Proc. Natl. Acad. Sci. USA Vol. 80, pp. 3411-3415, June 1983 Genetics

Fine structure analysis and nucleotide sequence of the vaccinia virus thymidine kinase gene (recombinant DNA/hybrid-arrested translation/nuclease SI/DNA sequence analysis) DENNIS E. HRUBY*, RICHARD A. MAKIt, DONNA B. MILLERS, AND L. ANDREW BALL: *Department of Microbiology, University of Texas, Austin, Texas 78712; 'Basel Institute for Immunology, Grenzacherstrasse 487, Postfach, CH-4005 Basel, Switzerland; and tBiophysics Laboratory, University of Wisconsin, Madison, Wisconsin 53706 Communicated by Esmond E. Snell, February 22, 1983 ABSTRACT The thymidine kinase (ATP:thymidine 5'-phos- is necessary to postulate the existence of several transcriptional photransferase, EC 2.7.1.21) gene of vaccinia virushas previously and post-transcriptional regulatory mechanisms to account for been mapped near the middle of the viral DNA, within the 4.85- the varied patterns of gene expression that have been ob- kilobase Hindm J fragment, and shown to encode a Mr 19,000 served. polypeptide [Hruby, D. E. & Ball, L. A. (1982)J. ViroL 43, 403- To address the problem of gene regulation during VV in- 409]. To locate the gene more precisely and to determine the fection, a detailed analysis of the behavior of the viral thymi- structure of the basic transcriptional unit, the positions of cleav- dine kinase (tk) gene has been undertaken. During VV infection age sites for several restriction endonucleases were mapped within of mouse or human cells, viral tk accumulates for the first 4 hr the HindEI J DNA fragment. Four appropriate subfragments of shut of the levels of tk mRNA HindU J DNA were inserted into plasmid pBR322 derivatives and then is abruptly off..Assays and cloned in Escherichia coli. These recombinant plasmid DNAs throughout infection show that this shut-off of synthesis occurs were tested for their ability to inhibit the cell-free synthesis of despite the continued presence of functional mRNA in the cy- active thymidine kinase and to retain the mRNA for this enzyme toplasm-i.e., at the translational level. In addition, transcrip- when immobilized on nitrocellulose filters. The data showed that tional control is exerted by an early viral gene, and tk mRNA the gene spanned an EcoRI cleavage site that lies 850 base pairs turnover is influenced by a late viral gene (2, 3). To develop from the left-hand end of the Hindu J fragment (the Hind]][ L- physical assays to complement the functional assays already J boundary). Because hybridization of vaccinia virus DNA to par- available for the tk enzyme and its mRNA, the tk gene was tially purified thymidine kinase mRNA detected only a single 670- mapped to a 4.85-kilobase (kb) HindIII fragment (J) of VV DNA, nucleotide RNA species capable of hybridizing to this region of and it was determined that this locus encoded a Mr 19,000 poly- the genome, nuclease SI mapping experiments were carried out peptide (4). A protein of this size requires only about 0.52 kb with thymidine kinase mRNA to protect DNA fragments that were of the 4.85-kb coding capacity of HindIII J, and translational terminally labeled at this EcoRI site. The results indicated that the mapping studies indicate that several other early and late genes gene extended from about 550 to 1,150 base pairs from the left end are located in this DNA fragment (5). Therefore, experiments of HindU J, was transcribed in a rightward direction, and con- were designed to map the position and boundaries of the tk tained no intervening sequences. Hence, a 1.04-kilobase Ava II- gene more precisely. This information was used to subclone ap- Hpa II restriction fragment containing this region of DNA was propriate fragments of HindIII J DNA in Escherichia coli. These isolated and subjected to nucleotide sequence analysis. An ex- recombinant plasmid DNAs were then used to determine the amination of this nucleotide sequence revealed the presence of an structure of the VV tk transcriptional unit and the correspond- open reading frame of 531 nucleotides capable of encoding a pro- ing nucleotide sequence. tein of 177 amino acids with a Mr of 20,077. MATERIALS AND METHODS Vaccinia virus (VV), the prototype member of the orthopox- virus family, has a DNA genome of Mr 122 X 106 that encodes Cell-Free Translation. Polyadenylylated immediate-early vi- some 200-250 genes (1). These genes are expressed in a tightly ral mRNA was extracted and purified from Ltk- cells infected regulated developmental program during infection and can be with VV (WR strain) for 5 hr in the presence of 100 ,ug of cy- grouped into three control categories on the basis of the pattern cloheximide per ml, as described (2). VV tk mRNA was partially of their sensitivity to inhibitors of macromolecular synthesis. purified by centrifugation of total immediate-early VV mRNA Thus, the immediate-early genes, which comprise about half of on 5-20% sucrose gradients containing NaDodSO4 in a Beck- the genetic capacity of the virus, are transcribed by the virion man SW 40 rotor at 38,000 rpm for 8 hr at 25°C and isolation DNA-dependent RNA polymerase and are insensitive to in- of the fraction that was maximally effective in directing the cell- hibitors of protein or DNA synthesis. On the other hand, free synthesis of VV tk (4). Hybrid-selection and hybrid-arrest expression of the delayed-early genes, which are represented procedures were used in concert with cell-free translation of most clearly by a function required for the viral DNA poly- VV tk mRNA in rabbit reticulocyte lysates and subsequent as- merase activity, depends on the protein products of one or more say for tk activity as previously detailed (4). of the immediate-early genes and is therefore sensitive to in- Restriction Enzyme Digestions and Gel Electrophoresis. hibitors of-translation. Expression of the late genes requires DNA samples were digested with restriction enzymes at 37C replication of the viral DNA. However, an examination of the overnight under the conditions suggested by the manufacturer. synthesis of individual viral polypeptides shows that many more The reactions were terminated by adding an equal volume of than three kinetic categories can be distinguished. Indeed, it loading buffer (0.1% bromophenol blue/0. 1% xylene cyanol/ 60% sucrose/0.05 M EDTA, pH 8.3). DNA fragments were The publication costs of this article were defrayed.in part by page charge resolved by electrophoresis in TBE buffer (50 mM Tris/50 mM payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: VV, vaccinia virus; tk, thymidine kinase; kb, kilobase(s). 3411 3412 Genetics: Hruby et al. Proc. Natl. Acad. Sci. USA 80 (1983) boric acid/i mM EDTA, pH 8.3) on either 0.8% agarose ment of E. coli DNA polymerase I (10), or uniformly labeled (SeaKem) or 8% polyacrylamide gels. After electrophoresis at by nick-translation (11). The radiochemical purity of the 5'- and 60 V for 30 min and 200 V for 3 hr. DNA bands were visualized 3'-labeled probes was checked by gel electrophoresis and au- by staining with 1 pug of ethidium bromide per ml and were toradiography. photographed by using a UV transilluminator (Ultraviolet Prod- RNA Transfer. Analyses of RNA transcripts were carried ucts, San Gabriel, CA) and Polaroid camera with type 57 Po- out by electrophoresing the RNA on 1% agarose gels that con- laroid film. The sizes of the restriction fragments were calcu- tained 2.2 M formaldehyde (12), transferring the RNA to ni- lated by interpolation from a standard curve generated by trocellulose, and setting up hybridizations according to the con- coelectrophoresis of DNA fragments of known size, such as ditions of Southern (13). bacteriophage A:HindIII, 4X174 RF:Hae III, or pBR322:HinfI Nuclease S1 Mapping. Nuclease S1 mapping was carried out digests. essentially as described by Villarreal (14). 5' or 3' terminally To isolate DNA fragments of interest, the appropriate band labeled probes (10,000-20,000 dpm) were mixed with gradient- was excised and transferred to the large well of a sample cup enriched VV tk mRNA in 25 ,ul of 80% deionized formamide in an ISCO 1750 sample concentrator. The DNA was elec- containing 0.4 M NaCl and 0.04 M Pipes (pH 6.4), denatured troeluted in TBE buffer with a current of 1 W at 40C for 2 hr. at 90°C for 3 min, and hybridized at 50°C for 7 hr. The reactions The polarity was reversed for 15 sec, and then the DNA was were diluted with 9 vol of nuclease S1 buffer (30 mM sodium removed from the small well in 200 ,p1. The DNA was extracted acetate, pH 4.5/250 mM NaCI/1 mM ZnSO4/5% glycerol) at sequentially with isoamyl alcohol, phenol [saturated with 10 mM 420C, made 40 units/ml in nuclease S1, and incubated at 42°C Tris (pH 8) and 1 mM EDTA], and finally, ether. After being for30 min. Nuclease Sl-resistant hybrids were isolated by ethanol precipitated twice with ethanol, the DNA was sufficiently pure precipitation in the presence of 10 ,ug of carrier calf liver tRNA. for use in the hybrid-arrest procedure or for further enzymatic The hybrids were denatured by heating at 900C for 5 min in reactions. 90% formamide/10 mM EDTA, pH 8.3/0.02% xylene cyanol/ Plasmid Construction and Isolation. To construct the plas- 0.02% bromophenol blue and then were subjected to electro- mids described in the text, the desired DNA fragments of plas- phoresis at 400 V on 8% acrylamide gels containing 7 M urea mid pBR322 and the VV DNA fragments to be cloned were for 4 hr. purified by electroelution from agarose gel slices as described DNA Sequence Analysis. Individual DNA fragments were above. Approximately 0.1 pug each of plasmid and insert were isolated and terminally labeled, and the nucleotide sequence mixed with 2 Weiss units of DNA ligase (P-L Biochemicals) in was determined by the methods of Maxam and Gilbert (9). The 50 mM Tris HCl, pH 7.6/10 mM MgCl2/20 mM dithiothrei- sequences of both DNA strands were determined several times tol/1 mM ATP and were incubated at 13°C overnight. The re- in independent experiments before compilation of the final se- action mixtures then were used to transform recipient HB101 quence. E. coli from an ampicillin-sensitive to an ampicillin-resistant phenotype (6). All transformants were screened to ensure that RESULTS AND DISCUSSION they possessed an ampicillin-resistant/tetracycline-sensitive Localization of the VV tk Gene. The restriction endonucle- phenotype, and small-scale plasmid preparations were purified ase HindIII cuts the 185-kb DNA of VV into 15 fragments that by the procedures of Holmes and Quigley (7). Plasmid DNAs range in size from 45.5 kb (HindIII A) to 1.5 kb (HindIII 0). were analyzed by restriction enzyme digestion and gel elec- The relative positions of these fragments in the VV genome trophoresis. Plasmids that contained the inserts of interest were have been mapped (Fig. 1; refs. 5, 15). Of these 15 fragments, then grown in 4-liter cultures of HB101, amplified with 100 ,ug HindIII J is unique in its ability to inhibit the translation of VV of chloramphenicol per ml overnight, and purified by the tk mRNA into active enzyme, to hybrid-select VV tk mRNA methods of Hardies et aL (8). from a mixed population of viral messengers, and to rescue tk- Labeling of DNA Fragments. Isotopically labeled DNA was viral mutants from growth inhibition of methotrexate during prepared by digesting the plasmids containing the VV DNA VV infection of tk- cells (1, 16). On the basis of these lines of inserts with appropriate restriction enzymes and purifying the evidence, it appears that HindIII J contains the VV tk gene. inserts by gel electrophoresis and electroelution. The frag- To localize the tk gene more precisely, the positions of a ments then were 5' terminally labeled with ['y-32P]ATP and number of restriction endonuclease cleavage sites were mapped polynucleotide kinase (9), 3' terminally labeled with a mixture within HindIII J DNA by standard single- and double-digestion of all four nucleoside [a- 2P]triphosphates and the Klenow frag- procedures (Fig. 1). The orientation of the J map in Fig. 1 rel- Hind N Map of Vaccinia Virus Genome C NMK F E 01 G L J H D A B mF T TI w T TV I I 4 v I 0~~30 0I60 elP! 120 Iso ISO

-~ Restriction Map of VV HindN J DNA Fragment I _ _ r PvuR mndE Eco RI HpaI XI;ho Kpnl HincX HindN I I I I 11 IF a 1. - I i I;ww I 0 l 2 3 4 Kb FIG. 1. Maps of theHindM cleavage sites in VV DNA (5, 15) and the cleavage sites for several restriction endonucleases in theHindl J frag- ment. For Hpa II, only the cleavage sites to the left of the Xho I site are indicated. -r|~~~~~~~~~~~~~~~~~~~ Genetics: Hruby et aL Proc. Natd Acad. Sci. USA 80 (1983) 3413 Table 1. Hybrid-arrested translation of VV tk mRNA fragment (R. Condit, Buffalo, NY), which spans the HindI11 L, [3H]dThd, J, and H regions of VV DNA. Advantage was taken of the single cpm X 10-3 Xho I and Kpn I cleavage sites in the HindIII J DNA fragment % hybrid- to preparatively isolate specific portions of this region. When DNA Hybrid Melt arrest* these subfragments were assayed for their ability to hybridize DNA fragmentst to tk mRNA, as determined by inhibition of VV tk enzyme syn- 49.8 40.8 0 thesis in a reticulocyte lysate, it became apparent that the tk Hindml J 1.9 41.9 95.5 gene resided in the left one-third of HindIII J, within the Xho I-HindUr 65.6 40.4 0 HtndIII-Xho I fragment (Table 1). This conclusion was con- HindlI-Xho I 2.3 62.0 96.3 firmed and extended by construction of recombinant plasmids HindlI-Kpn I 11.8 50.7 76.7 that contained four different pieces of HindIll J DNA (Fig. 2). Kpn I-Hindu 44.5 31.8 0 When these plasmid DNAs were tested in the hybrid-arrest Plasmid DNAsM assay, pDH-3 (containing the HindIH-Xho I fragment) was very 42.6 39.6 0 efficient at inhibiting the cell-free translation of VV tk mRNA pBR322 36.8 37.2 1 (Table 1). Furthermore, plasmids pDH-6 and pDH-7, which Hindm J:pBR322 5.8 35.9 84 contained the right 1.0 kb and left 0.85 kb, respectively, of the pDH-2 39.8 40.8 2.5 HindIII-Xho I fragment (delineated by the unique EcoRI site), pDH-3 2.1 23.3 89 both arrested VV tk synthesis. This indicated that the VV tk pDH-6 0.5 24.9 98 gene spanned the EcoRI cleavage site. pDH-7 3.4 17.1 80 Identification of the W tk Transcriptional Unit. VV tk mRNA, which had been size-selected (500-900 nucleotides) by Active VV tk was synthesized and assayed in reticulocyte lysates sucrose was programed with VV tk mRNA that had been annealed to the indicated gradient velocity centrifugation, electrophoresed DNA (hybrid) or annealed and then denatured by boiling (melt). under denaturing conditions and subjected to a RNA transfer * % hybrid arrest was calculated as [(melt - hybrid)/melt] x 100. analysis by using nick-translated pDH-3 DNA as a hybridiza- t DNA fiagments were isolated and purified from preparative agarose tion probe (Fig. 3A). A single species of mRNA was apparent gels after digestion of VV Hindu J:pBR322 DNA with the appro- with a size of =670 nucleotides, which agreed well with pre- priate restriction enzymes. The orientations of the cleavage sites are vious estimates (4). If total immediate-early VV mRNA se- left to right, as indicated in Fig. 1. Plasmid DNA was linearized by restriction enzyme digestion prior to hybridization. A B 1 2 ative to the HindIII map of VV DNA was determined by ap- propriate digestions of a cosmid containing the 14.9-kb Sst I F - 1,631

-5.0 - 517 - 396 - 3.35 - 344 RIH \ - 3.0 - 298 3.0Kb -2.25 a ~- 1.85 - 220 pON-2 \ -0.7 0 Aval' lXhol -0.75 -150

-0.08

Eco RI 0.85 Kb WHid a - 75 /EcoRIIH \

pDH-7

FIG. 3. (A) RNA transfer analysis of VV tk mRNA by using nick- FIG. 2. Diagrammatic representations of four recombinant plas- translated pDH-3 DNA. The position and size (kb) of rRNA, Brome mids constructed by replacing regions ofpBR322 DNA with fiagments mosaic virus RNAs 1-4, and tRNA, which were run as markers, are of VV DNA. The circles represent pBR322, with the dashed regions indicated. (B) Nuclease S1 mapping of the VV tk gene. Lane 1, 5'-la- having been replaced by VV DNA fiagments that are represented by beled HindM-EcoRP fragment. Lane 2, 3T-labeled EcoRI-Xho I frag- straight arrows. The arrows indicate the left-to-right orientation ofthe ment. The positions of pBR322:HinfI restriction fragments run as fragments relative to the HindIII VV DNA map shown in Fig. 1. markers are indicated. 3414 Genetics: Hruby et al. Proc. Natl. Acad. Sci. USA 80 (1983) Xhol HpaK HindN PvuI (Hind, I L)I Eco RI Hpal (Hind X J continued) ______0 0.5 0.57 10 1.14 I.5 Kb 5' (A)n 3' VV tk mRNA- FIG. 4. Position and direction of transcription of the VV tk gene in the HindllI-Xho I subfragment of HindII J DNA.

co _ 0) %6-- It to - C cr C cr c c _ _ . 4U..- c U 0 -, I. _ > co CU co .) CcU u ..-I -I

I '( - I VVV- LWV- I V maimmfimw I I I Wf T -.L-

4 -~~~~~~~~~~ PI

4- _ 4

FIG. 5. Nucleotide sequence analysis strategy. The indicated fragments were isolated; terminally labeled, and subjected to sequence analysis in the direction indicated by the arrows. The bold.line represents the location of the VV tk gene.

LAST SEQ NO = TTMACCAGGTCCCTATTGTTACAGATGGAAGGGTCAAACTTAATAAAGGATATTTGTTCGACTTTGTGATTAGTTTGATGCGATTCAAAAMAGMTCCTCTCTAGCTACCACCGCMATAG 10 20 30 40 50 60 70 80 90 100 110 120

M N G G H ATCCTGTTAGATACATAGATCCTCGTCGCAATATCGCATTTTCTMCGTGATGGATATATTAAAGTCGMTMAGTGMCAATMTTMTTCTTTATTGTCATCATGMCGGCGGACATA 130 140 150 160 170 180 190 200 210 220 230 240

A Q L I I G P M F S G K S T E L I R- R V R R Y Q I A Q Y K C V T I K Y S N D N R TTCAGTTGATAATCGGCCCCATGTTTTCAGGTAAAAGTACAGETTYTTAGACGAGTTAGACGTTATCAAATAGCTCAATATAAATGCCTGACTATI SRTATTCTCATATAAT 250 260 270 280 290 300 310 320 330 340 350 360

Y C T G L W T H D K N N F E A L E A T K L C D V L E S I T D F S V I G I D E G Q ACGGAACGGGACTATGGACGCATGATAAGAATAATTTTGAAGCATTGGAAGCAACTAAACTATGTG.ATGTCTTGTAATCAATTACAGATTTCTCCGTGATAGGTATCGATGAAGGACAGT 370 380 390 400 410 420 430 440 450 460 470 480

F F P D I V E F C E R M A N E G K I V I V A A L D G T F Q R K P F N N I L N, L I TCTTCCAGACATTGTTG^AATTCTGTGAGCGTATGGCAAACGAAGGAAAAATAGTTATAGTAGCCGCACTCGATGGGACATTTCAACGTAAACCGTTTAATATAmTTTTAA'CTTrATI'C 490 500 510 520 530 540 550 560 570 580 590 600

P L S E M V V K L T A V C M K C F K E A S F S K R L G E E T E I E I I G G N D M 610 620 630 640 650 660 670 680 690 700 710 720

Y Q S V C R K C Y I D S ATCAATCGGTGTGTAGAAAGTGTTACATCGACTCATAATATTGTATTTTTTATCTAGAACTAAATAACATTGAT 730 740 750 760 770 780 790 6 16 26 36 46

FIG. 6. Nucleotide sequence of the VV tk gene and its flanking regions. The predicted amino acid sequence of the encoded polypeptide is in- dicated by the single letter amino acid code. The EcoRI cleavage site is underlined. quences were analyzed in this manner, three RNAs were de- scripts and the mature 670-nucleotide tk mRNA must be in- tected with sizes of 2,400, 1,700, and 670 nucleotides (data not vestigated further. shown). This is in agreement with recent transcriptional map- Because the tk gene spanned the EcoRI cleavage site and ping studies carried out on the HindIII J DNA fragment by specified a single mRNA of 670 nucleotides that should be en- Bajszar et al. (17). The relationship between these larger tran- tirely encoded within the HindIII-Xho I fragment, this af- Genetics: Hruby et al. Proc. Nati. Acad. Sci. USA 80 (1983) 3415 forded the opportunity to utilize nuclease SI mapping proce- mRNA-specific hybridization probe with which to follow the dures to locate the 5' and 3' ends of the primary tk transcriptional biogenesis and fate of tk mRNA within the infected cell. Sec- unit. pDH-6 and pDH-7 plasmid DNAs were cleaved with EcoRI ond, tk is a selectable marker which can be reintroduced into and then terminally labeled at either the 5' or 3' ends. These the viral genome by marker rescue techniques. Thus, it should end-labeled fragments were thermally denatured, hybridized be possible to employ directed genetic procedures to modify to VV tk mRNA, and digested with nuclease Si, and then the the nucleotide sequence of the regions surrounding the tk gene, protected fragments were analyzed by electrophoresis in dena- to insert these tailored sequences back into infectious VV, and turing polyacrylamide gels. 5'-Labeled pDH-6 DNA and 3'-la- to assay the effects of these modifications on the in vivo expres- beled pDH-7 DNA were not protected by VV tk mRNA, whereas sion and regulation of the tk locus. In this manner the nature 5'-labeled pDH-7 and 3'-labeled pDH-6 gave rise to protected of the VV regulatory elements may be identified. fragments of 280 and 290 nucleotides, respectively (Fig. 3B). If uniformly labeled pDH-3 DNA was hybridized to VV tk We thank L. Guarino for her aid with the computer analysis of nu- mRNA and then nuclease SI-digested, a labeled 580-nucleotide cleotide sequences. We also thank D. Kelley for her skilled technical fragment was protected (data not shown). These results are assistance, the members of S. Tonegawa's laboratory for help with many summarized in Fig. 4: the VV tk gene is transcribed in a right- of the methods employed here, and H. Holacka for typing this manu- ward direction to yield a mRNA whose 5' end is encoded about script. This work was supported by University of Texas Research In- 0.57 kb to the right of the HindIII L-J boundary. The tran- stitute Grant BRSG 26-1694-1278, U.S. Public Health Service Grant scribed region is 570-580 base pairs in length, and the mature A118270, and a Research Career DevelopmentAward (AI00378) to L.A.B. polyadenylylated tk mRNA contains about 670 nucleotides. It 1. Moss, B. (1974) in Comprehensive Virology, eds. Fraenkel-Con- encodes a polypeptide of Mr 19,000 (4). These measurements rat, H. & Wagner, R. R. (Plenum, New York), Vol. 3, pp. 405- suggest that tk mRNA may only contain about 50 untranslated 474. nucleotides and have a polyadenylylate tail of about 100 resi- 2. Hruby, D. E. & Ball, L. A. (1981) Virology 113, 594-601. dues. This value is typical for the polyadenylylate tail sizes of 3. Hruby, D. E. & Ball, L. A. (1981)J. Virot 40, 456-464. early VV mRNAs in general (18). Like all VV genes that have 4. Hruby, D. E. & Ball, L. A. (1982)J. Virol 43, 403-409. 5. Belle Isle, H. S., Venkatesan, S. & Moss, B. (1981) Virology 112, been analyzed to date, the tk gene contains no intervening se- 306-317. quences. This property probably reflects the cytoplasmic lo- 6. Hershfield, V., Boyer, H. W, Yanofsky, C., Lovett, M. A. & Hel- cation of VV replication, because only viruses that transcribe inski, D. (1974) Proc. Natl Acad. Sci. USA 71, 3455-3461. in the nucleus have so far been found to contain intervening 7. Holmes, D. S. & Quigley, M. (1981) Anal Biochem. 114, 193-197. sequences. 8. Hardies, S. C., Patient, R. K., Klein, R. D., Ho, F., Reznikoff, Nucleotide Sequence of the W tk Gene. A number of ad- W. S. & Wells, R. D. (1979)J. Biol Chem. 254, 5527-5534. 9. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol 65, 499- ditional restriction enzyme cleavage sites were mapped in the 560. region of DNA known to encode VV tk. This information was 10. Yang, R. C. A. & Wu, R. (1979) Virology 92, 340-352. used to isolate a series of overlapping DNA fragments that were 11. Berk, A. J. & Sharp, P. (1978) Proc. Nati Acad. Sci. USA 75, 1274- terminally labeled and subjected to sequence analysis (Fig. 5). 1278. In this manner the nucleotide sequence shown in Fig. 6 was 12. Lehrach, D. H., Diamond, D., Wozney, J. M. & Boedtker, H. generated. A computer analysis of this sequence revealed an (1977) Biochemistry 16, 4743-4751. open 13. Southern, E. M. (1975) J. Mol Biol 98, 503-517. reading frame of 531 nucleotides that would encode a 14. Villarreal, L. (1981) Virology 113, 663-671. polypeptide of 177 amino acids with a Mr of 20,077. As has been 15. DeFilippes, F. M. (1982)J. Virol 43, 136-149. noted in sequence analysis of other vaccinia virus genes (19), 16. Weir, J. P., Bajszar, G. & Moss, B. (1982) Proc. Natl Acad. Sci. concensus signals present at the 5' and 3' ends of many other USA 79, 1210-1214. eukaryotic genes are conspicuously absent from this sequence. 17. Bajszar, G., Wittek, R., Weir, J. P. & Moss, B. (1983)J. ViroL 45, Also, no obvious homology to the previously sequenced herpes 62-72. tk gene was evident (20). 18. Nevins, J. R. & Joklik, W. K. (1975) Virology 63, 1-14. 19. Venkatesan, S., Gershowitz, A. & Moss, B. (1982)J. ViroL 44, 637- The data reported here concerning the structure and nu- 646. cleotide sequence of the VV tk gene should have two important 20. Wagner, M. J., Sharp, J. A. & Summers, W. C. (1981) Proc. Natl consequences. First, it should now be possible to prepare a tk Acad. Sci. USA 78, 1441-1445.