Lambdoid phages that simplify the recovery of in vitro recombinants

Murray, Noreen E.; Brammar, William J.; Murray, Kenneth

doi:10.1007/bf02425325

Cited by 771 publications

(229 citation statements)

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“…For experiments comparing temperature-sensitive and wildtype int gene products, crude cell extracts of strains HN496 and HN497 were made by digestion with lysozyme at 0WC as described (2) except that the cells were grown at 29°C in broth containing ampicillin (Sigma) at 25 ,tg/ml. Strains HN496 and HN497 are derivatives of ED8654 (12); each carries a hybrid plasmid (constructed in this laboratory) in which the DNA between the lone EcoRI and HindIII restriction endonuclease sites of pBR322 (13) has been replaced by the 4.2-kilobase-pair HindIII-EcoRI fragment of X that includes the int gene. The X fragment in the plasmid of strain HN496 carries the int ts2004 (14) and intC226 (15) alleles, while that in strain HN497 carries the wild-type structural int gene and the intC226 promotor; the fragments are otherwise identical.…”

mentioning

confidence: 99%

Nicking-closing activity associated with bacteriophage lambda int gene product.

Kikuchi¹,

Nash²

1979

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

165

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Integrative recombination of bacteriophage X requires the action of the protein Int, the product of the phage int gene. In this paper we show that highly purified Int relaxes supercoiled DNA. The association of this nicking-closing activity with Int is shown by: (i) the cosedimentation o nicking-closing and recombination activities of purified Int, (ii) the parallel inactivation of the two activities in purified Int by both heat and a specific antiserum, and (iii) the alteration of both activities in crude extracts of a strain expressing a mutant int gene. The nicking-closing activity of Int functions in the absence of divalent cations and in the absence of an apparent source of chemical energy. The activity displays no obvious sequence specificity and is inhibited by Mg +, spermidine, and singlestranded DNA. Int relaxes positive as well as negative supercoils. We present a model for the mechanism of strand exchange that describes how the nicking-closing activity of Int might be used during recombination. Bacteriophage X forms lysogens of Escherichia coli by integrating viral DNA into the bacterial chromosome (reviewed in ref. 1). The integration occurs as a result of a reciprocal recombination between specific genetic loci, the viral and bacterial attachment sites (attP and attB, respectively). Integrative recombination readily takes place in a cell-free system (2), and the in vitro reaction has been used to characterize the overall pathway of this recombination. Briefly, it has been shown that DNA supercoiling is essential for recombination: at least attP must be located on a negatively supertwisted circle of DNA (3,4). Given an appropriately supertwisted substrate, the only low molecular weight substances required for full recombination activity are spermidine, KC1, and buffer. The products of integrative recombination are a reciprocal pair of continuous double-helical DNAs. This fact and the simplicity of the cofactor requirements suggest that recombination occurs by a very conservative mechanism-i.e., one that involves-no degradation or resynthesis of DNA and one that retains the bond energy of the phosphodiester backbone during breakage and reunion.In vitro integrative recombination has been used to assay the purification of the proteins involved in this reaction. We have previously reported the purification to near homogeneity of the X int gene product, the only virus-coded component of the integration system (5, 6). We showed that, in addition to supertwisted substrate DNA and the cofactors described above, purified int gene product (Int) requires the participation of protein(s) encoded by the bacterial host in order to carry out integrative recombination. In the absence of this host component and spermidine, highly purified Int binds to DNA, forming specific stable complexes with DNA containing attP. Thus, Int is a specificity element for integrative recombination, recognizing at least one of the two recombining sites. We now report that, in the absence of the other components of the recombinati...

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mentioning

confidence: 99%

Nicking-closing activity associated with bacteriophage lambda int gene product.

Kikuchi¹,

Nash²

1979

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

165

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“…Media and general methods have been described (Murray et al, 1977). P1 transductions were carried out according to Miller (1992) and conjugation experiments followed the procedures described by .…”

Section: Microbial Methodsmentioning

confidence: 99%

ClpX and ClpP are essential for the efficient acquisition of genes specifying type IA and IB restriction systems

Makovets

Titheradge

Murray

1998

Molecular Microbiology

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SummaryEfficient acquisition of genes that encode a restriction and modification (R-M) system with specificities different from any already present in the recipient bacterium requires the sequential production of the new modification enzyme followed by the restriction activity in order that the chromosome of the recipient bacterium is protected against attack by the restriction endonuclease. We show that ClpX and ClpP, the components of ClpXP protease, are necessary for the efficient transmission of the genes encoding EcoKI and EcoAI, representatives of two families of type I R-M systems, thus implicating ClpXP in the modulation of restriction activity. Loss of ClpX imposed a bigger barrier than loss of ClpP, consistent with a dual role for ClpX, possibly as a chaperone and as a component of the ClpXP protease. Transmission of genes specifying EcoKI was more dependent on ClpX and ClpP than transmission of the genes for EcoAI. Sensitivity to absence of the protease was also influenced by the mode of gene transfer; conjugative transfer and transformation were more dependent on ClpXP than transduction. In the absence of either ClpX or ClpP transfer of the EcoKI genes by P1-mediated transduction was impaired, transfer of the EcoAI genes was not.

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“…A complete digest EcoRI library from PER-255 DNA was prepared in X607 (Murray et al, 1977) and screened with the Co probe M 131BlOBB 1 (Sims et al, 1984). Two types of clone were found, corresponding to the two rearranged TCR43 alleles, and subcloned into the plasmid vector pUC (Vieira and Messing, 1982).…”

Section: Methodsmentioning

confidence: 99%