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Fine structure of rice dwarf virus
500
DISCUSSION TABLE
AND
PRELIMINARY
1
NUMBER OF PROGENY PH.~GE PER INFECTIVE CENTER IN THE PRESENCE AND ABSENCE OF THYMINE Phage T4v, T4vl T4vlthyTlvlthy-
Thymine a/ml 620 560 270 360
3
No thymine 540 430 11 14
picked. The resulting phage (T4vIthy-) grew almost as well as the original T4Dw1thy+ in the presence of 3 pg thymine per milliliter. Demonstration of thymine requirement by a one-step growth experiment. The thymine-requiring strain E. coli B3 was grown to 5 x 10’ cells per milliliter at 37” in M9, supplemented with 0.3% casamino acids (Bacto casamino acids, Difco, purified with charcoal) and 2 pg thymine per milliliter. The bacteria were centrifuged at room temperature, washed with buffer (0.02 M NaaHP04 , 0.02 M KH,P04, 0.07 M NaCl, 0.002 M MgS04), again centrifuged, resuspended in buffer, and starved with aeration at 37” for 20 minutes. Bacteriophage were then added at a multiplicity of 0.1 phage per bacterium. After 10 minutes at 37”, the bacteria and complexes were collected by centrifugation at room temperature. Adsorption was more than 90% complete. The complexes were resuspended in buffer at a concentration of lo7 infective centers per milliliter and diluted to a concentration of 10” infective centers per milliliter (lo4 bacteria per milliliter) into two tubes containing M9 casamino acid medium, one tube being supplemented with 3 pg thymine per milliliter. Samples were plated within the first 10 minutes to measure the concentration of infective centers and at 50 minutes to count the progeny phage. Under these conditions, the first progeny appear at about 20 minutes and all complexes have yielded progeny by 40 minutes. The results of such an experiment (Table 1) clearly demonstrate the thymine requirement of the mutant phage. The thymine for the few phage that are produced
REPORTS
in the absence of externally added thymine does not stem from the casamino acids since a similar result is obtained in ?119 without a casamino acid supplement. Sate added in pxjof. E. H. Simon and I. Tesaman (Proc. S&l. Acad. Sci., Wash., 1963, in press) ha1.e independently isolated thymidine-requiring mutants of phage T4. ACKNOWLEDGMENTS One of us (D. L. W.) is indebted to the Kulturministerium of the Land Nordrhein-Westfalen for a postdoctoral fellowship. REFERENCES 1. BARNER, H. D., and COHEN, S. S., J. Biol. Chem. 234,2987-2991 (1959). 2. HARM, W., Virolog?J 19,66-71 (1963). 91, 52-62 (1960). 3. HARM, W., 2. Vererbungslehre Interscience, 4. ADAMS, M. H., “Bacteriophages.” Yew York, 1959. DASIEL L. WULFF KARL METZGER Institut fiir Genetik der Universittit zu K61n K6ln-Lindenthal, Germany Accepted June 24, 1963
Fine Structure
of Rice Dwarf
Virus
As previously reported (I), rice dwarf virus particles partially purified by differential centrifugation are covered with thin membranous envelopes that appear to be firmly attached to the virus particles. Assuming that these envelopes may be of cytoplasmic origin and lipoproteinaceous in nature, we attempted to remove them by treating the isolated virus particles wit.h 10% chloroform for r/s hour, 25-50% et’her for 1 hour, or other substances, but failed to find any discernible effect. Probably the failure was due to the fact that these particles were not observed in negatively stained preparations. Quite recently, however, Kimura et al. (2) succeeded in removing this membranous envelope by treating the virus with snake venom and thus revealing numerous subunits on the surface of the virus particle. We have also found that the envelopes under consideration can be removed by chloroform or ether to reveal the fine structure of
DISCUSSION
AND PRELIMINARY
FIG. 1 Electron micrograph of rice dwarf virus particles sion
REPORTS
purified
by the chloroform
emul-
technique and mounted in phosphotungstate.
this virus as shown in the following illustrations. The virus particles shown in Fig. 1 were purified by the slightly modified chloroform emulsion technique (3). A small quantity of the virus suspension was mixed with an equal volume of 1% phosphotungstic acid (the pH of which had been adjusted to 7.0), and droplets of the solution were put, on collodion-coated grids. After the droplets had dried the preparation was examined in a JEM Type 5Y electron microscope, operating at a voltage of 100 kv instead of 80 kv as in former experiments. As to the virus particles shown in Figs. 2 and 3, the partially purified virus suspension was treated with twice its volume of ether at 37°C for 40 minutes. The aqueous phase was then removed and dialyzed for 2 hours at 5°C against 1% ammonium acetate. A small quantity of the virus suspension thus purified was mixed with an equal volume of 1% phosphotungstic acid as stated above.
As shown in Figs. 2 and 3, the individual virus particles appear hexagonal in outline and occasionally show the shape of an icosahedron which is discernible by means of shadowing. The capsomeres arranged on the surface of the virus particles can be clearly discerned and appear to be identical one with another in accord with the general concept that the protein shells or capsids of viruses are built up of identical subunits. Some particles appear centrally dark, implying that they contain phosphotungstate inside their hollow capsids. It will be noted that the rice dwarf virus particles show some resemblance to those of woundtumor virus (4). According to Bils and Hall the wound-tumor virus is about 60 rnp in diameter and has the shape of an icosahedron. The capsid of wound-tumor virus consists of 92 subunits or capsomeres about 7.5 mp in diameter. The rice dwarf virus is about 70 rnp in diameter as previously reported (1) ; it is likely that it has the shape
502
DISCUSSION
AND PRELIMINARY
REPORTS
FIGS. 2 and 3. Electron micrographs of rice dwarf virus particles treated with ether and mounted in phosphotungstate. They show numerous subunits arranged on the surface of the virus particles.
DISCCSSION AND PRELIMIXARY of an icosahedron, but the number of capsomeres arranged on the surface is not definitely known. REFERENCES T., SHIKATA, E., and KIXURA, I., Vi18, 192-205 (1962). 2. KIMURA, I., TOYODA, S., and SUZUKI, N., Ann. Phytopathol. Sot. Japan 28, 85 (Abstract) 1. FIXSU~HI,
rology
(1963). R., Science117,30-31 (1953). R. F., and HALL, C. E., Virology 17, 123-
3. SCHNEIDER, I. .$. BILS,
130 (1962).
T. E.
FUKUSHI SHIKATA
Botanical Institute Faculty of Agriculture Hokkaido Vniversit2/ Sapporo, Jupan Accepted August 8, 1963
Localization
of Rice Dwarf
Virus in
Its Insect Vector
It has been previously reported (1) that rice dwarf virus particles have been found in ultrathin sections of the salivary glands of infective leafhoppers, Nephotettix: cincticeps Uhl., but electron micrographs of these particles were not published. In Figs. 1 and 2 virus particles are illustrated in the salivary glands of infect,ive leafhoppers, the bodies of which were fixed for 24 hours in 1% buffered osmium tetroxide solution, dehydrated in graded dilutions of ethyl alcohol, embedded in methacrylate, and sectioned by means of a JUM-5 ultramicrotome equipped with glass knives. The virus particles are seen to be scattered or in small clusters in the cytoplasm but not in
REPORTS
503
the cell nuclei; the larger aggregates of virus particles in crystal-like arrangements that have been frequently found in the abdomens of infective leafhoppers were not encountered in the salivary glands. This may be partly attributable to the possibility that the virus does not multiply so rapidly in the salivary glands as in the abdomen. Figures 3 and 4 show the virus particles in the ovariole of an infective adult leafhopper, 13 days after the final molting. The ovarioles were quickly removed under a dissecting microscope and fixed at 4°C for 5 hours in 1% osmium tetroxide solution buffered at pH 7.4 and containing 0.1 M sucrose. After washing and dehydration the materials were embedded in methacrylate and sectioned in the same way as mentioned above. Up to the present time virus particles have not been definitely demonstrated in egg cells, notwithstanding the fact that we (9) have successfully demonstrated the presence of virus in the egg by inoculat.ing nonviruliferous leafhoppers with extracts from eggs laid by infective female leafhoppers. REFERENCES 1. FUKUSHI,
rology 2. FUKUSHI,
T., SHIKATA, E., and KIMURA, I., Vi18, 192-205 (1962). T., and KIMURA, I., Proc. Japan Acnd.
35,482-484
(1959). T. FUKUSHI
E. Botanical Institute Faculty of Agriculture Hokkaido University Sapporo, Japan Accepted August 8, 1963
SHIKATA
DISCUSSION TABLE
AND
PRELIMINARY
1
NUMBER OF PROGENY PH.~GE PER INFECTIVE CENTER IN THE PRESENCE AND ABSENCE OF THYMINE Phage T4v, T4vl T4vlthyTlvlthy-
Thymine a/ml 620 560 270 360
3
No thymine 540 430 11 14
picked. The resulting phage (T4vIthy-) grew almost as well as the original T4Dw1thy+ in the presence of 3 pg thymine per milliliter. Demonstration of thymine requirement by a one-step growth experiment. The thymine-requiring strain E. coli B3 was grown to 5 x 10’ cells per milliliter at 37” in M9, supplemented with 0.3% casamino acids (Bacto casamino acids, Difco, purified with charcoal) and 2 pg thymine per milliliter. The bacteria were centrifuged at room temperature, washed with buffer (0.02 M NaaHP04 , 0.02 M KH,P04, 0.07 M NaCl, 0.002 M MgS04), again centrifuged, resuspended in buffer, and starved with aeration at 37” for 20 minutes. Bacteriophage were then added at a multiplicity of 0.1 phage per bacterium. After 10 minutes at 37”, the bacteria and complexes were collected by centrifugation at room temperature. Adsorption was more than 90% complete. The complexes were resuspended in buffer at a concentration of lo7 infective centers per milliliter and diluted to a concentration of 10” infective centers per milliliter (lo4 bacteria per milliliter) into two tubes containing M9 casamino acid medium, one tube being supplemented with 3 pg thymine per milliliter. Samples were plated within the first 10 minutes to measure the concentration of infective centers and at 50 minutes to count the progeny phage. Under these conditions, the first progeny appear at about 20 minutes and all complexes have yielded progeny by 40 minutes. The results of such an experiment (Table 1) clearly demonstrate the thymine requirement of the mutant phage. The thymine for the few phage that are produced
REPORTS
in the absence of externally added thymine does not stem from the casamino acids since a similar result is obtained in ?119 without a casamino acid supplement. Sate added in pxjof. E. H. Simon and I. Tesaman (Proc. S&l. Acad. Sci., Wash., 1963, in press) ha1.e independently isolated thymidine-requiring mutants of phage T4. ACKNOWLEDGMENTS One of us (D. L. W.) is indebted to the Kulturministerium of the Land Nordrhein-Westfalen for a postdoctoral fellowship. REFERENCES 1. BARNER, H. D., and COHEN, S. S., J. Biol. Chem. 234,2987-2991 (1959). 2. HARM, W., Virolog?J 19,66-71 (1963). 91, 52-62 (1960). 3. HARM, W., 2. Vererbungslehre Interscience, 4. ADAMS, M. H., “Bacteriophages.” Yew York, 1959. DASIEL L. WULFF KARL METZGER Institut fiir Genetik der Universittit zu K61n K6ln-Lindenthal, Germany Accepted June 24, 1963
Fine Structure
of Rice Dwarf
Virus
As previously reported (I), rice dwarf virus particles partially purified by differential centrifugation are covered with thin membranous envelopes that appear to be firmly attached to the virus particles. Assuming that these envelopes may be of cytoplasmic origin and lipoproteinaceous in nature, we attempted to remove them by treating the isolated virus particles wit.h 10% chloroform for r/s hour, 25-50% et’her for 1 hour, or other substances, but failed to find any discernible effect. Probably the failure was due to the fact that these particles were not observed in negatively stained preparations. Quite recently, however, Kimura et al. (2) succeeded in removing this membranous envelope by treating the virus with snake venom and thus revealing numerous subunits on the surface of the virus particle. We have also found that the envelopes under consideration can be removed by chloroform or ether to reveal the fine structure of
DISCUSSION
AND PRELIMINARY
FIG. 1 Electron micrograph of rice dwarf virus particles sion
REPORTS
purified
by the chloroform
emul-
technique and mounted in phosphotungstate.
this virus as shown in the following illustrations. The virus particles shown in Fig. 1 were purified by the slightly modified chloroform emulsion technique (3). A small quantity of the virus suspension was mixed with an equal volume of 1% phosphotungstic acid (the pH of which had been adjusted to 7.0), and droplets of the solution were put, on collodion-coated grids. After the droplets had dried the preparation was examined in a JEM Type 5Y electron microscope, operating at a voltage of 100 kv instead of 80 kv as in former experiments. As to the virus particles shown in Figs. 2 and 3, the partially purified virus suspension was treated with twice its volume of ether at 37°C for 40 minutes. The aqueous phase was then removed and dialyzed for 2 hours at 5°C against 1% ammonium acetate. A small quantity of the virus suspension thus purified was mixed with an equal volume of 1% phosphotungstic acid as stated above.
As shown in Figs. 2 and 3, the individual virus particles appear hexagonal in outline and occasionally show the shape of an icosahedron which is discernible by means of shadowing. The capsomeres arranged on the surface of the virus particles can be clearly discerned and appear to be identical one with another in accord with the general concept that the protein shells or capsids of viruses are built up of identical subunits. Some particles appear centrally dark, implying that they contain phosphotungstate inside their hollow capsids. It will be noted that the rice dwarf virus particles show some resemblance to those of woundtumor virus (4). According to Bils and Hall the wound-tumor virus is about 60 rnp in diameter and has the shape of an icosahedron. The capsid of wound-tumor virus consists of 92 subunits or capsomeres about 7.5 mp in diameter. The rice dwarf virus is about 70 rnp in diameter as previously reported (1) ; it is likely that it has the shape
502
DISCUSSION
AND PRELIMINARY
REPORTS
FIGS. 2 and 3. Electron micrographs of rice dwarf virus particles treated with ether and mounted in phosphotungstate. They show numerous subunits arranged on the surface of the virus particles.
DISCCSSION AND PRELIMIXARY of an icosahedron, but the number of capsomeres arranged on the surface is not definitely known. REFERENCES T., SHIKATA, E., and KIXURA, I., Vi18, 192-205 (1962). 2. KIMURA, I., TOYODA, S., and SUZUKI, N., Ann. Phytopathol. Sot. Japan 28, 85 (Abstract) 1. FIXSU~HI,
rology
(1963). R., Science117,30-31 (1953). R. F., and HALL, C. E., Virology 17, 123-
3. SCHNEIDER, I. .$. BILS,
130 (1962).
T. E.
FUKUSHI SHIKATA
Botanical Institute Faculty of Agriculture Hokkaido Vniversit2/ Sapporo, Jupan Accepted August 8, 1963
Localization
of Rice Dwarf
Virus in
Its Insect Vector
It has been previously reported (1) that rice dwarf virus particles have been found in ultrathin sections of the salivary glands of infective leafhoppers, Nephotettix: cincticeps Uhl., but electron micrographs of these particles were not published. In Figs. 1 and 2 virus particles are illustrated in the salivary glands of infect,ive leafhoppers, the bodies of which were fixed for 24 hours in 1% buffered osmium tetroxide solution, dehydrated in graded dilutions of ethyl alcohol, embedded in methacrylate, and sectioned by means of a JUM-5 ultramicrotome equipped with glass knives. The virus particles are seen to be scattered or in small clusters in the cytoplasm but not in
REPORTS
503
the cell nuclei; the larger aggregates of virus particles in crystal-like arrangements that have been frequently found in the abdomens of infective leafhoppers were not encountered in the salivary glands. This may be partly attributable to the possibility that the virus does not multiply so rapidly in the salivary glands as in the abdomen. Figures 3 and 4 show the virus particles in the ovariole of an infective adult leafhopper, 13 days after the final molting. The ovarioles were quickly removed under a dissecting microscope and fixed at 4°C for 5 hours in 1% osmium tetroxide solution buffered at pH 7.4 and containing 0.1 M sucrose. After washing and dehydration the materials were embedded in methacrylate and sectioned in the same way as mentioned above. Up to the present time virus particles have not been definitely demonstrated in egg cells, notwithstanding the fact that we (9) have successfully demonstrated the presence of virus in the egg by inoculat.ing nonviruliferous leafhoppers with extracts from eggs laid by infective female leafhoppers. REFERENCES 1. FUKUSHI,
rology 2. FUKUSHI,
T., SHIKATA, E., and KIMURA, I., Vi18, 192-205 (1962). T., and KIMURA, I., Proc. Japan Acnd.
35,482-484
(1959). T. FUKUSHI
E. Botanical Institute Faculty of Agriculture Hokkaido University Sapporo, Japan Accepted August 8, 1963
SHIKATA