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PMID: 6340111 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Characterization of the Escherichia coli thyA gene and its amplified thymidylate synthetase product.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 80 ·No. 7 ·1983-04-00 ·Pages 1858-61

Belfort M, Maley GF, Maley F

Abstract

The 7.8-kilobase HindIII insert in phage lambda NM589thyA [Borck, K., Beggs, J.D., Brammar, W.J., Hopkins, A.S. & Murray, N. (1976) Mol. Gen. Genet. 146, 199] was confirmed as originating from Escherichia coli by hybridization analysis and was shown to encode the thymidylate synthetase (5,-10-methylenetetrahydrofolate:dUMP C-methyltransferase EC 2.1.1.45) of E. coli K-12 by using biochemical, structural, and immunologic criteria. The 7.8-kilobase insert was reduced in size to a quasi-random population of DNA subfragments by partial digestion with the 4-base-pair recognition enzymes Alu I and Hae III. A clone containing a 1.1- to 1.2-kilobase fragment that encompassed the gene was obtained from this mixture by selecting for Thy+ recombinants. Fusion of this DNA fragment to the phage lambda rho L promoter in plasmid pKC30 revealed the direction of transcription of the thyA gene, and, in a phage lambda lysogen containing a thermolabile repressor, intracellular synthetase levels were increased about 700-fold. The enzyme was purified to homogeneity from this source by affinity chromatography, and some of its properties are described.

MeSH Terms
Cloning, Molecular Escherichia coli/genetics Gene Amplification Gene Expression Regulation Genes Genetic Linkage Methyltransferases/genetics Thymidylate Synthase/genetics,immunology
Chemicals
Methyltransferases Thymidylate Synthase
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Belfort M
Maley G F
Maley F
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27 references, click to expand
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.80.7.1858
Published
1983-04-00
Pages
1858-61
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC393709
Subset
IM
Grants
NIGMS NIH HHS · GM26387 · United States
NIGMS NIH HHS · GM26645 · United States
External Links
PubMed source DOI: 10.1073/pnas.80.7.1858 PMC Full Text View journal details
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