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  • 1
    ISSN: 1617-4623
    Keywords: Protoplast fusion ; Nitrate reductase deficiency ; Kanamycin ; Nicotiana tabacum ; Nicotiana sylvestris
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The combination in the nuclear genome of a dominant resistance marker (to select against unfused wild-type cells) and a recessive deficiency marker (to select against unfused mutant cells) in a cell line should provide a system for selecting fusion hybrids between the mutant line and any wild-type line. To test this idea, we fused protoplasts from a non-morphogenic cell line of Nicotiana tabacum which was kanamycin resistant (by transformation) and deficient in nitrate reductase (NR-K+) with protoplasts from N. tabacum cv. Petit Havana clone SR1, which provided resistance against streptomycin as an additional selectable marker (NR+K-SR+). Putative hybrids were selected using a culture medium containing no available reduced nitrogen source and 50 mg/l kanamycin sulphate. After regeneration into plants, the hybrid character was demonstrated from: (i) the morphological variation of the regenerants; (ii) the chromosome number; (iii) the ability to grow on medium without a reduced nitrogen source and containing kanamycin sulphate at 50 mg/l; (iv) the presence of nitrate reductase activity; (v) the presence of the gene coding for neomycin phosphotransferase, which provides resistance to kanamycin sulphate; (vi) callus formation from leaves on medium containing 1 g/l streptomycin or 50 mg/l kanamycin sulphate; (vii) F1 plants containing nitrate reductase and the gene for neomycin phosphotransferase. Fusions between the mutant cell line (NR-K+) and three wild-type tobacco species and subsequent cultivation on medium containing no available nitrogen source but 50 mg/l kanamycin sulphate resulted in callus formation with all combinations, while hybrid plants were only regenerated when N. sylvestris was the fusion partner.
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  • 2
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Two clones of N. tabacum, transformed to kanamycin resistance by direct transfer to protoplasts of a hybrid gene, consisting of the protein coding region from the bacterial gene for aminoglycoside phosphotransferase under the control of 5′/3′ expression signals from cauliflower mosaic virus gene VI, in the bacterial plasmid pUC8, have been subjected to a detailed genetic crossing analysis accompanied by Southern blot analysis and enzyme activity assays of representative offspring. The genetic data obtained from large populations of R1/F1 and R2/F2 offspring as well as from more than 20 subclones of each of the original transformants confirm that (a) one functional copy of the hybrid gene was stably integrated into chromosomal DNA of the original transformants, (b) that the gene normally was stably maintained during clonal proliferation, (c) that normally it is transmitted in a regular fashion (with exceptions) to sexual offspring, and (d) that it is inherited as a single dominant trait. Data from DNA hybridisation and enzyme assays confirm this interpretation. The functional gene is integrated together with several non-functional copies and bacterial plasmid sequences, which are inherited as one block together with the functional gene.
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  • 3
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Sequences essential for the conjugal transfer of ColE1 can be divided into a cis-acting site and a region encoding trans-acting products. Each of these was successively cloned into a non-transmissible plasmid vector. The resulting chimera was transmissible by the conjugative plasmids F'lac,pro (incFI) and R64drd11 (incIα). The sequences encoding colicin E1, immunity, and incompatibility were absent from this chimera: therefore they are not essential for the conjugal transmission of the ColE1 plasmid. In contrast to ColE1, however, the same chimera was deficient in conjugal transfer initiated by R751 (incP) and R388 (incW). This suggests that ColEl sequences other than those cloned in the chimeric plasmid are necessary for its mobilization by R751 and R388. Three such regions were revealed by screening a series of ColE1 insertion mutants for transfer by R751 and R388. Two of these regions encode no other known function while the third is encoded by a region which overlaps the gene for colicin E1 itself.
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  • 4
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Definitive evidence is presented for the first time for stable gene transfer to cultured cells in a plant of the family Gramineae, Lolium multiflorum (Italian Ryegrass), using DNA transformation of protoplasts from a non-morphogenic cell culture. A construction consisting of expression signals from gene VI of Cauliflower Mosaic virus joined to the aminoglycoside (neomycin) phosphotransferase gene (APH(3′)II) from transposon Tn5 conferred resistance to the antibiotic G-418 to cell colonies arising from transformed protoplasts. By demonstrating a tight correlation between the resistant phenotype, the physical presence of the foreign gene and the presence of the active gene product we have shown that these colonies are true transformants and that a gene which is expressed well in dicotyle-denous plants is also expressed in cells of graminaceous monocots.
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  • 5
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] Synthesis of the large subunit polypeptide of ribulose bisphosphate carboxylase can be detected in Escherichia coli cells containing the chloroplast genes from maize and wheat. The chloroplast DNA sequences contain a ‘promoter’ that, in Escherichia coli, initiates transcription and ...
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  • 6
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Plasmid DNA clones containing repetitive DNA sequences were isolated from Hyoscyamus muticus and Nicotiana tabacum. Non cross-hybridizing probes from each species were used in a simple hybridization test with DNA isolated from presumptive somatic hybrids. This allowed unequivocal identification of DNA from both species in the hybrids.
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  • 7
    Electronic Resource
    Electronic Resource
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 11 (1990), S. 176-181 
    ISSN: 0192-253X
    Keywords: Direct gene transfer ; transgenic plants ; expression of transgenes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Direct gene transfer to protoplasts is one of several methods developed for the production of transgenic plants. This method utilizes the efficient uptake of DNA from the surrounding medium by protoplasts (cell wall-less plant cells). Where a suitable protoplast system exists large numbers of transformant clones can be efficiently produced and often regenerated to normal fertile plants. This review concentrates on the fate of the DNA which is taken up into the protoplasts. Particular emphasis is given to the factors which can influence the integration and form of the transferred DNA, the expression of transferred genes, and the inheritance in further generations of those genes. The information available suggests (1) that DNA is taken up by a large proportion of the cells in a transformation mixture, (2) that this DNA forms complexes sometimes involving carrier DNA, (3) that fewer cells actually take up DNA into the nucleus, and (4) that the complex may be rearranged and/or amplified and then integrated into the genome. If the DNA is arranged in such a way that a gene can be expressed it does so in a normal manner and is stably inherited both mitotically and meiotically.
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