DISCUSSION AND PRELIMINARY REPORTS G13 Comparison of the Tryptic Peptides of Wild Strains of Tobacco Mosaic Virus Mutants of tobacco mosaic virus (TMV) derived under experimental conditions from the normal TMV strain vulgare show only minute differ- ences, if any: one or only a few amino acids may be replaced by others. Besides these mutants a number of TMV-strains are known whose rela- tionship to vulgare is not clear because they were found in nature. These strains show many biological differences when compared with the normal TMY strain; it is therefore interesting to study what chemical differ- ences exist among them. One of these wild TMY strains is dahlemense, isolated by Melchers (1) from tomato plants about twenty years ago. In contrast to the mutants derived from vulgare, which all produce local lesions on Phaseolus vul- garis, dahlemense does not give lesions on this plant. It shows green sys- temic symptoms on “Samsun tobacco” and produces local lesions on “Java tobaececo’’. Its serological behavior is quite different from that of the vulgare strain or its mutants (2, 3). Figure 1 shows the electrophoretic mobility of whole virus and corresponding derived A protein of rulgare, +08 =e—t | +06|—— wonne | +04|-—=o Qe BIE +a2|-— 3S ae ly 5 a0 — 7 8 9 3 -ae\— -04\— al oe “06 ; + —_ : \ se teen g ~~ ---../] en F o AV \ “Ye ° AF Woe -g8|—— _ LL. a AD Bal a ee ----4 L-—— I : a 10 | a-~-b----4 -~--D Fic, 1. Electrophoretic mobility of vulgare (V). flavum (F), and dahlemense (D) and their corresponding A proteins (AV, AF, and AD) (ef. (9)). Reprinted from Virovocy, Volume 12, Ne. # December 1960 Copyright © 1960 by Academie Press Ine. Printed in CLS A. 614 DISCUSSION AND PRELIMINARY REPORTS flavum, and daklemense. From this it can be concluded that the number of acidic groups in dahlemense virus is higher than in rulgare and the munber of basic groups lower. The isoelectric point of pH 2.7 is lower than that of any other mutant or strain of TMV, The amino acid com- position has been previously studied by the DNP method (4), however, without splitting the protein into peptides. In order to determine the exact amino acid composition of dahlemense its tryptic peptides were studied. The virus was split into ribonucleic acid and protein, the protein was digested with trypsin, and the tryptic 15 T - Vulgare T o | _ Os j | eff ree 1 r 1 re “1 Q 30 60 30 120 150 180 210 240 270 300 Pia. 2. Elation curves of the tryptic peptides of ragare. Optical density after alkaline hydrolysis and ninhydrin reaction, plotted against number of fractions, 1S r Dahlemense | 10|---- a it bo 1 L a 30 60 90 120 1 1 Ld L 1 i 1 4. 4 4 4 150 780 270 240 270 300 Fig. 3. Elation curves of the tryptic peptides of dahlemense, Optical density after alkaline hydrolysis and ninhydrin reaction, plotted against number of frac- tions. DISCUSSION AND PRELIMINARY REPORTS 615 Thr Ser Ala Val Met Jleu Leu together with peptide No Phe T1121 275 120] 7 | | [9 | 37070 |19]19 [19 |79| 7 | 7 [7724 72 V=Vulgare, D=Dahlemense I ~ x ~ Fie. 4. Comparison of the tryptic peptides of eigare and dahlemense. peptides were separated by column chromatography; ligs. 2 and 3 show typical elution curves for radgare and daklemense. The peptides were purified if necessury by paper chromatography or high voltage elec- trophoresis and hydrolyzed, their amino acid composition was de- termined in an automatie amino acid analyzer. Details of the method have been deseribed previously (4, 6). Figure + shows the comparison of the tryptic peptides of vulgare and dahlemense. The composition of certain of the dahlemense peptides, e.g., No. EX with 7 amino acids, is very different from that of the correspond- ing rudgare peptides, whereas other peptides, e.g., VI with 10 amino acids, are exactly the same. It is interesting that daklemense peptide X con- tains an amino acid, namely methionine, which is not present in rulgare or in any of its many mutants, but only in other very distantly related TMY strains, e.g., Holmes’ ribgrass strain (7). In spite of the pronounced differences between the proteins of vulgare and dahlemense the number of amino acids within the protein chain of both strains is the same, namely 157. The number of peptides obtained from dahlemense is 10, compared with 12 from rulgare; this is because two arginines, at which amino acid trypsin splits the chain, are exchanged for two other amino acids, Dahle- mense peptide IL] with 20 amino acids corresponds to rulgare peptide TT] O16 DISCUSSION AND PRELIMINARY REPORTS with 15 plus peptide I with 5 amino acids. Similarly the C-terminal dahlemense peptide X with 24 amino acids (and no arginine and lysine) corresponds to the cxulgare peptide IV with 7 plus peptide X with 17 amino acids. By comparing the tryptic peptides of several TMV strains and by determining their N-terminal amino acid residues, it has been possible to elucidate the arrangement of most of the tryptic peptides within the protein chain of TMY (8). ACKNOWLEDGMENT The tecbnical assistance of Miss Ingrid Hindennach and Miss Brigitte Ostertag is gratefully acknowledged. REFERENCES 1. Mevcuers, G., Vaturwissenschaften 30, 48-49 (1942). 2, Friepricnu-Frexsa, H., Meccuers, G., and ScuramM. G., Biol. Zentr. 66, 187—- 222 (1946). 3. Aacnu, HG... 4. Naturforsch. 12b, 614-022 (1937). 4. Ascn, H.G.. Z. Naturforsch. 18b, 425-433 (1958). 5. Wrrrmann, H.G., and Braunirzer, G., Virology 9, 726-728 (1959). 6. Wirrmann, H. G., Z. Vererbungslehre 90, 463-475 (1959). y. Weranr, C. A. J. Biol. Chem. 171, 297-309 (1947). 8. Wirrmann, H.G., Virology 11, 505-508 (1960). 9. Kramer, [., and Wirrmann, H. G., Z. Naturforsch. 18b, 30-33 (1958). Max-Planck-Institut fiir Brologie H. G. WrtTMANN bt. Melchers Tiibingen, Germany Received October 21, £960.