Asymmetric segregation of the complementary sex-factor DNA strands during conjugation in Escherichia coli

Vapnek, Daniel; Rupp, W. Dean

doi:10.1016/0022-2836(70)90066-5

Cited by 87 publications

(43 citation statements)

References 27 publications

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“…This suggests that the substrate for oriT recombination is strand specific, a result that would be expected if a single, unique strand were transferred. This is has been found to be true for all conjugating plasmids that have been examined in sufficient detail (20,21), and it would therefore not be surprising if it were true for dependent, mobilizable plasmids as well.…”

Section: Resultsmentioning

confidence: 78%

“…5. If the plasmid DNA transferred during conjugation is linear and single stranded, as it is for the self-transmissible plasmids that have been examined in detail (20,21), then phage DNA replicative intermediates could resemble those generated during conjugation and could then be a substrate for the plasmid-encoded breaking-resealing system. One possibility, suggested originally by Gilbert and Dressier (7), is that single-stranded concatemers are generated during transfer by rolling-circle replication.…”

Section: Resultsmentioning

confidence: 99%

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Site-specific recombination at oriT of plasmid R1162 in the absence of conjugative transfer

Meyer

1989

J Bacteriol

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R1162 is efficiently comobilized during conjugative transfer of the self-transmissible plasmid R751. Bacteriophage M13 derivatives that contain two directly repeated copies of oriT, the site on R1162 DNA required in cis for mobilization, were constructed. Phage DNA molecules underwent recombination during infection of Escherichia coli, with the product retaining a single functional copy of oriT. Recombination was strand specific and depended on R1162 gene products involved in mobilization, but did not require the self-transmissible plasmid vector. Two genes were identified, one essential for recombination and the other affecting the frequency of recombination. Recombination of bacteriophage DNA could form the basis of a simple model for some of the events occurring during conjugation without the complexity of a true mating system. R1162 is a broad-host-range plasmid (essentially identical to RSF1010) that is efficiently cotransferred during conjugation by the self-transmissible plasmids of the IncP1 group (14). Transfer of R1162 requires a 38-base-pair (bp) site, oriT, on the plasmid as well as the products of several R1162 genes (2, 3). If two directly repeated copies of oriT are present on the plasmid, then after conjugation a large proportion of the plasmid molecules in recipient cells contain a single copy of oriT and have lost the DNA located between the two copies. The deletion is transfer dependent and presumably reflects nicks or cleavages at each oriT, transfer of one or both linear strands of the resulting molecule, and the rejoining of ends derived from each of the two oriT copies on the original plasmid.I show here that when two copies of oriT are cloned into derivatives of bacteriophage M13, oriT-specific recombination can occur in infected cells in the absence of conjugation. Recombination is dependent on the products of R1162 genes involved in transfer. Bacteriophage molecules can be designed so that the frequency of recombination between two copies of oriT can be easily scored. Recombination is therefore a simple and useful assay for some conjugationrelated processes because mating-pair formation and selection for exconjugants are not required. MATERIALS AND METHODSStrains and plasmids. The Escherichia coli K-12 strains used were JM103 [zA(lac pro) strA thi supE endA sbcB hsdR (F' traD36 proAB laclq lacZAM15)] (12), M182 (AlacIPOZY lacX24 galK galU strA) (4), and RV (lacX42 Sup-) (11), containing F' lac (F-42), from I. Molineux. Plasmids used contain segments of the naturally occurring plasmid R1162 (1; Fig. 1A). Plasmid pMS40 (14) consists of a Kmr DNA fragment from Tn9O3 linked to R1162 DNA located between 0.9 and 6.6 kbp of DNA. A portion of this plasmid was cloned into pBR322 to construct pMS63 (14). Construction of pUT206, pUT208, pUT209, and pUT257, pBR322 derivatives containing cloned fragments of R1162 DNA, has also been described (2). The segments of R1162 DNA cloned in these plasmids are shown in Fig. 1B. Plasmids pUT856, pUT857, and pUT858 contain a 12-bp oligonucleotide, 5'-CTAGCTAGCT...

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Section: Resultsmentioning

confidence: 78%

Section: Resultsmentioning

confidence: 99%

Site-specific recombination at oriT of plasmid R1162 in the absence of conjugative transfer

Meyer

1989

J Bacteriol

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“…Several workers have isolated F' factor DNA as supercoiled circular molecules from mated and unmated E. coli host cells by sedimentation through alkaline sucrose gradient and by dye-buoyant density methods (1,11,12). Extracts of strains KL5/7 and 7F-were examined by these two methods.…”

Section: Resultsmentioning

confidence: 99%

Characterization by Electron Microscopy of Fused F-Prime Factors in Escherichia coli

Palchaudhuri

Mazaitis

Maas

et al. 1972

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

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During previous attempts to find mutants in which incompatibility between two F-prime (F′) factors of E. coli is abolished, we found that two F′ factors harbored in the same cell occasionally recombined to form a single genetic unit. Thus, a fused F′ trp + arg + factor was obtained from a mating of a strain carrying recA F′ trp + with a strain carrying recA F′ arg + . In the present paper physical fusion of the two F′ factors, inferred from genetic studies, is confirmed by electron microscopy. F DNA was extracted, after detergent lysis, from four fused F′ strains: the parental F′ trp + and F′ arg + strains and three F + strains. This supercoiled DNA was separated on ethidium bromide-cesium chloride gradients and converted to the open circular from before electron microscopy. The contour lengths in μm of the various DNA preparations were: F′ trp + , 59.4; F′ arg + , 33.2; the four F′ trp + arg + factors, 38.5, 39.6, 63.2, and 73.5: the three F factors, 31.1, 31.1, and 31.0. Thus, different fused F′ factors formed from the same parental F′ factors vary in length, but are shorter than the sum of the parental F′ factors.

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“…If transforming DNA is present in the cell in a double-stranded form the effect of in vivo restriction on transformation is expected to be similar to that observed in transduction or conjugation, i.e. An alternative model could be uptake of single strands of donor DNA and subsequent conversion to double-stranded DNA, as in conjugation (Vapnek & Rupp, 1970). Such effects are indeed found whenever non-modified DNA is introduced into a restricting host by transformation (Tables 2, 3).…”

Section: Discussion (I) the Nature Of Transforming Dna After Uptake Bmentioning

confidence: 99%

Transformation inEscherichia coli: studies on the nature of donor DNA after uptake and integration

1980

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Chromosomal E. coli DNA appears to be sensitive towards in vivo DNA restriction when transformed to a restrictive E. coli recipient. It is therefore concluded that transforming chromosomal donor DNA is present in a double-stranded form immediately after uptake.Genetic analysis of E. coli transformants, obtained with UV-irradiated donor DNA under conditions that exclude photorepair, show, especially in a uvrB recipient, loss of donor DNA information compared with the situation where DNA was not subjected to UV-irradiation. Similar conclusions were arrived at after genetic analysis of transductants obtained with UV-irradiated particles of the generalized transducing phage P i . The processing in E. coli of DNA after P i transduction is thus similar to that of transforming DNA. The observations are discussed and a possible explanation based on single-stranded DNA integration is presented in detail.

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Asymmetric segregation of the complementary sex-factor DNA strands during conjugation in Escherichia coli

Cited by 87 publications

References 27 publications

Site-specific recombination at oriT of plasmid R1162 in the absence of conjugative transfer

Site-specific recombination at oriT of plasmid R1162 in the absence of conjugative transfer

Characterization by Electron Microscopy of Fused F-Prime Factors in Escherichia coli

Transformation inEscherichia coli: studies on the nature of donor DNA after uptake and integration

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