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Generation of Deletion Derivatives by Targeted Transformation of Human Proc. Natl. Acad. Sci. USA Vol. 87, pp. 1300-1304, February 1990 Genetics Generation of deletion derivatives by targeted transformation of human-derived yeast artificial chromosomes (yeast artificial chromosome fragmentation/homologous recombination/repetitive DNA/macrorestriction map) WILLIAM J. PAVAN*, PHILIP HIETERt, AND ROGER H. REEVES*t Departments of *Physiology and tMolecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Communicated by John W. Littlefield, November 27, 1989 ABSTRACT Mammalian DNA 'segments cloned as yeast homologous recombination (HR)-based methods have in- artificial chromosomes (YACs) can be manipulated by DNA- volved DNA segments of about several hundred base pairs mediated transformation when placed in an appropriate yeast (bp) or more in length that are completely homologous to the genetic background. A "fragmenting vector" has been devel- genomic target sequence. The length of homologous se- oped that can introduce a yeast telomere and selectable marker quence required to target recombination, the degree of mis- into human-derived YACs at specific sites by means of homol- match tolerated, and the effects of these variables on the ogous recombination, deleting all sequences distal to the re- frequency of HR are not known. combination site. A powerful application of the method uses a Repetitive sequences are distributed throughout the ge- human Alu family repeat sequence to target recombination to nomes of mammals. The human genome contains -500,000 multiple independent sites on a human-derived YAC. Sets of copies of Alu family repetitive elements (8). Individual Alu deletion derivatives generated by this procedure greatly facil- elements are tandem direct repeats totaling "300 bp in itate restriction mapping oflarge genomic segments. Targeting length. They can be divided into families that vary in se- recombination with single copy sequences, such as cDNAs, will quence by >20% overall, although more highly conserved have many additional applications. This approach establishes regions occur within divergent Alu elements (9). Repetitive a paradigm for manipulation and characterization of mamma- sequences have been estimated to occur with an average lian DNA segments cloned as YACs. spacing of 3 kb in the human genome (10). Because repetitive elements will occur in virtually any human-derived YAC, Analysis of complex genomes has been facilitated by the they represent potential targets for manipulations mediated development of methods for cloning large DNA fragments as by HR in yeast. yeast artificial chromosomes (YACs) (1). YACs are con- Structural analysis of large cloned DNA segments by structed by adding to a segment of DNA the yeast signals for conventional methods represents a substantial undertaking. replication, segregation, and maintenance of a linear chro- To simplify this process, a fragmenting vector has been mosome, along with markers that allow the identification and developed that can introduce a yeast telomere and selectable selective growth of cells that take up the YAC (1). A major marker into chromosomes in yeast by means of HR. The advantage of this technology is the ability to clone intact method relies on a yeast strain developed for construction segments of DNA in the 300- to 1000-kilobase (kb) range, a and manipulation of YACs that has four nonreverting auxo- capacity 10 times greater than possible with cosmid technol- trophic markers for selection of transformants. The intro- ogy. However, physical mapping of such large DNA seg- duction of a telomere terminates the YAC at the site of ments still presents considerable challenge. Development of integration, deleting all distal sequences to form a deletion of YACs will derivative of the parental YAC. An Alu element was used in methods for rapid mapping and manipulation the fragmenting vector to target independent HR events to facilitate the mapping of complex genomes. In addition, the Alu segments distributed along a human YAC. The targeting sophisticated DNA-mediated transformation system of yeast of multiple elements suggests that some degree of mismatch allows site-specific alteration of cloned exogenous DNA by may be tolerated in the HR event. The use of deletion homologous recombination. derivatives greatly facilitated restriction mapping of the Integration of DNA transformed into yeast occurs almost cloned segment. Fragmentation with repetitive and unique exclusively by homologous recombination (2, 3). This obser- sequences has a number of applications for analysis, modi- vation has led to efficient gene transplacement techniques fication, and selection of mammalian sequences cloned as enabling replacement of genomic sequences by cloned DNA YACs. segments that have been altered in vitro. Free ends of DNA are highly "recombinogenic" in yeast and stimulate "target- ed" transformation of DNA with homologous sequences in MATERIALS AND METHODS the genome by several orders of magnitude (4, 5). A method Yeast Strains and Propagation. Saccharomyces cerevisiae called chromosome fragmentation has been described (6, 7). strain YPH252 (a, ura3-52, ade2-101, trpl-AI, lys2-801, his3- Fragmentation takes advantage ofthe recombinogenic nature A200, leu2-AI) was developed for the propagation and ma- of DNA transformed into yeast to determine chromosomal nipulation of YACs (11). Strain YPH510 carries a 360-kb position in terms of physical distance between a gene and human-derived YAC built in the pJS71 and pJS73 YAC each ofthe telomeric ends ofa linear chromosomal molecule. cloning vectors (J. Shero and P.H., unpublished work). The chromosome is broken at the site of the gene by Transformation was accomplished by the lithium acetate homologous integrative transformation, and the lengths of procedure (12). Approximately 500 colonies were obtained the two chromosome fragments (proximal and distal to the gene) are measured on pulsed-field gels. To date, these Abbreviations: YAC, yeast artificial chromosome; HR, homologous recombination; PFGE, pulsed-field gel electrophoresis. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Physiology, P202, The Johns Hopkins University School of Medi- in accordance with 18 U.S.C. §1734 solely to indicate this fact. cine, 725 North Wolfe Street, Baltimore, MD 21205. 1300 Downloaded by guest on October 1, 2021 Genetics: Pavan et al. Proc. Natl. Acad. Sci. USA 87 (1990) 1301 pBP62 pBP63A pBP63B 6.7 kb T3 7.3 kb 7.kb Sac! C$ LZ ~~~~~~~~~~SaCII Xal NOtI NVou Spel EcORV BLUR 8 1-finc ApaI BLUR 8 N ~~~~~~~~~~~~Y, FIG. 1. Fragmenting plasmids. pBP62 contains a polylinker cloning site in the bacterial IacZ gene for introduction of targeting sequences. A human Alu segment from BLUR8 plasmid was cloned in tandem into pBP62 to produce pBP63A, and a fragment containing the BLUR8 segment was reversed to make pBP63B. Arrows indicate 5' to 3' orientation of BLUR8 sequences. Any of several sites between the targeting segment and Y' can be used to linearize the plasmid before transformation. after transformation of 5 x 107 cells with 10 ,ug of linearized RESULTS plasmid DNA. Transformants were selected on SD plates lacking histidine but supplemented with uracil, tryptophan, The fragmentation vector pBP62 (Fig. 1) contains a telomere- leucine, lysine, and limiting adenine for color selection (13). adjacent Y' sequence of S. cerevisiae, the yeast HIS3 gene, His' transformants were colony purified and tested for the a cloning cassette with a polylinker inserted into the bacterial ability to grow in the absence of histidine and uracil or lacZ gene, and sequences for the growth and selection of the histidine and tryptophan. plasmid in bacteria. Fragmentation targeting sequences can Vector Construction. The fragmenting plasmid, pBP62 be conveniently inserted into the polylinker cloning site, and (Fig. 1), was constructed by blunt-end ligation of a 2.0-kb plasmid recombinants can be identified visually as a result of fragment including the Y' sequence from pJS89 (J. Shero and disruption ofthe lacZ gene. Fragmentation plasmids pBP63A P.H., unpublished work) into a Pvu II site in the HIS3- and -B are derivatives of pBP62 that contain two head-to-tail containing plasmid, pRS303 (11). Plasmid pBP63A was de- copies of the BLUR8 Alu segment in opposite orientations rived from pBP62 by insertion into the polylinker of tandem (Fig. 1). head-to-tail human Alu repeats from the BLUR8 plasmid The fragmentation paradigm is illustrated in Fig. 2. The (14). pBP63B contains the same Alu repeats in the opposite 360-kb human YAC used in these experiments contains orientation and was constructed by inverting the 870-bp Not -:=30-35 Alu sequences. Integration of a fragmentation plas- I fragment in pBP63A. mid at an Alu sequence will terminate the YAC by insertion Pulsed-Field Gel Electrophoresis (PFGE) and Restriction of a telomere at the site of recombination. About half of the Analysis. DNA for conventional analysis and high- Alu sequences in the YAC are expected to be in an orientation molecular-weight DNA for PFGE were prepared by standard such that integration will delete sequences distal to the procedures (15, 16). Electrophoretic karyotypes were as- recombination site, in which case the phenotype of the cell sessed using contour-clamped homogeneous electric field will change from Ura+,Trp+,His- to Ura+,Trp-,His+. To (17) or orthogonal field-alternation
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