- Email: [email protected]
Molecular cloning and comparative sequence analyses of bluetongue virus S1 segments by selective synthesis of specific full-length DNA copies of dsRNA genes
VIROLOGY
177,820~823
Molecular
(1990)
Cloning
and Comparative Sequence Analyses of Bluetongue Virus Sl Segments Synthesis of Specific Full-Length DNA Copies of dsRNA Genes’ TIMOTHY
*Departments
of Biology
and Khemistry
F.
KOWALIK,**’
and Biochemistry Received
YI-YUAN
YANG,t'+
and *Molecular
February
Biology
5, 1990; accepted
K.-K. LI*+~
AND JOSEPH Program, April
by Selective
Utah State University,
Logan,
Utah 84322-5500
70, 1990
Using primers complementary to the conserved sequences of the 3’ ends of bluetongue virus genomic dsRNA segments, full-length DNA clones of all 10 dsRNA genes from the five U.S. BTV serotypes were synthesized and amplified by a novel method (ClampR). This amounts to nearly 100,000 base pairs of dsRNA cloned as unique full-length DNA copies. This continuous one-tube procedure combined cloning of the denatured dsRNA with reverse transcriptase and the selective amplification of full-length DNA by the polymerase chain reaction. ClampR-derived clones of the genomic segment Sl of BTV-11 encoding the serogroup antigen, VP7, were sequenced and shown to be complete copy, containing a total of 1156 bp and a long open reading frame of 349 amino acids. Comparative sequence analyses of BTV-11 Sl with those of the other U.S. serotypes show that 95.2% of the nucleotides are conserved between BTV-11 and -10, while only 79.0% of the bases are identical between BTV-11 and -13. Comparison of the VP7 proteins demonstrates that 100% of the amino acids are conserved between BTV-11 and -10 and 93.7% of these residues are identical between VP7 of BTV-11 and -13. The adaptation of the polymerase chain reaction to the full-length cloning and amplification of dsRNA (ClampR) should greatly facilitate molecular studies within the Reoviridae family. o 1990 Academic Press, Inc.
The Reoviridae family contains viruses with genomes composed of segmented, double-stranded RNA (dsRNA). The dsRNAviruses are represented in all five taxonomic kingdoms with animal, plant, and fungal dsRNA viruses being the most studied and of greatest economic importance (1). The Orbivirus genus of Reoviridae is composed of arthropod-borne viruses of which bluetongue virus (BTV) is the prototype (2). BTV is spread by Culicoicfes spp. and is the causative agent of bluetongue, a degenerative and sometimes fatal disease of ruminants (3-9). At least 24 BTV serotypes exist worldwide, of which five (BTV2, -10, -1 1, -13, and -17) are found in the United States. As BTV is the model system for the Orbivirus genus and causes a debilitating condition in livestock, studies are underway to determine the molecular relationships among the BTV serotypes in the United States and abroad (10-16). To obtain sufficient genetic material for in-depth molecular studies of BTV, we coupled the polymerase chain reaction (17, 18) with a cDNA synthesis step as part of a continuous one-tube reaction to clone and amplify dsRNA (ClampR) into full-length DNA product. A mixture containing 50 to 500 ng of total genomic
dsRNA was mixed with primers (500 ng each) of which at least 13 bases were complementary to each of the 3’ ends of the target gene segment. The sample, in a final volume of 30 ~1, was boiled for 4 min to denature the dsRNA and quenched on ice. The nucleic acids were then combined with a 20-~1 reaction mixture containing 5 ~1 of 1 OX buffer (100 ml\/l Tris, pH 8.3, 500 mM KCI, 15 mM MgCI,, and 0.1% (w/v) gelatin), 1 ~1 of stock nucleotides (10 mM each of dATP, dCTP, dGTP, and dlTP), 0.5 ~1 (15 U) of AMV-RT, 0.5 ~1 (2.5 U) of Taq polymerase, and 13 ~1 of water. The resulting solution was overlayed with 50 ~1 of mineral oil, and the ClampR reaction carried out in a Programmable Cycle Reactor (Ericomp Corp.). It had been observed that the standard PCR buffer commonly used with Taq polymerase (18) resulted in the best yields of ClampR-derived DNA. This includes an optimal l-2 mM MgCI, concentration. The selection of primers used to clone specific dsRNA segments was based on the conservation of termini within each of the different serogroups within Reoviridae (79-21). A pair of primers specific for the termini of each set of cognate dsRNA segments (e.g., the Sl segments of all five serotypes) was used to clone and amplify that segment from total, genomic dsRNAof each of the five U.S. BTV serotypes. To facilitate ligation of the resultant DNA into vectors, these primers were engineered to contain Pstl sites upstream of the region complementary to the BTV sequences.
’ Sequence data from this article have been deposited with the EMEUGenBank Data Libraries under Accession No. M32102. ’ Present address: Lineberger Cancer Research Center, School of Medicine, Campus Box 7295, The Umversity of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295. 3 To whom requests for reprints should be addressed. 0042-6822190
$3.00
CopyrIght 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.
820
SHORT
COMMUNICATIONS
821
-3675bp
s3675bp
-2323bp -1929bp
::3922%$
1371bp 1264bp
-1371 bp -1264bp 702bp -702bp -702bp
FIG. 1. Analysis of ClampR reaction products representing each size class (L, M, or S) of BTV genomic dsRNA. Primers specific for segment (A) L3 (AGTCGACCTGCAGGTTAAAllTCCGTAGCC, AGTCGACCTGCAGGTAAGTGTGTTTCCGC), (B) M2 (AGTCGACCTGCAGGTTAAAAAATAAAAAATCGCATAG, AGTCGACCTGCAGGGTTCTCC, AGTCGACCTGCAGGTAAGTGTAAGCATCTC), or (C) S3 (AGTCGACCTGCAGGTT TAAGTGTGAAATCGCC) were mixed with 300 ng of total genomic dsRNA from BTV2, 10, 1 1, 13, or 17. cDNA was synthesized and amplified through 35 cycles. A 10% aliquot of each reaction product was analyzed by 1% (w/v) agarose gel electrophoresis.
The primed, denatured RNA molecules were reverse transcribed during an initial 60-min incubation at 42”. AMV-RT was used for this report, but mouse Moloney leukemia virus reverse transcriptase provided slightly reduced, but comparable results. Full-length DNA clones were amplified by denaturing the doublestranded nucleic acid complexes at 95” for 2 min, followed by a primer binding step at 35” for 2 min. Primer extension time and temperature were empirically determined using a reovirus Sl clone (Dearing strain; supplied by W. K. Joklik). Optimal extension occurred during incubation of the reaction at 72” for 10 min. Reducing or increasing this step resulted in lower yields, presumably due to incomplete extension of primers or DNA degradation, respectively (data not shown). The number of amplification steps was optimized empirically to 30 to 40 cycles with the elongation step of the last cycle changed to 72” for 20 min (data not shown). Upon completion of the ClampR reaction, each sample was extracted once with chloroform, ethanol precipitated, and pelleted by centrifugation. The pellet was resuspended in 20 ~1 of TE (10 mMTris, pH 8.0, 1 mM EDTA) and an aliquot examined by electrophoresis through 1 Yo (w/v) agarose gels. By subjecting the genomic RNA from each of the U.S. BTV serotypes to the ClampR reaction, all 50 dsRNA segments could be readily reverse transcribed and full-length cDNA amplified with representative results shown in Fig. 1. With all
but the largest segments, which are between 3000 and 4000 base pairs long, full-length DNAs are observed as single, discrete bands. The full-length cloning of these 50 dsRNA segments represents the conversion of approximately 100,000 bp of dsRNA into cDNA using less than 25 pg of starting dsRNA for all of these ClampR reactions. The sensitivity of the ClampR reaction was tested by using a 5-fold dilution of the starting total genomic dsRNA of which one segment, Sl , was the template. It was observed that even with less than 500 pg of total genomic dsRNA as starting material, a small aliquot (10%) of the ClampR reaction product was sufficient to demonstrate the presence of full-length DNA copies (data not shown). Since the Sl segment of BTV represents approximately 5% by length of the starting genomic dsRNA, less than 25 pg of input RNA (Sl) is sufficient as template under these conditions (data not shown). If it is assumed that all 25 pg of starting RNA template was made into complete-copy DNA, at least a 50,000-fold amplification has occurred based on observed yields. As prior cloning attempts have demonstrated that the vast majority of cDNA synthesized from BTV dsRNA is less than full-length (10, 72, 16) the actual ClampR amplification value is certainly much higher. Upon completion of the ClampR reaction, full-length cDNAs were isolated by agarose gel electrophoresis,
822
SHORT GTTAAAAATCTATAGAG
COMMUNICATIONS
ATG M
GAC Ll
ACT T
ATC I
GCT A
GCA A
AGA R
GCA A
CTC L
ACT T
GTG v
ATG M
CGA R
GCA A
59
TGT C
GCT A
ACG T
CTT L
CAA 0
GAA E
GCA A
AGA R
ATT I
GTG v
TTG L
GAA E
GCT A
AAT N
GTG v
ATG M
GAA E
ATA I
CTG L
GGG G
119
ATA I
GCT A
ATC I
AAC N
AGG R
TAC Y
AAT N
GGA G
CTA L
ACT T
CTA L
CGT R
GGG G
GTA v
ACG T
ATG M
CGC R
CCG P
ACC T
TCG S
179
TTA L
GCG A
CAA 0
AGA R
AAT N
GAG E
ATG M
TTT F
TTT F
ATG M
TGT C
CTT L
GAT D
ATG M
ATG M
TTG L
TCT S
GCT A
GCG A
GGA G
239
ATA I
AAT N
GTT V
GGA G
CCG P
ATA I
TCG S
CCA P
GAT D
TAT Y
ACT T
CAA 0
CAT H
ATG M
GCT A
ACA T
ATT I
GGT G
GTA V
CTA L
299
GCA A
ACG T
CCG P
GAA E
ATC I
CCT P
TTT F
ACA T
ACA T
GAA E
GCG A
GCG A
AAT N
GAA E
ATA I
GCA A
CGC R
GTG v
ACT T
GGG G
359
GAG E
ACT T
TCG S
ACA T
TGG W
GGG G
CCG P
GCG A
CGT A
CAG 0
CCT P
TAT Y
GGT G
TTT F
TTC F
CTT L
GAA E
ACT T
GAG E
GAG E
419
ACT T
TTC F
CAA 0
CCT P
GGA G
AGA R
TGG W
TTC F
ATG M
CGC R
GCC A
GCT A
CAA 0
GCG A
GTA V
GCT -T
GCA A
GTG V
GTG V
TGC C
479
GGT G
CCG P
GAT D
ATG M
ATT I
CAG 0
GTG V
TCA S
CTA L
AAT N
GCT A
GGA G
GCG A
AGA R
GGG G
GAT D
GTA v
CAA 0
CAG 0
ATA I
539
TTT F
CAG 0
GGT G
CGT R
AAT N
GAT D
CCC P
ATG M
ATG M
ATA I
TAT Y
TTG L
GTA V
TGG W
AGA R
AGA R
ATC I
GAA E
AAC N
TTT F
599
GCG A
ATG M
GCG A
CAA 0
GGT G
AAT N
TCA S
CAG 0
CAA 0
ACT T
CAA 0
GCA A
GGT G
GTG V
ACC T
GTT V
AGC S
GTT v
GGT G
GGA G
659
GTT V
GAC D
ATG M
AGA R
GCG A
GGA G
CGC R
ATT I
ATA I
GCG A
TGG W
GAT D
GGA G
CAG 0
GCC A
GCA A
TTG L
CAT H
GTG V
CAT H
71
AAC N
CCT P
ACA T
CAA 0
CAG 0
AAT N
GCG A
ATG M
GTG V
CAG 0
ATA I
CAG 0
GTT V
GTG V
TTC F
TAT Y
ATA I
TCT S
ATG M
GAT D
779
AAA K
ACT T
TTA L
AAC N
CAG 0
TAC Y
CCA P
GCT A
TTG L
ACT T
GCC A
GAA E
ATT I
TTT F
AAT N
GTT V
TAC Y
AGC S
TTC F
AGG R
839
GAT II
CAT H
ACA T
TGG W
CAT H
GGA G
TTA L
AGA R
ACG T
GCA A
ATA I
TTA L
AAC N
AGA R
ACC T
ACA T
CTG L
CCT P
AAC N
ATG M
099
CTG L
CCA P
CCA P
ATC I
TTC F
CCA P
CCA P
AAT N
GAT D
CGT R
GAT D
AGT S
ATC I
TTA L
ACC T
CTT L
CTA L
CTT L
TTA L
TCT S
959
ACA T
CTC L
GCT A
GAT D
GTT V
TAC Y
ACT T
GTG V
TTA L
AGA R
CCA P
GAG E
TTT F
GCG A
ATT I
CAC H
GGC G
GTA V
AAT N
CCG P
ATG M
CCA P
GGG G
CCG P
CTC L
ACA T
CGT R
GCT A
ATT I
GCA A
CGC R
GCC A
GCC A
TAT Y
GTG V
T&TCCACTTTGCACGGGT
GTGGGTTACATATGCGGTGTGTCGGTTGTGGGAAATATGTGACCCATTTAAACGTCTCTTAGATTACACTTAC
1019
1002 1156
FIG. 2. Nucleotide sequence of the plus strand of segment Sl and deduced codons of the ORF encoding VP7 are in boldface and the Pstl site that produced of the primers used to clone this segment were AGTCGACCTGCAGGTAAGTGTAATCT
digested with Pstl, ligated into pUC18, and transformed into competent fscherichia co/i (JM 109) cells. To confirm that clones made by the ClampR procedure resulted in full-length DNA, a cDNAof segment Sl from BTV-1 1 was digested with fstl producing two fragments of 470 and 686 bp. Each fragment was separately ligated into pUC18 and sequencing demonstrated that the resulting clones were collectively of the expected length of 1 156 bp (Fig. 2). Since the primers used in this ClampR reaction were derived from segment Sl of BTV-10 (22) the 5’ termini of dsRNA segment S 1 of BTV-1 1 were directly sequenced and determined to be identical to the BTV-derived primer sequence. The lengths of the 5’and 3’ noncoding regions as well as the 349 codon long open reading frame (ORF), which encodes VP7, were retained. The VP7
9
amino acid sequence of VP7 of BTV1 1. The start and stop the 470. and 686-bp fragments is underlined. The sequences and AGTCGACCTGCAGGTTAAAAATCTATAGAG.
protein of BTV-1 1 has a calculated molecular weight of 38,551 and a net charge of +l at neutral pH. Comparisons of BTVl 1 Sl with BTV-10 and -13 (12, 22) showed that 79.0% (BTV-1 1 :13) to 95.2% (BTV-11: 10) of the nucleotide sequences were conserved (Table 1). The mismatch percentage between BTVl 1 and -13 were similar to that observed with BTV-10 and -13 (79.6%). Alignments of the VP7 proteins showed that 93.7% of the amino acids between BTV-11 and -13, and BTVlO and -13 are conserved, whereas there is 100% identity between BTV-1 1 and -10 (Table 1). An in-depth analysis of the relatedness of five U.S. BTV serotypes based the sequences of segment Sl and VP7 has been discussed elsewhere (74). The ClampR technique described here is a major methodological advance in obtaining full-length DNA
SHORT
work was supported by Grant 4-20122, United CRCR-l-2251, and Utah and approved as Journal
TABLE1 PERCENTAGE HOMOLOGY OF Sl AND VP7 AMONG THREE U.S. BTV SEROTYPES’ BTV-10
11
823
COMMUNICATIONS
Utah State University Biotechnology Center States Department of Agriculture Grant 86Agricultural Experiment Station Project 537, Paper 3905.
13
REFERENCES
BTV- 10 11 13 a Nucleotide homologies acid homologies, below.
are shown
above
the diagonal
and amino
clones from dsRNA. This one-tube, one-reaction procedure is rapid, requiring only an overnight incubation to produce full-length cDNA compared with the months to years previously needed to clone and identify full-length cDNA from dsRNA. By manipulating the primer sequences upstream of the RNA-priming region, cDNA produced by the ClampR reaction can easily be cloned into any cloning/expression vector by the appropriate restriction endonuclease digestion and ligation. The availability of full-length DNA copies of all 50 BTV genes will facilitate site-directed mutagenesis and expression studies in heterologous hosts. The general applicability of ClampR will greatly simplify the cloning of dsRNAs from other sources such as plant and fungal viruses and should be adaptable to cloning specific single-stranded RNAs (i.e., mRNA), as well. The simplicity and generality of the ClampR procedure makes it amenable to automation for the cloning of large numbers of dsRNAs, such as the 10 genomic segments from the other 19 BTV serotypes found worldwide. Since the procedure is segment-specific, rapid (the reaction conditions can be shortened with a concomitant decrease in DNAyield), and requires minimal template RNA, ClampR should also prove to be useful in the diagnosis of RNA virus infections.
ACKNOWLEDGMENTS We thank Ms. Vlckl Shore for virus production and the Utah State University Biotechnology Center for oligonucleotide synthesis. This
(W. K. Joklik, Ed.), pp. 1-7. Ple1. JOKLIK, W. K., “The Reoviridae” num, New York, 1983. 2. GORMAN. B. M., TAYLOR, J., and WALKER, P. J., “The Reoviridae” (W. K. Joklik, Ed.), pp. 287-357. Plenum, New York, 1983. 3. DUTOIT, R. M., Onderstepoort/. Vet. Res. 19, 7-16 (1944). 4. HOFF, G. L., GRINER, L. A., and TRAINER, D. 0. /. Amer. Ver. Med. Assoc. 163,565-567 (1973). LUEDKE, A. J., JOCHIM. M. M., and JONES, R. H., Amer. /. Ver. Res. 37,1701-1704(1977). MAHRT, C. R., and OSBURN, B. I., Amer. /. Vet. Res. 47, 11981203(1986). OSBURN, B. I., SILVERSTEIN, A. M., PRENDERGAST, R. A., JOHNSON, R. T., and PARSHALL, C. J., Lab. Invest. 25, 197-205 (1971). 8. SLOTS. J. L., BARBER, T. L., and OSBURN, B. I., Amer. /. Vet. Res. 46,1043-1049(1985). 9. THOMAS, A. D., and NEITZ. W. O., OndersfepoorfI. Vet. Res. 22, 27-37 (1947). 10. HUISMANS, H., and CLOETE. M., Virology 158, 373-380 (1987). 11. KOWALIK, T. F., and LI, J. K.-K., Virology 158, 276-284 (1987). 12. KOWALIK, T. F., and LI, J. K.-K., Virology 172, 189-l 95 (1989). 13. KOWALIK, T. F., CHUANG, R. Y., DOI, R. H., and OSBURN, B. I., submitted for publication. 14. KOWALIK, T. F., and LI, J. K.-K., submitted for publication. 15. SQUIRE, K. R. E., CHUANG, R. Y., CHUANG, L. F., DOI, R. H., and OSBURN, B. I., Amer. /. Vet. Res. 47, 53-60 (1986). 16. SQUIRE, K. R. E., CHUANG, R. Y., DUNN, S. J., DANGLER, C. A., FALBO, M. T., CHUANG, L. F., and OSBURN, B. I., Amer. /. Vet. Res.47,1785-1788(1986). 17. GOBLET, C., PROST, E., and WHALEN, R. G., Nucleic Acids Res. 17,2144(1989). 18. SAIKI, R. K., GELFAND, D. H., STOFFEL, S.. SCHARF. S.. HIGUCHI, R., HORN, G. T.. MULLIS, K. B., and ERLICH, H. A., Science 239, 487-49 1 (1988). 19. LI, J. K.-K., KEENE, J. D., SCHEIBLE, P. P., and JOKLIK, W. K., Virology 105,41-51 (1980). 20. MERIENS, P. P. C., and SANGAR, D. V., LPology 140, 55-67 (1985). 21. RAO, C. D., KIUCHI, A., and ROY, P., /. Viral. 46, 378-383 (1983). 22. Yu, Y.. FUKUSHO, A., RITTER, D. G., and ROY, P., NucleiAcids Res. 16, 16-20(1988).
177,820~823
Molecular
(1990)
Cloning
and Comparative Sequence Analyses of Bluetongue Virus Sl Segments Synthesis of Specific Full-Length DNA Copies of dsRNA Genes’ TIMOTHY
*Departments
of Biology
and Khemistry
F.
KOWALIK,**’
and Biochemistry Received
YI-YUAN
YANG,t'+
and *Molecular
February
Biology
5, 1990; accepted
K.-K. LI*+~
AND JOSEPH Program, April
by Selective
Utah State University,
Logan,
Utah 84322-5500
70, 1990
Using primers complementary to the conserved sequences of the 3’ ends of bluetongue virus genomic dsRNA segments, full-length DNA clones of all 10 dsRNA genes from the five U.S. BTV serotypes were synthesized and amplified by a novel method (ClampR). This amounts to nearly 100,000 base pairs of dsRNA cloned as unique full-length DNA copies. This continuous one-tube procedure combined cloning of the denatured dsRNA with reverse transcriptase and the selective amplification of full-length DNA by the polymerase chain reaction. ClampR-derived clones of the genomic segment Sl of BTV-11 encoding the serogroup antigen, VP7, were sequenced and shown to be complete copy, containing a total of 1156 bp and a long open reading frame of 349 amino acids. Comparative sequence analyses of BTV-11 Sl with those of the other U.S. serotypes show that 95.2% of the nucleotides are conserved between BTV-11 and -10, while only 79.0% of the bases are identical between BTV-11 and -13. Comparison of the VP7 proteins demonstrates that 100% of the amino acids are conserved between BTV-11 and -10 and 93.7% of these residues are identical between VP7 of BTV-11 and -13. The adaptation of the polymerase chain reaction to the full-length cloning and amplification of dsRNA (ClampR) should greatly facilitate molecular studies within the Reoviridae family. o 1990 Academic Press, Inc.
The Reoviridae family contains viruses with genomes composed of segmented, double-stranded RNA (dsRNA). The dsRNAviruses are represented in all five taxonomic kingdoms with animal, plant, and fungal dsRNA viruses being the most studied and of greatest economic importance (1). The Orbivirus genus of Reoviridae is composed of arthropod-borne viruses of which bluetongue virus (BTV) is the prototype (2). BTV is spread by Culicoicfes spp. and is the causative agent of bluetongue, a degenerative and sometimes fatal disease of ruminants (3-9). At least 24 BTV serotypes exist worldwide, of which five (BTV2, -10, -1 1, -13, and -17) are found in the United States. As BTV is the model system for the Orbivirus genus and causes a debilitating condition in livestock, studies are underway to determine the molecular relationships among the BTV serotypes in the United States and abroad (10-16). To obtain sufficient genetic material for in-depth molecular studies of BTV, we coupled the polymerase chain reaction (17, 18) with a cDNA synthesis step as part of a continuous one-tube reaction to clone and amplify dsRNA (ClampR) into full-length DNA product. A mixture containing 50 to 500 ng of total genomic
dsRNA was mixed with primers (500 ng each) of which at least 13 bases were complementary to each of the 3’ ends of the target gene segment. The sample, in a final volume of 30 ~1, was boiled for 4 min to denature the dsRNA and quenched on ice. The nucleic acids were then combined with a 20-~1 reaction mixture containing 5 ~1 of 1 OX buffer (100 ml\/l Tris, pH 8.3, 500 mM KCI, 15 mM MgCI,, and 0.1% (w/v) gelatin), 1 ~1 of stock nucleotides (10 mM each of dATP, dCTP, dGTP, and dlTP), 0.5 ~1 (15 U) of AMV-RT, 0.5 ~1 (2.5 U) of Taq polymerase, and 13 ~1 of water. The resulting solution was overlayed with 50 ~1 of mineral oil, and the ClampR reaction carried out in a Programmable Cycle Reactor (Ericomp Corp.). It had been observed that the standard PCR buffer commonly used with Taq polymerase (18) resulted in the best yields of ClampR-derived DNA. This includes an optimal l-2 mM MgCI, concentration. The selection of primers used to clone specific dsRNA segments was based on the conservation of termini within each of the different serogroups within Reoviridae (79-21). A pair of primers specific for the termini of each set of cognate dsRNA segments (e.g., the Sl segments of all five serotypes) was used to clone and amplify that segment from total, genomic dsRNAof each of the five U.S. BTV serotypes. To facilitate ligation of the resultant DNA into vectors, these primers were engineered to contain Pstl sites upstream of the region complementary to the BTV sequences.
’ Sequence data from this article have been deposited with the EMEUGenBank Data Libraries under Accession No. M32102. ’ Present address: Lineberger Cancer Research Center, School of Medicine, Campus Box 7295, The Umversity of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295. 3 To whom requests for reprints should be addressed. 0042-6822190
$3.00
CopyrIght 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.
820
SHORT
COMMUNICATIONS
821
-3675bp
s3675bp
-2323bp -1929bp
::3922%$
1371bp 1264bp
-1371 bp -1264bp 702bp -702bp -702bp
FIG. 1. Analysis of ClampR reaction products representing each size class (L, M, or S) of BTV genomic dsRNA. Primers specific for segment (A) L3 (AGTCGACCTGCAGGTTAAAllTCCGTAGCC, AGTCGACCTGCAGGTAAGTGTGTTTCCGC), (B) M2 (AGTCGACCTGCAGGTTAAAAAATAAAAAATCGCATAG, AGTCGACCTGCAGGGTTCTCC, AGTCGACCTGCAGGTAAGTGTAAGCATCTC), or (C) S3 (AGTCGACCTGCAGGTT TAAGTGTGAAATCGCC) were mixed with 300 ng of total genomic dsRNA from BTV2, 10, 1 1, 13, or 17. cDNA was synthesized and amplified through 35 cycles. A 10% aliquot of each reaction product was analyzed by 1% (w/v) agarose gel electrophoresis.
The primed, denatured RNA molecules were reverse transcribed during an initial 60-min incubation at 42”. AMV-RT was used for this report, but mouse Moloney leukemia virus reverse transcriptase provided slightly reduced, but comparable results. Full-length DNA clones were amplified by denaturing the doublestranded nucleic acid complexes at 95” for 2 min, followed by a primer binding step at 35” for 2 min. Primer extension time and temperature were empirically determined using a reovirus Sl clone (Dearing strain; supplied by W. K. Joklik). Optimal extension occurred during incubation of the reaction at 72” for 10 min. Reducing or increasing this step resulted in lower yields, presumably due to incomplete extension of primers or DNA degradation, respectively (data not shown). The number of amplification steps was optimized empirically to 30 to 40 cycles with the elongation step of the last cycle changed to 72” for 20 min (data not shown). Upon completion of the ClampR reaction, each sample was extracted once with chloroform, ethanol precipitated, and pelleted by centrifugation. The pellet was resuspended in 20 ~1 of TE (10 mMTris, pH 8.0, 1 mM EDTA) and an aliquot examined by electrophoresis through 1 Yo (w/v) agarose gels. By subjecting the genomic RNA from each of the U.S. BTV serotypes to the ClampR reaction, all 50 dsRNA segments could be readily reverse transcribed and full-length cDNA amplified with representative results shown in Fig. 1. With all
but the largest segments, which are between 3000 and 4000 base pairs long, full-length DNAs are observed as single, discrete bands. The full-length cloning of these 50 dsRNA segments represents the conversion of approximately 100,000 bp of dsRNA into cDNA using less than 25 pg of starting dsRNA for all of these ClampR reactions. The sensitivity of the ClampR reaction was tested by using a 5-fold dilution of the starting total genomic dsRNA of which one segment, Sl , was the template. It was observed that even with less than 500 pg of total genomic dsRNA as starting material, a small aliquot (10%) of the ClampR reaction product was sufficient to demonstrate the presence of full-length DNA copies (data not shown). Since the Sl segment of BTV represents approximately 5% by length of the starting genomic dsRNA, less than 25 pg of input RNA (Sl) is sufficient as template under these conditions (data not shown). If it is assumed that all 25 pg of starting RNA template was made into complete-copy DNA, at least a 50,000-fold amplification has occurred based on observed yields. As prior cloning attempts have demonstrated that the vast majority of cDNA synthesized from BTV dsRNA is less than full-length (10, 72, 16) the actual ClampR amplification value is certainly much higher. Upon completion of the ClampR reaction, full-length cDNAs were isolated by agarose gel electrophoresis,
822
SHORT GTTAAAAATCTATAGAG
COMMUNICATIONS
ATG M
GAC Ll
ACT T
ATC I
GCT A
GCA A
AGA R
GCA A
CTC L
ACT T
GTG v
ATG M
CGA R
GCA A
59
TGT C
GCT A
ACG T
CTT L
CAA 0
GAA E
GCA A
AGA R
ATT I
GTG v
TTG L
GAA E
GCT A
AAT N
GTG v
ATG M
GAA E
ATA I
CTG L
GGG G
119
ATA I
GCT A
ATC I
AAC N
AGG R
TAC Y
AAT N
GGA G
CTA L
ACT T
CTA L
CGT R
GGG G
GTA v
ACG T
ATG M
CGC R
CCG P
ACC T
TCG S
179
TTA L
GCG A
CAA 0
AGA R
AAT N
GAG E
ATG M
TTT F
TTT F
ATG M
TGT C
CTT L
GAT D
ATG M
ATG M
TTG L
TCT S
GCT A
GCG A
GGA G
239
ATA I
AAT N
GTT V
GGA G
CCG P
ATA I
TCG S
CCA P
GAT D
TAT Y
ACT T
CAA 0
CAT H
ATG M
GCT A
ACA T
ATT I
GGT G
GTA V
CTA L
299
GCA A
ACG T
CCG P
GAA E
ATC I
CCT P
TTT F
ACA T
ACA T
GAA E
GCG A
GCG A
AAT N
GAA E
ATA I
GCA A
CGC R
GTG v
ACT T
GGG G
359
GAG E
ACT T
TCG S
ACA T
TGG W
GGG G
CCG P
GCG A
CGT A
CAG 0
CCT P
TAT Y
GGT G
TTT F
TTC F
CTT L
GAA E
ACT T
GAG E
GAG E
419
ACT T
TTC F
CAA 0
CCT P
GGA G
AGA R
TGG W
TTC F
ATG M
CGC R
GCC A
GCT A
CAA 0
GCG A
GTA V
GCT -T
GCA A
GTG V
GTG V
TGC C
479
GGT G
CCG P
GAT D
ATG M
ATT I
CAG 0
GTG V
TCA S
CTA L
AAT N
GCT A
GGA G
GCG A
AGA R
GGG G
GAT D
GTA v
CAA 0
CAG 0
ATA I
539
TTT F
CAG 0
GGT G
CGT R
AAT N
GAT D
CCC P
ATG M
ATG M
ATA I
TAT Y
TTG L
GTA V
TGG W
AGA R
AGA R
ATC I
GAA E
AAC N
TTT F
599
GCG A
ATG M
GCG A
CAA 0
GGT G
AAT N
TCA S
CAG 0
CAA 0
ACT T
CAA 0
GCA A
GGT G
GTG V
ACC T
GTT V
AGC S
GTT v
GGT G
GGA G
659
GTT V
GAC D
ATG M
AGA R
GCG A
GGA G
CGC R
ATT I
ATA I
GCG A
TGG W
GAT D
GGA G
CAG 0
GCC A
GCA A
TTG L
CAT H
GTG V
CAT H
71
AAC N
CCT P
ACA T
CAA 0
CAG 0
AAT N
GCG A
ATG M
GTG V
CAG 0
ATA I
CAG 0
GTT V
GTG V
TTC F
TAT Y
ATA I
TCT S
ATG M
GAT D
779
AAA K
ACT T
TTA L
AAC N
CAG 0
TAC Y
CCA P
GCT A
TTG L
ACT T
GCC A
GAA E
ATT I
TTT F
AAT N
GTT V
TAC Y
AGC S
TTC F
AGG R
839
GAT II
CAT H
ACA T
TGG W
CAT H
GGA G
TTA L
AGA R
ACG T
GCA A
ATA I
TTA L
AAC N
AGA R
ACC T
ACA T
CTG L
CCT P
AAC N
ATG M
099
CTG L
CCA P
CCA P
ATC I
TTC F
CCA P
CCA P
AAT N
GAT D
CGT R
GAT D
AGT S
ATC I
TTA L
ACC T
CTT L
CTA L
CTT L
TTA L
TCT S
959
ACA T
CTC L
GCT A
GAT D
GTT V
TAC Y
ACT T
GTG V
TTA L
AGA R
CCA P
GAG E
TTT F
GCG A
ATT I
CAC H
GGC G
GTA V
AAT N
CCG P
ATG M
CCA P
GGG G
CCG P
CTC L
ACA T
CGT R
GCT A
ATT I
GCA A
CGC R
GCC A
GCC A
TAT Y
GTG V
T&TCCACTTTGCACGGGT
GTGGGTTACATATGCGGTGTGTCGGTTGTGGGAAATATGTGACCCATTTAAACGTCTCTTAGATTACACTTAC
1019
1002 1156
FIG. 2. Nucleotide sequence of the plus strand of segment Sl and deduced codons of the ORF encoding VP7 are in boldface and the Pstl site that produced of the primers used to clone this segment were AGTCGACCTGCAGGTAAGTGTAATCT
digested with Pstl, ligated into pUC18, and transformed into competent fscherichia co/i (JM 109) cells. To confirm that clones made by the ClampR procedure resulted in full-length DNA, a cDNAof segment Sl from BTV-1 1 was digested with fstl producing two fragments of 470 and 686 bp. Each fragment was separately ligated into pUC18 and sequencing demonstrated that the resulting clones were collectively of the expected length of 1 156 bp (Fig. 2). Since the primers used in this ClampR reaction were derived from segment Sl of BTV-10 (22) the 5’ termini of dsRNA segment S 1 of BTV-1 1 were directly sequenced and determined to be identical to the BTV-derived primer sequence. The lengths of the 5’and 3’ noncoding regions as well as the 349 codon long open reading frame (ORF), which encodes VP7, were retained. The VP7
9
amino acid sequence of VP7 of BTV1 1. The start and stop the 470. and 686-bp fragments is underlined. The sequences and AGTCGACCTGCAGGTTAAAAATCTATAGAG.
protein of BTV-1 1 has a calculated molecular weight of 38,551 and a net charge of +l at neutral pH. Comparisons of BTVl 1 Sl with BTV-10 and -13 (12, 22) showed that 79.0% (BTV-1 1 :13) to 95.2% (BTV-11: 10) of the nucleotide sequences were conserved (Table 1). The mismatch percentage between BTVl 1 and -13 were similar to that observed with BTV-10 and -13 (79.6%). Alignments of the VP7 proteins showed that 93.7% of the amino acids between BTV-11 and -13, and BTVlO and -13 are conserved, whereas there is 100% identity between BTV-1 1 and -10 (Table 1). An in-depth analysis of the relatedness of five U.S. BTV serotypes based the sequences of segment Sl and VP7 has been discussed elsewhere (74). The ClampR technique described here is a major methodological advance in obtaining full-length DNA
SHORT
work was supported by Grant 4-20122, United CRCR-l-2251, and Utah and approved as Journal
TABLE1 PERCENTAGE HOMOLOGY OF Sl AND VP7 AMONG THREE U.S. BTV SEROTYPES’ BTV-10
11
823
COMMUNICATIONS
Utah State University Biotechnology Center States Department of Agriculture Grant 86Agricultural Experiment Station Project 537, Paper 3905.
13
REFERENCES
BTV- 10 11 13 a Nucleotide homologies acid homologies, below.
are shown
above
the diagonal
and amino
clones from dsRNA. This one-tube, one-reaction procedure is rapid, requiring only an overnight incubation to produce full-length cDNA compared with the months to years previously needed to clone and identify full-length cDNA from dsRNA. By manipulating the primer sequences upstream of the RNA-priming region, cDNA produced by the ClampR reaction can easily be cloned into any cloning/expression vector by the appropriate restriction endonuclease digestion and ligation. The availability of full-length DNA copies of all 50 BTV genes will facilitate site-directed mutagenesis and expression studies in heterologous hosts. The general applicability of ClampR will greatly simplify the cloning of dsRNAs from other sources such as plant and fungal viruses and should be adaptable to cloning specific single-stranded RNAs (i.e., mRNA), as well. The simplicity and generality of the ClampR procedure makes it amenable to automation for the cloning of large numbers of dsRNAs, such as the 10 genomic segments from the other 19 BTV serotypes found worldwide. Since the procedure is segment-specific, rapid (the reaction conditions can be shortened with a concomitant decrease in DNAyield), and requires minimal template RNA, ClampR should also prove to be useful in the diagnosis of RNA virus infections.
ACKNOWLEDGMENTS We thank Ms. Vlckl Shore for virus production and the Utah State University Biotechnology Center for oligonucleotide synthesis. This
(W. K. Joklik, Ed.), pp. 1-7. Ple1. JOKLIK, W. K., “The Reoviridae” num, New York, 1983. 2. GORMAN. B. M., TAYLOR, J., and WALKER, P. J., “The Reoviridae” (W. K. Joklik, Ed.), pp. 287-357. Plenum, New York, 1983. 3. DUTOIT, R. M., Onderstepoort/. Vet. Res. 19, 7-16 (1944). 4. HOFF, G. L., GRINER, L. A., and TRAINER, D. 0. /. Amer. Ver. Med. Assoc. 163,565-567 (1973). LUEDKE, A. J., JOCHIM. M. M., and JONES, R. H., Amer. /. Ver. Res. 37,1701-1704(1977). MAHRT, C. R., and OSBURN, B. I., Amer. /. Vet. Res. 47, 11981203(1986). OSBURN, B. I., SILVERSTEIN, A. M., PRENDERGAST, R. A., JOHNSON, R. T., and PARSHALL, C. J., Lab. Invest. 25, 197-205 (1971). 8. SLOTS. J. L., BARBER, T. L., and OSBURN, B. I., Amer. /. Vet. Res. 46,1043-1049(1985). 9. THOMAS, A. D., and NEITZ. W. O., OndersfepoorfI. Vet. Res. 22, 27-37 (1947). 10. HUISMANS, H., and CLOETE. M., Virology 158, 373-380 (1987). 11. KOWALIK, T. F., and LI, J. K.-K., Virology 158, 276-284 (1987). 12. KOWALIK, T. F., and LI, J. K.-K., Virology 172, 189-l 95 (1989). 13. KOWALIK, T. F., CHUANG, R. Y., DOI, R. H., and OSBURN, B. I., submitted for publication. 14. KOWALIK, T. F., and LI, J. K.-K., submitted for publication. 15. SQUIRE, K. R. E., CHUANG, R. Y., CHUANG, L. F., DOI, R. H., and OSBURN, B. I., Amer. /. Vet. Res. 47, 53-60 (1986). 16. SQUIRE, K. R. E., CHUANG, R. Y., DUNN, S. J., DANGLER, C. A., FALBO, M. T., CHUANG, L. F., and OSBURN, B. I., Amer. /. Vet. Res.47,1785-1788(1986). 17. GOBLET, C., PROST, E., and WHALEN, R. G., Nucleic Acids Res. 17,2144(1989). 18. SAIKI, R. K., GELFAND, D. H., STOFFEL, S.. SCHARF. S.. HIGUCHI, R., HORN, G. T.. MULLIS, K. B., and ERLICH, H. A., Science 239, 487-49 1 (1988). 19. LI, J. K.-K., KEENE, J. D., SCHEIBLE, P. P., and JOKLIK, W. K., Virology 105,41-51 (1980). 20. MERIENS, P. P. C., and SANGAR, D. V., LPology 140, 55-67 (1985). 21. RAO, C. D., KIUCHI, A., and ROY, P., /. Viral. 46, 378-383 (1983). 22. Yu, Y.. FUKUSHO, A., RITTER, D. G., and ROY, P., NucleiAcids Res. 16, 16-20(1988).