Summary
The Salmonella typhimurium LT2 metF gene, encoding 5,10-methylenetetrahydrofolate reductase, has been cloned. Strains with multicopy plasmids carrying the metF gene overproduce the enzyme 44-fold. The nucleotide sequence of the metF gene was determined, and an open reading frame of 888 nucleotides was identified. The polypeptide deduced from the DNA sequence contains 296 amino acids and has a molecular weight of 33 135 daltons. Mung bean nuclease mapping experiments located the transcription start point and possible transcription termination region for the gene. There is a 25bp nucleotide sequence between the translation termination site and the possible transcription termination region. This region possesses a GC-rich sequence that could form a stable stem and loop structure once transcribed (ΔG=-9 kcal/mol), followed by an AT-rich sequence, both of which are characteristic of rho-independent transcription terminators. The nucleotide and deduced amino acid sequences of the S. typhimurium metF gene are compared with the corresponding sequences of the Escherichia coli metF gene. The nucleotide sequences show 85% homology. Most of the nucleotide differences found do not alter the amino acid sequences, which show 95% homology. The results also show that a change has occurred in the metF region of the S. typhimurium chromosome as compared to the E. coli chromosome.
Similar content being viewed by others
Abbreviations
- 5-methylTHF:
-
5-methyltetrahydrofolate
- 5, 10-methylene THF:
-
5,10-methylenetetrahydrofolate
- ():
-
designates plasmid-carrier state
- Tc:
-
tetracycline
- GM:
-
glucose minimal
- Ap:
-
ampicillin
- ORF:
-
open reading frame
References
Ayling PD, Chater KF (1968) The sequence of four structural and two regulatory methionine genes in the Salmonella typhimurium linkage map. Genet Res 12:341–354
Belfaiza J, Guillou Y, Margarita D, Perrin D, Saint-Girons I (1987) Operator-constitutive mutations of the Escherichia coli metF gene. J Bacteriol 169:670–674
Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, Falkow S (1977) Construction and characterization of new cloning vehicles, II. A multipurpose cloning system. Gene 2:95–113
Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156
Dawes J, Foster MA (1971) Vitamin B12 and methionine synthesis in Escherichia coli. Biochim Biophys Acta 237:455–464
Duchange N, Zakin MM, Ferrara P, Saint-Girons I, Park I, Tran SV, Psy M-C, Cohen GN (1983) Structure of the metJBLF cluster in Escherichia coli K12. J Biol Chem 258:14868–14871
Emmett RM, Johnson JR (1986) Control of metF gene expression in maxicell preparations of Escherichia coli K-12: reversible action of the metJ protein and effect of vitamin B12. J Bacteriol 168:1491–1494
Freier SM, Kierzek R, Jaeger JA, Sugimoto N, Caruthers MH, Neilson T, Turner DH (1986) Improved free-energy parameters for predictions of RNA duplex stability. Proc Natl Acad Sci USA 83:9373–9377
Greene RC, Williams RD, Kung H-F, Spears C, Weissbach H (1973) Effects of methionine and vitamin B12 on the activities of methionine biosynthetic enzymes in metJ mutants of Escherichia colio K12. Arch Biochem Biophys 158:249–256
Ikemura T (1985) Codon usage and tRNA content in unicellular and multi-cellular organisms. Mol Biol Evol 2:13–34
Kung H-F, Spears C, Greene RC, Weessbach H (1972) Regulation of the terminal reactions in methionine biosynthesis by vitamin B12 and methionine. Arch Biochem Biophys 150:23–31
Kutzbach C, Stokstad ELR (1971) Mammalian methylenetetrahydrofolate reductase: partial purification, properties, and inhibition by S-adenosylmethionine. Biochim Biophys Acta 250:459–477
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York
Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65:499–560
Milner L, Whitfield C, Weissbach H (1969) Effect of l-methionine and vitamin B12 on methionine biosynthesis in Escherichia coli. Arch Biochem Biophys 133:413–419
Mulligan JT, Margolin W, Krueger JH, Walker GC (1982) Mutations affecting regulation of methionine biosynthetic genes isolated by use of met-lac fusions. J Bacteriol 151:609–619
Rosenberg M, Court D (1979) Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet 13:319–353
Rowbury RJ (1983) Methionine biosynthesis and its regulation. In: Herrmann KM, Somerville RL (eds) Amino acids: biosynthesis and genetic regulation. Wesley, Reading, Mass, pp 191–211
Saint-Girons I, Duchange N, Zakin MM, Park I, Margarita D, Ferrara P, Cohen GN (1983) Nucleotide sequence of metF, the E. coli structural gene for 5–10 methylene tetrahydrofolate reductase and of its control region. Nucleic Acids Res 11:6723–6732
Sanger F, Coulson AR (1978) The use of thin acrylamide gels for DNA sequencing. FEBS Lett 87:107–110
Shine J, Dalgarno L (1975) Determinant of cistron specificity in bacterial ribosomes. Nature 254:34–38
Shoeman R, Redfield B, Coleman T, Greene RC, Smith AA, Brot N, Weissbach H (1985) Regulation of methionine synthesis in Escherichia coli: Effect of metJ gene product and S-adenosylmethionine on the expression of the metF gene. Proc Natl Acad Sci USA 82:3601–3605
Sollner-Webb B, Reeder RH (1979) The nucleotide sequence of the initiation and termination sites for ribosomal RNA transcription in X. laevis. Cell 18:485–499
Stauffer GV, Brenchley JE (1977) Influence of methionine biosynthesis on serine transhydroxymethylase regulation in Salmonella typhimurium LT2. J Bacteriol 129:740–749
Stauffer GV, Plamann MD, Stauffer LT (1981) Construction and expression of hybrid plasmids containing the Escherichia coli gly A gene. Gene 14:63–72
Thèze J, Margarita D, Cohen GN, Borne F, Patte JC (1974) Mapping of the structural genes of the three aspartokinases and of the two homoserine dehydrogenases of Escherichia coli K-12. J Bacteriol 117:133–143
Urbanowski ML, Stauffer GV (1985) Cloning and initial characterization of the metJ and metB genes from Salmonella typhimurium LT2. Gene 35:187–197
Urbanowski ML, Plamann LS, Stauffer GV (1987) Mutations affecting the regulation of the metB gene of Salmonella typhimurium LT2. J Bacteriol 169:126–130
Weaver RF, Weissmann C (1979) Mapping of RNA by a modification of the Berk-Sharp procedure: the 5′ termini of 15S β-globin mRNA precursor and mature 10S β-globin mRNA have identical map coordinates. Nucleic Acids Res 7:1175–1193
Author information
Authors and Affiliations
Additional information
Communicated by D. Goldfarb
Rights and permissions
About this article
Cite this article
Stauffer, G.V., Stauffer, L.T. Cloning and nucleotide sequence of the Salmonella typhimurium LT2 metF gene and its homology with the corresponding sequence of Escherichia coli . Mol Gen Genet 212, 246–251 (1988). https://doi.org/10.1007/BF00334692
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00334692