Effects of deletions on expression of the herpes simplex virus thymidine kinase gene from the intact viral genome: the amino terminus of the enzyme is dispensable for catalytic activity.

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0022-538X/84/060733-06$02.00/0

Effects

of

Deletions

on

Expression

of the

Herpes

Simplex

Virus

Thymidine

Kinase Gene from the Intact Viral Genome: the Amino

Terminus

of the

Enzyme

Is

Dispensable

for

Catalytic

Activity

M. E. HALPERNAND J. R. SMILEY*

Department ofPathology, McMaster University, Hamilton, Ontario, Canada L8N 3Z5 Received 17October1983/Accepted 21 February 1984

We havetransferredtwodeletionsaffectingthe 5' endoftheherpessimplexvirusthymidinekinase(TK)

geneinto the intact viralgenome.One,extending from -12 to+189, hadnoeffectonTKmRNAsynthesis andonlyasmalleffectonTKactivity, althoughthefirst27codons of theTKpolypeptideweredeleted. The other, extendingfrom -85 to +85, severely impaired TK mRNAsynthesis. We conclude that the amino terminus of the TK polypeptide is dispensable for catalytic activity, and that expression ofTK in viral infections requires someof the same promoterelements used in uninfected cells.

Theherpessimplex virus(HSV)geneforthymidinekinase (TK) is a delayed early, or a viral gene (2). As such, its expression during lytic viral infections requires the prior synthesisandcontinuousactivityoftheimmediateearly,or a,protein ICP4 (3, 6,7, 18,26, 27). ICP4appearsto act as a positive transcriptional activator for all ofthe non-a HSV

genes;however, its mechanism of action remains unknown. Thisrequirement for ICP4 function isremarkably tight,asno TK RNA canbe detected by RNA-driven

hybridization

in

the absence of a protein synthesis (7). In contrast to the situationduringlyticviralinfections,TKisexpressedinthe

absence of a proteins when microinjected into Xenopus oocytes (14) ortransfected intomousefibroblasts (29). The

reasonsfor this difference remain unclear andmayberelated

tothefactthatbothoftheseexpression systemsinvolve the delivery ofthe TK gene into cellsoutofcontext, andbyan artificial route. This a-independent expression has in fact

provided a valuable system for studying HSV promoter

function. Usingexpression inXenopusoocytes as anassay,

McKnight and co-workershave systematically assessed the

effects of mutations onthe expression of microinjectedTK genes (13-16). The results demonstrate that expression in uninfected cellsdependsonatleast threeseparate upstream

regions mappingbetween -105and -18 with respecttothe

major transcription initiation site. The question arises whetheror not these same sequences are alsorequired for expression during viral infections. Part of the answer has recently emerged from studies ofmutantTK genesresident

in biochemically transformed cells. It has been known for

some time that HSV TK genes present inuninfected cells remain responsive to the action of a proteins supplied by

superinfecting HSV (4, 5, 8). We have recently reported

results that,when takenin combinationwith those ofZipser

et al. (30), suggest that this transactivation phenomenon requires the integrity of at least two separate upstream

regions thatoverlap the constitutive promoter used in oo-cytes (23). In this paper, we report on the effects of two

deletions on theexpression ofthe TK gene from the intact

HSV chromosome. The data demonstrate that expression requires sequences mapping between -12 and -85. It is

thereforelikelythat thepromoterelements used inartificial

assay systems correspond to, or form a subset of, the

elementsusedduringnatural viralinfections. Inaddition,we

* Correspondingauthor.

find that theamino-terminal45residues of the TK polypep-tide are dispensable forcatalytic activity, and thatdeleting all of the nontranslated leader of the wild-type TKmRNA

haslittle effect on expression.

MATERIALS AND METHODS

Virus and cells. HSVtype 1(HSV-1)strain KOS wildtype and the TK-deficient deletion mutant d2 (21) were used in thisstudy. Virusstocks wereprepared and titrated onVero cells as previously described (22). Mouse LtA cells, an adenine phosphoribosyl transferase-deficient derivative of the thymidine kinase-deficient (TK-) line LM (TK-) (4), were obtained from R. Hughes, Roswell Park Memorial

Institute, Buffalo, N.Y. Vero and LtA cells were grownin Alpha minimal essential medium (GIBCO Laboratories) supplemented with 5 and 10% fetal bovine serum,

respec-tively.

Marker rescue. Mutations weretransferred from plasmids

into the viral genome exactly as previously described (21,

22). Eachmutantplasmidwasseparately cotransfectedwith

wild-type and d2 viral DNA into Vero cells. The resulting virus stocks were then screened for rescue products.

TK-deficientproducts were selected from the crosses with wild-type viral DNA byplaque purification on Vero cells in the presence of100 ,ugofaraT per ml (17).

TK+ productswere selected from the crosses with d2 viral DNA by serial passage in LtA cells in the presence of

hypoxanthine, aminopterin, andthymidine. After the initial

selections, DNAfrom each of the recombinants was

exam-inedby Southern blot hybridization (24) to verify the pres-enceoftheappropriate deletion; then the recombinant viral strains were plaque purified an additional three times and

expanded into high-titer stocks. Blotting and hybridization were as previouslydescribed (22).

Induction of TKactivity.Subconfluentmonolayers of LtA

cells growing in 75-ml flasks were infected with the virus strains andharvested 12 h later.TKactivity incellextracts was measured by using C14 thymidine, as previously de-scribed(23).

S1 nuclease transcript mapping. To map the transcripts produced in cells infected with the mutant virus strains, we used the transcript mapping techniques of Berk and Sharp (1), as modified by Weaver and Weissman (28). Vero cells were infected at a multiplicity of 10, and total cytoplasmic RNA was prepared 4 h later, using the procedure of Berk andSharp(1). Uniquely end-labeled, single-stranded hybrid-733

on November 10, 2019 by guest

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ization probes were prepared by using T4 polynucleotide

kinase and [.y32P]ATP as described by Maxam and Gilbert (11). Afterstrand separation, approximately 2 x 104 dpm of

the eluted probe was coprecipitated with 10

pxg

ofRNA in

ethanol. The dried pellet was suspended in 30 ,ulof0.6 M NaCl-0.05 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid)-0.001 MEDTA (pH 7.5) andhybridized

for 12 hat50°C. ForSi digestions,thesamplewasaddedto

0.20 ml of ice-cold 150 mM NaCl-50 mM sodium acetate-5 mM ZnSO4 (pH 4.6) containing 2 x 103to 4 x 103 Uof S1

nuclease (Boehringer Mannheim Biochemicals). After 1 h at 37°C,the reactions were stoppedbytheaddition ofEDTA to 20 mM, extracted twice with phenol-chloroform (1:1), and

ethanol precipitated. Digestions with exonucleaseVII were carried out in 200

[lI

of 0.3 M KCl-0.1 M

Tris-hydrochlo-ride-0.1 M EDTA (pH 7.4) for 1 h at 45°C, using 2 U of

exonuclease VII (BethesdaResearchLaboratories)per reac-tion.

To detect TK-related transcripts, we used two different probes: one was aHinfl-EcoRI fragment end labeled at the Hinfl site located at +811; the other was an RsaI-PvuII

fragment end labeled at the RsaI site at +351. ICP4 tran-scripts were assayed by using a 290-base SalI-EcoRI

frag-ment labeled at the Sall site (9). The portions of the probe

protected from nuclease digestion were sized on

polyacryl-amidesequencing gels. DNAsequencingwasby the method

ofMaxam andGilbert (11). RESULTS

Rescue of deletion mutations. The genome of HSV is 150

kilobases in length, making direct site-specific mutagenesis difficult. Therefore,to study theeffectsofprecisely defined

deletions on TKexpression from theintact viralgenome,we decidedto use a two-stepapproach that has beenpreviously described (21). Briefly, mutations were introduced into an HSV-1TK gene cloned into abacterialplasmid, then trans-ferredinto the viral genomethroughhomologous

recombina-tion invivo. The resulting rarerecombinant virusesbearing

themutant allele were thenisolated byusingan appropriate

selection system. We had previously isolated a number of

deletions affecting the TK upstream region (23). Some of

thesemutations had no effect ontheexpressionor

transacti-vation of the TK gene when introduced into mouse fibro-blasts; others reduced expression and eliminated the re-sponsetoproteinsofsuperinfectingTK-virus. Asitwas not clear in advance what effects these same mutations would

have on expression from the intact viral genome, each

deletion was recombined with both wild-type HSV and a TK-deficientdeletion mutantvirus, HSVd2(21; seeFig. 1). The products of the former cross were screened for TK-deficientrecombinants, and the products ofthe latterwere screenedforwild-typerecombinants, asdescribed above.In all, we have attempted to rescue six deletions in this way, but have succeeded withonly two: Al andA35 (Fig. 1). Al

lacksresiduesextending from -11to +189onthe

wild-type

sequence (23; Fig. 1). The deletion leaves intact the three promoter elementsdefinedbyMcKnightandcoworkers

(13-16) butremoves allofthewild-type nontranslatedleader and the firstpotential initiation codon ofthe TK

polypeptide.

In contrast, A35 (-85 to +85)leaves the TK

coding

sequence intactbut deletes all threeofthepromoter elements thatare required for expression in Xenopus oocytes. As described

below, the Al deletion has little effect on TK

expression

from the HSV genome, whereas A35 reduces expression approximately 50-fold. This result parallels the effects of these same deletions on expression in uninfected

cells,

in

A

HSVWT

Bam Pvu1 AUG PvuII Bam

x

,/&35

I

TK'VIRUS

B

HSV d2 *4

I

TK*VIRUS

c

-100 +1 +100 +200

AUG

WT AUG

AUG

A35

FIG. 1. Rescue ofdeletions. (A) TheA35 deletionwas rescued by recombination between wild-type HSV DNA and a plasmid

bearingthe deletion.(B)Alwasrescuedbyrecombination with TK-deficient HSV d2 DNA.(C)Thedeletionendpointsarediagrammed, using an expanded scale, with respect tothe threetranscriptional control elements identified byMcKnight and co-workers. In addi-tion, the major transcription initiation sites forwild-type and Al virusare indicated. For the sake ofconsistencywiththeliterature, the mapsarepresentedinthe ILorientationof the HSV genome. which Al had no effect on

expression

or

transactivation,

whereas A35 wasdefective for both (23).

TK+

recombinants were recovered from the Al x HSVd2 cross, and

TK-recombinantswere recoveredfrom the A35 x

HSV+

cross,

suggestingthat Al does not

dramatically

affect TK

expres-sion from the viral

chromosome,

but A35 does. To demon-stratethat themutantviruses recovered from thesecrosses

actually

had

acquired

the

input mutations,

DNAfrom each wascleaved withacombination of PvuII and

SmaI,

andthe

TK-related

fragments

were

analyzed

by

Southern blot

hy-bridization. Deleted versions of the

1,400-base

PvuII-SmaI TK

fragment

werepresent in both isolates

(Fig.

2),

and these novel

deletion-bearing fragments

comigrated

withthe

corre-sponding fragments

ofthe

input

plasmids.

Additional

analy-ses showedthat Alvirusretained the EcoRI site at

-80,

as

expected, and A35 virus lacked this site

(data

not

shown).

We conclude,

therefore,

that both rescue

products

have

incorporated

the mutations

originally

carried

by

the

plasmid

DNA in the cotransfections.

Although

we are not yet certain for the reasons for the failureto rescuetheotherfour

deletions,

it may be relatedto the fact that the

plasmids

bearing

these four mutations all contain less than 130 bases of

homology

with the viral chromosome upstream ofthe deletion

(these

deletions were

inducedin thePvuIITK

fragment).

Incontrast,the

plasmids

bearing Al and A35 both retainover600bases of

homology

withviralDNAupstreamofthedeletion.

Consequently,

one would expect a

higher

frequency

of recombination between the viral genome and these two

plasmids.

We cannot,

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[image:2.612.318.558.78.341.2]

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1 2 3 4 5 6

[image:3.612.115.232.75.315.2]

_

FIG. 2. Southernblotanalysis of the mutant genomes. Plasmid and viral DNAs werecleaved with a tnixture of PvuII and SmaI. After electrophoresis through a 1.4% agarose gel, the fragments were blotted onto nitrocellulose and detected by hybridization to nick-translated pTK173DNA. Lanes: 1,pXlbearing the wild-type TKgene; 2,wild-typeviral DNA; 3, Alplasmid;4,Alvirus; 5,A35 plasmid; 6, A35 virus.

however,rule out thepossibilitythat the other four deletions arelethal, althoughweconsiderthistobe unlikely.

Expression of TK from the mutant viral genomes. To

accurately quantitate the levels ofTK enzymatic activity inducedbyAlandA35, TK-deficientLtAcellswereinfected

with either virusandthelevels ofTKactivityweremeasured

12h later. Al induced approximately 40%of the wild-type levelsofenzyme, whereasA35 inducedonlyca.3%(Fig. 3). As Al isdeletedfor the first27codons ofthe TK polypep-tide,thisresult demonstrates thattheaminoterminus ofthe

proteinisnotrequired forcatalyticactivity. A35 retains the entire TKcodingsequence, yetisseverely impairedfor TK

expression. This suggests that the A35 deletion interferes

with TK mRNA synthesis.

The effects of these deletions on TK mRNA synthesis

were assessed by Sl mapping. In the first experiment, infected cellRNA washybridizedtoasingle-strandedprobe

end-labeled at aHinfl site(+811)andextendingtotheEcoRI

site at -80. After hybridization and treatment with S1 nuclease or exonuclease VII, theprotected portions ofthe

probe were sized on a 3% polyacrylamide sequencing gel (Fig. 4A). To provide an internal control for variations betweeninfections and betweenRNApreparations, portions ofthese same RNApreparations were also hybridizedto a probe forthe aICP4mRNA(Fig. 4B)(seeabove). All of the

infected cell RNA preparations used contained roughly comparable amounts ofICP4 mRNA. RNA from wild-type

infections gave a majorprotected species of approximately 810 residues corresponding to TK transcripts initiating at +1. In addition, a somewhat weakersignal was observed,

suggesting an additional transcript starting at ca. +70.

Al-though avariety of minorTK-related transcriptshave been

observed in biochemically transformed mouse cells (20),

comparable RNAs have not been reported during lytic

infections. Wewish topointout, however, that both ofthe

high-resolution Si mapping studies ofTKmRNApublished

to date (12, 25) have employed hybridization probes that

would not detect initiations at +70. Consequently, we con-sider it possible that the signal at +70 represents a real transcript, rather than an artifact of nucleasedigestion.

RNA from Al-infected cells lacked the +1 and +70

species, asexpected, and insteadgave a new major hybrid-ization signal at ca. +200. The intensity of this signal was

comparable to that of the wild-type, suggesting that the

novel Al transcript was produced in normal amounts. The initiation site forthe mutant RNAcouldnotbemappedwith confidence in this experiment as the probe was prepared from wild-type DNA and the resolution of the gel was

insufficient to allow us to decide whether the signal was

produced by initiationupstream ordownstream ofthe

dele-tion. The fact that the Si and exonuclease VII digestion products comigrated could be rationalized with upstream

initiations ifthe upstreamportion of thetranscript was too

short toform a stable hybrid with theprobe.. Therefore, to map the Alinitiationsiteprecisely,anotherwild-typeprobe,

end-labeledat anRsaI site(+351) andextendingto thePvuII siteat -200, wasused. Inthis

case,

the position of the Al hybridization signalwasdetermined with referenceto

mark-ers generated by the Maxam-Gilbert sequencing reactions

carried out on thesame probe. The results (Fig. 5)

demon-stratethat the Al transcript initiates atresidue +198ofthe wild-typesequence,downstreamfromthedeletion endpoint. Incontrast to theessentially wild-type levels ofRNAfound in Al-infected cells, we were not able to detect any

TK-related transcripts in cells infected with A35 virus. We

estimate from this that the mutant transcripts, if present,

accumulate to less than 5% of the wild-type levels. This is consistent with theverylow levelsof TKenzymatic activity inducedby this mutant. Since the sameA35 RNA

prepara-tions contained normal amounts of ICP4 mRNA (Fig. 4B), we conclude thatA35 deletionreduces TKtranscription, or

(less likely) the stabilityorprocessing ofthe RNA.

DISCUSSION

We have drawn two major conclusions from this work.

First, thetranscription ofTKfrom theintactHSV

chromo-x 0

LU

I--0

I

C') 0

I

C.) 5

4 3 2 1

TIME(minutes)

FIG. 3. TK enzymatic activity induced by mutant virus. LtA cells were infectedwith the indicated viruses and harvested 12 h later. TKactivity was measured inextracts prepared from equiva-lentnumbers of cells, asdescribed in the text.

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[image:3.612.313.550.515.693.2]

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P Al WT A35

S X S X S X

WT

*r 51The initiation site for the Al transcript is consistent with P WT Al A35 the proposed role of the Goldberg-Hogness sequence in

directing transcription initiation events to a site 25 to 30

bases downstream.The Al deletion leaves the TK Hogness * sequenceintact butdeletes the wild-type initiation site. The

>,_ mutant transcript, however, continues to initiate 28 bases

downstream, at asitethatordinarily lies within the body of the mRNA. Similar resultshave beenobtainedbyMcKnight - et al. withTK inuninfectedcells (15), and by others with a

variety of genes.

McKnight and co-workers (13-16) have identified three upstreamregionsnecessaryfor TK transcription in

uninfect-ed cellswithin the DNA sequences extendingfrom -105 to -18. A35 lacks two of these elements entirely and retains 4 g * ~ only a portion ofthe most distal element (-105 to -81),

qp

[image:4.612.70.304.76.448.2]

A B

FIG. 4. Analysis of transcripts produced by wild-type and

mu-tant virus. (A) For TK transcripts, RNA prepared from cells infected withthe indicated virus washybridizedtoaHinfl-EcoRI TKprobe (P). After digestion with S1 nuclease(S)orexonuclease

VII(X),sampleswere run on a3% polyacrylamide sequencing gel. Themajor initiation sites areindicated by triangles. (B) For ICP4

transcripts, the RNA preparations usedin(A)werehybridizedtoa

SalI-EcoRI ICP4 probe (P). Afterdigestionwith Si nuclease, the

productswere run on an8%sequencing gel. Thearrowindicates the

approximately 180-base-long fragment protected byICP4 mRNA.

some during natural viral infections requires sequences

located between -12 and -85. This conclusion is based on the factthatAl, lackingresidues -11 to +189, accumulates

wild-type levels ofa novel TK transcript and A35, lacking residues -85to+85,accumulates less than 5% of the normal levels. We believe that the defect in A35 is atthe level of transcription initiation, rather than at the level of mRNA processing,transport,orstability,fortworeasons. First,the

difference between Al and A35 strongly suggests that the

defect in A35 can be accounted for by the deletion of

sequenceslocated between -12 and-85. Thepositionof the

relevant sequences is therefore consistentwith a transcrip-tional defect and is more difficult to reconcile with other possibilities. Second, Al synthesizes normal amounts ofa truncated mRNA that lacks the first 197 residues of the wild-typetranscript. Therefore,thestructureofthe 5' endofTK

mRNA does notappeartobe critical for normalprocessing, transport, or stability.

WT A1 GG

XCT

C

_s"

Us.

FIG.5.Fn_apn~ fteA~ ntainst.TeRApeaa

tions~~~~

usdi

~~

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seunigraton are u n h aepoe

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whereas Al retains all three (Fig. 1). The fact that A35 is

impaired stronglysuggests thattranscriptionin natural HSV infectionsrequiresatleast some of the samesequences used inuninfectedcells. Thisconclusion isnotespecially surpris-ing in view of our recent demonstration that sequences

located between -70 and -12 are required for the ICP4-mediated activation of TK genes resident in mouse fibro-blasts (23). We emphasize that the results of the present

study wereobtained by assayingthe effectsof deletions on

expression ofTKfrom its natural location within the intact HSV genome, eliminating thepotential artifacts associated

with other assay systems. One question that remains is

whether expression from the intact viral genome requires

sequencesin additiontothose needed inXenopusoocytes. The results obtained with Al rule out a requirement for

sequences between -11 and +189. However, it remains possiblethat sequences upstream of themostdistal

constitu-tive promoterelement play a role during infection. To test

this possibility, it will be necessary to modify sequences upstream of -105 while leaving the -105 to -18 region intact. This may prove to be difficult in practice as the

modifications might affecta neighboring essentialgene. The second major setof conclusions that we have drawn from our data is that the amino-terminal 45 residuesofthe

wild-typeTKpolypeptidearedispensable for catalytic activ-ity,and that the nontranslated leader of theTKmRNAdoes

not play a major sequence-specific role in translational initiation. These conclusions are basedonthe properties of virus bearing the Al deletion. This deletion removes all of

thewild-type nontranslated leader ofTKmRNAand the first 27 codons forthe TK polypeptide, but leaves the promoter

unaltered. Consequently, transcription in the mutant virus

initiatesatresidue +198ofthewild-typesequence, 44 bases upstream from what is ordinarily the second methionyl codon ofthe polypeptide. Presumably, translation initiates at this second site, which is the first AUG codon in the mutantRNA. As faras we are able todetermine, Al virus

accumulates wild-type levels of the truncated transcript. Because it also inducesapproximately40% ofthewild-type levelsofTKenzymaticactivity,weconcludethatthe amino-terminal45 residues ofthe wild-type enzyme are not

abso-lutely required for catalytic activity. Since we have not

directly measured the levels of the translated mutant TK

polypeptide,we are unable to decide from the present data

whether the approximately twofold reduction of activity

compared with wild-type is due to reduced

translation,

reduced activity ofthe truncatedprotein, or acombination of both factors.Nevertheless, because thedeletionhas

only

a relatively minor effect on the overall level ofactivity, it

seems safe to conclude that the truncated protein product retainsappreciableactivity,andthat the truncated mRNA is

translated at closeto the wild-typerate. This latter

conclu-sion implies either that the wild-type leader plays no

indis-pensable, specific role in translation, or that the novel 44-base leader ofthe Al transcriptis able to substitute for this function. At present we are unable to distinguish between these twoalternatives.

Preston and co-workers (10, 19) have reported that in

addition tothe42,000-molecular-weight (42K)TK polypep-tide, infected cells alsosynthesizesmalleramountsofa39K anda38K TK-relatedprotein.Theseauthorshaveproposed thatthesmallerproteinsarisethroughtranslational initiation events at the second and third initiator codons of TK

mRNA, respectively. In studies to be reported elsewhere, Haar, Smiley, Marsden, and Preston have shown that Al virusfails to induce the 42K protein and overproduces the

39K species. Taken in combination with the present data, this demonstrates that the 39K protein retains close to full

catalytic activity. This conclusion agrees with that of

Rob-erts and Axel (20), who proposed on the basis of indirect evidence that anaberrantTKtranscript postulatedtoinitiate at +200encodesafunctionalenzyme. The functional signifi-cance of the 39K and 38K polypeptides produced during

wild-type infections remains unclear. ACKNOWLEDGMENTS

We thank HelenRudzroga for expert technical assistance, and RoyPerssonforagiftofanICP4plasmid.

This workwas supported by grantsfrom the Medical Research Council and the National Cancer Institute of Canada. J.R.S. is a

research scholarof the National CancerInstitute.

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