Reprinted from Vrrotoay, Volume 12, No. 4, December 1960 Copyright © 1960 by Academic Press Inc. Printed in U.S.A. Comparison of the Tryptic Peptides of Chemically Induced and Spontaneous Mutants of Tobacco Mosaic Virus The genetic information of tobacco mosaic virus (TMV) resides in the sequence of the bases within its ribonucleic acid (RNA). The infecting TMV-RNA induces the cell to produce new virus particles consisting of RNA and a specifie protein. There must therefore exist a structural re- lation between the RNA and the corresponding protein. One way to study this relation is to change the sequence of the RNA bases and to study the effect on the corresponding protein. Treatment of TMV-RNA with nitrous acid converts certain bases into others, e.g., uracil to cytosin (1); a change of one nucleotide is sufficient to produce a mutation (2). By variation of the experimental conditions it is possible to vary the statistically defined average number of nucleo- tide changes within the RNA. Investigation of the protein structure of these chemically induced mutants promises to give information on the coding problem between nucleic acids and the corresponding proteins. The results of a comparison of the tryptic peptides of 26 chemically 610 DISCUSSION AND PRELIMINARY REPORTS induced and spontaneous TMV mutants will be described here. Part of this work has been presented before the National Academy of Sciences, Washington, D. C. (3). Comparisons of the amino acid composition (4) and of the composition of the tryptic peptides (5) of the normal TMV strain and a mutant induced by nitrous acid treatment have been pub- lished. To solve the coding problem, it is essential to have a method capable of detecting with certainty a change in only one out of the 157 amino acids making up the TMV protein. Therefore the purified TMV was split into RNA and protein and (besides determining the amino acid composition of the virus protein in an automatic amino acid analyzer) the protein was digested with trypsin; the resulting 12 tryptic peptides were isolated and purified by column and paper chromatography and paper electrophoresis. Their amino acid composition was analyzed in amino acid analyzers according to Spackman et al. (6). The methods were essentially the same as those described previously in detail (4, 7). Figure 1 gives a scheme showing derivation of the mutants whose tryptic peptides have been studied. Those mutants designated by “Ni” were produced by nitrous acid treatment; all others were spontaneous mutants. The isolation of the spontaneous mutants A 7 to E83 was done in such a way as to reduce to a minimum the probability of getting more than one mutation step (8). This principle was not stressed as much in isolating the spontaneous mutants designated by Latin names [flarwm @ b c qd e i Z k { m A | vulgore | o Ss ? Yu v w x y 2 PIR IE IEE eee] Fig. 1. Scheme of the TMV mutants studied. The differences in the amino acid composition of the tryptie peptides are given in the text. DISCUSSION AND PRELIMINARY REPORTS G11 (flav.), necans, (nec.), rertrescens (rev.), and reflareseens (refl.)], some of which were isolated many yeurs ago (9). The amino acid composition of the latter mutants has been analyzed before, without splitting the pro- tein into tryptie peptides (74). When ¢ is the time of treatment and 7 the time after which the in- fectivity decreases to Lc of the value before treatment [sce (2)], then for both NiXX. band NiXX 8,0 = d.sfor Ni 11, Ni 18, and Ni ay T t = 5.2 [see (1/)|; for Ni 54/45 and Ni 44/20, t 2.0; for Ni 54 °20.34, ; T = 6.5; and for Ni 101 to Ni 118, —_ 3.0. The 6 mutants Ni 101 to Ni T 118 and the 2 Ni XX/... were produced by treatment of the virus nucleoprotein with nitrous acid whereas all other Ni mutants were isoluted after nitrous acid treatment of the TMV-RNA. The mutants flavum, Ni XX/8, Ni 54/20/34, and FE 83 are yellow strains which can- not be differentiated by symptoms on “Samsun tobaceo”’. The analysis of the tryptic peptides of the 26 chemically induced and spontaneous mutants listed, gives the following results (cf. Fig. 1; pep- tides numbered as in (7)]: Steps ace, doe, fg. su, vow, ye no change Steps b, hyo. a: Asp — Ala in peptide NIT! Steps i,j: Asp 2 Gly in NOL Step k: Glu > Gly in VII Step 1: Asp > Gly in IX Step im: Ser > Phe in IV; Glu -» Valin NIT Step ns Pro > Leu in NIL step p: Ala + Phe > Val + Leu in NIT; Ser > Phe in IV Step q: Val + Leu > Asp + Phe in NT Step r: Leu > Phe in NOT Step t: eu — Thr in NI Step x: Asp > Lys in IV The results confirm the earlier finding (5) that there exist mutants which have the same amino aeid composition of their tryptic peptides al- though they show very striking differences in symptoms; this is true for both spontaneous and chemically induced mutants. This ean be ex- plained by assuming that the region within the TMV-RNA which codes for the 157 amino acids of the virus protein ix smaller than the 6500 nucleotides making up the TMV-RNA. Only when nucleotides within this region are changed docs an alteration of amino acids in the viral protein oecur, whereas changes of nucleotides outside this region result 1 Peptide No. NUL Ovith 41 amino acids) contains 4 Asp [see (7)]. ASP means aspartic acid or asparagine. Ghi means glutamic acid or glutamine. 612 DISCUSSION AND PRELIMINARY REPORTS in alterations that lead to different symptoms without changing the viral protein, By investigation of more nitrous acid mutants than have been studied hitherto, the number of nucleotides within the “gene” for the viral protein could be determined and the question answered how many nucleotides code for one amino acid. The elucidation of the total sequence of the 157 amino acids of the TMV protein (72) makes it possi- ble to pinpoint the alteration of a certain amino acid at a certain posi- tion within the protein chain. This opens up the possibility of seeing whether the points of amino acid replacement. are distributed randomly over the protein chain or whether there exist regions with a high fre- quency of replacement, and also of answering the question whether a certain amino acid can be substituted only by a limited number of others; this information would give additional help in resolving the code. ACKNOWLEDGMENT The technical assistance of Miss Ingrid Hindennach, Miss Brigitte Ostertag, and Miss Julita Huf is gratefully acknowledged. REFERENCES 7. Scuuster, H., and ScuramM, G., Z. Naturforsch. 13b, 697-704 (1958). 2. Gierer, A., and Munpry, KK. W., Vefure 182, 1457-1458 (1958). 3. Wirrmann, H. G., Symposiian on Genetic Determination of Protein Structure, Ann. Meeting Natl. Acad. Sei. Washington April, 1960. 4. Tstaira, A., and FRAENKEL-Conrat, H., Proce. Natl. Acad. Sct. U. S., 46, 636-642 (1960). §. Wittmann, H.G., 2. Vererbungslehre 90, 468-475 (1959). 6. Spackman, DD. H., Srerx, W. H., and Moors, 8., Anal. Chem. 30, 1130-1206 (1958). 7. WitrmMann, H. G., and BRAUNITZER, G., Virology 9, 726-728 (1959). 8. Me.cuers, G., unpublished data. 9. Meccousrs, G., Vaturwissenschaften 36, 48-49 (1942). 10, Aacu, H.G.. Z. Naturforsch, 18b, 425-433 (1958). it. Munpry, K.W., and Gierer, A., Z. Vererbungslehre 89, 614-4030 (1958). 12. ANbeRER, A., Utttic, H., Weper, IX., and Scuraw, G.. Nature 186, 922-925 (1960). Max-Planck-Institut fiir Biologie H. G. WittMaNnn Abt. Melchers Tiibingen, Germany Reeeived October 21, 1960