DNA sequence of the<i>E. coli gyr</i>B gene: application of a new sequencing strategy

Adachi, Toshiro; Mizuuchi, Michiyo; Robinson, Elizabeth A.; Appella, Ettore; O’Dea, Mary H.; Gellert, Martin; Mizuuchi, Kiyoshi

doi:10.1093/nar/15.2.771

Cited by 159 publications

(72 citation statements)

References 33 publications

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“…Another utility of the Mu end primer would be the convenient sequencing of the reaction products. Not only is the location of the mutation deduced from the fragment size, but the nature of the mutations can be immediately identified by sequencing with a Mu end primer (14). These features will be especially useful for bulk detection and identification of singlenucleotide polymorphism.…”

Section: Discussionmentioning

confidence: 99%

Mismatch-targeted transposition of Mu: A new strategy to map genetic polymorphism

Yanagihara

Mizuuchi

2002

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

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Phage Mu DNA transposes to duplex target DNA sites with limited sequence specificity. Here we demonstrate that Mu transposition exhibits a strong target site preference for all single-nucleotide mismatches. This finding has implications for the mechanism of transposition and provides a powerful tool for genomic research. A single mismatch could be detected as a preferred target of Mu transposition in the presence of 300,000-fold excess of nonmismatched sites. We demonstrate the detection of both heterozygous and homozygous mutations in the cystic fibrosis transmembrane conductance regulator gene and single nucleotide polymorphism in HLA region by Mu transposition mismatch analysis procedure.

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Section: Discussionmentioning

confidence: 99%

Mismatch-targeted transposition of Mu: A new strategy to map genetic polymorphism

Yanagihara

Mizuuchi

2002

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

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“…For example, primer walking is not possible in such regions because the primer anneals to multiple locations within the template and generates a mixed sequence. Transposon-based sequencing strategies, on the other hand, use unique priming sites located near the termini of integrated transposons to recover DNA sequences (Ahmed 1985;Adachi et al 1987;Phadnis et al 1989;Strathmann et al 1991;Devine and Boeke 1994;Kimmel et al 1997; Fig. 3) and thus avoid this problem.…”

Section: Discussion Artificial Transposons For Sequencing Repetitive Dnamentioning

confidence: 99%

A Transposon-Based Strategy for Sequencing Repetitive DNA in Eukaryotic Genomes

Devine¹,

Chissoe²,

Eby³

et al. 1997

Genome Res.

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Repetitive DNA is a significant component of eukaryotic genomes. We have developed a strategy to efficiently and accurately sequence repetitive DNA in the nematode Caenorhabditis elegans using integrated artificial transposons and automated fluorescent sequencing. Mapping and assembly tools represent important components of this strategy and facilitate sequence assembly in complex regions. We have applied the strategy to several cosmid assembly gaps resulting from repetitive DNA and have accurately recovered the sequences of these regions. Analysis of these regions revealed six novel transposon-like repetitive elements,

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“…The gyrA and g r B genes from E. coli have been cloned, sequenced (Yamagishi et al, 1986;Adachi e t al., 1987;Swanberg & Wang, 1987), expressed (Hallet et al, 1990) and extensively characterized. Since then, there have been reports on the cloning of DNA gyrase genes from several bacterial species primarily to study the molecular basis of quinolone resistance.…”

Section: Introductionmentioning

confidence: 99%

Mycobacterium smegmatis DNA gyrase: cloning and overexpression in Escherichia coli

Madhusudan

Nagaraja²

1995

Microbiology

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The cloning and characterization of DNA gyrase genes from Mycobacterium smegmatis is described. The DNA sequence of 5119 bp encoding both g y B and gyrA genes was determined. The gene gyrB precedes gyrA with a short intergenic region of 29 nucleotides. The proteins encoded, GyrB and GyrA, exhibit 45-80 O/ O identity to gyrase polypeptides from other bacteria. The genes were further engineered for overexpression in Escherichia coli. Both genes were individually cloned into a phage T7 expression system and overexpressed. The expressed GyrB and GyrA proteins had molecular masses of 75 and 95 kDa, respectively, in agreement with that calculated from the ORFs.The extracts from the overexpressing clones were fractionated to enrich the subunits and assayed for enzyme activity. While the individual extracts showed no detectable activity, the combined extract exhibited a strong DNA supercoiling activity. This activity was ATP-dependent and novobiocinsensitive. The identity of the genes was also confirmed by complementation analysis.

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DNA sequence of theE. coli gyrB gene: application of a new sequencing strategy

Cited by 159 publications

References 33 publications

Mismatch-targeted transposition of Mu: A new strategy to map genetic polymorphism

Mismatch-targeted transposition of Mu: A new strategy to map genetic polymorphism

A Transposon-Based Strategy for Sequencing Repetitive DNA in Eukaryotic Genomes

Mycobacterium smegmatis DNA gyrase: cloning and overexpression in Escherichia coli

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