Generation of Deletion Derivatives by Targeted Transformation of Human

Home , Alu element

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 "targeted" 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 gel electrophoresis (18) obtain the desired deletion derivatives, plasmids pBP63A and devices. Probes used for hybridization included 0.3-kb pBP63B were linearized between Alu and Y' and transformed BamHI fragments recognizing Alu repeats from BLUR8 and into YPH510. The use of vectors with targeting Alu se- BLUR13 plasmids (14), a 1.0-kb fragment of HIS3 (19), a quences in opposite orientations ensures that all Alu seg- 1.5-kb fragment of TRPI (20), and pBluescript (Stratagene ments on the YAC are potential fragmentation targets. Cells Cloning Systems). Radiolabeled probes were prepared by that incorporated the HIS3 gene were grown selectively on primer extension (21). Filters were prepared and hybridized SD plates lacking histidine. Transformation products with a as described (22). His' phenotype were noted for color, colony purified, and Alu Y' Alu r Phenotype: TEL CEN HIS3 HIS3 TEL _-- White, Ura+, Trp+, His- URA3/SUPI 1 TRP1

TEL CEN White, Ura+, Trp-, His+ URA3/SUP1 1 HIS3

TEL CEN White, Ura+, Trp-, His+ URA3/SUPI 1 HIS3 FIG. 2. Fragmentation paradigm. Homologous recombination between an Alu segment on the YAC and the BLUR8 segment in pBP63A or -B will result in deletion of all distal sequences, depending on the orientation of the Alu element on the YAC. Events deleting distal sequences are depicted here. Transformants containing the desired YAC fragmentation products can be identified by the change in phenotype of the cell. The white colony color phenotype indicates the presence of the SUP,, gene; colonies are red in the absence of SUP11 (13). TEL, telomere; CEN, centromere. Downloaded by guest on October 1, 2021 1302 Genetics: Pavan et al. Proc. Natl. Acad. Sci. USA 87 (1990)

Table 1. Phenotype frequencies among transformed yeast The 35 fragmentation events recovered occurred at 18 Colonies analyzed, no. different sites on the YAC, as determined by the sizes of U+T-H+t fragmented YACs on PFGE (Fig. 4). These sites included Plasmid Total H+ U+T+H+* three Alu elements that were targeted 4 times each, two (%) U-T+H+ elements targeted 3 times, three elements targeted twice, and pBP62 150 150 0 (0) 0 ten elements that were targeted once. Within the limits of pBP63A 508 487 20 (3.9) 0 resolution of PFGE, the sites targeted by pBP63A did not pBP63B 322 311 15 (4.7) 0 serve as targets for pBP63B, and vice versa. This result was *U+T+H+, cell that is Ura+,Trp+,His+. expected with acentric vectors because recombination in the tDeletion of sequences distal to the integration site produces this direction of the distal end of the YAC would produce an phenotype in a cell containing a fragmented YAC. unstable acentric fragment. Productive fragmentation by one of the two vectors establishes orientation of the Alu segment streaked on plates that lacked histidine and tryptophan or at the recombination site. Alu segments of opposite orienta- histidine and uracil. Colonies of each phenotype were ex- tions were interspersed along the YAC. Most of the frag- panded, and their electrophoretic karyotypes were analyzed mentation events recovered targeted to the distal third of the by PFGE. chromosome. Several thousand His+ colonies were obtained after trans- The determination of restriction maps of the very large formation of YPH510 cells with the pBP63A and -B plasmids segments of DNA in YACs is greatly simplified by the use of (Table 1). Of 830 white colonies that were analyzed further, deletion derivatives. The parental YAC generated three 35 (4.2%) were stable white, Ura+,Trp-,His+, the phenotype fragments upon digestion with Mlu I (Fig. 5). Digests of expected from a fragmentation event. All 35 lacked the successively shorter deletion derivatives dropped out the 72-, 360-kb parental YAC and contained a new chromosome that 100-, and 185-kb segments sequentially, indicating their order ranged in size from 50 to 350 kb (Fig. 3A). All ofthese deletion on the chromosome (Fig. 5). A total of 26 restriction sites for derivatives hybridized to probes of BLUR8 and HIS3 but not BssHI, Aat II, Sac II, and Mlu I were readily ordered using TRPI. In other words, all colonies with the phenotype the deletion series (Fig. 5). expected of fragmentation contained a fragmented YAC. No Ura+,Trp-,His+ colonies were obtained in control experi- DISCUSSION ments in which YAC-containing YPH510 cells were transformed with pBP62, which does not contain an Alu targeting Mammalian DNA segments cloned as YACs can be subjected sequence. to the same types of manipulations as yeast chromosomes. A To show that the fragmented YACs represented the ex- system has been developed that takes advantage of the high pected nested deletion series, DNA from cells containing frequency of HR in yeast and the frequent occurrence of fragmented YACs was analyzed by conventional blot hybrid- repetitive elements in human DNA to generate deletion ization with the BLUR8 probe by using restriction endonu- derivatives of human-derived YACs. Isolation of the desired cleases that do not cut within reaction products was accomplished readily because the the Alu sequence (Fig. 3B). target YAC was cloned in yeast strain YPH252, which is Depending on the restriction endonuclease used for analysis, deleted for several genes that can be used as a basis for between 30 and 35 unique fragments that contained an Alu selective growth (11). Although many YACs have been sequence could be detected in the Alu profile of the 360-kb constructed in cells that do not allow for this type of selec- parental YAC. The smallest deletion derivative contained tion, individual YACs can be transferred into this genetic five parental bands when analyzed with Pst I, and these background by standard crosses or by direct transformation bands were seen in every other YAC. Each successively of chromosome-sized DNA in the presence of polyamines larger YAC included all the Alu-containing fragments seen in (23). smaller derivatives, demonstrating that the fragmented All transformed cells with the Ura+,Trp-,His+ phenotype YACs were produced by sequential deletions. contained YAC deletion derivatives. The overall frequency A B a b c d e f a a b c d e

23.1 - 9.4 - 445 - 6.7 - 350 - 4.4 - 280 - 240 -

2.3 - 2.0 -

1,3 - ..:, I.. FIG. 3. Cells with the Ura+,Trp-,His+ phenotype contain fragmented YACs. (A) Electrophoretic karyotype of parental and fragmented YACs. Lanes: a, yeast strain YPH250, used as a molecular mass marker; b, strain YPH510, which contains the 360-kb parental YAC; c-g, successively smaller deletion derivatives. Sizes of S. cerevisiae chromosomes are indicated in kb. (B) "Alu profiles" produced by hybridization of a BLUR8 probe with Pst I-digested DNA from YPH252 (lane a), which does not contain a YAC; YPH510 (lane b); and successively smaller deletion derivatives (lanes c-e). Molecular mass markers are indicated in kb. Downloaded by guest on October 1, 2021 Genetics: Pavan et al. Proc. Natl. Acad. Sci. USA 87 (1990) 1303

M 185 kb M 100 kb M 72 kb M I I I I I J1JI I I I S LiIIII S --dB SS IB B SB SI SB S A AA A AA AA

t t f t t ff t pBP63A (4) (4) (3) (3)

~; 44 4 pBP63B (4) (2) (2) (2) I II 10 50 100 FIG. 4. Restriction and fragmentation maps of YPH510. Sites of fragmentation with BP63A or -B are indicated by arrows. Sites targeted more than once are noted in parentheses. M, Mlu I; B, BssHI; A, Aat II; and S, Sac 11.

of His' colonies that contained fragmented YACs was 4.2%. differently. Fragmentation by the acentric Alu vectors re- Because several hundred colonies are recovered in a single ported here may result in reduplication when Alu sequences experiment, a deletion-derivative series can be obtained in a near the centromere of the YAC are targeted. This type of week. While the frequencies obtained make generation of a event would confer a Ura',His',Trp+ phenotype. In the deletion-derivative series practical, other potential applica- experiments with pBP63A and -B, fragmentation plus redu- tions of the fragmenting plasmids will require a higher per- plication would not have been detected above the high centage offragmented YACs among the His+ transformants. background of His' cells. The high frequency of His+ cells containing nonfragmented YAC deletion derivatives produced by fragmenting with a YACs may be attributed to weak autonomously replicating repetitive DNA sequence are useful in the construction of sequence activity in the Y' element after it is healed to form restriction maps of very large DNA segments. These deriv- a telomere, which allows derivatives of the fragmenting atives will have additional application in delineating genes plasmid to replicate autonomously. In preliminary experi- within YACs that have been identified by a functional assay ments, replacement of this sequence with a portion of the S. (e.g., transformation or complementation of an enzyme de- cerevisiae telomere that has no autonomously replicating ficiency in cultured cells) because fragmentation events that sequence activity (24) eliminated completely the background disrupt the coding region would inactivate the gene. Our of autonomously replicating plasmids. results suggest that fragmentation can be targeted to many The 35 HR events analyzed occurred at 18 sites on the different families of highly repetitive elements in the mam- YAC. The targeting of multiple repeats with the arbitrarily malian genome to produce deletion derivatives to any desired chosen BLUR8 Alu repeat suggests some tolerance for level of resolution. Targeting specific sequences with the mismatch in the targeting by HR, because considerable fragmentation plasmid will also have useful applications. HR sequence diversity is known to exist among individual Alu using a cDNA as the targeting sequence will provide a means elements (14). The fragmentation events recovered targeted of mapping exons and determining gene orientation within a primarily to Alu segments in the distal third of the YAC. By YAC. The tolerance for sequence divergence at the target site using a centromere-containing fragmentation vector, which indicates that genes from one species may target homologues fragments containing all sequences distal yields chromosome from another. Fragmentation by cDNAs will also be useful in to the recombination site (see refs. 6, 7), we have identified "mini- HR events with Alu elements in the proximal region of the identifying the 5' ends of genes and in constructing and exons. chromosome, showing that they can also serve as targets. genes" with intact promoter regions spliced to Although experiments on yeast chromosome III have shown Finally, YACs spanning a region of the genome known that fragmentation by acentric vectors usually results in loss contain a gene of interest might be fragmented with cDNA of the targeted chromosome, YACs may behave somewhat libraries constructed in the fragmenting plasmid to identify a specific gene based on a knowledge of the tissue in which it a b c d is expressed and its position in the genome. We thank Carla Connelly for significant technical contributions. The BLUR8 and BLUR13 plasmids were provided by Dr. Prescott This work was supported by U.S. Public Health Service 185 - _ Deininger. Grants CA16519-14 (P.H.), HD22262-02 (R.R.), and HD24605-01 (P.H. and R.R.). V. Science lo o - 1. Burke, D. T., Carle, G. F. & Olson, M. (1987) 236, AL40 806-812. 72 -. FM A 2. Hinnen, A., Hicks, J. & Fink, G. (1978) Proc. Natl. Acad. Sci. USA 75, 1929-1933. _i. 3. Botstein, D. & Fink, G. (1988) Science 240, 1439-1442. 4. Orr-Weaver, T., Szostak, J. & Rothstein, R. (1981) Proc. Natl. Acad. Sci. USA 78, 6354-6358. 5. Orr-Weaver, T. & Szostak, J. (1983) Proc. Natl. Acad. Sci. USA 80, 4417-4421. 6. Vollrath, D. M., Davis, R. W., Connelly, C. & Hieter, P. (1988) FIG. 5. Restriction mapping of cloned segments in YACs is Proc. Natl. Acad. Sci. USA 85, 6027-6031. facilitated by the use of deletion derivatives. Hybridization of the 7. Gerring, S., Connelly, C. & Hieter, P. (1990) Methods Enzy- BLUR8 Alu probe to Mlu I-digested DNA from successively shorter mol., in press. deletion derivatives detects alteration of the 72-, 100-, and 185-kb 8. Schmid, C. W. & Shen, C.-K. J. (1986) in Molecular Evolu- fragments in that order. Lanes: a, YPH510; b-d, successively shorter tionary Genetics, ed. MacIntyre, R. J. (Plenum, New York), deletion derivatives. pp. 323-358. Downloaded by guest on October 1, 2021 1304 Genetics: Pavan et al. Proc. Nati. Acad. Sci. USA 87 (1990)

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