the cells were grown until the stationary phase and then washed with 10% glycerol electroporation solution. The transformation efficiency was inversely proportional to the quantity of plasmid DNA. The transformation efficiency reached 10(5) colony-forming units (cfu)/mug plasmid DNA. This protocol would be useful for genetic studies of C. pseudotuberculosis.

Corynebacterium pseudotuberculosis↗
PubMed1999

Spontaneous mutation, oxidative DNA damage, and the roles of base and nucleotide excision repair in the yeast Saccharomyces cerevisiae.

The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine (8-OxoG). When compared to wild-type, ogg1 mutants show an increase in the frequency of GC to TA transversions, indicating a role for Ogg1 in the repair of 8-OxoG. Here we report an increased frequency of forward mutation to canavanine resistance in mutants defective in the nucleotide excision repair (NER) gene RAD14. This was not increased further in strains additionally defective in OGG1. However, when compared to strains solely defective in OGG1, ogg1radl4 mutants displayed an increase in spontaneous GC to TA transversions. Intriguingly, reversion of the lys1-1 ochre allele was not increased in rad14 mutants, suggesting that oxidative base damage may only represent a substrate for NER in certain regions of the genome. We also examined repair of oxidative DNA damage by transforming mutant strains with plasmid DNA treated with methylene blue plus visible light. Mutants defective in OGG1 showed no significant reduction in transformation efficiency compared with wild-type strains. In contrast, disruption of RAD14 reduced the efficiency of transformation, yet there was no further decrease in an ogg1rad14 mutant. This strongly supports a role for NER in the repair of oxidative base damage in yeast, and differs from similar experiments carried out in E. coli, where transformation efficiency is only reduced in mutants defective in both fpg and uvrA. Finally, the repair of Fpg-sensitive sites was examined at the MATalpha and HMLalpha mating type loci, and NER was found to play a role in their removal.

Canavanine↗
PubMed2005

Parameters affecting the efficiency of Agrobacterium tumefaciens-mediated transformation of Colletotrichum graminicola.

We have developed an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola, the cause of anthracnose leaf blight and stalk rot of corn. The ATMT results in higher transformation efficiencies than previously available polyethylene glycol-mediated protocols, and falcate spores can be used instead of protoplasts for transformation. Various experimental parameters were tested for their effects on transformation efficiencies. The parameters with the greatest influence were the A. tumefaciens strain used and the Ti-plasmid it carried, the ratio of bacterium to fungus during cocultivation, and the length of cocultivation. Southern analysis demonstrated that most transformants (80%) contained tandem integrations of plasmid sequences, and at least 36% had integrations at multiple sites in the genome. In a majority of cases (70%), the whole Ti-plasmid, and not just the T-DNA, had integrated as a series of tandem repeats. Tandem integrations, especially of the whole plasmid, make it difficult to rescue DNA from both flanks of the integrations with standard PCR-based approaches. Thus, ATMT may be unsuitable for insertional mutagenesis of C. graminicola without further modification.

Agrobacterium tumefaciens↗
PubMed1992

Efficient electropulse transformation of intact Candida maltosa cells by different homologous vector plasmids.

Conditions for efficient and quick transformation by electroporation were developed in Candida maltosa. To investigate the efficiency of transformation with integrative as well as with autonomously replicating plasmids, a series of vectors was constructed for homologous transformation of this species. Transformants were obtained with different plasmids as covalently closed circular molecules and as linearized DNA. The influence of recipient strain and plasmid type as well as of cell number and parameters of the supplied electrical pulse on the transformation efficiency have been investigated. A maximum of 7000 transformants per 100 ng of plasmid DNA was reached. The efficiency of transformation was compared with that of the LiCl method.

Candida↗
PubMed1990

Transformation of Pseudomonas aeruginosa by electroporation.

Optimum conditions were defined for the electrotransformation of Pseudomonas aeruginosa PAO1 with plasmid pLAFR1, resulting in a 1500-fold increase in transformation efficiency compared to conventional chemical transformation with MgCl2. In addition, PAO236 and two out of three recent clinical isolates of P. aeruginosa from the sputum of cystic fibrosis patients were successfully transformed with plasmid pUC19 1.8. The applied voltage and the electroporation buffer composition were shown to have the greatest effect on transformation efficiency. Freezing the cells and prolonged storage at -70 degrees C did not significantly affect the transformation efficiency. The clinical isolates tested had lower transformation efficiencies than PAO1.

DNA, Bacterial↗
PubMed2005

Efficiency of transformation of Polish cultivars of pea (Pisum sativum L.) with various regeneration capacity by using hypervirulent Agrobacterium tumefaciens strains.

An Agrobacterium-mediated transformation method of pea has been developed for several edible and fodder cultivars of pea (Pisum sativum L.), characterized previously in their potential for regeneration via organogenesis. The most appropriate explant, which was susceptible to Agrobacterium infection and capable of regenerating transgenic plants, turned out to be a slice of an immature embryo, including the embryo axis and the basal part of a cotyledon. Three hypervirulent strains of A. tumefaciens were tested: AgL0, AgL1 and EHA105. Each carried the binary vector pP35SGIB containing the uid gene, with an intron under control of the 35S promoter, and the bar gene conferring resistance to phosphinotricin. Strain AgL0 was found to be efficient for the majority of cultivars, followed by AgL1 and EHA105. Transformation efficiency varied from 0.7 to 4.1%, depending on cultivar and Agrobacterium strain. The transformation efficiency of particular pea cultivars did not clearly correspond to their regeneration capacity, which--although indispensable--was not a critical parameter of successful transformation. The presence of integrated genes in pea genomic DNA was detected by the PCR. T-DNA was stably transmitted to the progeny, as it was confirmed by Southern hybridization. The activity of introduced genes was analysed by the histochemical GUS assay and by painting leaves or by spraying transgenic plants with the herbicide Basta.

Aminobutyrates↗
PubMed1990

Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis.

Recent development of vectors and methodologies to introduce recombinant DNA into members of the genus Mycobacterium has provided new approaches for investigating these important bacteria. While most pathogenic mycobacteria are slow-growing, Mycobacterium smegmatis is a fast-growing, non-pathogenic species that has been used for many years as a host for mycobacteriophage propagation and, recently, as a host for the introduction of recombinant DNA. Its use as a cloning host for the analysis of mycobacterial genes has been limited by its inability to be efficiently transformed with plasmid vectors. This work describes the isolation and characterization of mutants of M. smegmatis that can be transformed, using electroporation, at efficiencies 10(4) to 10(5) times greater than those of the parent strain, yielding more than 10(5) transformants per microgram of plasmid DNA. The mutations conferring this efficient plasmid transformation (Ept) phenotype do not affect phage transfection or the integration of DNA into the M. smegmatis chromosome, but seem to be specific for plasmid transformation. Such Ept mutants have been used to characterize plasmid DNA sequences essential for replication of the Mycobacterium fortuitum plasmid pAL5000 in mycobacteria by permitting the transformation of a library of hybrid plasmid constructs. Efficient plasmid transformation of M. smegmatis will facilitate the analysis of mycobacterial gene function, expression and replication and thus aid in the development of BCG as a multivalent recombinant vaccine vector and in the genetic analysis of the virulence determinants of pathogenic mycobacteria.

DNA, Bacterial↗
PubMedDate not supplied

Efficient malignant transformation of rat embryo fibroblasts by genomic DNA from Walker carcinoma cells.

DNA isolated from Walker carcinoma ascites cells was transfected into primary rat embryo fibroblasts (REF), selecting transformed cells by growth in soft agar after prolonged propagation in monolayer. Both high molecular weight genomic DNA and a partially purified mitochondrial DNA fraction were able to transform REF with high efficiency, whereas pure mitochondrial DNA failed to elicit a transformed phenotype. Hybridization experiments showed that the mitochondrial DNA fraction contained DNA species of presumably extramitochondrial origin. Colonies were cloned into morphologically transformed, foci-forming, immortalized cell lines, showing different degrees of chromosomal alterations, tumorigenicity, and production of cell growth factors. These results indicate that although REF are refractory to genomic neoplastic DNA or to single cloned oncogenes in the absence of enhancers, they can be efficiently transformed by chromosomal DNA from a highly malignant tumor under conditions selecting against the remaining normal cells.

Animals↗