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The use of selectable markers for the isolation of plant-DNA/T-DNA junction fragments in a cosmid vector

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Summary

DNA of the crown gall tumor line W38T37:: Tn7-1 was partially digested with Sau3A to an average molecular weight of 25 Md and ligated either directly or after size fractionation to BamHI cut cosmid pJC81 DNA. After in vitro packaging in phage λ particles and transduction to E. coli HB101, recombinants that expressed the Tn7 coded resistances to spectinomycin and trimethoprim were selected. The recombinant plasmids thus isolated contained part or the whole of Tn7 together with adjacent T-DNA. Four independent, large clones are described, three containing the left border of the T-DNA, one containing the right border and an intact copy of the Tn7 transposon. In this case all the Tn7 encoded genes were shown to have remained fully functional since the reisolated Tn7 was found to be capable of normal transposition in E. coli. The T-DNA in the W38T37::Tn7 tumor line is flanked both to the left and to the right by highly AT rich repetitive plant sequences. These results further demonstrate that foreign genes can be transferred, integrated and stably maintained in chromosomes of plant cells without undergoing any observable rearrangements. This method of cosmid cloning combined with direct selection for the desired recombinant colonies is of general application for the genomic cloning of transformed eukaryotic cells.

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Authors and Affiliations

  1. Laboratorium voor Genetica, Rijksuniversiteit Gent, B-9000, Gent, Belgium

    M. Holsters, R. Villarroel, M. Van Montagu & J. Schell

  2. Laboratorium voor Genetische Virologie, Vrije Universiteit Brussel, B-1640, St.-Genesius-Rode, Belgium

    M. Van Montagu

  3. Max-Planck-Institut für Züchtungsforschung, D-5000, Köln 30, Federal Republic of Germany

    J. Schell

Authors
  1. M. Holsters
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  2. R. Villarroel
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  3. M. Van Montagu
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  4. J. Schell
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Communicated by G. Melchers

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Holsters, M., Villarroel, R., Van Montagu, M. et al. The use of selectable markers for the isolation of plant-DNA/T-DNA junction fragments in a cosmid vector. Molec. Gen. Genet. 185, 283–289 (1982). https://doi.org/10.1007/BF00330799

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