Transposon Tn916 insertional mutagenesis of Pasteurella multocida and direct sequencing of disruption site

Microbial Pathogenesis
P L DeAngelis

Abstract

The transposon Tn916, when introduced into Pasteurella multocida by electroporation on a nonreplicating plasmid, integrates into the bacterial chromosome. Efficiencies of approximately 8x10(4) mutants/microg of plasmid DNA were obtained. Restriction digestion and Southern analysis indicate that the Tn916 element integrates in a quasi-random fashion throughout the genome. Most transformants had a single copy of the transposon but approximately 5% had two copies. Furthermore, the nucleotide sequence at the disruption site of any desired mutant was obtained by capitalizing on the differential sensitivity of the transposon and the genome to the restriction enzyme HhaI; molecular cloning or amplification by polymerase chain reaction was not required. The Tn916 element has a single HhaI site. On the other hand, this restriction enzyme frequently cleaves the P. multocida chromosome with the vast majority of the resulting genomic fragments being less than 7 kb in length. Tn916 integration adds a 12 kb segment to the genomic HhaI fragment at the site of disruption. The resulting chimeric DNA fragment was isolated on the basis of size from digests of mutant genomic DNA separated on agarose gels. DNA sequencing with primers corresponding ...Continue Reading

References

Oct 1, 1992·Journal of Bacteriology·J R Scott
Jan 1, 1992·Microbial Pathogenesis·L JablonskiG R Carter
Jan 1, 1989·Cold Spring Harbor Symposia on Quantitative Biology·C A Janeway
Jan 1, 1988·Journal of Bacteriology·E SenghasD B Clewell
Jul 28, 1995·Science·R D FleischmannJ M Merrick
May 1, 1996·Veterinary Microbiology·M D Lee, A D Henk
Feb 12, 2008·Current Protocols in Molecular Biology·K Wilson

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Citations

Jul 5, 2013·Clinical Microbiology Reviews·Brenda A Wilson, Mengfei Ho
May 9, 1998·The Journal of Biological Chemistry·P L DeAngelisA M Achyuthan
Mar 4, 2000·Veterinary Microbiology·M L HuntK M Townsend

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