Ribonuclease isozymes in Chinese cabbage systemically infected with turnip yellow mosaic virus

VIROLOGY

36, 556-563 (1968)

Ribonuclease

lsozymes with

in Chinese Turnip

Cabbage

Yellow

Mosaic

Systemically

Infected

Virus

J. W. RANDLES Department

of Cell Biology,

University of Auckland, Accepted August

Auckland,

iVew Zealand

14, 1968

An electrophoretic method has been developed for carrying out rapid and semiquantitative assays of ribonuclease (RNase) isozyme activity in crude extracts of Chinese cabbage leaves. The method has been used to study the nature of increases which have been observed in the level of RNase activity in leaves systemically infected with turnip yellow mosaic virus (TYMV). Three isozymes (RNases I, II, and III) were detected in Chinese cabbage leaves, and all were found at highest concentration in the 105,000 g cytoplasmic supernatant. Very little activity was associated with organelles. In healthy leaves, RNase III activity was high during the early stages of leaf growth, but declined to a virtually WIdetectable level when length exceeded 10 cm. RNases I and II showed no marked changes in activity with time. In TYMV-infected plants, the first leaf to develop systemic symptoms was studied. The RNase isozyme pattern in this leaf was identical to that of healthy leaf until after virus had begun to increase rapidly. RNase III then showed renewed strong activity, and RNases I and II also showed a small increase in activity. No new isozymes were observed in systemically infected leaves. Systemic infection with turnip mosaic virus (TuMV) caused changes in isozyme patterns very similar to those observed in leaf systemically infected with TYMV. Leaves wounded by rubbing with Carborundum showed increased RNase III activity, but those mechanically inoculated with TYMV retained high RNase III activity for a longer time than rubbed control leaves, and at a level comparable with that in systemically infected leaves of the same size. INTRODUCTION

Observations that ribonuclease (RNase) activity is raised in virus-infected leaf (Reddi, 1959; Diener, 1961; Shinde et al., 1964; Shinde and Santilli, 1967) have led to the conclusion that virus infection can cause new synthesis of RNase. However, all work to date describes RNase activity in mechanically inoculated leaf, and there is ample evidence that wounding can cause rapid, prolonged, and nonspecific increases in the RNase activity of leaves (Diener, 1961; Bagi and Farkas, 1967). To avoid complications arising from the use of mechanically inoculated leaf, the effect of virus infection on RNase activity has been studied in Chinese cabbage leaf sys-

temically infected with turnip yellow mosaic virus (TYMV). The levels of RNase activity in systemically infected leaf and the changes in R,Nase isozyme patterns during the development of systemic symptoms are described, and compared with isozyme patterns in inoculated leaf. The method of assaying total RNase activity differs from that commonly used. In addition a technique for fractionating and assaying low concentrations of RNase isozymes in vertical flat sheet polyacrylamide gels (Reid and Bieleski, 1968) has been developed as a modification of the method used by Boyd and Mitchell (1965) for fractionating deoxyribonuclease isozymes. 556

I:IBOYUCLEASE ~IrlTEII.IAI,S

AND

ISOZYlIES

METHODS

Plant and uiwses. Two isolates of TYXV, one causing yellow-green and one pale-yellow symptoms (Chalcroft and Matthews, 1967) were used in studies of interactions bekeen T\XIV and its host. Turnip mosaic virus (TulLIV) was a New Zealand isolate supplied by Dr. I’. R. Fry, Plant Diseases Division, D.S.I.R., Auckland. Both viruses were cultured in Chinese cabbage (Rlassica pekinensis, Rupr. cu. Wong Bok). For time course studies of changes in isozyme patt,erns, plant,s of the same age were pa,ired on the basis of the size of the smallest unrolled leaf and one of each pair was allocated to one of two groups. Expanded leaves of plants in one group were rubbed with water, and t$he ot’her with inoculum. The control and inoculated groups were grown in daylight supplemented with continuous fluorescent lighting in a glasshouse at lS”-24°C. To avoid any effects of damage from sampling procedures, separate plants were sampled at each t,ime. One leaf was taken from each of four plants at early times, and later from t,hree, as leaf area increased. I’iqual weights of t’issue were taken from the middle third of one half of each leaf and bIlllied for assay. The leaf sampled was approximately 2-4 cm long 4 days after inoculation. In infected plants this was the first, leaf to show syst’emic symptoms. The ndvnnt~age of using t’his first leaf is that secondary effects of infection on growth were minimized over t,he period during which RSnse activit’y was studied. Area measurements of thii: leaf from infected and healt’hy plants at, each harvest’ showed t#hat negligible change in rate of expansion occurred until about, 3 days after the first appearance of symptomk When total RSase activity was estimated on sy,~tcmically infect,ed leaf showing mosaic symptoms, t’he dark green areas of the mosaic which are substantially free of virus (Reid and RIatthews, 1966) were used as control areas. Equal fresh weights of tissue were taken from dark green and yellow areas OIL the same leaf where these occurred at identical positions on adjacent half leaves. Fractionation of organelbs. Cell-free homogenates were prepared from Chinese cab-

IN C:HIh-ESE

CABBAC;K

--” .):I i

bage leaf by chopping fresh tissue in sucrosedextran-Ficoll (SFD) medium (Spencer and Wildman, 1964) modified by the uddit,ion of bovine serum albumin (SFDP mediumP Honda et al., 1966) and straining t*hrough four layers of muslin. Fractions \vere prepared by sedimentat,ion over a range of increasing centrifugal forws: 1000 g 15min pelletPchloroplasts + nuclei (CS) ; 15,000 g 15min pellet-- -mitochondriu (:\I); 105,000 g :3-hr pellet--ribosomes ;md virus particles (I~) ; 105,000 g supernut:rncytoplasmic supernatant (S). CN and 11 fract,ions were washed once tq resuspension in the SF’DI’ medium, and resediment#ntion. R n-as rinsed by running ;I, small volume of dist’illed water over the surface of the pellet and wiping the inside of the tube dry. CX and 11 were suspended in a small volume of SE’DI’ medium; 11 \v:ts suspended in 0.01 31 i\IgCl, . Chloroplasts and nuclei were fractionated from the CIZ- fraction on a discontinuous gradient of 60 % (2 ml), 45% (2 ml), and 20 % (1 ml) sucrose in SFD medium, by cent’rifuging for 90 min at 36,000 rpm in the Spinco SW 39 rotor (Tewari and \~ildman, 1966). Two chloroplxst~ fractious (C ) and :m enriched nuclear pellet (K) wrr oht:Grwd. Fractions were stored frozen. I,ight microscopic cxamim~tion she\\-ed that .\ I contained numerous veq’ small spherical colourless purt~icles at t)hcalimit of resolution, C comprised npparent,ly lvell INserved single chloroplnsts, ant1 N w:ts :I mixture of nuclei, cell debris, :md n fen apparently broken chloroplasts. Kstiulatiorl.s oJ’total RNase adivif!g. RKme activit,y ~vas calculated :IS the rate of loss of trichloroacetic acid (TCA) precipitable &w/rerichia coli 14C-sRS:1 (uracil labeled- +tqplied by Dr. 1’. I,. Bergquist,). 20 mg of fresh lea,f tissue was homogenized \vith 0.2 ml of 0.1 M sodium :~cet’;tte buffer (pH .5.2) :md clarified at 1000 g for 10 miri; 0.14 ml of the supermkant was diluted -\vith 0.3 ml of the acetate buffer, then 0.4 ml of t,he substrate at, a concentration of 50 pg sRSa ‘ml (xpecific activit,y = 475 dpm/& uxs added at zero time. 1\Iixtures were incubated at 37”. Aliquots of 0.05 ml were taken at’ intervalq mixed with O.Oj ml of albumin (at 1 mg ‘ml),

558

RANDLES

and the reaction was stopped by adding 0.1 ml of cold 10 % TCA. Controls were run with buffer replacing leaf extracts. TCA-insoluble 14C-sRNA was collected on cellulose acetate membranes, and radioactivity was determined in a Packard Tri-Carb scintillation spectrometer. RNase activity is expressed as the initial rate of loss of radioactivity in counts per minute per minute. Fractionation and estimation of ribonuclease isoxymes. Seven percent polyacrylamide gels buffered at pH 8.9 with 0.38 M Tris-HCl (Davis, 1964) and modified by incorporating 0.30 mg of high molecular weight RNA (yeast ribosomal RNA) per milliliter as substrate and 2 X 1O-3 fM cupric chloride as a RNase inhibitor (Davis and Allen, 195,5) were formed in the apparatus of Reid and Bieleski (1968). Leaf extracts were prepared for electrophoresis by homogenizing 100 mg of fresh tissue with 0.17 ml of 0.1 M Tris (pH 8.3) containing 0.2 % Triton X-100 and 10 mg of enzyme-free sucrose (Mann Research Laboratories) and centrifuging the homogenates at 10,000 g for 5 min at 4”. Then lo-50 cl1 aliquots of the supernatant were layered on gels, and electrophoresis was carried out in a 2” room with the gel temperature at 8-10”~ for 2.5-3 hours at a current of 20-25 mA. The Tris-glycine running buffer (0.018 M Tris, pH 8.3) also contained 2 X 10m3M cupric chloride. At the completion of electrophoresis, the gel sheet was cut into longitudinal strips; one strip from each sample was fixed immediately with 50 % TCA to check whether RNase activity had been effectively inhibited during electrophoresis, and the remainder were washed in 0.1 M sodium acetate buffer (pH 5.4) containing 0.01 M EDTA at 0” for 45-60 min to chelate the cupric ion. Strips were then incubated in 0.1 M sodium acetate buffer (pH 5.4) with 0.002 M EDTA at 37“ over a range of times to allow RNase to react with the RNA in the gel. They were then fixed for 10 min in 50 % TCA, were washed in water, stained in 0.02 % aqueous toluidine blue, and destained in water. Zones of RNase activity appeared as clear bands against the blue background of stained

RNA fixed in the gel (Fig. 1). Toluidine blue gave more contrast than acridine orange, and did not stain any protein that was present. Since band width increased linearly with time of incubation (Fig. l), it was used for comparing the activities of RNase isozymes in gels incubated under identical conditions. Estimations of protein in samples were carried out according to Lowry et al. (1951). RESULTS

Total

RNase Actitity During Growth of Healthy and First Systemically Injected Leaves

The RNase activity of expanding healthy Chinese cabbage leaf was at a maximum (8.6 cpm per minute per 10 mg fresh weight) when the leaf was 3-5 cm long, falling to a minimum (2.2 cpm per minute per 10 mg fresh weight) 5 days later when the leaf was 9-10 cm long. Over the next 9 days activity remained low, but showed small fluctuations. In the first systemically infected leaf, no change was observed in the normal pattern of total RNase activity when TYMV commenced its initial rapid replication (5-7 days after inoculation of the plant). Total RNase Activity in TYMV Leaves Showing Mosaic

Infected

With leaves showing mosaic symptoms, the total RNase activity in yellow areas was 1.7-2.8 times greater than in comparable dark green tissue (Table 1). In experiments comparing the total RNase activity of healthy leaves with yellow areas TABLE

1

TOTAL RNASE ACTIVITY OF COMPARABLE DARKGREEN AND YELLOW AREAS OF SYSTEMICALLY INFECTED CHINESE CABBAGE LEAF SHOWING MOSAIC SYMPTOMP Expt. No. 1 2 3

Dark green 9.5 5.0 2.5

Yellow 16.5 14.0 5.5

Yellow/ dark green 1.70 2.80 2.20

a Activity is expressed as loss of radioactivity in counts per minute per minute from ‘GsRNA substrate.

11IBONUCLEASE

ISOZY?VIES

IN

CHISESII:

CABBSGE

.i.jU

of mosaic-exhibiting leaves of the same size, from different plants, the infected leaves showed between two and six times more RXase activit,y on an equal fresh weight basis. pH optima of RNase a&vity in crude clarified homogenates from infected and healthy leaves were compared over the range pH 4.0-7.0. The optimum in each case occurred between pH 5.5 and 6.0, with no indication of optima at other pH’s in this range. Changes in RNase Isozymes in Leaf Systenzicably Infected with TYX V Time course st’udies of changes in RNase isozyme patterns mere carried out with two strains of TYRIV of differing symptom severity. Four days after inoculation (before any virus could be dete&ed in uninoculated leaves) leaves approximately 3 cm long from both healthy and inoculated plants showed the presence of three RNase isozymes-designated as Rn’ases I, II, and III in order of increasing electrophoretic mobility (Figs. 1 and 2). (,4 diffuse zone of activity adjacent to the origin was ignored here because it appeared before the gel was incubated.) In

Plo. 1. KNasc isozyme activity (I, ZZ, ZZZ) in ext,racts of healthy and systemically infected leaves 12-15 cm long. Strips labeled 0 were not incubated and show clearing of the gel adjacent t,o the origin. Other strips were incubated after electrophoresis for 30 min, 60 min, and 150 min and show the increase in width of the zones with inrreasing time of incubation.

FIG. 2. Densitomet,er traces of photographic negatives of gels, showing relative concentrations of RNase isozymes (I, ZZ, ZZZ) iu expanding leaves of Chinese cabbage systemically infected with a pale yellow strain of TYMV, and itl eqrlivalent healthy leaves. Assays are shown of tissue harvested 4 days (a, O), 8 days (c), 14 days (cl), and 16 days (e) after t,he time of inoculation. Gel a was fixed immediately after electrophoresis, gels b-e were incubated before fixing. IIealt,hy and infected leaf extracts at each time were nssayed under identical conditions. Origin and bluffer front are marked by arrows, alld movemckllt is from right to left

the healthy leaf, RKase III declined in activity during growth, and became undetectable (except after prolonged incubat,ion) when leaves were 12-15 cm long. In leaves infected with either of the tIyo isolates of TTMV, RNase III act,ivity declined as in healthy leaf until 7 or S days after inoculation. From then until the last sample at 16 days, RNase III showed a resurgence of activity (Fig. 2). RNases I and II were slightly more active in infected Ieaf compared on an equal fresh weight basis with healthy leaf. No marked differences were

560

RANDLES

observed in the patterns obtained with the two strains. Changes in RNase Isoxymes in TuMV-Infected Leaf

TABLE

3

RNASE ISOZYMES ASSOCIATED WITH CELL FRACTIONS FROM HEALTHY CHINESE CABBAGE ASSAYED ON POLYACRYLAMIDE GELS” CN

M

R

s

To study the effect of a virus unrelated to TYMV, RNase isozymes were examined in Protein content 1450 1020 260 70 of sample (fig) the first leaf to show systemic symptoms of 4 4 4 1 TuMV. Ten days after symptoms first ap- Time of incubation (hr) peared, infected and healthy leaves of the Origin ++ ++ ++ ++ same length (10 cm) were sampled. R,ibonuclease As with TYMV at this stage of infection, I + + ++ RNase III appeared only in extracts from II + +ithe TuMV infected leaf. Bands I and II III + ++ were again broader in the infected extract, showing that on an equal fresh weight basis, 0 Symbols: ++, Positive activity; +, trace achigher RNase activity is associated with tivity; -, no activity. systemic TuMV infection. RNase Isozymes in TYMV-Inoculated

Leaf

To study the effect of inoculation, and to provide a comparison between inoculated and systemically infected leaf, RNase isosymes were assayed just before, and 0.5 hour, 8 hours, 1 day, 2 days, 3 days, 4 days, and 7 days after mechanical inoculation of fully expanded half-leaves with TYMV. Adjacent control half-leaves were rubbed with water. RNase III was almost undetectable in leaves assayed before rubbing and one-half hour after rubbing. At 8 hours, both inoculated and control half-leaves showed greatly increased RNase III activity, and a smaller rise in RNase II activity. Isozyme patterns of the inoculated and control half-leaves then remained identical until day 3, when the control tissue showed a decline in RNase III activity that continued until day 7. In infected tissue, RNase III activity remained high for the 3-7 days after inoculation. TABLE

2

RNASE ACTIVITY IN CN AND SUPERNATANT FRACTIONSOF HEALTHY AND SYSTEMICALLY INFECTED CHINESE

CABBAGE LEAF”

Fraction

Infected

Supernatant CN

10.8 0.67

Healthy 5.1 0.40

0 Activity is expressed as loss of radioactivity in counts per minute per minute from WI!-sRNA substrate.

Total RNase Activity Nuclei

of Chloroplasts

and

CN fractions were prepared from cell-free extracts of healthy and TYMV-infected leaf as described in Materials and Methods. The surface of the pellet was rinsed once with water, then the pellet was resuspended in 0.1 M sodium acetate buffer (pH 5.2). All the 1000 g supernatant from the CN fractionation was adjusted to pH 5.2 by adding 1.0 M sodium acetate buffer. The CN and supernatant fractions were made up to the same volume and were incubated before assay for 45 min at 37” to allow lysis of organelles and some hydrolysis of RNA which might compete with the E. coli 14CsRNA substrate. The CN fraction from both healthy and infected leaf had much lower activity than the supernatant (Table 2). In another experiment, the unwashed CN pellet from healthy leaf was fractionated on a discontinuous density gradient (see Materials and Methods). Fractions obtained by dripping from the bottom of the tube were assayed for RNase activity. No detectable activity was associated with the nuclear pellet, either of the two chloroplast fractions, or the clear zones between them. All activity was confined to the zone above the upper chloroplast band. RNase Isozymes Associated with ganelles

Cell Or-

Washed CN, M, R, and S fractions from both healthy and TYMV-infected leaf of

IlIBORUCLEASE

ISOZYMES

Ih- CXIINESE

CABBAGI:

.iti 1

inoculation is approximately t’he same ati in systemically infected leaf of the same size. It is possible t’hat RKase III has a function when cells are actively synthesizing RKA. Levels of RSA are raised in actively growing leaf, in leaf injured by rubbing (Fry and Matthews, 1963), and in leaf supporting virus multiplicat’ion. Increased RSwe III activity may be a response t’o events that, cause rapid increases in cell metabolic a(:tivity. There is a possibility that the technique of inhibiting plant RKase activity with Cu2+ ions during electrophoresis, then removing the Cu2+ (and other divalent cations) with EDTA before incubat’ion may have inacDISCUSSION tivated a virus-induced isozyme. There are report#s of different ionic requirements for Results described here shorn that systemic Rxases, and EDTA has been reported as infection with TTJIV, where no mechanical having varying effect)s on RKase act’ivity damage is implicat’ed, causes increased RKase activity. The increase appears to be (Jlatsushit,a and Ibuki, 1960; Wilwn and Shannon, 1963; Rrishnan, 1964). It may he confined to parts of the leaf containing virus, advisable to incubate gels in the presence of since dark green “virus-free” areas (Reid and Matthews, 1966) possess activity of the different ions in further searches for virusinduced RSase isozymes. same order as healthy leaf. Apart from R??A replicase (As&r-JIaniSeveral suggestions can be made to explain facier and Cornuet,, 1965), no virus-coded this increased RSase activity in infected enzymes have been found in infected plan&. leaf: (1) One or more of the RNase isozymes normally present in the host becomes more UMP kinase (Gilliland el al., 1966; Gilliland and Symons, 1967) has been shown to inactive. (2) A host isozyme not normally crease after virus infection, but this is approduced in leaves is induced by the virus. parently due to the virus-induced i;t imula(3) a new virus-specific isozyme, coded on t,ion of host enzymes, not to the synthesis of the virus genome is produced. 1\\To new isozyme is detectable in sys- a new virus specific enzyme. A newly syntemically infected leaf. The increase in thesized peroxidase isozyme has heen deRNase activit!y t)hat follows infection does tected in infected bean leaf (Farka:: and Stahmann, 1966; Solymosy et al., 1967), but not occur unt’il after TlYlV begins to multhis is also a host enzyme, and it appears tiply rapidly, and it appears t’o be attributable only to changes in the activity of host after infection wit’h different viruses. In agreement’ with studies on pen cotyleRKase isozymes. Of t#he three isozymes, dons (Barker and Hollinshead, 1967) and RNase III shows the greatest increase in tobacco (Ragi and Farkas, 1967), most, of the activity aft’er infection. This increase is not RKase activit)y of the Chinese cabbage is specific to infection of Chinese cabbage with associat’ed with the cxtoplasmic supernatant, TYI\IV, because (1) the isozyme is also very active in the early st’ages of rapid leaf and all three R?Y’aselsozymes appear in this fract8ion. Slost’ of t,he activity assoc&ed with growth, (2) it) appears after systemic infecorganelles is relat,ively immobile in Ihe gel tion with Tu;\IV, a virus unrelated to and may be bound to large molecules or TYhIV, and (3) it increases after mechanical membrane fragments. Contamination wit,h injury. Virus infection and injury combined (as in mechanically inoculated leaf) prolongs soluble RNase may account for some of this the period of high Rn’ase III activity over activity (Wilson and Shannon, 1963). t,ha-Ltoccurring in injured healthy leaf. The Gel electrophoresis permit,s t’he reprolevel of RNase III activity 4-7 days after ducible fractionation and rapid semiquantimean length 9 cm were homogenized with an equal volume of 0.1 M Tris (pH 8.3) containing 2.0 % TritonX-100, and were assayed for RNase isozyme activity. All three isozymes occurred in the 105,000 g supernatant from both healthy and infected leaf at a much greater concentration per unit weight of protein than they did in any of the organelle fractions. The activit’y that was detected in the CK, AI, and R fractions was largely confined to the sample origin of the gel, wit’h only a trace in the other positions (Table 3). Infection caused no change in the isozyme pattern of any of the organelle fraci.ions.

562

RANDLES

tative assay of small amounts of RNase isozymes in crude plant extracts, and should facilitate studies on changes in RNase-for example, during growth and development of plants. By varying factors such as pH, temperature, and the metal ion inhibitors of RNase, it is possible that one could adapt the method to other studies, with the advantage of better resolution over that achieved by Ressler et al. (1966) for assaying RNase isozymes. Two drawbacks to assaying RNase isozyme activity in crude extracts with this system should be mentioned. In some gels, faint bands corresponding in position to bands of acid phosphatase activity have been observed. These could be confused with low activity RKase isozymes, and so it is advisable to check the distribution of phosphatase and possibly phosphodiesterase in material assayed on polyacrylamide gels. Secondly, the clearing near the sample origin of the gel can interfere with the assay of RNase I activity. High-speed clarification of extracts can reduce this. This activity at the origin has not been attributed to a separate isozyme in this study. Other workers have isolated from one (Bagi and Farkas, 1967) to three (Wilson, 1963) RNases from plant tissue, using other fractionation procedures. The relationship of the isozymes fractionated by electrophoresis to RNases obtained by chromatographic methods is yet to be established. ACKNOWLEDGMENTS

I wish to thank Dr. P. R. Fry for providing inoculum, and Dr. P. L. Bergquist for providing E. coli W-sRNA. I am indebted to Professor R. E. F. Matthews, Dr. R. L. Bieleski, and Mr. M. S. Reid for helpful discussion, and gratefully acknowledge assistance gained from a New Zealand Postgraduate Scholarship. This work was supported in part by U.S.P.H.S. grant AI-04973.

TuMV

damaged

tobacco

leaf

tissues.

The degradation of ribonucleic acid in the cotyledons of Pisum arvense. Biochem. J. 103, 230-237.

BOYD, J. B., ~~~MITCHELL, H. K. (1965). Identification of deoxyribonucleases in polyacrylamide gel following their separation by disk electrophoresis. Anal. Biochem. 13, 28-42. CHALCROFT, J. P., and MATTHEWS, R. E. F. (1967). Role of virus strains and leaf ontogeny in the production of mosaic patterns by turnip yellow mosaic virus. Virology 33, 659-673. DAVIS, B. J. (1964). Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acarl. Sci. 121, 404-427. DAVIS, F. F., and ALLEN, F. W. (1955). The action of ribonuclease on synthetic substrates. J. Biol. Chem. 217, 13-21. DIENER, T. 0. (1961). Virus infection and other factors affecting ribonuclease activity of plant leaves. Virology 14, 177-189. FARKAS, G. L., and STAHMANN, M. A. (1966). On the nature of changes in peroxidase isozymes in bean leaves infected by southern bean mosaic virus. Phytopathology 56, 669-677. FRY, P. R., ~~~MATTHEWS, R. E. F. (1963). Timing of some early events following inoculation with tobacco mosaic virus. Virology 19, 461-469. GILLILAND, J. M., and SYMONS, R. H. (1967). Partial purification and properties of ribonucleotide kinases in virus-infected and healthy plants. Virology 33, 221-226. GILLILAND, J. M., LANGMAN, R. E., and SYMONS, R. H. (1966). Properties of the ribonucleotide kinases after infection

of cucumber

with tobacco

ringspot virus. Virology 30, 716-723. HONDA, S. I., HONGLADAROM, T. and LATIES, G. G. (1966). A new isolation medium for plant organelles. J. Exptl. Botany 17, 460-472. KRISHNAN, P. S. (1964). Enzymes of phosphate metabolism. “Modern Methods of Plant Analysis” (H. F. Linskens, B. D. Sanwal, and M. V. Tracey, eds.), Vol. 7, pp. 21-66. Springer-Verlag, Berlin. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275.

REFERENCES ASTIER-MANIFACIER, S., and CORNUET, P. (1965). Isolation of turnip yellow mosaic virus RNA replicase and asymmetrical synthesis of polynucleotides identical to TYMV-RNA. Biochem. Biophys.

Res. Commun. 18, 283-287. BAGI, G., and FARICAS, G. L. (1967). On the

nature of increase in ribonuclease

mechanically

Phytochemistry 6, 161-169. BARKER, G. R., and HOLLINSHEAD, J. A. (1967).

activity

in

MATSUSHITA, S., and IBUKI, F. (1960). Ribonucleases in the microsomes from pea seedlings. Biochim. Biophys. Acta 40, 358-359. REDDI, K. K. (1959). Tobacco leaf ribonuclease.

III. Its role in the synthesis of tobacco mosaic virus nucleic acid. Biochim. Biophys. Acta 33, 164-169. REID, M. S., and BIELESKI, R. L. (1968). A simple

RIBONUCLEASE

ISOZYMES

apparatus for vertical flat-sheet polyacrylamide Anal. Biochem. 22, 374-381. gel electrophoresis. REID, $1. S., and MATTHEWS, R. E. F. (1966). 011 the origin of the mosaic induced by turnip yellow mosaic virlls. Virology 28, 563-570. RESST~EIL, N., OLIVERO,~~., THOMPSON, G. R., and JOSEPH, 1~. K. (1966). Investigation of ribonuclease isozymes by an electrophoretic ultraviolet method. ,\‘atlhre 210, 695698. SHISDE, B. G., and SASTILLI, \.. (19G7). Effect of actinomycin I) 011 the tobacco mosaic virus illfeci.ioll-irldrlced increase in ribonuclease ac1ivity. Z’h?/2opathology 37, 315. SHINUE, B. G., CHANDRASEKHAR, B. K., and SANYILLI, V. (1964). Distribution of ribonuclease in sltbcellular fractions of Imtreated, wounded, :illd T&11- infected Pinto bean leaves. Phytop”hh~g~/ 5.4, 908.

IX

CHINESE

CABBAGE

x3

SOLYMOSY, F., SZIRMAI, J., BECZNER, L., and FARKAS, G. L. (1967). Changes in peroxidaseisozyme patterns induced by virus infection. Virology 32, 117-121. SPENCER, L)., and WILDMAX, H. G. (1964). The incorporation of amino acids into protein by cell-free extracts from tobacco leaves. Riochemi.slr~/ 3, 954-959. TEV-AKI, J%. li., and WILDMAN, S. (;. (19(i6). Chloroplast, l)?jil from khacro leaves. Scipncc 153, 1269S1271. WILSON, C. 11. (1963). Chromatographic scparation of ribonucleases in corn. Biochiur. Biophys. iicla 68, lii~18~. WILYOX. C. >I., and SHANSON, J. C. (1963). The distribllt ion of ribonucleases in corn, CIICIIIIIher, and soybean seedlings. Effects of isolar iota media. RiocAi~,. Biophyx. ,Icln 68, 311~31:1.