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Sequence-independent amplification and cloning of large dsRNA virus genome segments by poly(dA)-oligonucleotide ligation
Journal of Virological Methods 72 (1998) 243 – 247
Short communication
Sequence-independent amplification and cloning of large dsRNA virus genome segments by poly(dA)-oligonucleotide ligation F.T. Vreede *, M. Cloete 1, G.B. Napier 2, A.A. van Dijk, G.J. Viljoen Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa Received 15 October 1997; received in revised form 26 January 1998; accepted 26 January 1998
Abstract A strategy was developed for sequence-independent synthesis and amplification of full-length cDNA of 3 – 4 kb genes of dsRNA viruses. The method of single primer amplification (Lambden et al., 1992) was adapted by the inclusion of a 3% poly(A) tail to an oligonucleotide ligated to dsRNA genome segments as a template for oligo(dT)-primed cDNA synthesis. Full-length copies of the largest genome segments, 1 (4 kb) and 2 (3 kb), of African horse sickness virus (AHSV) have been cloned, terminally sequenced and expressed in vitro. © 1998 Elsevier Science B.V. All rights reserved. Keywords: dsRNA viruses; African horse sickness virus (AHSV); Single primer amplification
Investigation of the molecular biology of segmented dsRNA viruses, such as the Reoviridae, requires the construction of genomic cDNA * Corresponding author. Private Bag X5, Onderstepoort 0110, South Africa. Tel.: +27 12 5299442; fax: + 27 12 5299249; e-mail: [email protected] 1 Present address: Roodeplaat Vegetable and Ornamental Plant Institute, Pretoria 0001, South Africa. 2 Present address: Boehringer Mannheim South Africa, Randburg 2125, South Africa.
clones. It is, however, often difficult to synthesize and clone full-length cDNA of the large (3–4 kb) genome segments. This has led to the development of a wide array of methods for the molecular cloning of these genomes. Historically, the polyadenylation strategy of Cashdollar et al. (1982) has served as the foundation for much of the cloning of Reoviridae genes carried out to date, including reoviruses (Cashdollar et al., 1984), rotaviruses (Both et al., 1982), and orbiviruses (Purdy et al., 1984; Fukusho et
0166-0934/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0166-0934(98)00031-7
244
F.T. Vreede et al. / Journal of Virological Methods 72 (1998) 243–247
al., 1989). This approach involves polyadenylation of genomic dsRNA and cDNA synthesis with oligo(dT) primers, followed by either blunt-ended cloning into a suitable vector or dC tailing and cloning into dG-tailed Pst1-cut pBR322. This strategy has important limitations. Firstly, it biases the cloning towards smaller genome segments or truncated cDNAs (unpublished observations). Cashdollar et al. (1984) reported surmounting this problem by fractionation of the cDNA by alkaline agarose gel electrophoresis to optimise the cloning of complete gene copies. Secondly, bluntended ligation is notoriously inefficient, whereas the addition of homopolymeric G/C tails has been shown to inhibit the expression of cloned genes (Galili et al., 1986). A method for the removal of homopolymer tails, through an additional PCR amplification step with termini-specific primers, has been described (Nel and Huismans, 1991). A recently published modification of the polyadenylation method uses an adaptor oligo(dT) primer for cDNA synthesis (Shapouri et al., 1995). Restriction enzyme sequences were incorporated at the 5% end of the oligo(dT) primer to simplify cloning of the synthesized cDNA. However, the largest clone obtained using this approach represented a truncated gene of 1505 bp. Use of the PCR for the amplification of cDNA has increased the efficiency of cloning methods, but this requires prior availability of flanking sequence information of the gene of interest. The selective synthesis and PCR amplification of specific full-length cDNA of dsRNA genes using segment termini-specific primers (based on the conservation of termini within the serogroups of the Reoviridae) has been described (Kowalik et al., 1990; Cooke et al., 1991). However, researchers amplifying cDNAs of genome segments larger than 3 kb have resorted to an overlapping RT-PCR approach, using primers specific for sequences internal in the gene (Hwang and Li, 1993; Hwang et al., 1994; Huang et al., 1995). In order to overcome a lack of terminal sequence information, Lambden et al. (1992) devised a novel strategy for the cloning of non-cultivatable rotavirus through single primer amplification; a universal oligonucleotide ligated to genomic dsRNA serves as template for cDNA
synthesis and amplification with a single complementary primer. In our hands, this approach has only yielded full-length clones of the smaller dsRNA segments (unpublished results), as also reported by other researchers (Lambden et al., 1992; Bigot et al., 1995). Bigot et al. (1995) used internal segment primers for genes larger than 1.7
Fig. 1. Schematic representation of the strategy for synthesis and amplification of full-length cDNA of large dsRNA segments.
F.T. Vreede et al. / Journal of Virological Methods 72 (1998) 243–247
kb, thereby obtaining overlapping clones of the 5% and 3% ends of the gene. In this paper, a strategy is described (schematically represented in Fig. 1) that was devised from the above methods to clone 3 – 4 kb dsRNA genome segments with convenient flanking restriction enzyme sites, without any prior sequence information. This approach was used to obtain full-length clones of genome segments 1 (4 kb) and 2 (3.2 kb) of African horse sickness virus (AHSV), a member of the orbivirus genus. Genomic AHSV dsRNA was purified as described by Sakamoto et al. (1994). An oligonucleotide (primer-1) comprising convenient restriction enzyme sequences with a poly(dA) tail to facilitate oligo(dT) priming, specifically 5% -GGATCCCGGGAATTCGG(A)17- 3%, was ligated to the dsRNA using T4 RNA ligase as described by Lambden et al. (1992). A 3% terminal NH2 group was incorporated in the oligonucleotide to prevent concatenation. Unligated oligonucleotides were removed by spin column chromatography on Sephacryl S400. The oligonucleotide-ligated dsRNA was then enriched for the larger genome segments by centrifugation on a 5-ml gradient of 5 – 40% sucrose in 1×TE buffer (10 mM Tris pH 7.4, 1 mM EDTA pH 8.0). Centrifugation was carried out for 16 h at 48000 rpm in a Beckman SW50.1 rotor at 4°C. Gradients were fractionated using a gradient tube fractionator (Hoefer Scientific Instruments) and collecting 8 – 10 drops per fraction. Following agarose gel electrophoretic analysis (results not shown), fractions containing predominantly ( \ 80%) genome segments 1 – 3 were pooled, diluted in an equal volume of water and ethanol precipitated. The protocols for cDNA synthesis and labelling using oligo(dT) primers and 32P-dCTP incorporation, followed by size fractionation on alkaline sucrose gradients have been described previously (Huismans and Cloete, 1987). cDNA was purified from the sucrose gradient fractions by NENsorb column (New England Nuclear) chromatography. Alternatively, size fractionation of the 32P-labelled cDNA could also be achieved by vertical alkaline agarose gel electrophoresis. Autoradiography of the gel enabled the isolation of cDNA of individual genome seg-
245
Fig. 2. Electrophoretic analysis of cDNA synthesized from AHSV dsRNA enriched for the large genome segments. Lyophilised cDNA was resuspended in 10 × alkaline buffer (0.3 M NaOH, 20 mM EDTA) and resolved on a 1.5% agarose gel in 1 × alkaline buffer. The gel was then dried and autoradiographed. The AHSV genome segments are labelled on the left.
ments, which was purified by Geneclean™ II extraction. An autoradiograph of cDNA separated by alkaline agarose gel electrophoresis is shown in Fig. 2. Purified cDNA was allowed to anneal in a 50 mM Tris–HCl pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT buffer by heating to 80°C for 5 min, incubating at 65°C for 16 h and finally cooling to 30°C over 3 h. Partial duplexes were filled in using Klenow as described by Sambrook et al. (1989) prior to PCR amplification. A single primer (primer-2), complementary to primer-1, with the sequence 5% -CCGAATTCCCGGGATCC- 3%, was used for the PCR. AHSV cDNA was incubated in a reaction mixture containing 10 mM Tris pH 8.8, 50 mM KCl, 1.5 mM MgCl2 0.1% Triton X-100, 0.2 mM each dNTP, 500 ng primer-2 and 2.5 units DyNAZyme™ II (Finnzymes Oy). Optimization of PCR conditions
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F.T. Vreede et al. / Journal of Virological Methods 72 (1998) 243–247
allowed amplification of distinct cDNA species representing full-length 3 – 4 kb AHSV genome segments. This entailed 30 cycles of denaturation at 95°C for 30 s (120 s on the first cycle), annealing at 67°C for 30 s and extension at 72°C for 270 s (extended to 420 s on the final cycle). PCR amplification product from AHSV genome segment 2 and 3 cDNA is shown in Fig. 3. Amplified material was either first purified from agarose gels by Geneclean™ II or directly cloned into the pMOSBlue T-vector (Amersham Life Science) according to the manufacturer’s instructions. We were interested in cloning the genes encoding the putative RNA-dependent RNA polymerase, genome segment 1, of AHSV-1, and the outer capsid protein, genome segment 2, of AHSV-5. Full-length clones of both these genes were obtained by poly(dA)-oligonucleotide ligation. The identity and full-length status of these
Fig. 4. Sequences of the pMOSBlue vector/AHSV segment 2 gene junctions confirming the full-length status of the cloned gene and demonstrating the incorporation of terminal flanking restriction enzyme sequences. The AHSV gene sequence on the left (GTTTAT...) represents the 5% terminus of the coding strand of segment 2 and that on the right (GTAAGT...) the 5% terminus of the non-coding strand.
Fig. 3. Electrophoretic analysis of products of AHSV segments 2 and segments of AHSV genomic dsRNA the left. The photograph represents a bromide-stained 1% agarose gel.
the PCR amplification 3 cDNA (lane 2). The (lane 1) are labelled on negative of an ethidium
clones were confirmed by Northern blot hybridization (results not shown) and sequencing of the termini (Fig. 4). The conserved terminal hexanucleotides of AHSV genome segments (Roy et al., 1994) were identified abutting directly onto primer-1 and -2 specific restriction enzyme sequences. The presence of intact open reading frames was confirmed by in vitro transcription and translation of the genes with the Promega wheat germ extract (VP1) or rabbit reticulocyte lysate (VP2) systems according to the manufacturer’s protocols. Proteins of the expected sizes (150 and 120 kDa respectively) were obtained on SDSPAGE (results not shown). A protocol is described that permits the synthesis and amplification of cDNA of 3–4 kb dsRNA
F.T. Vreede et al. / Journal of Virological Methods 72 (1998) 243–247
genes without any prior sequence information. In addition, this protocol yields clones that are flanked by preferred restriction enzyme sites, enabling direct further molecular manipulation. The protocol represents an amalgamation of methods developed previously to allow optimal synthesis and cloning of dsRNA genes through oligo(dT) priming of poly(dA)-oligonucleotide ligated dsRNA enriched for larger genome segments.
Acknowledgements The authors would like to thank Drs. Ian Clarke and Paul Lambden for their support and contributions, Mr. John Putterill for assistance with the photography and Prof. Henk Huismans for valuable discussions during this study.
References Bigot, Y., Drezen, J.-M., Sizaret, P.-Y., Rabouille, A., Hamelin, M.-H., Periquet, G., 1995. The genome segments of DpRV, a commensal reovirus of the wasp Diadromus pulchellus (Hymenoptera). Virology 210, 109–119. Both, G.W., Bellamy, A.R., Street, J.E., Siegman, L.J., 1982. A general strategy for cloning double-stranded RNA: nucleotide sequence of the Simian-11 rotavirus gene 8. Nucleic Acids Res. 10, 7075–7088. Cashdollar, L.W., Esparza, J., Hudson, G.R., Chmelo, R., Lee, P.W.K., Joklik, W.K., 1982. Cloning the doublestranded RNA genes of reovirus: sequence of the cloned S2 gene. Proc. Natl. Acad. Sci. USA 79, 7644–7648. Cashdollar, L.W., Chmelo, R., Esparza, J., Hudson, G.R., Joklik, W.K., 1984. Molecular cloning of the complete genome of reovirus serotype 3. Virology 133, 191–196. Cooke, S.J., Lambden, P.R., Caul, E.O., Clarke, I.N., 1991. Molecular cloning, sequence analysis and coding assignment of the major inner capsid protein gene of human group C rotavirus. Virology 184, 781–785. Fukusho, A., Yu, Y., Yamaguchi, S., Roy, P., 1989. Completion of the sequence of bluetongue virus serotype 10 by the
.
247
characterization of a structural protein, VP6, and a nonstructural protein, NS2. J. Gen. Virol. 70, 1677 – 1689. Galili, G., Kawata, E.E., Cuellar, R.E., Smith, L.D., Larkins, B.A., 1986. Synthetic oligonucleotide tails inhibit in vitro and in vivo translation of SP6 transcripts of maize zein cDNA clones. Nucleic Acids Res. 14, 1511 – 1524. Huang, I.-J., Hwang, G.-Y., Yang, Y.-Y., Hayama, E., Li, J.K.-K., 1995. Sequence analyses and antigenic epitope mapping of the putative RNA-directed RNA polymerase of five U.S. bluetongue viruses. Virology 214, 280 – 288. Huismans, H., Cloete, M., 1987. A comparison of different cloned bluetongue virus genome segments as probes for the detection of virus-specified RNA. Virology 158, 373 – 380. Hwang, G.-Y., Li, J.K.-K., 1993. Identification and localization of a serotypic neutralization determinant on the VP2 protein of bluetongue virus 13. Virology 195, 859 – 862. Hwang, G.-Y., Xiang, M., Li, J.K.-K., 1994. Analyses and conservation of sequences among the cognate L3 segments of the five United States bluetongue viruses. Virus Res. 32, 381 – 389. Kowalik, T.F., Yang, Y.-Y., Li, J.K.-K., 1990. Molecular cloning and comparative sequence analyses of bluetongue virus s1 segments by selective synthesis of specific fulllength DNA copies of dsRNA genes. Virology 177, 820 – 823. Lambden, P.R., Cooke, S.J., Caul, E.O., Clarke, I.N., 1992. Cloning of noncultivatable human rotavirus by single primer amplification. J. Virol. 66, 1817 – 1822. Nel, L.H., Huismans, H., 1991. Synthesis of the virus-specified tubules of epizootic haemorrhagic disease virus using a baculovirus expression system. Virus Res. 19, 139 – 152. Purdy, M., Petre, J., Roy, P., 1984. Cloning of the bluetongue virus L3 gene. J. Virol. 51, 754 – 759. Roy, P., Mertens, P.P.C., Casal, I., 1994. African horse sickness virus structure. Comp. Immun. Microbiol. Infect. Dis. 17, 243 – 273. Sakamoto, K., Mizukoshi, N., Apiwatnakorn, B., Iwata, A., Tsuchiya, T., Ueda, S., Imagawa, H., Sugiura, T., Kamada, M., Fukusho, A., 1994. The complete sequences of African horsesickness virus serotype 4 (vaccine strain) RNA segment 2 and 6 which encode outer capsid protein. J. Vet. Med. Sci. 56, 321 – 327. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Shapouri, M.R.S., Kane, M., Letarte, M., Bergeron, J., Arella, M., Silim, A., 1995. Cloning, sequencing and expression of the S1 gene of avian reovirus. J. Gen. Virol. 76, 1515 – 1520.
Short communication
Sequence-independent amplification and cloning of large dsRNA virus genome segments by poly(dA)-oligonucleotide ligation F.T. Vreede *, M. Cloete 1, G.B. Napier 2, A.A. van Dijk, G.J. Viljoen Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa Received 15 October 1997; received in revised form 26 January 1998; accepted 26 January 1998
Abstract A strategy was developed for sequence-independent synthesis and amplification of full-length cDNA of 3 – 4 kb genes of dsRNA viruses. The method of single primer amplification (Lambden et al., 1992) was adapted by the inclusion of a 3% poly(A) tail to an oligonucleotide ligated to dsRNA genome segments as a template for oligo(dT)-primed cDNA synthesis. Full-length copies of the largest genome segments, 1 (4 kb) and 2 (3 kb), of African horse sickness virus (AHSV) have been cloned, terminally sequenced and expressed in vitro. © 1998 Elsevier Science B.V. All rights reserved. Keywords: dsRNA viruses; African horse sickness virus (AHSV); Single primer amplification
Investigation of the molecular biology of segmented dsRNA viruses, such as the Reoviridae, requires the construction of genomic cDNA * Corresponding author. Private Bag X5, Onderstepoort 0110, South Africa. Tel.: +27 12 5299442; fax: + 27 12 5299249; e-mail: [email protected] 1 Present address: Roodeplaat Vegetable and Ornamental Plant Institute, Pretoria 0001, South Africa. 2 Present address: Boehringer Mannheim South Africa, Randburg 2125, South Africa.
clones. It is, however, often difficult to synthesize and clone full-length cDNA of the large (3–4 kb) genome segments. This has led to the development of a wide array of methods for the molecular cloning of these genomes. Historically, the polyadenylation strategy of Cashdollar et al. (1982) has served as the foundation for much of the cloning of Reoviridae genes carried out to date, including reoviruses (Cashdollar et al., 1984), rotaviruses (Both et al., 1982), and orbiviruses (Purdy et al., 1984; Fukusho et
0166-0934/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0166-0934(98)00031-7
244
F.T. Vreede et al. / Journal of Virological Methods 72 (1998) 243–247
al., 1989). This approach involves polyadenylation of genomic dsRNA and cDNA synthesis with oligo(dT) primers, followed by either blunt-ended cloning into a suitable vector or dC tailing and cloning into dG-tailed Pst1-cut pBR322. This strategy has important limitations. Firstly, it biases the cloning towards smaller genome segments or truncated cDNAs (unpublished observations). Cashdollar et al. (1984) reported surmounting this problem by fractionation of the cDNA by alkaline agarose gel electrophoresis to optimise the cloning of complete gene copies. Secondly, bluntended ligation is notoriously inefficient, whereas the addition of homopolymeric G/C tails has been shown to inhibit the expression of cloned genes (Galili et al., 1986). A method for the removal of homopolymer tails, through an additional PCR amplification step with termini-specific primers, has been described (Nel and Huismans, 1991). A recently published modification of the polyadenylation method uses an adaptor oligo(dT) primer for cDNA synthesis (Shapouri et al., 1995). Restriction enzyme sequences were incorporated at the 5% end of the oligo(dT) primer to simplify cloning of the synthesized cDNA. However, the largest clone obtained using this approach represented a truncated gene of 1505 bp. Use of the PCR for the amplification of cDNA has increased the efficiency of cloning methods, but this requires prior availability of flanking sequence information of the gene of interest. The selective synthesis and PCR amplification of specific full-length cDNA of dsRNA genes using segment termini-specific primers (based on the conservation of termini within the serogroups of the Reoviridae) has been described (Kowalik et al., 1990; Cooke et al., 1991). However, researchers amplifying cDNAs of genome segments larger than 3 kb have resorted to an overlapping RT-PCR approach, using primers specific for sequences internal in the gene (Hwang and Li, 1993; Hwang et al., 1994; Huang et al., 1995). In order to overcome a lack of terminal sequence information, Lambden et al. (1992) devised a novel strategy for the cloning of non-cultivatable rotavirus through single primer amplification; a universal oligonucleotide ligated to genomic dsRNA serves as template for cDNA
synthesis and amplification with a single complementary primer. In our hands, this approach has only yielded full-length clones of the smaller dsRNA segments (unpublished results), as also reported by other researchers (Lambden et al., 1992; Bigot et al., 1995). Bigot et al. (1995) used internal segment primers for genes larger than 1.7
Fig. 1. Schematic representation of the strategy for synthesis and amplification of full-length cDNA of large dsRNA segments.
F.T. Vreede et al. / Journal of Virological Methods 72 (1998) 243–247
kb, thereby obtaining overlapping clones of the 5% and 3% ends of the gene. In this paper, a strategy is described (schematically represented in Fig. 1) that was devised from the above methods to clone 3 – 4 kb dsRNA genome segments with convenient flanking restriction enzyme sites, without any prior sequence information. This approach was used to obtain full-length clones of genome segments 1 (4 kb) and 2 (3.2 kb) of African horse sickness virus (AHSV), a member of the orbivirus genus. Genomic AHSV dsRNA was purified as described by Sakamoto et al. (1994). An oligonucleotide (primer-1) comprising convenient restriction enzyme sequences with a poly(dA) tail to facilitate oligo(dT) priming, specifically 5% -GGATCCCGGGAATTCGG(A)17- 3%, was ligated to the dsRNA using T4 RNA ligase as described by Lambden et al. (1992). A 3% terminal NH2 group was incorporated in the oligonucleotide to prevent concatenation. Unligated oligonucleotides were removed by spin column chromatography on Sephacryl S400. The oligonucleotide-ligated dsRNA was then enriched for the larger genome segments by centrifugation on a 5-ml gradient of 5 – 40% sucrose in 1×TE buffer (10 mM Tris pH 7.4, 1 mM EDTA pH 8.0). Centrifugation was carried out for 16 h at 48000 rpm in a Beckman SW50.1 rotor at 4°C. Gradients were fractionated using a gradient tube fractionator (Hoefer Scientific Instruments) and collecting 8 – 10 drops per fraction. Following agarose gel electrophoretic analysis (results not shown), fractions containing predominantly ( \ 80%) genome segments 1 – 3 were pooled, diluted in an equal volume of water and ethanol precipitated. The protocols for cDNA synthesis and labelling using oligo(dT) primers and 32P-dCTP incorporation, followed by size fractionation on alkaline sucrose gradients have been described previously (Huismans and Cloete, 1987). cDNA was purified from the sucrose gradient fractions by NENsorb column (New England Nuclear) chromatography. Alternatively, size fractionation of the 32P-labelled cDNA could also be achieved by vertical alkaline agarose gel electrophoresis. Autoradiography of the gel enabled the isolation of cDNA of individual genome seg-
245
Fig. 2. Electrophoretic analysis of cDNA synthesized from AHSV dsRNA enriched for the large genome segments. Lyophilised cDNA was resuspended in 10 × alkaline buffer (0.3 M NaOH, 20 mM EDTA) and resolved on a 1.5% agarose gel in 1 × alkaline buffer. The gel was then dried and autoradiographed. The AHSV genome segments are labelled on the left.
ments, which was purified by Geneclean™ II extraction. An autoradiograph of cDNA separated by alkaline agarose gel electrophoresis is shown in Fig. 2. Purified cDNA was allowed to anneal in a 50 mM Tris–HCl pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT buffer by heating to 80°C for 5 min, incubating at 65°C for 16 h and finally cooling to 30°C over 3 h. Partial duplexes were filled in using Klenow as described by Sambrook et al. (1989) prior to PCR amplification. A single primer (primer-2), complementary to primer-1, with the sequence 5% -CCGAATTCCCGGGATCC- 3%, was used for the PCR. AHSV cDNA was incubated in a reaction mixture containing 10 mM Tris pH 8.8, 50 mM KCl, 1.5 mM MgCl2 0.1% Triton X-100, 0.2 mM each dNTP, 500 ng primer-2 and 2.5 units DyNAZyme™ II (Finnzymes Oy). Optimization of PCR conditions
246
F.T. Vreede et al. / Journal of Virological Methods 72 (1998) 243–247
allowed amplification of distinct cDNA species representing full-length 3 – 4 kb AHSV genome segments. This entailed 30 cycles of denaturation at 95°C for 30 s (120 s on the first cycle), annealing at 67°C for 30 s and extension at 72°C for 270 s (extended to 420 s on the final cycle). PCR amplification product from AHSV genome segment 2 and 3 cDNA is shown in Fig. 3. Amplified material was either first purified from agarose gels by Geneclean™ II or directly cloned into the pMOSBlue T-vector (Amersham Life Science) according to the manufacturer’s instructions. We were interested in cloning the genes encoding the putative RNA-dependent RNA polymerase, genome segment 1, of AHSV-1, and the outer capsid protein, genome segment 2, of AHSV-5. Full-length clones of both these genes were obtained by poly(dA)-oligonucleotide ligation. The identity and full-length status of these
Fig. 4. Sequences of the pMOSBlue vector/AHSV segment 2 gene junctions confirming the full-length status of the cloned gene and demonstrating the incorporation of terminal flanking restriction enzyme sequences. The AHSV gene sequence on the left (GTTTAT...) represents the 5% terminus of the coding strand of segment 2 and that on the right (GTAAGT...) the 5% terminus of the non-coding strand.
Fig. 3. Electrophoretic analysis of products of AHSV segments 2 and segments of AHSV genomic dsRNA the left. The photograph represents a bromide-stained 1% agarose gel.
the PCR amplification 3 cDNA (lane 2). The (lane 1) are labelled on negative of an ethidium
clones were confirmed by Northern blot hybridization (results not shown) and sequencing of the termini (Fig. 4). The conserved terminal hexanucleotides of AHSV genome segments (Roy et al., 1994) were identified abutting directly onto primer-1 and -2 specific restriction enzyme sequences. The presence of intact open reading frames was confirmed by in vitro transcription and translation of the genes with the Promega wheat germ extract (VP1) or rabbit reticulocyte lysate (VP2) systems according to the manufacturer’s protocols. Proteins of the expected sizes (150 and 120 kDa respectively) were obtained on SDSPAGE (results not shown). A protocol is described that permits the synthesis and amplification of cDNA of 3–4 kb dsRNA
F.T. Vreede et al. / Journal of Virological Methods 72 (1998) 243–247
genes without any prior sequence information. In addition, this protocol yields clones that are flanked by preferred restriction enzyme sites, enabling direct further molecular manipulation. The protocol represents an amalgamation of methods developed previously to allow optimal synthesis and cloning of dsRNA genes through oligo(dT) priming of poly(dA)-oligonucleotide ligated dsRNA enriched for larger genome segments.
Acknowledgements The authors would like to thank Drs. Ian Clarke and Paul Lambden for their support and contributions, Mr. John Putterill for assistance with the photography and Prof. Henk Huismans for valuable discussions during this study.
References Bigot, Y., Drezen, J.-M., Sizaret, P.-Y., Rabouille, A., Hamelin, M.-H., Periquet, G., 1995. The genome segments of DpRV, a commensal reovirus of the wasp Diadromus pulchellus (Hymenoptera). Virology 210, 109–119. Both, G.W., Bellamy, A.R., Street, J.E., Siegman, L.J., 1982. A general strategy for cloning double-stranded RNA: nucleotide sequence of the Simian-11 rotavirus gene 8. Nucleic Acids Res. 10, 7075–7088. Cashdollar, L.W., Esparza, J., Hudson, G.R., Chmelo, R., Lee, P.W.K., Joklik, W.K., 1982. Cloning the doublestranded RNA genes of reovirus: sequence of the cloned S2 gene. Proc. Natl. Acad. Sci. USA 79, 7644–7648. Cashdollar, L.W., Chmelo, R., Esparza, J., Hudson, G.R., Joklik, W.K., 1984. Molecular cloning of the complete genome of reovirus serotype 3. Virology 133, 191–196. Cooke, S.J., Lambden, P.R., Caul, E.O., Clarke, I.N., 1991. Molecular cloning, sequence analysis and coding assignment of the major inner capsid protein gene of human group C rotavirus. Virology 184, 781–785. Fukusho, A., Yu, Y., Yamaguchi, S., Roy, P., 1989. Completion of the sequence of bluetongue virus serotype 10 by the
.
247
characterization of a structural protein, VP6, and a nonstructural protein, NS2. J. Gen. Virol. 70, 1677 – 1689. Galili, G., Kawata, E.E., Cuellar, R.E., Smith, L.D., Larkins, B.A., 1986. Synthetic oligonucleotide tails inhibit in vitro and in vivo translation of SP6 transcripts of maize zein cDNA clones. Nucleic Acids Res. 14, 1511 – 1524. Huang, I.-J., Hwang, G.-Y., Yang, Y.-Y., Hayama, E., Li, J.K.-K., 1995. Sequence analyses and antigenic epitope mapping of the putative RNA-directed RNA polymerase of five U.S. bluetongue viruses. Virology 214, 280 – 288. Huismans, H., Cloete, M., 1987. A comparison of different cloned bluetongue virus genome segments as probes for the detection of virus-specified RNA. Virology 158, 373 – 380. Hwang, G.-Y., Li, J.K.-K., 1993. Identification and localization of a serotypic neutralization determinant on the VP2 protein of bluetongue virus 13. Virology 195, 859 – 862. Hwang, G.-Y., Xiang, M., Li, J.K.-K., 1994. Analyses and conservation of sequences among the cognate L3 segments of the five United States bluetongue viruses. Virus Res. 32, 381 – 389. Kowalik, T.F., Yang, Y.-Y., Li, J.K.-K., 1990. Molecular cloning and comparative sequence analyses of bluetongue virus s1 segments by selective synthesis of specific fulllength DNA copies of dsRNA genes. Virology 177, 820 – 823. Lambden, P.R., Cooke, S.J., Caul, E.O., Clarke, I.N., 1992. Cloning of noncultivatable human rotavirus by single primer amplification. J. Virol. 66, 1817 – 1822. Nel, L.H., Huismans, H., 1991. Synthesis of the virus-specified tubules of epizootic haemorrhagic disease virus using a baculovirus expression system. Virus Res. 19, 139 – 152. Purdy, M., Petre, J., Roy, P., 1984. Cloning of the bluetongue virus L3 gene. J. Virol. 51, 754 – 759. Roy, P., Mertens, P.P.C., Casal, I., 1994. African horse sickness virus structure. Comp. Immun. Microbiol. Infect. Dis. 17, 243 – 273. Sakamoto, K., Mizukoshi, N., Apiwatnakorn, B., Iwata, A., Tsuchiya, T., Ueda, S., Imagawa, H., Sugiura, T., Kamada, M., Fukusho, A., 1994. The complete sequences of African horsesickness virus serotype 4 (vaccine strain) RNA segment 2 and 6 which encode outer capsid protein. J. Vet. Med. Sci. 56, 321 – 327. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Shapouri, M.R.S., Kane, M., Letarte, M., Bergeron, J., Arella, M., Silim, A., 1995. Cloning, sequencing and expression of the S1 gene of avian reovirus. J. Gen. Virol. 76, 1515 – 1520.