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Rapid mapping of transposon insertion and deletion mutations in the large Ti-plasmids of Agrobacterium tumefaciens.
Nucleic Acids Res 1979; 7(7):1837-49NA

Abstract

A procedure is presented, that has allowed the rapid assignment of transposon Tn1 and Tn7 insertion sites in the large (130 Md) nopaline Ti-plasmid pTiC58, to specific restriction enzyme fragments. Total bacterial DNA is isolated from Agrobacterium tumefaciens strain C58 mutants that carry a transposon in their Ti-plasmid, and digested with an appropriate restriction endonuclease. The fragments are separated on an agarose gel, denatured and transferred to nitrocellulose filters. These are hybridized against purified wild type pTiC58, or against segments of PTiC58, cloned in E. coli using pBR322 as a vector plasmid. DNA sequences homologous to the probe are detected by autoradiography, thus generating a restriction enzyme pattern of the plasmid from a digest of total bacterial DNA. Mutant fragments can be readily identified by their different position compared to a wild type reference. This protocol eliminates the need to separate the large plasmid from chromosomal DNA for every mutant. In principle, it can be applied to the restriction enzyme analysis of insertion or deletion mutants in any plasmid that has no extensive homology with the chromosome.

Authors

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Pub Type(s)

Journal Article

Language

eng

PubMed ID

231764

Citation

Dhaese, P, et al. "Rapid Mapping of Transposon Insertion and Deletion Mutations in the Large Ti-plasmids of Agrobacterium Tumefaciens." Nucleic Acids Research, vol. 7, no. 7, 1979, pp. 1837-49.
Dhaese P, De Greve H, Decraemer H, et al. Rapid mapping of transposon insertion and deletion mutations in the large Ti-plasmids of Agrobacterium tumefaciens. Nucleic Acids Res. 1979;7(7):1837-49.
Dhaese, P., De Greve, H., Decraemer, H., Schell, J., & Van Montagu, M. (1979). Rapid mapping of transposon insertion and deletion mutations in the large Ti-plasmids of Agrobacterium tumefaciens. Nucleic Acids Research, 7(7), pp. 1837-49.
Dhaese P, et al. Rapid Mapping of Transposon Insertion and Deletion Mutations in the Large Ti-plasmids of Agrobacterium Tumefaciens. Nucleic Acids Res. 1979 Dec 11;7(7):1837-49. PubMed PMID: 231764.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Rapid mapping of transposon insertion and deletion mutations in the large Ti-plasmids of Agrobacterium tumefaciens. AU - Dhaese,P, AU - De Greve,H, AU - Decraemer,H, AU - Schell,J, AU - Van Montagu,M, PY - 1979/12/11/pubmed PY - 1979/12/11/medline PY - 1979/12/11/entrez SP - 1837 EP - 49 JF - Nucleic acids research JO - Nucleic Acids Res. VL - 7 IS - 7 N2 - A procedure is presented, that has allowed the rapid assignment of transposon Tn1 and Tn7 insertion sites in the large (130 Md) nopaline Ti-plasmid pTiC58, to specific restriction enzyme fragments. Total bacterial DNA is isolated from Agrobacterium tumefaciens strain C58 mutants that carry a transposon in their Ti-plasmid, and digested with an appropriate restriction endonuclease. The fragments are separated on an agarose gel, denatured and transferred to nitrocellulose filters. These are hybridized against purified wild type pTiC58, or against segments of PTiC58, cloned in E. coli using pBR322 as a vector plasmid. DNA sequences homologous to the probe are detected by autoradiography, thus generating a restriction enzyme pattern of the plasmid from a digest of total bacterial DNA. Mutant fragments can be readily identified by their different position compared to a wild type reference. This protocol eliminates the need to separate the large plasmid from chromosomal DNA for every mutant. In principle, it can be applied to the restriction enzyme analysis of insertion or deletion mutants in any plasmid that has no extensive homology with the chromosome. SN - 0305-1048 UR - https://www.unboundmedicine.com/medline/citation/231764/Rapid_mapping_of_transposon_insertion_and_deletion_mutations_in_the_large_Ti_plasmids_of_Agrobacterium_tumefaciens_ L2 - https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/7.7.1837 DB - PRIME DP - Unbound Medicine ER -