Proc. Nati. Acad. Sci. USAVol. 82, pp. 4773-4777, July 1985Genetics

Application of the mini-Mu-phage for target-sequence-specificinsertional mutagenesis of the herpes simplex virus genome

(thymidine kinase selection/random insertion/genetic engineering)

FRANK J. JENKINS*t, MALCOM J. CASADABANt, AND BERNARD ROIZMAN*t**The Marjorie B. Kovler Viral Oncology Laboratories and tDepartment of Molecular Genetics and Cell Biology, The University of Chicago, 910 East 58thStreet, Chicago, IL 60637

Contributed by Bernard Roizman, March 28, 1985

ABSTRACT An earlier technique for insertionalmutagenesis of large viral genomes involved the insertion of thethymidine kinase (TK) gene at a specific target site,cotransfection of the fragment carrying the insertion with theintact viral genome, and selection of the progeny for viralrecombinants expressing the TK gene. The inserted TK genecould then be replaced by cotransfection of the recombinantDNA with fragments carrying a foreign sequence or a deletionin the target sequence. To enable the probing of larger targetdomains and facilitate insertional mutagenesis, we extendedthis technique by insertion of a 2.2-kilobase-pair (kbp) herpessimplex virus 1 (HSV-1) chimeric aTK gene into the 7.5-kbpmini-Mu-phage (aTK-mini-Mu) and lysogenized Escherichiacol with the helperMu phage and the aTK-mini-Mu. Inductionof phage multiplication of the lysogenized E. coli after trans-formation with plasmids carrying HSV-1 DNA and subsequentinfection of E. coli RecA' lysogenized with Mu phage yieldedplasmid populations carrying randomly inserted aTK-mini-MuDNA. Application of this procedure for insertional mutagenesisof the BamHl B fragment, which spans the junction betweenthe unique and reiterated sequences of the L component of viralDNA, yielded two types of recombinants. Viral recombinantdesignated RBMul contained the intact aTK-mini-Mu insertedinto unique sequences of BamfH B fragment. In recombinantRBMu2, the aTK-mini-Mu was inserted at or in the repeatedsequences, but -14 kbp comprising most of the internalreiterations of the viral genome in the prototype arrangementwere deleted.

Post and Roizman (1) have described a generalized techniquefor site-specific insertion or deletions in the chromosomes oflarge viral genomes, applied to herpes simplex virus 1(HSV-1) DNA. The technique consists of two steps. First,HSV-1 thymidine kinase (TK) gene is inserted into a clonedDNA fragment at the desired site. The amplified chimericfragment is then cotransfected with intact DNA of a HSV-1recombinant virus carrying a deletion in the TK gene, and theprogeny of the transfection is selected for TK' phenotype. Inthe second step, an appropriate insertion or deletion is madein the target sequence at the site of insertion of the TK gene.The fragment is amplified and cotransfected with the DNA ofthe recombinant carrying the TKgene in the target sequence,and the progeny is selected for TK- phenotype. In eachinstance, the recombination of the DNA fragments carryingthe TK gene or the deletion in the target sequence isfacilitated by the presence ofhomologous sequences flankingthe insert or deletion. The TK gene was used as a selectablemarker because of the availability of powerful selection bothfor and against the gene. In the case ofHSV-1 DNA, a simplemodification of the TK gene used for insertional inactivationof the target gene can insure that the recombination occurs

predominantly if not exclusively into the target sequencerather than restores the activity of the TK gene in its naturallocation in the HSV-1 genome (2, 3). This procedure has beenapplied to construct recombinants carrying a deletion in an agene (a22) of HSV-1 and to delete noninformational se-quences located in the internal inverted repeats in the HSV-1genome (1, 3). Application of this procedure resulted in theisolation ofa viable HSV-1 virus from which 15 kilobase pairs(kbp) comprising the internal inverted repeat sequences hadbeen deleted and which is frozen in one of the four isomericarrangements of the viral genome (2).Although this technique has been used successfully in

several applications, it has two disadvantages. The minor oneis that site-specific insertion of the TK gene into a targetsequence probes only the function of the immediate environ-ment of the inserted site. In many instances there are noreadily available sites for insertion, or perturbation of thedesired site results in extensive rearrangements that renderthe insertion technically complex or defeat the purpose of theinsertion. A major disadvantage is that to ensure selection ofviable recombinants, some knowledge of the underlying genearchitecture is essential. Thus, attempts to insert the TKgeneinto the target gene domains containing gene overlaps mayinactivate both the target gene and another as yet unidentifiedor poorly defined gene. In this paper we report a newprocedure based on the mini-Mu-phage system of Castilho etal. (4). We inserted the selectable marker, an a-gene pro-moter-regulated TK gene, into the mini-Mu. Under appro-priate manipulation of the system, the aTK-mini-Mu (aTK-mM) is inserted randomly into a DNA fragment in thebacterial vector. The DNA fragment carrying the selectablemarker is then cotransfected with intact viral DNA. Althoughtheoretically all chimeric fragments could recombine into theHSV-1 genome, only viable progeny containing the select-able marker will grow. Therefore, the procedure allows rapidscanning of the genome for genes or sequences that can beinterrupted without affecting the growth of HSV-1 in cellculture.

METHODS AND MATERIALSCells and Viruses. African green monkey (Vero) cells were

used for propagation ofvirus. A continuous line ofrabbit skincells was used for cotransfection with viral and plasmidDNA. The human 143 tk- cells (5) were used for selection ofTK' viruses. The recombinant virus HSV-1(F)A305 wasconstructed to contain a 700-bp deletion in the TK gene (6);it was derived from HSV-1(F), the prototype HSV-1 strainused in this laboratory (7).

Abbreviations: HSV-1, herpes simplex virus type 1; TK, thymidinekinase; kbp, kilobase pair(s); aTK-mM, aTK-mini-Mu; Kanr,kanamycin resistance; Ampr, ampicillin resistance.*To whom reprint requests should be addressed.

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Bacteria and Plasmids. The following Escherichia colistrains were used in this study: MC1040 [F- araD139ara::(Mu cts)3 A(lac)X74 galU galK rpsL]; M8820 [F-,araD139 A(ara-leu)7697 A(proAB-argF-lacIPOZYA)XIIIrpsL]; M882OMucts (M8820 with Mu cts62); M8820TRMucts[M882OMucts with recA56 srl::TnlO Tcr (tetracycline resist-ance)]. The plasmid pBC4041 contains the mini-Mu elementMudII4041 (Mucts62 A+B+ Kan9 inserted into a pUC9vector with the Hae II-lac fragment removed (4). pRB364contains the Bgl II-BamHI fragment of the HSV-1 TK genefused to the HSV-1 BamHI N fragment from which theS-component origin of HSV-1 DNA replication was deletedby a Sma I collapse (8). pRB112 and pRB114 contain theBamHI B and N fragments respectively, of HSV-1 clonedinto pBR322 (9).

Cloning of DNA. The procedures for the construction,cloning, screening, and purification of recombinant plasmidswere as described (9).

Analysis of Recombinant Virus. Isolation, purification, andrestriction enzyme analysis of recombinant virus DNA wereas described (1-3). The BamHI N probe used to detect theright terminus of the BamHI N fragment consisted of the3-kbp Pvu II-BamHI subfragment obtained from Pvu II andBamHI digests ofpRB114. The procedures for labeling DNAfragments with 32P by nick-translation and hybridization toelectrophoretically separated DNA fragments transferred tonitrocellulose sheets were as described (8).

RESULTSConstruction of the aoTK-mM Chimeric Plasmid pRB2500.

The Pvu II fragment from pRB364 containing a portion of the5' transcribed noncoding and coding sequences of the HSV-1(F) TK gene fused to the promoter-regulatory domain of thea4 gene was inserted in the orientation shown in Fig. 1 intothe BamHI site of the mini-Mu-phage MudII4041 containedin pBC4041. The resulting aTK-mM cloned as pRB2500 was9.7 kbp in size (Fig. 1). The plasmid was maintained at 30'Cin the E. coli strain MC1040, which contains a Mu ctsprophage preventing transposition of the mini-Mu genome tothe E. coli chromosome.

Construction of Double Lysogen and Principles of Insertion-al Mutagenesis of Plasmid Molecules by aTK-mM. The pro-cedure for insertional mutagenesis of HSV DNA fragmentswith aTK-mM consisted of three steps as illustrated in Fig. 2and detailed in the legend. The first step involved (i)transfection of E. coli lysogenized with both the helper Mucts phage and the aTK-mM phage with plasmids containingspecific HSV-1 DNA fragments and (ii) induction of phagemultiplication. The resulting lysates included phages con-taining DNA molecules consisting of the transfected plasmid

p RB364

pBC4041H

c nor A

pRI 2500H

P P

TK a4e_g , J

_II to

B Konr

H !i ih

c nor A B Konr T K 4

FIG. 1. Restriction endonuclease maps of mini-Mu-phage andconstruction of aTK-mM. The aTK chimeric gene from plasmidpRB364 was inserted in the orientation shown into the BamHI site ofpBC4041 carrying the mini-Mu. A, B, ner, and c are mini-Mu genes;Kanr refers to the gene for kanamycin resistance, and a4 refers to thepromoter-regulatory domain of the a4 gene. H, HindIll; Bg, Bgl II;Ba, BamHI; P, Pvu II cleavage sites.

A E.coli (Rec A;, Mucts)

helper Mu

.coiplaSmp

HSV DNAaTK-Mini Mu

(mM)Induction

B E.coli(Rec A+, Mucts)

Phep DNA

mM)Amp

HomologousRecombinotion

Eosmid

>/mp

mm

C. Rabbit skin ceUs

TK- HSV DNAC(otronsfectionTK4 Selection

mM Amp mA

Phoge DNA aTKmM-HSV Recombinants

FIG. 2. Schematic representation of insertional mutagenesis ofHSV-1 DNA by recombination with plasmid DNA molecules car-rying aTK-mM. (A) Construction of the double lysogen and mini-Mu-phages carrying HSV-1 DNA sequences. E. coli strain MC1040carrying the Mu cts prophage was transformed with pRB2500 asdescribed elsewhere (9). Induction at 420C (4, 10, 11) resulted in theproduction of both helper Mu phage and aTK-mM phage. The lysatewas used to transduce E. coli (RecA-) M8820TRMucts cells to Kan'as described (4, 10, 11). Cells containing an integrated aTK-mM wereselected by screening the transductants for a Kanr, Amps (ampicillinsensitive) phenotype indicative of the presence of the aTK-mMprophage and loss of the plasmid DNA sequences. The presence ofan integrated Mu cts prophage was confirmed by inducing the cellsat 420C, infecting strain M8820 with the resulting lysate, andscreening for plaque formation (not shown). The aTK-mM prophageis defective for replication; therefore, only the helper Mu ctsprophage is capable of producing plaques. The formal designation ofthis E. coli strain is M8820TRMucts MudII4041::aTK abbreviated asMucts/aTK-mM. In the second step, E. coli Mucts/aTK-mM weretransformed with plasmids containing HSV-1 DNA fragments asdescribed (9). Induction at 420C results in the formation of cointe-grate structures (4). The relevant structure would consist of theplasmid molecule flanked by single copies of the aTK-mM prophage(Fig. 2A). The Mu DNA sequences are packaged into Mu phageheads starting with the left end ofMu and proceeding until a headfulofDNA (-38 kbp) has been packaged. If the length ofthe cointegratestructure (plasmid and two copies of aTK-mM) is <38 kbp, thenflanking E. coli DNA sequences are packaged. A cointegrate struc-ture whose length is >38 kbp would lack the mini-Mu sequences atthe right end. (B) Conversion of the phage DNA carrying HSV-1DNA sequences into plasmids. The lysates containing packagedcointegrate structures, and helper Mu phage were used to infect theRecA' strain M882OMucts at 30'C. Infection at 30'C preventstransposition of any infecting Mu phage sequences and results inhomologous recombination between the two copies of the aTK-mMprophage flanking the plasmid DNA sequences. After infection,ampicillin and kanamycin were added to select cells containingplasmid molecules with aTK-mM insertions. Since transposition inMu phage is essentially a random event (12), plasmid DNA isolatedfrom the Ampr, Kan' cells should contain a pool ofplasmid moleculeswith single copies ofthe aTK-mM at different sites within the plasmid

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A o

B Be VI L I sI iKp II 0 1 J t lU aIQB~~~L KBog

a4

P Y6 N | iI * X F

I

II II

l' K

Ace-C is i l 6 iSI P FthN I J

Kp 0 -h F

1 23 ,

5 L-~

a27 ciao e a4

D dL I SI PIYbN J 1IXfiY1PKpI 0' J1 ,Q I F Il K

Ig BS

E Be L I N i J iXZYi PKpIIOI0 F I I K

1B9 BlN4tB8 9_

1011 12

13 14

FIG. 3. Sequence arrangement in wild-type and recombinantHSV-1 viruses. (A) Sequence arrangement in HSV-1 DNA. The openboxes represent terminal sequences of the L (ab) and S (ca)components, which are reiterated internally in an inverted orienta-tion and shown as baac. The dashed vertical line represents thejunction of the L and S components. The hatched box shows thelocation of the TK gene in the prototype arrangement of the HSV-1DNA. (B) Expanded portion of the HSV-1 DNA in prototypearrangement ofL and S components showingBamHl, Kpn I, and BglII cleavage sites and the site of the insertion of the aTK-mM. Themini-Mu-phage sequences are shown as a thick black line, the a4promoter as an open box, and the TK sequences as a hatched box.The location of the homologous a4 promoter sequences in BamHI Nfragment are also shown as an open box. The lines above the mapsrepresent the location of a27, a4, and a) genes. (C) Site andorientation of aTK-mM in the DNA of recombinant RBMu1. Thelines below labeled 1-7 represent the novel DNA fiagments gener-ated by cleavage with BamHI (lines 1 and 2), Kpn I (line 3), BgI II(lines 4 and 5), and Bgi ll/Kpn I (lines 6 and 7). (D) Site of insertionof aTK-mM in DNA of recombinant RBMu2. (E) Orientation ofaTK-mM and sequence arrangement of the DNA of recombinantRBMu2. The lines below the maps show the novel fragmentsgenerated by cleavage with BamHI (lines 8 and 9), Kpn I (line 10),Bgl II (lines 11 and 12) and Bgl II/Kpn I (lines 13 and 14).

DNA linearized randomly and flanked by direct copies of theaTK-mM DNA. In the second step, these lysates were usedto infect E. coli RecA' lysogenized with Mu cts prophage. Inthis strain, the phage DNA carrying the mini-Mu-phagecannot integrate and forms a plasmid by homologous recom-bination through the flanking aTK-mM DNA sequences. Theplasmid confers resistance to kanamycin (Kan) and ampicil-lin (Amp), which can be used to select the bacteria carryingthe plasmids. In the third step, the amplified plasmid DNA

DNA sequences. (C) Insertional mutagenesis of HSV-1(F) DNAfragments. Plasmid DNA purified from lysates ofE. coli selected forAmpr and Kanr resistance were cotransfected with intact HSV-1A305DNA into rabbit skin cells as described (1). Recombinant TK+progeny were selected in human 143 TK- cells overlaid with mediumcontaining hypoxanthine, aminopterin, and thymidine.

carrying the HSV-1 DNA sequences and the inserted aTK-mM were extracted and cotransfected with HSV-1(F)A305DNA carrying a deletion in the TK gene. The TK' progenywas then tested for the presence of aTK-mM in the targetDNA fragment.

Construction ofRBMul and RBMu2 Recombinant Viruses.The power of the technique and the consequences of inser-tion of aTK-mM are readily illustrated by the insertionalmutagenesis of the 10.4-kbp BamHI B fragment ofHSV-1(F)DNA. This fragment spans the junction between the uniqueand the inverted repeat sequences of the L component nearthejunction between L and S components ofthe viral genome(Fig. 3) and contains the entire domain of the a27 gene anda portion of the domain of the aO gene. Fig. 4 shows theelectrophoretic profile of the plasmid DNA carrying theaTK-mM after cleavage with BgI II and Cla I, which cleaveonly once, within the aTK-mM and the plasmid vectorsequences, respectively. As would be predicted if aTK-mMwere to insert randomly, the cleaved plasmid DNA includingthe HSV-1 BamHIDNA sequences formed a ladder ofbands.Of the HSV-1(F) recombinants carrying the aTK-mM, two

were selected for detailed analysis. The first, designatedRBMu1, carried the entire aTK-mM inserted 4.8 kbp from theleft terminus of the BamHI B fragment. This was deduced asfollows. Digestion of aTK-mM with BamHI should yield the2.2-kbp aTK insert and two mini-MuDNA fragments, 7.4 and0.1 kbp in size, flanking the TK insert. Hybridization of

S ta i ne d BomHI B Probe

up~

Insert pRB112 InsertPool Pool

FIG. 4. Photograph of electrophoretically separated fragments inrestriction enzyme digests ofpRB112 plasmid DNA pools containingrandomly inserted aTK-mM sequences. pRB112 plasmid DNA andpRB112 DNA containing randomly inserted aTK-mM sequences(designated as insert pool) were removed from cesium chloridedensity gradients, digested with the restriction enzymes Bgi II andCla I, and electrophoretically separated in agarose gels. (Left)Photograph of ethidium bromide-stained gel. (Right) Autoradiogramof the bands transferred to a nitrocellulose sheet and hybridized to32P-labeled BamHI B fragment DNA as described (8).

Genetics: Jenkins et al.

IXIZ]Yi PI I K19

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electrophoretically separated BamHI fragments with mini-Mu DNA revealed a 12.2-kbp fragment (band 1 in Fig. 3C andFig. 5) that had to carry 7.4 kbp ofmini-Mu-phage and 4.8 kbpofBamHI B DNA from either its left or right terminus. Theexact orientation, as shown in Fig. 3, was deduced from KpnI and Bgi II/Kpn I digests. Thus, the entire aTK-mM wasinserted into the Kpn I J fragment (band 3 in Figs. 3 and 5).Cleavage of that fragment with Bgl II within the mini-Musequences yielded 12.2- and 6.0-kbp fragments (bands 6 and7 in Figs. 3 and 5), which defined the orientation of theaTK-mM and positioned the insert 4.8 kbp from the leftterminus ofBamHI B fragment inasmuch as, if the aTK-mMinsert were inverted and located 4.8 kbp from the rightterminus ofBamHI B fragment, the Kpn I/Bgl II digest wouldhave yielded 9.4- and 7.3-kbp fragments hybridizing with themini-Mu DNA probe.The second recombinant, designated RBMu2, presented

an entirely different picture. In this recombinant, the aTK-mM was inserted 5.3 kbp from the left terminus of the BamHIB fragment and either near or within the internal copy of theinverted repeats flanking the L component of HSV-1 DNA.In this recombinant, however, all ofthe sequences to the rightofthe site of insertion ofthe aTK-mM comprising the internalinverted repeats flanking the unique sequences of the L andS components were deleted. This conclusion is based on thefollowing considerations. The digests ofRBMu2 DNA failedto show the fragments BamHI B and N and Kpn I J and I attheir usual positions (Figs. 3 and 5). The absence ofBamHIB and Kpn I J fragments was expected inasmuch as theywould be predicted to contain the aTK-mM. The absence ofBamHI N and Kpn I I fragments (Fig. 5) suggested that theinternal inverted repeats were deleted. Such a deletion couldhave occurred if the promoter regulatory sequences of the a4gene that was fused to the aTK gene in the aTK-mM were torecombine with the homologous sequences in their naturallocation in BamHI N fragment such that all ofthe interveningsequences were deleted. The deleted sequences do not

StainedCL CL CL

£e h£eno 0.w 00.- 0

1e 0) -

IL

appear to be essential for virus growth inasmuch as arecombinant virus carrying a similar deletion was obtained inan earlier study (2). Fig. 3E shows the predicted structure ofthe region of RBMu2 DNA spanning the novel junctionbetween the L and S components. If this were the case, theBamHI N probe described in Materials and Methods shouldhybridize with the novel bands designated 9, 10, 12, and 14containing fragments 6.6, 22.2, 17.7, and 7 kbp in size. Thesefragments are readily seen in Fig. 5. Inasmuch as mini-MuDNA hybridized with a 12.7-kbp BamHI fragment ofRBMu2DNA, which must contain 5.3 kbp ofBamHI B DNA and 7.4kbp of mini-Mu DNA, the insertion of aTK-mM must be asshown diagrammatically in Fig. 3E.

DISCUSSIONHSV-1 DNA encodes an excess of 50 genes (13), not all ofwhich appear to be essential for growth in cell culture (14, 15).Another characteristic of herpes-virus DNAs is the presenceof numerous reiterated sequences whose function is poorlyunderstood. Examples of such reiterations in HSV-1 DNAare the left-terminal 9 kbp (sequence ab, Fig. 3) and right-terminal 6.5 kbp (sequence ca, Fig. 3), which are reiteratedinternally in an inverted orientation. Both detection andanalysis of the function ofgenes not essential for viral growthin cell culture and of the reiterations in the genomic DNArequire techniques for site-specific insertions and deletions.As noted in the Introduction, the procedure described byPost and Roizman (1) utilizes a selectable marker, the TKgene, for this purpose. This report describes an extension ofthis technique. Instead of inserting the TKgene into a desiredsite within the target sequence, E. coli doubly lysogenizedwith a helperMu phage and a mini-Mu-phage carrying the TKgene is transformed with a plasmid carrying the targetsequence. Straightforward steps in the procedure yield =24hr after the transformation a population of plasmids in whichthe aTK-mM is randomly inserted into the plasmid se-

Probe A Probe BCL a- CL L L CLa. 0mmX:. .

Y0. lemmCB a. It*6 0-aa. CD C a00CD0-a .. co 0.. 5)0m~eccomx m v c cO I' w ml m anm hea

1010w_ ~~~~4I

114 cmvQl 12am

so

1 A&

9da 14d7N

N4

A305 RBMu2 I RBMu1 A305 RBMu2 I RBMul

FIG. 5. Photograph of ethidium bromide-stained gels (Left) and autoradiograms (Right) of electrophoretically separated digests ofrecombinant virus DNAs hybridized with mini-Mu DNA (probe A) or the Pvu II-BamHI 3-kbp fragment from BamHI N fragment DNA (probeB). Ba, BamHI; Kp, Kpn I; Bg, BgI II; Bg/Kp, both Kpn I and BgI II. The numbers to the left of specific bands refer to the fragments identifiedby the same number in Fig. 3. The letters to the left of the figure identify the adjacent BamHI bands of the parent virus [HSV-1(F)A305)] DNAcarrying a deletion in the TK gene (AQ). The letters to the left of the second lane identify the Kpn I S component terminal fragment Kpn I I,

the L-S component junction fragment Kpn I Q1, and the fragment Kpn I F, whose map location is shown in Fig. 3. The letter N in theautoradiogram of DNA hybridized with probe B identifies the BamHI N fragment.

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Proc. Natl. Acad. Sci. USA 82 (1985) 4777

quences. This population can then be cotransfected into TK-cells with intact HSV-1 TK- DNA, and the viral progeny thatrecombined the aTK-mM by homologous recombinationthrough flanking sequences is readily selected under condi-tions favoring the growth of viruses expressing the TK'phenotype. The purpose offusing the ca4 promoter regulatorysequence to the TK coding sequence in the mini-Mu-phageDNA is to reduce the probability that the TK gene ip theaTK-mM will restore the deleted sequences in the natural TKgene of HSV-1(F)A305 virus inasmuch as the inserted aTKcarries homologous flanking sequences on only one side ofthe deleted sequences in HSV-1(F)A305. The advantages ofthis modification of the original technique rest on both thesimplicity of the insertion of the marker gene into the targetsequence as well as on the fact that the entire domain of thetarget sequence rather than the specific site ofinsertion oftheTK gene is probed by insertional mutagenesis.Relevant to the procedure described in this report are the

following considerations.(t) The size of the aTK-mM constructed for this study is

9.7 kbp. Since the size ofthe DNA packaged by the Mu phageis 38 kbp and since the packaged DNA should contain twocopies of the mini-Mu-phage for generation of the recombi-nant plasmid (Fig. 2B), the size of the plasmid should notgreatly exceed 18.6 kbp. We should note, however, thatsmaller mini-Mu constructs are feasible, although they maybe less efficient, and that both copies ofthe mini-Mu need notbe intact (unpublished data). Thus, we have obtained inser-tions of aTK-mM in plasmids in excess of 21 kbp.

(ii) As would be expected, we obtained a population ofHSV-1(F) recombinants differing in the site ofinsertion ofthecaTK-mM within the domain of the BamHI B fragment. Theinsertions are within the sequences between the 3' termini ofthe a27 and a0 genes, indicating that this domain of theBamHI B fragment is not essential for DNA replication. Thetwo recombinants described in this report illustrate the likelyoutcome of most insertions of aTK-mM. Thus, the recom-binant RBMul contains the entire aTK-mM inserted into thetarget fragment without generating apparent deletions, sug-gesting that the HSV.1 genome can readily accommodate theadditional 9.7 kbp of the aTK-mM DNA. The second recom-binant carries a spontaneous deletion that appears to extendfrom the site of the insertion across the L-S componentjunction. The deletion may have resulted from recombinationbetween the a4 gene promoter fused to the TK inserted intothe mini-Mu-phage and the corresponding homologous se-quences in the domain ofthe c4 gene. The deleted sequences,-"14 kbp, appear to consist entirely of the internal invertedrepeats ("'15 kbp). In this regard, RBMu2 resembles therecombinant 1358 carrying a similar deletion (2). That recom-binant also was generated by insertional mutagenesis, butinto the S rather than L component.

(iii) The L and S components of HSV-1 DNA invertrelative to each other, giving rise to four isomers present inequimolar concentrations both in the virions and the infectedcells (16). Moreover, the a4 gene is contained entirely withinthe inverted repeats ofthe S component (sequence c, Fig. 3A)and, therefore, is present in two copies per genome (17, 18).The domain ofthe a4 promoter-regulatory region fused to theTK gene in mini-Mu maps in BamHI N and Z fragments (18).This feature ofHSV-1 genome is relevant with respect to two

observations. First, although in both recombinants the aTKgene in the inserted mini-Mu was in the same, direct,orientation relative to the nearest homologous aTK promot-er, the spontaneous deletion occurred only in the recombi-nant carrying the aTK-mM nearest or within the L compo-nent inverted repeats. It remains to be seen whether thehomologous recombination resulting in the deletion of theinverted repeats is favored when the inverted repeats areperturbed by insertional mutagenesis. Second, the deletionsoccurred between the a4 sequences fused to the TK gene andthe corresponding sequences inBamHI N rather than BamHIZ fragment. Because of the inversions of the L and Scomponents, the a4 sequences in both BamHI N and BamHIZ fragments would bejuxtaposed in direct orientation relativeto the homologous sequences in the aTK-mM, To date, alldeletions of internal inverted repeats occurred betweensequences in BamHI B and BamHI N fragments-i.e., theone offour isomers designated as the prototype (19). In all ofthese instances, the DNA was frozen in prototype arrange-ment (2). The significance of this observation discussed atlength elsewhere remains to be determined.

These studies were aided by Grants CA-08494 and CA-19264 fromthe National Cancer Institute, Grant MV-2T from the AmericanCancer Society, and Grant GM29067 from the National Institutes ofHealth. F.J.J. is a United States Public Health Service PostdoctoralTrainee (PHS-5-T32-CAO9241), and M.J.C. is the recipient of aNational Institutes of Health Research Career Development Award(AI00468).

1. Post, L. E. & Roizman, B. (1981) Cell 25, 227-232.2. Poffenberger, K. L., Tabares, E. & Roizman, B. (1983) Proc.

Natl. Acad. Sci. USA 80, 2690-2694.3. Voss, J. H. & Roizman, B. (1985) J. Virol., in press.4. Castilho, B. A., Olfson, P. & Casadaban, M. J. (1984) J.

Bacteriol. 158, 488-495,5. Campione.-Piccardo, J., Rawls, W. E. & Bacchetti, S. (1979) J,

Virol. 31, 281-287.6. Post, L. E., Mackem, S, & Roizman, B. (1981) Cell 24,

555-565.7. Ejercito, P. M., Kieff, E. D. & Roizman, B. (1968) J. Gen.

Virol. 2, 357-364.8. Mackem, S. & Roizman, B. (1982) J. Virol. 44, 939-949.9. Post, L. E., Conley, A. J., Mocarski, E. S. & Roizman, B.

(1980) Proc. Natl. Acad. Sci. USA 77, 4201-4205.10. Groisman, E. A., Castilho, B. A. & Casadaban, M. J. (1984)

Proc. Natl. Acad. Sci. USA 81, 1480-1483.11. Bukhari, A. I. & Ljungquist, E. (1977) in DNA Insertion

Elements, Plasmids and Episomes, eds. Bukhari, A. I., Sha-piro, J. A. & Adhya, S. L. (Cold Spring Harbor Laboratory,Cold Spring Harbor, NY), pp. 749-756.

12. Bukhari, A. I. (1976) Annu. Rev. Genet. 10, 389-412.13. Honess, R. W. & Roizman, B. (1974) J. Virol. 14, 8-19.14. Dubbs, D. R. & Kit, S. (1964) Virology 22, 493-502.15. Heine, J. W., Honess, R. W., Cassai, E. & Roizman, B. (1974)

J. Virol. 14, 640-651.16. Hayward, G. S., Jacob, R. J., Wadsworth, S. C. & Roizman,

B. (1975) Proc. Natl. Acad. Sci. USA 72, 4243-4247.17. Knipe, D. M., Ruyechan, W. T., Roizman, B. & Halliburton,

I. W. (1978) Proc. Natl. Acad. Sci. USA 75, 3896-3900.18. Mackem, S. & Roizman, B. (1980) Proc. Natl. Acad. Sci. USA

77, 7122-7126.19. Morse, L. S., Pereira, L., Roizman, B. & Schaffer, P. A.

(1978) J. Virol. 26, 389-410.

Genetics: Jenkins et al.

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