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PubMed1976

Genetic Transformation as a tool for detection of Neisseria gonorrhoeae.

A rapid method for the detection of Neisseria gonorrhoeae, making use of the ability of deoxyribonucleic acid samples from clinically isolated strains of this organism to transform nutritional mutants of a particular strain of N. gonorrhoeae, has been described. In addition to using isolated cultures, transforming deoxyribonucleic acid can be obtained directly from the material that adheres to swabs of the cervix or the urethra. The time interval for transfer of swabs to the diagnostic laboratory is not a significant factor. It is not necessary to use pure cultures on primary isolation plates to obtain definitive results. Nongonorrhoeae neisserias, as well as a large variety of commonly encountered unrelated bacteria, do not react or interfere in the transformation assay when using one of the mutant strains under a standardized set of conditions. The entire assay can be completed in less than 24 h. It has also been shown that type T4 cells of the strain of N. gonorrhoeae employed in the present study are competent for genetic transformation, although type T4 cells are transformed at a significantly lower frequency than are type T2 cells of the same strain.

Amino Acids↗

PubMed1979

Haemophilus influenzae periplasmic protein which binds deoxyribonucleic acid: properties and possible participation in genetic transformation.

A protein which binds to either single-stranded or double-stranded deoxyribonucleic acid (DNA) but not to ribonucleic acid has been isolated by osmotic shock treatment of growing cells. This periplasmic protein differs from the principal intracellular binding protein in its greater thermolability and by the absence of salt-induced cooperativity in its interaction with single-stranded DNA. Certain mutant strains of Haemophilus influenzae defective in the DNA suptake steps of genetic transformation were found to be deficient in periplasmic DNA-binding protein, suggesting that this protein participates in the uptake of DNA in transformation.

Bacterial Proteins↗

PubMed1990

Neisseria lactamica and Neisseria polysaccharea as possible sources of meningococcal beta-lactam resistance by genetic transformation.

We studied the susceptibilities of relatively penicillin G-resistant and -susceptible strains of Neisseria meningitidis, as well as Neisseria lactamica and Neisseria polysaccharea, to penicillin, ampicillin, and several cephalosporins. The MICs of penicillin, ampicillin, cephalothin, and cefuroxime for moderately resistant meningococci have increased two- to sixfold in relation to MICs for susceptible strains. For these strains of meningococci, N. lactamica, and N. polysaccharea, penicillin, ampicillin, cephalothin, and cefuroxime MICs for 50 and 90% of strains were similar. By genetic transformation of a penicillin-susceptible strain of N. meningitidis to low-level penicillin resistance with DNA from penicillin-resistant strains of N. meningitidis, N. lactamica, N. polysaccharea, and N. gonorrhoeae, isogenic strains with the same pattern of resistance to beta-lactams were obtained, suggesting that these commensal Neisseria spp. could be the source of meningococcal resistance genes.

Anti-Bacterial Agents↗

PubMed1982

Genetic transformation of Rhizobium leguminosarum by plasmid DNA.

We demonstrated the genetic transformation of Rhizobium leguminosarum by R68.45 plasmid DNA by freezing and thawing cell suspensions in the presence of R68.45 plasmid DNA and 20 mM MgCl2. Clones resistant to kanamycin and tetracycline were recovered at a frequency of 10(-8) per recipient cell. No colonies that were doubly drug resistant were recovered in parallel control experiments.

Kanamycin↗

PubMed1983

Diagnosis of gonorrhoea using a genetic transformation test at a venereal disease clinic in Thailand.

A genetic transformation test (GTT), a technique in which gonococcal DNA is detected in clinical specimens, was used to search for Neisseria gonorrhoeae infections in 37 men and 159 women at the Venereal Disease clinic in Cholburi, Thailand. Swabs were collected in duplicate from cervical specimens from 159 women and from urethral specimens from 37 men. One of each specimen was cultured on Thayer-Martin media while the other was mailed to the United States at room temperature for the GTT which involved a delay of 10 to 14 days. With the urethral specimens N. gonorrhoeae was identified in 84% (31/37) of specimens and there was 100% concordance between the results of the GTT and culturing specimens directly on Thayer-Martin media. With cervical specimens N. gonorrhoeae was isolated from 26% (41/159) by the standard culture technique and 19% (13/159) by the GTT. Seventy-six percent of the culture positive specimens were positive with the GTT and two specimens from which N. gonorrhoeae were not isolated were positive in the GTT. The GTT technique enables physicians to send swab collected from patient with suspected gonorrhoea without any special transport media to a central laboratory for laboratory diagnosis of gonorrhoeal infections. This technique which uses reagents which are available in most bacteriology laboratories, should facilitate surveillance of gonorrhoea especially when specimens are collected in clinics where bacteriology laboratory facilities are not available.

Biological Assay↗

PubMed1962

A defined agar medium for genetic transformation of Neisseria meningitidis.

Catlin, B. Wesley (Marquette University, Milwaukee, Wis.) and Gertrude M. Schloer. A defined agar medium for genetic transformation of Neisseria meningitidis. J. Bacteriol. 83:470-474. 1962.-An agar medium was developed for use in quantitative genetic studies of Neisseria meningitidis strain 15. It contains eight inorganic salts, sodium citrate, sodium lactate, arginine, cysteine, glycine, sodium glutamate, and purified agar. Abundant surface growth in the absence of supplemental carbon dioxide was obtained during 50 serial subcultures. A close correspondence was found between numbers of parental type colonies developing on the defined medium and on a complex medium. Cells subcultured serially three or four times on defined agar medium and placed directly into a solution of transforming deoxyribonucleic acid in defined liquid medium were susceptible to transformation without additional supplements. Of the treated population, 0.1 to 0.3% of the cells were transformed to streptomycin resistance.

Agar↗

PubMed1992

Stable genetic transformation of a beneficial arthropod, Metaseiulus occidentalis (Acari: Phytoseiidae), by a microinjection technique.

A microinjection technique has resulted in stable transformation of the western predatory mite Metaseiulus occidentalis. Early preblastoderm eggs within gravid females were microinjected. The needle was inserted through the cuticle of gravid females into the egg, or the tissue immediately surrounding the egg. This maternal injection method resulted in relatively high levels of survival and transformation. Transformation was achieved without the aid of any transposase-producing helper plasmid. The predatory mite was transformed with a plasmid containing the Escherichia coli beta-galactosidase gene (lacZ) regulated by the Drosophila hsp70 heat-shock promoter. Putatively transformed lines were isolated based on beta-galactosidase activity in first-generation larvae. Transformation was confirmed in the sixth generation by polymerase chain reaction amplification of a region spanning the Drosophila/E. coli sequences. Amplification of a nested region, also spanning the interspecific boundary, provided further evidence for stable transformation. Maternal microinjection may be adaptable to other beneficial arthropods, particularly other phytoseiid mites. Genetic transformation of M. occidentalis may improve its efficiency as a biological control agent as well as provide a method for investigating details of its physiology and ecology.

Animals↗

PubMed2006

Genetic transformation of Populus trichocarpa genotype Nisqually-1: a functional genomic tool for woody plants.

We report here the Agrobacterium-mediated genetic transformation of Nisqually-1, a Populus trichocarpa genotype whose genome was recently sequenced. Several systems were established. Internodal stem segments from vigorously growing greenhouse plants are the explants most amenable to transformation. For the most efficient system, approximately 40% of the stem segments infected with pBI121-containing Agrobacterium tumefaciens C58 produced transgenic calli, as confirmed by beta-glucuronidase (GUS) staining. The regeneration efficiency of independent transgenic plants was approximately 13%, as revealed by genomic Southern analysis. Some transgenic plants were produced in as little as 5 months after co-cultivation. This system may help to facilitate studies of gene functions in tree growth and development at a genome level.

Agrobacterium tumefaciens↗

PubMed1999

Optimization of conditions for growth of wild-type and genetically transformed Trypanosoma cruzi on agarose plates.

Growth of Trypanosoma cruzi as colonies on solid medium has not been widely used as an experimental procedure. We therefore sought to establish a reliable and routine plating method. The optimal results were achieved with a matrix of 0.65% low melting point agarose onto which epimasigotes from the mid-to-late logarithmic phase of growth were spread. Colonies could be isolated after incubation for 21 days in a humidified 5% CO2 environment at 28 degrees C. Plating efficiencies in the range of 40% were obtained by this method and clones could be recovered into liquid medium or onto blood-agar slopes with a high success rate. The procedure has also been adapted for the isolation of genetically transformed clones after electroporation of epimastigotes with either plasmid or cosmid vectors. This was best achieved by inclusion of the electroporated cell inoculum in a 0.6% agarose overlay containing G418 as the selective drug, on top of a 0.8% agar base. Transformation efficiencies were as high as 10(-5) cells per microgram of DNA. A reliable plating method for T. cruzi will have many applications and is a significant step towards the use of 'shotgun transformation' to generate libraries of T. cruzi recombinants.

Animals↗

PubMed2002

Extracellular-peptide control of competence for genetic transformation in Streptococcus pneumoniae.

Bacteria, which often are subjected to fluctuations in nutrients, temperature, radiation, pH, etc., adapt to the physico-chemical environment they live in by making the appropriate changes in their gene expression patterns. During the last decades it has become increasingly clear that bacteria, in addition, have a "social life", and that changes in gene expression can also be elicited by the presence of other bacteria. Traditionally bacteria have been viewed as solitary organisms that in general do not interact with other bacteria in a coordinated manner. Recent advances in the field of bacterial cell-to-cell communication has proved this to be a misconception, and mounting evidence now show that bacterial group behaviour is ubiquitous in nature. Competence for natural genetic transformation in Streptococcus pneumoniae, which has been studied for more than seventy years, has become a paradigm for intercellular communication and cell density dependent regulation of gene expression in Gram-positive bacteria. There has been rapid progress recently in elucidating the molecular mechanisms behind regulation of natural competence in S. pneumoniae. In this review, we describe the current status of our knowledge of natural competence in this bacterium, with particular emphasis on the early phase of competence induction.

Amino Acid Sequence↗

PubMed1983

[Amplification of portions of the genome in the somatic cells of mammals resistant to colchicine. IV. Genetic transformation using amplified genes from Djungarian hamster cells highly resistant to colchicine].

DNA-mediated transfer of colchicine-resistance from Djungarian hamster DM5/7 cell line, 750-fold resistant to the drug, was studied. The resistance to colchicine of DM5/7 cells is due to amplification of the genes, possibly coding for the polypeptide p22. Both high-molecular weight DNA (presumably, chromosomal DNA) and low-molecular weight DNA (presumably, extrachromosomal DNA) effectively transferred the colchicine-resistance to Djungarian hamster and mouse cells. DNA of sensitive to colchicine but resistant to ouabain mouse cells CAK-143OuaR was not capable to transfer colchicine-resistance, but effectively transferred ouabain-resistance connected with a mutation in Na+/K+-dependent ATP-ase locus. The differences in genetic transformation with amplified p22 genes and mutant Na+/K+-dependent ATP-ase genes were revealed. All cells of 3 colchicine-resistant transformants of DM-15 cells and all 10 spontaneously derived resistant clones contain the additional chromosome 4. The role of trisomy 4 in the development of colchicine-resistance in DM-15 cells is discussed.

Animals↗

PubMed1981

Genetic transformation of Streptococcus sanguis. Further studies on the production and isolation of the competence factor.

Two new media were developed, containing only the dialyzable components of Todd Hewitt broth (TH) with (medium II) or without (medium IV) inactivated horse serum. The two media were used to detect activity of the competence factor (CF) and the competence factor inactivator (CGI) of Streptococcus sanguis, and in preliminary experiments of CF isolation. Because all the dissolved substances with molecular weight (mol. wt.) less than 12,000 can be removed from these media by dialysis, leaving the CF and possible other high mol. wt. substances formed by growth in the dialysis tube, the use of these media should facilitate the isolation of CF and other high mol. wt. substances involved in genetic transformation of S. sanguis. Dialysis experiments suggest a mol. wt. greater than 12,000 of the CFs of the strains Challis and 13b. The CF of strain 13b was further isolated by Sephadex gel filtration and was eluted in the fractions of low mol. wt. compounds.

Bacterial Proteins↗

PubMed1989

Entrapment of high-molecular-mass DNA molecules in liposomes for the genetic transformation of animal cells.

We modified the Ca/EDTA procedure for the production of liposomes [Papahadjopoulos, Vail, Jacobson & Poste (1975) Biochim. Biophys. Acta 394, 483-491] to entrap intact DNA molecules of very high molecular mass into large unilamellar phospholipid vesicles. The use of DNA-protein complexes and phage particles instead of naked linear DNA increases the efficiency of entrapment and protects the integrity of DNA molecules. We investigated the interaction of mammalian cells with liposome-encapsulated recombinant lambda bacteriophages carrying marker genes. The liposomes bind surprisingly fast to the cellular surface and are taken up by the cells. A significant proportion of the encapsulated DNA is transported to and soon located in or around the nuclei. Experiments prove that these liposomes can be used for the genetic transformation of mammalian cells.

Animals↗

PubMed2001

Genetic transformation of the housefly Musca domestica with the lepidopteran derived transposon piggyBac.

The piggyBac transposable element was successfully used for stable genetic transformation of the housefly Musca domestica. The construct contains the EGFP marker under the control of Pax-6 binding sites, which can drive eye-specific expression in insect species as distantly related as Drosophila melanogaster and Tribolium castaneum [Berghammer, A.J., Klingler, M. and Wimmer, E.A. (1999) Nature 402: 370-371]. We obtained seven independent integration events among 41 fertile G0 Musca flies. Most of the transformed lines contained two or more chromosomal insertions of the EGFP marker which were stably inherited over more than 15 generations. piggyBac-mediated transposition was verified by identifying the characteristic TTAA duplication at the insertion sites. This first report of stable transmission of a genetic marker in Musca confirms the use of this vector-marker system for effective gene transfer in a broad range of insect species.

Animals↗

PubMed2002

In vitro organogenesis and genetic transformation in popular Cucumis sativus L. through Agrobacterium tumefaciens.

The effect of growth regulators and culture conditions on the morphogenetic response of cotyledonary leaf discs was studied in popular cucumber variety (Cucumis sativus cv. Sheetal). Organogenesis was induced directly without any intervening callus phase on Murashige and Skoog medium supplemented with different concentrations of benzyladenine and indole propionic acid. Best results (93%) were obtained in the presence of the 4 mg/L benzyladenine and 1 mg/L IPA. The elongated shoots were rooted in basal medium with 1 mg/L indole butyric acid, hardened and transferred to the field conditions. Genetic transformation system has been established for Cucumis sativus cv. Sheetal, plants by infecting cotyledonary explants with Agrobacterium tumefaciens strain LBA4404 carrying binary plasmid pBI121, which contains scorable marker, beta-glucuronidase and selectable marker nptII under the CaMV 35S promoter. Infection was most effective when explants were infected with Agrobacterium for 15 min and co-cultivated for 2 days in the co-cultivation medium. Shoots were regenerated directly from cotyledonary leaf explants in the presence of kanamycin (50 microg/ml) and analysed. Southern blot analysis confirmed that transformation had occurred. This method will allow genetic improvement of this crop by the introduction of agronomically important genes.

Agrobacterium tumefaciens↗

PubMed2000

[Genetic transformation of autotetraploid Isatis indigotica fort. induced by Ri T-DNA and plant regeneration].

OBJECTIVE: To establish an effective system for genetic transformation of autotetraploid Isatis indigotica by Agrobacterium rhizogenes. METHOD: Agrobacterium rhizogenes strains R1601, ATCC15834 and A4 were employed to induce hairy roots from autotetraploid Isatis indigotica, and the obtained hairy roots were developed into regenerated plants on solid MS media with different Kinds of plant growth regulators. RESULT: Root induction differed obviously. By three Agrobacterium strains. Hairy roots grew rapidly on solid MS medium without plant growth regulators and showed the typical hairy root phenotype: profuse branching, high density of root hairs and plagiotropism. And the transformation of Ri T-DNA was confirmed by opine analysis. The biomass of hairy roots increased nearly 35 times regulator-free MS after suspended in plant growth two weeks. On solid MS medium with BA, adventitious buds were differentiated directly from hairy roots without callus formation. All of the adventitious buds were rooted on root induction medium and developed into regenerated plants. Opine analysis indicated the integration of Ri T-DNA in the transformed plants. CONCLUSION: Agrobacterium rhizogene can induce hairy roots from autotetraploid Isatis indigotica, and the obtained hairy roots can develop into regenerated plants.

Coculture Techniques↗

PubMed2001

A convenient and rapid method for genetic transformation of E. coli with plasmids.

A convenient and rapid method for the genetic transformation of Escherichia coli with plasmids is proposed. By mixing the recipient cells and plasmid DNA and spreading them directly on selective medium plates containing Ca2+, the so-called 'plate transformation' could achieve almost the same transformation efficiency as the classical transformation method with calcium. The whole protocol takes only about 2 min, its simplicity compared favorably, not only to the usual protocol, but also to all other documented modifications.

Calcium Chloride↗

PubMed2005

A reproducible genetic transformation system for cultivated Phaseolus acutifolius (tepary bean) and its use to assess the role of arcelins in resistance to the Mexican bean weevil.

A reproducible Agrobacterium tumefaciens-mediated genetic transformation method that delivers fertile and morphologically normal transgenic plants was developed for cultivated tepary bean (Phaseolus acutifolius L. Gray). Factors contributing to higher transformation efficiencies include (1) a low initial concentration of bacteria coupled with a longer cocultivation period with callus, (2) an initial selection of callus on a medium containing low levels of the selectable agent, (3) omission of the selectable agent from the medium during callus differentiation to shoots and (4) the efficient conversion of transgenic shoots into fertile plants. All plants regenerated with this procedure (T0) were stably transformed, and the introduced foreign genes were inherited in a Mendelian fashion in most of the 33 independent transformants. Integration, stable transmission and high expression levels of the transgenes were observed in the T1 and/or T3 progenies of the transgenic lines. The binary transformation vectors contained the beta-glucuronidase reporter gene, the neomycin phosphotransferase II selectable marker gene and either an arcelin 1 or an arcelin 5 gene. Arcelins are seed proteins that are very abundant in some wild P. vulgaris L. genotypes showing resistance to the storage insect Zabrotes subfasciatus (Boheman) (Coleoptera, Bruchidae). Transgenic beans from two different cultivated P. acutifolius genotypes with high arcelin levels were infested with Z. subfasciatus, but they were only marginally less susceptible to infestation than the non-transgenic P. acutifolius. Hence, the arcelin genes tested here are not major determinants of resistance against Z. subfasciatus.

Agrobacterium tumefaciens↗