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Properties of the products of UTP incorporation by cell-free extracts of leaves infected with bromegrass mosaic virus or with broadbean mottle virus
VIROLOGY
40, 244-250 (1970)
Properties
of the Products
of Leaves
Infected
of UTP Incorporation with
Bromegrass
Broad bean
Mottle
Mosaic
by Cell-Free Virus
Extracts
or with
Virus
J. SEMAL des Sciences agronomiques, Gembloux, Belgium
Faculth
Accepted September 9, 1969
After deproteinization with phenol and detergent, the product of UTP-3H incorporation by a particulate fraction prepared from barley leaves infected with bromegrass mosaic virus displayed properties consistent with those of a double-stranded RNA. The major part of the label incorporated in the product was resistant to pancreatic ribonuclease (RNase) (5 pg/ml) in 1 X SSC, but was made acidsoluble by keatment with RNase in 0.1 X SSC. Similar results were obtained with the deproteinized product of UTP-3H incorporation by extracts from broadbean leaves infected with broadbean mottle virus. When the deproteinixed product synthesized by extracts from bromegrass mosaic virus-infected leaves was heated in 1 X SSC at increasing temperatures, a sharp transition from RNase resistance to RNase sensitivit.y was observed, with a T, of 99-100”. With both bromegrass mosaic virus and broadbean mottle virus, the particlebound product of UTP-3H incorporation was double-stranded in its native state, as suggested by RNase resistance in 1 X SSC and RNase sensitivity in 0.05 X SSC, respectively, of the radioactive product associated with the particulate leaf fraction. The native products of UTP-3H incorporation were sensitive to RNase in 0.05 X SSC without any pretreatment of the leaf extracts. MATERIALS
INTRODUCTION Upon incubation in the presence of actinomycin D (AMD) and the necessary ingredients for RNA synthesis, UTP-3H was incorporated into an acid-insoluble product by a particulate fraction prepared from cell-free of barley leaves infected with extracts
bromegrass mosaic virus (BMV) Hamilton,
1968).
Similar
results
(Semal and mere ob-
tained with a comparable fraction prepared from broadbean leaves infected with broadbean mottle virus (BBMV) (Semal, 1969). The products of UTP-3H incorporation have been characterized further, using the two plant-virus systems mentioned above; they displayed properties consistent with those of double-stranded RNA, either in their native state or after deproteinization.
AND METHODS
Xtandard assay for UTP-3H incorporation and study of RNase resistance. The leaf extracts were prepared and incubated with UTP-3H, AMD, and the necessary ingredients for RNA synthesis, as previously described (Semal and Hamilton, 1968; Semal, 1969). The leaf homogenates were filtered and centrifuged for 5 min at 1000 g; the supernatants were collected and centrifuged for 15 min at 10,000 g; the pellets thus obtained were resuspended in the incubation medium to give the fraction 1000-S/10,000-P, which was used for the incorporation experiments. Study of the ribonuclease (RNase) resistance of the deproteinized products was performed as follows: after 20 min incubation with UTP-3H, the leaf material was mixed with 244
VIRUSES
AND
UTP
245
INCORPORATION
Laboratories, Rahway, New Jersey. Tricyclohexamine salt of phosphoenolpyruvic acid, pyruvate kinase, and ribonuclease A were purchased from Sigma Chemicals Co. UTP-3H and the unlabeled nucleotides were obtained from Schwarz BioResearch Inc. RESULTS
E$ect of Salt Concentration on the RNase Sensitivity of the Labeled Product
SALT
CONCENTRATION
(X
SSC)
FIG. 1. Effect of salt concentration on the RNase resistance of the labeled product of UTPfH incorporation by the fraction 1000-S/10,009-P prepared from leaves of barley plants infected for 7 days with BMV. The deproteinized RNA product was resuspended in increasing salt concentration (up to 1 X SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7) and incubated with RNase (5 rg/ml, 30 min at 37”). Control tubes were treated similarly but without RNase. The acid-insoluble radioactivity was determined by Millipore filtration in the presence of carrier protein. Combined results of two experiments.
unlabeled UTP and treated with phenol and sodium lauryl sulfate; the RNA’s were precipitated with ethanol, resuspended in appropriate solution, incubated with RNase, mixed with carrier protein, and tested for acid-insoluble radioactivity. For studying the RNase resistance of the particle-bound native product, the material was treated as follows: after incubation of the extract (20 min at 30”) with UTP-3H in the presence of 20 pg/ml of AMD, unlabeled UTP (0.2 ml at 12.5 mg/ml) was added to each tube; the extracts were cooled and centrifuged at 2” for 15 min at 10,000 g. The pellets (fraction “P”) were resuspended and incubated with pancreatic RNase, as described under “Results” for individual experiments. Blanks were treated the same way, except that the leaf fraction was omitted during incubation with UTP-3H and added immediately thereafter; they consistently gave a final radioactivity of lo-20 cpm per tube, which was substracted from the other results. Actinomycin D was a gift from Merck, Sharp & Dohme Research
The effect of RNase on the deproteinized labeled product of UTP-3H incorporation was studied under various conditions of ionic strength. As shown in Fig. 1 for the BMV-barley system and in Table 1 for the BBMV-broadbean system, the labeled products were largely resistant to RNase (5 pg/ml, 30 min at 37”) in 1 X SSC (0.15 M NaC1, 0.015 M sodium citrate, pH 7), but were rendered acid-soluble by treatment with the enzyme at low salt concentration. TABLE
1
EFFECT OF SALT CONCENTRATION ON THE RNASE RESISTANCE OF THE RADIOACTIVE PRODUCT OF UTP-3H INCORPORATION IIY EXTRACTS OF BROADBEAN LEAVES INFECTED WITH BBMVa Treatment concentration ot : d’ eproteinized esuspending mediur
=g. srbit 1
2
1.00 0.30 0.10 0.01 2.06 1.00 0.50 0.25 0.12 0.06
x x x x x x x X x X
sscc ssc ssc ssc ssc ssc ssc SSC ssc SSC
of the
product b
RNase
Control
449d 47 6 20 731 540 353 156 25 42
634 408 413 421 1.038 1.052 1.028 883 918 927
* _-
L
71 12 1 5 70 51 34 18 3 5
a Fraction 1000-S/10,000-P was prepared from broadbean plants infected for 7 days with BBMV; it was incubated with UTP-3H, AMD (20 rg/ml), and the necessary ingredients for RNA synthesis (Semal, 1969). b The deproteinized RNA product was treated with 5 pg/ml of RNase for 30 min at 37”; controls were treated similarly, but without RNase. c SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7. d Results expressed as acid-insoluble counts per minute in the RNA fraction.
246
SEMAL
TEMPERATURE
FIG. 2. Thermal transition of the RNase resistance of the labeled product of UTP-aH incorporation by the fraction 1000-S/10,000-P prepared from leaves of barley plants infected for 7 days with BMV. The deproteinized RNA product in 1 ml of 1 X SSC was sealed in glass tubes, heated for 10 min at the temperature indicated, cooled rapidly, and subjected to digestion with RNase (5 rg/ml, 30 min at 37”). Control tubes were treated similarly but without RNase. The acid-insoluble radioactivity was determined by Millipore filtration in the presence of carrier protein. Combined results of two experiments.
cabbage leaves infected with turnip yellow mosaic virus (TYMV) was shown to be double-stranded prior to any phenol treatment (BovB et al., 1968). It was of interest to study this aspect with the plant-virus systems under investigations in the present research. It was found that more than 80% of the label incorporation by the 1000-S/10,000-P fraction from BMV-infected barley sedimented at 10,000 g when centrifugation was performed immediately after 20 min incubation with UTPJH; most of subsequent experiments were carried out with this 10,000 g pellet (fraction “P”). After resuspension in either 1 X SSC, or in 0.05 X SSC, the fraction “P” was incuTABLE
IIResuspending medium Pretreatment of fraction “P
Eflect of Temperature on the RNase Sensitivity of the Labeled Product Synthesized by the BMV-Barley System
A, None
The effect of temperature on the RNase sensitivity of the labeled product was studied with the BMV-barley system. As shown in Fig. 2, heating the RNA product for 10 min at increasing temperatures in 1 X SSC, followed by rapid cooling, led to a sharp thermal transition from RNase resistance to RNase sensitivity, with a T,,, of 99-100”.
B. Freezing thawing
RNase Sensitivity of the Native Labeled Product with the BMV-Barley System It has been suggested with RNA phages that the RNase resistance of the doublestranded deproteinized product of viral RNA polymerase resulted from deproteinization with phenol and detergent, the product being mostly single-stranded and RNase sensitive in its native state (Borst and Weissmann, 1965; Feix et cd., 1968). However, the nascent RNA synthesized by a cell-free particulate fraction extracted from
2
EFFECT OF SALT CONCENTRATION AND EFFECT OF PREFREEZING OF FRACTION lip” ON THE RNASE RESISTANCE OF THE NATIVE PRODUCT OF UTP INcORPORATIONQ
and
0.05 x ssc +
Sic”+ RNase 779c 819 703 807 773 863 842 848
of fraction “P”
RNase
761 948 735 840 817 846 817 827
113 137 97
‘%” 772 898
110
48 53 53 63
772 849 738 795
a Fraction 1000-S/10,000-P was prepared from barley plants infected for 7 days with BMV, and incubated with UTP-3H as previously described (Semal and Hamilton, 1968). b After incubation with UTP-3H, the leaf extract was centrifuged for 15 min at 10,000 g; the was frozen for 15 min at pellet (fraction “P”) -15”, thawed, resuspended in appropriate salt solution, and treated with 20 fig/ml of RNase (30 min at 37”); control tubes were treated similarly but without RNase. After deproteinization with phenol and detergent, the RNA was precipitated with ethanol in the presence of carrier yeast RNA. c Twin experiments with duplicate aliquots; results are expressed as acid-insoluble cpm in the RNA fraction after deproteinization and ethanol precipitation.
VIRUSES
AND
UTP
bated with RNase (20 or 40 pg/ml, 30 min at 37”), and the reaction was terminated by addition of phenol and detergent. Deproteinization and isolation of the RNA’s by ethanol precipitation were performed as previously described (Semal and Hamilton, 1968) with addition of carrier yeast RNA (50 b&ml) ; control tubes were treated similarly but without RNase. The acidinsoluble radioactivity associated with the RNA fraction was then determined by Millipore filtration and scintillation counting. The results thus obtained are presented in Table 2 A; they indicate that the native product of UTP-3H incorporation was resistant to RNase when the fraction “P” was incubated with the enzyme in 1 X SSC, but that it was made acid soluble upon RNase treatment of the fraction “P” in 0.05 X SSC. Bov6 et al. (1968) reported that, with the TYMV-cabbage system, prefreezing and thawing of the leaf extract was necessary to induce a pronounced sensitivity of the native double-stranded RNA product to RNase in 0.05 X SSC. As indicated in Table 2 B, freezing and thawing of the fraction “P” with the BMV-barley system did not change notably the effect of RNase in 1 X SSC, and the residual RNase resistance in 0.05 X SSC was decreased. E$ect of Pretreatment of the Fraction “P”, on the Subsequent RNase Resistance of the Native or the Deproteinized Product of UTP-3H Incorporation, with the BMVBarley System Deproteinization of the labeled fraction “P” in 0.05 X SSC, as performed in Table 2, could possibly lead to the digestion of the deproteinized product of UTP-3H incorporation by traces of RNase carried through the steps of phenol treatment and ethanol precipitation. The possibility was also considered that the access of RNase to the site of the native product could be modified by exposing fraction “P” to low salt concentration: under such conditions, a singlestranded RNA could appear as resistant to RNase in 1 X SSC (site closed to RNase action) while being sensitive to the enzyme in 0.05 X SSC (site open to RNase action). Experiments were carried out to clarify these questions.
INCORPORATION
247
In order to test the possible effect of residual RNase on the deproteinized total product of UTP incorporation, fraction 1000-S/10,000-P was incubated for 20 min with UTP-3H; incubation was followed by addition of unlabeled UTP to minimize further incorporation. The total incubated material was then adjusted to 1 X SSC, and aliquots were treated for 30 min at 37” with 20 pg/ml of RNase (controls were treated similarly without RNase). After deproteinization, the RNA was isolated, resuspended, and incubated further for 30 min at 37”. When resuspension of the RNA was in 1 X SSC, the acid-insoluble radioactivity was 992 cpm for the control sample and 841 cpm for the sample previously treated with RNase; when resuspension was in 0.05 X SSC, the values were 840 and 686, respectively. This result indicates that upon deproteinization, the labeled native product preincubated in 1 X SSC + RNase (20 pg/ml) yields an RNA which is stable in 0.05 X SSC. At this stage of the procedure, however, this RNA was made entirely acid-soluble upon incubation in 0.05 X SSC with 5 pg/ml of RNase (25 and 10 cpm, respectively). In further experiments, the fraction “P” material in 0.05 X SSC was adjusted to 1 X SSC immediately after RNase treatment, prior to addition of phenol and detergent, in order to ensure the stability of the deproteinized product and to obtain similar conditions of deproteinization for all samples. The RNA product thus isolated was tested for its resistance to RNase digestion in 1 X SSC. The effect of preincubating the fraction “P” in 0.05 X SSC, on its subsequent resistance to RNase in 1 X SSC, was also investigated. When compared with those of Table 2, the results (Table 3) show little effect of adjusting the salt concentration to 1 X SSC prior to deproteinization, or of preincubating the fraction “P” material in 0.05 X SSC before RNase treatment in 1 X SSC. RNase Resistance of the Native Product of UTP-3H Incorporation with the BBMVBroadbean System The fraction 1000-S/10,000-P was prepared from young leaves of broadbean
248
SEMAL
TABLE
3
EFFECTS OF PRETREATMENT OF FRACTION “P” ON THE SUBSEQUENT RNASE RESISTANCE OF THE NATIVE AND DEPROTEINIZED PRODUCT OF UTP-JH INCORPORATIONS
-
Treatment of the leproteinized prod&
Preincub?tion of %,&0n P
-
-
1X BC -IRNase
None
1 X + 1 x 0.05
ssc RNase ssc x ssc
-
bs”
_-
665
699
683 50
699
545 528
680 645
560 63
710 71
652
690
110
+ RNase Preincubation in 0.05 x ssc
0.05 1 x + 1 x 0.05
x ssc ssc RNase ssc x ssc
-
None
Preincubation in 0.05 x ssc
x ssc ssc RNase ssc x ssc RNase x ssc ssc RNase ssc x ssc RNase x ssc
-
-
81
incubation,
fraction 1000-S/10,000-P from leaves of Chinese
The pared
TABLE
was precabbage
4
RNASE RESISTANCE OF THE NATIVE PRODUCT OF UTP-3H INCORPORATION WITH THE BBMV-BROADHEAN SYSTEY~ -
40 69
60 -
-
-
-
-
all samples
were adjusted
to 1 X SSC and deproteinized with pheno1 and detergent, The RNA was precipitated with ethanol in the presence of carrier yeast RNA. d The RNA product was resuspended in 1 X SSC and treated as indicated (no treatment, or incubation for 30 min at 37”, either in 1 X SSC or in 1 X SSC + 5 pg/ml RNase).
plants infected for 5-7 days with BBMV, and was incubated for 20 min with UTP-3H as previously described (Semal, 1969). As shown in Table 4, the labeled product
-
?retreatment
I’
of fraction “PI>
0.05x ssc + RNase
5 I
Preincubation in 0.05 x ssc -
* Fraction
427 499 390 469 413 419 347
372 447 393 429 401 488 357
None -
06 55
Expt. Nab
None
a Fraction “P” was prepared from leaves of barley plants infected for 9 days with BMV. Results are presented as acid-insoluble counts per minute associated with the RNA fraction. b Fraction “P” was preincubated for 15 min in 0.05 X SSC before being subjected to treatment with RNase (40 pg/ml) in either 1 X SSC or 0.05 X ssc. c Fraction ‘(P” was incubated for 30 min at 37”. After
RNase Resistance of the Native Product of UTP-3H Incorporation with the TYMVCabbage System
Treatment of fraction “P”c -
+ RNase 0.05 1 x + 1 x 0.05 + 0.05 1 x + 1 x 0.05 + 0.05
associated with the fraction “P” of BB?(‘rTVinfected broadbean displayed properties of RNase resistance in 1 X SW and of RNase sensitivity in 0.05 X SSC, similar to those observed with BkIV-infected barley.
-
0.05 x SSC
--
95
343 426 560 566 393 468 378
98 68 84 80 96 64
-
1000-S/10,000-P was prepared from
leaves of broadbean plants infected for 5 days (experiment 2) or for 7 days (experiments 1 and 3) with BBMV; it was incubated with UTP-3H as previously described (Semal, 1969). b Experiment 1: Results of duplicate aliquots; samples were adjusted to 1 X SSC before deproteinization. Experiment 2: Twin experiments with duplicate aliquots; samples not adjusted to 1 X SSC before deproteinization. Experiment 3: fraction “P” was preincubated for 15 min in 0.05 X SSC prior to RNase treatment; samples were adjusted to 1 X SSC before deproteinization. c After incubation with UTP-*H, the leaf extract was centrifuged for 15 min at 10,000 g, to yield a pellet (fraction “P”). Fraction “P” was resuspended in 1 X SSC or 0.05 X SSC and incubated for 30 min at 37” with RNase (40pg/ml); control tubes were incubated without RNase. Fraction “P” was then deproteinized with phenol and detergent, the RNA was precipitated with ethanol in the presence of carrier RNA, and the acid-insoluble radioactivity (cpm) of the isolated RNA was determined.
VIRUSES TABLE
AND
5
RNASE
RESISTANCE OF THE NATIVE PRODUCT UTP-3H INCORPORATION WI’IH THE TYMVCABBAGE SYSTEM” Treatment
Expt. No.
1 2
UTP
OF
of fraction “P”5
1 x ssc
590 782 594
594 766 548
407 458 312
664 772 586
a Fraction 1000-S/10,000-P was prepared from leaves of Chinese cabbage systemically infected and incubated with UTP-3H as with TYMV, described for the BM\-barley system. (Semal and Hamilton, 1968). b After incubation, the leaf extract was centrifuged for 15 min at 10,000 9, to yield a pellet (fraction “P”). Fraction “P” was resuspended in 1 X SSC or 0.05 X SSC and incubated for 30 min at 37” with RNase (40 pg/ml); control tubes were incubaled without RNase. Fraction “P” was then adjusted to 1 X SSC and deproteinized with phenol and detergent; the RNA was precipitated with ethanol in the presence of carrier RNA, and the acid-insoluble radioactivity (cpm) of the isolated RNA was determined.
systemically infected with TYJIV, and was incubated with UTP3H as described for the BhIV-barley system. Table 5 presents the results of two independent experiments, which indicate that the labeled product associated with fraction “P” was completely resistant to RNase in 1 X SSC and 50-60 % resistant in 0.05 X SSC. DISCUSSION
The deproteinized product of UTPJH incorporation by a cell-free particulate fraction prepared from BMV-infected barley leaves, displayed properties similar to those of an RNA associated with a complementary polynucleotide into a double-stranded structure. According to the data presented by Guschlbauer et al. (1968), the thermal transition temperature TrnlXss~obtained for the product synthesized by the BMV-barley system (99-100”) fits rather well with that expected for a fresh preparation of a doublestranded RNA with a GC content of 0.464, as determined for the large component of BMV-RNA by Bockstahler and Kaesberg (1965). Part of the deproteinized product with the BB>IV-broadbean system behaved
INCORPORATION
249
also as an RNA associated into a doublestranded structure. With the BMV-barley and the BBMVbroadbean systems, the behavior of the native RNA product of nucleotide incorporation toward the action of RNase was in agreement with the properties of the product obtained for the TYMV-cabbage system by Bove et al. (1968), in that the particle-bound product of UTP incorporation, in its native state, was almost completely resistant to RNase ihn 1 X SSC. This situation differs from that described for an RNA phage by Borst and Weissmann (1965) and by Feix et al. (1968), where the product of nucleotide incorporation was RNase-sensitive in the native state, but became RNase-resistant upon deproteinization. However, comparisons in this matter between plants and RNase-deficient bacteria are made difficult because of differences in the amounts of endogenous RNase and in the efficiency of nucleotide incorporation. It may be that a specific RNase-resistant RNA product is selectively isolated with the plant system, which corresponds only to a minute fraction of the total product measured with the phage system. Of interest is the fact that the major part of the particle-bound native product of UTP3H incorporation was directly accessible to RNase in 0.05 X SSC with the BMV and the BB?(lV systems, whereas prefreezing of the labeled particulate leaf material was essential to induce a pronounced sensitivity to RNase in 0.05 X SSC with the extracts from TYJ\IV-infected cabbage (Bove et al., 1968). My experiments with the TYMV-cabbage system showed 50-60% resistance of the product of UTP incorporation to RNase in 0.05 X SSC, as compared with 65-71% resistance in the experiments by Bove et al. (1968); the lower resistance to RNase in the present work may result from various discrepancies in the experimental procedure. It remains that, using similar conditions for the preparation and the incubation of the leaf extracts, the native product of UTP-3H incorporation was more readily hydrolyzed by RNase in 0.05 X SSC with the BMVbarley and the BBMV-broadbean systems than with TY1IV in cabbage. This may
250
SEMAL
reflect a different subcellular structure for the synthesis of the double-stranded RNA product. Note Added in Proof: A study of the properties of RNA synthesized in vivo in barley leaves infected with bromegrass mosaic virus has been published recently by C. Hiruki (1969) J. Vi’irol. 3, 498-505. ACKNOWLEDGMENTS I thank Dr. W. Fiers for stimulating discussions and for reading the manuscript. I. also appreciated the assistance of Mrs. A. M. Pollart, Mrs. P. Janssens, and Mr. J. Kummert, and the help of Messrs E. Franyois and A. Riga with the radioactivity measurements. This work was supported by a grant from the “Fends National de la Recherche Scientifique,” Brussels Belgium. REFERENCES BOCKBTAHLER, L. E., and KAESBERG, P. (1965). Isolation and properties of RNA from bromegrass mosaic virus. J. MoZ. Biol. 13, 127-137. BORST, P., and WEISSMANN, C. (1965). Replication
of viral RNA. VIII. Studies on the enzymatic mechanism of replication of MS2 RNA. Proc. Natl. Acad. Sci. U.S. 54,982-987. Bovfi, J. M., Bovk, C., and MOCQUOT, B. (1968). Turnip yellow mosaic virus-RNA synthesis in vitro: Evidence for native double-stranded RNA. Biochem. Biophys. Res. Commun. 32, 480486. FEIX, G., POLLET, It., and WEISSMANN, C. (1968). Replication of viral RNA. XVI. Enzymatic synthesis of infectious viral RNA with noninfectious Qp minus strands as template. Proc. Natl. Acad. Sci. U.S. 59, 145-152. GUSCHLBAUER, W., COURTOIS, Y., BovB, C., and Bov~, J. M. (1968). Optical investigations on double-stranded ribonucleic acid from turnip yellow mosaic virus. Mol. Gen. Genet. 103, 150-158. SEMAL, J., and HAMILTON, R. I. (1968). RNA synthesis in cell-free extracts of barley leaves infected with bromegrass mosaic virus. Vi’irology 36, 293-302. SEMAL, J. (1969). Synthesis of ribonucleic acid in cell-free extracts of broadbean leaves infected with broadbean mottle virus. Phytopathology 59, 881-882.
40, 244-250 (1970)
Properties
of the Products
of Leaves
Infected
of UTP Incorporation with
Bromegrass
Broad bean
Mottle
Mosaic
by Cell-Free Virus
Extracts
or with
Virus
J. SEMAL des Sciences agronomiques, Gembloux, Belgium
Faculth
Accepted September 9, 1969
After deproteinization with phenol and detergent, the product of UTP-3H incorporation by a particulate fraction prepared from barley leaves infected with bromegrass mosaic virus displayed properties consistent with those of a double-stranded RNA. The major part of the label incorporated in the product was resistant to pancreatic ribonuclease (RNase) (5 pg/ml) in 1 X SSC, but was made acidsoluble by keatment with RNase in 0.1 X SSC. Similar results were obtained with the deproteinized product of UTP-3H incorporation by extracts from broadbean leaves infected with broadbean mottle virus. When the deproteinixed product synthesized by extracts from bromegrass mosaic virus-infected leaves was heated in 1 X SSC at increasing temperatures, a sharp transition from RNase resistance to RNase sensitivit.y was observed, with a T, of 99-100”. With both bromegrass mosaic virus and broadbean mottle virus, the particlebound product of UTP-3H incorporation was double-stranded in its native state, as suggested by RNase resistance in 1 X SSC and RNase sensitivity in 0.05 X SSC, respectively, of the radioactive product associated with the particulate leaf fraction. The native products of UTP-3H incorporation were sensitive to RNase in 0.05 X SSC without any pretreatment of the leaf extracts. MATERIALS
INTRODUCTION Upon incubation in the presence of actinomycin D (AMD) and the necessary ingredients for RNA synthesis, UTP-3H was incorporated into an acid-insoluble product by a particulate fraction prepared from cell-free of barley leaves infected with extracts
bromegrass mosaic virus (BMV) Hamilton,
1968).
Similar
results
(Semal and mere ob-
tained with a comparable fraction prepared from broadbean leaves infected with broadbean mottle virus (BBMV) (Semal, 1969). The products of UTP-3H incorporation have been characterized further, using the two plant-virus systems mentioned above; they displayed properties consistent with those of double-stranded RNA, either in their native state or after deproteinization.
AND METHODS
Xtandard assay for UTP-3H incorporation and study of RNase resistance. The leaf extracts were prepared and incubated with UTP-3H, AMD, and the necessary ingredients for RNA synthesis, as previously described (Semal and Hamilton, 1968; Semal, 1969). The leaf homogenates were filtered and centrifuged for 5 min at 1000 g; the supernatants were collected and centrifuged for 15 min at 10,000 g; the pellets thus obtained were resuspended in the incubation medium to give the fraction 1000-S/10,000-P, which was used for the incorporation experiments. Study of the ribonuclease (RNase) resistance of the deproteinized products was performed as follows: after 20 min incubation with UTP-3H, the leaf material was mixed with 244
VIRUSES
AND
UTP
245
INCORPORATION
Laboratories, Rahway, New Jersey. Tricyclohexamine salt of phosphoenolpyruvic acid, pyruvate kinase, and ribonuclease A were purchased from Sigma Chemicals Co. UTP-3H and the unlabeled nucleotides were obtained from Schwarz BioResearch Inc. RESULTS
E$ect of Salt Concentration on the RNase Sensitivity of the Labeled Product
SALT
CONCENTRATION
(X
SSC)
FIG. 1. Effect of salt concentration on the RNase resistance of the labeled product of UTPfH incorporation by the fraction 1000-S/10,009-P prepared from leaves of barley plants infected for 7 days with BMV. The deproteinized RNA product was resuspended in increasing salt concentration (up to 1 X SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7) and incubated with RNase (5 rg/ml, 30 min at 37”). Control tubes were treated similarly but without RNase. The acid-insoluble radioactivity was determined by Millipore filtration in the presence of carrier protein. Combined results of two experiments.
unlabeled UTP and treated with phenol and sodium lauryl sulfate; the RNA’s were precipitated with ethanol, resuspended in appropriate solution, incubated with RNase, mixed with carrier protein, and tested for acid-insoluble radioactivity. For studying the RNase resistance of the particle-bound native product, the material was treated as follows: after incubation of the extract (20 min at 30”) with UTP-3H in the presence of 20 pg/ml of AMD, unlabeled UTP (0.2 ml at 12.5 mg/ml) was added to each tube; the extracts were cooled and centrifuged at 2” for 15 min at 10,000 g. The pellets (fraction “P”) were resuspended and incubated with pancreatic RNase, as described under “Results” for individual experiments. Blanks were treated the same way, except that the leaf fraction was omitted during incubation with UTP-3H and added immediately thereafter; they consistently gave a final radioactivity of lo-20 cpm per tube, which was substracted from the other results. Actinomycin D was a gift from Merck, Sharp & Dohme Research
The effect of RNase on the deproteinized labeled product of UTP-3H incorporation was studied under various conditions of ionic strength. As shown in Fig. 1 for the BMV-barley system and in Table 1 for the BBMV-broadbean system, the labeled products were largely resistant to RNase (5 pg/ml, 30 min at 37”) in 1 X SSC (0.15 M NaC1, 0.015 M sodium citrate, pH 7), but were rendered acid-soluble by treatment with the enzyme at low salt concentration. TABLE
1
EFFECT OF SALT CONCENTRATION ON THE RNASE RESISTANCE OF THE RADIOACTIVE PRODUCT OF UTP-3H INCORPORATION IIY EXTRACTS OF BROADBEAN LEAVES INFECTED WITH BBMVa Treatment concentration ot : d’ eproteinized esuspending mediur
=g. srbit 1
2
1.00 0.30 0.10 0.01 2.06 1.00 0.50 0.25 0.12 0.06
x x x x x x x X x X
sscc ssc ssc ssc ssc ssc ssc SSC ssc SSC
of the
product b
RNase
Control
449d 47 6 20 731 540 353 156 25 42
634 408 413 421 1.038 1.052 1.028 883 918 927
* _-
L
71 12 1 5 70 51 34 18 3 5
a Fraction 1000-S/10,000-P was prepared from broadbean plants infected for 7 days with BBMV; it was incubated with UTP-3H, AMD (20 rg/ml), and the necessary ingredients for RNA synthesis (Semal, 1969). b The deproteinized RNA product was treated with 5 pg/ml of RNase for 30 min at 37”; controls were treated similarly, but without RNase. c SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7. d Results expressed as acid-insoluble counts per minute in the RNA fraction.
246
SEMAL
TEMPERATURE
FIG. 2. Thermal transition of the RNase resistance of the labeled product of UTP-aH incorporation by the fraction 1000-S/10,000-P prepared from leaves of barley plants infected for 7 days with BMV. The deproteinized RNA product in 1 ml of 1 X SSC was sealed in glass tubes, heated for 10 min at the temperature indicated, cooled rapidly, and subjected to digestion with RNase (5 rg/ml, 30 min at 37”). Control tubes were treated similarly but without RNase. The acid-insoluble radioactivity was determined by Millipore filtration in the presence of carrier protein. Combined results of two experiments.
cabbage leaves infected with turnip yellow mosaic virus (TYMV) was shown to be double-stranded prior to any phenol treatment (BovB et al., 1968). It was of interest to study this aspect with the plant-virus systems under investigations in the present research. It was found that more than 80% of the label incorporation by the 1000-S/10,000-P fraction from BMV-infected barley sedimented at 10,000 g when centrifugation was performed immediately after 20 min incubation with UTPJH; most of subsequent experiments were carried out with this 10,000 g pellet (fraction “P”). After resuspension in either 1 X SSC, or in 0.05 X SSC, the fraction “P” was incuTABLE
IIResuspending medium Pretreatment of fraction “P
Eflect of Temperature on the RNase Sensitivity of the Labeled Product Synthesized by the BMV-Barley System
A, None
The effect of temperature on the RNase sensitivity of the labeled product was studied with the BMV-barley system. As shown in Fig. 2, heating the RNA product for 10 min at increasing temperatures in 1 X SSC, followed by rapid cooling, led to a sharp thermal transition from RNase resistance to RNase sensitivity, with a T,,, of 99-100”.
B. Freezing thawing
RNase Sensitivity of the Native Labeled Product with the BMV-Barley System It has been suggested with RNA phages that the RNase resistance of the doublestranded deproteinized product of viral RNA polymerase resulted from deproteinization with phenol and detergent, the product being mostly single-stranded and RNase sensitive in its native state (Borst and Weissmann, 1965; Feix et cd., 1968). However, the nascent RNA synthesized by a cell-free particulate fraction extracted from
2
EFFECT OF SALT CONCENTRATION AND EFFECT OF PREFREEZING OF FRACTION lip” ON THE RNASE RESISTANCE OF THE NATIVE PRODUCT OF UTP INcORPORATIONQ
and
0.05 x ssc +
Sic”+ RNase 779c 819 703 807 773 863 842 848
of fraction “P”
RNase
761 948 735 840 817 846 817 827
113 137 97
‘%” 772 898
110
48 53 53 63
772 849 738 795
a Fraction 1000-S/10,000-P was prepared from barley plants infected for 7 days with BMV, and incubated with UTP-3H as previously described (Semal and Hamilton, 1968). b After incubation with UTP-3H, the leaf extract was centrifuged for 15 min at 10,000 g; the was frozen for 15 min at pellet (fraction “P”) -15”, thawed, resuspended in appropriate salt solution, and treated with 20 fig/ml of RNase (30 min at 37”); control tubes were treated similarly but without RNase. After deproteinization with phenol and detergent, the RNA was precipitated with ethanol in the presence of carrier yeast RNA. c Twin experiments with duplicate aliquots; results are expressed as acid-insoluble cpm in the RNA fraction after deproteinization and ethanol precipitation.
VIRUSES
AND
UTP
bated with RNase (20 or 40 pg/ml, 30 min at 37”), and the reaction was terminated by addition of phenol and detergent. Deproteinization and isolation of the RNA’s by ethanol precipitation were performed as previously described (Semal and Hamilton, 1968) with addition of carrier yeast RNA (50 b&ml) ; control tubes were treated similarly but without RNase. The acidinsoluble radioactivity associated with the RNA fraction was then determined by Millipore filtration and scintillation counting. The results thus obtained are presented in Table 2 A; they indicate that the native product of UTP-3H incorporation was resistant to RNase when the fraction “P” was incubated with the enzyme in 1 X SSC, but that it was made acid soluble upon RNase treatment of the fraction “P” in 0.05 X SSC. Bov6 et al. (1968) reported that, with the TYMV-cabbage system, prefreezing and thawing of the leaf extract was necessary to induce a pronounced sensitivity of the native double-stranded RNA product to RNase in 0.05 X SSC. As indicated in Table 2 B, freezing and thawing of the fraction “P” with the BMV-barley system did not change notably the effect of RNase in 1 X SSC, and the residual RNase resistance in 0.05 X SSC was decreased. E$ect of Pretreatment of the Fraction “P”, on the Subsequent RNase Resistance of the Native or the Deproteinized Product of UTP-3H Incorporation, with the BMVBarley System Deproteinization of the labeled fraction “P” in 0.05 X SSC, as performed in Table 2, could possibly lead to the digestion of the deproteinized product of UTP-3H incorporation by traces of RNase carried through the steps of phenol treatment and ethanol precipitation. The possibility was also considered that the access of RNase to the site of the native product could be modified by exposing fraction “P” to low salt concentration: under such conditions, a singlestranded RNA could appear as resistant to RNase in 1 X SSC (site closed to RNase action) while being sensitive to the enzyme in 0.05 X SSC (site open to RNase action). Experiments were carried out to clarify these questions.
INCORPORATION
247
In order to test the possible effect of residual RNase on the deproteinized total product of UTP incorporation, fraction 1000-S/10,000-P was incubated for 20 min with UTP-3H; incubation was followed by addition of unlabeled UTP to minimize further incorporation. The total incubated material was then adjusted to 1 X SSC, and aliquots were treated for 30 min at 37” with 20 pg/ml of RNase (controls were treated similarly without RNase). After deproteinization, the RNA was isolated, resuspended, and incubated further for 30 min at 37”. When resuspension of the RNA was in 1 X SSC, the acid-insoluble radioactivity was 992 cpm for the control sample and 841 cpm for the sample previously treated with RNase; when resuspension was in 0.05 X SSC, the values were 840 and 686, respectively. This result indicates that upon deproteinization, the labeled native product preincubated in 1 X SSC + RNase (20 pg/ml) yields an RNA which is stable in 0.05 X SSC. At this stage of the procedure, however, this RNA was made entirely acid-soluble upon incubation in 0.05 X SSC with 5 pg/ml of RNase (25 and 10 cpm, respectively). In further experiments, the fraction “P” material in 0.05 X SSC was adjusted to 1 X SSC immediately after RNase treatment, prior to addition of phenol and detergent, in order to ensure the stability of the deproteinized product and to obtain similar conditions of deproteinization for all samples. The RNA product thus isolated was tested for its resistance to RNase digestion in 1 X SSC. The effect of preincubating the fraction “P” in 0.05 X SSC, on its subsequent resistance to RNase in 1 X SSC, was also investigated. When compared with those of Table 2, the results (Table 3) show little effect of adjusting the salt concentration to 1 X SSC prior to deproteinization, or of preincubating the fraction “P” material in 0.05 X SSC before RNase treatment in 1 X SSC. RNase Resistance of the Native Product of UTP-3H Incorporation with the BBMVBroadbean System The fraction 1000-S/10,000-P was prepared from young leaves of broadbean
248
SEMAL
TABLE
3
EFFECTS OF PRETREATMENT OF FRACTION “P” ON THE SUBSEQUENT RNASE RESISTANCE OF THE NATIVE AND DEPROTEINIZED PRODUCT OF UTP-JH INCORPORATIONS
-
Treatment of the leproteinized prod&
Preincub?tion of %,&0n P
-
-
1X BC -IRNase
None
1 X + 1 x 0.05
ssc RNase ssc x ssc
-
bs”
_-
665
699
683 50
699
545 528
680 645
560 63
710 71
652
690
110
+ RNase Preincubation in 0.05 x ssc
0.05 1 x + 1 x 0.05
x ssc ssc RNase ssc x ssc
-
None
Preincubation in 0.05 x ssc
x ssc ssc RNase ssc x ssc RNase x ssc ssc RNase ssc x ssc RNase x ssc
-
-
81
incubation,
fraction 1000-S/10,000-P from leaves of Chinese
The pared
TABLE
was precabbage
4
RNASE RESISTANCE OF THE NATIVE PRODUCT OF UTP-3H INCORPORATION WITH THE BBMV-BROADHEAN SYSTEY~ -
40 69
60 -
-
-
-
-
all samples
were adjusted
to 1 X SSC and deproteinized with pheno1 and detergent, The RNA was precipitated with ethanol in the presence of carrier yeast RNA. d The RNA product was resuspended in 1 X SSC and treated as indicated (no treatment, or incubation for 30 min at 37”, either in 1 X SSC or in 1 X SSC + 5 pg/ml RNase).
plants infected for 5-7 days with BBMV, and was incubated for 20 min with UTP-3H as previously described (Semal, 1969). As shown in Table 4, the labeled product
-
?retreatment
I’
of fraction “PI>
0.05x ssc + RNase
5 I
Preincubation in 0.05 x ssc -
* Fraction
427 499 390 469 413 419 347
372 447 393 429 401 488 357
None -
06 55
Expt. Nab
None
a Fraction “P” was prepared from leaves of barley plants infected for 9 days with BMV. Results are presented as acid-insoluble counts per minute associated with the RNA fraction. b Fraction “P” was preincubated for 15 min in 0.05 X SSC before being subjected to treatment with RNase (40 pg/ml) in either 1 X SSC or 0.05 X ssc. c Fraction ‘(P” was incubated for 30 min at 37”. After
RNase Resistance of the Native Product of UTP-3H Incorporation with the TYMVCabbage System
Treatment of fraction “P”c -
+ RNase 0.05 1 x + 1 x 0.05 + 0.05 1 x + 1 x 0.05 + 0.05
associated with the fraction “P” of BB?(‘rTVinfected broadbean displayed properties of RNase resistance in 1 X SW and of RNase sensitivity in 0.05 X SSC, similar to those observed with BkIV-infected barley.
-
0.05 x SSC
--
95
343 426 560 566 393 468 378
98 68 84 80 96 64
-
1000-S/10,000-P was prepared from
leaves of broadbean plants infected for 5 days (experiment 2) or for 7 days (experiments 1 and 3) with BBMV; it was incubated with UTP-3H as previously described (Semal, 1969). b Experiment 1: Results of duplicate aliquots; samples were adjusted to 1 X SSC before deproteinization. Experiment 2: Twin experiments with duplicate aliquots; samples not adjusted to 1 X SSC before deproteinization. Experiment 3: fraction “P” was preincubated for 15 min in 0.05 X SSC prior to RNase treatment; samples were adjusted to 1 X SSC before deproteinization. c After incubation with UTP-*H, the leaf extract was centrifuged for 15 min at 10,000 g, to yield a pellet (fraction “P”). Fraction “P” was resuspended in 1 X SSC or 0.05 X SSC and incubated for 30 min at 37” with RNase (40pg/ml); control tubes were incubated without RNase. Fraction “P” was then deproteinized with phenol and detergent, the RNA was precipitated with ethanol in the presence of carrier RNA, and the acid-insoluble radioactivity (cpm) of the isolated RNA was determined.
VIRUSES TABLE
AND
5
RNASE
RESISTANCE OF THE NATIVE PRODUCT UTP-3H INCORPORATION WI’IH THE TYMVCABBAGE SYSTEM” Treatment
Expt. No.
1 2
UTP
OF
of fraction “P”5
1 x ssc
590 782 594
594 766 548
407 458 312
664 772 586
a Fraction 1000-S/10,000-P was prepared from leaves of Chinese cabbage systemically infected and incubated with UTP-3H as with TYMV, described for the BM\-barley system. (Semal and Hamilton, 1968). b After incubation, the leaf extract was centrifuged for 15 min at 10,000 9, to yield a pellet (fraction “P”). Fraction “P” was resuspended in 1 X SSC or 0.05 X SSC and incubated for 30 min at 37” with RNase (40 pg/ml); control tubes were incubaled without RNase. Fraction “P” was then adjusted to 1 X SSC and deproteinized with phenol and detergent; the RNA was precipitated with ethanol in the presence of carrier RNA, and the acid-insoluble radioactivity (cpm) of the isolated RNA was determined.
systemically infected with TYJIV, and was incubated with UTP3H as described for the BhIV-barley system. Table 5 presents the results of two independent experiments, which indicate that the labeled product associated with fraction “P” was completely resistant to RNase in 1 X SSC and 50-60 % resistant in 0.05 X SSC. DISCUSSION
The deproteinized product of UTPJH incorporation by a cell-free particulate fraction prepared from BMV-infected barley leaves, displayed properties similar to those of an RNA associated with a complementary polynucleotide into a double-stranded structure. According to the data presented by Guschlbauer et al. (1968), the thermal transition temperature TrnlXss~obtained for the product synthesized by the BMV-barley system (99-100”) fits rather well with that expected for a fresh preparation of a doublestranded RNA with a GC content of 0.464, as determined for the large component of BMV-RNA by Bockstahler and Kaesberg (1965). Part of the deproteinized product with the BB>IV-broadbean system behaved
INCORPORATION
249
also as an RNA associated into a doublestranded structure. With the BMV-barley and the BBMVbroadbean systems, the behavior of the native RNA product of nucleotide incorporation toward the action of RNase was in agreement with the properties of the product obtained for the TYMV-cabbage system by Bove et al. (1968), in that the particle-bound product of UTP incorporation, in its native state, was almost completely resistant to RNase ihn 1 X SSC. This situation differs from that described for an RNA phage by Borst and Weissmann (1965) and by Feix et al. (1968), where the product of nucleotide incorporation was RNase-sensitive in the native state, but became RNase-resistant upon deproteinization. However, comparisons in this matter between plants and RNase-deficient bacteria are made difficult because of differences in the amounts of endogenous RNase and in the efficiency of nucleotide incorporation. It may be that a specific RNase-resistant RNA product is selectively isolated with the plant system, which corresponds only to a minute fraction of the total product measured with the phage system. Of interest is the fact that the major part of the particle-bound native product of UTP3H incorporation was directly accessible to RNase in 0.05 X SSC with the BMV and the BB?(lV systems, whereas prefreezing of the labeled particulate leaf material was essential to induce a pronounced sensitivity to RNase in 0.05 X SSC with the extracts from TYJ\IV-infected cabbage (Bove et al., 1968). My experiments with the TYMV-cabbage system showed 50-60% resistance of the product of UTP incorporation to RNase in 0.05 X SSC, as compared with 65-71% resistance in the experiments by Bove et al. (1968); the lower resistance to RNase in the present work may result from various discrepancies in the experimental procedure. It remains that, using similar conditions for the preparation and the incubation of the leaf extracts, the native product of UTP-3H incorporation was more readily hydrolyzed by RNase in 0.05 X SSC with the BMVbarley and the BBMV-broadbean systems than with TY1IV in cabbage. This may
250
SEMAL
reflect a different subcellular structure for the synthesis of the double-stranded RNA product. Note Added in Proof: A study of the properties of RNA synthesized in vivo in barley leaves infected with bromegrass mosaic virus has been published recently by C. Hiruki (1969) J. Vi’irol. 3, 498-505. ACKNOWLEDGMENTS I thank Dr. W. Fiers for stimulating discussions and for reading the manuscript. I. also appreciated the assistance of Mrs. A. M. Pollart, Mrs. P. Janssens, and Mr. J. Kummert, and the help of Messrs E. Franyois and A. Riga with the radioactivity measurements. This work was supported by a grant from the “Fends National de la Recherche Scientifique,” Brussels Belgium. REFERENCES BOCKBTAHLER, L. E., and KAESBERG, P. (1965). Isolation and properties of RNA from bromegrass mosaic virus. J. MoZ. Biol. 13, 127-137. BORST, P., and WEISSMANN, C. (1965). Replication
of viral RNA. VIII. Studies on the enzymatic mechanism of replication of MS2 RNA. Proc. Natl. Acad. Sci. U.S. 54,982-987. Bovfi, J. M., Bovk, C., and MOCQUOT, B. (1968). Turnip yellow mosaic virus-RNA synthesis in vitro: Evidence for native double-stranded RNA. Biochem. Biophys. Res. Commun. 32, 480486. FEIX, G., POLLET, It., and WEISSMANN, C. (1968). Replication of viral RNA. XVI. Enzymatic synthesis of infectious viral RNA with noninfectious Qp minus strands as template. Proc. Natl. Acad. Sci. U.S. 59, 145-152. GUSCHLBAUER, W., COURTOIS, Y., BovB, C., and Bov~, J. M. (1968). Optical investigations on double-stranded ribonucleic acid from turnip yellow mosaic virus. Mol. Gen. Genet. 103, 150-158. SEMAL, J., and HAMILTON, R. I. (1968). RNA synthesis in cell-free extracts of barley leaves infected with bromegrass mosaic virus. Vi’irology 36, 293-302. SEMAL, J. (1969). Synthesis of ribonucleic acid in cell-free extracts of broadbean leaves infected with broadbean mottle virus. Phytopathology 59, 881-882.