Skip Navigation

This Article
Right arrow Full Text (PDF)
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 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 Request Permissions
Google Scholar
Right arrow Articles by IWAKURA, M.
Right arrow Articles by TSUDA, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by IWAKURA, M.
Right arrow Articles by TSUDA, K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

J. Biochem, 1982, Vol. 91, No. 4 1205-1212
© 1982 Japanese Biochemical Society

research-article

Cloning of Dihydrofolate Reductase Gene of Escherichia coli K12

Masahiro IWAKURA, Yukio SHIMURA and Keishiro TSUDA

Research Institute for Polymers and Textiles Yatabe-Higashi, Tsukuba, Ibaraki 305

Resistant strains for trimethoprim, a potent inhibitor of dihydrofolate reductase, were obtained by transforming the ligated products ofEscherichia coli K12 DNA and plasmid pBR 322 BamH I fragments. The strains carry a 13.6 kbp plasmid, pTP 1, which contains the trimethoprim- and ampicillin-resistance determinant genes. The trimethoprim-resistance determinant gene was estimated to consist of more than 500 nucleotides and less than 1,500 nucleotides and was restricted by EcoR I and Sal I.

Trimethoprim-, ampicillin-, and tetracycline-resistant plasmids were made in the following way, and the resultant plasmids contained a unique EcoR I "insertional inactivation" site for trimethoprim resistance: the DNA sequences extraneous to the determinant gene of the trimethoprim resistance on BamH I fragment of pTP 1 were eliminated by digestion with a double-strand-specific exonuclease BAL 31, and the resultant fragments were ligated with pBR 322 which had been digested by EcoR I and a single-strand-specific nuclease S1.

The strains carrying pTP 1 or trimethoprim-resistant plasmids produced about 10 times more dihydrofolate reductase than control strains. The enhancement of the enzyme production, which is due to an increase in the copy number of the enzyme gene, seems to be responsible for the trimethoprim resistance of the transformed cells.


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
 J. Bacteriol.Home page
N. Vázquez-Laslop, H. Lee, R. Hu, and A. A. Neyfakh
Molecular Sieve Mechanism of Selective Release of Cytoplasmic Proteins by Osmotically Shocked Escherichia coli
J. Bacteriol., April 15, 2001; 183(8): 2399 - 2404.
[Abstract] [Full Text]

Home page
 Microbiol. Mol. Biol. Rev.Home page
M. K. B. Berlyn
Linkage Map of Escherichia coli K-12, Edition 10: The Traditional Map
Microbiol. Mol. Biol. Rev., September 1, 1998; 62(3): 814 - 984.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. Fujita, T. Koguma, J. Ohkawa, K. Mori, T. Kohda, H. Kise, S. Nishikawa, M. Iwakura, and K. Taira
Discrimination of a single base change in a ribozyme using the gene for dihydrofolate reductase as a selective marker in Escherichia coli
PNAS, January 21, 1997; 94(2): 391 - 396.
[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.