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Fingerprint analyses of cytoplasmic adenovirus 2 RNA size classes synthesized late in productive infection
VIROLoGY68,547-549(
Fingerprint
lY75)
Analyses
of Cytoplasmic
Classes Synthesized JACOV TAL, PETER
JACOBI,’
Adenovirus
Late in Productive
HESCHEL
J. RASKAS,
2 RNA Size Infection
AND
ECKARD
WIMMER’
Departments o/Pathology and Microbiology, Washington University School o/Medicine, St. Louis, Missouri State University of New York at Stony Brook, Stony Brook, New York 63110, and Department of Microbiology, 11790 Accepted
August, 8, 1975
Three major size classes (27, 24 and 19 S) of adenovirus 2 RNA synthesized late m productive infection were digested with Tl RNase and analyzed by two-dimensional electrophoresis. Each size class yielded a different distribution of oligonucleotide spots.
During the late phase of adenovirus 2 replication in cultured human cells, the viral genome dominates macromolecular synthesis. By 12 hr after infection at least 80% of the newly synthesized cytoplasmic mRNA is virus specified (l-3)) and most of the peptides synthesized are either virion peptides or virus-induced proteins (4-6). Studies of the cytoplasmic viral RNAs synthesized at 18 hr after infection have identified four major size classes of cytoplasmic viral RNA (I, 3, 7). These RNA size classes migrate in polyacrylamide gels as 27, 24, 19 and 12-15 S. The three larger size classes have been identified in denaturing formamide gels and therefore do not represent aggregates (3). Direct evidence that these RNA size classes contain different RNA sequences has not yet been obtained. For this purpose we performed fingerprint analyses of the oligonucleotides generated by digestion with Tl RNase. Cultures infected with adenovirus 2 were labeled with 32P from 18 to 21 hr after infection, as described previously (3). The cytoplasmic RNA was purified (8) and poly(A)-containing molecules were selected by oligo(dT)-cellulose chromatography (8). The poly(A)-containing RNA, which hybridized to adenovirus DNA more than 80% (3), was fractionated by polya’ State University
crylamide-gel electrophoresis through denaturing formamide gels. As shown in Fig. lA, the three major size classes were resolved by this procedure. For preparative gels, the location of the three size classes was determined by Cerenkov counting of gel slices. Although the acrylamide used for the preparative gels had been recrystallized, RNA eluted from the gels by high voltage electrophoresis contained impurities that could not be removed by ethanol precipitation or extensive dialysis. Therefore the RNA in appropriate gel slices was electrophoresed through agarose (see legend to Fig. l), a procedure that removed the impurities sufficiently to allow fingerprint analysis. As shown in Figs. lB-D, the size-fractionated RNA prepared by this procedure migrated as peaks of approximately 27, 24 and 19 S when reanalyzed by polyacrylamide-gel electrophoresis. The RNA in the three major size classes was treated with Tl RNase and the digestion products analyzed by two-dimensional electrophoresis. The fingerprint analyses are shown in Fig. 2 and the relative counts per minute in selected spots are presented in Table 1. Spots unique to one of the three size classes were not detected. This result is not unexpected because of the large size of the mRNA’s and because the separation procedure leaves unresolved the larger oligonucleotides. However, quantitative anal-
of New York at Stony Brook. 547
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
TABLE
I
RELATIVE RADIOACTIVITY IN SELECTED OLIGONUCLEOTIDESPOTS AFTERTl DIGESTIONS spot
SLICE
NUMBER
FIG. 1. Polyacrylamide-gel electrophoresis of oligo(dT) selected cytoplasmic RNA synthesized late in infection. Cytoplasmic RNA was purified from a culture labeled with 32P 18-21 hr after infection (3). Poly(A)-containing RNA was selected by oligo(dT)cellulose chromatography; 2 x 10’ cpm of this RNA were treated with 90% formamide for 5 min at room temperature and placed on a 3.2% polyacrylamide gel containing 95% formamide. Electrophoresis was for 6 hr at 5mA at room temperature (A). RNA from the peak regions of a gel identical to that described for (A) was collected by electrophoresis through 7-cm 2% agarose gels for 2 hr at 5mA per gel. The eluted RNA was collected in dialysis tubing fastened to the gel tube. A sample of the eluted RNA’s was re-electrophoresed on a 3.2% acrylamide gel for 2 hr at 5mA. *‘C-labeled ribosomal RNA was added as marker. 27 S RNA was analyzed in (B), 24 S in (Cl, and 19 S in CD).
1 2 3 4 5 6 7 8 9 10 11 12 13
Base composition
G CG AG C& CAG, ACG A&
A& UG UCG UAG
Relative radioactivity (cpm) 19 s RNA
24 S RNA
27 s RNA
1.0 0.69 0.65 0.38 0.44 0.32 0.35 0.29 0.30 0.27 0.37 1.1 0.33
1 0.64 0.39 0.15 0.35 0.11 0.13 0.08 0.07 0.05 0.23 0.28 0.16
1 0.22 0.61 0.07 0.04 0.17 0.03 0.02 0.01 0.05 0.11 0.15 0.22
u Autoradiograms were aligned with PEI-cellulose plates and corresponding spots were cut out and counted in scintillation vials for 10 min. The positions of the spots on the PEI-cellulose plates are shown in Fig. 2. Counts per minute for G spots were 180 in 19 S, 464 in 24 S and 683 in 27 S. Background of 10 cpm was subtracted for all spots. The approximate molecular weights of these size classes are 1.5 x 106, 1.0 x lo6 and 0.8 x lo8 for the 27, 24 and 19 S RNA’s, respectively. 1. Cdlulow
Auktat
FIG. 2. Two-dimensional electrophoresis of Tl digests of size-fractionated RNA. RNA samples were treated with Tl RNase as follows: After elution from gel slices by electrophoresis, samples were dried in uacuo, dissolved in 20 pl of 15 mM Tris-HCl buffer, pH 7.4, 1.5 mM EDTA containing 10 pg of Tl RNase. Digestion was for 60 min at 37”. The digest was applied to cellulose acetate strips, 3 x 100 cm (Schleicher & Schuell), pre-wet with 5% acetic acid in 7 M urea, then adjusted to pH 3.5 with pyridine. Electrophoresis was for 95 min at 5000 V. The spots were transferred to PEI-cellulose plates, 20 x 40 cm (Macherey and Nagel, MN 300 PEI cellulose), by blotting with water with pressure, according to Sanger et al. (12). The plates were prechromatographed with water for 12 hr and then with methanol up to the top. This procedure was used to remove impurities from the plates (13). The plates were then dried and chromatographed in buffer (1.15 M pyridinium formate in 7 M urea, pH 3.5). The plates were autoradiographed for 14 days. The counts used per sample were 300,000 cpm for 27 S, 400,000 for 24 S, and 150,000 for 19 S. The left-hand panel shows the location of spots for which the actual counts per minute were determined (see Table 1). 548
SHORT
549
COMMUNICATIONS
ysis of the amounts of each nucleotide generated by Tl digestion yielded significant differences among each of the three size classes. Analyses of the relative abundance of some Tl spots demonstrated that each of these size classes contains at least some RNA sequences that are present in very different amounts in the other major size classes. For example, the amount of CCG (spot 4) relative to G (spot 1) is 0.07 for the 27 S size class, 0.15 for 24 S, and 0.38 for 19 S. Likewise, spot 5 (CAG,ACG) is present in relatively small amounts in 27 S RNA (0.04 relative to G) and much larger amounts (0.35 and 0.44) in 24 and 19 S RNA. If the sequences in the 19 and 24 S RNA’s which yield spots 4 and 5 are also present in the 27 S size class, then they must be present in much lower concentrations. Spot 10 (A,G) is an example of an oligonucleotide that is relatively abundant in the 19 S RNA (0.27 relative to G) and present only in very small amounts in 24 and 27 S RNA (0.05 each). This oligonucleotide is unlikely to be part of a sequence which is overlapping between the 19 S RNA and the other two size classes. Two related areas of investigation provide evidence that the three major RNA size classes are the products of different viral genes. Studies of the translation products of size-fractionated RNA have demonstrated that different peptides are coded for by the different RNAs (9, 10). Independent data are provided by hybridization of size-fractionated cytoplasmic RNA’s to unique viral DNA fragments generated by cleavage with restriction enzymes (II). This procedure provides an approach for correlating viral RNA’s with genome locations. Studies with EcoRl (I I) as well as other sets of adenovirus 2 DNA fragments (McGrogan and Raskas, in preparation) have demonstrated that some of these size classes contain more than one RNA species.
ACKNOWLEDGMENTS We appreciate the technical assistance provided by Mark Telle and Joe Kelley. This study was supported by a Public Health Service grant from the National Cancer Institute No. (CA125601 to HJR. a grant from the American Cancer Society (No. VC-94A) to HJR and, in part, by a National Cancer Institute grant (No. CAl4151) to EW. Jacov Tal is the recipient of a Fogarty International Fellowship from the National Institutes of Health. Cell culture media were prepared in a facility supported by a National Science Foundation grant (No. GB38657). This study was also supported by a grant from the following companies: Brown & Williamson Tobacco Corporation: Larus and Brother Company, Inc.; Liggett & Myers. Incorporated; Lorillard, a Division of Loews Theatres, Incorporated; Philip Morris, Incorporated; R. J. Reynolds Tobacco Company; United States Tobacco Company; and Tobacco Associates, Inc. REFERENCES
1. LINDBERG, U.. PERSSON,T.. and PHILIPSON, L.. J. Viral. 10, 909-919 (197%). 2. BHADURI. S.. RASKAS. H. J.. and GREEN, M.. J. Viral. 10, 1126-1129 (1972). 3. TAL, J., CRAIG, E. A., and RASKAS, H. J., J. Viral. 15, 137-144 (1975). 4. HORWITZ, M. S., SCHARFF, M. D., and MAIZEL, J. V., JR., Virology 39, 682-694 (1969). 5. ANDERSON, C. W., BAUM, P. R., and GESTELAND, R. F. J. Viral. 12, 241-252 (1973). 6. WALTER, G., and MAIZEL. J. V.. JR., Virology 57, 402-408 ( 1974). 7. PARSONS, J. T.. GARDNER, J.. and GREEN, M., Proc. Nat. Acad. Sci. USA 68, 557-560 (1971). 8. CRAIG, E. A., and RASKAS, H. J.. J. Viral. 14, 26-32 (1974). 9. ANDERSON, C. W.. LEWIS, J. B.. ATKINS, J. F., and GESTELAND, R. F., Proc. N&l. Acad. Sci. USA 71, 2756-2760 (1974). 10. OBERC, B., SABORIO,J., PERSSON,T., EVERITT, E., and PHILIPSON, L.. J. Viral. 15, 199-207 (1975). II. TAL, J., CRAIG, E. A., ZIMMER, S., and RASKAS. H. J. hoc. Nat. Acad. Sci. USA 71, 4057-4061 (1974). 12. SANGER,F., BROWNLEE.G. G., and BARRELL, B. G.. J. Mol. Biol. 13, 373-398 (1965). 13. SOUTHERN, E. M.. and MITCHELL, A. R., Biochem. J. 123, 613-617 (1971).
Fingerprint
lY75)
Analyses
of Cytoplasmic
Classes Synthesized JACOV TAL, PETER
JACOBI,’
Adenovirus
Late in Productive
HESCHEL
J. RASKAS,
2 RNA Size Infection
AND
ECKARD
WIMMER’
Departments o/Pathology and Microbiology, Washington University School o/Medicine, St. Louis, Missouri State University of New York at Stony Brook, Stony Brook, New York 63110, and Department of Microbiology, 11790 Accepted
August, 8, 1975
Three major size classes (27, 24 and 19 S) of adenovirus 2 RNA synthesized late m productive infection were digested with Tl RNase and analyzed by two-dimensional electrophoresis. Each size class yielded a different distribution of oligonucleotide spots.
During the late phase of adenovirus 2 replication in cultured human cells, the viral genome dominates macromolecular synthesis. By 12 hr after infection at least 80% of the newly synthesized cytoplasmic mRNA is virus specified (l-3)) and most of the peptides synthesized are either virion peptides or virus-induced proteins (4-6). Studies of the cytoplasmic viral RNAs synthesized at 18 hr after infection have identified four major size classes of cytoplasmic viral RNA (I, 3, 7). These RNA size classes migrate in polyacrylamide gels as 27, 24, 19 and 12-15 S. The three larger size classes have been identified in denaturing formamide gels and therefore do not represent aggregates (3). Direct evidence that these RNA size classes contain different RNA sequences has not yet been obtained. For this purpose we performed fingerprint analyses of the oligonucleotides generated by digestion with Tl RNase. Cultures infected with adenovirus 2 were labeled with 32P from 18 to 21 hr after infection, as described previously (3). The cytoplasmic RNA was purified (8) and poly(A)-containing molecules were selected by oligo(dT)-cellulose chromatography (8). The poly(A)-containing RNA, which hybridized to adenovirus DNA more than 80% (3), was fractionated by polya’ State University
crylamide-gel electrophoresis through denaturing formamide gels. As shown in Fig. lA, the three major size classes were resolved by this procedure. For preparative gels, the location of the three size classes was determined by Cerenkov counting of gel slices. Although the acrylamide used for the preparative gels had been recrystallized, RNA eluted from the gels by high voltage electrophoresis contained impurities that could not be removed by ethanol precipitation or extensive dialysis. Therefore the RNA in appropriate gel slices was electrophoresed through agarose (see legend to Fig. l), a procedure that removed the impurities sufficiently to allow fingerprint analysis. As shown in Figs. lB-D, the size-fractionated RNA prepared by this procedure migrated as peaks of approximately 27, 24 and 19 S when reanalyzed by polyacrylamide-gel electrophoresis. The RNA in the three major size classes was treated with Tl RNase and the digestion products analyzed by two-dimensional electrophoresis. The fingerprint analyses are shown in Fig. 2 and the relative counts per minute in selected spots are presented in Table 1. Spots unique to one of the three size classes were not detected. This result is not unexpected because of the large size of the mRNA’s and because the separation procedure leaves unresolved the larger oligonucleotides. However, quantitative anal-
of New York at Stony Brook. 547
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
TABLE
I
RELATIVE RADIOACTIVITY IN SELECTED OLIGONUCLEOTIDESPOTS AFTERTl DIGESTIONS spot
SLICE
NUMBER
FIG. 1. Polyacrylamide-gel electrophoresis of oligo(dT) selected cytoplasmic RNA synthesized late in infection. Cytoplasmic RNA was purified from a culture labeled with 32P 18-21 hr after infection (3). Poly(A)-containing RNA was selected by oligo(dT)cellulose chromatography; 2 x 10’ cpm of this RNA were treated with 90% formamide for 5 min at room temperature and placed on a 3.2% polyacrylamide gel containing 95% formamide. Electrophoresis was for 6 hr at 5mA at room temperature (A). RNA from the peak regions of a gel identical to that described for (A) was collected by electrophoresis through 7-cm 2% agarose gels for 2 hr at 5mA per gel. The eluted RNA was collected in dialysis tubing fastened to the gel tube. A sample of the eluted RNA’s was re-electrophoresed on a 3.2% acrylamide gel for 2 hr at 5mA. *‘C-labeled ribosomal RNA was added as marker. 27 S RNA was analyzed in (B), 24 S in (Cl, and 19 S in CD).
1 2 3 4 5 6 7 8 9 10 11 12 13
Base composition
G CG AG C& CAG, ACG A&
A& UG UCG UAG
Relative radioactivity (cpm) 19 s RNA
24 S RNA
27 s RNA
1.0 0.69 0.65 0.38 0.44 0.32 0.35 0.29 0.30 0.27 0.37 1.1 0.33
1 0.64 0.39 0.15 0.35 0.11 0.13 0.08 0.07 0.05 0.23 0.28 0.16
1 0.22 0.61 0.07 0.04 0.17 0.03 0.02 0.01 0.05 0.11 0.15 0.22
u Autoradiograms were aligned with PEI-cellulose plates and corresponding spots were cut out and counted in scintillation vials for 10 min. The positions of the spots on the PEI-cellulose plates are shown in Fig. 2. Counts per minute for G spots were 180 in 19 S, 464 in 24 S and 683 in 27 S. Background of 10 cpm was subtracted for all spots. The approximate molecular weights of these size classes are 1.5 x 106, 1.0 x lo6 and 0.8 x lo8 for the 27, 24 and 19 S RNA’s, respectively. 1. Cdlulow
Auktat
FIG. 2. Two-dimensional electrophoresis of Tl digests of size-fractionated RNA. RNA samples were treated with Tl RNase as follows: After elution from gel slices by electrophoresis, samples were dried in uacuo, dissolved in 20 pl of 15 mM Tris-HCl buffer, pH 7.4, 1.5 mM EDTA containing 10 pg of Tl RNase. Digestion was for 60 min at 37”. The digest was applied to cellulose acetate strips, 3 x 100 cm (Schleicher & Schuell), pre-wet with 5% acetic acid in 7 M urea, then adjusted to pH 3.5 with pyridine. Electrophoresis was for 95 min at 5000 V. The spots were transferred to PEI-cellulose plates, 20 x 40 cm (Macherey and Nagel, MN 300 PEI cellulose), by blotting with water with pressure, according to Sanger et al. (12). The plates were prechromatographed with water for 12 hr and then with methanol up to the top. This procedure was used to remove impurities from the plates (13). The plates were then dried and chromatographed in buffer (1.15 M pyridinium formate in 7 M urea, pH 3.5). The plates were autoradiographed for 14 days. The counts used per sample were 300,000 cpm for 27 S, 400,000 for 24 S, and 150,000 for 19 S. The left-hand panel shows the location of spots for which the actual counts per minute were determined (see Table 1). 548
SHORT
549
COMMUNICATIONS
ysis of the amounts of each nucleotide generated by Tl digestion yielded significant differences among each of the three size classes. Analyses of the relative abundance of some Tl spots demonstrated that each of these size classes contains at least some RNA sequences that are present in very different amounts in the other major size classes. For example, the amount of CCG (spot 4) relative to G (spot 1) is 0.07 for the 27 S size class, 0.15 for 24 S, and 0.38 for 19 S. Likewise, spot 5 (CAG,ACG) is present in relatively small amounts in 27 S RNA (0.04 relative to G) and much larger amounts (0.35 and 0.44) in 24 and 19 S RNA. If the sequences in the 19 and 24 S RNA’s which yield spots 4 and 5 are also present in the 27 S size class, then they must be present in much lower concentrations. Spot 10 (A,G) is an example of an oligonucleotide that is relatively abundant in the 19 S RNA (0.27 relative to G) and present only in very small amounts in 24 and 27 S RNA (0.05 each). This oligonucleotide is unlikely to be part of a sequence which is overlapping between the 19 S RNA and the other two size classes. Two related areas of investigation provide evidence that the three major RNA size classes are the products of different viral genes. Studies of the translation products of size-fractionated RNA have demonstrated that different peptides are coded for by the different RNAs (9, 10). Independent data are provided by hybridization of size-fractionated cytoplasmic RNA’s to unique viral DNA fragments generated by cleavage with restriction enzymes (II). This procedure provides an approach for correlating viral RNA’s with genome locations. Studies with EcoRl (I I) as well as other sets of adenovirus 2 DNA fragments (McGrogan and Raskas, in preparation) have demonstrated that some of these size classes contain more than one RNA species.
ACKNOWLEDGMENTS We appreciate the technical assistance provided by Mark Telle and Joe Kelley. This study was supported by a Public Health Service grant from the National Cancer Institute No. (CA125601 to HJR. a grant from the American Cancer Society (No. VC-94A) to HJR and, in part, by a National Cancer Institute grant (No. CAl4151) to EW. Jacov Tal is the recipient of a Fogarty International Fellowship from the National Institutes of Health. Cell culture media were prepared in a facility supported by a National Science Foundation grant (No. GB38657). This study was also supported by a grant from the following companies: Brown & Williamson Tobacco Corporation: Larus and Brother Company, Inc.; Liggett & Myers. Incorporated; Lorillard, a Division of Loews Theatres, Incorporated; Philip Morris, Incorporated; R. J. Reynolds Tobacco Company; United States Tobacco Company; and Tobacco Associates, Inc. REFERENCES
1. LINDBERG, U.. PERSSON,T.. and PHILIPSON, L.. J. Viral. 10, 909-919 (197%). 2. BHADURI. S.. RASKAS. H. J.. and GREEN, M.. J. Viral. 10, 1126-1129 (1972). 3. TAL, J., CRAIG, E. A., and RASKAS, H. J., J. Viral. 15, 137-144 (1975). 4. HORWITZ, M. S., SCHARFF, M. D., and MAIZEL, J. V., JR., Virology 39, 682-694 (1969). 5. ANDERSON, C. W., BAUM, P. R., and GESTELAND, R. F. J. Viral. 12, 241-252 (1973). 6. WALTER, G., and MAIZEL. J. V.. JR., Virology 57, 402-408 ( 1974). 7. PARSONS, J. T.. GARDNER, J.. and GREEN, M., Proc. Nat. Acad. Sci. USA 68, 557-560 (1971). 8. CRAIG, E. A., and RASKAS, H. J.. J. Viral. 14, 26-32 (1974). 9. ANDERSON, C. W.. LEWIS, J. B.. ATKINS, J. F., and GESTELAND, R. F., Proc. N&l. Acad. Sci. USA 71, 2756-2760 (1974). 10. OBERC, B., SABORIO,J., PERSSON,T., EVERITT, E., and PHILIPSON, L.. J. Viral. 15, 199-207 (1975). II. TAL, J., CRAIG, E. A., ZIMMER, S., and RASKAS. H. J. hoc. Nat. Acad. Sci. USA 71, 4057-4061 (1974). 12. SANGER,F., BROWNLEE.G. G., and BARRELL, B. G.. J. Mol. Biol. 13, 373-398 (1965). 13. SOUTHERN, E. M.. and MITCHELL, A. R., Biochem. J. 123, 613-617 (1971).