Summary
We report the construction of recombinant plasmids containing the dihydrofolate reductase structural gene (fol) from several trimethoprim-resistant mutants of Escherichia coli. Strains carrying some of these plasmids produced approximately 6% of their soluble cell protein as dihydrofolate reductase and are therefore excellent sources of the purified enzyme for inhibitor binding or mechanistic studies. The nucleotide sequence of the fol region from each of the plasmids was determined. A plasmid derived from a Ki mutant which produced a dihydrofolate reductase with lowered affinity for trimethoprim contained a mutation in the structural gene that altered the sequence of the polypeptide in a conserved region which is adjacent to the dihydrofolate binding site. Two other independently-isolated mutants which overproduced dihydrofolate reductase had a mutation in the-35 region of the fol promoter. One of them, strain RS35, was also temperature-sensitve for growth in minimal medium. This phenotype was shown to be the result of an additional mutation in a locus unlinked to fol by P1 transduction. The fol regions from two temperature-independent revertants of strain RS35 were sequenced. One of these had a mutation within the dihydrofolate reductase structural gene which altered some properties of the enzyme. This confirmed some previous enzymological data which suggested that some revertants of strain RS35 had mutations in fol (Sheldon 1977). These results suggest that dihydrofolate reductase interacts physically with some other essential gene product in E. coli.
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References
Alt FW, Kellems RE, Bertino JR, Schimke RT (1978) Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. J Biol Chem 253:1357–1370
Amyes SGB, Smith JT (1978) R-factor mediated dihydrofolate reductases which confer trimethoprim resistance. J Gen Microbiol 107:263–271
Barnes W (1977) Plasmid detection and sizing in single colony lysates. Science 195:393–394
Bertino J, Johns D (1972) Folate antagonists. In: Bradsky I (ed) Cancer chemotherapy, vol 2. Grune and Stratton, New York, pp 9–22
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Breeze AS, Simms P, Stacey KA (1975) Trimethoprim-resistant mutants of E coli K12: preliminary genetic mapping. Genet Res 25:207–214
Calvo JM, Fink GR (1971) Regulation of biosynthetic pathways in bacteria and fungi. Annu Rev Biochem 40:943–968
Chang SE, Littlefield JW (1976) Elevated dihydrofolate reductase messenger RNA levels in methotrexate resistant BHK cells. Cell 7:391–396
Ferone R, Roland S (1980) Dihydrofolate reductase: thymidylate synthase, a bifunctional polypeptide from Crithidia fasciculata. Proc Natl Acad Sci 77:5802–5806
Friedkin M (1973) Thymidylate synthetase. Adv Enzymol 38:235–292
Freisheim JH, Bitar KG, Reddy, AV, Blankenship DT (1978) Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei. J Biol Chem 253:6437–6444
Gronenborn AM, Davies RW (1981) DNA binding by dihydrofolate reductase from Lactobacillus casei. J Biol Chem 256:12152–12155
Matthews DA, Alden RA, Bolin JT, Freer ST, Hamlin R, Yuong N, Kraut J, Poe M, Williams M, Hoogsteen K (1977) Dihydrofolate reductase: X-ray structure of the binary complex with methotrexate. Science 197:452–455
Melera PW, Lewis JA, Biedler JL, Hession C (1980) Antifolateresistant chinese hamster cells. J Biol Chem 255:7024–7028
Miller JH (1972) In: Experiments in molecular genetics. Cold Spring Harbor Press, New York
Nunberg JH, Kaufman RJ, Schimke RT, Urlaub G, Chasin LA (1978) Amplified dihydrofolate reductase genes are localized to a homogeneously staining region of a single chromosome in a methotrexate-resistant Chinese Hamster ovary cell line. Proc Natl Acad Sci USA 75:5553–5556
Osborn MJ, Huennekens FM (1958) Enzymatic reduction of dihydrofolic acid. J Biol Chem 233:969–974
Reddy GPV, Pardee AB (1980) Multienzyme complex for metabolic channeling in mammalian DNA replication. Proc Natl Acad Sci USA 77:3312–3316
Rosenberg M, Court D (1979) Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet 13:319–353
Rosner J (1972) Formation, induction, and curing of bacteriophage P1 lysogens. Virology 48:679–684
Sheldon R (1977) Altered dihydrofolate reductase in fol regulatory mutants of Escherichia coli K-12. Mol Gen Genet 151:215–219
Sheldon R, Brenner S (1976) Regulatory mutants of dihydrofolate reductase in Escherichia coli K-12. Mol Gen Genet 147:91–97
Sirotnak FM (1970) Increased dihydrofolate reductase synthesis in Diplococcus pneumoniae following translatable alteration of the structural gene. III. Further evidence on the extent of genic involvement. Genetics 65:391–406
Sirotnak FM, McCuen RW (1973) Hyperproduction of dihydrofolate reductase in Diplococcus pneumoniae after mutation in the structural gene. Evidence for an effect at the level of transcription. Genetics 74:543–556
Smith DR, Calvo JM (1979) Regulation of dihydrofolate reductase synthesis in Escherichia coli. Mol Gen Genet 175:31–38
Smith DR, Calvo JM (1980) Nucleotide sequence of the E. coli gene coding for dihydrofolate reductase. Nucl Acids Res 8:2255–2274
Smith DR, Rood JI, Bird PI, Sneddon MK, Calvo JM, Morrison JF (1982) Amplification and modification of dihydrofolate reductase in Escherichia coli: nucleotide sequence of fol genes from mutationally altered plasmids. J Biol Chem (in press)
Tyler-Smith C, Bostock CJ (1981) Gene amplification in methotrexate-resistant mouse cells. III. Interrelationships between chromosome changes and DNA sequence amplification or loss. J Mol Biol 153:237–256
Werkheiser WC (1961) Specific binding of 4-amino folic acid analogues by folic acid reductase. J Biol Chem 236:888–893
Wahba AJ, Friedkin M (1961) Direct spectroscopic evidence for the oxidation of tetrahydrofolate during the enzymatic synthesis of thymidylate. J Biol Chem 236:PC11-PC12
Williams MN, Poe M, Greenfield NJ, Hirshfield JM, Hoogsteen K (1973) Methotrexate binding to dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli. J Biol Chem 248:6375–6379
Wu R, Jay E, Roychoudhury R (1976) Nucleotide sequence analysis of DNA. In: Busch H (ed) Methods in cancer research, vol 12. Academic Press, New York, pp 87–176
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Smith, D.R., Calvo, J.M. Nucleotide sequence of dihydrofolate reductase genes from trimethoprim-resistant mutants of Escherichia coli . Molec. Gen. Genet. 187, 72–78 (1982). https://doi.org/10.1007/BF00384386
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DOI: https://doi.org/10.1007/BF00384386