- Email: [email protected]
Electrophoretic separation of dsRNA genome segments from Fiji disease and maize rough dwarf viruses
VIROLOGY
63, 287-291 (1975)
Electrophoretic
Separation
Fiji Disease
of dsRNA
and Maize Rough
Genome
Department
of Genetics and Development, Accepted
from
Dwarf Viruses’
D. V. R. REDDY, GUIDO BOCCARD0,2 R. OUTRIDGE, AND L. M. BLACK Provisional
Segments
University
of Illinois,
D. S. TEAKLE,3
Urbana, Illinois
61801
October 14, 1974
By employing two different buffer solutions for polyacrylamide gel electrophoresis, all genome segments of Fiji disease and maize rough dwarf virus were separated. Fiji disease virus contains ten genome segments with approximate genome molecular weights of 19.26 x loo and 19.85 x lOa, depending on the buffer employed for electrophoresis. Maize rough dwarf virus possesses ten dsRNA segments and according to the buffer employed for electrophoresis the approximate molecular weights of the genome were 18.91 x lo6 and 19.61 x 10”. In the samples of maize rough dwarf virus analysed, the evidence indicates that two types of virions were present which were distinguished by a slight difference in molecular weight for segment 10. The electrophoretic patterns for all dsRNA components of Fiji disease and maize rough dwarf viruses were very similar suggesting a close relationship between them.
Fiji disease virus (FDV) and maize rough dwarf virus (MRDV) are similar to wound tumor virus (WTV) (1, 2). The presence of dsRNA’ in the case of FDV was indicated by reactions between extracts of gall tissue from infected sugarcane and antisera prepared against synthetic poly(I):poly(C) dsRNA (3). However, we know of no previous report on the electrophoretic analysis of the RNA of FDV. MRDV was recently shown to possess dsRNA that could be fractionated into nine distinct zones (4). I This research was supported in part. by a grant from the National Science Foundation (GB 36220X). 2 Laboratorio di Fitovirologia applicata de1 C.N.R., Via 0. Vigliani 104, 10135 Torino, Italy. 3 Department of Microbiology, University of Queensland, St. Lucia, Brisbane, Australia 4067. ’ The following abbreviations are used: ds, doublestranded; 0.1 M His-Mg, 0.1 M histidine, 0.01 MgCI,; SDS, sodium dodecyl sulfate; Bis, N,N’-methylenebisacrylamide; Tris, tris(hydroxymethyl)aminomethane; EDTA, disodium dihydrogen ethylenediaminetetraacetate dihydrate; 0.045 M PTE, 0.045 M sodium phosphate, 0.030 M Tris, 0.002 M EDTA (pH 7.98.0). Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
287
By the use of appropriate buffer systems all dsRNA components of FDV and MRDV viruses have been resolved. FDV was purified from gall tissue on infected sugarcane leaves by repeated cycles of differential centrifugation or by employing polyethylene glycol (5). The purified virus in a 40% sucrose solution was diluted in 0.1 M His-Mg buffer, pH 7.0, collected on a Millipore filter of 0.05~pm pore diameter, resuspended in 0.2 ml of distilled water containing 7.5% sucrose and then treated with 2% SDS. FDV-RNA was also obtained by Kirby’s method as described before (6). MRDV was purified by a method described by Redolfi and Boccardo (4) and its RNA was extracted employing the doublephase phenol-SDS procedure (7) and by the perchlorate method (13). Electrophoresis of the RNA was performed at room temperature (about 25”) in 5% acrylamide gels containing 0.1% Bis with 900 ml of buffer in each compartment of the apparatus. The best resolution of the RNA c.omponents was achieved when both
288
SHORT
COMMUNICATIONS
Loening’s Tris-phosphate-EDTA buffer, Loening’s buffer. When electrophoresis was pH 7.7-7.8 (8) and 0.045 M PTE buffer, pH first performed in Loening’s buffer for 5 hr 7.9-8.0, were used. The 0.045 PTE buffer at 3.5 V/cm and the buffer was then gave as good or better resolution of the dsRNA components of reovirus type 3, FDV, MRDV and WTV as did the 0.04 M PTE buffer of Reddy and Black (6). Details of the preparation of PTE buffer were as follows. A 1 M phosphate buffer containing 69 g NaH,PO,. H,O and 71 g of Na,HPO, in a liter of distilled water was prepared and stored at room temperature. Stock solutions of 1 M Tris and 0.2 M EDTA were prepared separately and stored at 4”. PTE buffer (.045 M, pH 7.9-8.0) was prepared in distilled water just before the run. About 15-30 pg of RNA in lo-30-~1 volumes was loaded on each gel column and electrophoresis was performed at 3.5 V/cm for about 30 hr, unless otherwise specified. After completion of a run, gels were stained in 0.1% toluidine blue 0 and scanned at 540 nm in a Gilford spectrophotometer equipped with a linear transport. The electrophorograms were used to compute approximate molecular weights of the segFIG. 1. Electrophoresis of dsRNA genome segments. All molecular weight determinaments from (a) reovirus type 3, (b) WTV, ic) FDV and tions were made in coelectrophoretic runs (d) MRDV. Approximately 15-30 wg of RNA was run of reovirus type 3, FDV and MRDV. The in Loening’s buffer through 5% acrylamide gel at -3.5 molecular weights of reovirus type 3 RNA V/cm for 19 hr, stained in 0.1% toluidine blue 0 and segments adopted here as standards were destained in distilled water. Movement was from top the same as those employed by Martin and to bottom. Zweerink (9). All RNA components migrated as sharp zones in 5% acrylamide gels in accord with their double-stranded nature. In addition RNA from MRDV and FDV was incubated for 30 min at 37” in 1 pg RNase ‘A’ (Worthington Biochemicals) per ml as described by Reddy and Black (10). The RNA preparation treated with RNase and control untreated RNA samples yielded the same electrophoretic pattern for genome segments. Nine genome segments of FDV were resolved when. Loening’s buffer was used for electrophoresis (Figs. 1 and 2). SegFIG. 2. Electrophoresis of dsRNA genome segments 9 and 10 could not be resolved even ments from FDV (a, b, cl and MRDV (d, e, f). by prolonged electrophoresis in this buffer. Electrophoresis was at -3.5 V/cm for 30 hr in Although the PTE buffer resolved all ten PTE buffer (a,d), in Loening’s buffer (b,e) and for 5 segments of FDV (Fig. 2), the separation of hr in Loening’s buffer followed by 24 hr in PTE buffer segments 2, 3 and 4 were superior in (c and 0. Other details same as in Fig. 1.
SHORT
replaced with PTE before continuing electrophoresis at 3.5 V/cm for an additional 24 hr, results indicated that the desirable resolving features of both buffers could be incorporated in the same gel (Fig. 2). The molecular weight for the FDV genome was estimated as -19.26 x lo8 in PTE and -19.85 x lo6 in Loening’s buffer (Table 1 and Fig. 3). The correspondence between the observed weights of areas under the peaks and theoretical values indicates that all virions contain all RNA segments in equimolar amounts. The genome of MRDV showed anomalous results. The observed and theoretical values for areas under the peaks of segments l-9 indicated that these segments are present in all virions in equimolar amounts. On the other hand the area under the last peak comprising two segments (Fig. 2) indicates one molecular weight equivalent, and the two segments are thought to be two forms (a and b) of component 10. The near equality of peaks 10a and lob (Fig. 5) indicates that 10a appears to be present in about half of the virion population and lob in the remaining half. The separation of all segments could TABLE
289
COMMUNICATIONS
be accomplished with either buffer (Fig. 2). The molecular weight of the MRDV genome was -18.91 x lo8 in PTE and -19.61 x lo6 in Loening’s buffer (Table 1 and Fig. 4). A close relationship between MRDV and FDV is indicated by their very similar electrophoretic patterns. It was interesting to note that segments 5 and 7 of FDV and MRDV occupied virtually the same position in the gel irrespective of the buffer employed for electrophoresis. Since serological reactions have been reported between MRDV and rice black streaked dwarf virus (RBSDV) (II) it seems possible that the RNA components of RBSDV resemble those of MRDV. The dsRNA pattern for FDV and MRDV appears to be very different from that reported for WTV
REOWRUS
TYPE 3
1
APPROXIMATE MOLECULAR WEIGHTS OF dsRNA SEGMENTS OF FDV AND MRDV DETERMINED IN LOENING’S AND PTE BUFFERS
Segment
Loening’s buffep FDV
1 2 3 4 5 6 7 8 9 10
MRDV
PTE buffer’ FDV
MRDV
2.93” 2.53 2.50 2.48 2.25 1.90 1.50 1.32 1.22 -1.22
2.85 2.52 2.50 2.48 2.25 1.82 1.50 1.32 1.24 1.13 -
2.90 2.50 2.48 2.48 2.12 1.85 1.45 1.21 1.15 1.12 __
2.88 2.50 2.35 2.35 2.12 1.75 1.45 1.25 1.18 1.08 __
19.85
19.61
19.26
18.91
a Molecular weight x10-“. Molecular weights for the dsRNA of FDV and MRDV were calculated by using dsRNA segments of reovirus type 3 as standards.
MAIZE
ROUGH
DWARF
VIRUS
FIG. 3. Electrophorogram of dsRNA segments after electrophoresis in Loening’s buffer at -3.5 V/cm for 20 hr. staining in 0.1% toluidine blue 0, destaining in distilled water and scanning at 540 nm.
290
SHORT
COMMUNICATIONS
and RDV by Reddy et al. (12) (Fig. 1). Thus it appears that there are at least two main electrophoretic patterns for the dsRNA’s in this group of plant viruses.
REOVIRUS
FIJI
DISEASE
TYPE
E FIJI
DISEASE
DWARF
VIRUS
VIRUS
3
MAIZE
VIRUS
ROUGH
FIG. 5. Electrophoresis of dsRNA segments from FDV and MRDV for 5 hr in Loening’s buffer followed by 24 hr in PTE buffer. Other details same as in Fig. ACKNOWLEDGMENT We thank Dr. C. M. Wilson for the use of his Gilford spectrophotometer. REFERENCES MAIZE
ROUGH
DWARF
VIRUS
2.
3. 4. 5.
FIG. 4. Electrophorogram of dsRNA after electrophoresis in PTE buffer. Conditions of electrophoresis were as described for Fig. 3.
P. B.. and FRANCKI, R. I. B., “Commonwealth Mycological Institute Descriptions of Plant Viruses,” No. 119, 3 pp. 1973. LOVISOLO, 0.. “Commonwealth Mycological Institute Descriptions of Plant Viruses,” No. 72. 4 pp. 1971. IKEGAMI, M., and FRANCKI, R. I. B., Virology 56, 404-406 (1973). REDOLFI, P., and BOCCAFCDO, G., Virology 59, 319-322 (1974). OUTRIDGE, R., and TEAKLE, D. S.. (in preparation). REDDY, D. V. R., and BLACK, L. M., Virology 54, 557-562 (1973). BOCKSTAHLER, L. E., and KAESBERG, P., J. Mol.
1. HUTCHINSON,
6. 7.
SHORT
COMMUNICATIONS
Biol. 13, 127-137 (1965). 8. LOENING, U. E., Biochem. J. 113, 131-138 (1969). 9. MARTIN, S. A., and ZWEERINK, H. J., Virology 50, 495-506 (1972). IO. REDDY, D. V. R., and BLACK, L. M., Virology 61,458-473 (1974).
291
11. LUISONI, E., LOVISOLO, O., KITAGAWA, Y., and SHIKATA, E., Virology 52. 281-283 (1973). 12. REDDY, D. V. R., KIMIJRA, I., and BLACK, L. M. Virology 60, 293-296 (1974). 13. IGLEWSKI, W. J., and FRANKLIN, R. M., J. Viral. 1, 302-307 (1976)
63, 287-291 (1975)
Electrophoretic
Separation
Fiji Disease
of dsRNA
and Maize Rough
Genome
Department
of Genetics and Development, Accepted
from
Dwarf Viruses’
D. V. R. REDDY, GUIDO BOCCARD0,2 R. OUTRIDGE, AND L. M. BLACK Provisional
Segments
University
of Illinois,
D. S. TEAKLE,3
Urbana, Illinois
61801
October 14, 1974
By employing two different buffer solutions for polyacrylamide gel electrophoresis, all genome segments of Fiji disease and maize rough dwarf virus were separated. Fiji disease virus contains ten genome segments with approximate genome molecular weights of 19.26 x loo and 19.85 x lOa, depending on the buffer employed for electrophoresis. Maize rough dwarf virus possesses ten dsRNA segments and according to the buffer employed for electrophoresis the approximate molecular weights of the genome were 18.91 x lo6 and 19.61 x 10”. In the samples of maize rough dwarf virus analysed, the evidence indicates that two types of virions were present which were distinguished by a slight difference in molecular weight for segment 10. The electrophoretic patterns for all dsRNA components of Fiji disease and maize rough dwarf viruses were very similar suggesting a close relationship between them.
Fiji disease virus (FDV) and maize rough dwarf virus (MRDV) are similar to wound tumor virus (WTV) (1, 2). The presence of dsRNA’ in the case of FDV was indicated by reactions between extracts of gall tissue from infected sugarcane and antisera prepared against synthetic poly(I):poly(C) dsRNA (3). However, we know of no previous report on the electrophoretic analysis of the RNA of FDV. MRDV was recently shown to possess dsRNA that could be fractionated into nine distinct zones (4). I This research was supported in part. by a grant from the National Science Foundation (GB 36220X). 2 Laboratorio di Fitovirologia applicata de1 C.N.R., Via 0. Vigliani 104, 10135 Torino, Italy. 3 Department of Microbiology, University of Queensland, St. Lucia, Brisbane, Australia 4067. ’ The following abbreviations are used: ds, doublestranded; 0.1 M His-Mg, 0.1 M histidine, 0.01 MgCI,; SDS, sodium dodecyl sulfate; Bis, N,N’-methylenebisacrylamide; Tris, tris(hydroxymethyl)aminomethane; EDTA, disodium dihydrogen ethylenediaminetetraacetate dihydrate; 0.045 M PTE, 0.045 M sodium phosphate, 0.030 M Tris, 0.002 M EDTA (pH 7.98.0). Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
287
By the use of appropriate buffer systems all dsRNA components of FDV and MRDV viruses have been resolved. FDV was purified from gall tissue on infected sugarcane leaves by repeated cycles of differential centrifugation or by employing polyethylene glycol (5). The purified virus in a 40% sucrose solution was diluted in 0.1 M His-Mg buffer, pH 7.0, collected on a Millipore filter of 0.05~pm pore diameter, resuspended in 0.2 ml of distilled water containing 7.5% sucrose and then treated with 2% SDS. FDV-RNA was also obtained by Kirby’s method as described before (6). MRDV was purified by a method described by Redolfi and Boccardo (4) and its RNA was extracted employing the doublephase phenol-SDS procedure (7) and by the perchlorate method (13). Electrophoresis of the RNA was performed at room temperature (about 25”) in 5% acrylamide gels containing 0.1% Bis with 900 ml of buffer in each compartment of the apparatus. The best resolution of the RNA c.omponents was achieved when both
288
SHORT
COMMUNICATIONS
Loening’s Tris-phosphate-EDTA buffer, Loening’s buffer. When electrophoresis was pH 7.7-7.8 (8) and 0.045 M PTE buffer, pH first performed in Loening’s buffer for 5 hr 7.9-8.0, were used. The 0.045 PTE buffer at 3.5 V/cm and the buffer was then gave as good or better resolution of the dsRNA components of reovirus type 3, FDV, MRDV and WTV as did the 0.04 M PTE buffer of Reddy and Black (6). Details of the preparation of PTE buffer were as follows. A 1 M phosphate buffer containing 69 g NaH,PO,. H,O and 71 g of Na,HPO, in a liter of distilled water was prepared and stored at room temperature. Stock solutions of 1 M Tris and 0.2 M EDTA were prepared separately and stored at 4”. PTE buffer (.045 M, pH 7.9-8.0) was prepared in distilled water just before the run. About 15-30 pg of RNA in lo-30-~1 volumes was loaded on each gel column and electrophoresis was performed at 3.5 V/cm for about 30 hr, unless otherwise specified. After completion of a run, gels were stained in 0.1% toluidine blue 0 and scanned at 540 nm in a Gilford spectrophotometer equipped with a linear transport. The electrophorograms were used to compute approximate molecular weights of the segFIG. 1. Electrophoresis of dsRNA genome segments. All molecular weight determinaments from (a) reovirus type 3, (b) WTV, ic) FDV and tions were made in coelectrophoretic runs (d) MRDV. Approximately 15-30 wg of RNA was run of reovirus type 3, FDV and MRDV. The in Loening’s buffer through 5% acrylamide gel at -3.5 molecular weights of reovirus type 3 RNA V/cm for 19 hr, stained in 0.1% toluidine blue 0 and segments adopted here as standards were destained in distilled water. Movement was from top the same as those employed by Martin and to bottom. Zweerink (9). All RNA components migrated as sharp zones in 5% acrylamide gels in accord with their double-stranded nature. In addition RNA from MRDV and FDV was incubated for 30 min at 37” in 1 pg RNase ‘A’ (Worthington Biochemicals) per ml as described by Reddy and Black (10). The RNA preparation treated with RNase and control untreated RNA samples yielded the same electrophoretic pattern for genome segments. Nine genome segments of FDV were resolved when. Loening’s buffer was used for electrophoresis (Figs. 1 and 2). SegFIG. 2. Electrophoresis of dsRNA genome segments 9 and 10 could not be resolved even ments from FDV (a, b, cl and MRDV (d, e, f). by prolonged electrophoresis in this buffer. Electrophoresis was at -3.5 V/cm for 30 hr in Although the PTE buffer resolved all ten PTE buffer (a,d), in Loening’s buffer (b,e) and for 5 segments of FDV (Fig. 2), the separation of hr in Loening’s buffer followed by 24 hr in PTE buffer segments 2, 3 and 4 were superior in (c and 0. Other details same as in Fig. 1.
SHORT
replaced with PTE before continuing electrophoresis at 3.5 V/cm for an additional 24 hr, results indicated that the desirable resolving features of both buffers could be incorporated in the same gel (Fig. 2). The molecular weight for the FDV genome was estimated as -19.26 x lo8 in PTE and -19.85 x lo6 in Loening’s buffer (Table 1 and Fig. 3). The correspondence between the observed weights of areas under the peaks and theoretical values indicates that all virions contain all RNA segments in equimolar amounts. The genome of MRDV showed anomalous results. The observed and theoretical values for areas under the peaks of segments l-9 indicated that these segments are present in all virions in equimolar amounts. On the other hand the area under the last peak comprising two segments (Fig. 2) indicates one molecular weight equivalent, and the two segments are thought to be two forms (a and b) of component 10. The near equality of peaks 10a and lob (Fig. 5) indicates that 10a appears to be present in about half of the virion population and lob in the remaining half. The separation of all segments could TABLE
289
COMMUNICATIONS
be accomplished with either buffer (Fig. 2). The molecular weight of the MRDV genome was -18.91 x lo8 in PTE and -19.61 x lo6 in Loening’s buffer (Table 1 and Fig. 4). A close relationship between MRDV and FDV is indicated by their very similar electrophoretic patterns. It was interesting to note that segments 5 and 7 of FDV and MRDV occupied virtually the same position in the gel irrespective of the buffer employed for electrophoresis. Since serological reactions have been reported between MRDV and rice black streaked dwarf virus (RBSDV) (II) it seems possible that the RNA components of RBSDV resemble those of MRDV. The dsRNA pattern for FDV and MRDV appears to be very different from that reported for WTV
REOWRUS
TYPE 3
1
APPROXIMATE MOLECULAR WEIGHTS OF dsRNA SEGMENTS OF FDV AND MRDV DETERMINED IN LOENING’S AND PTE BUFFERS
Segment
Loening’s buffep FDV
1 2 3 4 5 6 7 8 9 10
MRDV
PTE buffer’ FDV
MRDV
2.93” 2.53 2.50 2.48 2.25 1.90 1.50 1.32 1.22 -1.22
2.85 2.52 2.50 2.48 2.25 1.82 1.50 1.32 1.24 1.13 -
2.90 2.50 2.48 2.48 2.12 1.85 1.45 1.21 1.15 1.12 __
2.88 2.50 2.35 2.35 2.12 1.75 1.45 1.25 1.18 1.08 __
19.85
19.61
19.26
18.91
a Molecular weight x10-“. Molecular weights for the dsRNA of FDV and MRDV were calculated by using dsRNA segments of reovirus type 3 as standards.
MAIZE
ROUGH
DWARF
VIRUS
FIG. 3. Electrophorogram of dsRNA segments after electrophoresis in Loening’s buffer at -3.5 V/cm for 20 hr. staining in 0.1% toluidine blue 0, destaining in distilled water and scanning at 540 nm.
290
SHORT
COMMUNICATIONS
and RDV by Reddy et al. (12) (Fig. 1). Thus it appears that there are at least two main electrophoretic patterns for the dsRNA’s in this group of plant viruses.
REOVIRUS
FIJI
DISEASE
TYPE
E FIJI
DISEASE
DWARF
VIRUS
VIRUS
3
MAIZE
VIRUS
ROUGH
FIG. 5. Electrophoresis of dsRNA segments from FDV and MRDV for 5 hr in Loening’s buffer followed by 24 hr in PTE buffer. Other details same as in Fig. ACKNOWLEDGMENT We thank Dr. C. M. Wilson for the use of his Gilford spectrophotometer. REFERENCES MAIZE
ROUGH
DWARF
VIRUS
2.
3. 4. 5.
FIG. 4. Electrophorogram of dsRNA after electrophoresis in PTE buffer. Conditions of electrophoresis were as described for Fig. 3.
P. B.. and FRANCKI, R. I. B., “Commonwealth Mycological Institute Descriptions of Plant Viruses,” No. 119, 3 pp. 1973. LOVISOLO, 0.. “Commonwealth Mycological Institute Descriptions of Plant Viruses,” No. 72. 4 pp. 1971. IKEGAMI, M., and FRANCKI, R. I. B., Virology 56, 404-406 (1973). REDOLFI, P., and BOCCAFCDO, G., Virology 59, 319-322 (1974). OUTRIDGE, R., and TEAKLE, D. S.. (in preparation). REDDY, D. V. R., and BLACK, L. M., Virology 54, 557-562 (1973). BOCKSTAHLER, L. E., and KAESBERG, P., J. Mol.
1. HUTCHINSON,
6. 7.
SHORT
COMMUNICATIONS
Biol. 13, 127-137 (1965). 8. LOENING, U. E., Biochem. J. 113, 131-138 (1969). 9. MARTIN, S. A., and ZWEERINK, H. J., Virology 50, 495-506 (1972). IO. REDDY, D. V. R., and BLACK, L. M., Virology 61,458-473 (1974).
291
11. LUISONI, E., LOVISOLO, O., KITAGAWA, Y., and SHIKATA, E., Virology 52. 281-283 (1973). 12. REDDY, D. V. R., KIMIJRA, I., and BLACK, L. M. Virology 60, 293-296 (1974). 13. IGLEWSKI, W. J., and FRANKLIN, R. M., J. Viral. 1, 302-307 (1976)