Biomaterial Database

Transposon Tn916 insertional mutagenesis of Pasteurella multocida and direct sequencing of disruption site. 1998

P L DeAngelis

Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., Oklahoma City, OK, 73104, USA.

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 to the terminus of the Tn916 element was used to determine the sequence at the disruption site. In summary, Tn916 can be used to disrupt and to clone genes of P. multocida in a rapid and facile fashion.

UI	MeSH Term	Description	Entries
D004251	DNA Transposable Elements	Discrete segments of DNA which can excise and reintegrate to another site in the genome. Most are inactive, i.e., have not been found to exist outside the integrated state. DNA transposable elements include bacterial IS (insertion sequence) elements, Tn elements, the maize controlling elements Ac and Ds, Drosophila P, gypsy, and pogo elements, the human Tigger elements and the Tc and mariner elements which are found throughout the animal kingdom.	DNA Insertion Elements,DNA Transposons,IS Elements,Insertion Sequence Elements,Tn Elements,Transposable Elements,Elements, Insertion Sequence,Sequence Elements, Insertion,DNA Insertion Element,DNA Transposable Element,DNA Transposon,Element, DNA Insertion,Element, DNA Transposable,Element, IS,Element, Insertion Sequence,Element, Tn,Element, Transposable,Elements, DNA Insertion,Elements, DNA Transposable,Elements, IS,Elements, Tn,Elements, Transposable,IS Element,Insertion Element, DNA,Insertion Elements, DNA,Insertion Sequence Element,Sequence Element, Insertion,Tn Element,Transposable Element,Transposable Element, DNA,Transposable Elements, DNA,Transposon, DNA,Transposons, DNA
D004252	DNA Mutational Analysis	Biochemical identification of mutational changes in a nucleotide sequence.	Mutational Analysis, DNA,Analysis, DNA Mutational,Analyses, DNA Mutational,DNA Mutational Analyses,Mutational Analyses, DNA
D004269	DNA, Bacterial	Deoxyribonucleic acid that makes up the genetic material of bacteria.	Bacterial DNA
D014169	Transformation, Bacterial	The heritable modification of the properties of a competent bacterium by naked DNA from another source. The uptake of naked DNA is a naturally occuring phenomenon in some bacteria. It is often used as a GENE TRANSFER TECHNIQUE.	Bacterial Transformation
D016254	Mutagenesis, Insertional	Mutagenesis where the mutation is caused by the introduction of foreign DNA sequences into a gene or extragenic sequence. This may occur spontaneously in vivo or be experimentally induced in vivo or in vitro. Proviral DNA insertions into or adjacent to a cellular proto-oncogene can interrupt GENETIC TRANSLATION of the coding sequences or interfere with recognition of regulatory elements and cause unregulated expression of the proto-oncogene resulting in tumor formation.	Gene Insertion,Insertion Mutation,Insertional Activation,Insertional Mutagenesis,Linker-Insertion Mutagenesis,Mutagenesis, Cassette,Sequence Insertion,Viral Insertional Mutagenesis,Activation, Insertional,Activations, Insertional,Cassette Mutagenesis,Gene Insertions,Insertion Mutations,Insertion, Gene,Insertion, Sequence,Insertional Activations,Insertional Mutagenesis, Viral,Insertions, Gene,Insertions, Sequence,Linker Insertion Mutagenesis,Mutagenesis, Linker-Insertion,Mutagenesis, Viral Insertional,Mutation, Insertion,Mutations, Insertion,Sequence Insertions
D016979	Pasteurella multocida	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria normally found in the flora of the mouth and respiratory tract of animals and birds. It causes shipping fever (see PASTEURELLOSIS, PNEUMONIC); HEMORRHAGIC BACTEREMIA; and intestinal disease in animals. In humans, disease usually arises from a wound infection following a bite or scratch from domesticated animals.
D018274	Electroporation	A technique in which electric pulses, in kilovolts per centimeter and of microsecond-to-millisecond duration, cause a loss of the semipermeability of CELL MEMBRANES, thus leading to ion leakage, escape of metabolites, and increased uptake by cells of drugs, molecular probes, and DNA. Depending on the dosage, the formation of openings in the cell membranes caused by the electric pulses may or may not be reversible.	Electric Field-Mediated Cell Permeabilization,Irreversible Electroporation,Reversible Electroporation,Electropermeabilisation,Electric Field Mediated Cell Permeabilization,Electroporation, Irreversible,Electroporation, Reversible