Comparison of the Tryptic Peptides of Chemically induced and
       Spontaneous Mutants of Tobacco Mosaic Virus
The genetic inform&ion of t'obacco mosaic virus (TRW) resides in the
sequence of the bases within its ribonucleic acid (RXA). The infect'ing
TMV-RKA induces t,he cell t#o produce new virus particles conskking of
RNA and a specific prot,ein. There must therefore exist a struct,ural re-
lnt,iou between the RKA and the corresponding protein. One way to
study this relation is to change the sequence of the R,NA bases and to
study the effect on the corresponding protein.
Treatment, of T?tZV-RXA with nitrous acid convert,s certain bases into
ot,hers, e.g., uracil to cytosin (1); a change of one nucleotide is sufficient
t,o produce a mut,at,ion (2). Ry variation of t*he experimental conditions
it is possible to vary the st,atistically defined average number of nucleo-
tide changes wit'hin the RiYA. Investigation of the protein structure of
t,hese chemically induced mutants promises to give information on the
coding problem bet'ween nucleic acids and t)he corresponding proteins.
The results of a comparison of the trypbic peptides of 26 chemically


610          I)lS(`lXSIOS .iSI) PKELIMISARY IZEPOltTS

induced and spontaucous TMV mutants will be described hew. Part of
this work has been presented before t,he National Academy of Scithnces,
Washington, I). C. (3). Comparisons of the amino acid compoaitioll (,$)
and of the composition of the tryptic pcptides (5) of the normal T.\I\'
st)rain alld a mutant induced by nitrous acid t,restment 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 1.5i amino
acids making up the TJIV protein. Therefore the purified TA\I\- \\-as
split into It?;:2 and protein and (besides detjermining the amino acid
composition of t,he virus protein in an automatic amino acid nnalyzc~)
the protein was digest,ed with trypsin; the resulting 12 tryptic peptidrs
ivcre isolated and purified by column and paper chromatography and
paper clectrophorwis. Their amino acid composition was analyzed in
amino acid analyzers according to Spackman d al. (6). The met hods ww
rsselltially the same as those described previously in detail (a, 7).

Figure 1 gives a scheme showing derivation of the mutants whose
tryptic pcpt,ides have been studied. Those mlltnnts designated 1)~ "Si"
were produced by uitrous acid treatment,; all others were spontaneotw
mutants. The isolation of the spontaneous mrltunts ;Z 7 to E 83 was done
in such a way as to reduce to a minimum the probability of getting mow
than one mutation sttlp (8). This principle was not stressed as much ill
isolating the spontaneous mutants designated by Latin names [flarlttnl

Ni 101 Ni IO3 Ni 109 N, 11.5  Ni 117 Ml18

A7 AU   83   ES..      E66  E77   EC93



in alterations that lead to different symptoms without changing the
\rir:il protein. Uy investigation of more nitrous acid mutants than have
been studied hitherto, the number of nucleotides within the "gene" for
the viral protein could he determined and the questSion answered how
many n~~cleotides code for one amino acid. The elucidation of the total
sec~uel~w of the 137 amino acids of the TXIV protein (12) makes it' possi-
hle to pillpoint the alteration of a certain amino acid at a certain posi-
tion \vithin the protein chaill. This opens up the possibility of seeing
whether the points of amino Cd replacement are distributed randomly
over the protein chain or \vhrther there exist regions with a high fre-
q~wncy of replaccmrnt, and :dso of answering the (luest'ion whether a
certain amino acid can bc sutx&tuted only by a limit,ed number of others;
this informnt~ion would give additional help m resolving the code.

                ,4CI~?;OWI,EI~~;~~l:NT
`l'hr trrhnical :mrist:lrwe of Miss Ingrid Hindennwh, Rliss Brigittr Ostertng,
:LINI Miss .Jrilit:t Hrlf is gr:~irfrdIy :wkrmwledgrd.

                    I~I~FI~:ItI~:SCI~:S
I. SC*HI`STER, H., :urtl S(~HR,AMM, (i.. %. .VnI~~r~orsch. 13b, M7-704 (1058).
2. (iIORER. A., anti bII.SI)RY, I<. W., .Ycrtrrrr 182, 145im1458 (1958).
3. wITTnr.4sN, II. t;., S!/rrlpf'"i/ct,/ f,,L Getzelic f)Ptf,r~~trirtntior OJ f'twlrin sl/~/cctt/rP,
  :ltzt,. ;Iffwtirr{/ .\-tell. .lcd. Sri. Il~ashir/~/~o/r .Ipril, 2960.
4. TSI-GITA, A., :III~ I'R,~Es~~I.-(~~osR.~T. H., I'wr. .\`a//. :lmtl. Sri. I:. S., 46,
   (iXk(i42 (lR(i0 j
5. WITT~IASS, H. C:., %. I.c,~~,,h,rrr!/s/c~~~(, 90, l(i3~475 (1959).
(1. SPA~K~IAS, I). H.. STEIS, W. H., anti ~~IIORE, S., .Ind. (`hrur. 30, 11~~0~12O~i
   (1058).
7. WITT.VANN, H. C;., :md HRAI~NIWER. Ct., T7im/q~/ 9, 721iki28 ilM!)j.
8. MICI.(YIKHS, C;., urll)llt)lishwl d:~i:~.
9. MICI.~~II~:HS. C;., .\`~~/~/1~11~I'ss~n,srhn!`~etz36,48-4!~ (1042).
10. AAUI, H. (;.. Z. .Vcrlurj'om(.h. 13b, 425m4:13 (1!)58).
11. MI.XI)KY, I<. W., :m(l (;IERER, A., %. I'e~rrh,ln!/a(~hr~ 89, (il-!Ni80 (1$)5X).
12. .kNI),CRER, A.. ~:III.I(:. H., WER~K, II:., ant1 ~(~llRA\l\l, (:.. .~rclur~c 186, 022.-925
   (l!NiO).

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