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At least 73 records · Page 4Linked to original sources
PubMed1977

Restriction and modification in Bacillus species: genetic transformation of bacteria with DNA from different species, part I.

Host specific restriction was detected in 13 Bacillus strains, when 63 strains of Bacillus subtilis and 15 other Bacillus strains were tested with phage phi 105C. These 13 strains were classified into 8 groups (M,H,C,N,E,F,G,P) by the type of restriction. M-type strains (B. subtilis Marburg 168, its derivatives, and two other strains) showed relatively weak restriction, restricting phi 105C from other groups of Bacillus by ratios of 10(-1) to 10(-3). Strains of groups H,C,N,E,F,G, and P restricted phi 105C from other groups by ratios of 10(-2) to 10(-8). It was confirmed with some of the strains that type-specific modification was endowed only by the last host. Furthermore, we isolated one restriction deficient mutant of B. subtilis Marburg 168-YS11, which had also lost its modification phenotype.

Bacillus↗
PubMed1990

Genetic transformation of glutamine auxotrophy to prototrophy in the cyanobacterium Nostoc muscorum.

Glutamine auxotrophic (Gln-) and L-methionine D,L-sulfoximine (MSX) resistant (MSXr) mutants of N. muscorum were isolated and characterized for nitrogen nutrition, nitrogenase activity, glutamine synthetase (GS) activity and glutamine amide, alpha-keto-glutarate amido transferase (GOGAT) activity. The glutamine auxotroph was found to the GOGAT-containing GS-defective, incapable of growth with N2 or NH4+ but capable of growth with glutamine as nitrogen source, thus, suggesting GS to be the primary enzyme of both ammonia assimilation and glutamine formation in the cyanobacterium. The results of transformation and reversion studies suggests that glutamine auxotrophy is the result of a mutation in the gln A gene and that gln A gene can be transferred from one strain to another by transformation.

Ammonia↗
PubMed1995

Molecular genetic properties of the yeast Torulaspora pretoriensis: characterization of chromosomal DNA and genetic transformation by Saccharomyces cerevisiae-based plasmids.

Chromosomal DNA banding patterns were obtained for three strains of Torulaspora pretoriensis by contour-clamped homogeneous-electric-field gel electrophoresis. Chromosomes were resolved into six or seven bands in the range of 800 to 2000 kb, and a polymorphism of these lengths was observed. By Southern-blot analysis, the three strains were shown to lack the DNA sequences homologous to the URA3, LEU2, TRP1, and HO genes of Saccharomyces cerevisiae. A uracil auxotrophic mutant derived from T. pretoriensis was transformed with three plasmids (YEp24, YRpHI, and YCp50) carrying the URA3 gene of S. cerevisiae by the lithium acetate method.

Blotting, Southern↗
PubMed1992

Genetic transformation of the symbiotic basidiomycete fungus Hebeloma cylindrosporum.

The pAN7.1 plasmid containing the E. coli hygromycin B phosphotransferase gene was used to transform protoplasts of the ectomycorrhizal fungus Hebeloma cylindrosporum. Hygromycin-resistant transformants were selected at a frequency of one to five per micrograms of transforming DNA. Southern blot analyses revealed multiple copy integration of the transforming plasmid into the genome. The selection system was used to introduce other genes of interest by co-transformation. Two plasmids, one containing tryptophan biosynthesis genes and the other the NADP-glutamate dehydrogenase gene from the saprophytic basidiomycete Coprinus cinereus, were successfully introduced into the H. cylindrosporum genome with up to 70% efficiency of co-transformation. The hygromycin resistance phenotype was stably maintained during growth of transformants on hygromycin-free medium. All transformants retained their ability to form mycorrhizae with the habitual host plant Pinus pinaster, making them suitable for future physiological studies.

Basidiomycota↗
PubMed1981

Liposome-mediated genetic transformation of Neurospora crassa.

Transformation of Neurospora crassa spheroplasts is reported for three different genes, using uncloned Neurospora DNA, both naked and encapsulated in synthetic phosphatidylserine liposomes. Whereas transformation by naked DNA is DNase-sensitive, that by liposomes is not. Per unit of transforming DNA, liposome transformation is significantly more efficient than that with naked DNA, ranging from 19x for the am gene to 41x for pyr-3. Levels of activity of pyr-3 and am transformants, and segregation data on pyr-3 transformation are given.

DNA, Fungal↗
PubMed1985

Enhancement of genetic transformation frequencies of mammalian cell cultures by damage to the cell DNA.

Ultraviolet (UV)-light and 5-fluorodeoxyuridine (FUdR), two known DNA damaging agents, were found to enhance the frequency of stable plasmid transformations in several different animal cell lines. Combined treatment with the two agents was more effective than treatment with either agent alone. A correlation between the transformability of a cell line in the absence of treatment and its response to damaging treatment was also observed. Southern blot analysis of transformed clones indicated that the stimulation in transformation frequency was not due to an increased number of copies of the integrated plasmid in the transformed cells.

Animals↗
PubMed1980

Expression of human genes for adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase after genetic transformation of mouse cells with purified human DNA.

Human DNA purified from HeLa cells and from three strains of skin fibroblasts was precipitated with calcium phosphate and added to mouse cells that were deficient in adenine phosphoribosyltransferase (APRT) and hypoxanthine phosphoribosyltransferase (HPRT). Selection for cells possessing either of the phosphoribosyltransferases was imposed by blocking de novo synthesis of purine nucleotides with azaserine in a medium supplemented with adenine and hypoxanthine. The frequency of colony formation after selection was 1.7 x 10(-7)-3.3 x 10(-6). Excepting some azaserine-resistant colonies that appeared only in the first experiment and infrequent revertants expressing moust APRT, all characterized clones expressed the human forms of APRT or HPRT according to the criteria of specific immunoprecipitation and electrophoretic mobility. The frequency of transfer of the human APRT gene was much greater than that of HPRT. Transfer efficiency was not significantly reduced when HeLa DNA was sheared to 6.5-13.5 kb size or when the donor DNA was isolated from a transferent that expressed human APRT.

Adenine Phosphoribosyltransferase↗
PubMed1976

A possibility to achieve genetic transformation in the platyfish-swordtail system (Platypoecilus maculatus - Xiphophorus helleri).

In order to determine how informative homologous donor DNA might be made available to propigment cells of the recipient Xiphophorus helleri for transformation, labelled heterologous DNA from E. coli was injected into the neural crest region or the yolk sac of embryos of the recipient. On the basis of the degradation rate of the donor DNA and the incorporation rate of radioactivity into the recipient DNA, it is concluded that injection into the neural crest region may be a suitable method to make available informative homologous donor DNA for transformation.

Animals↗
PubMed1994

Genetic transformation of plants by protoplast electroporation.

This article describes an optimized protocol for the electroporation of tobacco mesophyll protoplasts together with notes and data on the effects of various parameters and suggestions for work with protoplasts of other species. In this protocol, electroporation is achieved by means of electrical pulses from a high-voltage, capacitive-discharge unit. Procedures are described for measurement of protoplast viability with Evan's blue, the detection of transient expression of CAT and GUS gene plasmid constructs, and for the recovery of stable transformants based on selection for kanamycin resistance.

Cell Survival↗
PubMed1975

Prevention of cell agglutination and competence in a genetically transformable strain of Pneumococcus by D-glucosamine and D-galactosamine.

In the presence of amino sugars D-glucosamine and D-galactosamine no spontaneous competence could be observed in the highly transformable R6bd strain of Pneumococcus or it was decreased by several orders of magnitude. The highest inhibition of competence was detected when the amino sugar at a concentration 5 mg/ml of the medium was added not only to the transformation but also to the pretransformation medium. After a 150 min growth in the transformation medium in the presence of the amino sugar a 3--4-fold greater number of cells (as a viable count) could be detected as compared with the control without the amino sugar. It was found microscopically that the amino sugar prevents natural agglutination, which normally occurs in the competent culture. The role of specific amino sugar determinants for binding of the competence factor on the cell surface and the resulting inhibitory effect of these sugars on the development of competence are discussed.

Agglutination↗
PubMed2000

Genetic transformation and biotechnological application of the yeast Arxula adeninivorans.

The relatively unknown, non-pathogenic, dimorphic, haploid, ascomycetous yeast Arxula adeninivorans exhibits some unusual properties which are of biotechnological interest. The yeast is able to assimilate and ferment many compounds as sole source of carbon and/or nitrogen, it utilises n-alkanes and degrades starch efficiently. A. adeninivorans features such as thermo- and haloresistance as well as the yeast's uncommon growth and secretion behaviour should be especially emphasised. In media containing up to 20% NaCl, A. adeninivorans is able to grow at cultivation temperatures up to 48 degrees C. Additionally, the dimorphism of the yeast is unusual. Arxula grows at up temperatures of up to 42 degrees C as budding cells, which turn into mycelia at higher temperatures. This environmentally conditioned dimorphism is reversible and budding is reestablished when the cultivation temperature is decreased below 42 degrees C. Alteration of morphology correlates with changes in secretion behaviour. Mycelium cultures accumulate two-fold higher protein concentrations and contain two- to five-fold higher glucoamylase and invertase activities in the medium than budding cells. Based on these unusual properties, Arxula adeninivorans is used for heterologous gene expression and as a gene donor to construct more suitable yeasts for biotechnology. For example the Arxula glucoamylase gene was successfully expressed in Saccharomyces cerevisiae and Kluyveromyces lactis. Both transformed yeasts are able to assimilate and ferment starch as carbon source. A transformation system is used for heterologous gene expression which is based on integration of linearised DNA fragments in two to ten copies, e.g. into the 25S rDNA of A. adeninivorans by homologous recombination. The obtained transformants are mitotically stable. The expression of the lacZ gene from E. coli as well as the XylE gene from Pseudomonas putida indicates the suitability of A. adeninivorans as host for heterologous gene expression.

Biotechnology↗
PubMed2001

Genetic transformation of Trametes versicolor to phleomycin resistance with the dominant selectable marker shble.

We have developed a stable, DNA-mediated transformation system for the white-rot basidiomycete Trametes versicolor based on the dominant selectable marker shble (phleomycin resistance). We employed a vector containing the selectable marker under control of expression sequences from the basidiomycete Schizophyllum commune and a polyethylene glycol/ CaCl2 protoplast-fusion technique to introduce the transforming DNA. This transformation system generated stable phleomycin-resistant transformants at a frequency of four to seven transformants/microg of transforming DNA.

Anti-Bacterial Agents↗
PubMed2001

Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic ascomycete Calonectria morganii.

Conidia of the phytopathogenic fungus Calonectria morganii were transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selective trait, governed by a heterologous fungal promoter and the Aspergillus nidulans trpC terminator. Agrobacterium tumefaciens-mediated transformation yielded stable hygromycin B-resistant clones (average number (106 per 10(7)) [corrected] conidia). Putative transformants appeared to be mitotically and meiotically stable. The presence of the hph gene was checked by PCR. In four randomly chosen transformants, single-copy integrations of the marker gene at different chromosomal sites were proven by Southern analysis.

Agrobacterium tumefaciens↗
PubMed2003

Genetic transformation of Pueraria phaseoloides with Agrobacterium rhizogenes and puerarin production in hairy roots.

An efficient transformation system for the medicinal plant Pueraria phaseoloides was established by using agropine-type Agrobacterium rhizogenes ATCC15834. Hairy roots could be obtained directly from the cut edges of petioles of leaf explants or via callus 10 days after inoculation with the bacteria. The highest frequency of explant transformation by A. rhizogenes ATCC15834 was about 70% after infection for 30 days. Hairy roots could grow rapidly on solid, growth regulator-free Murashige and Skoog medium and had characteristics of transformed roots such as fast growth and high lateral branching. Paper electrophoresis revealed that bacteria-free hairy roots of P. phaseoloides could synthesize agropine and mannopine. The polymerase chain reaction amplification of rooting locus genes showed that left-hand transferred DNA of the root inducing plasmid of A. rhizogenes was inserted into the genome of transformed P. phaseoloides hairy roots. The content of puerarin in hairy roots reached a level of 1.190 mg/g dry weight and was 1.067 times the content in the roots of untransformed plants.

Culture Techniques↗