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PubMed1986

Factors enhancing genetic transformation of intact yeast cells modify cell wall porosity.

Genetic transformation of intact cells of Saccharomyces cerevisiae, achieved by incubating the cells with plasmid DNA in the presence of PEG, could be enhanced, not only by pretreatment of the cells with Li+ and 2-mercaptoethanol, as has been reported previously, but also by pretreatment with proteolytic enzymes. The efficiency of transformation with 2-mercaptoethanol rose dramatically when the pretreated cells had been handled in osmotically stabilized media. Following all the pretreatments the cells became leaky for nucleic acids as detected by the presence of endogenous RNAs in the medium. The pretreatments evidently facilitated the passage of transforming DNA across the cell wall.

Cell Survival↗
PubMed1977

Factors affecting genetic transformation of Neisseria gonorrhoeae.

Piliated gonococci were competent in genetic transformation in all stages of growth in minimal and enriched media, but nonpiliated cells were almost totally incompetent. Uptake of deoxyribonucleic acid into a deoxyribonuclease-insensitive state was observed only in competent piliated cells. Competence was not affected by washing of competent cells or treatment of competent cells with proteolytic enzymes. Expression of competence required presence of any of several different monovalent or divalent cations, as well as a utilizable source of energy. Efforts to produce genotypically or phenotypically competent derivatives of nonpiliated cells were unsuccessful. These experiments are consistent with the idea that pili may play a role in the irreversible uptake of transforming deoxyribonucleic acid by the gonococcus, but fail to provide evidence for other types of competence factors.

Cations, Divalent↗
PubMed1979

Hybridization of DNA's from Neurospora crassa strains may indicate base sequence alterations as a consequence of genetic transformation.

Deoxyribonucleic acids of Neurospora crassa strains involved in genetic transformation experiments were studied by means of DNA-DNA hybridization. No significant difference was detected in the extent of hybridization reassociating 32P-DNA of an inositol-requiring recipient strain with an excess amount of unlabelled homologous DNA and that of the transformed, spontaneous revertant and wild-type strains. Studies on the thermal stability of hybrids revealed 1.2-1.7% heterology between the recipient and transformant DNA's. The spontaneous revertant and wild-type strains proved to be homologous with the recipient strain. We suppose that the heterology we measured is the result of the alteration of the nucleotide sequences caused by the multilocal integration of transforming DNA into the recipient genome.

Base Sequence↗
PubMed2000

[Bacillus subtilis undergoes natural genetic transformation on agar plates].

This paper conducted preliminary investigation on natural genetic transformation of Bacillus subtilis on agar plates. The results showed that, under same conditions, the natural transformation of the strain on agar plate was much more efficient than that of standard liquid method, and the transformation system could sustain higher DNase concentration. In addition, that the LB culture, which usually did not support the strain acquire competency, could undergo transformation as soon as it being spread on agar plate, suggested that the natural genetic transformation of Bacillus subtilis on solid plates may be different form the standard liquid transformation method. The cell-to-cell transformation between strains having different genetic makers could also be observed on agar plates.

Agar↗
PubMed2004

Early events in Agrobacterium-mediated genetic transformation of citrus explants.

BACKGROUND AND AIMS: Genetic transformation of plants relies on two independent but concurrent processes: integration of foreign DNA into plant cells and regeneration of whole plants from these transformed cells. Cell competence for regeneration and for transformation does not always fall into the same cell type/developmental stage, and this is one of the main causes of the so-called recalcitrance for transformation of certain plant species. In this study, a detailed examination of the first steps of morphogenesis from citrus explants after co-cultivation with Agrobacterium tumefaciens was performed, and an investigation into which cells and tissues are competent for regeneration and transformation was carried out. Moreover, the role of phytohormones in the co-cultivation medium as possible enhancers of gene transfer was also studied. METHODS: A highly responsive citrus genotype and well-established culture conditions were used to perform a histological analysis of morphogenesis and cell competence for transformation after co-cultivation of citrus epicotyl segments with A. tumefaciens. In addition, the role of phytohormones as transformation enhancers was investigated by flow cytometry. KEY RESULTS: It is demonstrated that cells competent for transformation are located in the newly formed callus growing from the cambial ring. Conditions conducive to further development of this callus, such as treatment of explants in a medium rich in auxins, resulted in a more pronounced formation of cambial callus and a slower shoot regeneration process, both in Agrobacterium-inoculated and non-inoculated explants. Furthermore, co- cultivation in a medium rich in auxins caused a significant increase in the rate of actively dividing cells in S-phase, the stage in which cells are more prone to integrate foreign DNA. CONCLUSIONS: Use of proper co-cultivation medium and conditions led to a higher number of stably transformed cells and to an increase in the final number of regenerated transgenic plants.

Agrobacterium tumefaciens↗
PubMed1983

Genetic transformation of murine bone marrow cells to methotrexate resistance.

Genetic transformation of murine bone marrow stem cells to methotrexate resistance was achieved using a modified calcium phosphate-DNA coprecipitation procedure. Bone marrow cells were transformed by DNA derived from methotrexate-resistant mouse 3T6 cells. In vivo selection of drug-resistant bone marrow cells resulted from thrice weekly injections of methotrexate (MTX) for a period of 6-8 wk. Following selection, dihydrofolate reductase activity encoded by the donor DNA species was easily detectable in extracts of recipient mouse spleens. In addition, selection of methotrexate-resistant cells was indicated by the persistence of spleen colony-forming units (CFU-S) in drug-treated animals. Also, changes in ratios of mixed syngeneic bone marrow cells derived from CBA and CBA/T6T6 mice resulted from initial treatment of either cell type with 3T6 DNA. These results confirm and extend the observations of Cline and coworkers that normal bone marrow cells can be genetically transformed to methotrexate resistance.

Animals↗
PubMed1979

Lethal effect of protamine and histone on competent Bacillus subtilis cells. Inhibition of genetic transformation by protamine in sublethal concentration.

Under experimental conditions of genetic transformation, protamine and total histone were bactericidal for Bacillus subtilis cells. The abilities to cause lethality were very similar for both, either protamine or histone, with no antagonistic effects amongst these natural polycations. With both basic proteins acting simultaneously the enhancement was higher than a summation of the separate lethal effects. Sublethal concentration of protamine added at the beginning of transformation time, produced a strong inhibition of transforming efficiency. The same concentration added later than 10 min from the start of transformation had no inhibitory effect. These facts together with the absence of inhibition by simple pretreatment of DNA alone as well as the cell protection by protamine against lytic activity of lysozyme, suggest a protamine-cell surface interaction which impedes DNA uptake events.

Bacillus subtilis↗
PubMed1982

Genetic transformation in chicken by the use of irradiated male gametes.

Genetic transformation for egg and feather colour hs been obtained in the whole animal, chicken, by the use of irradiated male gametes. In this technique recipient females are first inseminated with irradiated semen from the donor and 24 h later are reinseminated with unirradiated, normal semen of the recipient strain. The transformed progeny are identical to the female strain but for one or a few genes derived from the irradiated semen. The expression of the transferred gene is irregular and may occur either in the first generation, after insemination with irradiated semen, or may occur in the following backcross generation. It is suggested that integration of a normally recessive, transferred gene at non-homologous sites in the recipient chromosomes might affect not only its regularity of expression but also its dominance relationship with the original maternal gene, "releasing" it from the dominance of the latter. The technique potentially offers a rapid method of improving an already established strain of an animal.

Animals↗
PubMed1997

Oligonucleotide-mediated genetic transformation of Borrelia burgdorferi.

We have used short oligonucleotides to genetically transform the Lyme disease spirochaete Borrelia burgdorferi. The oligonucleotides are derived from the sequence of an Arg-133 to Ile mutant gyrB (chromosomal) gene that confers resistance to the antibiotic coumermycin A1. Oligonucleotides were about 10,000-fold less efficient at transformation, on a molar basis, than longer PCR-generated substrates. All of the transformants tested contained the predicted site-directed silent mutation in their gyrB genes. Antisense oligonucleotides were more efficient at transformation than either sense or double-stranded oligonucleotides. This is the first demonstration of oligonucleotides used to introduce site-directed mutations directly into the genome of a bacterium.

Aminocoumarins↗
PubMed1981

Genetic transformation in Streptococcus sanguis. Competence factor and competence factor inactivator.

Genetic transformation in Streptococcus sanguis is a complex, multi-step process, involving several factors. The competence factor (CF), occurring in the culture filtrates of some strains, is essential in streptococcal transformation, but other factors are also involved. The presence of serum during growth is not obligatory for the preparation of active culture filtrate, but serum increases the amount and duration of CF activity of some strains. The stability of the CF activity differs distinctly in filtrates prepared from various strains. This observation can be explained by the presence of a previously undescribed factor--names the competence factor inactivator (CFI)--which inactivates the CF in culture filtrates during the transforming experiment as well as during storage. The CFI is thermolabile and is estimated from the filtrates by heating (65 degrees C for 15 min). The properties of the CFI suggest that it may be a protein with enzymatic activity. The CFs from the strains Challis and 13b resist heating to 100 degrees C for 2 h, and are stable for at least 72 h if the CFI is absent or has been inactivated by heating. The CF activity in the filtrates is inactivated by pronase and chymotrypsin, suggesting that the CF may be of a polypeptide nature.

Bacterial Proteins↗
PubMed2005

Agrobacterium-mediated genetic transformation of the desiccation tolerant resurrection plant Ramonda myconi (L.) Rchb.

In this paper we describe the first procedure for Agrobacterium tumefaciens-mediated genetic transformation of the desiccation tolerant plant Ramonda myconi (L.) Rchb. Previously, we reported the establishment of a reliable and effective tissue culture system based on the integrated optimisation of antioxidant and growth regulator composition and the stabilisation of the pH of the culture media by means of a potassium phosphate buffer. This efficient plant regeneration via callus phase provided a basis for the optimisation of the genetic transformation in R. myconi. For gene delivery, both a standard (method A) and a modified protocol (method B) have been applied. Since the latter has previously resulted in successful transformation of another resurrection plant, Craterostigma plantagineum, an identical protocol was utilized in transformation of R. myconi, as this method may prove general for dicotyledonous resurrection plants. On this basis, physical and biochemical key variables in transformation were evaluated such as mechanical microwounding of plant explants and in vitro preinduction of vir genes. While the physical enhancement of bacterial penetration was proved to be essential for successful genetic transformation of R. myconi, an additional two-fold increase in the transformation frequency was obtained when the above physical and biochemical treatments were applied in combination. All R0 and R1 transgenic plants were fertile, and no morphological abnormalities were observed on the whole-plant level.

Agrobacterium tumefaciens↗
PubMedDate not supplied

[Genetic transformation of animals by the insertion of foreign genes into the zygotes].

A review of literature on genetic transformation of animal (mouse and Drosophila) germ cells by microinjection of the cloned genes into the pronuclei or into the early embryos. The fate of foreign genes and mechanisms of their integration into the recipient genome are considered in detail, as well as genetic consequences of the exogenic DNA insertion. Expression of the donor genes in transgenic animals is analyzed, its irregular and unusual pattern is noted in transgenic mice. An attempt has been undertaken not only to stress obvious advances in genetic transformation of animals, but also to outline unsolved problems related to inheritance of exogenous genes, insertion mutagenesis and irregular expression of the donor genes.

Animals↗
PubMedDate not supplied

[Transgene transfer and inheritance under genetic transformation in maize].

Achievements are discussed regarding in genetic transformation of maize is examined from that point of view what cells, tissues and organs can be the targets for the alien gene transfer. The review of available data on genotypical peculiarities of transgenesis and inheritance of alien genes included in the maize genome are presented.

Gene Transfer Techniques↗
PubMed1994

The assembly of cytochrome b6/f complexes: an approach using genetic transformation of the green alga Chlamydomonas reinhardtii.

As an approach to the study of the biogenesis of the cytochrome b6/f complex, we characterized the behaviour of its constitutive subunits in mutant strains of Chlamydomonas reinhardtii bearing well-defined mutations. To this end, we have constructed three deletion mutant strains, each lacking one of the major chloroplast pet genes: the delta petA, delta petB and delta petD strains were unable to synthesize cyt f, cyt b6 and subunit IV (suIV) respectively. Western blotting analysis, pulse-labelling and pulse-chase experiments allowed us to compare the cellular accumulation, the rates of synthesis and the turnover of the cyt b6/f subunits remaining in the various strains. We show that the rates of synthesis of cyt b6 and suIV are independent of the presence of the other subunits of the complex but that their stabilization in the thylakoid membranes is a concerted process, with a marked dependence of suIV stability on the presence of cyt b6. In contrast, mature cyt f was stable in the absence of either suIV or cyt b6 but its rate of synthesis was severely decreased in these conditions. We conclude that the stoichiometric accumulation of the chloroplast-encoded subunits of the cyt b6/f complex results from two regulation processes: a post-translational regulation leading to the proteolytic disposal of unassembled cyt b6 and suIV and a co-translational (or early post-translational) regulation which ensures the production of cyt f next to its site of assembly.

Animals↗