ColE1 plasmid incompatibility: localization and analysis of mutations affecting incompatibility

Hashimoto-Gotoh, Tamotsu; Inselburg, Joseph

doi:10.1128/jb.139.2.608-619.1979

Cited by 35 publications

(25 citation statements)

References 25 publications

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“…To say that the required regions are identical is premature; such a conclusion will require comparison of the DNA sequences. Since pl5A and ColEl are compatible (G. Selzer, personal communication) and the region between -200 and -555 bp from the origin of replication has been implicated in incompatibility (9), it is likely that the two sequences differ in a significant manner in this region.…”

Section: Resultsmentioning

confidence: 99%

Homology between Escherichia coli plasmids ColE1 and p15A

Bird¹

1981

J Bacteriol

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The location and extent of the homology between plasmids ColEl and pl5A were determined by analysis of heteroduplexes formed between them as well as with a related plasmid, pBR322, and by hybridization of radioactive deoxyribonucleic acids to restriction fragments of pl5A and ColEl. The homology between the plasmids contained the entire region of ColE1 required for its replication as well as an additional 400 base pairs downstream from the origin of replication. This region on pl5A, which was 980 ± 43 base pairs, started at 0.1 of the molecular length from one end formed by cleavage with the restriction endonuclease BglI and extended to 0.54 of the molecular length from the same end. Restriction cleavage maps for the enzymes BglI, HpaI, HaeII, HaeIII, and HinclI are also presented.The small Escherichia coli plasmid pl5A has a molecular weight of about 1.5 x 106 (7). The plasmid replicates in the absence of protein synthesis but not in the presence of rifampin, a known inhibitor ofE. coli DNA-dependent RNA polymerase (12). In addition, this plasmid replicates in bacterial extracts which support the replication of plasmid CoIEl (16). The principal products of the in vitro system, when pl5A DNA is used as the template in the presence of 10% glycerol, are molecules containing newly replicated 6S fragments (unpublished data), as is observed with CoIEl DNA as the template (17). Thus, pl5A appears to replicate by a mechanism very much like that of plasmid CoIEl.Replication of ColEl DNA is unidirectional from a fixed origin (10,11). The info.rmation required for ColEl replication is contained in a 580-base-pair (bp) segment of its DNA. This segment extends from 567 bp before the origin of replication to a point 13 bp past the origin (2), where the origin of replication is defined as the point at which the first deoxyribonucleotide is added to an RNA primer (21). Previous DNA-DNA hybridization studies have shown that there is some homology (approximately 30%) between pl5A and ColEl (8), and heteroduplex analysis between derivatives of pl5A and ColEl has shown considerable homology, which was suggested to be at the origin of replication (4). To examine pl5A for regions of homology with ColEl, I carried out heteroduplex analysis of DNA-DNA hybrids formed between pl5Aand both ColEl and pBR322. Plasmid pBR322 was constructed from pMB1, whose sequence is similar to ColEl in the region required for replication (3). The region of pBR322 homologous to ColEl lies between nucleotides 1,762 and 3,146, with the region required for its replication lying between nucleotides 2,523 and approximately 3,103 on the sequence of Sutcliffe (20). This plasmid is useful in heteroduplex studies because pBR322 made linear by cutting at the single EcoRI site has sequences at one end (0 through 1,761) derived from plasmid pSC101, whereas those at the other end (3,147 through 4,362) are derived from Tn3. These two regions of ColEl nonhomology can be used to orient the pBR322 DNA present in heteroduplexes. In this paper, I locate the sites on pi5A DNA...

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Section: Resultsmentioning

confidence: 99%

Homology between Escherichia coli plasmids ColE1 and p15A

Bird¹

1981

J Bacteriol

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“…These latter plasmids differ from the pCGBPV constructs in at least three points. The origin of plasmid replication in pMGBPV is supplied by pML2 (pMBl/pBR322 derivative; Lusky and Botchan, 1981) whereas in pCGBPV it is supplied by pHSG262, a ColEl derivative (Brady et al, in preparation;Hashimoto-Gotoh and Inselburg, 1979). The pMGBPV plasmids contain an ad-ditional antibiotic resistance gene (the 3-lactamase gene from pML2) and do not contain the cos site region of bacteriophage X.…”

Section: Discussionmentioning

confidence: 99%

A bovine papilloma virus vector with a dominant resistance marker replicates extrachromosomally in mouse and E. coli cells.

et al. 1983

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We describe the construction of a bovine papilloma virus‐based vector (pCGBPV9) which contains a dominant selectable marker and replicates autonomously in both mouse and Escherichia coli cells. This vector contains the complete bovine papilloma virus genome, a ColE1 replication origin and a dominant selectable marker conferring resistance to kanamycin in bacteria and G418 in eukaryotic cells. A high number of G418R colonies are obtained after transfer of pCGBPV9 into mouse C127 cells. These G418R colonies contain vector DNA which replicates autonomously at approximately 10‐30 copies per cell. The molecules are in most cases unrearranged and can be rescued into E. coli cells by bacterial transformation.

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“…Bacteria, plasmids, and plasmid derivations. The strains of E. coli which were used were P678-54 (thr leu thi rpsT Sm') (5,6) and Om84 [tyr(Am) trp(Am) thy his ilv supD] (5,6). Each strain contained appropriate plasmids which were introduced by standard DNA transformation methods (see below).…”

Section: Materils and Methodsmentioning

confidence: 99%

“…1) is a chimera formed by ligating the BamHI-cleaved pDMS6642 and pHSG1, which is a temperature-sensitive DNA replication mutant of pSC101 (5,7) that was previously used in constructing chimeras with ColEl plasmid derivatives (5). Such chimeras exhibit temperature-resistant replication so long as the ColEl component can replicate normally (3,5,6). Such chimeras have been shown to exhibit the copy number of the ColEl plasmid component.…”

Section: Materils and Methodsmentioning

confidence: 99%

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ColE1 copy number mutants

Schmidt¹,

Inselburg²

1982

J Bacteriol

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A deletion mutant of the colicin E1-derived plasmid, pDMS6642, exhibited an approximately fourfold increase in copy number. We subsequently isolated hydroxylamine-induced mutants of that plasmid that had a further increase in copy number. Analysis of them suggests that the increased copy number of pDMS6642 is associated with transcriptional readthrough from a Tn3 transposon into the region of ColE1 containing information that influences plasmid replication. The hydroxylamine mutation in one copy number mutant appeared to increase the plasmid copy number by stimulating readthrough transcription from the Tn3 transposon into the ColE1 replication control region, whereas the other hydroxylamine mutation acts by another mechanism.

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ColE1 plasmid incompatibility: localization and analysis of mutations affecting incompatibility

Cited by 35 publications

References 25 publications

Homology between Escherichia coli plasmids ColE1 and p15A

Homology between Escherichia coli plasmids ColE1 and p15A

A bovine papilloma virus vector with a dominant resistance marker replicates extrachromosomally in mouse and E. coli cells.

ColE1 copy number mutants

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