A novel method for the rapid cloning in Escherichia coli of Bacillus subtilis chromosomal DNA adjacent to Tn917 insertions (original) (raw)
Summary
A rapid and general procedure has been devised for the pBR322-mediated cloning in Escherichia coli of Bacillus subtilis chromosomal DNA extending in a specified direction from any Tn_917_ insertion. Derivatives of Tn_917_ have been constructed that contain a pBR322-derived replicon, together with a chloramphenicol-resistance (Cmr) gene of Gram-positive origin (selectable in B. subtilis), inserted by ligation in two orientations into a Sal_I restriction site located near the center of the transposon. When linearized plasmid DNA carrying such derivatives was used to transform to Cmr B. subtilis bacteria already containing a chromosomal insertion of Tn_917, the pBR322 sequences efficiently became integrated into the chromosomal copy of the transposon by homologous recombination. It was then possible to clone chromosomal sequences adjacent to either transposon insertion junction into E. coli, using a selection for ampicillin-resistance, by transforming CaCl2-treated cells with small amounts of insert-containing DNA that had been digested with various restriction enzymes and then ligated at a dilute concentration. Because pBR322 sequences may be inserted by recombination in either orientation with respect to the transposon arms, a single restriction enzyme (such as _Eco_Ri or Sph_I) that has a unique recognition site in pBR322 DNA may be used to separately clone chromosomal DNA extending in either direction from the site of any transposon insertion. A family of clones generated from the region of an insertional spo mutation (spoIIH::Tn_917) was used in Southern hybridization experiments to verify that cloned material isolated with this procedure accurately reflected the arrangement of sequences present in the chromosome. Strategies are discussed for taking advantage of certain properties inherent in the structure of clones generated in this way to facilitate the identification and study of promoters of insertionally mutated genes.
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- Department of Cellular and Developmental Biology, The Biological Laboratories, Harvard University, 16 Divinity Avenue, 02138, Cambridge, MA, USA
Philip Youngman, John B. Perkins & Richard Losick
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- Philip Youngman
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Communicated by G.R. Fink
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Youngman, P., Perkins, J.B. & Losick, R. A novel method for the rapid cloning in Escherichia coli of Bacillus subtilis chromosomal DNA adjacent to Tn_917_ insertions.Mol Gen Genet 195, 424–433 (1984). https://doi.org/10.1007/BF00341443
- Received: 08 March 1984
- Issue Date: July 1984
- DOI: https://doi.org/10.1007/BF00341443