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Sequence of the black beetle virus subgenomic RNA and its location in the viral genome
VIROLOGY
139,199-203(19&i)
Sequence of the Black Beetle Virus Subgenomic RNA and Its Location in the Viral Genome LINDA A. GUARMO,’ AMIT GHOSH,~ BIMALENDU DASMAHAPATRA, RANJIT DASGUPTA, AND PAUL KAESBERG~ Biophysics Iabodaq
and Biochemistry ReckvedJune
Department,
University of Wkonsin,
Madison, Wisconsin ~$706
1.4, 198g acceptedAugust 15,1984
BBV (black beetle virus) RNAI, the subgenomic messenger RNA for BBV protein B and its double-stranded form (dsRNA3) were purified from cells infected with BBV and were sequenced. RNA3 is 339 bases long. The sequence is homologous to that of the 3’terminal region of virion RNAl. RNA3 has a very limited homology to virion RNAt RNA3 is capped at its 5’ terminus and has a structural feature at its 3’ terminus that renders it inert to the action of the enzymes RNA ligase and poly(A) polymerase. RNA3 has two overlapping reading frames for putative proteins of size 10,763 and 11,633 Da. The positive and negative strands of dsRNA3 are not capped and correspond in length and sequence to RNA3 itself. Q 1986 Academic Press. 1nc.
The genome of black beetle virus (BBV) is comprised of two RNAs. RNA1 (3105 bases) is the messenger for 104-kDa protein A believed to be involved in viral RNA synthesis (I, 2). RNA2 (1399 bases) is the messenger for alpha, the 47-kDa virion capsid protein precursor (I). Cells infected with BBV produce an additional RNA designated RNA3 and a lo-kDa protein B (3). Cells transfected with RNAl, alone, also produce RNA3 and protein B indicating that their gene(s) reside in RNA1 (4) or possibly in host cells. Translation systems derived from Lh-osophda melanogastw cells or from rabbit reticulocytes, when programmed with RNA3, synthesize only protein B and when programmed with RNA1 synthesize only protein A (I, S) indicating that RNA3 (but not RNAl) is a messenger for protein B. In this paper we show that infected cells contain an RNA with properties to be expected for a double-stranded form of ’ Present address: Department of Entomology, Texas A & M University, College Station, Tex. 77343. ‘Present address: Indian Institute of Chemical Biology, Calcutta, India. ’ Author to whom requests for reprints should be addressed. 199
RNA3 (designated dsRNA3) and present the sequence of RNA3 and dsRNA3. We proceeded as follows to identify and to purify RNAB. Polysomal RNA, isolated from BBV-infected Drosophila cells, was subjected to sucrose density gradient sedimentation (3). Isolation and subsequent translation of the 9 S fraction yielded a single major product identified as protein B. That translation was inhibited in the presence of the cap analog m7GMP indicating that the mRNA for protein B is capped. The 9 S fraction was treated with calf-intestinal phosphatase and the 5’ ends of RNAs therein were 32Plabeled with T4 polynucleotide kinase (5) with or without prior enzymatic decapping (6) with tobacco acid pyrophosphatase (TAP). Gel electrophoretic patterns of the labeled material derived from infected or from uninfected cells were the same in the absence of prior treatment with TAP and consisted of a collection of ill-defined bands. Prior treatment with TAP resulted in a single additional band in the material extracted from infected cells. We infer that the additional band is RNA3 and that it has a 5’-capping group. These results also indicated that RNA3 is synthesized as a specific subgenomic RNA and is not de0042-6822/&l $3.00 Copyright
0 1984 by Academic
All rights
of reproduction
Press,
in any form
Inc. resewed.
200
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rived from nucleolytic degradation of virion RNA. The 9 S fraction was then electrophoresed for 20 hr at 400 V on an 8% acrylamide gel (0.15 X 20 X 40 cm) according to Peacock and Dingman (7’). The RNA3 band was cut from the gels, homogenized, and the RNA was eluted in 0.25 M NaCl. Such 5’-labeled RNA was sequenced by the enzymatic cleavage method (8-10) through base 322. Under a variety of conditions, including dephosphorylation and heat treatment, we were unable to bond pCp or adenylate residues to the RNA3 3’ terminus with RNA ligase or poly(A) polymerase, respectively, and consequently we were unable to obtain sequence information on such RNA commencing at the 3’ end. We thus sought a double-stranded form of RNA3 whose positive strand 3’ terminus might be more accessible. Thus double-stranded RNA was purified from Drosophila cells harvested 7 hr after their infection with BBV. Cells were collected by centrifugation (500 ~,2 min) and resuspended in 10 mM Tris-Cl (pH 7.4), 10 mM KCl, 1.5 m&f MgClz, 0.1% Triton X-100. Intact nuclei were pelleted at 10,000 g. The resultant supernatant was extracted twice with an equal volume of phenol and RNA was precipitated from the aqueous phase with 2 vol of ethanol and was resuspended in water. Single-stranded RNA was removed by two cycles of 2.2 M LiCl precipitation (II). The material in the supernatant liquid was precipitated with ethanol and double-stranded RNA3 was separated from other dsRNA and from tRNA by electrophoresis in lowmelting point agarose in Tris-borate buffer (7) containing 1 pg/ml ethidium bromide. Such preparations showed dsRNAl, dsRNA2, a double-stranded RNA approximately 1750 bases long which has not yet been well characterized, and a band in the predicted position for a double-stranded form of RNA3. The band material designated dsRNA3 was located with ultraviolet light, eluted, and was characterized.
To determine whether dsRNA3 has capped 5’ ends it was treated with calf intestinal phosphatase and then labeled with T4 polynucleotide kinase with or without prior treatment with TAP. Gel electrophoretic analyses showed that both strands were labeled after the phosphatase treatment. The negative strand was labeled about 60% more efficiently than the positive strand. TAP treatment had no effect on the efficiency of labeling of either strand. Thus dsRNA3 is phosphorylated at its 5’ termini and does not have a cap structure analogous to ssRNA3. The lesser incorporation of label into the positive strand suggests that its 5’ terminus is not as readily accessible to the enzyme as is the negative strand 5’ terminus. Both 3’ termini were readily labeled with 32pCp and T4 RNA ligase (12). The strands of dsRNA3, labeled at their 5’ termini, or alternatively at their 3’ termini were then separated by polyacrylamide gel electrophoresis (1.3) and were sequenced by the enzymatic cleavage method as described above and by the chemical cleavage method (14). Also, DNA complementary to negativestrand RNA was prepared as follows and was sequenced by the chemical cleavage method (13). A deoxyoligonucleotide corresponding to bases 283-294 was synthesized by the phosphite triester method with the DNA synthesis kit of New England Biolab. Its 5’ end was labeled with
FIG. 1. The BBV RNA3 sequence and the amino acid sequence of putative proteins Bl and B2 (see text).
SHORT
COMMUNICATIONS
T4 polynucleotide kinase and [T-~‘P]ATP. It was annealed in molar excess to negative-strand RNA3 by heating for 5 min at 65”, 20 min at 57”, and cooling to 44’ over a period of 20 min. The cDNA was synthesized as described (15) except that no labeled triphosphate was included. Primer-extended cDNA was electrophoresed in 8% polyacrylamide sequencing gels, located by autoradiography, and excised for sequencing. The consensus of the sequence determinations is given in Fig. 1. The consensus consists of enzymatic sequencing of RNA3, chemical and enzymatic sequencing of dsRNA3, and chemical sequencing of cDNA3. Where the procedures overlapped they gave identical results. Although each procedure gave some uncertain positions, arising primarily in enzymatic sequencing where U and C were sometimes not clearly distinguished and in chemical sequencing where extraneous bands were sometimes observed, the consensus has no ambiguous or uncertain positions. Because initially we could not be certain that RNA3 was a
201
single species we avoided cloning methods which might arbitrarily select particular RNAs among several RNA&like molecules. RNA2 (1399 bases) has been sequenced (16) and the first 3022 bases of BBV RNA1 (3105 bases) are known (our unpublished work). The sequence of RNA1 from base 2717 to base 3022 is identical to the sequence of the first 306 bases of RNA3 given in Fig. 1. The sequence of RNA3 has a very limited homology with that of RNAZ, not great enough to warrant conclusions in the absence of detailed statistical analyses. RNA3 has two large open reading frames, bases lo-318 and 20-340 for predicted proteins of 10,768 and 11,633 Da (proteins Bl and B2, respectively, in Fig. 1). Protein sequence analysis will be required to determine which of these corresponds to protein B. Indeed, both proteins Bl and B2 could have functional significance. In RNA1 protein B2 has the same reading frame as protein A and begins just 3 bases beyond protein A’s
UCG C A 1aoc 3 c A G AA G-C G-cc40 A-U
FIG. 2. Computer-generated
secondary
structure
pattern
of BBV RNA3.
SHORT COMMUNICATIONS
202 TABLE 1
STABILITY OF STEMAND LOOPREGIONS
Loop
No. of bases
Energy (kcal)
Energy/base (kcal/base)
1 2 3 4 5 6 7 8 9
48 21 27 17 41 11 32 49 28
-12.1 -12.5 -11.8 -7.9 -23.3 -5.5 -12.6 -21.4 -16.0
0.25 0.60 0.44 0.46 0.57 0.50 0.39 0.44 0.57
Note. Table 1 gives the predicted stability of the nine stem and loop regions of Fig. 2.
terminus. Protein Bl overlaps the protein A cistron. We have used the computer program of Zuker and Stiegler (17’) with base pairing and stacking energies recommended by Salser (18) to generate the secondary structure shown in Fig. 2. Such structures should be regarded primarily as guides for using enzymatic and other procedures for the determination of spatial structure. Nevertheless such hypothetical structures identify regions of sequence which have or lack the potential for strong base pairing and stacking interaction. The secondary structure diagram of Fig. 2 consists of nine stem and loop regions connected to each other by single-stranded regions or by sequence regions which have associated to form double-stranded segments. The predicted stability of these regions is given in Table 1. It is noteworthy that stem region 1, which includes the initiation sites for synthesis of putative proteins 1 and 2, has a number of bulge loops so that the overall structure is held together only minimally by base stacking. It is a structure that is likely to be readily accessible to ribosomes. Conversely, the structure of stem and loop region 9, corresponding to the 3’ terminus, has the highest stability per base of any of the nine stem and loop regions. Such a structure could hinder enzymatic interaction
with the 3’ terminus. The set of four GC pairs closing loop 9 is particularly stable and should persist in all but extreme denaturing conditions. ACKNOWLEDGMENTS This work was supported by NIH Grants AI1466 and AI15342 and NIH Career Award AI21942. REFERENCES 1. FRIESEN, P. D., and RUECKERT, R. R. (1981). Synthesis of black beetle virus proteins in cultured Drosophila cells: Differential expression of RNAs 1 and 2. J. Viral 37,876-886. P GUARINO, L. A. and JSAESBERG, P. (1981).Isolation and characterization of an RNA dependent RNA polymerase for black beetle virus infected Drosophila melumgaster cells. J. Vim! 40, 379-386.
3. FRIESEN, P. D., and RUECKERT.R. R. (1982). Black beetle virus: Messenger for protein B is a subgenomic message. J. Viral 42, 986-995. 4 GALLAGHER,T. M., FRIESEN,P. D., and RUECKERT, R. R. (1983). Autonomous replication and expression of RNA1 from black beetle virus. J. Viral 46,481-489. 5. DASGUPTA,R., AHJ&UIST, P., and KAESBERG,P. (1980). Sequence of the 3’ untranslated region of brome mosaic virus coat protein messenger RNA. Virdogy 104,339-346. 6. EFSTRATIADIS, A., VOURNAKIS, J. N., DONISKELLER, H., CHACONAS,G., DOUGAL, D. K., and KAFATOS, C. F. (1977). End labelling of enzymatically decapped mRNA. Nucl Acids Res. 4,4165-4174. 7. PEACOCK,A. C., and DINGYAN, C. W. (1968). Molecular weight estimation and separation of ribonueleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistry 7.668-674. 8. DONIS-KELLER,H. (1980). PhyM: An RNase activity specific for U and A residues useful in RNA sequence analysis. Null Ads Re.s. 8, 3133-3142. 9. DONIS-KELLER,H., MAXAM, A. M., and GILBERT, W. (1977). Mapping adenines, guanines, and pyrimidines in RNA. Nucl Acids Res 4,25272538. 10. SIMONCSITS, A., BROWNLEE, G. G., BROWN,R. S., RUBIN, J. R., and GUILLEY, H. (1977). New rapid gel sequencing method for RNA. Nature @imdon) 269, 833-836.
SHORT COMMUNICATIONS IL BOTH, G. W., BELLAMY, A. R., STREET,J. E., and SIECMAN,L. J. (1982). A general strategy for cloning double-stranded RNA: Nucleotide sequence of the Simian-11 rotavirus gene 8. NUCL AcidaRes 10,7075-7088. 1.2.ENGLAND,T., and UHLENBECK,0. (1978). 3’ Terminal labeling of RNA with T4 RNA ligase. Nature @m&m) 275,560-561. 1% MAXAM, A. M., and GILBERT,W. In “Methods in Enzymology” (L. Grossman and K. Moldave, eds.), Vol. 65, pp. 499-560. Academic Press, New York, 1980. 14 PEA’ITIE, D. A. (1979). Direct chemical method for sequencing RNA. PTOC Nat1 Acad Sci USA 76, 1760-1764. 15. AHLQUIST, P., LUCKOW, V., and KAESBERG, P.
203
(1981). Complete nucleotide sequence of brome mosaic virus RNAI. J. Mel Bid 153,23-38. 16. DASGUPTA,R., GHOSH, A., DASMAHAPATRA,B., GUARINO, L. A., and KAESBERG, P. (1984). Primary and secondary structure of black beetle virus RNAB, the genomic messenger for BBV coat protein precursor. Nu.el Acids Res. (in press). 17. ZUKER, M., and STIEGLER, P. (1981). Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucl Acids Res. 9.133-148. 18. SALSER, W. (1977). Globin mRNA sequences: Analysis of base pairing and evolutionary implications. Cdd Sprint Harbor Spp. Quant Biol 42, 985-1002.
139,199-203(19&i)
Sequence of the Black Beetle Virus Subgenomic RNA and Its Location in the Viral Genome LINDA A. GUARMO,’ AMIT GHOSH,~ BIMALENDU DASMAHAPATRA, RANJIT DASGUPTA, AND PAUL KAESBERG~ Biophysics Iabodaq
and Biochemistry ReckvedJune
Department,
University of Wkonsin,
Madison, Wisconsin ~$706
1.4, 198g acceptedAugust 15,1984
BBV (black beetle virus) RNAI, the subgenomic messenger RNA for BBV protein B and its double-stranded form (dsRNA3) were purified from cells infected with BBV and were sequenced. RNA3 is 339 bases long. The sequence is homologous to that of the 3’terminal region of virion RNAl. RNA3 has a very limited homology to virion RNAt RNA3 is capped at its 5’ terminus and has a structural feature at its 3’ terminus that renders it inert to the action of the enzymes RNA ligase and poly(A) polymerase. RNA3 has two overlapping reading frames for putative proteins of size 10,763 and 11,633 Da. The positive and negative strands of dsRNA3 are not capped and correspond in length and sequence to RNA3 itself. Q 1986 Academic Press. 1nc.
The genome of black beetle virus (BBV) is comprised of two RNAs. RNA1 (3105 bases) is the messenger for 104-kDa protein A believed to be involved in viral RNA synthesis (I, 2). RNA2 (1399 bases) is the messenger for alpha, the 47-kDa virion capsid protein precursor (I). Cells infected with BBV produce an additional RNA designated RNA3 and a lo-kDa protein B (3). Cells transfected with RNAl, alone, also produce RNA3 and protein B indicating that their gene(s) reside in RNA1 (4) or possibly in host cells. Translation systems derived from Lh-osophda melanogastw cells or from rabbit reticulocytes, when programmed with RNA3, synthesize only protein B and when programmed with RNA1 synthesize only protein A (I, S) indicating that RNA3 (but not RNAl) is a messenger for protein B. In this paper we show that infected cells contain an RNA with properties to be expected for a double-stranded form of ’ Present address: Department of Entomology, Texas A & M University, College Station, Tex. 77343. ‘Present address: Indian Institute of Chemical Biology, Calcutta, India. ’ Author to whom requests for reprints should be addressed. 199
RNA3 (designated dsRNA3) and present the sequence of RNA3 and dsRNA3. We proceeded as follows to identify and to purify RNAB. Polysomal RNA, isolated from BBV-infected Drosophila cells, was subjected to sucrose density gradient sedimentation (3). Isolation and subsequent translation of the 9 S fraction yielded a single major product identified as protein B. That translation was inhibited in the presence of the cap analog m7GMP indicating that the mRNA for protein B is capped. The 9 S fraction was treated with calf-intestinal phosphatase and the 5’ ends of RNAs therein were 32Plabeled with T4 polynucleotide kinase (5) with or without prior enzymatic decapping (6) with tobacco acid pyrophosphatase (TAP). Gel electrophoretic patterns of the labeled material derived from infected or from uninfected cells were the same in the absence of prior treatment with TAP and consisted of a collection of ill-defined bands. Prior treatment with TAP resulted in a single additional band in the material extracted from infected cells. We infer that the additional band is RNA3 and that it has a 5’-capping group. These results also indicated that RNA3 is synthesized as a specific subgenomic RNA and is not de0042-6822/&l $3.00 Copyright
0 1984 by Academic
All rights
of reproduction
Press,
in any form
Inc. resewed.
200
SHORT
COMMUNICATIONS
rived from nucleolytic degradation of virion RNA. The 9 S fraction was then electrophoresed for 20 hr at 400 V on an 8% acrylamide gel (0.15 X 20 X 40 cm) according to Peacock and Dingman (7’). The RNA3 band was cut from the gels, homogenized, and the RNA was eluted in 0.25 M NaCl. Such 5’-labeled RNA was sequenced by the enzymatic cleavage method (8-10) through base 322. Under a variety of conditions, including dephosphorylation and heat treatment, we were unable to bond pCp or adenylate residues to the RNA3 3’ terminus with RNA ligase or poly(A) polymerase, respectively, and consequently we were unable to obtain sequence information on such RNA commencing at the 3’ end. We thus sought a double-stranded form of RNA3 whose positive strand 3’ terminus might be more accessible. Thus double-stranded RNA was purified from Drosophila cells harvested 7 hr after their infection with BBV. Cells were collected by centrifugation (500 ~,2 min) and resuspended in 10 mM Tris-Cl (pH 7.4), 10 mM KCl, 1.5 m&f MgClz, 0.1% Triton X-100. Intact nuclei were pelleted at 10,000 g. The resultant supernatant was extracted twice with an equal volume of phenol and RNA was precipitated from the aqueous phase with 2 vol of ethanol and was resuspended in water. Single-stranded RNA was removed by two cycles of 2.2 M LiCl precipitation (II). The material in the supernatant liquid was precipitated with ethanol and double-stranded RNA3 was separated from other dsRNA and from tRNA by electrophoresis in lowmelting point agarose in Tris-borate buffer (7) containing 1 pg/ml ethidium bromide. Such preparations showed dsRNAl, dsRNA2, a double-stranded RNA approximately 1750 bases long which has not yet been well characterized, and a band in the predicted position for a double-stranded form of RNA3. The band material designated dsRNA3 was located with ultraviolet light, eluted, and was characterized.
To determine whether dsRNA3 has capped 5’ ends it was treated with calf intestinal phosphatase and then labeled with T4 polynucleotide kinase with or without prior treatment with TAP. Gel electrophoretic analyses showed that both strands were labeled after the phosphatase treatment. The negative strand was labeled about 60% more efficiently than the positive strand. TAP treatment had no effect on the efficiency of labeling of either strand. Thus dsRNA3 is phosphorylated at its 5’ termini and does not have a cap structure analogous to ssRNA3. The lesser incorporation of label into the positive strand suggests that its 5’ terminus is not as readily accessible to the enzyme as is the negative strand 5’ terminus. Both 3’ termini were readily labeled with 32pCp and T4 RNA ligase (12). The strands of dsRNA3, labeled at their 5’ termini, or alternatively at their 3’ termini were then separated by polyacrylamide gel electrophoresis (1.3) and were sequenced by the enzymatic cleavage method as described above and by the chemical cleavage method (14). Also, DNA complementary to negativestrand RNA was prepared as follows and was sequenced by the chemical cleavage method (13). A deoxyoligonucleotide corresponding to bases 283-294 was synthesized by the phosphite triester method with the DNA synthesis kit of New England Biolab. Its 5’ end was labeled with
FIG. 1. The BBV RNA3 sequence and the amino acid sequence of putative proteins Bl and B2 (see text).
SHORT
COMMUNICATIONS
T4 polynucleotide kinase and [T-~‘P]ATP. It was annealed in molar excess to negative-strand RNA3 by heating for 5 min at 65”, 20 min at 57”, and cooling to 44’ over a period of 20 min. The cDNA was synthesized as described (15) except that no labeled triphosphate was included. Primer-extended cDNA was electrophoresed in 8% polyacrylamide sequencing gels, located by autoradiography, and excised for sequencing. The consensus of the sequence determinations is given in Fig. 1. The consensus consists of enzymatic sequencing of RNA3, chemical and enzymatic sequencing of dsRNA3, and chemical sequencing of cDNA3. Where the procedures overlapped they gave identical results. Although each procedure gave some uncertain positions, arising primarily in enzymatic sequencing where U and C were sometimes not clearly distinguished and in chemical sequencing where extraneous bands were sometimes observed, the consensus has no ambiguous or uncertain positions. Because initially we could not be certain that RNA3 was a
201
single species we avoided cloning methods which might arbitrarily select particular RNAs among several RNA&like molecules. RNA2 (1399 bases) has been sequenced (16) and the first 3022 bases of BBV RNA1 (3105 bases) are known (our unpublished work). The sequence of RNA1 from base 2717 to base 3022 is identical to the sequence of the first 306 bases of RNA3 given in Fig. 1. The sequence of RNA3 has a very limited homology with that of RNAZ, not great enough to warrant conclusions in the absence of detailed statistical analyses. RNA3 has two large open reading frames, bases lo-318 and 20-340 for predicted proteins of 10,768 and 11,633 Da (proteins Bl and B2, respectively, in Fig. 1). Protein sequence analysis will be required to determine which of these corresponds to protein B. Indeed, both proteins Bl and B2 could have functional significance. In RNA1 protein B2 has the same reading frame as protein A and begins just 3 bases beyond protein A’s
UCG C A 1aoc 3 c A G AA G-C G-cc40 A-U
FIG. 2. Computer-generated
secondary
structure
pattern
of BBV RNA3.
SHORT COMMUNICATIONS
202 TABLE 1
STABILITY OF STEMAND LOOPREGIONS
Loop
No. of bases
Energy (kcal)
Energy/base (kcal/base)
1 2 3 4 5 6 7 8 9
48 21 27 17 41 11 32 49 28
-12.1 -12.5 -11.8 -7.9 -23.3 -5.5 -12.6 -21.4 -16.0
0.25 0.60 0.44 0.46 0.57 0.50 0.39 0.44 0.57
Note. Table 1 gives the predicted stability of the nine stem and loop regions of Fig. 2.
terminus. Protein Bl overlaps the protein A cistron. We have used the computer program of Zuker and Stiegler (17’) with base pairing and stacking energies recommended by Salser (18) to generate the secondary structure shown in Fig. 2. Such structures should be regarded primarily as guides for using enzymatic and other procedures for the determination of spatial structure. Nevertheless such hypothetical structures identify regions of sequence which have or lack the potential for strong base pairing and stacking interaction. The secondary structure diagram of Fig. 2 consists of nine stem and loop regions connected to each other by single-stranded regions or by sequence regions which have associated to form double-stranded segments. The predicted stability of these regions is given in Table 1. It is noteworthy that stem region 1, which includes the initiation sites for synthesis of putative proteins 1 and 2, has a number of bulge loops so that the overall structure is held together only minimally by base stacking. It is a structure that is likely to be readily accessible to ribosomes. Conversely, the structure of stem and loop region 9, corresponding to the 3’ terminus, has the highest stability per base of any of the nine stem and loop regions. Such a structure could hinder enzymatic interaction
with the 3’ terminus. The set of four GC pairs closing loop 9 is particularly stable and should persist in all but extreme denaturing conditions. ACKNOWLEDGMENTS This work was supported by NIH Grants AI1466 and AI15342 and NIH Career Award AI21942. REFERENCES 1. FRIESEN, P. D., and RUECKERT, R. R. (1981). Synthesis of black beetle virus proteins in cultured Drosophila cells: Differential expression of RNAs 1 and 2. J. Viral 37,876-886. P GUARINO, L. A. and JSAESBERG, P. (1981).Isolation and characterization of an RNA dependent RNA polymerase for black beetle virus infected Drosophila melumgaster cells. J. Vim! 40, 379-386.
3. FRIESEN, P. D., and RUECKERT.R. R. (1982). Black beetle virus: Messenger for protein B is a subgenomic message. J. Viral 42, 986-995. 4 GALLAGHER,T. M., FRIESEN,P. D., and RUECKERT, R. R. (1983). Autonomous replication and expression of RNA1 from black beetle virus. J. Viral 46,481-489. 5. DASGUPTA,R., AHJ&UIST, P., and KAESBERG,P. (1980). Sequence of the 3’ untranslated region of brome mosaic virus coat protein messenger RNA. Virdogy 104,339-346. 6. EFSTRATIADIS, A., VOURNAKIS, J. N., DONISKELLER, H., CHACONAS,G., DOUGAL, D. K., and KAFATOS, C. F. (1977). End labelling of enzymatically decapped mRNA. Nucl Acids Res. 4,4165-4174. 7. PEACOCK,A. C., and DINGYAN, C. W. (1968). Molecular weight estimation and separation of ribonueleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistry 7.668-674. 8. DONIS-KELLER,H. (1980). PhyM: An RNase activity specific for U and A residues useful in RNA sequence analysis. Null Ads Re.s. 8, 3133-3142. 9. DONIS-KELLER,H., MAXAM, A. M., and GILBERT, W. (1977). Mapping adenines, guanines, and pyrimidines in RNA. Nucl Acids Res 4,25272538. 10. SIMONCSITS, A., BROWNLEE, G. G., BROWN,R. S., RUBIN, J. R., and GUILLEY, H. (1977). New rapid gel sequencing method for RNA. Nature @imdon) 269, 833-836.
SHORT COMMUNICATIONS IL BOTH, G. W., BELLAMY, A. R., STREET,J. E., and SIECMAN,L. J. (1982). A general strategy for cloning double-stranded RNA: Nucleotide sequence of the Simian-11 rotavirus gene 8. NUCL AcidaRes 10,7075-7088. 1.2.ENGLAND,T., and UHLENBECK,0. (1978). 3’ Terminal labeling of RNA with T4 RNA ligase. Nature @m&m) 275,560-561. 1% MAXAM, A. M., and GILBERT,W. In “Methods in Enzymology” (L. Grossman and K. Moldave, eds.), Vol. 65, pp. 499-560. Academic Press, New York, 1980. 14 PEA’ITIE, D. A. (1979). Direct chemical method for sequencing RNA. PTOC Nat1 Acad Sci USA 76, 1760-1764. 15. AHLQUIST, P., LUCKOW, V., and KAESBERG, P.
203
(1981). Complete nucleotide sequence of brome mosaic virus RNAI. J. Mel Bid 153,23-38. 16. DASGUPTA,R., GHOSH, A., DASMAHAPATRA,B., GUARINO, L. A., and KAESBERG, P. (1984). Primary and secondary structure of black beetle virus RNAB, the genomic messenger for BBV coat protein precursor. Nu.el Acids Res. (in press). 17. ZUKER, M., and STIEGLER, P. (1981). Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucl Acids Res. 9.133-148. 18. SALSER, W. (1977). Globin mRNA sequences: Analysis of base pairing and evolutionary implications. Cdd Sprint Harbor Spp. Quant Biol 42, 985-1002.