Mutagenesis by insertion of a drug-resistance element carrying an inverted repetition

Kleckner, Nancy; Chan, Russell K.; Tye, Bik Kwoon; Botstein, David

doi:10.1016/s0022-2836(75)80059-3

Cited by 319 publications

(159 citation statements)

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“…Transductions were performed with a nonlysogenizing, high frequency transducing mutant of phase P22 (11). A Tn10-carrying phage (P22-Tnl0) and its manipulations have been described (4). Bacterial and phage techniques have been described (12).…”

Section: Methodsmentioning

confidence: 99%

“…TnlO contains about 9.3 kb consisting of a 6.7-kb central core flanked by two inverted repeats of 1.3 kb (2,4).…”

mentioning

confidence: 99%

“…TnlO shares two features with most if not all Tn elements: (i) they can translocate in a recA -host, and (ii) they generate deletions with a high frequency (4,7). One end point of these deletions lies within or at the borders of the transposon, the other is frequently located outside (7).…”

mentioning

confidence: 99%

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A genetic approach to analysis of transposons.

Beck¹

1979

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

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Integration of the tetracycline resistance transposon TnlO into lacI of a lacI-lacZ gene fusion permits the isolation of deletions that excise DNA from one end of TnlO and fuse TnlO genes with lacZ in such a manner that chimeric proteins with fB-galactosidase activity are produced. The synthesis of the chimeric proteins is under the same control as the transoson genes. Thus, regulation of expression of TnlO genes can be investigated by measuring r-galactosidase activity.Analysis of TnlO-lacZ fusions revealed different deletion endpoints within TnlO; lacZ has been fused to at least three different TnlO genes or operons. Two of these genes are under the control of a tetracycline repressor. Genetic elements of defined nucleotide sequence that are able to translocate (reviewed in refs. 1 and 2) can be divided into two classes: (i) short ones termed insertion sequences (IS) that are smaller than about 2 kilobases (kb) and do not encode any known function, and (ii) longer ones termed transposons (Tn) that carry genes encoding resistance to antimicrobial agents (3).

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Section: Methodsmentioning

confidence: 99%

“…TnlO contains about 9.3 kb consisting of a 6.7-kb central core flanked by two inverted repeats of 1.3 kb (2,4).…”

mentioning

confidence: 99%

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A genetic approach to analysis of transposons.

Beck¹

1979

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

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“…. C'B'A' have been described in the leader sequences in both prokaryotes (Lee & Yanofsky, 1977) and animal viruses (Chow et al, 1977), at the ends of insertion elements (Ohtsubo & Ohtsubo, 1978) andin several transposable DNA segments carrying antibiotic resistance genes (Kleckner et al, 1975;Heffron et al, 1975). It is possible that many modes of recombination occur through self-complementary sequences or that they are involved in the regulation of transcription.…”

Section: Discussionmentioning

confidence: 99%

Intrastrand Self-complementary Sequences in Bacillus subtilis DNA

Galloway

Rudner

1979

Journal of General Microbiology

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Intrastrand self-complementary sequences have been isolated from the DNA of Bacillus subtilis by hydroxyapatite (HA) chromatography following thermal renaturation of strands separated by chromatography on methylated albumin-kieselguhr (MAK). The intrastrand structures derived from the MAK H strand (HA HII) were biologically active showing transforming activity for a wide variety of markers, as well as hybridization to both pulselabelled and ribosomal RNA. Removal of regions of single-strand DNA with S1 nuclease did not significantly alter the biological activity of the self-annealed molecules. The overall efficiency of transformation and hybridization of the intrastrand self-annealing DNA was low suggesting that many sequences in the population are neither active in transformation to prototrophy nor transcribed into RNA.

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“…F' factors, ampicillin resistance, and tetracycline resistance genetic elements carrying an IS can integrate, excise, or translocate independently of the function of recA gene (13)(14)(15)(16). It has also been proposed that palindromic sequences at promoter regions or at other specific sites of DNA are capable of forming a characteristic structure (palindromic loop), which promotes recombination (17)(18)(19).…”

Section: Discussionmentioning

confidence: 99%

Involvement of DNA-dependent RNA polymerase in a recA-independent pathway of genetic recombination in Escheria coli.

Ikeda¹,

Kobayashi²

1977

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

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Recombinant DNA molecules of phage X formed in Escherichia coli in the presence of chloramphenicol and/or rifampin can be assayed by their biological activity.recA -cells were found to be capable of forming recombinant A phage DNA in the presence of chloramphenicol. The relatively high recA-independent recombination observed in this system contrasts with the relatively low recA-independent recombination when recombinant phage particles rather than recombinant DNA are titrated. Formation of the recombinant DNA was suppressed by the addition of rifampin. The introduction of the rifr mutation into host bacteria made their recombination activity rifaimpin-resistant. These results show that DNA-dependent RNA polymerase (EC 2.7.7.6) is involved in this recAindependent pathway of recombination, which is named the "Rpo pathway." This is distinct from Red, Int, RecBC, RecE, or Der pathways of recombination. Crossover was much more frequent in the N-PL-cI and cI-PR-0 regions than in the A-D and O-S regions. The crossover seems to occur in the regions that are transcribed actively. Some local change of DNA structure caused by transcription might be required for the Rpo pathway of recombination. In order to understand the roles of Escherichia coli functions in genetic recombination, we have developed a system that has enabled us to study recombination separated from DNA replication, transcription, translation, and progeny formation (1). E. coli was mixedly infected with two mutants of A phage in the presence of thloramphenicol and rifampin, and the recombinant DNA formed within the cell was detected by in vitro packaging. In this. system, the recA gene product was required for the formation of recombinant DNA molecules, in agreement with studies on recA mutations by other investigators (2). During the course of this study, we encountered an unexpected observation that recA mutations did not block completely the formation of recombinant X DNA when mixed infection was carried out in the presence of chloramphenicol without addition of rifampin. This fact suggested that the recombinant DNA was formed in recA -cells by a rifampin-sensitive mechanism. In the present study, we show that RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6) is involved in this recA-independent pathway of recombination and that the transcription process per se plays an essential role in this recombination. MATERIALS AND METHODSBacterial and Phage Strains. The Escherichia coli K-12 and bacteriophage A strains used are listed in Tables 1 and 2, respectively.Media and Chemicals. Media, buffer, and chemicals used are generally those described previously (1). The final concentrations of chloramphenicol (Sankyo) and rifampin (B grade, Calbiochem) are 150 and 200 ,ug/ml, respectively, unless otherwise stated.Mixed Infection. The procedure was the same as that described previously (1). In brief, bacteria were incubated with inhibitor(s) (chloramphenicol only or chloramphenicol and rifampin) in A dilution buffer [10 mM MgSO4/10 mM Tris-HCl (pH 7.4...

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Mutagenesis by insertion of a drug-resistance element carrying an inverted repetition

Cited by 319 publications

References 36 publications

A genetic approach to analysis of transposons.

A genetic approach to analysis of transposons.

Intrastrand Self-complementary Sequences in Bacillus subtilis DNA

Involvement of DNA-dependent RNA polymerase in a recA-independent pathway of genetic recombination in Escheria coli.

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