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. 1986 Jul;167(1):205–209. doi: 10.1128/jb.167.1.205-209.1986

Cloning and expression in Escherichia coli of the gene for 10-formyltetrahydrofolate synthetase from Clostridium acidiurici ("Clostridium acidi-urici").

T R Whitehead, J C Rabinowitz
PMCID: PMC212862  PMID: 3013834

Abstract

The gene for 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) from the purinolytic anaerobic bacterium Clostridium acidiurici ("Clostridium acidi-urici") was cloned into Escherichia coli JM83 with plasmid pUC8. A C. acidiurici genomic library was prepared in E. coli from a partial Sau3A digest and screened with antibody against the synthetase. Of 10 antibody-positive clones, 1 expressed a high level of synthetase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis demonstrated that the protein synthesized in E. coli had the same subunit molecular weight as the C. acidiurici enzyme. The gene was located on an 8.3-kilobase genomic insert and appeared to be transcribed from its own promoter. Analysis of genomic digests with a fragment of the synthetase gene indicated that one copy of the gene was present in the C. acidiurici chromosome.

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Selected References

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  1. Alvarado-Urbina G., Sathe G. M., Liu W. C., Gillen M. F., Duck P. D., Bender R., Ogilvie K. K. Automated synthesis of gene fragments. Science. 1981 Oct 16;214(4518):270–274. doi: 10.1126/science.6169150. [DOI] [PubMed] [Google Scholar]
  2. Appling D. R., Rabinowitz J. C. Evidence for overlapping active sites in a multifunctional enzyme: immunochemical and chemical modification studies on C1-tetrahydrofolate synthase from Saccharomyces cerevisiae. Biochemistry. 1985 Jul 2;24(14):3540–3547. doi: 10.1021/bi00335a023. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Caperelli C. A., Benkovic P. A., Chettur G., Benkovic S. J. Purification of a complex catalyzing folate cofactor synthesis and transformylation in de novo purine biosynthesis. J Biol Chem. 1980 Mar 10;255(5):1885–1890. [PubMed] [Google Scholar]
  5. Clarke L., Carbon J. A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome. Cell. 1976 Sep;9(1):91–99. doi: 10.1016/0092-8674(76)90055-6. [DOI] [PubMed] [Google Scholar]
  6. Delk A. S., Nagle D. P., Jr, Rabinowitz J. C. Methylenetetrahydrofolate-dependent biosynthesis of ribothymidine in transfer RNA of Streptococcus faecalis. Evidence for reduction of the 1-carbon unit by FADH2. J Biol Chem. 1980 May 25;255(10):4387–4390. [PubMed] [Google Scholar]
  7. Dev I. K., Harvey R. J. A complex of N5,N10-methylenetetrahydrofolate dehydrogenase and N5,N10-methenyltetrahydrofolate cyclohydrolase in Escherichia coli. Purification, subunit structure, and allosteric inhibition by N10-formyltetrahydrofolate. J Biol Chem. 1978 Jun 25;253(12):4245–4253. [PubMed] [Google Scholar]
  8. Graves M. C., Mullenbach G. T., Rabinowitz J. C. Cloning and nucleotide sequence determination of the Clostridium pasteurianum ferredoxin gene. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1653–1657. doi: 10.1073/pnas.82.6.1653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Helfman D. M., Feramisco J. R., Fiddes J. C., Thomas G. P., Hughes S. H. Identification of clones that encode chicken tropomyosin by direct immunological screening of a cDNA expression library. Proc Natl Acad Sci U S A. 1983 Jan;80(1):31–35. doi: 10.1073/pnas.80.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Himes R. H., Harmony J. A. Formyltetrahydrofolate synthetase. CRC Crit Rev Biochem. 1973 Sep;1(4):501–535. doi: 10.3109/10409237309105441. [DOI] [PubMed] [Google Scholar]
  11. Jones E. W. Bipartite structure of the ade3 locus of Saccharomyces cerevisiae. Genetics. 1977 Feb;85(2):209–223. doi: 10.1093/genetics/85.2.209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Ljungdahl L., Brewer J. M., Neece S. H., Fairwell T. Purification, stability, and composition of formyltetrahydrofolate synthetase from Clostridium thermoaceticum. J Biol Chem. 1970 Sep 25;245(18):4791–4797. [PubMed] [Google Scholar]
  14. MacKenzie R. E., Rabinowitz J. C. Cation-dependent reassociation of subunits of N10-formyltetrahydrofolate synthetase from Clostridium acidi-urici and Clostridium cylindrosporum. J Biol Chem. 1971 Jun 10;246(11):3731–3736. [PubMed] [Google Scholar]
  15. McGuire J. J., Rabinowitz J. C. Studies on the mechanism of formyltetrahydrofolate synthetase. The Peptococcus aerogenes enzyme. J Biol Chem. 1978 Feb 25;253(4):1079–1085. [PubMed] [Google Scholar]
  16. O'Brien W. E., Brewer J. M., Ljungdahl L. G. Purification and characterization of thermostable 5,10-methylenetetrahydrofolate dehydrogenase from Clostridium thermoaceticum. J Biol Chem. 1973 Jan 25;248(2):403–408. [PubMed] [Google Scholar]
  17. Paukert J. L., Straus L. D., Rabinowitz J. C. Formyl-methyl-methylenetetrahydrofolate synthetase-(combined). An ovine protein with multiple catalytic activities. J Biol Chem. 1976 Aug 25;251(16):5104–5111. [PubMed] [Google Scholar]
  18. Paukert J. L., Williams G. R., Rabinowitz J. C. Formyl-methenyl-methylenetetrahydrofolate synthetase (combined); correlation of enzymic activities with limited proteolytic degradation of the protein from yeast. Biochem Biophys Res Commun. 1977 Jul 11;77(1):147–154. doi: 10.1016/s0006-291x(77)80176-9. [DOI] [PubMed] [Google Scholar]
  19. RABINOWITZ J. C., PRICER W. E., Jr Formyltetrahydrofolate synthetase. I. Isolation and crystallization of the enzyme. J Biol Chem. 1962 Sep;237:2898–2902. [PubMed] [Google Scholar]
  20. Schirch L. Formyl-methenyl-methylenetetrahydrofolate synthetase from rabbit liver (combined). Evidence for a single site in the conversion of 5,10-methylenetetrahydrofolate to 10-formyltetrahydrofolate. Arch Biochem Biophys. 1978 Aug;189(2):283–290. doi: 10.1016/0003-9861(78)90214-x. [DOI] [PubMed] [Google Scholar]
  21. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  22. Staben C., Rabinowitz J. C. Immunological crossreactivity of eukaryotic C1-tetrahydrofolate synthase and prokaryotic 10-formyltetrahydrofolate synthetase. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6799–6803. doi: 10.1073/pnas.80.22.6799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Staben C., Rabinowitz J. C. Nucleotide sequence of the Saccharomyces cerevisiae ADE3 gene encoding C1-tetrahydrofolate synthase. J Biol Chem. 1986 Apr 5;261(10):4629–4637. [PubMed] [Google Scholar]
  24. Tan L. U., Drury E. J., MacKenzie R. E. Methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase. A multifunctional protein from porcine liver. J Biol Chem. 1977 Feb 10;252(3):1117–1122. [PubMed] [Google Scholar]
  25. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  26. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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