Proc. Natl. Acad. Sci. USAVol. 91, pp. 6186-6190, June 1994Genetics

Efficient manipulation of the human adenovirus genome as aninfectious yeast artificial chromosome cloneGARY KETNER*t, FORREST SPENCER§, STUART TUGENDREICH§, CARLA CONNELLY§, AND PHILIP HIETER§*Department of Immunology and Infectious Diseases, Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD 21205; $Center forMedical Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 212874922; and *Department of Molecular Biology and Genetics,Johns Hopkins University School of Medicine, Baltimore, MD 21205

Communicated by Hamilton 0. Smith, March 9, 1994

ABSTRACT A yeast artIal chromosome (YAC) contain-ing a complete human adenovirus type 2 genome was con-structed, and viralDNA derived from the YAC was shown to beinfectious upon introduction into m cells. The adeno-virus YAC could be mi eicintl using ho usrecombination-based methods in the yeast host, and mutantviruses, ing a variant that expresses the human analog ofthe Saccharomyces cerevuia CDC27 gene, were readily recov-ered from modified derivatives of the YAC. The applaon ofpowerful yeast genetic teniques to an infectious adenovirusclone promises to s ftly enhn the genetic analysis ofadenovirus and to simple the construction ofadenovirus-basedvector for vaccines or for gene trner to ma an c orwhole animals. The adenovirus YAC was produced by homol-ogous recombination w between adenovirus 2 virion DNAandYAC vector plamids cying segments othe viral left andright ic termini. This recombional cloning strategy isgenerally applicable to the construction of YACs containother DNA segments, such as the genms of other viruses.Further, It is very i t and may permit the targ dcloningof segments of the genes of higher orgasms directly fromgeno DNA.

Adenoviruses are among the most widely exploited experi-mental model systems for studies of basic eukaryotic molec-ular biology. The splicing of eukaryotic mRNAs was firstobserved in studies of adenoviruses (1), the in vitro adeno-viral DNA replication system was the first for a eukaryoticreplicon (2), and binding of the product of the RB gene to aviral oncoprotein was first demonstrated in adenovirus-infected cells (3). Adenoviruses also promise to be valuableas vectors for gene transfer to whole organisms or to indi-vidual cells for the purposes ofvaccination and gene therapy.For example, an adenovirus-based recombinant rabies vac-cine has been shown to be efficacious in dogs (4), andadenovirus-based gene transfer vectors have been used forthe transfer ofa human gene to the lung epithelium ofrats (5).The study ofbasic adenoviral biology and the development

of adenoviral vectors both require manipulation of the viralgenome: in the first case for the production of viral mutantsfor genetic studies and in the second to incorporate exoge-nous DNA into the viral genome and to optimize its expres-sion. Manipulation of adenoviral DNA is possible usingcurrent in vitro and Escherichia coli-based technology, butthe schemes employed are time-consuming. In contrast, avariety ofmethods are available for the rapid manipulation oflarge DNA segments in yeast. To make it possible to applyyeast genetic methods to the adenoviral genome, we haveconstructed in Saccharomyces cerevisiae a yeast artificialchromosome (YAC) that contains a complete copy of thelinear 36-kb adenovirus type 2 (Ad2) genome. The cloned

adenoviral genome is infectious, the viral sequences can bemodified efficiently using conventional yeast genetic tech-niques and vectors described below, and mutant viruses canbe recovered from modified YAC clones. Remarkably, therecombinational strategy used to construct the adenovirusYAC proved to be very efficient. Our data suggest that it willbe of use in the targeted cloning of specific genomic DNAsegments from higher organisms.

MATERIALS AND METHODSPlasds. The YAC vector pair pRML1 and pRML2 are

described in Spencer et al. (6). Together, these plasmidscontain the cis-acting genetic elements required for YACmaintenance in yeast (telomeres, a centromere, and origins ofreplication), genetic markers used to select yeast cells con-taining the YAC (TRPI and URA3), and a system derivedfrom pCGS966 (7) that permits amplification of the YAC bygrowth in selective medium (a conditional centromere and theherpes simplex virus thymidine kinase gene underthe controlof the yeast DEDI promoter). pRMLlAd2L was made byinserting a segment ofthe left end ofthe Ad2genome betweenEcoRI and Cla I sites ofpRML1; pRML2Ad2R was made byinserting a segment of the right end of the Ad2 genomebetween the EcoRI and Bgl II sites of pRML2. Both viralfiagments were produced from Ad2 virionDNA by PCR. Theviral portion of the genomic terminal primer used to amplifyboth fragments (5'-CCGAATTCTACGTACATCALL TAITACC-3'; adenoviral sequence is underlined)differed from the published Ad2 sequence (8) by the insertionof an adenosine residue (boldface type) between positions 7and 8, and the primer contained both a SnaBI site immedi-ately adjacent to the viral terminal sequence and an EcoRIsite used for cloning. The internal primer used to produce theleft-hand fiagment included Ad2 nt 988-1005; the primerusedto produce the right-hand fragment covered Ad2 nt 34,357-34,374. The Cla I and Bgl II sites used for cloning occurnaturally in Ad2 DNA at nt 916 and 34,390, respectively.

p680, an integrating plasmid containing theLEU2 gene andthe dominant cycloheximide-sensitivity allele (CYH2') ofCYH2 (9), is described in Spencer et al. (6). p680E3A wasconstructed by insertion of two PCR fragments (Ad2 nt27,410-28,404 and 30,801-31,825) between Xho I and Sal Isites and between Xba I and Sac II sites, respectively, in thepolylinker of p680. For construction of H2subCDC72Hs, acDNA copy of the CDC27Hs gene (10) fused at its 3' end tosequences encoding two repetitions of the C-terminal 28amino acids of the avian infectious bronchitis virus Elglycoprotein (11) was inserted between the Sal I and Xba Isites in p68OE3A. Construction details of the epitope-taggedversion of CDC27Hs will be presented elsewhere.

Abbreviations: YAC, yeast artificial chromosome; Ad2, adenovirustype 2; E3, early region 3.1To whom reprint requests should be addressed.

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Yeast Methods. Yeast selective media are described inRose et al. (12). For the preparation of YACs, spheroplastsof the yeast strain YPH857 (MATa, ade2-101, cyh2r, leu2A1,lys2-801, his3A200, trp1A63, ura3-52; ref. 6) were trans-formed with equimolar quantities of Ad2 virion DNA,pRML1Ad2L linearized with Cla I, and pRML2Ad2R linear-ized with Bgl II (10 pug, total). For two-step gene replace-ments, YAC-containing strains were transformed by thelithium acetate method. Detailed protocols are presented inSpencer et al. (6). Amplification of the adenovirus YAC wasby growth of YAC-containing strains in selective mediumcontaining thymidine (800 ug/ml), sulfanilamide (1 mg/ml),and methotrexate (10 gg/ml), with galactose as the carbonsource (7). Cultures were inoculated with 105 cells per ml;growth to saturation took 3-5 days at 30'C. Amplified cul-tures were diluted 1:50 into YPD and grown overnight beforeanalysis by pulsed-field gel electrophoresis or preparation ofDNA for transfection. High molecular weight yeast DNA wasprepared by a protocol developed by A. Wilmen, M. Funk,and J. Hegemann (personal communication). Cells werecollected by centrifugation from 500-ml YPD cultures of cellsat 5x 107 cells per ml, washed once in water, suspended in20 ml of 1 M sorbitol/4.25 mM KH2PO4/42.75 mMK2HPO4/20 mM dithiothreitol, and incubated for 1 hat 370C.Cells were collected and incubated for 1 h at 370C in 20 ml of1 M sorbitol/1 mM EDTA (DB) containing zymolyase 20T(200 pg/ml). The resulting spheroplasts were collected,washed once in DB, resuspended in 15 ml of 20 mM Mops/0.84 mM spermine/2.16 mM spermidine/10 mM K2EDTA/2mM EGTA/1 mM aminoacetonitrile/1 mM phenylmethyl-sulfonyl fluoride/0.2% Triton X-100/1% thiodiglycol, anddisrupted by five strokes of a glass Potter B type homoge-nizer. Undisrupted spheroplasts were removed by centrifu-gation at 120x g for 10 min; nuclei were collected bycentrifugation at 3000x g for 10 min, resuspended in 0.5 mlof 20 mM EDTA/1% SDS/20 mM TrisHCl, pH 8.0/proteinase K (50 pug/ml), and incubated at 65°C for 2-5 h.Potassium acetate (0.5 ml of 5 M) was added and the sampleswere placed at4°C for 30 min. The precipitate was removedby centrifugation, and nucleic acids were precipitated fromthe supernatant by addition of an equal volume of isopro-panol. The pellet was gently dissolved in 10mM Tris HCl, pH8.1/1 mM EDTA (TE) containing RNase A (200 ug/ml) andincubated for 2 hat37°C; DNA was precipitated with ethanoland gently dissolved in TE.

RESULTSRecombinatal C ing of the Adenovfrus Genome. Ade-

novirus YACs were constructed by recombination in vivobetween Ad2 virion DNA and linear forms of the YAC vectorplasmids pRML1Ad2L and pRML2Ad2R after transforma-tion of the three DNAs into yeast spheroplasts (Fig.1A).After transformation, TRPI URA3 transformants were se-lected, colony-purified, and examined to identify YAC-containing strains.

In strains that contain an adenovirus YAC, the TRPI andURA3 genes will be linked, and as a consequence, URA3 willbe retained under conditions that select for TRP1. To test thestability of URA3, transformants were plated on medium thatselects for TRPI and the resulting colonies were replica-plated on medium that selects for TRPI and against URA3[with 5-fluoroorotic acid (FOA) at 1 mg/ml (13)]. Theseconditions reveal transformants in which URA3 can be lostwhile TRPI remains: such strains will produce FOA-resistant(TRPI, ura3) segregants. Forty-seven ofthe 48 transformantstested yielded no FOA-resistant segregants, suggesting thatURA3 and TRP1 are linked in those strains.Ten transformants in which TRPI and URA3 appeared to

be linked were then examined by pulsed-field gel electropho-

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FIG. 1. (A) Construction of an adenovirus YAC by recombina-tional cloning. (i) Maps of the plasmids pRMLlAd2L andpRML2Ad2R.(ii) Recombination events ( H ) required to assemblean adenovirus YAC from Ad2 virion DNA and linear pRMLlAd2Land pRML2Ad2R DNAs. (iii) Map of the expected adenovirus YAC.CEN, centromere; TEL, telomere; ARS, yeast autonomously repli-cating sequence; amp and oni,ampicillin-resistance gene and plasmidorgin of replication; TK, herpes simplex virus thymidine kinasegene; DED) and GAL], yeastDEDI and GALI promoters; Cla I,Bg1H, and SnaBI, restriction enzyme cleavage sites. The positions of theinternal restriction sites used to clone the adenovirus segments areindicated. (B) Construction of an adenovirus E3 deletion mutant bytwo-step gene replacement. YAC-containing yeast strains are trans-formed with p68OE3A linearized at an Nde I site within the Ad2sequences. Recombination between the YAC and the plasmid at thesite of linearization (dashed line)(i) integrates theplasmid into theYAC, producing a YAC with two copies of E3 bracketing theremaining plasmid sequences(ii). Homologous recombination be-tween the duplicated portions of E3 (dashed lines) can excise theplasmid leaving a single copy of E3 in the YAC. Depending uponwhere the recombination event occurs, either a mutant or a wild-typecopy remains (iii). Excision also removes the plasmid LEU2 andCYH2S genes. A, A' and B, B' are homologous segmentsflanking theregion of E3 deleted in p68OE3A. V, Deletion mutation. The origin(plasmid or YAC) of the E3 sequences is indicated by shading.

resis (14, 15) and Southern blot hybridization (16) to deter-mine whether they contained a YAC of the expected sizecarrying adenoviral DNA sequences. Seven of the transform-ants produced a single band close to the size of the predictedadenovirus YAC (53 kb) that hybridized to labeled Ad2 virionDNA, two produced the 53-kb band and additional bands of

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90-150 kb, and the final strain produced a faint band of =150kb (not shown). DNA prepared (6) from the seven strainscontaining only a 53-kb band produced the bands expectedfrom an adenovirus YAC of the proposed structure afterdigestion with Afl II and SnaBI or with Afl II alone andanalysis by Southern blot hybridization (not shown). Thesedata are consistent with the interpretation that nearly all ofthe original TRP1 URA3 transformants contain an adenovirusYAC with the structure diagramed in Fig. 1A, created byhomologous recombination between viral sequences carriedon the linearized YAC vector plasmids and Ad2 virion DNA.

Adenovirus YAC DNA Is Infectious. High molecular weightDNA was prepared from three YAC-containing strains afteramplification. Asjudged by comparison ofthe intensity oftheamplified YAC band to that of the smallest yeast chromo-some on a pulsed-field electrophoresis gel (Fig. 2), amplifi-cation in these strains is consistently p40-fold. The DNAextracted from each strain was digested with SnaBI to excisethe viral genome from the YAC and the digested DNAs wereintroduced into 293 cells (17), an adenoviral host cell line, bycalcium phosphate transfection (18). Adenoviral plaquesarose on transfected dishes at a frequency of2-10 plaques perpg of total yeast DNA. By assuming 10 copies of theadenovirus YAC per cell after amplification, this correspondsto a specific infectivity of100-500 plaque-forming units/pg ofviral DNA, comparable to that obtained for deproteinizedAd2 virion DNA in parallel transfections. The viral DNAproduced by cleavage of a YAC with SnaBI will contain a3-base remnant of the SnaBI site at each end; apparently,these extra bases do not substantially reduce the infectivityof the excised genome. No plaques have been observed onplates transfected with a total of 20 pLg of undigested DNA.Thus, SnaBI digestion increases the infectivity ofadenovirusYAC DNA at least 40- to 200-fold.

Construction of an Adenovfrus Mutant by Two-Step GeneReplacement. A variety of techniques have been developedfor the genetic manipulation of YACs and natural chromo-somes in yeast cells. One of these, two-step gene replace-ment, exploits targeted homologous recombination in trans-

formed cells to replace a selected segment ofyeastDNA witha mutant or exogenous sequence (19). To confirm the suit-ability of the adenovirus YAC for manipulation by suchtechniques, two-step gene replacement was used to introducean early region 3 (E3) deletion mutation into the viral DNA.An Ad2 DNA segment encompassing E3 (Ad2 nt 27,410-31,825, with sequences from nt 28,404 to 30,801 replaced bya polylinker) was constructed in p680, a plasmid specificallydesigned for use in two-step gene replacements in YACs inconjunction with the leu2 and cyh2r markers present inYPH857. The resulting plasmid, p680E3A (Fig. lBi) waslinearized by cleavage at a unique Nde I site in E3 (Ad2residue 31,076) and was introduced into a YAC-containingstrain. LEU2 transformants were selected. Linearization ofp680E3A within the Ad2 sequence targets recombination tothat site, and transformants should contain YACs with acomplete copy of p680E3A integrated at the Ad2 E3 Nde Isite. These YACs will carry two copies of E3, one wild-typeand one mutant, separated by an integrated copy of the p680plasmid (Fig. lBii).YPH857 (cyh2r) is resistant to cycloheximide. However,

p680 includes the dominant sensitivity allele (CYH2') ofCYH2 (9) and strains carrying aYAC with an integrated copyof p680E3A are cycloheximide-sensitive. During growth ofsuch strains, spontaneous homologous recombination eventsbetween the tandemly duplicated E3 segments can excise thep680 sequences and one copy of E3 from the YAC. Thoseevents restore cycloheximide resistance and, dependingupon the site at which recombination occurs, generate aYACbearing either wild-type E3 or a deletion mutant derivative(Fig. lBiii). Thus, E3 mutant YACs should be found amongcycloheximide-resistant segregants of strains transformed byp680E34. Several LEU2 transformants were grown over-night in YPD broth to permit excision of the plasmid byrecombination, and 100l- portions of the cultures wereplated on medium that selects for URA3, TRPI, and cyh2r.Sixteen segregants derived from three transformants wereexamined by Southern blot hybridization (Fig. 3). Ten con-tained a wild-type YAC and 4 carried E3 deletion mutant

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FiG. 3. (Left) Identification of E3 deletion mutant YACs aftertwo-step gene replacement. DNAs prep from 16 strains gener-ated by the two-step gene replacement protocol from p680AE3 andYAC strains 28, 34, and 43 were digested with EcoRI and examinedby Southern blot hybridization. Symbols beneath each lane of theautoradiograms indicate the genotype of the YAC present in thecorresponding strain: A, E3 deletion mutant; +, wild type; *, YACretaining the E3 duplication and an integrated copy of p680. Thepositions and sizes of the EcoRI fiagments of wild-type Ad2 areindicated on the left; the bullets indicate the two new frgentsproduced by EcoRI digestion of E3 deletion mutant DNA. (Right)Ethidium bromide-stained agarose gel displaying EcoRI restrictionfragments of H2dI810, an E3 mutant virus derived from YAC strain34.7, and wild-type Ad2.

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YACs. Two strains carried YACs that retained the integratedp680E3A sequences and may have become cycloheximide-resistant by mutation or conversion of CYH2'.

Viruses were recovered from two strains carrying deletionmutant YACs by transfection of 293 cells with amplifiedSnaBI-digested DNA. All DNAs prepared from 21 viralisolates examined by restriction enzyme digestion and gelelectrophoresis showed the restriction pattern expected foran E3 mutant (data not shown). These experiments confirmthat the adenovirus YAC can be manipulated by conventionalyeast genetic techniques and that viral mutants can be readilyrecovered from a modified YAC.Conston of an Adenovirus Expression Vector. Adeno-

viruses have been used as vectors for the expression offoreign DNAs in cells and whole animals (4, 5). To demon-strate the feasibility of producing vectors by manipulation ofthe adenovirus YAC, an adenovirus derivative that expressesan exogenous gene was constructed. The p680E3A polylinkerreplaces the majority of E3 coding sequences (ref. 20; Fig.4A), and adenovirus mutants containing insertions at that siteshould express the inserted DNA under the control of E3regulatory sequences. An epitope-tagged version of the hu-man homolog of the yeast CDC27 gene (CDC27Hs; ref. 10)was inserted in the p680E3A polylinker, the resulting E3::CDC27Hs substitution mutation was introduced into theadenovirus YAC by two-step gene replacement, and a viruscontaining the substitution (H2subCDC27Hs) was recov-ered. Extracts prepared 22 h after infection ofHeLa cells byH2subCDC27Hs were examined by SDS/PAGE and immu-noblot analysis using polyclonal antisera directed againsteither the epitope tag or bacterially expressed CDC27Hs.Both antisera detected a protein at the expected position ofepitope-tagged CDC27Hs in cells infected with H2sub-CDC27Hs but not in cells infected with wild-type adenovirus

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FIG. 4. Expression of the human CDC27 gene by H2sub-CDC27Hs. (A) The positions of the E3 cap and polyadenylylationsites and the common E3 splice donor and acceptor sites areindicated on a diagram ofE3 mRNA c (20). The adenoviral segmentspresent in p680E3A (E3A and E3B) are shown below. Numbers referto nucleotide position on the Ad2 genome. (B) Extracts from HeLacells infected with H2subCDC27Hs or with adenovirus type 5 (Ad5)and extracts from uninfected cells were fractionated on an SDS/10%opolyacrylamide gel, transferred to a nylon membrane filter, andprobed with antisera directed against the epitope tag El or the intactprotein CDC27Hs.

or in uninfected cells (Fig. 4B). Material reactive with bothantisera was also found in cells infected with H2sub-CDC27Hs and incubated in the presence of cytosine arabi-noside, which blocks the replication of adenoviral DNA andrestricts expression of the viral genome to early regions (21).The antiserum against the intact protein reacted with thetagged CDC27Hs in H2subCDC27Hs-infected cells and withendogenous CDC27 present all extracts. These data demon-strate that H2subCDC27Hs expresses the CDC27Hs geneand suggest that the expression ofCDC27Hs is driven at leastin part by E3 regulatory signals.Recombinatonly Targeted Coning. The recombinational

cloning method used to construct the adenovirus YAC couldin principle be applied to the cloning ofany DNA segment asaYAC ifDNA fragments that bracket the target segment canbe obtained. Further, the extremely efficient production ofadenovirus YACs in our original experiments suggested to usthat it might be possible to use recombination in vivo to clonespecific segments ofDNA from sources such as the genomicDNA of higher organisms, in which the target is a minorcomponent. To determine the efficiency of YAC formationfrom a rare target segment, a series of experiments wasperformed in which decreasing amounts of Ad2 DNA mixedwith mouse DNA carrier were introduced, along with linear-ized pRML1Ad2L and pRML2Ad2R DNAs, into YPH857spheroplasts. The total amount of the DNA mixture (virusplus mouse) was kept constant, while the proportion of viralDNA in the mixture was varied from exclusively Ad2 to 1/105by mass. The latter is approximately equivalent to one copyof the viral genome (3.6 x 104 bp) per mouse haploid genomeequivalent (3 x 109 bp). TRPI URA3 transformants arisingfrom each transformation were tested for stability of URA3under selection for TRPI, for the presence of Ad2 DNAsequences by colony hybridization to an internal Ad2 probe(22), and for the presence of an adenovirus YAC by pulsed-field electrophoresis and conventional Southern blot hybrid-ization (Table 1).The frequency of transformation to TRPI URA3 was

independent of the amount ofadenovirus DNA present in thetransformation. The number oftransformants in which URA3was stable under selection for TRPI decreased with decreas-ing Ad2 DNA to a minimum of -25% of transformants at a

Table 1. Efficiency of recombinationally targeted cloningFraction

Ad2 URA3 Hybridization Authentic EquivalentDNA stable to Ad2 probe Ad2 YACs genome1 0.96

10-1 0.75 0.98 (52)10-2 0.56 E. coli10-3 0.24 0.07 (156) C. albicans10-4 0.31 0.007 (444) 3/3 Drosophila,

C. elegans10s 0.25 None (384) MammalsNone 0.23YPH857 spheroplasts were transformed with linearized pRML-

1Ad2L (2.8 ,Ag) and pRML2Ad2R (1.2 lAg) DNAs and with a total of6.7 pg of a mixture of Ad2 and mouse DNAs. In different transfor-mations, the fractions of viral DNA in this mixture varied from 1 to10-5 (as indicated). The fraction of transformants in which URA3was mitotically stable under selection for TRPI, the fraction thathybridized to an internal Ad2 DNA fragment, and the proportion ofhybridization-positive strains that harbored an authentic adenovirusYAC Budged by size or structure) were determined. For the ora-nisms listed in the final column, a single-copy 36-kb DNA segmentconstitutes a fraction of the haploid genome equal to or larger thanthe fraction of the input DNA made up of Ad2 in the correspondingtransformation. Number oftransformants screened is in parentheses.C. albicans, Candida albicans; C. elegans, Caenorhabditis elegans.

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level of viral DNA corresponding to -100 viral DNA mole-cules per mouse genome equivalent (Table 1, line 4) andremained constant at lower levels and in the absence of Ad2DNA. The fraction of transformants that hybridized to theAd2 probe also decreased with decreasing input Ad2 DNA toa frequency of 0.7% at 10 viral DNA molecules per mousegenome equivalent (line 5). No hybridizing transformantswere detected among 384 colonies screened in the transfor-mation performed at 1 viral DNA molecule per mousegenome equivalent (line 6). Of the three strains from the10-copy transformation identified by colony hybridization ascontaining viral DNA, all contained a YAC of the expectedsize and/or with the expected structure as assessed bySouthern blot hybridization (data not shown). It is thereforepossible with modest effort to recover segments ofDNA byrecombinational YAC cloning at levels of representation aslow as 10 copies per mammalian genome equivalent.

DISCUSSIONRecombination-based methods for the manipulation of theadenoviral genome in yeast have several advantages overconventional methods for the construction of mutants: theyare applicable to any region of the viral genome, they areindependent of the placement of restriction sites in the viralDNA and, since microbiological cloning is accomplished inthe yeast host, they eliminate the need for time-consumingplaque purification of mutant virus. Because constructionsinvolving several successive modifications of the viral ge-nome can be carried out without the isolation of viral inter-mediates, such methods will be particularly useful in thepreparation ofmultiple mutants and complex viral vectors forgene delivery. It should be noted that mutant virus containinginternal SnaBI sites cannot easily be produced from theexisting adenovirus YAC.The recombinational strategies used to make and manip-

ulate the adenovirus YAC have obvious application to otherviral genomes. For example, we have used recombinationalcloning to construct YAC clones of simian immunodeficiencyvirus (SIV) and human immunodeficiency virus and haveproduced a SIV mutant by two-step gene replacement (D.Hauer, G.K., and J. E. Clements, unpublished data). YACscontaining cDNA copies of conventional RNA viruses couldalso be constructed. For each viral YAC, a method forproducing infectious nucleic acid is required. For the aden-ovirus YAC, infectious DNA can be. excised from the YACby restriction enzyme digestion, and the SIV YAC is itselfinfectious. For large viral DNAs, it may be possible to use anexcisional strategy and a nuclease with a long recognitionsequence. For positive-strand viruses whose genomic RNAis infectious, transcription of the cDNA from an appropri-ately positioned yeast promoter might produce infectiousviral RNA in the yeast cell. It may even be possible to extendthe technique to negative-strand RNA viruses by co-producing the viral replicase and genome-like negative-strand RNA in yeast and by transferring both components toanimal cells by spheroplast fusion.The high efficiency offormation ofadenovirus YACs in the

original transformations suggested that the targeted produc-tion ofYAC clones of specific segments from complex DNAscould be accomplished by recombinational cloning. In re-construction experiments that used small amounts of aden-oviralDNA in large excesses ofmammalianDNA to simulaterareDNA segments, it proved possible to recover adenovirusYACs from mixtures that contained as few as 10 copies ofadenoviral DNA per haploid mammalian genome equivalent.Because the efficiency of recombinational cloning may beaffected by factors such as the size of the target and positionof the physical ends of the target segment, the data obtainedin the reconstruction may only approximate the results that

can be expected when cloning a segment from genomic DNA.Nevertheless, the recovery of adenovirus YACs Erom DNAmixtures containing very small proportions of Ad2 DNAstrongly indicates that for genomes of lower complexity thanthat of the mouse (such as Drosophila melanogaster, Cae-norhabditis elegans, and Candida albicans), single-copygenes can be obtained directly by recombinationally targetedYAC cloning. In the reconstruction experment, YAC recov-ery declined in a roughly linear fashion with decreasing Ad2DNA content, suggesting that single-copy segments in mam-malian DNA may be recoverable simply by increasing thescreening effort (the expected yield is a1 YAC per 1500transformants). Alternatively, enrichment of target se-quences (for example, by pulsed-field gel electrophoresis) orrefinement of the technique (for example, optimization oftheratios of YAC vector plasmids to target DNA) may permitmore efficient recovery ofYACs from mammalian DNA. Theability to rapidly produce YAC clones of specific segments ofa mammalian genome could be of value for several applica-tions including the isolation of genomic clones of multipleallelic variants of a cloned gene, for obtaining cognates ofcloned genomic regions from related species, or for obtainingclones covering gaps in existing libraries.

We thank Dr. J. H. Hegemann, Institute for Microbiology andMolecular Biology, Justus Liebig University, Giessen, Germany, forkindly furnishing his protocol for the preparation of high molecularweight yeast DNA prior to publication. This work was supported bygrants from the National Institutes of Health to G.K. (A126239) andP.H. (CA16519 and HD24605) and from the American CancerSociety to F.S. (CD509).

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