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J Bacteriol. Feb 1989; 171(2): 1068–1074.
PMCID: PMC209703

A recB recC sbcB recJ host prevents recA-independent deletions in recombinant cosmid DNA propagated in Escherichia coli.

Abstract

Segments of DNA are deleted from recombinant cosmid DNAs with high frequency during propagation in standard recA Escherichia coli hosts. An attempt has been made to derive an appropriate strain of E. coli, suitable for cosmid cloning, in which such deletions do not occur. We examined the effects of a series of host recombinational mutations on the deletion process, using six independent recombinant cosmids that carry inserts of mouse, Chinese hamster, or human DNA. Various E. coli host cells carrying the recombinant cosmids were cultured serially in liquid medium, and the recombinant cosmid DNAs were extracted from the host cells and analyzed by agarose gel electrophoresis and by gene transfer of the DNAs into cultured mammalian cells. Of the mutations examined, only a recB recC sbcB recJ (or recN) quadruple combination of host mutations prevented the deletion of DNA segments. The recombinant cosmid DNAs propagated in E. coli hosts that carried this combination of mutations were functionally as well as structurally intact. We propose that the recJ (and/or recN) gene is involved in some aspect of the events that lead to deletions of cosmid DNA in a recB recC sbcB genetic background.

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  • Albertini AM, Hofer M, Calos MP, Miller JH. On the formation of spontaneous deletions: the importance of short sequence homologies in the generation of large deletions. Cell. 1982 Jun;29(2):319–328. [PubMed]
  • Birnboim HC. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 1983;100:243–255. [PubMed]
  • Boissy R, Astell CR. An Escherichia coli recBCsbcBrecF host permits the deletion-resistant propagation of plasmid clones containing the 5'-terminal palindrome of minute virus of mice. Gene. 1985;35(1-2):179–185. [PubMed]
  • Bolivar F, Backman K. Plasmids of Escherichia coli as cloning vectors. Methods Enzymol. 1979;68:245–267. [PubMed]
  • Boyer HW, Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. [PubMed]
  • Brutlag D, Fry K, Nelson T, Hung P. Synthesis of hybrid bacterial plasmids containing highly repeated satellite DNA. Cell. 1977 Mar;10(3):509–519. [PubMed]
  • Chang AC, Cohen SN. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. [PMC free article] [PubMed]
  • Chia W, Scott MR, Rigby PW. The construction of cosmid libraries of eukaryotic DNA using the Homer series of vectors. Nucleic Acids Res. 1982 Apr 24;10(8):2503–2520. [PMC free article] [PubMed]
  • Cohen A, Ram D. Deletions within E. coli plasmids carrying yeast rDNA. Gene. 1978 Apr;3(2):135–147. [PubMed]
  • Collins J. Instability of palindromic DNA in Escherichia coli. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):409–416. [PubMed]
  • Collins J, Volckaert G, Nevers P. Precise and nearly-precise excision of the symmetrical inverted repeats of Tn5; common features of recA-independent deletion events in Escherichia coli. Gene. 1982 Jul-Aug;19(1):139–146. [PubMed]
  • Dewitt SK, Adelberg EA. The Occurrence of a Genetic Transposition in a Strain of Escherichia Coli. Genetics. 1962 May;47(5):577–585. [PMC free article] [PubMed]
  • Graham FL, van der Eb AJ. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. [PubMed]
  • Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. [PubMed]
  • Holmes DS, Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. [PubMed]
  • Horii Z, Clark AJ. Genetic analysis of the recF pathway to genetic recombination in Escherichia coli K12: isolation and characterization of mutants. J Mol Biol. 1973 Oct 25;80(2):327–344. [PubMed]
  • Howard-Flanders P, Theriot L. Mutants of Escherichia coli K-12 defective in DNA repair and in genetic recombination. Genetics. 1966 Jun;53(6):1137–1150. [PMC free article] [PubMed]
  • Ishiura M, Hirose S, Uchida T, Hamada Y, Suzuki Y, Okada Y. Phage particle-mediated gene transfer to cultured mammalian cells. Mol Cell Biol. 1982 Jun;2(6):607–616. [PMC free article] [PubMed]
  • Jones IM, Primrose SB, Ehrlich SD. Recombination between short direct repeats in a recA host. Mol Gen Genet. 1982;188(3):486–489. [PubMed]
  • Kolodner R, Fishel RA, Howard M. Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli. J Bacteriol. 1985 Sep;163(3):1060–1066. [PMC free article] [PubMed]
  • Leach DR, Stahl FW. Viability of lambda phages carrying a perfect palindrome in the absence of recombination nucleases. Nature. 305(5933):448–451. [PubMed]
  • Lilley DM. In vivo consequences of plasmid topology. Nature. 1981 Jul 23;292(5821):380–382. [PubMed]
  • LITTLEFIELD JW. STUDIES ON THYMIDINE KINASE IN CULTURED MOUSE FIBROBLASTS. Biochim Biophys Acta. 1965 Jan 11;95:14–22. [PubMed]
  • Liu LS, Lark KG. The Red function of phage lambda mediates the alteration of an interspersed repeated DNA sequence from the kangaroo rat Dipodomys ordii. Mol Gen Genet. 1982;188(1):27–36. [PubMed]
  • Lloyd RG, Benson FE, Shurvinton CE. Effect of ruv mutations on recombination and DNA repair in Escherichia coli K12. Mol Gen Genet. 1984;194(1-2):303–309. [PubMed]
  • Lloyd RG, Buckman C. Identification and genetic analysis of sbcC mutations in commonly used recBC sbcB strains of Escherichia coli K-12. J Bacteriol. 1985 Nov;164(2):836–844. [PMC free article] [PubMed]
  • Lovett ST, Clark AJ. Genetic analysis of the recJ gene of Escherichia coli K-12. J Bacteriol. 1984 Jan;157(1):190–196. [PMC free article] [PubMed]
  • Lovett ST, Clark AJ. Cloning of the Escherichia coli recJ chromosomal region and identification of its encoded proteins. J Bacteriol. 1985 Apr;162(1):280–285. [PMC free article] [PubMed]
  • Morrison DA. Transformation and preservation of competent bacterial cells by freezing. Methods Enzymol. 1979;68:326–331. [PubMed]
  • Murray NE, Brammar WJ, Murray K. Lambdoid phages that simplify the recovery of in vitro recombinants. Mol Gen Genet. 1977 Jan 7;150(1):53–61. [PubMed]
  • Nader WF, Edlind TD, Huettermann A, Sauer HW. Cloning of Physarum actin sequences in an exonuclease-deficient bacterial host. Proc Natl Acad Sci U S A. 1985 May;82(9):2698–2702. [PMC free article] [PubMed]
  • Nakayama H, Nakayama K, Nakayama R, Irino N, Nakayama Y, Hanawalt PC. Isolation and genetic characterization of a thymineless death-resistant mutant of Escherichia coli K12: identification of a new mutation (recQ1) that blocks the RecF recombination pathway. Mol Gen Genet. 1984;195(3):474–480. [PubMed]
  • Picksley SM, Attfield PV, Lloyd RG. Repair of DNA double-strand breaks in Escherichia coli K12 requires a functional recN product. Mol Gen Genet. 1984;195(1-2):267–274. [PubMed]
  • Ponticelli AS, Schultz DW, Taylor AF, Smith GR. Chi-dependent DNA strand cleavage by RecBC enzyme. Cell. 1985 May;41(1):145–151. [PubMed]
  • Stanners CP, Eliceiri GL, Green H. Two types of ribosome in mouse-hamster hybrid cells. Nat New Biol. 1971 Mar 10;230(10):52–54. [PubMed]
  • Willetts NS, Clark AJ. Characteristics of some multiply recombination-deficient strains of Escherichia coli. J Bacteriol. 1969 Oct;100(1):231–239. [PMC free article] [PubMed]
  • Wyman AR, Wolfe LB, Botstein D. Propagation of some human DNA sequences in bacteriophage lambda vectors requires mutant Escherichia coli hosts. Proc Natl Acad Sci U S A. 1985 May;82(9):2880–2884. [PMC free article] [PubMed]
  • Yelton MM, Timberlake WE, Hondel CA. A cosmid for selecting genes by complementation in Aspergillus nidulans: Selection of the developmentally regulated yA locus. Proc Natl Acad Sci U S A. 1985 Feb;82(3):834–838. [PMC free article] [PubMed]

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