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PMID: 2825136 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Rapid sequencing of cloned DNA using a transposon for bidirectional priming: sequence of the Escherichia coli K-12 avtA gene.

Nucleic acids research ·Vol. 15 ·No. 22 ·1987-11-25 ·Pages 9461-9

Liu L, Whalen W, Das A, Berg CM

Abstract

A new approach to determining the sequence of cloned DNA is described. Unique regions near each end of the transposable element gamma-delta provide a pair of "portable" primer-specific sites for bidirectional sequencing by the dideoxy chain termination method. A set of gamma-delta insertions positioned about 200 bp apart over the entire cloned DNA allowed us to determine the sequence of both strands in a single parental plasmid without subcloning. The avtA (alanine-valine transaminase) gene of E. coli K-12 was sequenced by this approach. Surprisingly, gamma-delta insertions downstream of the coding region were found to significantly reduce avtA expression. We suggest that these nondisruptive insertions probably change the DNA topology and thereby alter gene expression.

MeSH Terms
Amino Acid Sequence Base Sequence DNA/isolation & purification DNA Transposable Elements Escherichia coli/enzymology,genetics Gene Expression Regulation Genes Genes, Bacterial Molecular Sequence Data Promoter Regions, Genetic Transaminases/genetics
Chemicals
DNA Transposable Elements DNA Transaminases valine-pyruvate transaminase
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Liu L
Department of Molecular and Cell Biology, University of Connecticut, Storrs 06268.
Whalen W
Das A
Berg C M
References (21)
21 references, click to expand
  1. The gamma delta sequence of F is an insertion sequence.
    J Mol Biol. 1978 Dec 15;126(3):347-65 PMID: 745233
  2. DNA sequencing with chain-terminating inhibitors.
    Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463-7 PMID: 271968
  3. Transposition of the Escherichia coli insertion element gamma generates a five-base-pair repeat.
    Proc Natl Acad Sci U S A. 1979 Oct;76(10):4882-6 PMID: 388421
  4. Analysis of an avtA::Mu d1(Ap lac) mutant: metabolic role of transaminase C.
    J Bacteriol. 1982 May;150(2):739-46 PMID: 7040341
  5. Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes.
    Cell. 1982 Nov;31(1):43-51 PMID: 6297752
  6. Role of alanine-valine transaminase in Salmonella typhimurium and analysis of an avtA::Tn5 mutant.
    J Bacteriol. 1983 Sep;155(3):1009-14 PMID: 6309735
  7. New M13 vectors for cloning.
    Methods Enzymol. 1983;101:20-78 PMID: 6310323
  8. Uses of the transposon gamma delta in the analysis of cloned genes.
    Methods Enzymol. 1983;101:362-9 PMID: 6310336
  9. Kilo-sequencing: creation of an ordered nest of asymmetric deletions across a large target sequence carried on phage M13.
    Methods Enzymol. 1983;101:98-122 PMID: 6310345
  10. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing.
    Gene. 1984 Jun;28(3):351-9 PMID: 6235151
  11. Topological repression of gene activity by a transposable element.
    Proc Natl Acad Sci U S A. 1984 Oct;81(19):6115-9 PMID: 6091136
  12. Biology of bacterial deoxyribonucleic acid topoisomerases.
    Microbiol Rev. 1984 Dec;48(4):273-89 PMID: 6097803
  13. Cloning and expression of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli.
    J Bacteriol. 1985 Mar;161(3):896-903 PMID: 2982793
  14. A rapid procedure for DNA sequencing using transposon-promoted deletions in Escherichia coli.
    Gene. 1985;39(2-3):305-10 PMID: 3005115
  15. Rapid and reliable dideoxy sequencing of double-stranded DNA.
    Gene. 1985;40(2-3):317-23 PMID: 3007296
  16. DNA sequence of the E. coli gyrB gene: application of a new sequencing strategy.
    Nucleic Acids Res. 1987 Jan 26;15(2):771-84 PMID: 3029692
  17. Analysis of E. coli promoter sequences.
    Nucleic Acids Res. 1987 Mar 11;15(5):2343-61 PMID: 3550697
  18. High frequency generalized transduction by miniMu plasmid phage.
    Genetics. 1987 Jun;116(2):201-6 PMID: 3301525
  19. Cloning and characterization of the Escherichia coli K-12 alanine-valine transaminase (avtA) gene.
    J Bacteriol. 1987 Sep;169(9):4228-34 PMID: 3040683
  20. Determinant of cistron specificity in bacterial ribosomes.
    Nature. 1975 Mar 6;254(5495):34-8 PMID: 803646
  21. Cloning of uvrA, lexC and ssb genes of Escherichia coli.
    Biochem Biophys Res Commun. 1979 Sep 12;90(1):123-9 PMID: 387033
Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/15.22.9461
Published
1987-11-25
Pages
9461-9
Language
English
Region
England
NLM ID
0411011
PMCID
PMC306480
Subset
IM
Grants
NIAID NIH HHS · AI-19919 · United States
NIGMS NIH HHS · GM-28946 · United States
Databases
GENBANK
Y00490
External Links
PubMed source DOI: 10.1093/nar/15.22.9461 PMC Full Text View journal details
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