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Molecular cloning of heterologous chromosomal DNA by recombination between a plasmid vector and a homologous resident plasmid in Bacillus subtilis

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Summary

The difficulty experienced in the shotgun cloning of chromosomal DNA on plasmid vectors in Bacillus subtilis is analyzed and an explanation for this difficulty is offered based on an inherent property of competent cells which imposes a requirement of plasmid multimers in transformation of plasmidfree recipients (Canosi et al., 1978). A stratagem which uses cloning by recombination between the vector and a resident homologous plasmid is tested and shown to be successful. Several recombinant plasmids are obtained containing Bacillus licheniformis DNA fragments which complement aromatic amino acid mutants of Bacillus subtilis. The yield of recombinant clones ranges from 6.7 to 210 per μg of chromosomal DNA, depending on the selection and the restriction endonuclease. The various trp clones obtained after cutting chromosomal DNA with BglII and BclI do not complement trpE and exhibit both orientations with respect to the vector. The location of several restriction endonuclease cleavage sites in the cloned trp fragments is presented, and their relationship to the genetic map of Bacillus licheniformis is described.

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Abbreviations

Km:

kanamycin

Cm:

chloramphenicol

Em:

erythromycin

CCC:

covalently closed circular

OC:

open circular

г:

resistant

MDal:

megadalton

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Authors and Affiliations

  1. Department of Microbiology, The Public Health Research Institute of The City of New York, Inc., 455 First Avenue, 10016, New York, New York, USA

    Thomas Gryczan, Sara Contente & David Dubnau

Authors
  1. Thomas Gryczan
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  2. Sara Contente
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  3. David Dubnau
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Additional information

Communicated by H.W. Boyer

In partial fulfillment of the requirements for the doctoral degree in the Department of Microbiology at the New York University School of Medicine, for S.C.

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Gryczan, T., Contente, S. & Dubnau, D. Molecular cloning of heterologous chromosomal DNA by recombination between a plasmid vector and a homologous resident plasmid in Bacillus subtilis . Molec. Gen. Genet. 177, 459–467 (1980). https://doi.org/10.1007/BF00271485

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  • DOI: https://doi.org/10.1007/BF00271485

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