Expression of Ti-plasmid DNA in E. coli: Comparison of homologous fragments cloned from Ti plasmids of Agrobacterium strains C58 and Ach5

Active DNA helicase I (Mr 180,000) can be isolated from Escherichia coli F+ strains but not F strains. The transfer of the F sex factor to F strains by conjugation permits the purification of the enzyme from the transconjugant strains. We conclude from this that helicase I is coded for by a portion of the F factor. Results also obtained by using recombinant plasmids carrying different DNA fragments of the F factor transfer region suggest that DNA helicase I is identical to the product of tral, one of the transfer genes of the F factor.

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“…were purified and labeled with 40 1LCi/ml [35S]methionine (Amersham; 1,300 Ci/mmol) as described (18).…”

mentioning

confidence: 99%

Identification of Escherichia coli DNA helicase I as the traI gene product of the F sex factor.

Abdel-Monem¹,

Taucher-Scholz²,

Klinkert³

1983

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

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“…1 [5,16]. This fragment was cloned before into a pBRvector resulting in plasmid pSS83 [27] and further cloned into plasmid pfdA2 resulting in plasmid pfdA2-83 [10]. The latter plasmid was cleaved with restriction enzymes EcoRI and XbaI which yields, besides the vector, a large fragment B with the nopaline-synthase gene and the right border of the T-DNA and a smaller fragment A covering the left part of the EcoRI fragment no.…”

Section: Analysis Of Cellular Dnamentioning

confidence: 99%

Transformation of soybean protoplasts from permanent suspension cultures by cocultivation with cells of Agrobacterium tumefaciens

1987

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Cell wall regenerating protoplasts from soybean cells kept in suspension culture were cocultivated with bacteria which were derived from the nopaline strain C58 of Agrobacterium tumefaciens. When the bacteria carried an oncogenic Ti-plasmid, about 5% of the surviving protoplasts were able to form calli on hormone-free agar in contrast to controls, where bacteria without Ti-plasmid were applied, and where no calli were formed. After isolation of DNA from hormone-independently growing cells further evidence for transformation was obtained by hybridization to Ti-plasmid specific RNA and by rescue of a segment with a bacterial resistance gene which had been inserted before into the T-DNA. Transfer of T-DNA harboring a neomycin-resistance gene activated by the nos-promoter resulted in calli growing on kanamycin. Verification of segments located at the left and the right part of the T-DNA indicated the presence of its entire length in transformed soybean cells. Expression of T-DNA genes was measured by the assay of nopaline-synthase. Cells cultured on agar had a much higher level of nopaline-synthase than fast growing cells in suspension culture. Transferring them to agar or treatment with azacytidine strongly increased synthesis of nopaline-synthase indicating a reversible repression presumably via a methylation mechanism.

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“…These data suggest, that for these plasmids an information encoded by Ti plasmid DNA sequences was recognized only in A.twnefaciens extracts. The specificity of A.twnefaciens extracts for Ti plasmid DNA sequences was further analysed by studying the expression of the EcoRI fragment 7 from the octopine plasmid pTi-Ach5, cloned in both possible orientations in the EcoRI site of a pBR325 derivative (plasmids pSS155 and pSS156) (12). In E.coli extracts both plasmids gave a similar protein pattern, except for one protein of 15.5 K, which was shown earlier to be a fusion product, encoded partly by the vector and partly by the insertion (12).…”

Section: Optimum Conditions For Atumefaciens Cell-free Systemmentioning

confidence: 99%

“…Generally, bacteria that induce octopine tumors catabolize octopine, whereas bacteria that induce nopaline tumors catabolize nopaline (1,9). Until now, little is known about expression into proteins of genes encoded by Ti plasmid DNA sequences in bacterial and plant cells (11,12,13).…”

Section: Introductionmentioning

confidence: 99%

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A coupled transcription-translation system from Agrobacterium tumefaciens and its applicationto study Ti plasmid expression in vitro

1981

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A coupled transcription-translation system was isolated from A. tumefaciens. Expression of plasmids pBR322 and pKT212 from E.coli, cloned fragments of Ti plasmid (plasmids pSS155 and pSS156) and Ti plasmid derivatives pAL2802, pAL2811, pAL2821 and pAL2832 was analysed in an A. tumefaciens cell-free system and compared with their expression in an E.coli cell-free system. New proteins of 41K and 44K appeared in A tumefaciens extracts, as the result of Ti plasmid expression. These proteins were not found in E.coli extracts.

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Expression of Ti-plasmid DNA in E. coli: Comparison of homologous fragments cloned from Ti plasmids of Agrobacterium strains C58 and Ach5

Cited by 15 publications

References 24 publications

Identification of Escherichia coli DNA helicase I as the traI gene product of the F sex factor.

Identification of Escherichia coli DNA helicase I as the traI gene product of the F sex factor.

Transformation of soybean protoplasts from permanent suspension cultures by cocultivation with cells of Agrobacterium tumefaciens

A coupled transcription-translation system from Agrobacterium tumefaciens and its applicationto study Ti plasmid expression in vitro

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