Studies on the relation between protein and nucleoprotein particles in turnip yellow mosaic virus infections

VIROLOGY 6, l%-%.!i (1958)

Studies on the Relation Between Protein and Nucleoprotein Particles in Turnip Yellow

Mosaic Virus Infections

R. E. F. IMATTHEWS'

A.R.C. Virus Research Unit, Molteno Institute, Cambridge, England

Accepted November M, 1967

The ratio of numbers of virus nucleoprotein to protein particles in Chinese cabbage plants infected with turnip yellow mosaic virus (TYMV) remained close to 2:l under a wide range of growing conditions. This ratio did not change significantly from the earliest stage of infection at which crystalline virus could be isolated to the time when plants contained maximal amounts of virus, and were approaching senescence. Total ribonucleic acid (RNA) content of healthy and infected Chinese cabbage leaf tissue was examined by a method involving very little loss of polyribonucleotides. Healthy leaf tissue may contain up to 3 mg/g fresh weight of RNA. In leaves infected with TYMV the total RNA is increased. The increase over comparable healthy leaf is entirely accounted for by the amount of virus RNA that can be isolated in combination with the virus protein antigen.

INTRODUCTION

Turnip yellow mosaic virus is of particular interest among the “spher- ical” plant viruses so far studied in that two types of particle are found in infected plants. One is an infectious nucleoprotein containing about 38% ribonucleic acid. The other is an apparently identical protein containing no nucleic acid and which is not infectious (Markham and K. M. Smith, 1949). These authors found that in mature infections approximately one in three of the particles contain no nucleic acid. It has been suggested that the protein particle could be a precursor of the complete virus, a degradation product of the virus, or a mistake in the process of virus synthesis (Markham, 1953). Jeener (1954) followed the incorporation of Cl402 into the protein of the two types of particle over short times. His results supported the view that the protein particle is

1 Present address: Plant Diseases Division, Department of Scientific and Industrial Research, Auckland, New Zealand.

192

RELATION BETWEEN PROTEIN AND NUCLEOPROTEIN PARTICLES 193

.a precursor of the nucleoprotein. In the work described here the ratio of the two types of particle under a wide range of conditions has been determined. This ratio is remarkably constant from the earliest detect- able stage of infection to the time when infected plants are almost senescent. There is no evidence for the existence of any “free” virus nucleic acid corresponding in amount to the virus protein. The data suggest that although up to 6 mg of virus per gram fresh weight of leaf tissue may accumulate, this has very little effect on the amount or composition of the leaf ribonucleic acid.

MATERIALS AND METHODS

The “type” strain of turnip yellow mosaic virus (I‘YMV) described by Markham and K. M. Smith (1949) was used in all experiments. The virus was cultured in Chinese cabbage Brass&a chinensis var. Wong Bok., grown in 4- or 6-inch pots under glass.

Isolation of virus. Virus was prepared from infected leaves by alcohol clarification of expressed sap followed by three crystallizations of the virus with ammonium sulfate according to the method of Markham and K. M. Smith (1949). Slight variations in the degree of saturation with ammonium sulfate can affect the composition of the product (Matthews, 1955). All the preparations for the experiments shown in Figs. 3a and 3b were carried out simultaneously t.o avoid differences in composition caused by changes in room temperature affecting the con- centration of salt:

Analytical procedures. The methods of chromatography and electrophoresis on filter paper were those developed by Markham and J. D. Smith, (1949,195O a, b, 1952). Phosphorus was determined by the method of Allen (1940). Optical densities of virus preparations were measured at 260 rnp in water using a cell of 1 cm path length f&0).

Estimation of virus in leaf discs. The serological-chromatographic procedure (Matthews, 1955) was used to estimate the amount of nucleic acid combined with virus protein in leaf discs.

Estimation of totaL ribonucleic acid in leaf discs. For the experiments described here a method was required which would give an accurate and specific estimate of total ribonucleie acid (RNA) in small samples of leaf (less than one gram fresh weight). Procedures invoIving grinding of the fresh leaves resulted in large and variable losses in R.NA presumably caused by the action of leaf nucleases. The following method based on a procedure suggested by Markham (1955) avoids this difficulty.

194 MATTHEWS

Discs about 10 mm in diameter were cut from leaves with a cork borer (10-20 discs per sample) avoiding the midrib and large veins. Discs were placed in a vapor-tight weighing bottle and weighed as soon as possible after sampling. Discs were then plunged into 100 ml of boiling 70% ethanol made 0.1 iV with respect to acetic acid. After boiling 3-5 minutes the 70 % ethanol was poured off. Samples were then extracted for 3 to 5 minutes in 50 ml of boiling absolute ethanol. These two extrac- tions effectively remove from the discs substances of small molecular weight which absorb ultraviolet light. Polynucleotides are insoluble and remain in the discs. Finally discs were soaked in 20 ml acetone for about 30 minutes, followed by drying in warm air. The dry discs were then extracted with a measured volume (0.2-0.5 ml) of 2 N KOH overnight at room temperature in a small tube. The KOH was neutralized with perchloric acid, and the precipitate of potassium perchlorate together with the leaf discs was centrifuged to the bottom of the tubes. Measured portions of the supernatant fluid were placed on sheets of Whatman No. 3 MM filter paper and subjected to chromatography in the isopropanol-ammonia solvent of Markham and Smith (1952). The two spots containing the mononucleotides of guanine and of adenine, cytosine, and uracil were located by ultraviolet photography (Markham and J. D. Smith, 1949) eluted in N/10 HCl and the optical density of the eluate measured at 260 rnp. From the specific extinctions of the nucleotides, and the composition of the nucleic acids the amount of ribonucleic acid in the original sample could be calculated. The following lines of evidence show that this procedure measures optical density arising only from polyribonucleotides.

1. Only two ultraviolet-absorbing spots could be detected following chromatography of the neutralized leaf disc digest in the isopropanol- ammonia solvent. These corresponded to the guanylic acid and the mixed adenylic, cytidylic, and uridylic acid spots.

2. On electrophoresis of the neutralized digest on filter paper a& pH 3.5 in formate buffer only four ultraviolet-absorbing spots could be detected. These had the expected mobilities for uridylic, guanylic, adenylic, and cytidylic acids, and on elution were found to have the expected ultraviolet absorption spectra for these four compounds in acid and in alkaline solution.

3. When a ‘portion of the digest was subjected to chromatography in the ammonium sulfate-water solvent (Markham and Smith, 1952) five ultraviolet-absorbing spots were observed with mobilities corre-


sponding to the two adenylic acids (3’ and 2’ phosphates) and the two guanylic acids (3’ and 2’ phosphates) and the spot cantaining the mixed pyrimidine mononucleotides. From alkaline hydrolysis of polyribonucleotides a slight excess of the 3’ phosphates is to be expected. In these tests the ratios of 3’ to 2’ phosphates were: guanylie, 1.17, adenylic, 1.23.

Incorporation of P32 into nucleic acid. The rate of incorporation of P32 into the virus RNA was used to estimate the relative amounts of virus being produced at different stages of infection. After soil had been washed from the roots, Chinese cabbage plants were placed in conical flasks containing culture solution. One liter of the culture solution contained: 116 mg CaCL; 437mg MgS04.7H20; 276 mg (NH&SOd. P32 was supplied as orthophosphate in quantities of about 1 mC per lit,er of solution, each plant receiving 25-50 ml. Uptake of P32 into leaves was measured on 5-mm diameter discs punched from leaves. Discs were collected, weighed and the radioactivity (counts per minute) determined on the intact discs. To ensure that the virus for activity measurements came from comparable positions in the leaf, a larger punch (11 mm) was used to obtain rings of tissue surrounding the discs taken for measurement of leaf activity. The rings of tissue were weighed, ground finely in a small agate pestle with about 5 ml of 0.14 M saline. The sample was then heated to 55” for 3 minutes and centrifuged at 2500 rpm for 10 minutes. Excess of a turnip yellow mosaic virus rabbit antiserum was added to the supernatant fluid. The mixture was shaken well and allowed to stand l-2 hours at room temperature and overnight at 4”. The virus-antibody precipitate was centrifuged down, washed three times with about 2 ml 0.05 M KHZ PO+ followed by one wash with 0.14 M NaCI. The precipitate was dried in an oven at 90” and digested overnight in a small volume of 2 N KOH. The KOH was neutralized with HCl04 and the precipitate of KC104 and protein centrifuged down. Portions of the superna.tant fluid were subjected to chromatography in the isopropanol-ammonia solvent. The two spots containing the mixed nudeotides were eluted in 0.1 N HCI. After measurement of the optical density at 260 rnp, portions of the eIuate were dried down on planchettes for radioactivity measurements. That this procedure measured activity only from the mononucleotides is shown by the following evidence: After chromatography in isopropanol-ammonia, nearly all the radioactivity on the paper was associated with the two ultraviolet-absorbing spots. A small amount of radioactivity remained near the origin and

196 MATTHEWS

was presumably from inorganic phosphate. When the nucleotide spots were eluted after isopropanol-ammonia chromatography and subjected to electrophoresis on filter paper at pH 3.5 in formate buffer, all the radioactivity on the paper was associated with the four mononueleotide spots. Table 1 shows that the four mononucleotides were labeled to a similar extent and that the composition had the typical high cytidylic acid content of TYMV RNA. The values for cytidylic acid are higher than those of Markham and Smith (1951). This is probably due to some trailing of adenylic acid during the electrophoretic separation.

RESULTS

Composition as a measure of the ratio of number of protein and nucleoprotein particles. In attempting to detect small differences in the ratio of protein to nudeoprotein, the phosphorus content and the optical density at 260 rnp were used, the total amount of purified material being determined by dry weights. Optical density at 260 rnp and the phosphorus content are closely correlated as is shown in Fig. 1. The four points with a phosphorus content of about 2.5% are from different preparations or groups of preparations made according to the procedure outlined above. The two extreme points are for “pure” preparations of the proteins and the nucleoprotein made by the centrifugation procedure of Markham and K. M. Smith (1949). The nucleoprotein preparation contained 3.53 % phosphorus. This is equivalent to 38% ribonucleic acid by weight (as the ammonium salt). If we assume this is the composition of the nucleoprotein, we can determine the percentage of protein and nucleoprotein particles in a preparation from the relationship :

x= 0.62 Y 3.53 - 0.38Y

TABLE 1

INCORPORATION OF P32 INTO RNA OF TURNIP YELLOW MOSAIC VIRUS;.MOLAR RATIOS. DETERMINED FROM DzGO AND RADIOACTIVITY

On Dm On cpma Ratios after Marlsham and Smith (1951)

Cytidylic acid 1.70 1.71 1.52 Adenylic acid 0.76 0.70 0.92 Guanylic acid 0.60 0.67 0.68 Uridylic acid 0.94 0.91 0.88

a cpm = Counts per minute.


“&, P on Dry weight

FIG. 1. Relation between percentage phosphorus in a preparation of TYMV and the optical density at 260 mp for 0.1 mg/ml of preparation (based on dry weight).

where X = proportion of particles with nucleic acid and Y = percentage of phosphorus in preparation. This relationship together with the range of values found for unfractionated preparations is shown in Fig. 2.

Proportion of protein and nucleoprotein during the course of infection and at di$erent seasons. In two experiments, one carried out in summer sod the other during winter, groups of whoIe plants were cut at soil level at weekIy intervals after inoculation and frozen. At the end of the period, virus was isolated from each sample by the ammonium sulfate procedure. The phosphorus content and optical density at 200 rnp for

3.6 - I I I I I I I I I I-

so-

2.4 -

0 IO 20 30 40 50 60 70 80 90 100

ojn 01 oarticler with nucleic acid

FIG. 2. Relationship between percentage of particles containing nucleic acid in a TYMV preparation, and. the phosphorus content (per cent of dry weight) assum- ing the pure nucleoprotein contains 3.5370 phosphorus. The range and mean for 14 preparations are shown by arrows.

0.1 mg,/ml were determined on the purified products. Results are summarized in Figs. 3a and 3b.

For the first two weeks or so, very little virus is produced. After this initial period, yield of virus rose rapidly reaching a maximum about the seventh week. Thereafter there was a fall in virus content, very probably caused by marked senescence of the plants at this stage. For both experiments the composition of the virus isolated remained remarkably constant. The mean value for 14 preparations was 63.6 % of nucleoprotein particles (by number) with a range from 57 % to 69 %. The distri- bution of the values grouped in 3 % ranges was as follows:

RELATlON BETWEEN PROTEIN AND NUCLEOPROTEIN PARTICLES 199

Y. Nucleoprotein particles Number of preparations

56-59 I

59-62 3 62-65 4 65-68 5 68-71 1

Five of the fourteen preparations fell in the range 6%68%. If in fact the ratio of nucleoprotein to protein particles is exactly 2:1, the preparations with a lower ratio may have contained some impurity with a low content of nucleic acid. To reduce the apparent percentage of nucleoprotein particles (by number) from 67 to 57% would require a contamination of 11.5% by weight with an impurity containing no phosphorus.

Search for protein antigen at early stages of infection. In tobacco leaves

o-3 - - 3*0 0.6

0.2- - 2-o 0.4

0*1- - I.0 0.2

0 I 2 3 4 5 6 7 8 9

weeks after inoculation

FIG. 3. a. 3. The amount and composition of TYMV preparations containing bot.h

nucleoprotein and protein particles isolated by the ammonium sulfate procedure at various times after infection of Chinese cabbage plants. (a) Experiment during summer. (b) Experiment during winter.

200 MATTHEWS

H

0.6 -

52 h-A z 0*5- % c _ P

2 >0*4- d m

.E o.,- - 3.0

6 0 $ 0.2 - - 2.0

0-I - - I-0

I I I I I I I I I I I I I I I I I I I _

w 0260 for 0-I mg/ml

0.9

08

0.7

0.6

0.5

0.4

0’3

0.2

0-I

I 2 3 4 5 6 7 5 9 weeks after inoculation

FIG. 3. b.

inoculated with tobacco mosaic virus, substantial increase in virus can be detected after a few days. The final yield of virus is usually about 2 g/liter of sap from whole plants. Although similar maximum yields are obtained from Chinese cabbage plants infected with TYMV, no virus can be isolated for the first lo-14 days after inoculation. To check on the possibility that virus protein in some form which was not isolated by the ammonium sulfate procedure might be accumulating during this period, leaves were sampled and tested serologically for the presence of antigen. Eight Chinese cabbage plants were inoculated on all leaves with TYMV. The leaves were washed thoroughly immediately after inoculation. One leaf from each plant was harvested at various times and stored in a frozen condition. When all samples had been collected they were tested for amount of virus antigen present by the virus endpoint method using twofold dilutions. Tests were made on expressed sap, clarified by heating to 55” for 3 minutes. These dilution endpoint measurements are subject to an error of 50%, but they are the most sensitive method available for detecting the virus protein. Results summarized in Table 2 show that there is no accumulation of protein antigen in the early stages of infection. There is a steady rise up to the


TA3LE 2

AMOUNTS OF TYMV ANTIGEN IN SAP FROM CHINESE CABBAGE LEAVES AT

VARIOUS TIMES AFTER INOCULATION, ESTIMATED BY TNE PRECIPITATION END POINT METHOD

Days after inoculation Virus dilution end point

0 2 4 6 9

12 15 21

time when virus can first be isolated in sufficient quantities for analysis. A solution of TYMV containing 1 mg/ml of purified virus usually has a dilution endpoint of l/256 ox about 4 pgg/ml. Thus by 12-15 days about 120 pg/ml of virus had accumulated, by 3 weeks about 1 mg/ml, which is substantially more than the amounts isolated at 3 weeks in the experiments of Figs. 3a and 3b. This is caused by a combination of the following factors: Firstly, in the experiments of Figs. 3a and 3b whole plants were harvested, in which only about 1 leaf in 5 had been inoculated. In the present experiment only inoculated leaves were used. Secondly, there is always some loss of virus with the ammonium sulfate precipitation procedure. Although it has a large error owing to the dilution steps, the virus endpoint method measures all the virus antigen in the solution.

The RNA content of healthy Chinese cabbage leaves and leaves infected with TYA!W. One possible explanation for the presence of the virus protein particles containing no nucleic acid in TYMV infections might be that free virus RNA and protein were in equilibrium with complete virus. If this were so we would expect to find an amount of free virus RNA equivalent to the free protein.

To test this possibility we have examined the RNA content of Chinese cabbage leaves, by the methods described earlier. The RNA content of normal leaves varies substa,ntially with age and nutrition, ranging in these experiments from about 0.3 to 3.0 mg RNA per gram fresh weight of ‘leaf disc. This is substantially higher than the value of 0.1 mg/g given by Markham (1955) and it is probable that the figure of 400 mg

202 MATTHEWS

of nucleic acid of plant origin falling on each square mile of soil per year given by Leslie (1955) is something of an underestimate. The values found here are higher because the met.hod used involves virtually no loss of polyribonucleotides, and because we used selected leaf discs containing no midrib or large veins.

In some comparisons between healthy and TYMV-infected tissues separate plants grown under the same conditions were analyzed. How- ever, a closer comparison can be made using half leaves (attached to the plant), since TY,MV does not move from one side of a leaf to the other for several weeks. Results are summarized in Table 3. For the increase in total RNA in infected tissue the mean value was 1.17 mg/g fresh weight. The mean value for RNA associated with virus antigen in the same leaves was 1.22 mg/g fresh weight, Thus all the increase in RNA in infected leaves is accounted for by the RNA associated with virus protein.

Table 4 summarizes the base ratios obtained on samples from healthy and virus-infected leaves. When the values for the healthy leaf are sub- tracted from those for the virus-infected material, ratios are obtained which are clearly of the unusual TYMV type, although guanine is low and uracil is high compared with the data of Markham and Smith (1951).

Rate of incorporation of Pa2 into TYMV RNA at di$erent times after infection. If the protein particle is a precursor of the whole virus, the constant ratio between the two types of particle might be due to a steady synthesis and breakdown of virus at all stages of infection.

TABLE 3

RNA CONTENT OF HEALTHY AND TYMV-INFECTEV CIXINESE CABBAGE LEAVER

Experiment type

Total RNA content of leaves (mg/g fresh weight) 3 Virus AA mg/g

Time after Infected minus fresh weight inoculation healthy (column 2 (measured by sew

Heaihy - column 1) logical-cbromato-

graphic method)

Separate plants 5 weeks 1.68 4.13 2.45 2.59 Half leaves 4 weeks 1.43 3.63 2.20 2.30 Separate plants 3 weeks 1.76 2.54 0.78 0.90 Half leaves 1 week 1.36 1.39 0.03 0.00 Half leaves 3 weeks 0.66 1.52 0.86 0.75 Half leaves 4 weeks 0.34 1.02 0.68 0.76

Mean = 1.17 Mean = 1.22


TABLE 4 MOLAR RATIOS OF BASES IN CHINESE CABBAGE LEAF RNA AND TYMV RNA. I___-

Health: leaf Leaf i&ten ai’ch 3

TYMV 5 weeks after inoculation

DiffFe;my irus-

Guanine Adenine Cytosine Uracil

a 6 a b- a b

0.48 1.17 0.74 0.81 0.26 0.52 0.68 0.38 0.93 0.84 0.92 0.46 0.92 0.92 0.37 0.91 1.16 1.28 0.79 1.57 1.52 0.41 1.00 0.91 1.00 0.50 0.99 0.88

NOTE: Columns la and 2a give the optical densities found for the four bases from RNA from equivalent fresh weights of leaf tissue divided by the following extinction coefficients: Guanine Ez50 = 11.0; Adenine EzE~ = 13.0; Cytidylic acid EZSO = 12.3; Uridylic acid Ezio = 9.45. Columns lb and 2b are the corresponding molar ratios of bases. Column 3a = 2a - la. Column 3b gives the corresponding molar ratios.

a From Markham and Smith (1951).

TABLE 5 INCORPORATION OF P32 INTO CHINESE CABBAGE LEAF TISSUE AND TYMV RNA

Time aft~$$,tior~ with Time afFeE;;dded to cpm/mg leaf tissue cpm/&g virus RNA

3 weeks 24 hours 2400 134 48 hours 4200 300

8 weeks 24 hours 1070 7 48 hours 3100 50

The rate of uptake of P32 into Ieaves and into virus RNA was measured at various times after infection with virus. The results of such an experiment are shown in Table 5. About 95 % of the activity added to the nutrient solution was taken up by the plants after 48 hours. If it is assumed that by this time the activit.y was uniformly distributed among virus precursor compounds containing phosphorus then these figures suggest a substantially greater rate of production of new virus RKA at the earlier stage of infection.

DISCUSSION

The noninfective protein particles produced during multiplication of TYMV differ from the abnormal protein or proteins found in tobacco

204 MATTHEWS

plants infected with tobacco mosaic virus in that they are very similar to the complete virus in size, shape, and surface properties. The anom- alous tobacco mosaic virus proteins, recently discussed in detail by Pirie (1957), are more comparable with subunits of the protein part of the complete virus rod. So far there is no decisive evidence on the nature of the abnormal proteins for either of these viruses. The analyses summarized in Table 3 give no support to the idea that free virus protein and free virus nucleic acid are in equilibrium with complete virus. How- ever, they do not rule out the possibility that the host plant RNA in infected cells is reduced in quantity and composition in a way that would just allow for the appropriate amount of “free” virus nucleic acid.

It can be calculated from yields of virus and from the approximate number of cells per unit weight of leaf that each mesophyll cell contains about 2 million virus nucleoprotein particles when fully infected. If we assume that there is a negligible amount of “free” virus RNA in mature infections then the results in Table 3 show that the production of this large number of particles has very little effect on the amount of host cell RNA.

Jeener (1954) exposed Chinese cabbage plants in which TYMV was multiplying to an atmosphere of Cl402 for short periods. He then found that the protein of the protein particles was more heavily labeled than that of the nucleoprotein. If the protein particle is a precursor of complete virus, as these results suggest, it is difficult to see why there should be the same ratio of precursor to complete virus under the wide range of conditions examined in our experiments. From the earliest stage of infection at which crystalline virus can be isolated to the time when plants are senescent and almost fully infected with virus, the ratio of number of complete virus particles to virus protein particles is close to 2:l. A drop in the proportion of a precursor might be expected at the later stages of infection as the rate of virus production falls. A roughly constant proportion of precursor could be explained if virus was being degraded and resynthesized at all stages of infection. However the data on the uptake of P3* into the virus RNA indicate that the rate of production of new virus RNA is much greater in the early stages.

Likewise if the protein particle is a “mistake” of virus synthesis it is difficult to see why such a constant proportion should be produced under a wide range of conditions. There remains the possibility that the protein particle is a product of virus breakdown. As a first step in each


cycle of multiplication the RNA might escape, leaving the protein shells as stable particles which accumulate as multiplication proceeds. While there is no direct evidence in favor of this hypothesis, it could explain the constant ratio of the two types of particle. If we assume that in each cycle of multiplication the RNA from the parent gives rise to three new nucleoprotem particles, leaving one stable empty shell, then the observed ratio of two nucleoprotein to one protein particle would be achieved.

REFERENCES

ALLEN, R. J. L. (1940). The estimation of phosphorus. Biochem. J. 34, 858-865. JEENER, R. (1954). A preliminary study of the incorporation in growing turnip

yellow mosaic virus and its related non-infective antigen of labelled amino acids. Biochim. et Biophys. Acta 13, 307-308.

LESLIE, I. (1955). The nucleic acid content of tissues and cells. In The Nucleic Acids (E. Chargaff and J. N. Davidson, eds.), VoI. IT, pp. l-44. Academic Press, New York.

MARKHAM, R. (1953). Nucleic acids in virus multiplication. Symposium Sot. Gen. Microbial. 2, 86-98.

MARKRAM, R. (1955). Nucleic acids, their components and related compounds. In Modern Methods of Plant Analysis (K. Paech and M. V. Tracey, eds.), Vol. IV, pp. 246-304. Springer-Verlag, Berlin.

MARKHAM, R., and SMITH, J. D. (1949). Chromatographic studies on nucleic acids. 1. A technique for the identification and estimation of purine and pyrimidine bases, nueleosides and related substances. Rio&em. J. 45,294-298.

MARKHAM, R., and SMITH, J. D. (1950a). Chromatographic studies on nucleic acids. 2. The quantitative analysis of ribonucleic acids. Biochem. J. 46, 509- 513.

MARKHAM, R., and SMITH, J. D. (19505). Chromatographic studies on nucleic acids. 3. The nucleic acids of five strains of tobacco mosaic virus. Biochem. J. 46, 513-516.

MARKHAM, R., and SIVIITH, J. D. (1951). Chromatographic studies on nucleic acids. 4. The nucleic acid of turnip yellow mosaic virus, including a note on the nucleic acid of tomato bushy stunt virus. Biochem. J. 49, 401-406.

MARKHAM, R., and SMITH, J. D. (1952). The structure of ribonuoleic acids. 1. Cyclic nucleotides produced by ribonuclease and by alkaline hydrolysis. Bio- them. J. 62, 552-557.

MARKHAM, R., and SMITH, K. M. (1949). Studies on the virus of turnip yellow mosaic. Parasitology 39, 330-342.

MATTREWS, R. E. F. (1955). Infectivity of turnip yellow mosaic virus containing 8azaguanine. Virology 1, 165-175.

PIRIE, N. W. (1957). The anatomy of tobacco mosaic virus. Advances in Virus Research 4, 159L190.

Documents

Studies on the relation between protein and nucleoprotein particles in turnip yellow mosaic virus infections