Size distribution and In vitro translation of the RNAs isolated from turnip yellow mosaic virus nucleoproteins

84, 153-161 (1978)

VIROLOGY

Size Distribution and in Vitro Translation of the RNAs Isolated Turnip Yellow Mosaic Virus Nucleoproteins T. J. V. HIGGINS,* *Diwsion

P. R. WHITFELD,*j

1 AND

from

R. E. F. MATTHEWS?

of Plant Industry, CSIRO, Canberra City, A.C.T. 2601, Australia, and tDepartment Biology, University ofduckland, Auckland, New Zealand

of Cell

Accepted August 22,1977 Purified preparations of TYMV contain a number of minor nucleoprotein components distinguishable on the basis of their density in CsCl gradients (R. E. F. Matthews, Virology 12, 521-539, 1960). When analysed by polyacrylamide-gel electrophoresis, the RNA from each of these various components was found to consist of a characteristic set of molecular-weight species. Full-size RNA (2 x 10” daltons) was present only in nucleoproteins B, and BZ. Nucleoproteins B,, Boo, and Beoo contained RNAs ranging in size from approx 1.3 to 0.28 x lo6 daltons. The 0.28 x 106-dalton RNA species was detectable in all nucleoprotein fractions, but was a significant proportion of the RNAs of B,,, and B,,. Translation of the RNA from each of the nucleoproteins in the wheat germ cell-free protein synthesizing system yielded essentially similar patterns of polypeptides which ranged in size from 5000 to 70,000 daltons. The major radioactive product migrated on SDS-polyacrylamide gels to the same position as TYMV coat protein. The data suggest that the 0.28 x 106-dalton RNA component is the cistron for coat protein, and that it and other RNAs associated with TYMV infection are encapsidated. INTRODUCTION

When purified preparations of turnip yellow mosaic virus (TYMV) are fractionated on cesium chloride density gradients, a series of minor nucleoprotein bands can be isolated in addition to the infectious virus and the empty protein shells (T component) (Matthews, 1960; Johnson, 1964; Derosier and Haselkorn, 1966). The infectious virus nucleoprotein (B1), containing 36% RNA, makes up about 70% of a virus preparation. The T component with 0% RNA makes up about 20%. The minor nucleoprotein fractions are made up as follows (in order of increasing enective buoyant density in CsCl gradients): B,,, with about 5% RNA and making up about 1% of the preparations; B,, with about 10% RNA, and B, with about 20% RNA, each making up about 3% of the preparation; and B, with 36% RNA making up about 4% of the preparation. None ’ Send reprint requests Division of Plant Industry,

to P. R. Whitfeld CSIRO.

at the

of the minor nucleoprotein fractions, or the RNA isolated from them, is infectious nor do they enhance infectivity when added to B, nucleoprotein or to RNA isolated from B, (Faed et al., 1972). The coat protein of all components appears to be identical as judged by serological tests (Matthews and Ralph, 1966) and by tryptic peptide mapping (Faed et al., 1972). The Bz nucleoprotein is, at least in large part, an artifact formed from B, in the CsCl gradient (Matthews, 1974). The BmO, Bon, and B, nucleoproteins do not appear to be artifacts of the isolation procedure, nor do they appear to form part of a multicomponent virus. In the work presented here we have investigated the size distribution of the RNAs in the various nucleoprotein components, and the ability of these RNAs to act as templates for protein synthesis in the wheat germ system. The results provide evidence for the presence of a lowmolecular weight RNA species in TYMV preparations which is a very efficient mes-

153 0042-6822/78/0841-0153 $02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

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WHITFELD,

senger for coat protein. While these experiments were in progress Klein et al. (1976), Pleij et al. (1976), and Ricard et al. (1977) reported that when RNA isolated from purified but presumably unfractionated TYMV preparations was heated briefly it could be separated into two distinct components of MWs 2 x lo6 and approximately 0.3 x lo6 and that the smaller of these RNAs contained the coat protein cistron. Our data complement these observations by establishing that the coat protein cistron and probably other viral-related RNAs can become encapsidated into particles which lack full-length RNA but which copurify with TYMV. MATERIALS

AND

METHODS

Virus. The standard mixed-strain culture of TYMV maintained in Auckland was cultured in Chinese cabbage plants (Brussicu pekinensis Rupr., var. Wong Bok) grown in pots in the glasshouse. Virus was isolated from plants that had been infected from 3 to 6 weeks as follows: Expressed sap from infected leaves was brought to pH 4.8 with 1.0 N acetic acid and clarified by centrifugation at 8000 rpm for 10 min. Saturated ammonium sulphate (0.5 vol) was added and after l-2 days the crystalline virus was collected by centrifugation and dialysed against 0.05 M sodium acetate, pH 4.8. The virus was then further purified by three cycles of low-speed (10,000 rpm for 10 min) and high-speed (45,000 rpm for 1 hr) centrifugation (Matthews, 1974). Isolation and characterization of nucleoprotein components. The nucleoprotein components were isolated on CsCl density gradients as previously described (Matthews, 1960) using two to four cycles of gradient fractionation to achieve bands free of visible cross-contamination as judged by light scattering. Infectivity of nucleoprotein components was assayed in 0.01 M phosphate, pH 7.0, on Chinese cabbage leaves using half-leaf comparisons. A solution of B, nucleoprotein at approximately 5 pg/ml was used as a standard on each half-leaf. As an alternative means of assessing cross-contamination between the nucleo-

AND

MATTHEWS

protein fractions, samples were examined in a Spinco Model E analytical ultracentrifuge using Schlieren optics. Samples (1 mg/ml) were centrifuged at 40,000 rpm and photographs were recorded at 4-min intervals. Isolation and fractionation of RNA by polyucrylamide-gel electrophoresis and sucrose gradient centrifugution. RNA was isolated from purified nucleoprotein fractions and analysed under nondenaturing conditions on 2.2% polyacrylamide gels as previously described (Whitfeld and Higgins, 1976). The molecular weights of the various RNA components were determined under denaturing conditions on 4% polyacrylamide gels in 99% formamide (Boedtker et al., 1973) using cucumber mosaic virus RNA (kindly provided by&-. R. H. Symons, University of Adelaide), Escherichiu coli RNA, and cowpea strain TMV-RNA as molecular weight markers. Fractionation of RNA on 520% sucrose gradients was carried out as detailed by Whitfeld and Higgins (1976). In vitro protein synthesis and charucterization of the labelled polypeptide products. The wheat germ cell-free protein synthesizing system was prepared as described (Higgins et al., 1976) and protein synthesis was monitored using either [35S]methionine or a mixture of 14C-labelled amino acids (data not shown). Reaction mixtures were as described previously except that 20 mM KC1 and 130 mM potassium acetate replaced the 105 mM KCl. The labelled polypeptides were separated on slab gels (15 x 20 x 0.15-cm) of 14% acrylamide, 0.19% bisacrylamide. The separating gel contained 0.375 M Tris-HCI (pH 8.9) and electrophoresis was carried out for 16 hr at constant amperage (16 mA); otherwise conditions for staining and fluorography were as previously specified (Higgins et al., 1976). In some experiments, viral RNA fractions were also translated in a rabbit reticulocyte system which had been pretreated with micrococcal nuclease as described by Pelham and Jackson (1976). RESULTS

RNA

species present

in nucleoprotein

RNAS

AND

TRANSLATION

PRODUCTS

OF TYMV

155

fractions. Figure 1 shows the pattern of ‘Boo, Boo,,. A series of seven major RNA species as well as at least three minor RNA bands obtained following polyacrylcomponents (numbered l-10 for reference) amide-gel electrophoresis of RNA from the five nucleoprotein components Bs, B,, BO, and a proportion of polydisperse, presumably randomly degraded, RNA were observed. Band 1, which is full-length RNA (2 x lo6 daltons), occurred only in B, and B,. The small peak which was occasionally apparent in the equivalent region of the gel patterns of B,, and Boo0RNAs (arrows in Fig. 1) was shown to be due to contaminating DNA. The RNA preparations from 11 0 55.106 B, and B, also contained a series of minor bands which, although not resolved in A 260nm scans of the gels, were apparent in gels stained with toluidine blue. Although it was not possible to relate these minor bands to those that occurred in Boo and Boo0 (and B, after heating in formamide), it is likely that some were common to all nucleoprotein fractions. However, band 10 (MW 0.28 x 106) was sufficiently prominent to allew us to conclude that it was present in all nucleoproteins. When B, RNA was heated at 65” for 5 min prior to electrophoresis, the proportion of polydisperse material was increased substantially, indicating the presence of hidden breaks in some “full-length” molecules as has already been reported (Matthews, 1974). Heating, however, did not appear to increase the relative proportion of band 10 RNA (data not shown). Nucleoprotein B, yielded what appeared to be a single major RNA species (band 2, Fig. 11, of MW 1.3 x 106, as well as a considerable amount of polydisperse RNA and a small distinct amount of band 10 RNA. Analysis of the RNA on formamidepolyacrylamide gels, however, showed that band 2 was composed of a number of lower-molecular-weight RNA species in addition to a small amount of 1.3 x lo”-

DISTANCE

MIGRATED

(cm)

FIG. 1. Polyacrylamide-gel (2.2%) electrophoresis patterns of the RNAs from each of the TYMV

nucleoprotein fractions run under nondenaturing conditions. MW markers were E. coli ribosomal RNAs. Major RNA species are denoted by numerals (l-10). The presence of two components in the band designated (4, 5) of Boo RNA and the identification of all minor RNA species was confirmed by subsequently staining the gels with toluidine blue. The high-molecular-weight bands (denoted by arrows) in B,, and B,,,, are due to contaminating host DNA.

156

HIGGINS,

WHITFELD,

AND

MW RNA. Whether band 2 RNA as depicted in Fig. 1 represents an aggregate of RNA species or is really a 1.3 x 106-MW species containing a large number of hidden breaks has not been ascertained. Nucleoproteins B,, and Boo0each yielded a distinctive mixture of discrete RNA species (Fig. 1). There were five major RNA species in Boo, (bands 3-6 and 10, Fig. 1) and three minor species (7-9). RNA species 4 and 5 and the minor species 7-9 were not well resolved in the A,,, nm gel scans but were clearly distinguishable when the gels were stained with toluidine blue. There were two major RNA species in B,,, (bands 6 and 10, Fig. 1) and three minor species (7-9) which appeared to correspond to the three minor species observed in Boo RNA. It can be seen that band 10 RNA is a major component of Boo,, and a significant fraction of B, RNA also. Electrophoresis under denaturing conditions did not affect the patterns of Boo and Boo,,RNAs. Table 1 summarizes some of the properties of the nucleoprotein components and gives the molecular weights of the RNA species as well as an indication of their relative distribution among the five TYMV components. An estimate of the extent of cross-contamination of the various nucleoprotein fractions was obtained from ultracentrifugation analyses. Within the levels of detection of the method, Boo preparations appeared to be uncontaminated. B,,, contained some B,, which might account for

the presence of some very minor RNA species which coincided with bands 3 and 4 of Boo RNA. However, the extent of this contamination could not account for the levels of bands 6 to 10 in Boo,,which presumably are components of both nucleoproteins Boo and B,,,. Bz, B1, and B,, preparations were apparently free of any contaminating Boo and Boo,,. Activity of RNA species as messengers for protein synthesis. RNA from each of the nucleoproteins was tested for its activity as a messenger in the wheat germ cellfree system using [35Slmethionine or a 14Clabelled amino acid mixture as radioactive precursors. A dose-response curve was set up for each RNA preparation and the TCA-insoluble counts per minute incorporated were plotted versus RNA level (Fig. 2). All the RNA preparations saturated the system at concentrations less than 2 pg of RNA per assay, with Boo0 RNA saturating at the lowest concentration (0.3 pg per assay). When a mixture of messenger RNAs is used to program a cell-free system there is a greater opportunity for all species to be expressed under nonsaturating conditions. On reaching saturation the more efficient messengers take over and the others are translated with lower efficiency or not at all (see, e.g., Higgins et al., 1976). The radioactive products synthesized by the wheat germ system in response to the various RNAs, each of which contained more than one component, were therefore compared at saturating and non-

TABLE PROPERTIES Component

B* B, J% BIN BWO

-%o,wa

114 114 81 75 60

(117) (114) (92) (72)

Infectivityb

9.00 100.00 0.80 0.04 0.00

OF TYMV

RNA as percent1 ageofto(2.00) tal RNA’ 5.2 91.3 2.2 1.1 0.2

+ + -

1

NUCLEOPROTEINS 2 (1.30)

t -

3 (0.96)

t + -

MATTHEWS

AND THEIR RIBONUCLEIC

(MW x lOme) RNA specie& 4 6 7 5 (0.76) (0.71) (0.59) (0.39)

+

+

+ +

+ +

ACIDS 0 (0.36)

+ +

9 (0.34)

10 (0.28)

+ +

+ + + + t

Polydisperse RNA t + + + t

0 Figures in brackets are the spO,wvalues previously reported (Matthews, 1970). b Number of local lesions per half-leaf compared with B, standardized to 100 lesions. B, wa8 inoculated at 5 @g/ml; other components were inoculated at equivalent or higher concentrations and lesion numbers were corrected to the same A Pmnm value for each component. c Calculated from the data of Matthews (1970) 88 given in the introduction. d MWs were determined from migration on formamide-acrylamide gels using E. coli rRNA, cowpea strain TMV-RNA, and CCMV-RNA as markers. A blank space in the table signifies that it was not possible to decide unequivocally whether the RNA species was present or not against the background of polydisperse RNA.

RNAS

AND

TRANSLATION

PRODUCTS

OF TYMV

157

6

5

-4 a v x3 E u”

2

1

0

1 c1g RNA

FIG. 2. Dose-response curve of the RNAs of TYMV nucleoprotein particles in the wheat germ cell-free protein synthesizing system. The amino acid precursor was [%]methionine, 3 &i per 50-~1 reaction volume: incubation was at 25” for 90 min.

saturating levels of RNA. After fractionation of the products by SDS-polyacrylamide-gel electrophoresis (SDS-PAGE), radioactivity was located by fluorography of the dried gel for 2-4 days (Fig. 3). In each case the major region of radioactivity coincided with the TYMV coat protein marker which migrates as a double band in this gel system (Matthews, 1974). It was estimated by densitometric analysis of the fluorographs that 30-40% of the radioactivity was in this region. The remainder of the radioactivity was associated with polypeptides ranging in size from 5000 to 70,000 daltons. Some higher-molecular-weight products (up to 150,000) were also formed in response to B, RNA, but in very small amounts. Saturating levels of the RNAs resulted in a greater proportion of the radioactivity being associated with coat protein and lower-molecular-weight polypeptides. Conversely, more higher-molecular-weight products were apparent where subsaturating amounts of the RNAs were used. Although minor differences in the spectrum of polypeptides generated by the

FIG. 3. Fluorograph of SDS-PAGE-fractionated Wlmethionine-labelled in uitro translation products. Two concentrations of each of the RNAs from the five TYMV nucleoproteins were added to the cell-free wheat germ system. The position of molecular weight markers (X 10m3) is shown on the left and the arrows indicate the position of the two bands of TYMV coat protein. (a and b) Programmed with B,,, RNA; (c and d) with B,, RNA; (e and D with B. RNA; (g and h) with B, RNA; and (i and j) with B, RNA. In each pair the first slot shows the products formed in the presence of subsaturating levels of RNA and the second slot those formed at saturating levels of RNA.

RNAs of the five nucleoprotein fractions could be seen in the fluorographs, the overall similarity of the patterns suggested that most of those RNA species which were being translated were common to all RNA preparations. When B, RNA was centrifuged on a sucrose gradient and fractions across the gradient were translated in the wheat germ system, it was found that full-length RNA did not program the synthesis of a polypeptide comigrating with TYMV coat protein whereas RNA recovered from the top half of the gradient did (Fig. 4A). High-molecular-weight RNA from the lower portion of the gradient, however, stimulated the synthesis of many large polypeptides (up to 150,000; Fig. 4A). In order to test whether the multiplicity of products formed in the wheat germ system could be due to the premature termination

158

HIGGINS,

WHITFELD,

AND

MATTHEWS

wheat germ system, are an indication of the extent of variation which can occur between translation experiments. DISCUSSION

a

b

c

d

e

a

b

FIG. 4. Fluorographs of SDS-PAGE-fractionated 135S1methionine-labelled in vitro translation products. (A) Products formed using fractionated B, RNA. RNA (2 mg) was centrifuged on a 5-20% (w/ v) sucrose gradient (20 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 8.2; 18 hr, 25,000 rpm) and the A 280nm peak was divided into four fractions. The recovered RNAs were used to program the cell-free wheat germ system. (a) Programmed by unfractionated B, RNA; (b-e) programmed by fractionated RNA, (bl being the most rapidly sedimenting and (e) the least rapidly sedimenting fraction. (Bl Comparison of the products formed by (a) the rabbit reticulocyte cell-free system and (b) the wheat germ system, each being programmed by the most rapidly sedimenting fraction of B, RNA from the sucrose gradient. The arrows indicate the position of the two bands of TYMV coat protein and the number 68 indicates the positions of a 68,000molecular weight protein marker (BSA).

of polypeptide chains, full-length B, RNA was also translated in a rabbit reticulocyte system, which is generally regarded as not giving rise to incomplete polypeptides. It can be seen in Fig. 4B that the patterns of polypeptides formed in the two systems are very similar, both with respect to the total number of bands formed and also with respect to the equivalence of electrophoretic mobility of the major products. The slight differences observable between the patterns of slot b, Fig. 4A and slot b, Fig. 4B, both of which represent translation of the same RNA fraction by the

The results presented above show that the RNAs of the minor nucleoproteins Boo and Boo,, (and possibly B,J associated with TYMV preparations are not a random selection of heterogeneous RNAs but are sets of molecules of discrete sizes. B, and B,,, share a number of these RNAs in common, most notably those of bands 6 to 10 (Fig. 1). The situation with regard to B, is at present not entirely clear because much of its RNA is polydisperse and the fraction which migrated as a single species of MW 1.3 x lo6 on polyacrylamide gels largely dispersed into a number of subspecies when examined in denaturing conditions. Previous evidence has shown that the RNA of the minor nucleoproteins has the cytidylic acid content characteristic of the virus itself (Matthews, 1960). Further support for the view that the RNA of these nucleoproteins is TYMV related comes from our analysis of the polypeptides synthesized in vitro in the wheat germ system programmed by these RNAs. The products formed using RNAs from Bz, BO, Boo, and B,,,, were very similar to those formed using viral RNA (BJ as message. Thus, there is no evidence for the encapsidation of any translatable host messenger RNA in the minor nucleoproteins. RNAs from each of the nucleoproteins contained a template for a polypeptide of the size expected for TYMV coat protein and, in view of the conclusive evidence of Benicourt and Haenni (1976) that the major product of the TYMV-RNA-programmed wheat germ system is coat protein, we have assumed that the product we observe is also the coat protein. The one RNA species which is definitely common to all five nucleoprotein fractions is the 0.28 x 106-dalton (band 10) RNA, and this size RNA would clearly be adequate to serve as the coat protein cistron. Klein et al. (1976) and Pleij et al. (1976) have both recently shown that heated TYMVRNA can be fractionated to yield a lowmolecular-weight RNA species (0.25-0.30

RNAS

AND

TRANSLATION

x 106) which directs the synthesis of coat protein in the wheat germ system, and full-length TYMV-RNA which is incapable of programming the synthesis of coat protein, although it can function in the production of other polypeptides. We have confirmed the observation that full-length TYMV-RNA from B, which has been freed of low-molecular-weight RNA species by fractionation on a sucrose gradient does not direct the synthesis of coat protein (Fig. 4A). From our data it would appear that a situation exists in TYMV similar to that in the TMV system. In TMV-infected tobacco plants a low-molecular-weight RNA (MW 0.28 x 106) is formed (Siegel et al., 1973; Beachy and Zaitlin, 1975) which has been identified as the TMV coat protein cistron (Hunter et al., 1976; Siegel et al., 1976). Furthermore, in the case of the cowpea strain of TMV, this RNA species may become encapsidated and can then be isolated as a nucleoprotein particle fraction (Whitfeld and Higgins, 1976; Higgins et al., 1976; Bruening et al., 1976). Thus, the presence of the 0.28 x 106-MW RNA (band 10) in the TYMV nucleoproteins probably reflects the encapsidation of some coat protein cistron RNA which has been formed in the infected cell. Likewise, some of the other RNA species present in the TYMV nucleoproteins may reflect the encapsidation of various viral RNA messengers of less than full genome length, which are active in the cell during the process of viral replication. Such RNA species would be equivalent to the series of less-thanfull-length TMV-RNA species that have recently been detected in preparations of three different strains of TMV (Bruening et al., 1976; Beachy et al., 1976; Beachy and Zaitlin, 1977). However, the possibility that the encapsidated minor RNA species represent fragments of TYMV-RNA which do not play a direct role in virus replication cannot at present be excluded. Encapsidation of RNA species of less than full genome size is presumably a chance process and is unimportant in so far as the future survival of the virus is concerned. The various species detected in B,, and B,,,, are not present in equimolar

PRODUCTS

OF TYMV

159

amounts and the sum of their molecular weights exceeds that of full-length RNA. Thus, it is likely that heterogeneity exists within each nucleoprotein fraction and Matthews (1974) has in fact shown that each of the minor nucleoproteins can be resolved into two bands in CsCl gradients if the pH is maintained in the range 5-7 and high centrifugal forces are used. Heterogeneity within the minor nucleoprotein preparations may also be responsible for the discrepancy between the measured RNA concentrations of BO, Boo, and B,,, (Table 1) (Matthews, 1970) and those which can be calculated from the molecular weights of the RNA species and their distribution in the particles. Thus, for B,,oo to have an RNA content of 5 to 6%, the average molecular weight of its RNA should be about 200,000 and, to compensate for the presence of RNAs more than twice that size (e.g., band 6) one must assume that some empty protein shells are present as well. However, it is possible that the RNA content of the minor nucleoproteins used in the current series of experiments is higher than that of the preparations analysed some years ago. Analysis of the RNA content has not been carried out again because of the difficulty of accumulating sufficient amounts of the purified minor fractions to permit accurate determinations to be made. In view of the relative contributions of the various nucleoproteins to the total RNA of a TYMV preparation and because of the absence of detectable levels of crosscontamination, it is unlikely that the presence of band 10 RNA in B, can be attributed to contamination by B,, or B,,,. Furthermore, because the amount of band 10 RNA in B, is submolar with respect to full-length RNA, it cannot result from the encapsidation of one coat protein cistron in every B, particle. Whether only a few B, particles contain a molecule of band 10 RNA as well as full-length RNA or whether B, preparations are contaminated with an occasional unidentified particle containing a number of band 10 RNA molecules is not known. However, it is clear that preparations of TYMV which have not been processed specifically to

160

HIGGINS,

WHITFELD,

remove minor nucleoproteins would contain significant amounts of the coat protein cistron and such undoubtedly is the case in the experiments reported by Klein et al. (1976) and Pleij et al. (1976). The extreme efficiency with which the messenger RNA for coat protein dominates the wheat germ translation system can be gauged by consideration of the relative amounts of band 10 RNA present in the B1, B2, and B,, preparations (Fig. 1). It comprises far less than 1% of the total RNA of the fractions and yet it is the preferred messenger species. Such a characteristic, however, is clearly in accord with its presumed role in the infected cell. The fact that the non-coat-protein products synthesized in response to B, and B, RNAs are little different from those synthesized in response to BO, BOO,and B,,,, RNAs would suggest that they are programmed by minor RNA species which are common to all the nucleoprotein fractions and which, like the coat protein cistron, are efficient messengers. RNAs 3-6, for instance, could be present in all five nucleoprotein particles, but not detectable in the gel scans of B2, B1, and B,,, (Fig. 1) because of the background of heterogeneous RNA. It should be possible to establish whether this is indeed the case by fractionating the various RNA species of Booand B,,, and identifying their translation products. ACKNOWLEDGMENT We thank Dr. Pleij and his colleagues for supplying us with a manuscript of their paper before publication. REFERENCES BEACHY, R. N., and ZAITLIN, M. (1975). Replication of tobacco mosaic virus. VI. Replicative intermediate and TMV-RNA-related RNAs associated with polyribosomes. Virology 63, 84-97. BEACHY, R. N., and ZAITLIN, M. (1977). Characterization and in vitro translation of the RNAs from less-than-full-length, virus-related, nucleoprotein rods present in tobacco mosaic virus preparations. Virology 81, 160-169. BEACHY, R. N., ZAITLIN, M., BRUENING, G., and ISRAEL, H. W. (1976). A genetic map for the cowpea strain of TMV. Virology 73, 498-507. BENICOURT, C., and HAENNI, A. L. (1976). In oitro synthesis of turnip yellow mosaic virus coat pro-

AND

MATTHEWS

tein in a wheat germ cell-free system. J. Virol. 20,196-202. BOEDTKER, H., CRKVENJAKOV, R. B., DEWEY, K. F., and LANKB, K. (1973). Some properties of high molecular weight ribonucleic acid isolated from chick embryo polysomes. Biochemistry 12, 43564360. BRUENING, G., BEACHY, R. N., SCALLA, R., and ZAITLIN, M. (1976). In vitro and in viuo translation of the ribonucleic acids of the cowpea strain of tobacco masaic virus. Virology 71, 498-517. DEROBIER, D., and HASELKORN, R. (1966). Minor components associated with turnip yellow mosaic virus. Virology 30, 705-715. FAED, E. M., BURNS, D. J. W., and MATTHEWS, R. E. F. (1972). Properties of minor nucleoprotein components found in TYMV preparations. Virology 48, 627-629. HIGGINS, T. J. V., GOODWIN, P. B., and WHITFELD, P. R. (1976). Occurrence of short particles in beans infected with the cowpeaitrain of TMV. II. Evidence that the short particles contain the cistron for coat-protein. Virology 71, 486-497. HUNTER, T. R., HUNT, T., KNOWLAND, J., and ZIMMERN, D. (1976). Messenger RNA for the coat protein of tobacco mosaic virus. Nature (London) 260, 759-764. JOHNSON, M. W. (1964). The binding of metal ions by turnip yellow mosaic. Virlogy 24, 26-35. KLEIN, C., FRITSCH, C., BRIAND, J. P., RICHARDS, K. E., JONARD, G., and HIRTH, L. (1976). Physical and functional heterogeneity in TYMV RNA: Evidence for the existence of an independent messenger coding for coat protein. Nucleic Acids Res. 3, 3043-3061. MATTHEWS, R. E. F. (1960). Properties of nucleoprotein fractions isolated from turnip yellow mosaic virus preparations. Virology 12, 521-539. MATTHEWS, R. E. F. (1970). “Plant Virology.” Academic Press, New York. MATTHEWS, R. E. F. (1974). Some properties of TYMV nucleoproteins isolated in cesium chloride density gradients. Virology 60, 54-64. MATTHEWS, R. E. F., and RALPH, R. K. (1966). Turnip yellow mosaic virus. Aduan. Virus Res. 12, 273-328. PELHAM, H. R. B., and JACKSON, R. J. (1976). An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem. 67, 247-256. PLEIJ, C. W. A., NEELEMAN, A., VAN VLOTEN-DOTING, L., and BOSCH, L. (1976). Translation of turnip yellow mosaic virus RNA in uitro: A closed and an open coat-protein cistron. Proc. Nat. Acad. Sci. USA 73, 4437-4441. RICARD, B., BARREAU, C., RENAUDIN, H., MOUCHES, C., and Bov&, J. M. (1977). Messenger properties of TYMV-RNA. Virology 79, 231-235. SIEGEL, A., HARI, V., MONTGOMERY, I., and KOLACZ,

RNAS

AND

TRANSLATION

K. (19’76). A messenger RNA for capsid protein isolated from tobacco mosaic virus-infected tissue. Virology 73, 363-371. SIEGEL, A., ZAITLIN, M., and DUD.+, C. T. (1973). Replication of tobacco mosaic virus. IV. Further characterization of the viral related RNAs. Virol-

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ogy 53, 75-83. WHITFELD, P. R., and HIGGINS, T. J. V. (1976). Occurrence of short particles in beans infected with the cowpea strain of TMV. I. Purification and characterization of short particles. Virology 71, 471-485.