In a previous paper, we showed that tobacco mosaic virus (TMV) produced by TMV-infected leaf consists of rods of various lengths.1 In long-infected leaf, rods 3,000 A + 300 A long predominate and represent the maximum natural length of the virus, longer rods resulting from end-to-end polymerization during purification of the virus. However, rods shorter than the maximum length (i.e., those less than 2,700 A long) are also characteristic products of the biosynthetic process induced by inoculation with TMV. It becomes of interest, therefore, to determine the relationship between this heterogeneity in the product of TMV biosynthesis and the mechanism of TMV biosynthesis. The importance of such a study is emphasized by our earlier experiments on C14 incorporation into TMV protein and RNA, which suggest that the virus rod is synthesized by a process of linear extension.2 In the present paper, we examine the relative rates at which rods of maximum length (MI) and short rods (S) appear in extracts of TMV-infected leaf during the course of the infection and some of the characteristic properties of the short rods.Methods.-For studies on the rates of synthesis of TMV rods, infected tissue was prepared as follows: A group of 20 mature plants of N. tabacum (variety White Burley) grown concurrently in the greenhouse under conditions of maximal nitrogen supply was removed from the greenhouse and maintained under constant temperature (240C) and constant illumination. At the start of the experiment, 5-6 mature leaves on each plant were inoculated with 300 ,ug/ml of purified TMV by rubbing, using carborundum powder and a glass rod. One hr later, the leaves were washed. At daily intervals, samples of leaves, equivalent with respect to size and location on the plant, were removed from the plants. Leaf blade tissue was cut out, each sample yielding about 50 grams wet weight. The blade tissue was frozen in liquid nitrogen and stored frozen.In certain experiments, several plants were inoculated as above and harvested simultaneously at 24 hr after inoculation. In these instances, special procedures, which are described below, were required to isolate the very small quantity of TMV present at this stage of the infection process.TMV was isolated from the frozen leaf by grinding in an equal volume of pH 0.05 M phosphate buffer, in a blendor. The homogenate was then clarified by filtration through cheesecloth and the virus isolated from the extract by means of a 3-stage process (one isoelectric precipitation and 2 ultracentrifugations, with intervening clarification by means of low-speed centrifugation). All operations were performed in the cold. Each of the purified TMV preparations achieved in this way was analyzed as follows: Absorption spectra were obtained with a recording UV spectrophotometer. Folin protein determinations were carried out by a method which has been described elsewhere.2 Specific infectivity was determined on leaves of N. glutinosa and Pinto bean by methods also described earlier.2 Finally, electron micrographs of ind...

This paper considers whether the ribonucleoprotein rods that are the distinctive product of the tobacco mosaic virus (TMV) infection process are inherently variable in length. The problem is of interest because it influences conclusions regarding the mechanisms of TMV biosynthesis and replication.In previous studies of the incorporation of C14 into TMV in the infected tobacco leaf we have shown that the virus protein and RNA components are synthesized concurrently from the leaf's pools of free amino acids and ribonucleotides.' This observation, which has now been confirmed in two other RNA-bearing viruses, poliovirus2 and fowl plague virus,3 mitigates against the possible occurrence of free RNA and protein precursors of TMV, and suggests instead that incomplete or alternative forms of the virus are more likely to consist of ribonucleoprotein. Since the virus' protein subunits and RNA fiber form coaxial structures in the TMV rod, any ribonucleoprotein alternative to the predominant 3,000 A rod is likely to take the form of a rod of some different length.The problem of TMV rod length has been the subject of considerable study, but there is little agreement as to the significance of the results. While it is now clear that rods longer than 3,000 A are preparative artefacts,4 5 there is wide disagreement regarding the origin of the rods shorter than 3,000 A that are always observed in TMV preparations. Two recent reviews of this question reach opposite conclusions. Pirie6 supports the view that short rods are natural products of TMV bio-

1. Exhaustive fractionation of leaves from tobacco plants systemically infected with TMV has led to the isolation of two non-virus proteins, B3 and B6, and the detection of a third, A4, which do not occur in comparable uninfected plants. 2. Components B3 and B6 have been found consistently in a series of ten extracts from plants grown over an 18 month period in all seasons of the year. It is concluded that these components are as characteristic of the infected plant as TMV itself. 3. As they occur in the initial extracts, the non-virus proteins are of low molecular weight (S20 ca. 3). On treatment, each component tends to form a high molecular weight polymer with an electrophoretic mobility considerably greater than that of the starting material. The high molecular weight derivatives of A4, B3, and B6 have been designated A8, B8, and B7 respectively. There is no evidence that these high molecular weight components occur as such in the infected leaf. 4. The non-virus proteins are free of nucleic acid and are not infectious. They cross-react immunochemically with TMV. 5. Compared with TMV content, the amounts of the non-virus proteins found in infected leaf are relatively small, falling in the range of 10 to 150 micrograms per gm. of tissue.

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