Journal o f General Virology (1992), 73, 323-328.

Printed in Great Britain

323

The myxoma virus thymidine kinase gene: sequence and transcriptional mapping
Ronald J. Jackson* and Harold G. B u i t s
CSIRO Division of Wildlife and Ecology, PO Box 84, Lyneham, A C T 2602, Australia

The myxoma virus thymidine kinase (TK) gene is encoded on a 1-6 kb S a c I - S a l I restriction fragment located between 57-7 and 59-3 kb on the 163 kb genomic map. The nucleotide sequence of this fragment as well as 228 bp from the adjacent S a l I - A A 2 fragment was determined and found to encode four major open reading frames (ORFs). Three of these ORFs are similar in nucleotide sequence to ORFs L5R and J 1R, and the TK gene of vaccinia virus (VV). The fourth ORF, MF8a, shows similarity to the ORFs found in the same position relative to the TK genes of Shope fibroma virus, Kenya sheep-1 virus and swinepox virus. A search of the complete VV nucleotide

sequence for regions of similarity to MF8a identified the host specificity gene C7L. Northern blot analysis of early viral RNA identified transcripts of approximately 700 nucleotides for both the TK gene and O R F MF8a. The 5' ends of the TK gene and O R F MF8a early mRNAs were mapped by primer extension to initiation sites 13 nucleotides downstream of sequences with similarity to the VV early promoter consensus. The sizes of the TK and MF8a mRNAs are consistent with transcription termination and polyadenylation occurring downstream of the sequence TTTTTNT, which is identical to the consensus sequence for the VV transcription termination signal.

Introduction
The myxoma virus (MV) belongs to the family Poxviridae, genus leporipoxvirus (Fenner, 1976). The MV genome is a linear dsDNA molecule of approximately 163 kb, with inverted terminal repeats (ITRs) of 11.5 kb which are covalently cross-linked at the ends (Block et al., 1985; Russell & Robbins, 1989). Only limited gene mapping and nucleotide sequence information are available for MV, but include the identification of a gene whose product is related to the epidermal growth factor family (Upton et al., 1987b), a gene encoding a serine protease inhibitor-like protein involved in virus virulence (Upton et al., 1990) and genes similar in nucleotide sequence to those encoding the 22K R N A polymerase subunit and overlapping open reading frame (ORF), J3R, of the orthopoxvirus vaccinia virus (VV) (Jackson & Bults, 1990). In this paper we report the location, nucleotide sequence and transcriptional mapping of the MV thymidine kinase (TK) gene. Mapping of the gene indicates that there are an additional 2 kb of D N A sequence immediately downstream from the TK gene relative to the VV genome.
The sequence data reported have been deposited with the EMBL database under the accession numbers X52655 and X58790. 0001-0540 1992 SGM

Methods
General molecular methods. These were performed essentially as described by Ausubel et al. (1987), except where otherwise stated. Cells and virus. SIRC cells (Oryctolagus cuniculus cornea, ATCC CCL60) were maintained in MEM supplemented with 5 ~ (v/v) foetal calf serum (FCS). MV, strain Uriarra (Ur) (Mykytowycz, 1953; Russell & Robbins, 1989) was grown on confluent monolayers of SIRC ceils in MEM supplemented with 2 ~ (v/v) FCS. Cloning and nucleotide sequence analysis. The identification of MV ORFs MF9 and MF10 (Jackson & Bults, 1990) indicated that the MV 163 kb restriction map of Russell & Robbins (1989) is inverted with respect to the accepted prototypic VV genomic map (Goebel et al., 1990). Throughout this manuscript we use the map coordinates in the opposite orientation to those published by Russell & Robbins (1989). A 1-6 kb SacI-SalI restriction fragment (positions 57.7 to 59-3 kb on the 163 kb MV map) was excised from pUrE-C (Russell & Robbins, 1989) and ligated between the EcoRI and SalI sites of the plasmid vector pGem3 to generate pUrTK1. The adjacent 0.6 kb S a l I - A A 2 fragment (59.3 to 59-9 kb) was also excised from pUrE-C and ligated into the XhoI site of p G e m 7 Z f ( - ) to generate pUrS-AA2. Plasmids containing nested deletions were generated using the Erase-a-Base System (Promega). The nucleotide sequences were completely determined from both strands by the dideoxynucleotide chain termination procedure using dsDNA templates (Chen & Seeburg, 1985), SP6 and T7 primers (Promega), and the T7 sequencing kit (Pharmacia) following the manufacturer's instructions. The nucleotide sequence of the junction between the two restriction fragments was determined using custom oligonucleotide-primed sequencing of pUrE-C. Oligonucleotide primers were constructed using a Pharmacia Gene Assembler Plus DNA synthesizer. Analyses of the DNA and deduced amino acid

324

R. J. Jackson and H. G. Bults

sequences were performed using the IBI Pustell Sequence Analysis Software (V2.02). ORFs were considered to be any contiguous stretch of DNA that could be translated into a polypeptideof 65 or more amino acids and was initiated by a methionine codon.
Virus mRNA preparation. Total early and late viral RNA w a s isolated from virus-infectedmonolayersof SIRC cells in the presence or absence of cycloheximide (100 ~tg/ml; Sigma), respectively, as described by Smith et al. (1989). Nucleic acid hybridization. Northern blots were probed with purified DNA fragments which were labelled with [ct-32p]ATP following electrophoresis and isolation in low gelling temperature agarose (FMC SeaPlaque). The probes used in the hybridization experiments were SacI-SalI (spanning nucleotides 2 to 1637 of the sequence published here), MF7 (367 to 718), TK (719 to 1258) and MF8a (1259 to 1637). Primer extension assays. Primer extension reactions were performed using 32p 5' end-labelled primers for TK (ACGTTTAACTAGACGGATTAGTTCCGTGCT;810 to 781)and MF8a (CTCTTTGATGGCGATCCCTTCGCTCACGAG; 1392 to 1363). Transcription initiation sites were determined by comigration of the primer extension products with DNA sequencing ladders of pUrTKI generated using the respective primers.

Table 1. Identity at the nucleotide and amino acid sequence* levels between M V ORFs and the equivalent ORFs o f SFV, KS-l, SPV, V V and F P V Identical nucleotide/amino acid residues (%) Virus SFV KS-1 SPV VV FPV F6 SNAI" 66/51~/ SNA 52/44:~ 41/27:~ F7 88/90~: 62/61 53/48:~ 58/51 48/37 F8 (TK) 85/82 65/68 64/66 68/67 (55/48)tl F8a 77/71:~ 49/38 51/41 GNP GNP

* Nucleotide sequence alignments are based on the amino acid alignments generated using the IBI Pustell Sequence AnalysisSoftware (V2.02). Sequence data are from Plucienniczak et al. (1985), Upton & McFadden (1986), Boyleet al. (1987), Drillien et al. (1987), Gershon & Black (1989a) and Schnitzlein & Tripathy (1991). t SNA,Sequence not available. :~Incomplete ORF in one or both strands. GNP, Gene not present. IIThe TK gene is situated elsewhere in the FPV genome.

Homology o f M F 8 a to other sequences

Results
Cloning and D N A sequencing

The nucleotide sequence of the MV 1.6 kb S a c I - S a l I restriction fragment contained in p U r T K 1 was determined. The complete nucleotide sequence of the O R F spanning the S a i l site was determined by partial nucleotide sequencing of the adjacent S a l I - A A 2 fragment. The deduced 1865 nucleotide sequence of this region is shown in Fig. 1. Four major O R F s are encoded by the sequence, MF6 (partial), MF7, MF8 (TK gene) and MF8a. The first three O R F s are similar to VV O R F s L 5 R (VF6), J 1 R (VF7) and J2R (VF8, T K gene) (Plucienniczak et al., 1985; Goebel et al., 1990), respectively (Table 1). The remaining O R F , MF8a, is similar to O R F s SF8a, C F 8 a and SwF8a, which are found in the same positions relative to the T K genes of Shope fibroma virus (SFV) (Upton & McFadden, 1986), K e n y a sheep-1 virus (KS-I) (Gershon & Black, 1989a) and swinepox virus (SPV) (Schnitzlein & Tripathy, 1991), respectively (Table 1). Two short ORFs, O R F A and ORFB, occur on the opposite strand to that containing the O R F s described above. A short O R F is found in the KS-1 sequence, and corresponds in position and reading frame to MV O R F A , although more significantly O R F A is not conserved in the SFV sequence. Neither O R F A or O R F B is likely to encode a product as they are not generally conserved in the corresponding sequences of other characterized poxviruses.

Comparison of the M F 8 a nucleotide sequence and translation product with sequences in the G e n B a n k (R62.0) and N B R F - P I R (R22.0) databases failed to identify any significant similarity to known sequences, excluding the expected m a t c h to the SFV sequence of U p t o n & M c F a d d e n (1986). The sequence of the translation product of O R F M F 8 a was compared with the translation products of the complete VV (Copenhagen strain) nucleotide sequence (Goebel et al., 1990); similarity to the product of O R F C7L was observed. Alignments of the amino acid sequences of the MF8a, SF8a (partial sequence), CF8a, SwF8a and C7L O R F products are presented in Fig. 2. Over the entire nucleotide and amino acid sequences of M F 8 a and CL7 48% and 31% of residues are identical. Allowing for conservative amino acid replacement, the overall similarity between the translation products is 46%, with the first 60 residues of the M F 8 a product showing 66% similarity to the corresponding region of the C7L product.
Transcription mapping

Northern blot analysis of total viral early and late R N A using the MF7, T K and M F 8 a probes identified early transcripts of approximately 700 nucleotides hybridizing to the T K and M F 8 a gene probes, whereas all three probes hybridized to heterogeneous populations of late transcripts (Fig. 3). The presence of the smear of hybridization below the T K and M F 8 a early m R N A s could be the result of either R N A degradation or the presence of non-polyadenylated nascent R N A .

Myxoma virus thymidine kinase gene

325

I GAGCTCGAGTGATCGCTTTGTTGATTTTCcTcTTTTTCAAAACCGAGTTTAAcATGTTACTGCGGAAcAcCGTCGATcTGGCGAACGATCcTATAAAACAGTTTAccTCAACGAGTTG MF6> E L A V I A L L I F L F F K T E F N M L L R N T V D L A N D P I K Q F T S T S L> -40 121 A G G A G G A C C c A T A C A C C T C T A `C A G A T A A C G C c C C A T A A A A C A T G C A G G A T C C G T T T A A T A C T T A G A A G G T T T T A A A A G C A T G C G G T A A C G G T G G T A T G A A A A A T C C A A C G G G C C T T C A C T C K G N A L F I K H M P N S T E L K P A L A ! N R K Q I V H E N C A A L L Q $> -1 +4 241 ATAAATGGATCACGGAAAGTATCTCTTAACGATATTCTTGAA~GACGATGATTCGTTCTTCAAGTATTTGGCGGCACAGGAGGACGACGTCGCGATGTCGGA~GTTCACACGATCGTAGA I N G S R K V S L N D I L E R R * MF7> M D H G K Y L L T I F L N D D D S F F K Y L A A Q E D D V A M S D V H T I V D> 361 CTAT TTAAACT TT T TACTAGCCCTAT TAATTAAATCGAAAGATAAACTAGAAGCCGTAGGATATTATTACGCTCCT CTGTCGGAGGAATACAAAGCCGT GTT T GAT TT CACCGACACAAA * A G R D S S Y L A T N S K V S V F Y L N F L L A L L I K S K D K L E A V G Y Y Y A P L S E E Y K A V F D F T D T K> 481GTCGTTGAAACAATTGTTTAACAAACAACCCGGTACGTTGAGAGCGACTCTCCCATTTGCGTGGATAAGGGATATTTGGCAGATTTTGTTCTCGCAACGACTAGATTAAAGAAACAATT < D N F C N N L L C G T Y T S L S E G M Q T S L P Y K A S K T R A V V L N F F C N S L K O L F N g O P V Y V E S P S P I C V D K G Y L A D F V L A T T R L K K Q L> -28 -13 601ACCTTTGGTGTTGGATAAGGAAGTTACGTACGTAGATCCGTATAAGGATAAACGATTCGCAAATATCCTGTCTATATTGAAAAAACTGAAGCATTAAAAACTATTATTACAAGTTTAA < G K T N S L S T V Y T S G Y L S L R N A F I R D I N C L F Q L M < O R F A P L V L D K E V T Y V D P Y K D K R F A N I L S I L H K N * 721 ATCATGGCCATGTACGGGGGACAGAT TCACCTCATTATAGGACCCATGTTTGCCGGTAAAAGCAC GGAACTAATCCGTCTAGTTAAACGTTATCAGATAGCGAGATACAAATGTCTCGTT TK> M A M Y G G Q I H L [ [ G P M F A G K S T E L [ R L V K R Y Q I A R Y K C L V> 841 GTAAATACGAAAAAGACGCCCGTTACGGAAAGGCGTACGCACTCACGATAACACATGCATCTCCGCCGTACAACCGCGTCCCTAGACGAGTAGAATCGATATCCGAACAGTCGAA V N Y E K D A R Y G N G V R T H D N T C I S A V P T A S L D D V E S I S E E>H 961 GTGATCGGGATAGAGAGGGACAGTTTTTCCCCAACATCGTATCGTTTTGCGAACGTATGGCGAATGCCGGAAAGGTGTTGATCGGGCCGCGTTGGACGGAACGTTTCAACGTAAACCG V I G I D E G Q F F P N I V S F C E R M A N A G K V L I V A A L D G T F Q P>R 1081 TTTACCAACATTTGTGAACTGATACGTTGGCCGAAAACGTAACAAAACTGAAGCGGTATGTATGTACTGTTACAAGGACGATCTTTCTCTAAACGGTTGGGAAACGAGACAGAGATC F T N I C E L I P L A E N V T K L N A V C M Y C Y K D A S F S K R L G N E T -28 -13 # 1201 GAAATAATAGGAGGTAGCGACAAGTACAAATCGGTGTGTAGAAAAGTTATTTTTTTTAAAATACTGAAAAAATAAATGGATATTTAGATTAGGTTGATCGTCTCCGACAGCGACCTC E ] I G G S D K Y K S V C R K C Y F F * 1321 TAGGACGTCGGTATGGGCGTGCAACACAAATTGGAATATTCTCGTGAGCGAAGGGATCGCATCAAAGAGGCAAATCTACTCAAGGGGGACAGTTAGGGTGCACCATCAAAATAAAA MFSa> M G V Q H K L D I F L V S E G I A I K E A N L L K G D S Y G C K> T 1441 T TGGATAAGGAAAAGACGTTCAAAT T CGTCATCGTATTGGAACCGGAGTGGATAGATGAGATAAAACCTATATACATGAAAGTTAACGACGAGTCGGTGGAGTTA GAATTAGACTATAAA I D K E K T F K F V I V L E P E W [ D E I K P I Y M K V N D E S V E L E L K>D 1561

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Fig. 1. Nucleotide sequence of the 1.6 kb SacI-Sall fragment and 228 bp of the adjacent SalI-AA2 genomic fragment mapping between 57-7 kb and 59-6 kb from the left end of the 163 kb genomic map. The amino acids encoded by ORFs MF6, MF7, MF8 (TK gene), MF8a, ORFA and ORFB are indicated below the sequence. The putativeearly promoters(P,k and Paa)and late promoter(PFT) are underlined.Transcriptioninitiationsites for the TK gene and ORF MF8a early mRNAs are indicatedabove the sequence(#). The putative early transcription terminationsignals are doubly underlined.

Primer extension products generated using the TK primer hybridized to total early R N A correspond to major transcription initiation sites at positions 711 ( + 1) and 713 ( + 1), with a minor initiation site at position 712 ( + 1) on the D N A sequence (Fig. 4). A primer extension assay of early viral transcripts using the MF8a primer generated extension products corresponding to a major initiation site at position 1288 ( + 1) and a possible minor transcript initiating at position 1289 ( + 1), although it is impossible to exclude the latter as an artefact due to premature termination of the extension product (Fig. 4).

Discussion
Comparison of the MV TK gene region with the equivalent genomic regions of the orthopoxvirus VV (Plucienniczak et al., 1985), avipoxvirus fowlpox virus (FPV) (Drillien et al., 1987; Binns et al., 1988), capripoxvirus KS-1 (Gershon & Black, 1989a), suipoxvirus SPV (Schnitzlein & Tripathy, 1991) and the leporipoxvirus SFV (Upton & McFadden, 1986) indicates that although the viruses resemble one another with regard to their genetic organization there are substantial

326

R. J. Jackson and H. G. Bults

MFSa SFSa CFSa SwFSa C7L

MGvQHkLDIFLVSEgIAiKeanLLKGDSYGCTIKIKldkeKtfKFvIVLePeW.,IDEIKPIyMkvNd MGIQHkLDvFIVSENIAiKdanLLnGDSYGCTIKIKINtkKsvrFvvlLePe ........................................ ^ .... MGIRHELDIILVSENIALKNVELLKGDSYGCTINIKvNqqKKLdFIIILRPDWTEvrnVKkInMvcNG MGIRHELDIFLVSEDIAm~hVELHKGnSYGCVLNIKSscrKqmKiIFVLkPDWTEIDsIKPIqMeAdG ...................... ^. . . . . . . . . . . . . . . . . . . . MGIQHEfDI.IingDIALrNIqLHKGDnYGCkLKIiSNdyKKLKFrFYiRPDWsEIDEVKgltvfANn

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Fig. 2. Alignment of the amino acid sequences of the MV MFSa, SFV SF8a (partial sequence), KS-1 CF8a, SPV SwF8a and VV C7L polypeptides. Alignments were performed using the IBI Pustell Sequence Analysis Software (V2.02) allowing for similar amino acid substitutions. Similarity scores of > + I are indicated below the sequences ( A ). Amino acid residues conserved in two or more genera are indicated by upper case characters.

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Fig. 3. Northern blot of 50 ~tg R N A isolated from uninfected SIRC cells (lane 1), or SIRC cells infected with MV early in infection (lanes 2, 4, 6 and 8) or late in infection (lanes 3, 5, 7 and 9). The D N A probes used were a 1.6 kb SacI-SalI fragment (lanes 1, 2 and 3), a TK gene probe (lanes 4 and 5), an MF8a gene probe (lanes 6 and 7) and an MF7 gene probe (lanes 8 and 9). The positions of R N A markers are indicated.

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differences. Most notably, the genetic locus of the FPV TK gene is found elsewhere in the viral genome (Boyle & Coupar, 1986; Boyle et al., 1987; Binns et al., 1988) and an additional gene, F8a, is present between the TK gene and ORF F9 of KS-1 and SPV. MV also encodes an ORF F8a, but this is situated approximately 1.5 kb upstream of ORF MF9 (Jackson & Bults, 1990). The organization of the ORFs surrounding the MV TK gene suggests that the leporipoxviruses are more closely related to the genera capripoxvirus and suipoxvirus than to the other genera characterized. This relationship is further supported by the studies of

Fig. 4. Primer extension analysisofthe TK gene and ORF MF8aearly m R N A s . Total MV early R N A (50 p.g) was hybridized with the 5' endlabelled TK (lane 1) and MF8a (lane 10) primers, and extended using avian myeloblastosis virus reverse transcriptase. Sequencing ladders (order GATC) of pUrTK1 generated using the TK (lanes 2 to 5) and MFSa (lanes 6 to 9) primers.

Gershon & Black (1989b), who have identified ORFs of the capripoxvirus ]TR with similarity to ORFs found in the SFV ITR (Upton et al., 1987a). Examination of the MV D N A sequence for the presence of sequences related to the characterized VV transcription regulation sequences reveals the presence of a consensus sequence for a late promoter, TAAAT(G)

M y x o m a virus thymidine kinase gene

327

(H/inggi et aL, 1986; Rosel et al., 1986; Davison & Moss, 1989b), overlapping the initiation codon of ORF MF7. The presence of a smear of hybridization to MV late mRNAs suggests that such transcripts have heterogeneous 3' termini, comparable to VV late mRNAs (Cooper et al., 1981; Mahr & Roberts, 1984b). Examination of the D N A seqeunces upstream of the TK gene and ORF MF8a reveals the presence of putative early promoter motifs (Fig. 1) which are similar in sequence to the VV early promoter consensus (Davison & Moss, 1989a). Both the MV TK gene and ORF MF8a early mRNAs appear to initiate preferentially with adenosine residues located 13 nucleotides downstream of their respective promoter elements. VV early transcripts initiate with purine residues located in approximately the same position downstream of early promoter motifs (Boone & Moss, 1977; Keith et al., 1980; Davison & Moss, 1989a). The sizes of the 700 nucleotide TK and MF8a mRNAs are consistent with transcriptional termination occurring 20 to 50 bases downstream of overlapping early transcription termination signals, TsNT (Rohrmann et al., 1986; Yuen & Moss, 1986, 1987), situated near the 3' ends of the genes (Fig. 1). This suggests that the TK and MF8a early transcripts could contain 3' poly(A) tails of approximately 100 to 150 nucleotides, which is in agreement with the predicted size of the poly(A) tail of VV early transcripts (Nevins & Joklik, 1975; Hruby et al., 1983). No transcripts greater than 700 nucleotides hybridized to the TK gene probe, suggesting that significant readthrough of the transcription termination signals does not occur, in contrast to VV and FPV TK mRNAs (Bajszar et al., 1983; Hruby et al., 1983; Mahr & Roberts, 1984a; Boyle et al., 1987). The MV TK gene has the potential to encode a 178 amino acid polypeptide (Mr 19930). Transcription initiation occurs upstream of the 5' in-frame methionine codon. Translation from this initiation codon would result in the MV TK protein containing two additional N-terminal amino acids relative to the predicted TK proteins of other poxviruses. ORF MF8a has the potential to encode a 158 amino acid protein (Mr 18391). This is substantially smaller than the predicted KS-1 CF8a (Mr 23048) and the SPV SwF8a (Mr 21750) proteins. The major difference in size is due to the absence of the large stretch of acid-rich residues present at the C terminus of the CF8a and SwF8a products (Gershon & Black, 1989 a; Schnitzlein & Tripathy, 1991). We have observed that ORF MF8a is similar to VV O R F C7L. The size of the predicted C7L product (Mr 17997) is similar to that of the MF8a product and is also devoid of a highly disproportionate level of acidic amino acid residues. The similarity between the N-terminal regions of the C7L and the F8a

proteins suggests a possible functional relationship. The C7L product has been implicated in host range specificity (Perkus et al., 1990); whether the F8a gene products determine host range specificity remains to be elucidated. While this manuscript was in preparation, Schnitzlein & Tripathy (1991) also reported similarity between SPV ORF SwF8a and VV C7L. The poxvirus TK gene locus has been used successfully in the development of recombinant viruses as expression vectors and potential living vaccines (for review see Piccini & Paoletti, 1988). The identification of the MV TK gene has allowed procedures to be developed for the insertion of foreign D N A sequences into the genome of MV (R. J. Jackson & H. G. Bults, unpublished results). Recombinant MVs may be useful agents in the development of strategies to enhance the effic'~cy of myxomatosis in the biological control of the wild European rabbit in Australia.
We are grateful to R. Joseph and M. Carson for technical assistance, and H. Garnett, M. Holland and P. Kerr for critically reading the manuscript. This research was supported by a grant from the Australian Wool Research and Development Fund.

References
AUSUBEL,F. M., BRENT,R., KINGSTON,R. E., MOORE,D. D., SEIDMAN, J. G., SMITH, J. A. & STRUHL, K. (1987). Current Protocols in Molecular Biology. New York: John Wiley & Sons. B~SZAR, G., W~YrEK,R., WEIR, J. P. & MOSS,B. (1983). Vaccinia virus thymidine kinase and neighbouring genes: mRNAs and polypeptides of wild-type virus and putative nonsense mutants. Journal of Virology 45, 62-72. BINNS, M. M., TOMLEY, F. M., CAMPBELL,J. & BOURSNELL,M. E. (1988). Comparison of a conserved region in fowlpox virus and vaccinia virus genomes and the translocation of the fowlpox virus thymidine kinase gene. Journal of General Virology 69, 1275-1283. BLOCK, W., UPTON, C. & MCFADDEN, G. (1985). Tumorigenic poxviruses: genomic organization of malignant rabbit virus a recombinant between Shope fibroma virus and myxoma virus. Virology 140, 113-124. BOOr,m, R. F. & Moss, B. (1977). Methylated 5'-terminal sequences of vaccinia virus mRNA species made in vivo at early and late times after infection. Virology 79, 67-80. BOYLE,D. B. & COUPAR,B. E. H. (1986). Identification and cloning of the fowlpox virus thymidine kinase gene using vaccinia virus. Journal of General Virology 67, 1591-1600. BOYLE,D. B., COUPAR,B. E. H., GlasS, A. J., SEIGMAN,L. J. d~ BOTH, G. W. (1987). Fowlpox virus thymidine kinase: nucleotide sequence and relationship to other thymidine kinases. Virology 156, 355-365. CI-mN, E. Y. & SEEnURG,P. H. (1985). Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4, 165-170. COOPER, J. A., WITTEK, R. & MOSS, B. (1981). Extension of the transcriptional and translational map of the left end of the vaccinia virus genome to 21 kilobase pairs. Journal of Virology 39, 733-745. DAVtSON, A. J. & MOSS, B. (1989a). Structure of vaccinia virus early promoters. Journal of Molecular Biology 210, 749-769. DAVtSON, A. J. & MOSS, B. (1989b). Structure of vaccinia virus late promoters. Journal of Molecular Biology 210, 771-784. DRILLmN, R., SPErrNER, D., VILLEVAL,D. & LECOCQ, J.-P. (1987). Similar genetic organization between a region of fowlpox virus DNA and the vaccinia virus HindlII J fragment despite divergent location of the thymidine kinase gene. Virology 160, 203-209.

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(Received 9 August 1991; Accepted 2 October 1991)