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PubMed1978

Genetic transformation in Rhizobium trifolii.

Markers controlling the synthesis of amino acids and organic bases as well as streptomycin resistance and sensitivity to acriflavine were transformed in Rhizobium trifolii. The results indicate that the str marker was transformed independently of leu, his, ade and trp markers. Co-transformation of leu and utra markers ranged from 3 to 7%, whereas that of thi and acr was 10%.

Acriflavine↗
PubMed2002

[In vitro culture and the Agrobacterium-mediated genetic transformation of Morinda officinalis].

OBJECTIVE: To establish an effective system for the Agrobacterium-mediated genetic transformation of M. officinalis, for laying a foundation for the improvement of breeds and introduction of foreign objective genes. METHOD: The explants used for culture were the nodular stem segments from M. officinalis. Agrobacterium tumefaciens strain was EHA101, containing vector plasmid pGA482GG. The GUS gene and NPT II gene were introduced into the plasmid. RESULT: MT basal medium with BA 1 mg.L-1 was effective to inducing the direct shoot formation, and the frequency of shoot formation was 97.8%. As BA concentrations increased, the ability of shoot formation decreased. The explants oriented with their apical ends protruding from the medium produced more shoots than when they were placed with their basal end upright or were placed horizontally. The optimal rooting medium for regenerating shoots was MT basal medium supplemented with 0.2 to 0.5 mg.L-1 NAA, and a root induction rate over 80.0% was observed. The selection pressure for kanamycin was 50 mg.L-1. Cefotaxime was used as antibiotics, and the concentration was 300 mg.L-1. After 1.5 months, 14.8% resistant shoots were emerged from the explants. Histochemical GUS assay showed that 22.2% of the resistant plants were GUS-positive. CONCLUSION: Plant regeneration system and Agrobacterium-mediated genetic transformation have been established for M. officinalis in vitro.

Agrobacterium tumefaciens↗
PubMed1970

Cellular metabolism in genetic transformation of pneumococci: requirement for protein synthesis during induction of competence.

Metabolic inhibitors have differential effects on various phases of genetic transformation in pneumococci. Evidence is presented suggesting that, in addition to the competence factor, another specific protein or class of proteins is essential for the development of cellular "competence." The precise role of this protein(s) in genetic transformation is not known, but it seems essential for some function subsequent to the interaction of competence factor and cells.

Amino Acids↗
PubMed1989

RNA-mediated genetic transformation in Aspergillus nidulans.

We developed a model-system for correcting genetic alterations of an Aspergillus nidulans strain (ribo, paba, bio, w, Acr) by treating protoplasts with total RNA extracted from another A. nidulans strain bearing wild type alleles for the same genetic markers. The results revealed the occurrence of a true genetic transformation. The phenomenon was RNA-dependent since it was abolished by pancreatic ribonuclease treatment. The term retrotransformation is proposed since the RNA messages artificially inserted into the mutant protoplast cells restore the wild type chromosomal information.

Aspergillus nidulans↗
PubMed1974

Simple genetic transformation assay for rapid diagnosis of Moraxella osloensis.

A genetic transformation assay for unequivocal identification of strains of Moraxella osloensis is described. In this assay a stable tryptophan auxotroph is transformed to prototrophy by deoxyribonucleic acid (DNA) samples from other strains of M. osloensis but not by DNA samples from unrelated bacteria. The test is simple to perform and definitive results can be obtained in less than 24 h. The procedure, which is suitable for routine diagnosis in a clinical laboratory, involves a rapid method for preparation of crude transforming DNA from small quantities of bacterial cells and permits simultaneous examination of large numbers of isolated cultures. The assay was shown to correctly identify 27 strains previously classified as M. osloensis. Forty-five other gram-negative, oxidase-positive, nonmotile coccobacilli, which might be confused with M. osloensis unless subject to more extensive testing, were shown to be unrelated genetically to M. osloensis. The transformation assay clearly distinguishes M. osloensis from Acinetobacter. Although most strains of M. osloensis are nonfastidious, being able to grow in a mineral medium supplemented with a single organic carbon source, one of the strains tested was only able to grow on fairly complex media and could not be transformed to grow on simple media. Inability to alkalize Simmons citrate agar was shown not to be characteristic of all strains of M. osloensis.

Acinetobacter↗
PubMed2025

A genotype-independent and highly efficient Agrobacterium-mediated soybean genetic transformation system.

A stable and efficient transformation system is crucial for functional genomics and trait improvement in soybean. This study developed a tissue culture based genetic transformation system incorporating dual selection (Spectinomycin and RUBY). This system significantly enhances transformation efficiency, shortens the transformation cycle, and demonstrates broad genotype independence, providing a powerful tool for soybean research and breeding.

Glycine max↗
PubMed1974

Early stages in DNA binding and uptake during genetic transformation of pneumococci.

Ethylenediaminetetraacetate and other divalent-cation-complexing agents greatly stimulate the cellular binding of DNA molecules to competent pneumococci, while the appearance of genetic transformants and nuclease-resistant DNA binding are completely inhibited. Based on this finding, we developed an experimental system in which three early and consecutive stages of genetic transformation can be experimentally separated: (i) attachment of DNA molecules to cell surface sites that are only demonstrable in the competent state; (ii) a divalent-cation-dependent nucleolytic splitting and release of the adsorbed molecules to the medium; and (iii) emergence of potential transformants accompanied by an energy-requiring and divalent-cation-dependent process in which the cell-associated DNA molecules become inaccessible to shearing forces, nucleases, anti-DNA serum, and polycations.

Binding Sites↗
PubMed1989

Genetic transformation of the mosquito Aedes aegypti by micro-injection of DNA.

We report the successful introduction of heterologous DNA sequences into embryos of the mosquito Aedes aegypti (L.) by microinjection. The injected DNA carried P transposable element sequences, derived from and known to facilitate transformation in Drosophila melanogaster. Two plasmids, one of which carried a dominant selectable marker, were introduced into the posterior of embryos prior to pole cell formation and subsequently taken up into the germ line of transformed individuals. Stable transfer of the selectable marker (G418 resistance) was demonstrated over two generations. The precise nature of these putative P mediated integration events is currently being investigated. However, the results presented here establish the technique of DNA transformation for the genetic manipulation of Aedes aegypti.

Aedes↗
PubMed1997

The com locus controls genetic transformation in Streptococcus pneumoniae.

Genetic exchange by natural transformation in Streptococcus pneumoniae occurs in a cell-density dependent process and is initiated by a small extracellular signalling molecule, the competence-stimulating peptide (CSP). comC, the gene for this peptide, has previously been identified and encodes a 44 amino acid pre-peptide that is apparently processed to an active molecule that consists of the C-terminal 17 amino acids. We have sequenced the region adjacent to comC and shown that it is the first gene of an operon, com, consisting of two downstream elements, comD and comE, which encode members of the two-component family of sensor regulators. Null mutants with defects in either comC or comD were transformation deficient and failed to respond to exogenous CSP. A comC mutant did not exhibit any detectable CSP activity, while a comD mutant that contained an intact comC produced minimal CSP activity. In mixed-culture experiments consisting of isogenic pairs of pneumococci (Csp+ and Csp-), we showed that induction of competence by quorum sensing was independent of CSP. Northern analysis showed that com was transcribed as a single polycistronic message, while analysis of strains with transcriptional fusions showed that com was constitutively expressed under conditions that both promoted or repressed the development of competence. Finally, we showed genetically and biochemically a CSP-dependent transcription of rec, a competence-induced locus, and that ComD and ComE are required for this CSP-dependent expression.

Bacterial Proteins↗
PubMed1990

Identification of Haemophilus aphrophilus and Actinobacillus actinomycetemcomitans by DNA-DNA hybridization and genetic transformation.

DNA-DNA hybridization was used to identify clinical isolates as Haemophilus aphrophilus or Actinobacillus actinomycetemcomitans. Some of the isolates were naturally competent for genetic transformation and were also used as DNA recipients for identification of other isolates. The results obtained by hybridization were supported by interstrain-to-intrastrain transformation ratios. Distinction between the closely related species H. aphrophilus and A. actinomycetemcomitans was generally clear-cut by both methods. Distinction of H. aphrophilus and A. actinomycetemcomitans from type and reference strains of a diversity of species in the family Neisseriaceae and other gram-negative species was also demonstrated by both methods. This is the first description of the identification of clinical isolates of H. aphrophilus or A. actinomycetemcomitans by using them as recipients in genetic transformation. The results suggest that this is a reliable system for identification of new clinical isolates belonging to these taxonomic entities.

Actinobacillus↗
PubMed2000

Genetic transformation of Coccidioides immitis facilitated by Agrobacterium tumefaciens.

Agrobacterium tumefaciens was used to facilitate genetic transformation of Coccidioides immitis. A gene cassette containing the gene encoding hygromycin phosphotransferase (hph) was cloned into a T-DNA vector plasmid and introduced into A. tumefaciens, and the resultant strain was used for cocultivation with germinated arthroconidia. This procedure produced numerous colonies 60- to >500-fold more resistant to hygromycin than untransformed mycelia. Both polymerase chain reaction and Southern blot analysis of the transformants indicated that all contained hph, usually as a single genomic copy. A transformation frequency of 1 per 10(5) arthroconidia was obtained by varying the germination time prior to cocultivation and altering the bacterium: fungus ratio. This approach requires no special equipment that might complicate biocontainment. Furthermore, transformation does not require digestion of fungal cell walls, further simplifying this procedure. A. tumefaciens-facilitated transformation should make possible the development of tagged mutagenesis and targeted gene disruption technology for C. immitis and perhaps other fungi of medical importance.

Agrobacterium tumefaciens↗
PubMedDate not supplied

[Genetic transformation of Streptomyces globisporus strain 1912: restriction barrier and plasmid compatibility].

Low efficiency of genetic transformation of protoplasts of different strains of Streptomyces globisporus 1912 by means of DNA preparations of three vectors pIJ487, pGM160 and pWHM4 is explained by presence of the restriction barrier in the recipients. This obstacle can be overcome by the use of modified DNA of the same vectors, isolated from not numerous transformants. The frequency of transformation by such modified vector DNA was increased by two-three orders in comparison with initial DNA, isolated from Streptomyces lividans TK24, loosing restriction-modification system. The vector pCNB4001, containing the replicon of endogeneous plasmid pSG1912-1, effectively transformed the protoplasts of all S. globisporus 1912 strains. Compatibility of pIJ487 and pSG1912-1 plasmids and incompatibility of the latter and pWHM4 was shown.

DNA, Bacterial↗
PubMed1991

[The effect of silicon metabolism on genetic transformation in Bacillus subtilis].

Germanium dioxide is found to increase the frequencies of the genetical transformation in Bacillus subtilis 30-40 fold. The increased frequency of transformation was registered in Sil- mutant in contrast to Sil+ strain having the decreased one. Bacillus megatherium strain KU-2 and Bacillus oligonitrophilus KU-1 were isolated from soil. These strains possess better ability to utilize the orthoclase and biotite. Germanium dioxide did not induce the transformation frequencies increase in these strains. Sil mutant of Bacillus oligonitrophilus demonstrated no competence to transformation.

Bacillus↗