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At least 109 records · Page 6Linked to original sources
PubMed1971

DNA uptake during genetic transformation and the growing zone of the cell envelope.

Continuous cellular incorporation of choline molecules is essential for DNA binding to competent pneumococci. The choline molecules are incorporated into the cell surface at the equatorial region of the cocci. As a working hypothesis, it is proposed that DNA molecules enter these bacteria at the growing zone(s) of the cell envelope.

Amino Alcohols↗
PubMed1974

Role of a deoxyribonuclease in the genetic transformation of Diplococcus pneumoniae.

Two steps in the uptake of DNA by Diplococcus pneumoniae were characterized by analyzing mutants defective in transformation. A strain deficient in the two major deoxyribonucleases of D. pneumoniae takes up DNA normally and converts it to single strands within the cell and oligonucleotide fragments outside the cell. Extracts of this strain contain a residual deoxyribonuclease that produces similar oligonucleotide fragments in vitro. This enzyme is missing in transformation-defective mutants blocked in the second or entry step. Cells of this mutant class bind large amounts of DNA to their surface in a form accessible to external agents. Another class of nontransformable mutants fails to bind DNA at all. Their deoxyribonuclease content is unchanged, and they are apparently blocked in the first or binding step of DNA uptake. The binding step requires a source of energy and prior activation of the cells by competence factor. Entry may be independent of these requirements and may come about by action of the deoxyribonuclease on one strand of DNA with energy for the transport of the intact strand deriving from hydrolysis of the degraded strand. The enzyme may thus act as a DNA translocase.

Binding Sites↗
PubMed1980

Genetic transformation of mouse embryos by microinjection of purified DNA.

A recombinant plasmid composed of segments of herpes simplex virus and simian virus 40 viral DNA inserted into the bacterial plasmid pBR322 was microinjected into pronuclei of fertilized mouse oocytes. The embryos were implanted in the oviducts of pseudopregnant females and allowed to develop to term. DNA from newborn mice was evaluated by the Southern blotting technique for the presence of DNA homologous to the injected plasmid. Two of 78 mice in one series of injections showed clear homology, though the injected sequences had been rearranged. Band intensities from the two positive mice were consistent with the presence of donor DNA in most or all of the cells of the newborns. These results demonstrate that genes can be introduced into the mouse genome by direct insertion into the nuclei of early embryos. This technique affords the opportunity to study problems of gene regulation and cell differentiation in a mammalian system by application of recombinant DNA technology.

Animals↗
PubMed1998

Natural genetic transformation of Pseudomonas stutzeri in a non-sterile soil.

Natural transformation of the soil bacterium Pseudomonas stutzeri JM300 in a non-sterile brown earth microcosm was studied. For this purpose, the microcosm was loaded with purified DNA (plasmid or chromosomal DNA, both containing a high-frequency-transformation marker, his+, of the P. stutzeri genome), the non-adsorbed DNA was washed out with soil extract and then the soil was charged with competent cells (his-1). Both chromosomal and plasmid transformants were found among the P. stutzeri cells recovered from the soil. The number of plasmid transformants increased in a linear fashion with the amount of DNA added [10-600 ng (0.7 g soil)-1]. The observed efficiency of transformation, the time course of transformant formation and the complete inhibition of transformation by DNase I, when added to the soil, were similar to that seen in optimized transformations in nutrient broth. Addition of cells as late as 3 d after loading the soil with plasmid DNA still yielded 3% of the initial transforming activity. This suggests that nucleases indigenous to the soil destroyed the transforming DNA, but at a rate allowing considerable DNA persistence. Transformants were also obtained when intact P. stutzeri cells were introduced into the soil to serve as plasmid DNA donors. Apparently, DNA was released from the cells, adsorbed to the soil material and subsequently taken up by recipient cells. The results indicate that competent cells of P. stutzeri were able to find access to and take up DNA bound on soil particles in the presence of micro-organisms and DNases indigenous to the soil.

DNA, Bacterial↗
PubMed1977

Genetic transformation in Methylobacterium organophilum.

Several mutants have been isolated from the facultative methylotroph, Methylobacterium organophilum, using either N-methyl-N'-nitro-N-nitrosoguanidine or ultraviolet light as mutagens. One of these isolates, a glutamate auxotroph lacking isocitrate dehydrogenase, has been transformed to prototrophy, using wild-type DNA, at a frequency of 0-5%. Competence and DNA uptake occur only in cultures which are near the end of exponential growth, and maximal transformation requires a DNA concentration of 100 mug ml-1.

DNA, Bacterial↗
PubMed1993

Genetic transformation and phylogeny of bacterial symbionts from tsetse.

Two isolates of bacterial endosymbionts, GP01 and GM02, were established in cell free medium from haemolymph of the tsetse, Glossina pallidipes and G. morsitans. These microorganisms appear similar to rickettsia-like organisms reported previously from various tsetse species. The 16S rRNA sequence analysis, however, placed them within the gamma subdivision of the Proteobacteria, phylogenetically distinct from most members of the Rickettsiaceae which align with the alpha subdivision. Distinct multiple endogenous plasmids are harboured by GP01 and GM02, suggesting that the two isolates are different. Restriction mapping analysis showed that one of the conserved plasmids is present in high copy number and is at least 80 kb in size. A heterologous plasmid pSUP204, which contains the broad host range oriV replication origin, was used to transfect bacterial cultures. The symbiont GM02 was transformed, and it expressed plasmid encoded resistance to the antibiotics ampicillin, tetracycline and chloramphenicol. Transformation of these symbionts may provide a novel means for expressing anti-parasitic genes within tsetse populations.

Animals↗