Complete Nucleotide Sequences of Two Adjacent Early Vaccinia Virus Genes Located Within the Inverted Terminal Repetition

0022-538X/82/110637-10$02.00/0

CopyrightC1982,American Society for Microbiology

Complete Nucleotide Sequences of Two Adjacent Early

Vaccinia

Virus

Genes

Located Within the Inverted Terminal

Repetition

SUNDARARAJANVENKATESAN,t ALANGERSHOWITZ, ANDBERNARDMOSS*

LaboratoryofBiology ofViruses,National InstituteofAllergyandInfectiousDiseases, National Institutes of

Health,Bethesda,

Maryland

Received28 May1982/Accepted9August 1982

Theproximal part ofthe 10,000-base pair (bp)inverted terminal repetition of vaccinia virus DNAencodesatleast threeearly mRNAs. A2,236-bpsegmentof the repetition was sequenced to characterize two of the genes. This task was

facilitated by constructing a series of recombinants containing overlapping deletions; oligonucleotidelinkers withsynthetic restriction sitesprovidedpoints forradioactive labelingbeforesequencing bythe chemicaldegradation method of Maxam and Gilbert (Methods Enzymol. 65:499-560, 1980). The ends of the transcripts were mapped by hybridizing labeled DNA fragments to early viral RNA and resolving nuclease Si-protected fragments in sequencing gels, by sequencing cDNA clones, and from the lengths of the RNAs. The nucleotide

sequencesforatleast 60 bpupstream ofbothtranscriptional initiation sites are more than 80% adenine * thymine rich and contain long runs of adenines and thymines with some homology to procaryotic and eucaryotic consensus se-quences. The gene transcribed in the rightward direction encodes an RNA of approximately 530 nucleotides with a single openreading frame of 420 nucleo-tides.PrecedingthefirstAUG,there isaheptanucleotidethatcanhybridizetothe 3' endof 18S rRNA with onlyonemismatch.Thederived amino acidsequenceof the proteinindicated amolecular weight of 15,500. The genetranscribed in the leftward direction encodesanRNA1,000to1,100nucleotides long withanopen

reading frame of 996nucleotidesandaleadersequenceofonly 5to6nucleotides. Thederivedamino acid sequenceof thisproteinindicated amolecularweightof 38,500. The 3' ends of the two transcripts were located within 100 bp of each other. Although there are adenine * thymine-rich clusters near the putative transcriptional termination sites, specific AATAAA polyadenylic acid signal sequences areabsent.

Poxviruses are large DNA viruses that

repli-catewithin thecytoplasm of infected cells. This extraordinary feat is accomplished partly by incorporating a complete viral transcriptional

system within the infectious particle (19, 23). Although apparentlynotspliced(12, 43, 45), the

mRNAs generated have typical eucaryotic

fea-tures including 5' terminal cap structures (39)

and 3' polyadenylic acid [poly(A)]tails (18, 24) enablingthem to usethe hosttranslational appa-ratus.Studies with vaccinia virus,the prototype

for thisgroup,revealed thathalfofthe genome

is transcribed early in infection (8, 14, 20, 26).

Since 100 or more early genes are scattered throughout thelength ofthe DNA (3, 10), they musthavecommonstructuralfeatures leadingto

tPresentaddress: Laboratoryof InfectiousDiseases,

Na-tional Institute of Allergy and Infectious Diseases, NaNa-tional Institutes ofHealth, Bethesda, MD 20205.

their selection by the viral multisubunit RNA polymerase (1,25, 32)orassociated factors. To identify such structural elements, we have

un-dertaken the sequencingof several earlygenes. Only recentlyhave technical advances made it possibletocharacterizeindividual vaccinia virus transcripts. Thus far, about a dozen early mRNAs mapping within and proximal to the invertedterminalrepetitionhave beenexamined (12, 43, 45). Theinverted terminal repetition of vaccinia virus is about 10,000 base pairs (bp) long (13, 42) and containsa novelflip-flop loop

at its distal end (2), followed by two sets of tandem 70-bp repeats (44) and then coding

re-gions for polypeptides of approximately 7,500 daltons(7.5K), 19K, and42K. The correspond-ing mRNAs are about 1,000, 600, and 1,050 nucleotides long, respectively, and are synthe-sized in vitrobydetergent-treatedvirusparticles (36)aswellasinvivo underconditions in which

637

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viral proteinorDNAsynthesis is prevented (11,

43).Furthermore,all three transcripts have mul-tiple 5' ends asjudged by the isolation ofcap structures containing adenine (A) and guanine (G)asthe penultimate nucleotide (36); the

reten-tionof the

P-phosphate

Limited nucleotide sequence analysis of the 7.5K polypeptide gene revealed an extremely adenine * thymine (A * T)-rich cluster of nucleo-tides immediatleyupstreamof thecapsite anda

tandemly repeated hexanucleotide near the 3' end (35). In the present communication, we

provide the sequence fora2,236-bp segmentof theinverted terminal repetitionthatincludes the entire 19K and 42Kpolypeptidegenes aswellas

locations of putative transcriptional initiation andtermination sites.

MATERIALS AND METHODS

Construction of recombinantplasmids. Recombinant pVG3, a pBR322derivativecontaininga3.4-kilobase pair (kbp)SaIl-EcoRIfragmentfrom theleft end of the vaccinia virus genome,wasderived fromapreviously described recombinant, pAG1 (36). A setof in vitro deletionmutants wasconstructedbycleaving pVG3at

theuniqueKpnIsite (seeFig.1). Afterphenol

extrac-tion and ethanol precipitation, 10 ,ug of linearized plasmid was digested with 1.25 U of Bal 31 (15). Samples were removedat 1, 2,and 3 min, atwhich time thereactions werestopped byheatingat65°Cin the presenceof 0.25% sodiumdodecyl sulfate-0.012M ethyleneglycol-bis(3-aminoethyl ether)-N,N-tetraace-tic acid(EGTA)-0.02M EDTA. After phenol-chloro-formextractionand ethanol precipitation, theextent

ofexonucleolytic cleavagewasmonitoredbyagarose gelelectrophoresis. Frayedends remainingafter this stepwerefilled in withreversetranscriptase (2.5Uper FgofDNA) and 0.25 mM deoxynucleoside triphos-phate(dNTP)(each type)at37°Cfor 30 min. HindIII oligonucleotide linkers were labeled attheir 5' ends with[,y-32P]ATPbyanexchange reactioncatalyzed by polynucleotidekinase(5).Linkers,at amolar ratioof 100:1,wereligatedtotheplasmidDNAat4°Cfor4h, and theligatedmixwasrecoveredbyethanol precipi-tation after phenol extraction. The DNA was then digested with HindIII, phenol-chloroform extracted, and renderedfreeof linkersby gelfiltrationthrougha

Sepharose 4B column. The plasmid DNA was then incubatedat aconcentration of 10,ug/mlwith 7.5 Uof T4 DNAligaseat4°Cfor4h.Approximately0.2 ,ug of DNA was used to transform competent Escherichia coli K-12 HB101cells, andcolonieswere selectedby growthonampicillin plates (7). The number of

trans-formants obtained varied from5,000 for limited Bal31

digestions to 500forextensive digestions.

Recombi-nantplasmidswerepurifiedbyasmall-scale isolation procedure (6) and screenedbyrestriction

endonucle-asedigestions and agarose gelelectrophoresis.Atleast 75% of the tested recombinants hadaHindIIIsite,and themajorityof them had deletions that varied from100 to1,300bp. Fiverecombinants withoverlapping dele-tions(Dl,D3, D4, D5, andD6)wereselected forDNA

sequencing.

End labelingDNA.Restriction fragments were treat-ed with alkaline phosphatase and labeltreat-ed at the 5' end with [y-32P]ATP and T4 polynucleotide kinase (22). Avian myeloblastosis reverse transcriptase or the Klenowfragment of DNA polymerase was used to add

asingle complementary [a-32P]dNTPtothe recessed 3'end of restriction fragments (47). When the 3' end was protruding, labeling was accomplished with

[a-32P]cordycepin triphosphate and terminal deoxynu-cleotidyltransferase (34).

DNA sequence analysis.End-labeled DNAfragments werecleaved with appropriate restriction endonucle-ases, purified by agarose gel electrophoresis, and recoveredwith glass powder (37) or by electrophoresis

ontoDEAE paper (41).The limitedchemical degrada-tion procedureof Maxam and Gilbert (22) was used for sequencing. Reaction products were resolved on 0.4-mm-thick gels with either 4, 6, 8, or20%o polyacryl-amide (30 by 160 or 40 by 80 cm) in 8 M urea (31).

cDNA sequencing. Alibrary of cDNA recombinants was generously provided by B. Roberts (Harvard UniversitySchool ofMedicine).cDNA wasprepared using early RNA from cells infected with vaccinia virus (strain WR). Oligodeoxythymidylic acid [oli-go(dT)] served as a primer; hairpin 5' ends were removed with nuclease S1, and the double-stranded cDNAs were cloned in pBR322 by deoxycytidylate and deoxyguanylate tailing (B. Roberts, personal com-munication). We screened thelibrary by colony hy-bridization (33) witha3-kbpBamHI-EcoRlfragment (seeFig. 1) labeled with32Pby nick translation (29). A total of 35 colonies were selected for further restriction endonuclease analyses, and 8 were sequenced after labeling the 3' ends with [a-32P]cordycepin triphos-phate(34).

Mapping 5' and 3' ends by nuclease Si protection. poly(A)-containing RNA was purifiedfrom the cyto-plasmof cells 4 h after infection withvacciniavirus (15 PFU percell)in the presence of cycloheximide or was made in vitro by vaccinia virus particles (36). The RNAwashybridized to DNAfragments containing a labelatthe 5'or3' end on thecoding strand (35). After nuclease S1 digestion(35),hybrids were analyzed by electrophoresisonsequencing polyacrylamide gels.

Materials. Restriction endonucleases were pur-chased from NewEnglandBiolabs (Beverly, Mass.)or

BethesdaResearch Laboratories (Gaithersburg, Md.). Nucleases Bal 31 and S1 were supplied by New England Biolabs and Miles Laboratories, Inc., Elk-hart, Ind.,respectively. Bothterminal deoxynucleoti-dyltransferase and T4 polynucleotide kinase came from P-L Biochemicals, Milwaukee, Wis., whereas DNApolymerase holoenzyme and the Klenow

frag-ment camefrom BoehringerMannheim Corp. India-napolis, Ind. T4 DNA ligase was purchased from Bethesda Research Laboratories, and avian myelo-blastosis virus reversetranscriptase wassupplied by J. W. Beard of Life Sciences, Coral Gables, Fla. Oligodeoxynucleotideswereobtained from Collabora-tive Research, Inc., Waltham, Mass., [a-32P]dNTP

and[y-32P]ATPfrom AmershamSearle,Chicago, Ill., and [a-32P]cordycepin triphosphate from New En-glandNuclearCorp., Boston, Mass.

RESULTS

Sequencing strategy. Three mRNAsencoding polypeptides ofapproximately 7.5K, 19K, and

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VACCINIA VIRUS GENES

42K were mapped within the inverted terminal repetitionof the vaccinia virus genome(11, 42, 43) (Fig. 1). A strategy, avoiding either

exten-sive restriction endonuclease mapping or shot-gun methods, was used to determine the geno-mic sequence of the 19K and42Kpolypeptides. This deletion linkerapproach requiredthe clon-ing in pBR322 ofthe 3.4-kbp SalI-EcoRI frag-mentlocated 5.6to9.0kbpfrom the end of the viral DNA(Fig. 1). Therecombinantis referred to aspVG3. AuniqueKpnI site, located within the viral DNA segment, was cleaved, and the endsofthelinearizedplasmidweresubjectedto

exonucleasedigestionwith Bal 31(15).After the addition of synthetic oligonucleotide linkers containing a HindIII site, the plasmids were

recircularized and used to transform E. coli. Recombinantplasmidswerescreened, anda set

withoverlappingdeletions was selected(Fig.1).

A

TR

Sequencing of each recombinantwasfacilitated by use of the mobile HindlIl site for 5' and 3' labeling. Additional sequencing was carriedout

using therestriction sites andconventional strat-egyindicated at the bottom ofFig. 1.

A sequence of2,236 nucleotides encompass-ing genes for the 19K and 42K polypeptides is shown inFig. 2. As will be discussedlater,both genes have long open translational reading frames ofoppositepolarity.

Location of the 5' ends of transcripts. The mRNAs for the 19K and 42K polypeptides are transcribed fromopposite strands of the invert-edterminal repetition(Fig. 1). Approximate map positions of these mRNAs were determined by use of restriction fragments for hybridization selection and cell-free translation and for prob-ing blots of electrophoretically separated RNAs. To more precisely locatetheir 5' ends, a

modifi-CONSTRUCTION OF RECOMBINANT

vtASMIDS

TR

7.5K 19K 42K

,----go

1 2 3 4 5 6 7 8 91

Dl D4 D3 D5 D6

6.5 7.0 7.5 8.0 8.5 9.0

SEQUENCING STRATEGY

B 19K 42K

6.5 7.0 7.5 8.0 8.5 9.0

1T1~

t

it

T

t

T

Y

T

tt

ao

-_~

9 - m-Dl

D6 Dl - o- -D4

- 0 o D4 v

D5D3 ° _ a _D3

5 co

FIG. 1. Construction of recombinants and sequencing strategy. (A) Part of the 10-kbp inverted terminal repetitionof vaccinia virus is shown. The dark blocks labeled TR represent the two sets of70-bptandemrepeats. Arrows represent theapproximate lengths, map positions, and directions of transcriptionof three early mRNAs encoding polypeptides of approximately 7.5K, 19K, and 42K. TheBamHI-EcoRI segment of therecombinant plasmid pVG3 is expanded. Additional recombinants were constructed from pVG3 using Bal 31 to make deletionsattheKpnIsite. The extent of the deletions in recombinantsDl,D4,D3, D5, and D6 is shownby gaps in theheavy bar. (B) The BamHI-EcoRI segment of the inverted terminal repetition with the mRNAs for the 19K and 42Kpolypeptides is shown. Symbols for relevant restriction endonuclease sitesare:AvaIl,9;BamHI, l; EcoRI,4;HincII,6;Hinfl, I; HpaII,4; KpnI,*;TaqI,T;XbaI, .Filled and unfilled circles represent sites of 3' and 5'labeling, respectively. Solid arrows indicate the extent of sequence determination; interrupted parts of thearrowindicateregions where the sequence was not determined. ThedesignationsDltoD6refertotheuseof deletionrecombinants; otherwise, pVG3 or a related recombinant pVG1 wasused.

VOL.44,1982

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J.VIROL.

10 20 30 40 50 60 70 80 90 100

TTTTTAACAG CAAACACATT CAATATTOTA TTGTTATTTT TATGTATTAT TTACACAATT AACAATATAT TATTAGTTTA TATTACTGAA TTAATAATAT AAAAATTGTC GTTTGTGTAA GTTATAACAT AACAATAAAA ATACATAATA AATGTGTTAA TTGTTATATA ATAATCAAAT ATAATGACTT AATTATTATA

'S 120 130 140 150 160 ...L 170 180 1 90 20 0

AAAATTCCCA ATCTTGTCAT AAACACACAC TGAGAAACAG CATAAACACA AAATCCATCA AAAATGTCGA TGAAATATCT GATGTTGTTG TTCGCTGCTA

TTTTAAGGGT TAGAACAGTA TTTGTGTGTG ACTCTTTGTC GTATTTGTGT TTTAGGTAGT TTTTACAGCT ACTTTATAGA CTACAACAAC AAGCGACGAT

210 220 250 240 250 260 270 280 290 300

TGATAATCAG ATCATTCGCC GATAGTGGTA ACGCTATCGA AACGACATCG CCAGAAATTA CAAACGCTAC AACAGATATT CCAGCTATCA GATTATGCGG ACTATTAGTC TAGTAAGCGG CTATCACCAT TGCGATAGCT TTGCTGTAGC GGTCTTTAAT GTTTGCGATG TTGTCTATAA GGTCGATAGT CTAATACGCC

510 320 330 340 350 360 370 380 390 400

TCCAGAGGGA GATGGATATT GT.TTACACGG TGACTGTATC CACGCTAGAG ATATTGACOG TATGTATTGT AGATGCTCTC ATGGTTATAC AGGCATTAGA AGGTCTCCCT CTACCTATAA CAAATGTGCC ACTGACATAG GTGCGATCTC TATAACTGCC ATACATAACA TCTACGAGAG TACCAATATG TCCGTAATCT

410 420 430 440 450 460 470 480 490 '500

TGTCAGCATG TAGTATTAGT AGACTATCAA CGTTCAGAAA ACCCAAACAC TACAACGTCA TATATCCCAT CTCCCGGTAT TATGCTTGTA TTAGTAGGCA

ACAGTCGTAC ATCATAATCA TCTGATAGTT GCAAGTCTTT TGGGTTTGTG ATGTTGCAGT ATATAGGGTA GAGGGCCATA ATACGAACAT AATCATCCGT

510 520 530 540 550 560 570 580 TL 590 600

TTATTATTAT TACGTGTTGT CTATTATCTO TTTATAGOTT CACTCOACGA ACTAAACTAC CTATACAAGA TATGGTTGTG CCATAATTTT TATAAATTTT AATAATAATA ATGCACAACA GATAATAGAC AAATATCCAA GTGAGCTGCT TGATTTGATG GATATGTTCT ATACCAACAC GGTATTAAAA ATATTTAAAA

610 620 630 13 650 660 6'70 680 690 700

TTTATGAGTA TTTTTACAAA AAAAATGTAT AAAGTOTATO TCTTATGTAT ATTTATAAAA ATGCTAAGTA TGCGATGTAT CTATGTTATT TGTATTTATC AAATACTCAT AAAAATGTTT TTTTTACATA TTTCACATAC AGAATACATA TAAATATTTT TACGATTCAT ACGCTACATA GATACAATAA ACATAAATAG

710 720 730 740 T1. 750 760 770 780 790 800

TAAACAATAC CTCTACCTCT AGATATTATA CAAAAATTTT TTATTTCGGC ATATTAAAGT AAAATCTAGT TACCTTGAAA ATGAATACAG TGGGTGGTTC

ATTTGTTATG GAGATGGAGA TCTATAATAT GTTTTTAAAA AATAAAGCCG TATAATTTCA TTTTAGATCA ATGGAACTTT TACTTATGTC ACCCACCAAG

810 820 830 840 850 860 870 880 890 900

CGTATCACCA GTAAGAACATAATAGTCGAA TACAGTATCC OATTGAGATT TTGCATACAA TACTAGTCTA OAAAGAAATT TGTAATCATC TTCTGTGACG GCATAGTGGT CATTCTTGTA TTATCAGCTT ATGTCATAGG CTAACTCTAA AACGTATGTT ATGATCAGAT CTTTCTTTAA ACATTAGTAG AAGACACTGC

910 920 930 940 950 960 970 980 990 1000

GGAGTCCATA TATCTGTATC ATCGTCTAGT TTATCAGTGT CCCATGCTAT ATTCCTGTTA TCATCATTAG TTAATGAAAA TAACTCTCGT GCTTCAGAAA

CCTCAGGTAT ATAGACATAG TAGCAGATCA AATAGTCACA GOGTACOATA TAAGGACAAT AGTAGTAATC AATTACTTTT ATTGAGAGCA CGAAGTCTTT

1010 1020 1030 1040 1050 1060 1070 1080 1090 1100

AGTCAAATAT TGTATCCATA CATACATCTC CAAAACTATC GCTTATACGT TTATCTTTAA CGATACCTAT ACCTAGATGG TTATTTACTA ACAGACATTT

TCAGTTTATA ACATAGGTAT GTATGTAGAG GTTTTGATAG CGAATATGCA AATAGAAATT GCTATGGATA TGGATCTACC AATAAATGAT TGTCTGTAAA

1110 1120 1130 1140 1150 1160 1170 1180 1190 1200

TCCAGATCTA TTGACTATAA CTCCTATAGT TTCCACATCA ACCAAGTAAT GATCATCTAT TGTTATATAA CAATAACATA ACTCTTTTCC ATTTTTATCA AGGTCTAGAT AACTGATATT GAGGATATCA AAGGTGTAGT TGGTTCATTA CTAGTAGATA ACAATATATT GTTATTGTAT TGAGAAAAGG TAAAAATAGT

1210 1220 1230 1240 1250 1260 1270 1280 1290 1300

GTATGTATAT CTATATCAAC OTCGTCGTTG TAGTGAATAG TAGTCATTOA TCTATTATAT GAAACGGATA TGTCTAGAAC GGCAATTGTT TTACGTCCAG CATACATATA GATATAGTTG CAGCAGCAAC ATCACTTATC ATCAGTAACT AGATAATATA CTTTGCCTAT ACAGATCTTG CCGTTAACAA AATGCAGGTC

1310 1320 1330 1340 1350 1360 1370 1380 1390 1400

TTAACACTTT CTTTGATTTA AAGTCTAGAG TCTTTGCAAA CATAATATCC TTATCCGACT TTATATTTCC TGTAGGGTGG TATAATTTTA TTTTGCCTCC

AATTGTGAAA GAAACTAAAT TTCAGATCTC AGAAACGTTT GTATTATAGG AATAGGCTGA AATATAAAGG ACATCCCACC ATATTAAAAT AAAACGGAGG

1410 1420 1430 1440 1450 1460 1470 1480 1490 0500

ACATATCGGT GTTTCCAAAT ATATTACTAG ACAATATTCC ATATAGTTAT TAGTTAAGGG TACCCAATTA GAACACGTAC GCTTATTATC ATCATTTGGA TGTATAGCCA CAAAGGTTTA TATAATGATC TGTTATAAGG TATATCAATA ATCAATTCCC ATGGGTTAAT CTTGTGCATG CGAATAATAG TAGTAAACCT

1510 1520 1530 1540 1550 1560 1570 1580 1590 1600

TCGTATTTCA TAAAAGTTAT TGTACTATCG ATGTCAACAC ATTCTACATT TTTTAATCGT CTATATAGTA TTTTTCTGAT ATTTTCTATA ATATCAGAAT

AGCATAAAGT ATTTTCAATA ACATGATAGC TACAGTTGTG TAAGATGTAA AAAATTAGCA GATATATCAT AAAAAGACTA TAAAAGATAT TATAGTCTTA

1610 1620 1630 1640 1650 1660 1670 1680 1690 1700

TGTCTTCCAT-CGGAAGTTGT ATACTATCGG AATCAGTTAC ATGTTTAAAT AATTCTCTGA TGTCATTCCT TATACAATCA AATTCATTAT TAAACAGTTT ACAGAAGGTA GCCTTCAACA TATGATAGCC TTAGTCAATG TACAAATTTA TTAAGAGACT ACAGTAAGGA ATATGTTAGT TTAAGTAATA ATTTGTCAAA

1710 1720 1730 -1 1750 1760 1770 1780 1790 1800

AATAGTCTGT AGACCTTTAT CGTCGTAAAT ATCCATTGTC TTATTAGTTA CGCTTATTTT TATGTGTTTT ACGTTGCTTT ATTATATTTT ATAAGAATGA

TTATCAGACA TCTGGAAATA GCAGCATTTA TAGGTAACAG AATAATCAAT GCGAATAAAA ATACACAAAA TGCAACGAAA TAATATAAAA TATTCTTACT

1810 1820 1830 1840 1850 1860 1870 1880 1890 1900

TTGTTTGACG AATCACGAGA ACTATTAAGACACATTATTA GGTATATATT ATAAAAAAGT TTTTGATTAC GATGTTATAA GAGGAAAGAG GACACATTAA AACAAACTGC TTAGTGCTCT TGATAATTCT GTGTAATAAT CCATATATAA TATTTTTTCA AAAACTAATG CTACAATATT CTCCTTTCTC CTGTGTAATT

1910 1920 1930 1940 1950 1960 1970 1980 1990 2000

CATCATACAT CAATTAACTA CATTCTTATA ACATCGTAAT CAAAAGAATT GCAATTTTGA TGTATAACAA CTGTCAATGG GTTATGAAAT TGTATATTAC

GTAGTATGTA GTTAATTGAT GTAAGAATAT TGTAGCATT4GTTTTCTTAA CGTTAAAACT ACATATTGTT GACAGTTACC CAATACTTTA ACATATAATG

2010 2020 2030 2040 2050 2060 2070 2080 2090 2100

ATATTATACG GTATGTTGGT AACGACAAAT ACCGATCGGT AATTGTCTGC CGGTGTACGA GAATTATATA TATCTATCTA TTACACCGGC TGAGTATGCA

TATAATATGC CATACAACCA TTGCTGTTTATGGCTAGCCA TTAACAGACG GCCACATGCT CTTAATATAT ATAGATAGAT AATGTGGCCG ACTCATACGT

2110 2120 2130 2140 2150 2160 2170 2180 2190 2200

TAATAATAAG TTGTGGTAGT ATGATCTCCA TATTTATAAT TTAGGACTTT GTATTCAGTA TTTTTGGAAT CATAAAAAAT AAAAAAAAGT TTTACTAATT ATTATTATTC AACACCATCA TACTAGAGGT ATAAATATTA AATCCTGAAA CATAAGTCAT AAAAACCTTA GTATTTTTTA TTTTTTTTCA AAATGATTAA

2210 2220 2230 TAAAATTTAA AAAGTATTTA CATTTTTTTC ACTGTT ATTTTAAATT TTTCATAAAT GTAAAAAAAG TGACAA

FIG. 2. Nucleotide sequence ofa2,236-bpsegment of DNA encompassingthegenesfor the 19K and 42K

polypeptides.Thenucleotide numbered 1 is approximately6,800nucleotides from the end of the genome, and nucleotide 2,236 is afew nucleotides before the first EcoRI site. The upper and lower lines represent the

noncodingstrandsfor mRNAsexpressingthe 19K and 42Kpolypeptides,respectively. Theputative

transcrip-tionalinitiation sites for thetwomRNAsareindicatedbyan arrowwith 5'overit. The 3'end(s)of the mRNA for the 19K polypeptide is shown by an arrow with 3' written over it. The 3' end of the mRNA for the 19K

polypeptideidentifiedbycDNAsequencingis shownby!.Iand Trefertothepresumptivetranslational initiation andterminationcodons,respectively, of the twomRNAs.

cationintroducedbyWeaver andWeissman(38) beled strand of this fragment was expected to of the nucleaseSi procedureof Berk andSharp containsequencescomplementary tothe 5' end

(4) was used. A HincII-AvaII fragment (6.6 to of the 19KpolypeptidemRNA. After hybridiza-7.1 kbpfrom the end of thegenome)with 5' 32P tion to early RNA, made in infected cellsor in label at the AvaII site was employed. The la- vitro by detergent-treated virus particles,

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maining single-strandedDNA wasdigested with

nuclease SI. The radioactively' labeled DNA segment that wasprotected from nuclease

diges-tion represented the distance from the RNA 5'

terminus to the 5' end of the DNAprobe. The

size ofthis segment was determined by

poly-acrylamide gelelectrophoresis under denaturing conditions. As shown in Fig. 3, at least six

closely spaced nuclease-resistant bands were resolved. By coelectrophoresis with the DNA sequence reaction products of the original

HinclI-Avall fragment, the nuclease-resistant bands werelinedup with a sequence ladder. As

pointed outpreviously (16), a 1.5-bp downward

displacementof the nuclease S1 bands is neces-sary for proper alignment. This placed the 5'

ends of the RNA complementary to the se-quence AGATT in Fig. 3. Since the

nuclease-protected bands were spaced one nucleotide

apart, the heterogeneity could result from a

1 2 3CTAG

:x0

A^ aw

ar

S. ,

U?E,Fa

.

0.

_a

i-.p

ZS

_,.sv,P G>hv,t,l

-1G-GIM

_rNG-T

FIG. 3. Map locations of the 5' end(s) of mRNA encoding the 19K polypeptide. Approximately 0.5 pmol of the AvaII-HincII fragment (7.0 to 6.64 kbp fromthe endof theDNA) 5' labeledatthe AvaIl site

wasdenatured and incubated under conditions

favor-ing RNA-DNA hybridization with 5 to 10 pmol of poly(A) selected early in vivo RNAorin vitroRNA. Afterhybridization, single-stranded DNAwas

digest-ed with nuclease S1 (750 U/ml). Resistant products after incubation with (1) in vivo RNA, (2) in vitro RNA, or(3)noRNAandsequencereaction products (C [cytosine]; Tis Tplus C; A is A plusG; G)were

resolved on an8%polyacrylamide gel (30by160cm) in8 Murea.

AJAA

;CA

rc04A

A

CTAG 1 2

.. _ w4

&-S

_ ,.2,d

@00

*a 1.

e~dP

4w

.A_e

S.

_p

a'#

sip

33 OTAGI 2

U, U'

..

'-r

!

S

"SE te

hA.v-'!

d- .1 -...!.ir Ut

A,%,cc-1'

FIG. 4. Map locations of the 5' ends of mRNA

encoding the 42K polypeptide. Approximately 0.5

pmol of(A) anXbaI-EcoRI fragment(8.1 to 9.0 kbp

from the end of the DNA) and (B) a TaqI-EcoRI fragment(8.3 to 9.0 kbp from the end of the DNA)5' labeledat theXbaIand TaqI sites, respectively, was

hybridizedtoapproximately 25 pmol of poly(A)

select-ed early cytoplasmic RNA. Nuclease Si-resistant products obtained with(1)in vivoRNA, (2) no RNA, or(3) in vitro RNA andsequence reactionproducts (C; Tis T plus C;AisAplus G; G) were resolved on an

8%polyacrylamide gel(30by160 cm)in 8 M urea.

tendency of nuclease Si to leave overhanging

ends or to nibble into a base-paired region.

Analysis ofthe capped 5' ends of this message

indicated that 80% ended in A and 20% in G, suggesting thatthe major endsare

complemen-tary to one orboth of the adjacent deoxythymi-dylate residues (positions110and111inFig. 2.). Themapposition of the 5' endwasconfirmed by usingas aprobe theHincII-HpaII fragment(6.6 to7.2 kbp from the end of the DNA) labeledat

the5' endatthe HpaII site(notshown). The 5' endsof themessageencodingthe42K

polypeptideweremappedin ananalogous man-ner. Two DNA fragments, XbaI-EcoRI (8.1 to

9.0 kbp from the end of the DNA) and

TaqI-EcoRI (8.3 to 9.0 kbp from the end of the DNA),

labeled attheXbaI and TaqI site, respectively,

were employed. The major nuclease-resistant bands lined up within a sequence TCTT(Fig.4).

Previous cap analysis indicated approximately equalamounts of G and A ends suggestingthat

they are complementary to the CT residues of the above sequence (numbered 1,740 and 1,741

in Fig. 2).

Location of the3'ends oftranscripts. Previous studies indicated that the mRNAs for the 19K

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A B

G AC C;' A -T C

A.e

-~,

I~~~~~~~~I

4

FIG. 5. Map locations of the 3' ends of mRNA

encoding the 19K polypeptide by cDNA sequencing

and nuclease Si protection. (A)A recombinant

plas-mid (pVBR12) was selected from a vaccinia cDNA

library by its ability to hybridize specifically to a

vaccinia DNAfragment encodingmRNAfor the 19K

polypeptide. The recombinant DNA was cleaved at

Axv i t [ .2pt

the Pstl site and 3' labeled with

kx3Pcordycepin

triphosphate and terminal

deoxynucleotidyltransfer-ase. After cleavage with Hpall, the smaller of the labeled DNA fragments was purified, sequenced by

the chemical degradation method, and analyzed by electrophoresis on 8% polyacrylamide gel. The

se-quenceof thenoncoding (i.e., RNA)strandis indicat-ed. The 15 adenylate residues representthe proximal portionof thepoly(A)tail of the mRNA.(B) Approxi-mately0.5pmolofanHpall-Hinflfragment(7.2to7.6

kbpfrom the end of theDNA)labeledatthe 3' endwas

hybridizedto 10pmol ofpoly(A) selectedearly

cyto-plasmicRNA in80% formamideat380C.Nuclease

Si-resistant products obtained with (1) or without (2) RNAand sequencereactionproducts (G;Ais Aplus

G;Tis TplusC;C)wereresolvedon an8%

polyacryl-amide gel (30 by 160 cm) in 8 M urea. The coding

strand DNA sequence isindicated.

and 42Kpolypeptidesareapproximately580 and 1,050 nucleotides long, respectively (36, 43). Using the map locations determined above for the 5' ends, this wouldplace the3' ends of the oppositely oriented 19K and 42K polypeptide messagesclosetoeach othernearpositions690

and 740, respectively, of the genomic sequence in Fig. 2.

Attempts were made to map the 3' ends of

bothmRNAs moreprecisely.The first approach involved a procedure analogous to that used for

mapping5'termini.AHpaII-Hinfl fragment(7.2 to7.6 kbp from the end of the DNA) labeled at the 3' end at the HpaII site was used as a probe for the 19K polypeptide message. After hybrid-ization to early RNA, the nuclease-protected DNA segmentrepresented the distance fromthe 3' end of the RNA to the labeled restriction site.

The size of this segment was determined by

polyacrylamide gel electrophoresis, again using a sequence ladder prepared from the original hybridization probefor comparison. Two major and several minor bands were detected (Fig.

SB). After making the appropriate correction, they appeared to line up with the complemen-tary AA residues of the sequence GAAT. This

corresponds to the two thymine (T) residues

located at positions 643 and 644 of Fig. 2 and

places the 3'end within 50 bpof the site

predict-ed from the length ofthe RNA using northern

blotanalysis. This slightdeviation from its orig-inally reported size might reflect the length of

thepoly(A)tail.

Similarattempts to mapthe 3' end of the42K

polypeptide mRNA were unsatisfactory, possi-bly because of the low abundance of the mes-sage, the existence oftranscripts with overlap-ping 3' ends that might compete for

hybridization, andpossible heterogeneity ofthe 3' ends (36,43).

An alternative procedure was investigated to

confirm the mappositions of the 3' endsofthe

19K and 42K polypeptide mRNAs. For this

approach we needed recombinants containing cDNAs prepared using anoligo(dT) primer hy-bridized to the poly(A) tails of early vaccinia

virus mRNAs. By sequencing the recombinant cDNAs we hoped to identify the nucleotides

adjacenttotheoligo(dT) primeror

complemen-tary oligodeoxyadenylate [oligo(dA)]. Such a

cDNA recombinant library, generously provid-edbyB.Roberts(Harvard Medical School),was screened by colony hybridization. Selected re-combinants were further characterized by re-striction endonuclease analyses, andeightwere

sequenced fromtheir PstI sites. The sequenceof

one is shown in Fig. SA. After a cluster of

deoxycytidylate residues, derived from the

deoxyguanylate and deoxycytidylate tailing

usedforconstruction oftherecombinants,there were 15 deoxyadenylate (dA)residues andthen a sequence corresponding to the genome near

the 3' endofthe19Kpolypeptide. The deoxyth-ymidylateresidueadjacenttotheoligo(dA)isat

position 639 ofFig. 2, four nucleotides down-streamfromthe 3' end deducedby nuclease S1

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SEQUENCES OF EARLY VACCINIA VIRUS GENES protection experiments. Further comparison of

the cDNAsequence with the genomicsequence

(position 678 to 638, Fig. 2) revealed twominor

discrepancies. There were seven dAs incDNA

in contrast toeightdAsin genomic DNA

(posi-tion 618, Fig. 2) and six dAs in cDNA

corre-spondingto seven dAs in the genome (position

603, Fig. 2). These differences might represent

slippage during reverse transcription or minor

variations inthe vaccinia virus WR isolatesused

for genome sequencing and cDNA cloning. Since these differencesoccurin the

nontranslat-edregion of thegene, theremightbe no

biologi-calconsequencesof such variations.

Unfortunately, the otherseven cDNA

recom-binants from thisregion of the genome as wellas mostfromasecondregion lackedtheoligo(dA)

sequence and therefore could not be used to

identify the precise 3'end ofthe message.

DISCUSSION

The majority ofDNAviruses engage cellular RNApolymerases forgene transcription within

the nucleus ofthe infected cell. Thus it is not

surprisingthattheirregulatory signalsresemble

those ofthe host (9). By contrast, poxviruses are

cytoplasmicandpossess aunique transcription-al systemmaking it likely that distinctive DNA sequencesinteract withthe viral RNA

polymer-ase. To elucidate structural features related to

the organization and expression ofthe vaccinia virus genome, several regions have been

se-quenced. These include the terminal loops that link thetwo DNAstrands together andadjacent

DNA (2) and portions of the most distal gene

coding for a 7.5Kpolypeptide (35). To these is

nowaddeda2,236-bpsegmentthat encodestwo

early polypeptides. Sequencingwasachievedby thechemical degradationmethodofMaxamand

Gilbert (22) using a strategy that involved the

preparation of a series of recombinants with

overlapping deletions and inserted restriction site linkers. This deletion linker approach

elimi-nated the need for extensive restriction site

mapping or shotgun sequencing.

A procedure of Weaver and Weissman (38)

was used to map the 5' ends of both mRNAs. After hybridization ofa DNAfragment labeled

atthe 5' endtoRNA, thenuclease S1-protected

segment and the nucleotide sequence reaction

productsof the originalfragmentwerecompared by electrophoresis on the same pol7yacrylamide gel. These data andourfinding ofm G(5')pppAm

and m7G(5')pppGm caps on both the 19K and

42Kpolypeptide mRNAs(36)placedtwomajor

5' ends at or near nucleotides 110 and 111 and

1,740 and1,741ofFig. 2, respectively.A

previ-ousanalysis of the5' ends of the 7.5K polypep-tide mRNA alsosuggestedthepresence of

multi-ple closely spacedpurine ends (35).

Since the cap structuresof both the 19Kand 42K mRNAs retain the ,B-phosphate of GTP

(36), the5'endsof their messagescorrespondto

sites oftranscriptional initiation. Accordingly, theadjacent DNA maycontainpromoter

recog-nition sequences. In Fig. 6, regionsupstreamof the 5' ends of the mRNAs coding for polypep-tides estimated to be 7.5K, 19K, and 42K are

shown. Foreach, the 60bppreceding the start

siteare atleast80%A * Trich with manyrunsof

A's and T's including some 14 to18nucleotides

long. Embeddedwithin the A * T-richregionare

possible equivalents of the Pribnow and Hog-ness-Goldberg boxes ofprocaryotesand eucary-otes,respectively (9, 30).Nevertheless,the sim-ilarity to the eucaryoticTATA sequence is not exact.Interestingly, thesequenceCGTAAAA is found starting at -28 and -29 ofthe 7.5K and

42Kpolypeptidegenes,respectively (Fig. 6). In

addition, each of the genes contains other ho-mologousA *T-rich clustersincludingCAAT40 to60 bpupstream ofthe capsites.

-100 -90 -80 -70 -60 -50 -40 -30 -20 -10 0

( !) AACTGATCACTAATTCCAAACC CAC C CGCTTTTTATAGTAAGTTTTTCACCCATAAATAATAAATACAATAATTAATTTCTCGTAAAMGTAGAAAATATATTCTAATTTA

... ... . ... ... . ... ... ...

(II) TTTTTAACAGCAAACACATTCAATATTGTATTGTTATTTTTATGTATTATTTACACAATTAACAATATATTATTAGTTTATATTACTGAATTAATAATATAAAATTCCCA

... ... ... ...

(111) TATAATATATACCTAATAATGTGTCTTAATAGTTCTCGTGATTCGTCAAACAATCATTCTTATAAAATATAATAAAGCAACGTAAAACACATAAAAATAAGCGTAACTAA

(I)~~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~... .. ... .. .

(IV)

(V) GGCCAATCT_T

TTGACA TATAATG

TATAAAA_{T T}

FIG. 6. Sequences upstream of three early transcriptional sites of vaccinia virus. I, II, and III refer

respectively to the upstream sequences of mRNAs encoding 7.5K, 19K, and 42K polypeptides. The most

proximal initiation site is indicated by 0. Lines IV and V represent the near upstream and far upstream sequences in procaryotes and eucaryotes, respectively.

VOL.44, 1982

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Examination ofmore geneswillbe necessary

to know whether to designate any of the

se-quences referred to above as specific "pox-boxes."Toassessthefunctional significance of

DNA structures, genetic experiments are re-quired. With this in mind, we have recently

mapped theselectablethymidinekinasegeneof

vacciniavirus (40). Ourplansareto mutatethe putativepromoter ofthe thymidinekinasegene withinplasmidorphage recombinants and then

to introduce the mutated DNA into infectious virus by transfection procedures. In this

man-ner, structure/function relationships can be ex-plored.

A sequenceof four or more nucleotides pre-ceding the translational initiation codon in pro-caryotic mRNA is complementary to the 3' terminal segment of 16S rRNA. This comple-mentarity is thought to facilitate the bindingof

mRNA to ribosomes. The situation in eucary-otes is less certain since the presence of a

sequence complementaryto 18SrRNA is

vari-able (17, 46). For this reason, itwas of interest

to examine the leader sequences ofearly vac-cinia virus mRNAs. Before the first ATG of

the19Kpolypeptidegene,thereisa

heptanucle-otide ATCCATC that has close sequence

complementarity to the 3' end of 18S rRNA (3'AUUACUAGGAAGGGCG,indicatedin

ital-ics; 17). Similarly, a complementary

tetranu-cleotide TCCTwasnoted(35)afewnucleotides

before the secondATG of the 7.5Kpolypeptide

gene. However, lesshomology to the 3' end of

18S rRNA is present in the 42K polypeptide

genewhichappearsto havealeader ofonlyfive to six nucleotides. Whetherthese differencesin

leadersequencesareresponsiblefor thegreater invitro synthesisof 19Kand 7.5Kpolypeptides

193 223 253

ATGTCG ATG AAA TAT CTG ATG TTG TTGTTC GCT GCT ATGATA ATC AGA TCA TTC GCC GAT AGT GGT AAC GCT ATC GAA ACGACA TCG CCA MET SER MET LYS TYR LEU MET LEU LEU PHE ALA ALA MET ILE ILE ARGSER PHE ALA ASP SER GLY ASN ALA ILEGLU THR THR SER PRO

283 313 343

GAA ATT ACA AAC GCT ACA ACA GAT ATT CCA GCT ATC AGATTA TGC GOT CCAGAG GGA GAT GGA TAT TGT TTA CAC GOT GAC TGTATC CAC GLU ILE THR ASH ALA THR THR ASP ILE PRO ALA ILEARG LEU CYS GLYPROGLU GLYASP GLY TYR CYS LEU HIS GLYASP CYS ILE HIS

373 403 433

GCT AGA GAT ATT GACGGT ATG TAT TGT AGA TGC TCT CAT GGT TAT ACA GGCATT AGA TGT CAG CAT GTA GTATTA OTA GAC TAT CAA CGT

ALA ARGASP ILE ASPGLYMET TYR CYS ARG CYS SER HIS GLYTYR THR GLY ILEARG CYS GLNHIS VAL VAL LEUVAL ASP TYRGLN ARG

463 493 523

TCA GAA AAC CCA AAC ACT ACA ACO TCA TAT ATC CCA TCT CCC GGTATT ATO CTTGTA TTA GTAGGCATT ATT ATT ATT ACG TGT TGTCTA SER GLU ASN PRO ASNTHR THR THR SER TYR ILE PRO SERPRO GLY ILEMET LEU VAL LEU VAL GLY ILE ILE ILE ILE THR CYSCYS LEU

553 583

TTA TCT GTT TAT AGG TTC ACT CGA CGAACT AAA CTACCT ATA CAA GAT ATG GTT GTGCCA TAA LEUSER VAL TYR ARG PHE THR ARG ARGTHR LYS LEU PRO ILE GLNASPMET VAL VAL PRO END

MOLECULAR WEIGHT - 15525

1707 1677 1647

ATGGAT ATT TAC GAC GAT AAA GGT CTA CAG ACT ATT AAA CTGTTT AAT AAT GAATTT GAT TOT ATA AGGAAT GAC ATC AGAGAA TTA TTT MET ASP ILE TYR ASP ASP LYS GLY LEU GLN THR ILE LYS LEU PHEASN ASNGLUPHE ASP CYS ILE ARG ASNASP ILE ARG GLU LEU PHE

1617 1587 1557

AAA CAT GTA ACT GAT TCC GAT AGTATA CAA CTT CCG ATG GAA GAC AAT TCT GAT ATT ATAGAA AAT ATC AGAAAA ATA CTA TAT AGA CGA

LYS HIS VAL THR ASP SER ASP SERILEOLN LEU PROMET GLUASP ASNSER ASP ILEILE GLU ASN ILE ARGLYS ILE LEU TYR ARG ARG

1527 1497 1467

TTA AAA AAT GTA GAA TGT GTT GACATC GAT AGT ACA ATA ACT TTT ATO AAA TAC GAT CCA AAT GAT GAT AAT AAG CGT ACO TGT TCT AAT LEU LYS ASN VAL GLU CYS VAL ASP ILEASP SERTHR ILETHRPHEMET LYS TYR ASP PRO ASH ASP ASPASN LYS ARG THRCYS SER ASN

1437 1407 1377

TGG GTA CCCTTA ACT AAT AAC TAT ATG GAA TAT TGT CTA GTA ATA TAT TTO GAA ACA CCOATA TGT GGA GGC AAA ATAAAA TTA TAC CAC TRPVAL PRO LEU THR ASN ASHTYR MET GLUTYR CYS LEU VAL ILETYR LEUGLU THRPRO ILE CYSGLY GLY LYS ILE LYS LEU TYR HIS

1347 1317 1287

CCTACA GGAAAT ATA AAG TCG GAT AAG GAT ATTATG TTT GCA AAGACT CTA GACTTT AAA TCA AAGAAA GTGTTA ACT GGA CGT AAA ACA PRO THR GLYASH ILE LYS SER ASP LYS ASP ILE MET PHE ALA LYS THR LEU ASP PHE LYS SER LYS LYS VAL LEUTHR GLY ARG LYS THR

1257 1227 1197

ATT GCC GTTCTA GAC ATA TCCGTT TCA TAT AAT AGA TCA ATG ACT ACT ATT CAC TAC AAC GACGACGTT GAT ATAGAT ATA CAT ACT GAT

ILE ALA VAL LEU ASP ILESER VAL SER TYRASN ARG SER MET THR THR ILE HIS TYR ASNASP ASP VAL ASP ILEASP ILE HIS THR ASP

1167 1137 1107

AAA AAT GGA AAA GAG TTA TGT TAT TGT TAT ATA ACA ATA GAT GAT CAT TAC TTG GTT GAT GTGGAA ACT ATA GGA GTT ATA GTC AAT AGA LYS ASH GLY LYS GLU LEU CYS TYR CYS TYR ILE THR ILEASP ASP HIS TYR LEU VAL ASP VAL GLU THR ILE GLY VAL ILE VAL ASN ARG

1077 1047 1017

TCT GGAAAA TGT CTG TTA GTAAAT AAC CAT CTA GGT ATA GGT ATCGTT AAA GAT AAA CGT ATA AGC GAT AGT TTT GGA GAT GTA TGT ATG

SER GLY LYS CYS LEU LEU VAL ASN ASNHIS LEU GLY ILE GLY ILE VAL LYS ASP LYSARG ILE SER ASP SER PHE GLYASP VAL CYS MET

987 957 927

GAT ACA ATA TTT GAC TTT TCT GAA GCA CGA GAGTTA TTT TCA TTA ACT AAT GAT GAT AAC AGGAAT ATA GCA TGGGAC ACT GAT AAA CTA ASP THR ILE PHE ASP PHE SERGLU ALA ARGGLU LEU PHE SER LEU THR ASN ASP ASP ASN ARGASH ILE ALA TRPASP THRASP LYS LEU

897 867 837

GACGAT GAT ACA GAT ATA TGG ACT CCCGTC ACA GAA GAT GAT TAC AAA TTT CTT TCT AGA CTAGTA TTGTAT GCA AAA TCT CAA TCG GAT

ASP ASP ASP THRASP ILE TRP THR PRO VAL THR GLU ASP ASP TYR LYS PHE LEU SER ARGLEU VAL LEU TYR ALA LYS SER GLN SER ASP

807 777 747

ACT GTA TTC GAC TAT TAT GTT CTT ACT GGT GAT ACG GAA CCA CCC ACT GTA TTC ATT TTCAAG GTA ACT AGA TTT TAC TTT AAT ATG CCG

THR VAL PHEASP TYR TYR VAL LEU THR GLY ASP THR GLU PRO PRO THR VAL PHE ILE PHE LYS VAL THR ARG PHE TYR PHE ASH MET PRO AAA TAA

LYS END

MOLECULAR WEIGHT = 38508

FIG. 7. Derived amino acidsequencesofthetwoearlypolypeptides. Open readingframesareshown with theirpredicted molecularweights.

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relativetothe 42Kpolypeptide(11) isnot under-stood.

Nuclease Si protection experimentswerealso

used to map the major 3' ends of the 19K

polypeptide mRNA at or near nucleotides 643 and 644 (Fig. 2). We considered that a more preciseidentificationof thesequenceadjacentto

thepoly(A)tail of themessagecould beobtained bysequencingcDNArecombinants. Indeed,aT residue atposition 639 wasfound adjacent toa

nongenomic oligo(dA) cluster. Thesequence of additional cDNA recombinants is necessary to

determine theextent of 3' heterogeneity. How-ever, as mentioned above, most of the cDNA recombinants did not have the oligo(dA) * oli-go(dT) terminus because thesecondpolymerase reaction didnotgo tocompletion orthis struc-ture was nibbled away during the nuclease Si step usedto remove the 5' hairpin. An alterna-tive method of cDNA cloning that avoids this

stepwouldseempreferable(21). Foravariety of

possiblereasons, we could notprecisely locate the 3' end of the 42K polypeptide message. However, fromthelocation of the 5' end and the length of the RNA, itmustoccurbetween nucle-otides 640 and 740.Thus, the3' ends of thetwo oppositely oriented mRNAsmustbeveryclose

toeach other.

Examination of the genomic sequence near

the 3' ends of thetwo mRNAs does notreveal the tandem CTATTC that was found with the 7.5Kpolypeptide'message (35). Evidently, this repeated structure is not a general termination

sequence. Just before the end of the 19K poly-peptide mRNA there is a possibly related se-quence CTTATG. The significance of the

tan-demly repeated sequence TAGAGGTAGAGG

beyond the coding regions of the 42K polypep-tide mRNA is questionable (Fig. 2). Although A * T-rich sequences are present, a precise

AATAAApoly(A)signal sequence (27)was not

found. It is not known whether the 3' ends of vaccinia virus mRNAs represent termination

sitesor sites ofRNAprocessing.

Thegenomicsequencein Fig. 2wasanalyzed with the aid of a computer program (28) for amino acid and stop codons in both directions and all three reading frames. The first ATG occurs at position 164, about 50 nucleotides downstream from the start site for the 19K

polypeptide mRNA. A second ATG occurs in-phase, six nucleotides further downstream.

Be-tweenthefirst ATG and the TAA stopcodonat

position 584, there is a 420-nucleotide open

reading frame sufficient to code for 140 amino acids (Fig. 2). Additional inphase stop codons occurbefore the RNAterminates. In both other reading frames, there are many stop codons throughout thegene.

Ontheopposite DNA strand, anATGoccurs

at position 1,736, only five to six nucleotides downstreamfromthe5'endof the 42K polypep-tide mRNA. A 993-nucleotide open reading

frame continues until the TAA at position 743 neartheendof the message. Thisregionwould code forapolypeptide of 331 amino acids (Fig. 2). Both of the other reading frames have stop

codons throughout.

Based on the predicted amino acid sequence (Fig. 7), the molecular weights of the two poly-peptides would be 15.5K and 38.5K. These

numbersare somewhat lower thanthe 19Kand

42K values determined by polyacrylamide gel electrophoresis (11). It seems likely that the latter values were overestimated since in that studytheendogenousreticulocyte43K polypep-tide appearedto be 48K.

Theamino acid sequences of thetwo polypep-tidesareunremarkable. Neither hasaclusterof hydrophobic amino acids indicative of a mem-brane transportfunction. A * T-rich codons are

usedprimarily, reflecting the 60%A * T content

of the genes. As yet, there is no information regarding the functions ofthese two early

pro-teins.

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