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Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens.
EMBO J. 1983; 2(3):411-7.EJ

Abstract

Transmission of ColE1/pMB1-derived plasmids, such as pBR322, from Escherichia coli donor strains was shown to be an efficient way to introduce these plasmids into Agrobacterium. This was accomplished by using E. coli carrying the helper plasmids pGJ28 and R64drd11 which provide the ColE1 mob functions and tra functions, respectively. For example, the broad host-range replication plasmid, pGV1150, a co-integrate plasmid between pBR322 and the W-type mini-Sa plasmid, pGV1106, was transmitted from E. coli to A. tumefaciens with a transfer frequency of 4.5 x 10(-3). As pBR322 clones containing pTiC58 fragments were unable to replicate in Agrobacterium, these clones were found in Agrobacterium only if the acceptor carried a Ti plasmid, thus allowing a co-integration of the pBR322 clones with the Ti plasmid by homology recombination. These observations were used to develop an efficient method for site-specific mutagenesis of the Ti plasmids. pTiC58 fragnents, cloned in pBR322, were mutagenized in vitro and transformed into E. coli. The mutant clones were transmitted from an E. coli donor strain containing pGJ28 and R64drd11 to an Agrobacterium containing a target Ti plasmid. Selecting for stable transfer of the mutant clone utilizing its antibiotic resistance marker(s) gave exconjugants that already contained a co-integrate plasmid between the mutant clone and the Ti plasmid. A second recombination can dissociate the co-integrate plasmid into the desired mutant Ti plasmid and a non-replicating plasmid formed by the vector plasmid pBR322 and the target Ti fragment. These second recombinants lose the second plasmid and they are identified by screening for the appropriate marker combination.

Authors+Show Affiliations

Laboratorium voor Genetica, Rijksuniversiteit Gent, Belgium.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

11894957

Citation

Van Haute, E, et al. "Intergeneric Transfer and Exchange Recombination of Restriction Fragments Cloned in pBR322: a Novel Strategy for the Reversed Genetics of the Ti Plasmids of Agrobacterium Tumefaciens." The EMBO Journal, vol. 2, no. 3, 1983, pp. 411-7.
Van Haute E, Joos H, Maes M, et al. Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. EMBO J. 1983;2(3):411-7.
Van Haute, E., Joos, H., Maes, M., Warren, G., Van Montagu, M., & Schell, J. (1983). Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. The EMBO Journal, 2(3), 411-7.
Van Haute E, et al. Intergeneric Transfer and Exchange Recombination of Restriction Fragments Cloned in pBR322: a Novel Strategy for the Reversed Genetics of the Ti Plasmids of Agrobacterium Tumefaciens. EMBO J. 1983;2(3):411-7. PubMed PMID: 11894957.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. AU - Van Haute,E, AU - Joos,H, AU - Maes,M, AU - Warren,G, AU - Van Montagu,M, AU - Schell,J, PY - 1983/1/1/pubmed PY - 2002/4/12/medline PY - 1983/1/1/entrez SP - 411 EP - 7 JF - The EMBO journal JO - EMBO J VL - 2 IS - 3 N2 - Transmission of ColE1/pMB1-derived plasmids, such as pBR322, from Escherichia coli donor strains was shown to be an efficient way to introduce these plasmids into Agrobacterium. This was accomplished by using E. coli carrying the helper plasmids pGJ28 and R64drd11 which provide the ColE1 mob functions and tra functions, respectively. For example, the broad host-range replication plasmid, pGV1150, a co-integrate plasmid between pBR322 and the W-type mini-Sa plasmid, pGV1106, was transmitted from E. coli to A. tumefaciens with a transfer frequency of 4.5 x 10(-3). As pBR322 clones containing pTiC58 fragments were unable to replicate in Agrobacterium, these clones were found in Agrobacterium only if the acceptor carried a Ti plasmid, thus allowing a co-integration of the pBR322 clones with the Ti plasmid by homology recombination. These observations were used to develop an efficient method for site-specific mutagenesis of the Ti plasmids. pTiC58 fragnents, cloned in pBR322, were mutagenized in vitro and transformed into E. coli. The mutant clones were transmitted from an E. coli donor strain containing pGJ28 and R64drd11 to an Agrobacterium containing a target Ti plasmid. Selecting for stable transfer of the mutant clone utilizing its antibiotic resistance marker(s) gave exconjugants that already contained a co-integrate plasmid between the mutant clone and the Ti plasmid. A second recombination can dissociate the co-integrate plasmid into the desired mutant Ti plasmid and a non-replicating plasmid formed by the vector plasmid pBR322 and the target Ti fragment. These second recombinants lose the second plasmid and they are identified by screening for the appropriate marker combination. SN - 0261-4189 UR - https://www.unboundmedicine.com/medline/citation/11894957/Intergeneric_transfer_and_exchange_recombination_of_restriction_fragments_cloned_in_pBR322:_a_novel_strategy_for_the_reversed_genetics_of_the_Ti_plasmids_of_Agrobacterium_tumefaciens_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0261-4189&date=1983&volume=2&issue=3&spage=411 DB - PRIME DP - Unbound Medicine ER -