Proc. Nati. Acad. Sci. USAVol. 77, No. 8, pp. 4559-4563, August 1980Biochemistry

Eukaryotic DNA segments capable of autonomous replicationin yeast

(transformation/eukaryotic origins of replication)

DAN T. STINCHCOMB*, MARJORIE THOMAS*, JEFFREY KELLY*, ERIC SELKERt, AND RONALD W. DAVIS**Department of Biochemistry, Stanford School of Medicine, and tDepartment of Biology, Stanford University, Stanford, California 94305

Communicated by I. Robert Lehman, May 5, 1980

ABSTRACT A selective scheme is presented for isolatingsequences capable of replicating autonomously in the yeastSaccharomyces cerevisiae. YIp5, a vector that contains the yeastgene ura3, does not transform a ura3 deletion mutant to Ura+.Hybrid YIp5-Escherichia coliDNA molecules also fail to pro-duce transformants. However, collections of molecular hybridsbetween YIpS and DNA from any of six eukaryotes tested (S.cerevisiae, Neurospora crassa, Dictyostelium discoideum,Ceanhorabditis elegans, Drosophila melanogaster, and Zeamays) do transform the deletion mutant. The Ura+ transfor-mants grow slowly, are unstable under nonselective conditions,and carry the transforming DNA as autonomously replicating,supercoiled circular molecules. Such a phenotype is qualitativelyidentical to that of strains transformed by molecules containinga yeast chromosomal origin of replication. Thus, these DNAhybrid molecules may contain eukaryotic origins of replication.Ile isolated sequences may be useful in determining the signalscontrolling DNA replication in yeast and in studying bothDNAreplication and transformation in other eukaryotic organ-isms.

The ability of extrachromosomal DNA molecules to replicateautonomously has been utilized to isolate prokaryotic originsof replication. Typically, DNA is introduced into bacteria viaphage infection, conjugation, or Ca2+-mediated transformation.A given DNA molecule will replicate independently of inte-gration into the host genome only if it contains an initiation siterecognized by the essential replication enzymes and factors.Propagation of such extrachromosomal DNA molecules can beensured by selecting for the expression of a linked marker-e.g.,a gene encoding drug resistance or a gene capable of comple-menting a host lesion. This rationale has been used to isolate anddefine the origins of replication of X (1-3), F and R factorplasmids (4-10), and the Salmonella typhimurium (11) andEscherichia coli chromosomes (12-16).The yeast Saccharomyces cerevisiae is the only eukaryote

in which a similar selection scheme is currently practical.Several yeast genes have been isolated as hybrid moleculescapable of complementing corresponding E. coli auxotrophs(17-19). Hinnen et al. (20) used chimeric molecules containingone of these yeast markers (leu2) to demonstrate transforma-tion; auxotrophic yeast mutants were complemented at lowfrequency (1-10 colonies per ,g of DNA) and the transformingDNA was found to be integrated into the host genome. Otherhybrid molecules containing segments of a yeast plasmid(21-23) or other segments of chromosomal DNA (23-25) werefound to transform yeast at high frequencies (5000-50,000colonies per ug of DNA). One such chromosomal segment(termed arsl for autonomously replicating sequence) wasshown to behave as an origin of replication, capable of auto-

nomous replication in the absence of recombination with theyeast genome (25). Thus, when transformed into yeast, achimeric molecule carrying an origin of replication has a readilyselectable property: high-frequency transformation. Here wereport the isolation of additional DNA sequences from S. cer-evssiae, Neurospora crassa, Dictyostelium discoideum, Cae-norhabditis elegans, Drosophila nelanogaster, and Zea maysthat allow autonomous replication in yeast cells. By analogy,such sequences may contain other eukaryotic origins of repli-cation.

MATERIALS AND METHODSBacterial and Yeast Strains. The strains used in this work

are shown in Table 1. YNN27 is a ura3-52 strain that is trans-formed by YRp12 (see Fig. 2) at a particularly high frequency(2000-10,000 colonies per gg of DNA). It was obtained bycrossing YNN6 and YNN34 and assessing the transformationability of strains grown from individual spores. Growth andstorage conditions used for all strains have been described(27).DNA. Bacterial plasmid DNA was purified by repeated

isopycnic centrifugation in CsCl (27). Chromosomal yeast DNAwas prepared by the method of Cameron (28). N. crassa DNAwas purified from conidia (unpublished method). E. coil, D.melanogaster, D. discoideum, C. elegans, and Z. mays DNAswere generous gifts of Lee Rowan, Louise Prestidge, Alan Ja-cobsen, David Hirsh, and Irwin Rubenstein, respectively.pSY317, a kanamycin-resistant plasmid carrying the E. coliorigin of replication, was provided by Seiichi Yasuda.Enzymes and Reagents. EcoRI endonuclease was purified

by the published procedure (29). T4 DNA ligase and DNApolymerase I were generously provided by Stewart Scherer. Allother enzymes and reagents were purchased from commercialsuppliers and were used as described (27).

Construction of Hybrid DNA Molecules. Random DNAfragments were inserted into YIp5 to produce pools of hybridmolecules. After digestion with the appropriate restrictionendonuclease(s) (EcoRI, HindIII, BamHI, or codigestion withEcoRI and HindIII), the YIp5 and chromosomal DNAs (eachat 15-20 ,ug of DNA per ml) were mixed and ligated with 0.1,g of T4 DNA ligase in 100 mM NaCl/50 mM Tris-HCl, pH7.4/10 mM MgSO4/1 mM ATP/10 mM dithiothreitol at 40Cfor 1-24 hr. This ligation mixture was directly used to transformyeast cells.

Hybrids were constructed between YIp5 and the E. coil or-igin of replication, oriC, by mixing and ligating EcoRI-digestedpSY317 and YIp5 DNAs (as described above). Two fragmentsof pSY317 are produced by EcoRI digestion. One fragment[approximately 5 kilobases (kb) long] contains oriC and the

Abbreviation: kb, kilobase(s).

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The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked "ad-vertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

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Table 1. Strains usedSource

Strain Synonym Genotype or ref.Bacteria:BNN20 SF8 C600 rK-mK- recBC- lopll lig+ F. SchachatBNN45 LE392 C600 rK-mK+ rec+ supE44 supF thy L. EnquistBNN70 Esrm recA F+ asn recA strr S. Yasuda

Bacteria containing plasmids:PNN33 trpC9830(YRp12) Trp+ tetr ampr 26PNN36 MB1000(YIp5) trp lac- Pyr+tetr ampr 23PNN52 BNN70(YIp5-Ec3l7a) Asn+ tetr ampr This studyPNN53 BNN70(YIp5-Ec3l7b) Asn+ tetr ampr This study

Yeast:YNN6 D13-1A a his3-532 trp1-289gal2 23YNN34 SX1-2 a trpl-289 ura3-52 gal2 gallO 26YNN27 M1-2B a trpl-289 ura3-52 gal2 This study

linked gene encoding asparagine synthetase asn (15); the otherfragment encodes kanamycin resistance (6). The asn bacterialstrain, BNN70, was then transformed to tetracycline resistancewith the ligated DNA. Clones carrying YIp5-oriC hybrids wereAsn+, tetracycline resistant, ampicillin resistant, and kanamycinsensitive. Two plasmid DNAs, YIp5-Ec317a and YIp5-Ec317b,were purified, and were demonstrated to contain the oriCfragment in each orientation as assessed by restriction endo-nuclease cleavage and agarose gel electrophoresis (30).

Yeast Transformations. Transformation of yeast strains wasperformed as described (23). Approximately 0.5 ,ug of YIp5

E. co/i DNA

+ EcoRIYI p5

DNA was mixed with 108 yeast spheroplasts and embedded inagar on a 9-cm plate.

Analysis of Transformants. Growth rates of yeast trans-formants in the standard yeast minimal medium were mea-sured by using a Klett-Summerson colorimeter. To assess thestability of the transformed phenotype, cultures grown underselective conditions were diluted 1:1000 into rich medium andwere grown until saturated. The percentage of cells that re-mained Ura+ was then determined by duplicate platings ontoselective and nonselective agar plates. E. coil transformations,rapid DNA preparations, agarose gel electrophoresis, transfer

Eukaryotic DNA

I EcoRI

Eco RI

00%

I-as

No Ura+colonies

R____ R(

+t s

R

ura3~~+

No Ura+colonies

Slow Growing Ura+colonies

FIG. 1. Scheme for isolating arss.Pools of hybrid DNA molecules wereconstructed by digesting DNAs with arestriction endonuclease (designated byarrow labeled "EcoRI") and then ligatingthe mixture of fragments (second verticalarrow marked "ligase"). The pools of hy-brid DNA molecules were mixed with theura3-52 yeast strain under transforma-tion conditions (labeled "Ca2+, PEG").Transformation to Ura+ results in colo-nies growing on the selective media. Thevector YIp5 fails to transform the ura3-52yeast mutant (diagrammed in the middleof the of figure). Likewise, hybrid YIp5-E.coli DNA molecules are insufficient (di-agrammed at left). However, hybrids be-tween YIp5 and six different eukaryoticDNAs will transform the yeast mutant toUra+ (diagrammed on the right). Openbars, pBR322 sequences; the squiggly line,ura3; stippled bars, E. coli DNA; solidbars, eukaryotic DNA; R, H, B, and S,cleavage sites for the restriction endonu-cleases EcoRI, HindIII, BamHI, and SalI, respectively.

= _ ~~~~~~~.

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to nitrocellulose paper, and hybridization with 32P-labeledpBR322 DNA were carried out with minor modifications of thepublished procedures (23, 27, 31, 32).

RESULTSSelective System for ars. The rationale for isolating DNA

sequences that allow autonomous replication in yeast (whichwe term ars for autonomously replicating sequence) is shownin Fig. 1. The yeast/E. colh vector YIp5 [a hybrid of pBR322DNA (33) and the yeast gene ura3] has not been observed totransform YNN27 or any other yeast strain carrying the ura-3-52 allele (26). In contrast, YRp12, a YIpS hybrid containingthe previously identified arsl locus (Fig. 2), will transformYNN27 at high frequency (2000-10,000 transformants per jugof DNA). The resulting transformants demonstrate the com-plete ars phenotype: they grow slowly, they are mitoticallyunstable (upon dilution and growth in rich medium the trans-formants rapidly lose the Ura+ phenotype and, concurrently,the transforming DNA), and they bear the hybrid DNA as ex-trachromosomal supercoiled molecules (25).The different behaviors of YIpS and YRpi2 provide the basis

for isolating other DNA fragments that permit autonomousreplication in yeast. When such sequences are inserted intoYIpS, the hybrid DNA should transform a ura3-52 strain at highfrequency and the transformants thereby produced should showthe ars phenotype.

Search for E. coli arss. Because the signals that control yeastand E. colh gene expression are sufficiently similar to allow theexpression of yeast genes in E. coil (17-19) as well as E. coilgenes in yeast (34), we asked whether E. col sequences coulddirect autonomous replication in yeast. This was answered intwo ways. A pool of hybrid molecules was constructed consistingof random EcoRI-generated E. col DNA fragments insertedinto YIp5. We used two different preparations of E. colh DNA;both had been used previously to isolate functional genes. Thetransformation regimen was followed with YNN27 cells andthe collection of chimeric molecules. As shown in Fig. 1, notransformants were obtained with E. coil DNA (4 Mg) but otherDNA preparations yielded Ura+ colonies at the expectedfrequencies. This result precludes the existence of several E. colhsequences capable of autonomous replication in yeast. However,

trpI .sI

se

YRpl2

Ylp5-E c 317a Ylp5&E c 317b

FIG. 2. Structure of YIp5-origin hybrids. YRp12 is a 7.0-kbplasmid carrying pBR322 sequences (33), the yeast genes, ura3 andtrpl, and a chromosomal origin of replication. YIp5-Ec317a andYIp5-Ec3l7b are hybrids containing the ura3 gene, the E. coli chro-mosomal origin of replication, oriC, and the asparagine synthetasegene asn. The two hybrids differ only in the orientation of the E. coliDNA as determined by the HindIll endonuclease cleavage site. Allsymbols areas in Fig. 1.

if only one such sequence exists, it may have escaped detection.The most likely candidate is theE. col origin of replication. Totest its ability to direct autonomous replication upon transfor-mation of yeast, we inserted an EcoRI-generated fragmentcarrying oriC into YIp5 in both orientations [YIpS-Ec317a andYIp5-Ec317b (Fig. 2)]. When transformation of YNN27 witheither YIp5-Ec317a or YIp5-Ec317b was attempted, notransformants were generated, indicating that the E. colichromosomal origin of replication does not support autonomousreplication of hybrid molecules in yeast.

Isolation of Eukaryotic arss. Eukaryotic DNA seemed apotentially fertile source of arss; the signals controlling chro-mosomal replication may be similar to those regulating auto-nomous replication in yeast (25, 35). Furthermore, arss may beabundant in eukaryotic DNAs because eukaryotic genomes aredivided into replication units that are, on the average, 10- to100-fold smaller than theE. col chromosome (35). Several poolsof hybrid molecules were made by inserting restriction endo-nuclease-generated segments of different eukaryotic chromo-somal DNAs into YIp5. EcoRI was used to fragment N. crassa,D. discoideum, C. elegans, D. melanogaster, and Z. maysDNA. D. melanogaster DNA was also cleaved with HindIIIand EcoRI simultaneously. Both the endogenous yeast plasmidScpl and the yeast ribosomal gene cluster are known to trans-form yeast with a high frequency (21-23, 36). We wished toexclude these sequences from our search for new yeast ars loci.Neither Scpl nor the rDNA repeat contains a cleavage site forBamHI. Therefore, we constructed YIp5-yeast hybrids by li-gating DNAs cleaved with BamHI. Under these conditions, noYIp5-rDNA or YIp5-Scpl hybrid molecules should form.YNN27 was transformed with each separate pool of YIp5-

eukaryotic DNA hybrids. As diagrammed in Fig. 1, all eukar-yotic pools generated Ura+ yeast colonies. The frequency atwhich YNN27 was transformed to Ura+ varied from approxi-mately 50 colonies per Mg of YIp5-N. crassa or YIp5-C. eleganshybrids to 2000 colonies per Mug for the pool of D. discoideumhybrids (all values represent Ura+ transformants per mass ofYIp5 DNA present in a hybrid pool and are corrected for thedifferent YRp12 transformation efficiencies observed in eachdifferent experiment). Two separate pools of YIp5-D. mela-nogaster hybrids constructed by EcoRI cleavage of differentDNA preparations yielded 800 and 1000 Ura+ transformantsper ug of DNA. Moreover, YIp5-D. melanogaster hybridsconstructed by using HindIII generated 600 Ura+ colonies perMug of hybrid DNA upon transformation of YNN-27. The sim-ilarity of results suggests that the frequency of Ura+ transfor-mants is an inherent property of the eukaryotic DNA insertedinto YIp5.

ars Phenotype of the Transformants. Approximately 10Ura+ transformants were picked randomly from each trans-formation and their phenotype was assessed. The doubling timefor a YRp12 transformant growing in a selective medium isapproximately 4 hr. YNN27 has a generation time of 2.5 hr inthe same medium supplemented with uracil. Doubling timesfor strains that have been transformed to Ura+ by the YIp5-yeast DNA hybrids showed generation times of 4-8.5 hr. Sur-prisingly, the N. crassa hybrid pool yielded transformants thatgrew slightly faster, with generation times of 3.0-4.2 hr. Thedoubling times for transformants generated by the D. discoi-deum, C. elegans, D. melanogaster, and Z. mays hybrids variedfrom 4.5 to 62 hr. The distribution of doubling times was highlyskewed with a cluster around 4-10 hr and with isolated hybridsrequiring days to double their cell number.

All of the Ura+ transformants were unstable. After growingapproximately 10 generations under nonselective conditions,95% or more of each transformed strain (transformed by YRp12DNA or a YIp5-eukaryotic hybrid DNA) lost the Ura+ char-

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acter. There seemed to be a rough correlation between relativeinstability and growth rate. Those transformants with longerdoubling times lost the Ura+ phenotype more quickly in richmedia (see below).The state of the DNA responsible for the Ura+ phenotype of

the transformants was determined. Yeast DNA was isolatedfrom each transformed strain. The circular, extrachromosomalDNA was separated from the linear, high molecular weight,chromosomal DNA by agarose gel electrophoresis. Aftertransfer to nitrocellulose paper, the yeast DNA was probed forthe YIp5 chimeric sequences by hybridization with 32P-labeledpBR322 DNA. Fig. 3 shows such an analysis of seven Ura+transformants: three transformed by YIp5-D. discoideumhybrid DNA, one by YRp12, and three resulting from trans-formation by a pool of YIp5-Z. mays hybrids. In each instance(the 7 transformants shown as well as 62 others representing allsources of hybrid DNAs), the transforming hybrid DNA mol-ecules migrated in unique positions, distinct from both the yeastchromosomal DNA and the endogenous yeast plasmid. Themultiple bands of hybridizing DNA are most simply explainedas supercoiled and nicked circles of monomer, dimer, and (insome cases) trimer forms of the transforming DNA. Suchmultimers are often produced by the recombination-proficientyeast (25). Again, a correlation could be drawn between theintensity of DNA hybridization and the growth rate of eachtransformant. The strains carrying Dd ars22, 23, and 24 haddoubling times of 5.5, 5, and 26 hr, respectively. Dd ars22 and23 hybridized almost as much 32P-labeled pBR322 as did arsl;Dd ars24 showed dramatically less hybridization. Likewise, the

_-->2S

( hr I P ),'

1)\ \

_~~~~~~~~_,s l ;-'ii:

FIG. 3. Autonomously replicating YIp5-ars hybrids. YeastDNAwas purified from Ura+ transformants. Electrophoresis of the undi-gested DNA was on 0.6% agarose in 40mM Tris/20mM acetic acid/2mM EDTA for 16 hr at 1 V/cm. The yeast chromosomal DNA andendogenous plasmid DNA migrated to the positions designated bythe arrows. The gel was transferred to nitrocellulose and then hy-bridized with approximately 5 X 106 cpm of 32P-labeled pBR322 DNAin 50% formamide/0.9 M NaCl/50 mM NaPO4, pH 7/5 mM EDTA/0.2% NaDodSO4 containing 100 ,ug of denatured salmon sperm DNAper ml. The washed and dried nitrocellulose filter was used to exposeKodak XR-5 x-ray film. Autoradiography was performed for a fewdays at -70°C using a DuPont Lightning Plus intensifying screen.

Lanes Dd ars24, 23, and 22 contained DNA isolated from three in-dependent yeast clones transformed by a pool of YIp5-D. discoideumhybrid DNA molecules. Lane Sc arsl contained yeast DNA purifiedfrom a transformant carrying an arsl hybrid. Lanes Zm ars20, 19, and18 contained DNA samples from three independent yeast clonestransformed by a pool of YIp5-Z. mays DNA hybrids. All lanes con-tained approximately equal amounts of yeast chromosomal DNA andthe endogenous yeast plasmid Scpl, as judged by ethidium bromidefluorescence.

Z. mays hybrids Zm arsl8 and 19 doubled in 7.5 and 8.5 hr andshowed less hybridization than Zm ars20, which doubled in 4.5hr. Because approximately equal amounts of yeast chromosomaland yeast plasmid (Scpl) DNA were applied to each well, thosestrains whose DNA annealed to more 32P-labeled pBR322carried more copies of the foreign hybrid sequences.

If the bacterial plasmid sequences encoding drug resistanceand replication functions are being propagated without alter-ation in the Ura+ transformants, it should be possible to trans-form bacteria (BNN20 or BNN45) directly with the yeast DNA.Indeed, 1 to 100 drug-resistant (tetracycline or ampicillin)bacterial clones were obtained from 53 Ura+ yeast transfor-mants. Bacterial clones were not obtained with DNA prepa-rations from 15 of the slowest growing Ura+ yeast transfor-mants. This is presumably due to the low transformation effi-ciency of the crude DNA preparations and the small amountof YIp5 hybrid DNA present. The drug-resistant bacterialclones carried YIp5-eukaryotic DNA hybrids as demonstratedby restriction endonuclease digestion and agarose gel electro-phoresis of rapid plasmid DNA preparations. Each hybridcontained intact YIp5 sequences with one or two insert DNAfragments varying from 2 to 9 kb in total length (data notshown). All of the 27 bacterial plasmid DNA preparations testedcould transform YNN27 to Ura+ at a high frequency, similarto YRpl2. The nature of the insert DNA in nine of the hybridplasmids was further examined. Four ars+ plasmids presumablybearing yeast DNA inserts were labeled with 32P by nick-translation and were hybridized to restriction endonuclease-cleaved yeast DNA fixed to nitrocellulose sheets. In each case,the hybrid plasmid DNA annealed to the yeast DNA fragmentsin patterns consistent with each plasmid's structure (data notshown). Thus, all four hybrids contain intact, unique, yeastDNA fragments. Likewise, of five EcoRI-generated YIp5-D.melanogaster hybrids, four hybridized to the expected D.melanogaster EcoRI fragments. One hybrid annealed to severaladditional fragments, indicating that this ars-bearing DNAfragment is associated with repetitive DNA sequences (data notshown).

Frequency of ars Loci. As mentioned above, eacheukaryoticDNA used to construct pools of YIpS hybrid molecules showeda characteristic frequency of transformation of YNN27 to Ura+.However, any calculation of the frequency of ars loci in a eu-karyotic genome from the transformation frequency wouldrequire assumptions regarding the efficiency of the enzymaticprocesses used in constructing the hybrids, as well as the effi-ciency of transformation, the number of transformation com-petent cells, and the average number of DNA molecules takenup by each cell.To investigate the frequency of ars loci further, we trans-

formed the bacterial strain BNN45 with a pool of YIp5-D.melanogaster hybrid DNAs. Fifteen bacterial clones, eachcontaining one DNA hybrid, were isolated. The ability of eachhybrid molecule to transform YNN27 was then tested. Thrgeof the 15 EcoRI-generated inserts allowed high-frequencytransformation of YNN27 associated with autonomous repli-cation of the hybrid molecules. The remaining hybrids did nottransform YNN27 to Ura+. The average D. melanogaster DNAfragment inserted by EcoRI digestion was 3 kb. Thus, we de-tected approximately 1 ars locus per 15 kb of D. melanogasterDNA.

DISCUSSIONYIp5 alone has never been successfully used to transform aura3-52 strain to Ura+. Because the ura3-52 mutant containsa small deletion, it is likely that the ura3 gene in YIp5 can notrecombine with its genomic counterpart at a frequency suffi-cient to observe transformants (26). Transformation of a

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ura3-52 strain does occur when DNA inserted into YIp5 allowsthe hybrid to integrate into the genome or to propagate itselfwithout integration. We have found no segment of E. coli DNAthat is capable of supporting autonomous replication in yeast.Likewise, the yeast structural gene his3 and many other randomeukaryotic DNA segments, when inserted into YIp5, do notallow high-frequency transformation of YNN27.

In contrast, YIp5 hybrids carrying arsi or certain DNA in-serts from all six eukaryotes tested will transform a ura3-52strain to Ura+. Invariably, the Ura+ yeast transformed withYIp5-eukaryotic DNA hybrids carried autonomously repli-cating molecules and demonstrated a phenotype qualitativelyindistinguishable from that observed in arsl transformants. Wehave shown that arsI fulfills the genetic criteria for an isolatedchromosomal origin of replication (25). The DNA sequencesresponsible for autonomous replication of the YIp5 hybridmolecules are likely to be origins of replication in yeast. Theforeign ars loci, however, may represent foreign chromosomalorigins of replication or fortuitous sites at which yeast DNAreplication is initiated. The frequency at which we detectedars loci supports the former possibility. D. melanogastercleavage nuclei have an average origin-to-origin spacing of 7.9kb as determined by electron microscopy. Cell culture nucleiorigins have an average spacing of 40 kb (37). Our finding ofapproximately 1 ars locus per 15 kb of D. melanogaster DNAfalls within this range of origin-to-origin distances.

Alternatively, the eukaryotic DNA inserts may activate apreviously silent ars locus in the vector (YIp5) sequences. Othershave used different vectors carrying the yeast gene leu2 toisolate S. cerevtsiae arss (ref 38; S. M. Chan and B. K. Tye,personal communication). Thus, ars function more likely is acharacteristic of the inserted DNA than a property of the YIp5vector.The three facets of the ars phenotype were qualitatively

interdependent. The transformed strains that grew more slowlywere also more unstable and contained fewer copies of thehybrid DNA. It is interesting to note that the YIp5-yeast andYIp5-N. crassa ars hybrids all were similar to arsl in theirbehavior. Some D. discoideum, C. elegans, D. melanogaster,and Z. mays hybrids also replicated as well as ars 1. However,others were clearly less proficient. Comparison of several eu-karyotic sequences that express the normal ars phenotype withthose that are less efficient arss should help to define the signalsresponsible for the initiation of DNA replication in yeast.

If the eukaryotic arss indeed contain chromosomal originsof replication, they may be useful as templates for studies of invitro DNA replication in these eukaryotic systems. In addition,the eukaryotic arss may be capable of autonomous replicationin their homologous host cells as well as in yeast. Thus, an ars-containing fragment linked to an appropriate selectable markeris a useful probe for studying transformation in these eukaryoticspecies.

We thank Stewart Scherer and Tom St. John for fruitful discussionsand useful DNA. We are grateful to Seymour Fogel and JudithJaehning for constructive and critical readings of the manuscript. Weappreciate Bik Tye's liberal communication of results prior to publi-cation. This work was supported in part by Grant GM21891 from theNational Institutes of Health, Grant 77-17859 from the NationalScience Foundation, and Grant 7800503 from the U.S. Departmentof Agriculture. D.T.S. is a National Science Foundation PredoctoralFellow. J.K. and E.S. are supported by training grants from the Na-tional Institutes of Health.

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