The site-specific deletion in plasmid pBR322

Garaev, M M; Bobkov, Aleksei; Bobkova, A. F.; Калинин, В. Н.; Smirnov, V D; Khudakov, Yu.E.; Tikchonenko, T.I.

doi:10.1016/0378-1119(82)90052-x

Cited by 16 publications

(9 citation statements)

References 13 publications

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“…The rejoining of complementary DNA ends in E. coli has been studied by transforming cultures with linearized plasmid DNA (10)(11)(12)(13). The most common rejoin product in transformed bacteria results from the faithful ligation of the DNA ends to restore the original plasmid.…”

Section: Introductionmentioning

confidence: 99%

“…The most common rejoin product in transformed bacteria results from the faithful ligation of the DNA ends to restore the original plasmid. End modifications, typically deletions, have been observed in several studies (10,13). Several systems have been developed to study the rejoining of complementary and noncomplementary restriction enzymeproduced DNA double-strand breaks in vertebrate cells.…”

Section: Introductionmentioning

confidence: 99%

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Noncomplementary DNA double-strand-break rejoining in bacterial and human cells

et al. 1993

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We examined the rejoining of noncomplementary restriction enzyme-produced DNA double-strand breaks in Escherichia coli and in cultured human cells. The enzymes used in this study, ClaI, BamHI and SalI, produce double-strand breaks with 5 protruding single strands. The joining of a ClaI-produced DNA end to a BamHI-produced end or to a SalI-produced end was examined at the DNA sequence level. End rejoining in E.coli was studied by transforming cultures with linear plasmid DNA that was gel purified from restriction digests, and end rejoining in cultured human cells was studied by introducing enzymes into the cells by electroporation. The human cells used contain an Epstein-Barr virus (EBV)-based shuttle vector, pHAZE, that was recovered and introduced into E.coli for further analysis. The major products of DNA end-joining processes observed in linear plasmid-transformed E.coli and in the human cells exposed to restriction enzymes were identical. Furthermore, the deletions observed in both systems and in the spontaneous mutant plasmids in untreated human cells had a common underlying feature: short stretches of directly repeated DNA at the junction sites.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Noncomplementary DNA double-strand-break rejoining in bacterial and human cells

et al. 1993

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“…Recently, it was reported that spontaneous deletions are easily isolated from pBR322 carrying cloned foreign DNA fragments (12,16). The other mechanism of deletion formation in Tn3411 is that deletion occurs at short repeated sequences in both recA+ and recAstrain backgrounds (1,7).…”

Section: Poh34 E' Orimentioning

confidence: 99%

“…The selective medium used for citrate utilization was Simmons citrate agar (Eiken) supplemented with appropriate nutritional requirements (15). The concentrations of antibiotics used were as follows: ampicillin, 50 ,ug/ml; chloramphenicol, 25 ,ug/ml; kanamycin, 25 ,ug/ml; rifampin, 50 ,ug/ml; streptomycin, 12.5 ,ug/ml for plasmid determinants and 500 p.g/ml for selecting chromosomal determinants of JC1557 or JC1569 in the mating experiments; sulfonamide, 800 ,ug/ml; tetracycline, 12 Rifr mutant of strain C600 thi thr leu lac SGll recA mutant of strain SG8 483 A(proAB-lac) rpsL nalA galTNIJ6::IS1 20 433 del mutant of strain 483 20 a Genotype symbols are the same as those used by Bachmann et al (2).…”

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confidence: 99%

Spontaneous deletion of citrate-utilizing ability promoted by insertion sequences

Ishiguro¹,

Sato²

1984

J Bacteriol

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The citrate utilization (Cit+) transposon Tn3411 was shown to be flanked by directly repeated sequences (IS3411L and IS3411R) by restriction enzyme analysis and electron microscope observation. Citdeletion mutants were frequently found to be generated in pBR322::Tn3411 by intramolecular recombination between the two copies of IS3411. The flanking IS3411 elements of Tn3411 were shown to be functional insertion sequences by Tn3411-mediated direct and inverse transposition. Tn3411-mediated inverse transposition from pBR322::Tn3411 to the F-plasmid derivative pED100 occurred more efficiently than that of direct transposition of the Cit+ determinant. This was thought to be due to the differential transposability of IS3411L and IS3411R in the transposition process. The frequency of transposition of IS3411 marked with a chloramphenicol resistance determinant was much higher than IS3411-mediated cointegrate formation, suggesting that replicon fusions are not essential intermediates in the transposition process of Tn3411 or IS3411. Spontaneous deletions occurred with high frequency in recA hosts. The spontaneous deletion promoted by homologous recombination between two IS3411 elements in Tn3411 was examined with deletion mutants.

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“…Here we describe a rapid gene-fusion method that 4 1.1, and 2 kb in size, have been successfully joined to yield a 3.5-kb large fusion product in 2 working days. 6 This DNA construct has been successfully used for disrupting the pyruvate dehydrogenase (pdh) E 1 b subunit 7 fragment fusion template for a second PCR with the two outside primers, 1 and 4 ( Fig.…”

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confidence: 99%