Molecular cloning of overlapping segments of the nopaline Ti-plasmid pTiC58 as a means to restriction endonuclease mapping

Depicker, Anna; Wilde, Michel De; Vos, Guido De; Vos, R. De; Montagu, Marc Van; Schell, J.

doi:10.1016/0147-619x(80)90109-2

Cited by 211 publications

(71 citation statements)

References 33 publications

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“…Open reading frames of genes virDI and virD2 (23) were amplified by the polymerase chain reaction (PCR) using the Ti-plasmid C58 subclone pGV0361 (24) as template, Taq DNA polymerase (Perkin-Elmer), and oligodeoxynucleotide primers 5'-CGCGGATCCATATGTCGCAAGGCAGTAG-GCC-3' and 5'-ATGGATCCCTACAAGGCGTCTTTCAG-CAG-3' for virDI and 5'-CGCGGATCCATATGCCCGATC-GAGCTCAAG-3' and 5'-ATGGATCCCTATCTCCTATT-TCCCCCACG-3' for virD2. To avoid generation of translational gene fusions during cloning ofthe PCR products in expression vector pET3b (25), the 5'-end-specific PCR primers were designed so that they contained both Nde I and BamHI sites, whereas the 3'-end primers included only a BamHI site.…”

Section: Methodsmentioning

confidence: 99%

“…Coding regions of virDI and virD2 (23) were amplified by PCR using plasmid pGV0361 DNA (24) as template and were cloned in pET3b to yield expression vectors pETvirDl and pETvirD2. To produce VirDl and VirD2 proteins in large quantities, plasmids pETvirDl and pETvirD2 were transformed into E. coli BL21(DE3) (25).…”

Section: Cloning and Overexpression Of Virdi And Vird2mentioning

confidence: 99%

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Agrobacterium T-strand production in vitro: sequence-specific cleavage and 5' protection of single-stranded DNA templates by purified VirD2 protein.

Jasper

Koncz

Schell

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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Virulence proteins VirDl and VirD2 are subunits of a relaxosome-like protein complex that mediates conjugational transfer of a Ti plasmid segment, the T-DNA, from Agrobacterium into higher plants. The VirDl-VirD2 complex binds to 25-bp repeats at the borders of the T-DNA and catalyzes sequence-specific nicking of the conijugative DNA strand (the T-strand) at the third base of these repeats. Nuclear localization signals present in VirD2 target the T-strand to plant ceil nuclei. In addition, VirD2 probably plays a role in the high-frequency integration of the T-DNA into the plant genome by illegitimate recombination. Whereas Agrobacterium transformation of dicots is very efficient, T-DNA integration in most monocots can barely be detected. To develop an artificial T-DNA delivery system for monocots, a technique for efficient in vitro production of T-strand DNAs was established by using VirDl and VirD2 proteins purified from overexpressing Escherichia coli strains. The topoisomerase-like VirD2 enzyme was shown to mediate precise, sequence-specific cleavage of T-DNA border sequences carried by single-stranded DNA templates, even in the absence of VirDl protein. During this reaction, VirD2 remains covalently bound to the 5' end of artificial T-strand DNAs. In contrast, VirD2, alone or in complex with VirDl, fails to nick linear double-stranded DNA templates in vitro.

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Section: Methodsmentioning

confidence: 99%

Section: Cloning and Overexpression Of Virdi And Vird2mentioning

confidence: 99%

Agrobacterium T-strand production in vitro: sequence-specific cleavage and 5' protection of single-stranded DNA templates by purified VirD2 protein.

Jasper

Koncz

Schell

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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“…To construct pRK415K, the kanamycin resistance cassette from the cosmid vector pCP13 (13) was excised as a BamHI fragment, the restriction sites were filled in with Klenow polymerase, and the DNA fragment was blunt-end ligated into the unique XmnI site of the broad-host-range vector pRK415 (30). Ti plasmid restriction fragment designations are those of Depicker et al (14).…”

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confidence: 99%

The oriT region of the Agrobacterium tumefaciens Ti plasmid pTiC58 shares DNA sequence identity with the transfer origins of RSF1010 and RK2/RP4 and with T-region borders

Cook

Farrand

1992

J Bacteriol

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Ti plasmids ofAgrobacterium tumefaciens are conjugal elements whose transfer is induced by certain opines secreted from crown galls. On transmissible plasmids, DNA transfer initiates within a cis-acting site, the origin of conjugal transfer, or oriT. We have localized an oriT on the A. tumefaciens plasmid pTiC58 to a region containing the conjugal transfer loci traI and traII and acc, which is the locus encoding catabolism of the two conjugal opines, agrocinopines A and B. The smallest functional oriT clone, a 65-bp BamHI-ApaI fragment in the recombinant plasmid pDCBA60-11, mapped within the traII locus. The nucleotide sequence for a 665-bp KpnI-EcoRI fragment with oriT activity was determined. DNA sequence alignments showed identities between the pTiC58 oriT and the transfer origins of RSF1010, pTF1, and RK2/RP4 and with the pTiC58 T-region borders. The RSF1010-like sequence on pTiC58 is located in the smallest active oriT clone of pTiC58, while the sequence showing identities with the oriT regions of RK2/RP4 and with T-region borders maps outside this region. Despite their sequence similarities, pTiC58 oriT clones were not mobilized by RP4; nor could vectors containing the RK2/RP4 oriT region or the oriT-mob region from RSF1010 be mobilized by pTiC58. In contrast, other Ti plasmids and a conjugally activeAgrobacterium opine catabolic plasmid, pAtK84b, efficiently mobilized pTiC58 oriT clones. In addition, the RSF1010 derivative, pDSK519, was mobilized at moderate frequencies by an Agrobacterium strain harboring only the cryptic plasmid pAtC58 and at very low frequencies by an Agrobacterium host that does not contain any detectable plasmids.The gram-negative phytopathogen Agrobacterium tumefaciens induces crown gall tumors on susceptible plant species by a novel gene-transfer process. Tumorigenicity is dependent upon a large plasmid, called Ti, present in the bacterium. During infection, a portion of the Ti plasmid called the T-region is excised and exported from the agrobacterial cell to a plant cell. Once the T-region is integrated into the plant nuclear genome, expression of transfer DNA (T-DNA)-encoded genes leads to the synthesis of phytohormones and unique nutritional compounds collectively named opines. The overproduction of the plant growth factors causes tumor formation. Opines are secreted from the tumors into the rhizosphere, where they become available as a nutritional source to A. tumefaciens strains carrying an appropriate Ti plasmid encoding functions for opine uptake and catabolism (39).In addition to carrying pathogenicity determinants, Ti plasmids also encode conjugal transfer functions which allow for Ti plasmid transmission to Ti plasmid-less agrobacteria (31). Ti plasmid conjugal transfer is normally repressed but is inducible by an opine subset, the conjugal opines (23, 38). Once a strain ofAgrobacterium has acquired a Ti plasmid, it is capable of utilizing opines as a sole nutritional substrate. Thus, the opines synthesized by the biologically engineered plant cells act as signals to s...

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“…1982). Several plasmids including pKC7 (Rao and Roger 1979), pUC9-nopneo 4 (Bevan 1984) and pGVO415 containing HindIIl fragment 23 of a nopaline type Ti plasmid (Depicker et at . 1980) were used.…”

Section: Methodsmentioning

confidence: 99%

Analysis of transcripts of aminoglycoside phosphotransferase II and nopaline synthease in Nicotiana glauca transformants mediated by recombinant Ti plasmid.

Ono

Hashimoto

Yoshioka

et al. 1986

The Japanese journal of genetics

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The vector possessing two marker genes encoding for, aminoglycoside phosphotransferase II (APH(3')II) and nopaline synthase (NOS) was inserted into the octopine-type Ti plasmid through homologous recombination.Following the confirmation of foreign gene expression in Nicotiana glauca tumor cells with respect to kanamycin resistance and nopaline synthesis, the analysis was made on the transcripts.There was a quantitative difference in the amount of mRNA between APH(3')II and NOS. The size of mRNA for APH(3')II and NOS were 1250 bases and 1550 bases, respectively.

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Molecular cloning of overlapping segments of the nopaline Ti-plasmid pTiC58 as a means to restriction endonuclease mapping

Cited by 211 publications

References 33 publications

Agrobacterium T-strand production in vitro: sequence-specific cleavage and 5' protection of single-stranded DNA templates by purified VirD2 protein.

Agrobacterium T-strand production in vitro: sequence-specific cleavage and 5' protection of single-stranded DNA templates by purified VirD2 protein.

The oriT region of the Agrobacterium tumefaciens Ti plasmid pTiC58 shares DNA sequence identity with the transfer origins of RSF1010 and RK2/RP4 and with T-region borders

Analysis of transcripts of aminoglycoside phosphotransferase II and nopaline synthease in Nicotiana glauca transformants mediated by recombinant Ti plasmid.

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