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Previous studies have indicated that essential amino acids must be supplied for the initiation of deoxyribonucleic acid (DNA) synthesis by amino acid auxotrophs (5, 7, 11). Analogues of required amino acids can be substituted in a number of instances (3). Thus, the methionine analogues, norleucine and ethionine, and the tryptophan analogue, 7-azatryptophan, allow reinitiation of DNA replication in E. coli 15Tafter starvation for methionine or tryptophan. These analogues, as well as 4-methyltryptophan, are incorporated into protein in place of their respective amino acid counterparts (1, 2, 12, 14). It is surprising, however, that 5-methyltryptophan (5-MT) also permits DNA replication after starvation for tryptophan (3). This suggests that 5-MT may be incorporated into protein despite previous reports to the contrary (9, 10). An indication that it might be incorporated was reported in studies on polynucleotide phosphorylase (6, 15). In the experiments below, I shall describe the incorporation of radioactive 5-MT into a hot trichloroacetic acid-insoluble fraction of the cell, presumably protein. A similar observation has been reported by D. H. Ezekiel and N. Carlson (Bacteriol. Proc., p. 116, 1967). All of the experiments were carried out with the quadruple auxotroph E. coli 15T-, met-, trp-, arg-. The growth of this organism in thymine and in amino acid-supplemented M9 medium has been described in detail (5, 13). Estimation of radioactivity by scintillation spectrometry has also been described (13). DL-5-MT was tritiated by Schwartz Bio Research Inc., Orangeburg, N.J., by use of catalytic exchange. This material was purified in our laboratory by two passages through Sephadex G-25 with 0.01 M NH40H as the solvent. Four additional passages through columns of amberlite IRC50 eluting with 0.3 M ammonium acetate (pH 5.28) at 50 C (4, 8) resulted in a product which appeared to be pure upon two-dimensional chromatography in tert-butyl alcohol-methyl ethyl ketone-water (4:4:2) and tert-butylalcoholmethanol-water (4:5:1). The specific activity of the product was 1.2 c/mmole. When cells of 15Twere incubated with this material, it was incorporated into the hot trichloroacetic acid-insoluble fraction (Table 1). The incorporation was eliminated by the addition of minute amounts of tryptophan, but was only moderately reduced by addition of nonradioactive 5-MT. Incorporation of L-tryptophan was not concentration-dependent above 0.5 ,g/ml, whereas incorporation of DL-5-MT decreased with concentrations below 6 ,g/ml. The incorporated material was not soluble in alcohol or ether. Pronase digestion followed by column chromatography was used to identify the incorporated radioactivity. Two preparations were examined: (i) a culture which had incorporated 3H-5-MT, and (ii) a culture which had been allowed to incorporate '4C-tryptophan for 40 min and then had been transferred, by collection and washing on a membrane filter (13), to medium in which 3H-5-MT was substituted for tryptophan. The labeled cells were harvested and washed by membrane filtration; then they were precipitated and washed twice with 10% trichloroacetic acid, washed twice with alcohol, and washed twice with ether. The dried material was dissolved in 1 ml of 0.05 M NH4HCO3 (pH 7.45), and Pronase was added to a concentration of 0.5 mg/ml. After 15 hr of incubation at 37 C, the material was chromatographed on a column (20 by 2 cm) of Sephadex G-25 eluting with 0.01 M NH40H. Fractions (1.5 ml) werecollected, andradioactivity could be measured in fractions 32 to 46. Pronase emerged with the solvent front in fractions 13 to 18. The radioactive fractions were pooled, dried, and chromatographed at 50 C on columns (24 by 1.2 cm) of amberlite IRC50 (4) eluting with 0.3 M ammonium acetate, pH 5.28. (The samples were dissolved in a volume of 2 ml of 0.01 M acetic acid and were immediately added to the amberlite columns; ammonium acetate buffer was used to wash the sample into the column.) Suitable nonradioactive standards of tryptophan or 5-MT were added to the samples for identification purposes. Figures 1A, 1B, and 2 present the results. Some channeling of the columns occurred; this may have been responsible for the observation that the peaks are asymmetric and emerge at somewhat different effluent volumes in different

Incorporation of 5-methyltryptophan into the protein of Escherichia coli 15T- (555-7)