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Ribonuclease-induced stabilization of an alkali-labile TMV strain and lack of stabilization of reaggregated TMV protein
DISCUSSION
AND
PRELIMINARY
9. ALLEN, D. W., Biochim. Biophys. Acta 114, 606-611 (1966). i0. EIDINOFF, M. L., BATES, B., KIM, S. H., and STEINGLASS,M., Proc. Am. Assoc. Cancer Res. 8, 15 (1967). 11. TEMIN, H. M., Proc. Natl. Acad. Sci. U.S. 52, 323329 (1964). lb. HAREL, L., HAREL, J., LACOUR, F., and HUPPERT, J., Compt. Rend. Acad. Sci. 263, 616-619 (1966). 1% BAUER, H., BAHNEMANN, H., AND SCHAEFER, W. 2. Naturforsch. 2Ob, 959-965 (1965). 14. GILLESPIE, D., and SPIEGELMBN, S., J. Mol. Biol. 12, 829-842 (1965). 25. BON.%R, R. A., SVERAK, L., BOLOGNESI, D. P., LANGLOIS, A. J., BEARD, D., and BEARD, J. W., Cancer Res. 27, 1138-1157 (1967). 16. SCHERRER, K., and DARNELL, J. E., Biochem. Biophys. Res. Commun. 7, 4W-490 (1962). 17. MIRSKY, A. E., and RIS, H., Nature 163, 666-
667 (1949). 18. BOLTON, E. T., BRITTEN, R. J., COWIE, D. B., ROBERTS,R. B., SZAFRANSKI,~.,~~~ WARING, M. J., Carnegie Institution of Washington Year Book No. 64,313316 (1965). D. E. WILSON H. BAUER Max-Planck-Institut fiir Virusforschung Biologisch-medizinische Ableilung Tiibingen, Germany Accepted September 22, 1967
Ribonuclease-Induced Alkali-Labile Stabilizahon
Stabilization TMV
Strain
of Reaggregated
and
of lack
an of
TMV
Protein
Addition of ribonuclease (RNase) to tobacco mosaic virus (TMV) solution protects the virus from degradation when the TMV-RNase mixtures are exposed to alkali (1,2). It is of interest to us whether addition of RNase also protects another strain of TMV or TMV protein polymer (nucleic acid free) from degradation when the pH of the medium is raised. The ordinary strain of TMV was propagated in plants of Nicotiana tabacum L. var. Bright Yellow and the bean strain in Phaseolus vulgaris L. var. Otebo or Kinugasa. The viruses were isolated by several cycles of low and high speed centrifugation followed by resuspensionin water.
REPORTS
7.57
Chromatography on ECTEOLA cellulose columns was the same as that used earlier (2, S) with the modification that 1.5 Jf NaCl in phosphate buffer (100 ml) was poured into a separa,tory funnel in place of 1 M NaCl in the same buffer. TMV prot’ein (TMV-P) of the ordinary strain was prepared by the method of Fraenkel-Conrat (4). The protein was finally suspended in water (pH 8), and its concentration was adjusted to about 1.4 mg/ml. The stability of the bean strain in alkaline solut’ions was compared with that of the ordina1.y strain. One milliliter of eit’her virus solution (l/3, 2/3 or 1 mg/ml) was mixed with 3 ml of sodium borate buffer (0.03 M, pH 10.0) and incubated at 0” for 90 minutes. The solutions were then centrifuged at 100,000 q for 60 minutes. The ODzGompof the supernatants was measured. It was clear from the result that the bean st,rain was less stable in the alkaline solution than the ordinary strain. The degradation of the two strains by alkali was then studied by chromatography. The elution patterns of control samples (nontreated) are shown in Fig. 1A (ordinary strain) and 1C (bean strain). The control sample was a mixture of 1 ml of either strain (3 mg/ml) and 3 ml of neutral phosphate buffer. The peak of the bean strain was always broader than that of the ordinary strain. TMV solution (1 ml) was mixed with 0.03 M, pH 10.0 borate buffer (1 ml) and incubated at 0” for 60 minutes. The alkalitreated virus solutions were then mixed with 1 ml of 0.1 N HCl and 1 ml of neutral phosphate buffer and were added on the top of the column. The pattern (Fig. 1D) obtained with the alkali-treated bean strain was different from that of the ordinary strain (Fig. 2B). The P3 component was detected with the ordinary strain, but not with the bean strain. The ratios of absorbancies at 260:280 mp of the rapidly eluted fractions in the peak P2 of bean strain was 1.2 and that of the slower eluted part in the same peak was higher than 1.20. Thus, the protein content of the slower eluted materials in the component P2 was lower than that of the rapidly eluted materials in the same peak.
DISCUSSION
758
AND
PRELIMINARY
REPORTS
P2
0 0
10
20
30
0
Fraction
10
20
30
No.
FIG. 1. Chromatography of ordinary strain of TMV (A and B) and bean strain (C and D). Both virus solutions were treated with alkali (pH 10.0) at 0°C for 60 minutes (B and D). A and C were not treated with alkali.
The phenomenon that RNase protects TMV (ordinary strain) against alkaline degradation (1, 2) was reexamined by another method with both strains of TMV. Results obtained with the ordinary strain are summarized in Table 1. Experiments were done 4 times, and the average values are listed in the table. One milliliter of TMV (0.8 mg/ml) was mixed with 3 ml of distilled water, and the mixture was centrifuged at high speed. The precipitate was suspended in 4 ml of water and its OD,,, mpwas measured (sample A). When TMV was mixed with pH 10.4, 0.03 M borate buffer and incubated at 0” for 60 minutes, the amount of the precipitated materials by high speed centrifugation was 75 % of that of sample A (sample B). When RNase (100 pg/ml in water,
Nutritional
Biochemicals
Co.)
was
added to the alkali-treated virus solution (sample C), the amount of the precipitate was smaller compared with that of sampleB. This decrease indicates that the partially stripped RNA of the degraded virus particle is digested by the enzyme. When RNase was added to the virus before alkaline treatment (sample D), the amount of the material increased again.
The same experiments were done with the bean strain (Table 1) ; the values are the averages of three experiments. When the strain was treated with alkali, about 90 % of the material was not precipitated by high speed centrifugation (samples B and C). However, virus pretreated with enzyme was more stable, and about 50% of the original TABLE
1
EFFFET OF RIBONUCLEASE ON DEGRADATION ORDINARY AND BEAN STRAIN PARTICLES TOBACCO MOSAIC VIRUS BY ALKALI
OF OF
r7ocedure*: I ml 1 iv1V (0.8 mg/ml) mi xed. with ..
precipitated materiais by high speed centrifugation Sample lml borate (ODssomr) 1 ml RNase, buffer, pH 0.1 mg/ml 10.4, 0.03 Ordinary Bean strain strain M A B
-
+
0.552
g
+* +
+ +
0.366 0.461
0.415
0.607 0.083 0.043 0.312
a Each sample was adjusted to 4 ml with water and finally centrifuged at 100,000 g for 60 minutes. * RNase was added after the addition of borate buffer.
DISCUSSlON
AND PRELIMINARY
REPORTS
759
TABLE 2 EFFECT OF RIBON~CLEASE ON DEPOLYMERIZATION OF TOBACCO MOSAIC VIRUS PROTEIN (TMV-P) Procedure”: 1 ml TMV-P
(1.4 mg/ml) with
Sample
1 ml phosphate bui$p; 5.9,
1 ml RNase 0.1 w/ml
0.7 ml NaOH, 0.1 N
Low speed centrifugation
High speed centrifution
A 3 C D E F
+ + + + +
+ +b
+ + +
+ -
+ + + +
ODao mlr of supernatant
0.440 0.502 0.116” 0.398” 0.416” 0.414”
= Each sample was adjusted to 3.7 ml with water before centrifugation. * RNase was added before the addition of phosphate buffer. c The pH of the final solution was 7.6.
was precipitated (sample D). These facts show that RNase protects the virus from degradation by alkali whether the strains are stable in alkaline solution or not. One milliliter of phosphate buffer (pH 5.9, 0.1 M), 1 ml of RNase, or 0.7 ml of 0.1 N NaOH solution were added to TMV protein (TMV-P) solution in the order shown in Table 2, and the final volume of each mixture was adjusted to 3.7 ml with water. After addition of each solution, the reaction mixtures were held for 10 minutes at room temperature, except after the addition of phosphate buffer, when incubation was for 20 minutes. Finally the reaction mixtures (except sample A) were centrifuged at low or high speed and the optical density of the supernatants was determined at 280 rnp. TMV-P which had been mixed with phosphate buffer followed by low speed centrifugation (sample B) showed an increase in absorbancy compared with that sample A. Formation of rod-shaped particles under these conditions was alsochecked by electron microscopy through the courtesy of Dr. H. Hibino (Institute for Plant Virus Research, Chiba). Moreover, when the protein solution at pH 5.9 was centrifuged at 100,000 g for 60 minutes, about four-fifths of the material was precipitated (sample C). On the other hand, when the pH of the solution was raised from 5.9 to 7.6 by addition of NaOH, a amount
greater
part
of the
material
was
not
pre-
cipitated by high speedcentrifugation (sam-
ple D). These results show that under the conditions used TMV-P polymerized at pH 5.9 and depolymerized at pH 7.6. When RNase was added to TMV-P after (sample E) or before (sample F) polymerization and then the mixture was adjusted to pH 7.6, the amount of the material in the supernatant obtained by high speed centrifugation was nearly equal to that of sample D which did not contain RNase. Thus, RNase did not protect the virus protein polymer from depolymerization. ACKNOWLEDGMENT The authors gratefully acknowledge a gift of the bean strain of TMV from Dr. H. H. Murakishi, Michigan State University. This investigation was partly supported by a fund of the Ministry of Education of Japan. REFERENCES 1. DIENER, T. O., and DESJARDINS, P. R., Viirology 29, 15-25 (1966). 2. TANIGUCHI, T., viz’rology 30,317-318 (1966). TANIGUCHI, T., ri’irology 22, 245-252 (1964). ;: FRAENKEL~ONRST, H., Viiirology 4, 14 (1957). TAKESHI TANIGUCHI AKIR~ YAMAGUCHI TOKUZO HIRII Plant Pathology Laboratory Faculty of Agriculture Nagoya University, Chikusa-Ku
Nagoya, Japan Accepted
October
4, 1967
AND
PRELIMINARY
9. ALLEN, D. W., Biochim. Biophys. Acta 114, 606-611 (1966). i0. EIDINOFF, M. L., BATES, B., KIM, S. H., and STEINGLASS,M., Proc. Am. Assoc. Cancer Res. 8, 15 (1967). 11. TEMIN, H. M., Proc. Natl. Acad. Sci. U.S. 52, 323329 (1964). lb. HAREL, L., HAREL, J., LACOUR, F., and HUPPERT, J., Compt. Rend. Acad. Sci. 263, 616-619 (1966). 1% BAUER, H., BAHNEMANN, H., AND SCHAEFER, W. 2. Naturforsch. 2Ob, 959-965 (1965). 14. GILLESPIE, D., and SPIEGELMBN, S., J. Mol. Biol. 12, 829-842 (1965). 25. BON.%R, R. A., SVERAK, L., BOLOGNESI, D. P., LANGLOIS, A. J., BEARD, D., and BEARD, J. W., Cancer Res. 27, 1138-1157 (1967). 16. SCHERRER, K., and DARNELL, J. E., Biochem. Biophys. Res. Commun. 7, 4W-490 (1962). 17. MIRSKY, A. E., and RIS, H., Nature 163, 666-
667 (1949). 18. BOLTON, E. T., BRITTEN, R. J., COWIE, D. B., ROBERTS,R. B., SZAFRANSKI,~.,~~~ WARING, M. J., Carnegie Institution of Washington Year Book No. 64,313316 (1965). D. E. WILSON H. BAUER Max-Planck-Institut fiir Virusforschung Biologisch-medizinische Ableilung Tiibingen, Germany Accepted September 22, 1967
Ribonuclease-Induced Alkali-Labile Stabilizahon
Stabilization TMV
Strain
of Reaggregated
and
of lack
an of
TMV
Protein
Addition of ribonuclease (RNase) to tobacco mosaic virus (TMV) solution protects the virus from degradation when the TMV-RNase mixtures are exposed to alkali (1,2). It is of interest to us whether addition of RNase also protects another strain of TMV or TMV protein polymer (nucleic acid free) from degradation when the pH of the medium is raised. The ordinary strain of TMV was propagated in plants of Nicotiana tabacum L. var. Bright Yellow and the bean strain in Phaseolus vulgaris L. var. Otebo or Kinugasa. The viruses were isolated by several cycles of low and high speed centrifugation followed by resuspensionin water.
REPORTS
7.57
Chromatography on ECTEOLA cellulose columns was the same as that used earlier (2, S) with the modification that 1.5 Jf NaCl in phosphate buffer (100 ml) was poured into a separa,tory funnel in place of 1 M NaCl in the same buffer. TMV prot’ein (TMV-P) of the ordinary strain was prepared by the method of Fraenkel-Conrat (4). The protein was finally suspended in water (pH 8), and its concentration was adjusted to about 1.4 mg/ml. The stability of the bean strain in alkaline solut’ions was compared with that of the ordina1.y strain. One milliliter of eit’her virus solution (l/3, 2/3 or 1 mg/ml) was mixed with 3 ml of sodium borate buffer (0.03 M, pH 10.0) and incubated at 0” for 90 minutes. The solutions were then centrifuged at 100,000 q for 60 minutes. The ODzGompof the supernatants was measured. It was clear from the result that the bean st,rain was less stable in the alkaline solution than the ordinary strain. The degradation of the two strains by alkali was then studied by chromatography. The elution patterns of control samples (nontreated) are shown in Fig. 1A (ordinary strain) and 1C (bean strain). The control sample was a mixture of 1 ml of either strain (3 mg/ml) and 3 ml of neutral phosphate buffer. The peak of the bean strain was always broader than that of the ordinary strain. TMV solution (1 ml) was mixed with 0.03 M, pH 10.0 borate buffer (1 ml) and incubated at 0” for 60 minutes. The alkalitreated virus solutions were then mixed with 1 ml of 0.1 N HCl and 1 ml of neutral phosphate buffer and were added on the top of the column. The pattern (Fig. 1D) obtained with the alkali-treated bean strain was different from that of the ordinary strain (Fig. 2B). The P3 component was detected with the ordinary strain, but not with the bean strain. The ratios of absorbancies at 260:280 mp of the rapidly eluted fractions in the peak P2 of bean strain was 1.2 and that of the slower eluted part in the same peak was higher than 1.20. Thus, the protein content of the slower eluted materials in the component P2 was lower than that of the rapidly eluted materials in the same peak.
DISCUSSION
758
AND
PRELIMINARY
REPORTS
P2
0 0
10
20
30
0
Fraction
10
20
30
No.
FIG. 1. Chromatography of ordinary strain of TMV (A and B) and bean strain (C and D). Both virus solutions were treated with alkali (pH 10.0) at 0°C for 60 minutes (B and D). A and C were not treated with alkali.
The phenomenon that RNase protects TMV (ordinary strain) against alkaline degradation (1, 2) was reexamined by another method with both strains of TMV. Results obtained with the ordinary strain are summarized in Table 1. Experiments were done 4 times, and the average values are listed in the table. One milliliter of TMV (0.8 mg/ml) was mixed with 3 ml of distilled water, and the mixture was centrifuged at high speed. The precipitate was suspended in 4 ml of water and its OD,,, mpwas measured (sample A). When TMV was mixed with pH 10.4, 0.03 M borate buffer and incubated at 0” for 60 minutes, the amount of the precipitated materials by high speed centrifugation was 75 % of that of sample A (sample B). When RNase (100 pg/ml in water,
Nutritional
Biochemicals
Co.)
was
added to the alkali-treated virus solution (sample C), the amount of the precipitate was smaller compared with that of sampleB. This decrease indicates that the partially stripped RNA of the degraded virus particle is digested by the enzyme. When RNase was added to the virus before alkaline treatment (sample D), the amount of the material increased again.
The same experiments were done with the bean strain (Table 1) ; the values are the averages of three experiments. When the strain was treated with alkali, about 90 % of the material was not precipitated by high speed centrifugation (samples B and C). However, virus pretreated with enzyme was more stable, and about 50% of the original TABLE
1
EFFFET OF RIBONUCLEASE ON DEGRADATION ORDINARY AND BEAN STRAIN PARTICLES TOBACCO MOSAIC VIRUS BY ALKALI
OF OF
r7ocedure*: I ml 1 iv1V (0.8 mg/ml) mi xed. with ..
precipitated materiais by high speed centrifugation Sample lml borate (ODssomr) 1 ml RNase, buffer, pH 0.1 mg/ml 10.4, 0.03 Ordinary Bean strain strain M A B
-
+
0.552
g
+* +
+ +
0.366 0.461
0.415
0.607 0.083 0.043 0.312
a Each sample was adjusted to 4 ml with water and finally centrifuged at 100,000 g for 60 minutes. * RNase was added after the addition of borate buffer.
DISCUSSlON
AND PRELIMINARY
REPORTS
759
TABLE 2 EFFECT OF RIBON~CLEASE ON DEPOLYMERIZATION OF TOBACCO MOSAIC VIRUS PROTEIN (TMV-P) Procedure”: 1 ml TMV-P
(1.4 mg/ml) with
Sample
1 ml phosphate bui$p; 5.9,
1 ml RNase 0.1 w/ml
0.7 ml NaOH, 0.1 N
Low speed centrifugation
High speed centrifution
A 3 C D E F
+ + + + +
+ +b
+ + +
+ -
+ + + +
ODao mlr of supernatant
0.440 0.502 0.116” 0.398” 0.416” 0.414”
= Each sample was adjusted to 3.7 ml with water before centrifugation. * RNase was added before the addition of phosphate buffer. c The pH of the final solution was 7.6.
was precipitated (sample D). These facts show that RNase protects the virus from degradation by alkali whether the strains are stable in alkaline solution or not. One milliliter of phosphate buffer (pH 5.9, 0.1 M), 1 ml of RNase, or 0.7 ml of 0.1 N NaOH solution were added to TMV protein (TMV-P) solution in the order shown in Table 2, and the final volume of each mixture was adjusted to 3.7 ml with water. After addition of each solution, the reaction mixtures were held for 10 minutes at room temperature, except after the addition of phosphate buffer, when incubation was for 20 minutes. Finally the reaction mixtures (except sample A) were centrifuged at low or high speed and the optical density of the supernatants was determined at 280 rnp. TMV-P which had been mixed with phosphate buffer followed by low speed centrifugation (sample B) showed an increase in absorbancy compared with that sample A. Formation of rod-shaped particles under these conditions was alsochecked by electron microscopy through the courtesy of Dr. H. Hibino (Institute for Plant Virus Research, Chiba). Moreover, when the protein solution at pH 5.9 was centrifuged at 100,000 g for 60 minutes, about four-fifths of the material was precipitated (sample C). On the other hand, when the pH of the solution was raised from 5.9 to 7.6 by addition of NaOH, a amount
greater
part
of the
material
was
not
pre-
cipitated by high speedcentrifugation (sam-
ple D). These results show that under the conditions used TMV-P polymerized at pH 5.9 and depolymerized at pH 7.6. When RNase was added to TMV-P after (sample E) or before (sample F) polymerization and then the mixture was adjusted to pH 7.6, the amount of the material in the supernatant obtained by high speed centrifugation was nearly equal to that of sample D which did not contain RNase. Thus, RNase did not protect the virus protein polymer from depolymerization. ACKNOWLEDGMENT The authors gratefully acknowledge a gift of the bean strain of TMV from Dr. H. H. Murakishi, Michigan State University. This investigation was partly supported by a fund of the Ministry of Education of Japan. REFERENCES 1. DIENER, T. O., and DESJARDINS, P. R., Viirology 29, 15-25 (1966). 2. TANIGUCHI, T., viz’rology 30,317-318 (1966). TANIGUCHI, T., ri’irology 22, 245-252 (1964). ;: FRAENKEL~ONRST, H., Viiirology 4, 14 (1957). TAKESHI TANIGUCHI AKIR~ YAMAGUCHI TOKUZO HIRII Plant Pathology Laboratory Faculty of Agriculture Nagoya University, Chikusa-Ku
Nagoya, Japan Accepted
October
4, 1967