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  • Ti-plasmid  (2)
  • DNA/RNA hybridization  (1)
  • 1
    Electronic Resource
    Electronic Resource
    Transient transfection of mammalian cells with DNA of the plant-pathogenic Ti-plasmid and expression of marker and resident sequences (1989)
    Springer
    Molecular and cellular biochemistry 85 (1989), S. 19-28 
    Details
    ISSN: 1573-4919
    Keywords: Ti-plasmid ; transfection ; mammalian cells ; T-DNA expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Summary The large Ti-plasmid from Agrobacterium tumefaciens strain C58 has been used for transfection experiments with mammalian cells. In DNA from Tupaia baby fibroblasts Ti-plasmid sequences could be identified by filter hybridization as long as four weeks after transfection including two cell passages. The hybridization signals decreased rapidly after addition of the Ti-plasmid DNA-coprecipitate to the cells. The signals were often not detected any more after the first day, but were visible one week after transfection. Nuclei prepared from Ti-plasmid-transfected cells hybridized to pTi-specific RNA. With the chloramphenicol acetyl transferase-gene as marker no discrimination in DNA uptake was found between the Ti-plasmid and much smaller plasmids. According to the number of nuclei with homology to pTi-sequences it is assumed that about 0.2% of the cells carry Ti-plasmid DNA in the nucleus. Analysis of RNA isolated from cells transfected with cloned segments of the Ti-plasmid revealed that the TDNA region of the Ti-plasmid was predominantly transcribed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00223510
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  • 2
    Electronic Resource
    Electronic Resource
    Transformation of soybean protoplasts from permanent suspension cultures by cocultivation with cells of Agrobacterium tumefaciens (1987)
    Springer
    Plant molecular biology 9 (1987), S. 135-145 
    Details
    ISSN: 1573-5028
    Keywords: azacytidine ; DNA/RNA hybridization ; gene expression ; marker rescue ; nopaline-synthase ; plant tumor cells ; Ti-plasmid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cell wall regenerating protoplasts from soybean cells kept in suspension culture were cocultivated with bacteria which were derived from the nopaline strain C58 of Agrobacterium tumefaciens. When the bacteria carried an oncogenic Ti-plasmid, about 5% of the surviving protoplasts were able to form calli on hormone-free agar in contrast to controls, where bacteria without Ti-plasmid were applied, and where no calli were formed. After isolation of DNA from hormone-independently growing cells further evidence for transformation was obtained by hybridization to Ti-plasmid specific RNA and by rescue of a segment with a bacterial resistance gene which had been inserted before into the T-DNA. Transfer of T-DNA harboring a neomycin-resistance gene activated by the nos-promoter resulted in calli growing on kanamycin. Verification of segments located at the left and the right part of the T-DNA indicated the presence of its entire length in transformed soybean cells. Expression of T-DNA genes was measured by the assay of nopaline-synthase. Cells cultured on agar had a much higher level of nopaline-synthase than fast growing cells in suspension culture. Transferring them to agar or treatment with azacytidine strongly increased synthesis of nopaline-synthase indicating a reversible repression presumably via a methylation mechanism.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00015646
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