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Efficient Manipulation of the Human Adenovirus Genome As an Infectious Yeast Artificial Chromosome Clone

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Efficient Manipulation of the Human Adenovirus Genome As an Infectious Yeast Artificial Chromosome Clone Proc. Natl. Acad. Sci. USA Vol. 91, pp. 6186-6190, June 1994 Genetics Efficient manipulation of the human adenovirus genome as an infectious yeast artificial chromosome clone GARY 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 for Medical 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- adenoviral genome is infectious, the viral sequences can be ing a complete human adenovirus type 2 genome was con- modified efficiently using conventional yeast genetic tech- structed, and viral DNA derived from the YAC was shown to be niques and vectors described below, and mutant viruses can infectious upon introduction into m cells. The adeno- be recovered from modified YAC clones. Remarkably, the virus YAC could be mi eicintl using ho us recombinational strategy used to construct the adenovirus recombination-based methods in the yeast host, and mutant YAC proved to be very efficient. Our data suggest that it will viruses, ing a variant that expresses the human analog of be of use in the targeted cloning of specific genomic DNA the Saccharomyces cerevuia CDC27 gene, were readily recov- segments from higher organisms. ered from modified derivatives of the YAC. The applaon of powerful yeast genetic teniques to an infectious adenovirus MATERIALS AND METHODS clone promises to s ftly enhn the genetic analysis of adenovirus and to simple the construction ofadenovirus-based Plasds. The YAC vector pair pRML1 and pRML2 are vector for vaccines or for gene trner to ma an c or described in Spencer et al. (6). Together, these plasmids whole animals. The adenovirus YAC was produced by homol- contain the cis-acting genetic elements required for YAC ogous recombination w between adenovirus 2 virion DNA maintenance in yeast (telomeres, a centromere, and origins of and YAC vector plamids cying segments othe viral left and replication), genetic markers used to select yeast cells con- right ic termini. This recombional cloning strategy is taining the YAC (TRPI and URA3), and a system derived generally applicable to the construction of YACs contain from pCGS966 (7) that permits amplification of the YAC by other DNA segments, such as the genms of other viruses. growth in selective medium (aconditional centromere andthe Further, It is very i t and may permit the targ dcloning herpes simplex virus thymidine kinase gene underthe control of segments of the genes of higher orgasms directly from of the yeast DEDI promoter). pRMLlAd2L was made by geno DNA. inserting a segment ofthe left end ofthe Ad2genome between EcoRI and Cla I sites ofpRML1; pRML2Ad2R was made by Adenoviruses are among the most widely exploited experi- inserting a segment of the right end of the Ad2 genome mental model systems for studies ofbasic eukaryotic molec- between the EcoRI and Bgl II sites of pRML2. Both viral ular biology. The splicing of eukaryotic mRNAs was first fiagments were produced from Ad2 virion DNA by PCR. The observed in studies of adenoviruses (1), the in vitro adeno- viral portion of the genomic terminal primer used to amplify viral DNA replication system was the first for a eukaryotic both fragments (5'-CCGAATTCTACGTACATCAL replicon (2), and binding of the product of the RB gene to a L TAITACC-3'; adenoviral sequence is underlined) viral oncoprotein was first demonstrated in adenovirus- differed from the published Ad2 sequence (8) by the insertion infected cells (3). Adenoviruses also promise to be valuable of an adenosine residue (boldface type) between positions 7 as vectors for gene transfer to whole organisms or to indi- and 8, and the primer contained both a SnaBI site immedi- vidual cells for the purposes ofvaccination and gene therapy. ately adjacent to the viral terminal sequence and an EcoRI For example, an adenovirus-based recombinant rabies vac- site used for cloning. The internal primer used to produce the cine has been shown to be efficacious in dogs (4), and left-hand fiagment included Ad2 nt988-1005; theprimerused adenovirus-based gene transfer vectors have been used for to produce the right-hand fragment covered Ad2 nt 34,357- the transfer ofa human gene to the lung epithelium ofrats (5). 34,374. The Cla I and Bgl II sites used for cloning occur The study ofbasic adenoviral biology and the development naturally in Ad2 DNA at nt 916 and 34,390, respectively. of adenoviral vectors both require manipulation of the viral p680, an integrating plasmid containing the LEU2 gene and genome: in the first case for the production of viral mutants the dominant cycloheximide-sensitivity allele (CYH2') of for genetic studies and in the second to incorporate exoge- CYH2 (9), is described in Spencer et al. (6). p680E3A was nous DNA into the viral genome and to optimize its expres- constructed by insertion of two PCR fragments (Ad2 nt sion. Manipulation of adenoviral DNA is possible using 27,410-28,404 and 30,801-31,825) between Xho I and Sal I current in vitro and Escherichia coli-based technology, but sites and between Xba I and Sac II sites, respectively, in the the schemes employed are time-consuming. In contrast, a polylinker of p680. For construction of H2subCDC72Hs, a variety ofmethods are available for the rapid manipulation of cDNA copy of the CDC27Hs gene (10) fused at its 3' end to large DNA segments in yeast. To make it possible to apply sequences encoding two repetitions of the C-terminal 28 yeast genetic methods to the adenoviral genome, we have amino acids of the avian infectious bronchitis virus El constructed in Saccharomyces cerevisiae a yeast artificial glycoprotein (11) was inserted between the Sal I and Xba I chromosome (YAC) that contains a complete copy of the sites in p68OE3A. Construction details of the epitope-tagged linear 36-kb adenovirus type 2 (Ad2) genome. The cloned version of CDC27Hs will be presented elsewhere. The publication costs ofthis article were defrayed in part by page charge Abbreviations: YAC, yeast artificial chromosome; Ad2, adenovirus payment. This article must therefore be hereby marked "advertisement" type 2; E3, early region 3. in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1To whom reprint requests should be addressed. 6186 Downloaded by guest on September 25, 2021 Genetics: Ketner et al. Proc. Nadl. Acad. Sci. USA 91 (1994) 6187 Yeast Methods. Yeast selective media are described in A pRMLIAd2L pRML2Ad2R Rose et al. (12). For the preparation of YACs, spheroplasts TEL X-- of the yeast strain YPH857 (MATa, ade2-101, cyh2r, leu2A1, ....\Ad2 lys2-801, his3A200, trp1A63, ura3-52; ref. 6) were trans- formed with equimolar quantities of Ad2 virion DNA, TK CEN pRML1Ad2L linearized with Cla I, and pRML2Ad2R linear- TR ARS ized with Bgl II (10 pug, total). For two-step gene replace- P ments, YAC-containing strains were transformed by the C/a lithium acetate method. Detailed protocols are presented in ARS Spencer et al. (6). Amplification of the adenovirus YAC was o- by growth of YAC-containing strains in selective medium TEL TK TRP1 CEN A TS TEL containing thymidine (800 ug/ml), sulfanilamide (1 mg/ml), Ad2 vIrion DNA J36kb and methotrexate (10 gg/ml), with galactose as the carbon source (7). Cultures were inoculated with 105 cells per ml; R ecorb traatrOr growth to saturation took 3-5 days at 30'C. Amplified cul- ARS --; ARS tures were diluted 1:50 into YPD and grown overnight before TEL TK TRPl CeA - Ad2 URA3 TEL analysis by pulsed-field gel electrophoresis or preparation of DNA for transfection. High molecular weight yeast DNA was Aderc, YACi53km.) prepared by a protocol developed by A. Wilmen, M. Funk, B Ad2 YAC -- ., -i and J. Hegemann (personal communication). Cells were 6 -0 M collected by centrifugation from 500-ml YPD cultures of cells A E at 5x 107 cells per ml, washed once in water, suspended in 20 ml of 1 M sorbitol/4.25 mM KH2PO4/42.75 mM K2HPO4/20 mM dithiothreitol, and incubated for 1 hat 370C. p68CE3 .\ Cells were collected and incubated for 1 h at 370C in 20 ml of 1 M sorbitol/1 mM EDTA (DB) containing zymolyase 20T "3YH.@ \ 2LEU4I (200 pg/ml). The resulting spheroplasts were collected, washed once in DB, resuspended in 15 ml of 20 mM Mops/ SeIecL LeLj- 0.84 mM spermine/2.16 mM spermidine/10 mM K2EDTA/2 mM EGTA/1 mM aminoacetonitrile/1 mM phenylmethyl- IEU2_MCYH2?-" 44- - -. / - - sulfonyl fluoride/0.2% Triton X-100/1% thiodiglycol, and A I~ P. disrupted by five strokes of a glass Potter B type homoge- nizer. Undisrupted spheroplasts were removed by centrifu- sSeiecCv.?- gation at 120 x g for 10 min; nuclei were collected by x centrifugation at 3000 g for 10 min, resuspended in 0.5 ml I of 20 mM EDTA/1% SDS/20 mM TrisHCl, pH 8.0/ 1 _>~~~~~~~~~~~~~~~~~~~~~~~ proteinase K (50 pug/ml), and incubated at 65°C for 2-5 h. MUtan7 YAC Potassium acetate (0.5 ml of 5 M) was added and the samples were placed at4°C for 30 min. The precipitate was removed Wild-type YAC by centrifugation, and nucleic acids were precipitated from the supernatant by addition of an equal volume of isopro- FIG. 1. (A) Construction of an adenovirus YAC by recombina- panol. The pellet was gently dissolved in 10mM Tris HCl, pH tional cloning. (i) Maps of the plasmids pRMLlAd2L and 8.1/1 mM EDTA (TE) containing RNase A (200 ug/ml) and pRML2Ad2R.(ii) Recombination events ( H ) required to assemble incubated for 2 hat37°C; DNA was precipitated with ethanol an adenovirus YAC from Ad2 virion DNA and linear pRMLlAd2L and gently dissolved in TE.
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