Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow Print PDF (183K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (6)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Dykxhoorn, D. M.
Right arrow Articles by Linn, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dykxhoorn, D. M.
Right arrow Articles by Linn, T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, Vol 25, Issue 21 4209-4218, Copyright © 1997 by Oxford University Press

ARTICLES

An efficient protocol for linker scanning mutagenesis: analysis of the translational regulation of an Escherichia coli RNA polymerase subunit gene

DM Dykxhoorn, R St Pierre, O Van Ham and T Linn
Department of Microbiology and Immunology, Faculty of Medicine, University of Western Ontario, London, Ontario N6A 5C1, Canada.

A protocol has been developed that is capable of saturating regions hundreds of basepairs in length with linker scanning mutations. The efficacy of this method stems from the design of the linker scanning mutagenesis (LSM) cassette which is composed of a selectable marker flanked by two oligonucleotides, each of which contains a recognition site for a different restriction endonuclease. The cleavage site for one endonuclease is within its recognition site, while the second endonuclease cleaves in the target DNA beyond the end of the cassette. Digestion with these endonucleases and subsequent ligation results in the replacement of 12 bp of the original target sequence with 12 bp of the linker scanning oligonucleotide. We have used this protocol to mutagenize a span of approximately 400 bp surrounding the start site of the gene for the beta subunit (rpoB) of Escherichia coli RNA polymerase. The translation of the beta mRNA has been shown previously to be regulated by the intracellular concentration of either beta or beta'. Analysis of the linker scanning mutations indicates that sequences extending a considerable distance both upstream and downstream of the start site are required for normal translation. Also a site that appears to be involved in translational repression by excess beta' has been identified.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Protein Eng Des SelHome page
T. Seitz, B. Berger, V. T. Nguyen, C. Tricot, V. Villeret, S. Schmid, V. Stalon, and D. Haas
Linker insertion mutagenesis based on IS21 transposition: isolation of an AMP-insensitive variant of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa
Protein Eng. Des. Sel., May 1, 2000; 13(5): 329 - 337.
[Abstract] [Full Text] [PDF]


Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.