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The nucleic acid content of the homologous nuclear and cytoplasmic polyhedrosis viruses
I76
BIOCHIMICA AT BIOPHYSICA ACTA
THE NUCLEIC ACID CONTENT OF THE HOMOLOGOUS NUCLEAR AND CYTOPLASMIC POLYHEDROSIS VIRUSES N. XEROS
A.R.C. Virus Research Unit, Cambridge (Great Britain)"
(Received May I3th, I961)
SUMMARY Chromatographic and electrophoretic analysis of the nucleic acid content of the cytopolyhedra from Sphinx populi showed that they contained 0.9 % RNA and no DNA. Chromatographic analysis of cytopolyhedra from a further 5 species showed the presence in them of similar quantities of RNA and the absence of DNA. The nuclear polyhedrosis viruses contain DNA. The homologous patterns of virogenesis caused by these viruses are therefore due to DNA and RNA viruses, respectively, and perhaps to their DNA and RNA molecules alone.
INTRODUCTION It has been known for some time that the virus rods of the nucleopolyhedroses of Lepidopterous larvae are deoxyribonucleoprotein in composition I-3. These viruses form from the proteinaceous virogenic stromata which arise de novo in the nuclei of infected cells~,6. As they mature the stromata become heavily loaded with DNA. No RNA has ever been detected in them, just as no RNA has ever been detected in their viruses. Recently it has been found s,6 that homologous stromata are the site of virus formation in the cytopolyhedroses also. These, however, form in the cytoplasm of the midgut cells and become heavily loaded with RNA in their development. No DNA can be detected in them at any stage of their development with Feulgen staining. In both nucleo- and cyto-polyhedroses the main constituent of the stroma is 6 m/~ diameter fibrils which must be viral nucleoprotein. These become incorporated in the virus rods and spheres into which the stromata more or less completely transform s. 6. Virogenic stromata have been observedS, 6 in cytopolyhedroses of the midgut oi
Thaumatopoea p.ityocarapa, Phalera bucephala, Arctia ca~a, Automeris aurantica, Actias luna, Actias cecropia, Aglais urticae, sphinx ligustri, Panaxia dominula, and Abraxas grossulariata. The virogenic stromata of the cytopolyhedroses of the first three of these species have been examined in thin sections in the electron microscope. The concentration of viral spheres in maturing stromata was such that if they contained DNA, and even if the percentage of DNA in them was far less than in the " Present address: Pathology Department, University of Melbourne (Australia).
Biochim. Biophys. Acta, 55 (1962) 176-18x
HOMOLOGOUS DNA
AND
RNA
POLYHEDROSIS VIRUSES
x77
cytoplasmic iridescent virus of Tipula paludosa ~, they should have been detected with Feulgen staining as was the iridescent virus. The conclusion was drawn from the cytological data that the viruses of the cytopolyhedroses contained only RNA and no DNA. Since the viral spheres of the cytopolyhedroses become occluded in crystalline polyhedral protein in the same way as the virus rods of the nucleopolyhedroses, the cytopolyhedra which may be readily isolated, could be analyzed for their nucleic acid content. As there was no reason to consider that normal nuclear DNA might become occluded in them, it was expected that chromatographic analysis would show the presence of RNA and absence of DNA in the cytopolyhedra, confirming the cytological conclusions. To this end cytopolyhedra from six species were examined chromatographically. MATERIALS AND METHODS
Seven lots of midgut cytopolyhedra were prepared from larvae of six species of Lepidoptera (Table I) experimentally infected by feeding suitable preparations of TABLE I SAMPLES
OF MIDGUT POLYHEDRA ANALYZED FOR NUCLEIC ACID CONTENT
Sowr~oft,oly~dra
Drymig~ (~)
x. s. pop~i 2. L. dispar(dp) 3. A. urticae
9.3 x9.9
4. 5.
M . neustria L. dispar(ddp)
H. 7 Io.6
7.
B. b~ularia
x4.o
6. B. mori
x2.o
8.8
cytoplasmic polyhedra. Care was taken throughout to determine that the inocula were free of nuclear polyhedra. Polyhedra which had been obtained on first (dp) and second (ddp) passage of the virus of Sphinx l~ofuli through L y ~ disl~ar were examined. Polyhedra of S. ~bo~/i, of the same sample as used to infect the larvae of L. disbar, were also analysed. The polyhedra of MMacosoma seustria were obtained from larvae infected with polyhedra of Arctia v///ica. I am indebted to Dr. K. M. SHITS for the naturally occurring cytoplasmic polyhedra of B i s ~ b e ~ r i a used here to infect larvae of the same species. The original source of the virus of the polyhedra twice passaged through Bombyx mori is uncertain. The larvae of Aglais ur~icae were infected with a stock of cytoplasmic polyhedra obtained from B. mori fed with polyhedra of .Pyrameis cardui. Full details of the above and further cross-infection studies are to be found elsewhere 6. The guts and other tissues of samples of control larvae of the stocks used in this work were examined microscopically before, during and after the course of the experiments. The numerous control animals of all species used here were free of nucleopolyhedra, and, except in the case of the larvae of A. urticae in which a small proportion of the controls developed them naturally, all were likewise free of cytopolyhedra in their larval and pupal life. The experimentally infected an~at~ were examined individually either while still:alive or after death from disease, as larvae or pupae, and Biockim. Biopkys. Acta, 55 (x962) x76--x8z
178
z~. x~Ros
in every case their tissues were examined microscopically for nuclear and cytoplasmic polyhedra, l~o nucleopelyhedra were ever found in them. l ~ r e cytopolyhedra were prepared from isolated diseased midguts, either immediately or after the conventional method of combined bacterial and autolytic digestion of the tissue in water for some days at room temperature. Samples of polyhedra virtually free of bacterial and tissue impurities were prepared by washing in water and o.85 % NaC1 and differential centrifugation. These preparations were then allowed to stand i~ water with a few drops of chloroform for some days. When being prepared for chromatography the polyhedra were further cleaned by washing and centrifugation. They were then allowed to stand for x-4 days at 37° in chloroform-saturated water to whichri bonuclease had been added, in order to clean the Surfaces of the polyhedra thoroughly and to lyze any bacteria that might still remain and destroy their RNA. After this treatment they were washed and differentiallycentrifuged a further three times. Cleanliness of the preparations was checked by microscopical examination of Giemsa-stained heat-dried smears. The cleaned polyhedra were dehydrated in weighed centrit[uge tubes with four changes of acetone. They were air-dri.ed overnight in a hot oven. The dry weight of each of the seven lots was between 9 and 2o rag. The total surface area of the viral spheres ill the cytopolyhedra, when examined in thin sections, was of the same order as that of the virus rods in the nuclear polyhedra, and the dense core of each was about half the diameter of the whole particle5. It was therefore expected t h a t the proportion of virus and probably of nucleic acid would be roughly of the same order (approx I%) as in the nuclear polyhedra, and that the amount of polyhedra used would be more than sufficient for analysis of their nucleic acid content. Since the results of cytochemical and cytological work made it virtually certain that the viruses of the cytopolyhedroses contained RNA and no DNA, the method of hydrolysis chosen for identifying and estimating the nucleic acid content of the polyhedra was that of SCHMIDT AND THANNHAUSER8 as modified by MARKHAM°. This method involves treating the nucleic acids with i N KOH at room temperature (2o°). The DNA's are not hydrolyzed by this treatment while the RNA's are hydrolyzed to their constituent mono-nucleotides. Hydrolysis of the polyhedra was allowed to proceed for 48 h at room temperature in o.x to o.3 ml I N KOH. At the completion of the hydrolysis the solutions were adjusted to not less than pH 4 with glacial acetic (lots 1-4) or perchloric acid (lots 5--7) and centrifuged. The supernatants and washings were combined, dried down in an air stream to less than o.I ml and with a little added water chromatographed (A) as single spots on Whatman No.3 MM pape rl° for 4o or 24 h. The residues from the neutralized KOH digests were treated for 3o min at 55 ° with I N HC1 in order to liberate the purines of any DNA which may have been present in the polyhedra. After cooling and concentrating the hydrolysates were centrifuged and the supernatants (B) chromatographed on Whatman No. I paper in isopropanol-HC1 (see ref. 1I) for 20 h. Small amounts of ultraviolet absorbing material remained at the origin of the chromatograrns (A) of the alkali.digests. The paper was cut diagonally and these spots( residues C) were rerun in isopropanol-HC1 for 17 h 1Loi~eck for DNA purines. Bage~ratio analysis and determination of the percentage of RNA was completed only J!or the polyhedra of S, populi (lot I). In this case the ribonucleotide spots on the Biochim. Biophys. Acta, 55 (x96z) x76-x8x
HOMOLOGOUS D N A AND R N A
POLYHEDROSIS VIRUSES
t79
isopropanol-NH s chromatogram (A) were eluted and separated electrophoretically using fol~nate buffer, pH 3.5 (see ref. 9). The four spots of the nucleotides of RNA were lo~ted, eluted overnight in 5 ml o.z N HCI and the abs6rbancies of the eluates read in an ultraviolet spectrophotometer in z - c m cells over the range 2oo-3oo rap. The ribonucleotide spots of the chromatograms (A) of the L. dispar (ddp, lot 5) and A. t~rticae (lot 3) polyhedra were extracted with o.z N HCI. After being reduced to dryness, they were hydrolyzed in o.25 rnl I N HC1 for 3o rain at zoo°, cooled, dried down, ts~en up in water and run on chromatograms (D) in isopropanol-HC1 for z 9 h to separate the free purines and pyrimidine nucleotides. Prints of all the chromatograms by absorption and by fluorescence, in acid and alkali, iv~ultraviolet light (near 26o rap) were made by themethods of MARKHAMAND SMITH ls. RESULTS
The chromatograms of the superuatants (A) of all the alkali digests each showed the two slowly moving spots of the ribonucleotides, of which the upper was guanylic acid. The four ribonudeotides of chromatogram IA (polyhedra of S. ~bofJdi) were well separated by eiectrophoresis. The shapes of the ultraviolet absorption curves of the eluates and the ratios of the absorbancies at the various wave lengths were close to those of chromatographically pure specimens of these substances. The calculated molar ratio of the nucleotide residues A: G: C: U equals4z: z3: z3:z3 (Table rI). The TABLE TI D ] ~ I ~ R M I N A T I O N O1r T H E MOLZCULAR RATIO OF T H E BAS]t.S A N D T H E T O T A L W ~ I G H T OF T H E N U C L E O T I DES IN 9.3
mg
D R Y W E I G H T O1• C Y T O P O L Y H E D R A OF ~ .
Tat4/ ~ , ~
A G C U Totals
Po~di
Ma/c#tdarv~/o (~)
36.2 2x.4 H.2 2o.z 88.9
o! ~
o.4z 0.23 o.x3 0.23 I .oo
total weight of the isolated nucleotides is calculated to be 88.9 pg. The total calculated I~NA content (v/z. the total weight of the nucleotide residues less the weight of an equal number of molecules of water) of the S. ~op~di polyhedra is therefore 84. 4 pg. Since this was present in 9.3 mg dry weight of polyhedra, the calculated RNA content of the S. poptdi polyhedra is o.9 %, as previously reported. This compares with o.84 % DNA content in the nuclear polyhedra of B. mo~TM. It would appear that the RNA content of the polyhedra of the other species is of the .,~me order, as judged roughly by the ultraviolet absorption of the mononucleotide spots from their alkali digests. In the case of the polyhedra of L. dispar (ddp, lot 5) and A. m,t/cae (lot 3), whose nucleotides were further separated by chromatography in isopropanol-HCl, the uridylic acid, cytidylic acid, adenine and guanine were well separated. Though these were not eluted, the absorption of the spots on Biochim. Biophys. Acta, 55 (x96a) x76-xSx
I80
N. XEROS
these chromstograms suggested that in the RNA of these two species, as in the S. p o ~ i virus, adenine predominates and there appears to be very little cytosine. In the chromatograms A of L. dispar (dp) and L. dispar (ddp) polyhedra, large ultraviolet absorbing spots were present at the bottom of the chromatogram where nucleosides might be expected. These spots were fluorescent, but their fluorescence was not abolished by ammonia vapour. They may indicate some other compound present in the stromata of L. dispar gut cells infected with S. populi virus. The chromatograms (C2-7) of the ultraviolet absorbing residues remaining at the origin after running the alkaline hydrolysates Showed no detectable ultraviolet absorption but only weakly fluorescing spots of which the fluorescence was extinguish° ed by ammonia vapour. These could be due to guanine and guanyh~c acid deriving from traces of guanylic acid trailing behind the original guanylic acid spot. In any case the quantities of guanine must be less than o.I/~g, i.e. less than I ~ of the amount of guanine present as mononucleotide in chromatograms A I - 7. The chromatograms (B I-7) of the acid-extracted residues of the KOH digests all merely showed the presence, in the fluorescent prints only, of minute amounts (of the order of o.x/~g) of guanine and gnanylic acid. The origifi of the guanylic acid could not have been DNA since on mild acid hydrolysis of DNA its purine bases are readily freed, but no purine nucleotides are liberated. The results indicate that some viral RNA had escaped complete hydrolysis in KOH, possibly because it adhered to the tube above the level of the KOH. These residues are not completely hydrolyzed to free bases in the subsequent mild acid hydrolysis and account for the presence of both guanine and guanylic acid in chromatograms B. An additional ultraviolet absorbing spot lying between guanine and guanylic acid was present on each of chromatograms B 5-7. They were green, not bluish-violet, when excited by ultraviolet light, and their fluorescence was not extinguished by ammonia vapour. It is therefore not a recognized nucleic acid base. The results indicate an absence of DNA in the cytopolyhedra, and it is concluded that the only nucleic acid present in the cytopolyhedra is therefore RNA. DISCUSSION
Since the earlier report that the place of the DNA of the nucleopolyhedrosis viruses was taken by RNA in the cytopolyhedroses6, KRIEG18has found by chemical analysis an absence of DNA and the presence of RNA in virus spheres extracted from cyo topolyhedra of Dasychira pudibunda. He found also an absence of nucleic acid in the dissolved polyhedral protein from which the virus spheres had been removed by ultraeentrifugation. The report 14 that BERGOLDhad found cytopolyhedra to contain DNA was without doubt due to contamination of his material with nuclear polyhedra, indicated by the presence of virus rods in it 16. All other authorsS,6,~,l*, le-~e have found only spherical particles in cytopolyhedra. Differential heat denaturation with subsequent stainings. IT to distinguish between nucleo- and cyto-polyhedra was not done b y BERGOLD. The cytopolyhedra or virus have been analyzed chromatographically or chemically for their nucleic acid in seven species. The cytopolyhedroses of a further ten species (listed in the introduction and including one whose polyhedra have been analyze d - A. urticae,) have been examined cytologically. Their vir()genic stromata have Biochira. Biophys. Acta, 55 (z962) x76--x8z
HOMOLOGOUS DNA AND R N A
POLYHEDROSIS VIRUSES
TSX
been id~mtified and their nucleic acid recognized cytochemicaHy to be RNA. In a further twelve species 5 examined for cytoplasmic DNA prior to the discovery of the stromata, no Feulgen positive material was ever folmd in the cytophsm of infected cells, and these also therefore appear to have RNA cytopolyhedral viruses. There can be no question therefore that whereas nucleo-polyhedrai viruses contain DNA," the homologous cytopolyhedral viruses contain RNA. ACKNOWLEDGEMENTS I w i s h t o a c k n o w l e d g e m y i n d e b t e d n e s s t o D r . R . MARKHAM, F . ' R . S. for his a d v i c e t h r o u g h o u t t h e c h r o m a t o g r a p h i c s t u d i e s , t o Dr. R. MARKHAM a n d Mr. J . V~. LEGGE for r e a d i n g a n d c o m m e n t i n g o n t h e m a n u s c r i p t , a n d to D r . K . M. SMITH f o r t h e v i r u s e s of s e v e r a l c y t o p o l y h e d r o s e s . T h e i n v e s t i g a t i o n w a s c a r r i e d o u t a n d s u b m i t t e d i n I 9 5 6 in p a r t f u l f i l m e n t of a P h . D . a t t h e U n i v e r s i t y of C a m b r i d g e . REFERENCES I t s * s e 7 s *
G. H. BERGOLD, Z. Natur/ofsch., 2b (z947) I22. A. G ~ A , J. BRACKETAND R. J~ESSR, Bull. Read. roy. reed. Belg., xo (x945) 72. j . D. SMITH AND G. R. WYATT, Biochem. J., 49 (I95 x) x44. N. XZROS, Nature, x75 (x955) 588. N. X~:ROS, P h . D . Thesis, University of Cambridge, x956. N. XEROS, Nature, x78 (x956) 4I~. N. X~:ROS, Nature, x74 (x954) 562. G. SCtIMIDT AND S. J. THANNHAUSER,J. Biol. Chem., i6x (x945) 83. It. MARKHAM,in K. PARCH AND M. V. TRACRY, Modern Methods o/ Plant .4nalysisj Vol. 4, Springer-Verlag, Berlin, 1955J P. 246. 10 R. MARKHAMAND J. D. SMITH, Biochem. J., 52 (x95~) 552. zl G. R. WYATT, Biochera. J., 48 (x95 x) 584 . it R. MARKHAMAND J. D. SMITH, Biochem. J., 45 (1949) 294. is A. KRIE~, Natnrwisser,scha/hra, 23 (i956) 537, xt F. T. IBIRD AND M. M. WHALES, Canad. J. Zool., 2x (x954) 82. xs G. It. BERGOLDj Ann. N. Y. Acad. Sci., 56 (I953) 495. 10 K. M. SMITH AND R. W. G. WYCKOIrF, Nature, x66 (x95 o) 86I, 17 N, XEROS, No,ture, I7o (I952) xo73. xs K. M. SMITH AND N. XEROS, Parasitology, 44 (x954) 400xs M. TSUIITA, Proc. Jap. Acad., 3 x (x955) 93. m K. M. SMITH AND N. XXROS, Nature, x73 (I954) 866.
Biochim. Biophys. Acta, 55 (x96a) x76-x8x
BIOCHIMICA AT BIOPHYSICA ACTA
THE NUCLEIC ACID CONTENT OF THE HOMOLOGOUS NUCLEAR AND CYTOPLASMIC POLYHEDROSIS VIRUSES N. XEROS
A.R.C. Virus Research Unit, Cambridge (Great Britain)"
(Received May I3th, I961)
SUMMARY Chromatographic and electrophoretic analysis of the nucleic acid content of the cytopolyhedra from Sphinx populi showed that they contained 0.9 % RNA and no DNA. Chromatographic analysis of cytopolyhedra from a further 5 species showed the presence in them of similar quantities of RNA and the absence of DNA. The nuclear polyhedrosis viruses contain DNA. The homologous patterns of virogenesis caused by these viruses are therefore due to DNA and RNA viruses, respectively, and perhaps to their DNA and RNA molecules alone.
INTRODUCTION It has been known for some time that the virus rods of the nucleopolyhedroses of Lepidopterous larvae are deoxyribonucleoprotein in composition I-3. These viruses form from the proteinaceous virogenic stromata which arise de novo in the nuclei of infected cells~,6. As they mature the stromata become heavily loaded with DNA. No RNA has ever been detected in them, just as no RNA has ever been detected in their viruses. Recently it has been found s,6 that homologous stromata are the site of virus formation in the cytopolyhedroses also. These, however, form in the cytoplasm of the midgut cells and become heavily loaded with RNA in their development. No DNA can be detected in them at any stage of their development with Feulgen staining. In both nucleo- and cyto-polyhedroses the main constituent of the stroma is 6 m/~ diameter fibrils which must be viral nucleoprotein. These become incorporated in the virus rods and spheres into which the stromata more or less completely transform s. 6. Virogenic stromata have been observedS, 6 in cytopolyhedroses of the midgut oi
Thaumatopoea p.ityocarapa, Phalera bucephala, Arctia ca~a, Automeris aurantica, Actias luna, Actias cecropia, Aglais urticae, sphinx ligustri, Panaxia dominula, and Abraxas grossulariata. The virogenic stromata of the cytopolyhedroses of the first three of these species have been examined in thin sections in the electron microscope. The concentration of viral spheres in maturing stromata was such that if they contained DNA, and even if the percentage of DNA in them was far less than in the " Present address: Pathology Department, University of Melbourne (Australia).
Biochim. Biophys. Acta, 55 (1962) 176-18x
HOMOLOGOUS DNA
AND
RNA
POLYHEDROSIS VIRUSES
x77
cytoplasmic iridescent virus of Tipula paludosa ~, they should have been detected with Feulgen staining as was the iridescent virus. The conclusion was drawn from the cytological data that the viruses of the cytopolyhedroses contained only RNA and no DNA. Since the viral spheres of the cytopolyhedroses become occluded in crystalline polyhedral protein in the same way as the virus rods of the nucleopolyhedroses, the cytopolyhedra which may be readily isolated, could be analyzed for their nucleic acid content. As there was no reason to consider that normal nuclear DNA might become occluded in them, it was expected that chromatographic analysis would show the presence of RNA and absence of DNA in the cytopolyhedra, confirming the cytological conclusions. To this end cytopolyhedra from six species were examined chromatographically. MATERIALS AND METHODS
Seven lots of midgut cytopolyhedra were prepared from larvae of six species of Lepidoptera (Table I) experimentally infected by feeding suitable preparations of TABLE I SAMPLES
OF MIDGUT POLYHEDRA ANALYZED FOR NUCLEIC ACID CONTENT
Sowr~oft,oly~dra
Drymig~ (~)
x. s. pop~i 2. L. dispar(dp) 3. A. urticae
9.3 x9.9
4. 5.
M . neustria L. dispar(ddp)
H. 7 Io.6
7.
B. b~ularia
x4.o
6. B. mori
x2.o
8.8
cytoplasmic polyhedra. Care was taken throughout to determine that the inocula were free of nuclear polyhedra. Polyhedra which had been obtained on first (dp) and second (ddp) passage of the virus of Sphinx l~ofuli through L y ~ disl~ar were examined. Polyhedra of S. ~bo~/i, of the same sample as used to infect the larvae of L. disbar, were also analysed. The polyhedra of MMacosoma seustria were obtained from larvae infected with polyhedra of Arctia v///ica. I am indebted to Dr. K. M. SHITS for the naturally occurring cytoplasmic polyhedra of B i s ~ b e ~ r i a used here to infect larvae of the same species. The original source of the virus of the polyhedra twice passaged through Bombyx mori is uncertain. The larvae of Aglais ur~icae were infected with a stock of cytoplasmic polyhedra obtained from B. mori fed with polyhedra of .Pyrameis cardui. Full details of the above and further cross-infection studies are to be found elsewhere 6. The guts and other tissues of samples of control larvae of the stocks used in this work were examined microscopically before, during and after the course of the experiments. The numerous control animals of all species used here were free of nucleopolyhedra, and, except in the case of the larvae of A. urticae in which a small proportion of the controls developed them naturally, all were likewise free of cytopolyhedra in their larval and pupal life. The experimentally infected an~at~ were examined individually either while still:alive or after death from disease, as larvae or pupae, and Biockim. Biopkys. Acta, 55 (x962) x76--x8z
178
z~. x~Ros
in every case their tissues were examined microscopically for nuclear and cytoplasmic polyhedra, l~o nucleopelyhedra were ever found in them. l ~ r e cytopolyhedra were prepared from isolated diseased midguts, either immediately or after the conventional method of combined bacterial and autolytic digestion of the tissue in water for some days at room temperature. Samples of polyhedra virtually free of bacterial and tissue impurities were prepared by washing in water and o.85 % NaC1 and differential centrifugation. These preparations were then allowed to stand i~ water with a few drops of chloroform for some days. When being prepared for chromatography the polyhedra were further cleaned by washing and centrifugation. They were then allowed to stand for x-4 days at 37° in chloroform-saturated water to whichri bonuclease had been added, in order to clean the Surfaces of the polyhedra thoroughly and to lyze any bacteria that might still remain and destroy their RNA. After this treatment they were washed and differentiallycentrifuged a further three times. Cleanliness of the preparations was checked by microscopical examination of Giemsa-stained heat-dried smears. The cleaned polyhedra were dehydrated in weighed centrit[uge tubes with four changes of acetone. They were air-dri.ed overnight in a hot oven. The dry weight of each of the seven lots was between 9 and 2o rag. The total surface area of the viral spheres ill the cytopolyhedra, when examined in thin sections, was of the same order as that of the virus rods in the nuclear polyhedra, and the dense core of each was about half the diameter of the whole particle5. It was therefore expected t h a t the proportion of virus and probably of nucleic acid would be roughly of the same order (approx I%) as in the nuclear polyhedra, and that the amount of polyhedra used would be more than sufficient for analysis of their nucleic acid content. Since the results of cytochemical and cytological work made it virtually certain that the viruses of the cytopolyhedroses contained RNA and no DNA, the method of hydrolysis chosen for identifying and estimating the nucleic acid content of the polyhedra was that of SCHMIDT AND THANNHAUSER8 as modified by MARKHAM°. This method involves treating the nucleic acids with i N KOH at room temperature (2o°). The DNA's are not hydrolyzed by this treatment while the RNA's are hydrolyzed to their constituent mono-nucleotides. Hydrolysis of the polyhedra was allowed to proceed for 48 h at room temperature in o.x to o.3 ml I N KOH. At the completion of the hydrolysis the solutions were adjusted to not less than pH 4 with glacial acetic (lots 1-4) or perchloric acid (lots 5--7) and centrifuged. The supernatants and washings were combined, dried down in an air stream to less than o.I ml and with a little added water chromatographed (A) as single spots on Whatman No.3 MM pape rl° for 4o or 24 h. The residues from the neutralized KOH digests were treated for 3o min at 55 ° with I N HC1 in order to liberate the purines of any DNA which may have been present in the polyhedra. After cooling and concentrating the hydrolysates were centrifuged and the supernatants (B) chromatographed on Whatman No. I paper in isopropanol-HC1 (see ref. 1I) for 20 h. Small amounts of ultraviolet absorbing material remained at the origin of the chromatograrns (A) of the alkali.digests. The paper was cut diagonally and these spots( residues C) were rerun in isopropanol-HC1 for 17 h 1Loi~eck for DNA purines. Bage~ratio analysis and determination of the percentage of RNA was completed only J!or the polyhedra of S, populi (lot I). In this case the ribonucleotide spots on the Biochim. Biophys. Acta, 55 (x96z) x76-x8x
HOMOLOGOUS D N A AND R N A
POLYHEDROSIS VIRUSES
t79
isopropanol-NH s chromatogram (A) were eluted and separated electrophoretically using fol~nate buffer, pH 3.5 (see ref. 9). The four spots of the nucleotides of RNA were lo~ted, eluted overnight in 5 ml o.z N HCI and the abs6rbancies of the eluates read in an ultraviolet spectrophotometer in z - c m cells over the range 2oo-3oo rap. The ribonucleotide spots of the chromatograms (A) of the L. dispar (ddp, lot 5) and A. t~rticae (lot 3) polyhedra were extracted with o.z N HCI. After being reduced to dryness, they were hydrolyzed in o.25 rnl I N HC1 for 3o rain at zoo°, cooled, dried down, ts~en up in water and run on chromatograms (D) in isopropanol-HC1 for z 9 h to separate the free purines and pyrimidine nucleotides. Prints of all the chromatograms by absorption and by fluorescence, in acid and alkali, iv~ultraviolet light (near 26o rap) were made by themethods of MARKHAMAND SMITH ls. RESULTS
The chromatograms of the superuatants (A) of all the alkali digests each showed the two slowly moving spots of the ribonucleotides, of which the upper was guanylic acid. The four ribonudeotides of chromatogram IA (polyhedra of S. ~bofJdi) were well separated by eiectrophoresis. The shapes of the ultraviolet absorption curves of the eluates and the ratios of the absorbancies at the various wave lengths were close to those of chromatographically pure specimens of these substances. The calculated molar ratio of the nucleotide residues A: G: C: U equals4z: z3: z3:z3 (Table rI). The TABLE TI D ] ~ I ~ R M I N A T I O N O1r T H E MOLZCULAR RATIO OF T H E BAS]t.S A N D T H E T O T A L W ~ I G H T OF T H E N U C L E O T I DES IN 9.3
mg
D R Y W E I G H T O1• C Y T O P O L Y H E D R A OF ~ .
Tat4/ ~ , ~
A G C U Totals
Po~di
Ma/c#tdarv~/o (~)
36.2 2x.4 H.2 2o.z 88.9
o! ~
o.4z 0.23 o.x3 0.23 I .oo
total weight of the isolated nucleotides is calculated to be 88.9 pg. The total calculated I~NA content (v/z. the total weight of the nucleotide residues less the weight of an equal number of molecules of water) of the S. ~op~di polyhedra is therefore 84. 4 pg. Since this was present in 9.3 mg dry weight of polyhedra, the calculated RNA content of the S. poptdi polyhedra is o.9 %, as previously reported. This compares with o.84 % DNA content in the nuclear polyhedra of B. mo~TM. It would appear that the RNA content of the polyhedra of the other species is of the .,~me order, as judged roughly by the ultraviolet absorption of the mononucleotide spots from their alkali digests. In the case of the polyhedra of L. dispar (ddp, lot 5) and A. m,t/cae (lot 3), whose nucleotides were further separated by chromatography in isopropanol-HCl, the uridylic acid, cytidylic acid, adenine and guanine were well separated. Though these were not eluted, the absorption of the spots on Biochim. Biophys. Acta, 55 (x96a) x76-xSx
I80
N. XEROS
these chromstograms suggested that in the RNA of these two species, as in the S. p o ~ i virus, adenine predominates and there appears to be very little cytosine. In the chromatograms A of L. dispar (dp) and L. dispar (ddp) polyhedra, large ultraviolet absorbing spots were present at the bottom of the chromatogram where nucleosides might be expected. These spots were fluorescent, but their fluorescence was not abolished by ammonia vapour. They may indicate some other compound present in the stromata of L. dispar gut cells infected with S. populi virus. The chromatograms (C2-7) of the ultraviolet absorbing residues remaining at the origin after running the alkaline hydrolysates Showed no detectable ultraviolet absorption but only weakly fluorescing spots of which the fluorescence was extinguish° ed by ammonia vapour. These could be due to guanine and guanyh~c acid deriving from traces of guanylic acid trailing behind the original guanylic acid spot. In any case the quantities of guanine must be less than o.I/~g, i.e. less than I ~ of the amount of guanine present as mononucleotide in chromatograms A I - 7. The chromatograms (B I-7) of the acid-extracted residues of the KOH digests all merely showed the presence, in the fluorescent prints only, of minute amounts (of the order of o.x/~g) of guanine and gnanylic acid. The origifi of the guanylic acid could not have been DNA since on mild acid hydrolysis of DNA its purine bases are readily freed, but no purine nucleotides are liberated. The results indicate that some viral RNA had escaped complete hydrolysis in KOH, possibly because it adhered to the tube above the level of the KOH. These residues are not completely hydrolyzed to free bases in the subsequent mild acid hydrolysis and account for the presence of both guanine and guanylic acid in chromatograms B. An additional ultraviolet absorbing spot lying between guanine and guanylic acid was present on each of chromatograms B 5-7. They were green, not bluish-violet, when excited by ultraviolet light, and their fluorescence was not extinguished by ammonia vapour. It is therefore not a recognized nucleic acid base. The results indicate an absence of DNA in the cytopolyhedra, and it is concluded that the only nucleic acid present in the cytopolyhedra is therefore RNA. DISCUSSION
Since the earlier report that the place of the DNA of the nucleopolyhedrosis viruses was taken by RNA in the cytopolyhedroses6, KRIEG18has found by chemical analysis an absence of DNA and the presence of RNA in virus spheres extracted from cyo topolyhedra of Dasychira pudibunda. He found also an absence of nucleic acid in the dissolved polyhedral protein from which the virus spheres had been removed by ultraeentrifugation. The report 14 that BERGOLDhad found cytopolyhedra to contain DNA was without doubt due to contamination of his material with nuclear polyhedra, indicated by the presence of virus rods in it 16. All other authorsS,6,~,l*, le-~e have found only spherical particles in cytopolyhedra. Differential heat denaturation with subsequent stainings. IT to distinguish between nucleo- and cyto-polyhedra was not done b y BERGOLD. The cytopolyhedra or virus have been analyzed chromatographically or chemically for their nucleic acid in seven species. The cytopolyhedroses of a further ten species (listed in the introduction and including one whose polyhedra have been analyze d - A. urticae,) have been examined cytologically. Their vir()genic stromata have Biochira. Biophys. Acta, 55 (z962) x76--x8z
HOMOLOGOUS DNA AND R N A
POLYHEDROSIS VIRUSES
TSX
been id~mtified and their nucleic acid recognized cytochemicaHy to be RNA. In a further twelve species 5 examined for cytoplasmic DNA prior to the discovery of the stromata, no Feulgen positive material was ever folmd in the cytophsm of infected cells, and these also therefore appear to have RNA cytopolyhedral viruses. There can be no question therefore that whereas nucleo-polyhedrai viruses contain DNA," the homologous cytopolyhedral viruses contain RNA. ACKNOWLEDGEMENTS I w i s h t o a c k n o w l e d g e m y i n d e b t e d n e s s t o D r . R . MARKHAM, F . ' R . S. for his a d v i c e t h r o u g h o u t t h e c h r o m a t o g r a p h i c s t u d i e s , t o Dr. R. MARKHAM a n d Mr. J . V~. LEGGE for r e a d i n g a n d c o m m e n t i n g o n t h e m a n u s c r i p t , a n d to D r . K . M. SMITH f o r t h e v i r u s e s of s e v e r a l c y t o p o l y h e d r o s e s . T h e i n v e s t i g a t i o n w a s c a r r i e d o u t a n d s u b m i t t e d i n I 9 5 6 in p a r t f u l f i l m e n t of a P h . D . a t t h e U n i v e r s i t y of C a m b r i d g e . REFERENCES I t s * s e 7 s *
G. H. BERGOLD, Z. Natur/ofsch., 2b (z947) I22. A. G ~ A , J. BRACKETAND R. J~ESSR, Bull. Read. roy. reed. Belg., xo (x945) 72. j . D. SMITH AND G. R. WYATT, Biochem. J., 49 (I95 x) x44. N. XZROS, Nature, x75 (x955) 588. N. X~:ROS, P h . D . Thesis, University of Cambridge, x956. N. XEROS, Nature, x78 (x956) 4I~. N. X~:ROS, Nature, x74 (x954) 562. G. SCtIMIDT AND S. J. THANNHAUSER,J. Biol. Chem., i6x (x945) 83. It. MARKHAM,in K. PARCH AND M. V. TRACRY, Modern Methods o/ Plant .4nalysisj Vol. 4, Springer-Verlag, Berlin, 1955J P. 246. 10 R. MARKHAMAND J. D. SMITH, Biochem. J., 52 (x95~) 552. zl G. R. WYATT, Biochera. J., 48 (x95 x) 584 . it R. MARKHAMAND J. D. SMITH, Biochem. J., 45 (1949) 294. is A. KRIE~, Natnrwisser,scha/hra, 23 (i956) 537, xt F. T. IBIRD AND M. M. WHALES, Canad. J. Zool., 2x (x954) 82. xs G. It. BERGOLDj Ann. N. Y. Acad. Sci., 56 (I953) 495. 10 K. M. SMITH AND R. W. G. WYCKOIrF, Nature, x66 (x95 o) 86I, 17 N, XEROS, No,ture, I7o (I952) xo73. xs K. M. SMITH AND N. XEROS, Parasitology, 44 (x954) 400xs M. TSUIITA, Proc. Jap. Acad., 3 x (x955) 93. m K. M. SMITH AND N. XXROS, Nature, x73 (I954) 866.
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