Eukaryotic DNA Segments Capable of Autonomous Replication in Yeast (Transformation/Eukaryotic Origins of Replication) DAN T

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

Eukaryotic DNA segments capable of autonomous replication in 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 isolating nomous replication in the absence of recombination with the sequences capable of replicating autonomously in the yeast yeast genome (25). Thus, when transformed into yeast, a Saccharomyces cerevisiae. YIp5, a vector that contains the yeast gene ura3, does not transform a ura3 deletion mutant to Ura+. chimeric molecule carrying an origin of replication has a readily Hybrid YIp5-Escherichia coli DNA molecules also fail to pro- selectable property: high-frequency transformation. Here we duce transformants. However, collections of molecular hybrids report the isolation of additional DNA sequences from S. cer- between YIpS and DNA from any of six eukaryotes tested (S. evssiae, Neurospora crassa, Dictyostelium discoideum, Cae- cerevisiae, Neurospora crassa, Dictyostelium discoideum, norhabditis elegans, Drosophila nelanogaster, and Zea mays Ceanhorabditis elegans, Drosophila melanogaster, and Zea that allow autonomous replication in yeast cells. By analogy, mays) do transform the deletion mutant. The Ura+ transformants grow slowly, are unstable under nonselective conditions, such sequences may contain other eukaryotic origins of repli- and carry the transforming DNA as autonomously replicating, cation. supercoiled circular molecules. Such a phenotype is qualitatively identical to that of strains transformed by molecules containing MATERIALS AND METHODS a yeast chromosomal origin of replication. Thus, these DNA hybrid molecules may contain eukaryotic origins of replication. Bacterial and Yeast Strains. The strains used in this work Ile isolated sequences may be useful in determining the signals are shown in Table 1. YNN27 is a ura3-52 strain that is trans- controlling DNA replication in yeast and in studying both DNA formed by YRp12 (see Fig. 2) at a particularly high frequency replication and transformation in other eukaryotic organ- (2000-10,000 colonies per gg of DNA). It was obtained by isms. crossing YNN6 and YNN34 and assessing the transformation The ability of extrachromosomal DNA molecules to replicate ability of strains grown from individual spores. Growth and autonomously has been utilized to isolate prokaryotic origins storage conditions used for all strains have been described of replication. Typically, DNA is introduced into bacteria via (27). phage infection, conjugation, or Ca2+-mediated transformation. DNA. Bacterial plasmid DNA was purified by repeated A given DNA molecule will replicate independently of inte- isopycnic centrifugation in CsCl (27). Chromosomal yeast DNA gration into the host genome only if it contains an initiation site was prepared by the method of Cameron (28). N. crassa DNA recognized by the essential replication enzymes and factors. was purified from conidia (unpublished method). E. coil, D. Propagation of such extrachromosomal DNA molecules can be melanogaster, D. discoideum, C. elegans, and Z. mays DNAs ensured by selecting for the expression of a linked marker-e.g., were generous gifts of Lee Rowan, Louise Prestidge, Alan Ja- a gene encoding drug resistance or a gene capable of comple- cobsen, David Hirsh, and Irwin Rubenstein, respectively. menting a host lesion. This rationale has been used to isolate and pSY317, a kanamycin-resistant plasmid carrying the E. coli define the origins of replication of X (1-3), F and R factor origin of replication, was provided by Seiichi Yasuda. plasmids (4-10), and the Salmonella typhimurium (11) and Enzymes and Reagents. EcoRI endonuclease was purified Escherichia coli chromosomes (12-16). by the published procedure (29). T4 DNA ligase and DNA The yeast Saccharomyces cerevisiae is the only eukaryote polymerase I were generously provided by Stewart Scherer. All in which a similar selection scheme is currently practical. other enzymes and reagents were purchased from commercial Several yeast genes have been isolated as hybrid molecules suppliers and were used as described (27). capable of complementing corresponding E. coli auxotrophs Construction of Hybrid DNA Molecules. Random DNA (17-19). Hinnen et al. (20) used chimeric molecules containing fragments were inserted into YIp5 to produce pools of hybrid one of these yeast markers (leu2) to demonstrate transforma- molecules. After digestion with the appropriate restriction tion; auxotrophic yeast mutants were complemented at low endonuclease(s) (EcoRI, HindIII, BamHI, or codigestion with frequency (1-10 colonies per ,g of DNA) and the transforming EcoRI and HindIII), the YIp5 and chromosomal DNAs (each DNA was found to be integrated into the host genome. Other at 15-20 ,ug of DNA per ml) were mixed and ligated with 0.1 hybrid molecules containing segments of a yeast plasmid ,g of T4 DNA ligase in 100 mM NaCl/50 mM Tris-HCl, pH (21-23) or other segments of chromosomal DNA (23-25) were 7.4/10 mM MgSO4/1 mM ATP/10 mM dithiothreitol at 40C found to transform yeast at high frequencies (5000-50,000 for 1-24 hr. This ligation mixture was directly used to transform colonies per ug of DNA). One such chromosomal segment yeast cells. (termed arsl for autonomously replicating sequence) was Hybrids were constructed between YIp5 and the E. coil or- shown to behave as an origin of replication, capable of auto- igin of replication, oriC, by mixing and ligating EcoRI-digested pSY317 and YIp5 DNAs (as described above). Two fragments of The publication costs of this article were defrayed in part by page pSY317 are produced by EcoRI digestion. One fragment charge payment. This article must therefore be hereby marked "ad- [approximately 5 kilobases (kb) long] contains oriC and the vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: kb, kilobase(s). 4559 Downloaded by guest on September 30, 2021 4560 Biochemistry: Stinchcomb et al. Proc. Natl. Acad. Sci. USA 77 (1980)

Table 1. Strains used Source Strain Synonym Genotype or ref. Bacteria: BNN20 SF8 C600 rK-mK- recBC- lopll lig+ F. Schachat BNN45 LE392 C600 rK-mK+ rec+ supE44 supF thy L. Enquist BNN70 Esrm recA F+ asn recA strr S. Yasuda Bacteria containing plasmids: PNN33 trpC9830(YRp12) Trp+ tetr ampr 26 PNN36 MB1000(YIp5) trp lac- Pyr+tetr ampr 23 PNN52 BNN70(YIp5-Ec3l7a) Asn+ tetr ampr This study PNN53 BNN70(YIp5-Ec3l7b) Asn+ tetr ampr This study Yeast: YNN6 D13-1A a his3-532 trp1-289gal2 23 YNN34 SX1-2 a trpl-289 ura3-52 gal2 gallO 26 YNN27 M1-2B a trpl-289 ura3-52 gal2 This study

linked gene encoding asparagine synthetase asn (15); the other DNA was mixed with 108 yeast spheroplasts and embedded in fragment encodes kanamycin resistance (6). The asn bacterial agar on a 9-cm plate. strain, BNN70, was then transformed to tetracycline resistance Analysis of Transformants. Growth rates of yeast trans- with the ligated DNA. Clones carrying YIp5-oriC hybrids were formants in the standard yeast minimal medium were mea- Asn+, tetracycline resistant, ampicillin resistant, and kanamycin sured by using a Klett-Summerson colorimeter. To assess the sensitive. Two plasmid DNAs, YIp5-Ec317a and YIp5-Ec317b, stability of the transformed phenotype, cultures grown under were purified, and were demonstrated to contain the oriC selective conditions were diluted 1:1000 into rich medium and fragment in each orientation as assessed by restriction endo- were grown until saturated. The percentage of cells that re- nuclease cleavage and agarose gel electrophoresis (30). mained Ura+ was then determined by duplicate platings onto Yeast Transformations. Transformation of yeast strains was selective and nonselective agar plates. E. coil transformations, performed as described (23). Approximately 0.5 ,ug of YIp5 rapid DNA preparations, agarose gel electrophoresis, transfer

Eukaryotic DNA E. co/i DNA YI p5 + EcoRI I EcoRI

Eco RI 00% ( R____ R = _ ~~~~~~~. FIG. 1. Scheme for isolating arss. Pools of hybrid DNA molecules were constructed by digesting DNAs with a restriction endonuclease (designated by arrow labeled "EcoRI") and then ligating the mixture offragments (second vertical I-as +t s arrow marked "ligase"). The pools ofhy- R brid DNA molecules were mixed with the ura3-52 yeast strain under transformation conditions (labeled "Ca2+, PEG"). Transformation to Ura+ results in colonies growing on the selective media. The ura3~~+ vector YIp5 fails to transform the ura3-52 yeast mutant (diagrammed in the middle ofthe offigure). Likewise, hybrid YIp5-E. coli DNA molecules are insufficient (diagrammed at left). However, hybrids between YIp5 and six different eukaryotic DNAs will transform the yeast mutant to Ura+ (diagrammed on the right). Open bars, pBR322 sequences; the squiggly line, ura3; stippled bars, E. coli DNA; solid bars, eukaryotic DNA; R, H, B, and S, cleavage sites for the restriction endonu- No Ura+ No Ura+ Slow Growing Ura+ cleases EcoRI, HindIII, BamHI, and Sal colonies colonies colonies I, respectively. Downloaded by guest on September 30, 2021 Biochemistry: Stinchcomb et al. Proc. Natl. Acad. Sci. USA 77 (1980) 4561 to nitrocellulose paper, and hybridization with 32P-labeled if only one such sequence exists, it may have escaped detection. pBR322 DNA were carried out with minor modifications of the The most likely candidate is theE. col origin of replication. To published procedures (23, 27, 31, 32). test its ability to direct autonomous replication upon transformation of yeast, we inserted an EcoRI-generated fragment RESULTS carrying oriC into YIp5 in both orientations [YIpS-Ec317a and Selective System for ars. The rationale for isolating DNA YIp5-Ec317b (Fig. 2)]. When transformation of YNN27 with sequences that allow autonomous replication in yeast (which either YIp5-Ec317a or YIp5-Ec317b was attempted, no we term ars for autonomously replicating sequence) is shown transformants were generated, indicating that the E. coli in Fig. 1. The yeast/E. colh vector YIp5 [a hybrid of pBR322 chromosomal origin of replication does not support autonomous DNA (33) and the yeast gene ura3] has not been observed to replication of hybrid molecules in yeast. transform YNN27 or any other yeast strain carrying the ura- Isolation of Eukaryotic arss. Eukaryotic DNA seemed a 3-52 allele (26). In contrast, YRp12, a YIpS hybrid containing potentially fertile source of arss; the signals controlling chro- the previously identified arsl locus (Fig. 2), will transform mosomal replication may be similar to those regulating auto- YNN27 at high frequency (2000-10,000 transformants per jug nomous replication in yeast (25,35). Furthermore, arss may be of DNA). The resulting transformants demonstrate the com- abundant in eukaryotic DNAs because eukaryotic genomes are plete ars phenotype: they grow slowly, they are mitotically divided into replication units that are, on the average, 10- to unstable (upon dilution and growth in rich medium the trans- 100-fold smaller than theE. col chromosome (35). Several pools formants rapidly lose the Ura+ phenotype and, concurrently, of hybrid molecules were made by inserting restriction endo- the transforming DNA), and they bear the hybrid DNA as ex- nuclease-generated segments of different eukaryotic chromo- trachromosomal supercoiled molecules (25). somal DNAs into YIp5. EcoRI was used to fragment N. crassa, The different behaviors of YIpS and YRpi2 provide the basis D. discoideum, C. elegans, D. melanogaster, and Z. mays for isolating other DNA fragments that permit autonomous DNA. D. melanogaster DNA was also cleaved with HindIII replication in yeast. When such sequences are inserted into and EcoRI simultaneously. Both the endogenous yeast plasmid YIpS, the hybrid DNA should transform a ura3-52 strain at high Scpl and the yeast ribosomal gene cluster are known to trans- frequency and the transformants thereby produced should show form yeast with a high frequency (21-23, 36). We wished to the ars phenotype. exclude these sequences from our search for new yeast ars loci. Search for E. coli arss. Because the signals that control yeast Neither Scpl nor the rDNA repeat contains a cleavage site for and E. colh gene expression are sufficiently similar to allow the BamHI. Therefore, we constructed YIp5-yeast hybrids by li- expression of yeast genes in E. coil (17-19) as well as E. coil gating DNAs cleaved with BamHI. Under these conditions, no genes in yeast (34), we asked whether E. col sequences could YIp5-rDNA or YIp5-Scpl hybrid molecules should form. direct autonomous replication in yeast. This was answered in YNN27 was transformed with each separate pool of YIp5- two ways. A pool of hybrid molecules was constructed consisting eukaryotic DNA hybrids. As diagrammed in Fig. 1, all eukar- of random EcoRI-generated E. col DNA fragments inserted yotic pools generated Ura+ yeast colonies. The frequency at into YIp5. We used two different preparations of E. colh DNA; which YNN27 was transformed to Ura+ varied from approxi- both had been used previously to isolate functional genes. The mately 50 colonies per Mg of YIp5-N. crassa or YIp5-C. elegans transformation regimen was followed with YNN27 cells and hybrids to 2000 colonies per Mug for the pool of D. discoideum the collection of chimeric molecules. As shown in Fig. 1, no hybrids (all values represent Ura+ transformants per mass of transformants were obtained with E. coil DNA (4 Mg) but other YIp5 DNA present in a hybrid pool and are corrected for the DNA preparations yielded Ura+ colonies at the expected different YRp12 transformation efficiencies observed in each frequencies. This result precludes the existence of several E. colh different experiment). Two separate pools of YIp5-D. mela- sequences capable of autonomous replication in yeast. However, nogaster hybrids constructed by EcoRI cleavage of different DNA preparations yielded 800 and 1000 Ura+ transformants

trpI .sI per ug of DNA. Moreover, YIp5-D. melanogaster hybrids se constructed by using HindIII generated 600 Ura+ colonies per Mug of hybrid DNA upon transformation of YNN-27. The sim- ilarity of results suggests that the frequency of Ura+ transformants is an inherent property of the eukaryotic DNA inserted into YIp5. ars Phenotype of the Transformants. Approximately 10 Ura+ transformants were picked randomly from each trans- YRpl2 formation and their phenotype was assessed. The doubling time for a YRp12 transformant growing in a selective medium is approximately 4 hr. YNN27 has a generation time of 2.5 hr in the same medium supplemented with uracil. Doubling times for 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 that grew slightly faster, with generation times of 3.0-4.2 hr. The doubling times for transformants generated by the D. discoi- Ylp5-E c 317a Ylp5&E c 317b deum, C. elegans, D. melanogaster, and Z. mays hybrids varied FIG. 2. Structure of YIp5-origin hybrids. YRp12 is a 7.0-kb from 4.5 to 62 hr. The distribution of doubling times was highly plasmid carrying pBR322 sequences (33), the yeast genes, ura3 and skewed with a cluster around 4-10 hr and with isolated hybrids trpl, and a chromosomal origin of replication. YIp5-Ec317a and requiring days to double their cell number. YIp5-Ec3l7b are hybrids containing the ura3 gene, the E. coli chro- All of the Ura+ transformants were unstable. After growing mosomal origin of replication, oriC, and the asparagine synthetase gene asn. The two hybrids differ only in the orientation of the E. coli approximately 10 generations under nonselective conditions, DNA as determined by the HindIll endonuclease cleavage site. All 95% or more of each transformed strain (transformed by YRp12 symbols areas in Fig. 1. DNA or a YIp5-eukaryotic hybrid DNA) lost the Ura+ char- Downloaded by guest on September 30, 2021 4562 Biochemistry: Stinchcomb et al. Proc. Natl. Acad. Sci. USA 77 (1980) acter. There seemed to be a rough correlation between relative Z. mays hybrids Zm arsl8 and 19 doubled in 7.5 and 8.5 hr and instability and growth rate. Those transformants with longer showed less hybridization than Zm ars20, which doubled in 4.5 doubling times lost the Ura+ phenotype more quickly in rich hr. Because approximately equal amounts of yeast chromosomal media (see below). and yeast plasmid (Scpl) DNA were applied to each well, those The state of the DNA responsible for the Ura+ phenotype of strains whose DNA annealed to more 32P-labeled pBR322 the transformants was determined. Yeast DNA was isolated carried more copies of the foreign hybrid sequences. from each transformed strain. The circular, extrachromosomal If the bacterial plasmid sequences encoding drug resistance DNA was separated from the linear, high molecular weight, and replication functions are being propagated without alter- chromosomal DNA by agarose gel electrophoresis. After ation in the Ura+ transformants, it should be possible to trans- transfer to nitrocellulose paper, the yeast DNA was probed for form bacteria (BNN20 or BNN45) directly with the yeast DNA. the YIp5 chimeric sequences by hybridization with 32P-labeled Indeed, 1 to 100 drug-resistant (tetracycline or ampicillin) pBR322 DNA. Fig. 3 shows such an analysis of seven Ura+ bacterial clones were obtained from 53 Ura+ yeast transfor- transformants: three transformed by YIp5-D. discoideum mants. Bacterial clones were not obtained with DNA prepa- hybrid DNA, one by YRp12, and three resulting from trans- rations from 15 of the slowest growing Ura+ yeast transfor- formation by a pool of YIp5-Z. mays hybrids. In each instance mants. This is presumably due to the low transformation effi- (the 7 transformants shown as well as 62 others representing all ciency of the crude DNA preparations and the small amount sources of hybrid DNAs), the transforming hybrid DNA mol- of YIp5 hybrid DNA present. The drug-resistant bacterial ecules migrated in unique positions, distinct from both the yeast clones carried YIp5-eukaryotic DNA hybrids as demonstrated chromosomal DNA and the endogenous yeast plasmid. The by restriction endonuclease digestion and agarose gel electro- multiple bands of hybridizing DNA are most simply explained phoresis of rapid plasmid DNA preparations. Each hybrid as supercoiled and nicked circles of monomer, dimer, and (in contained intact YIp5 sequences with one or two insert DNA some cases) trimer forms of the transforming DNA. Such fragments varying from 2 to 9 kb in total length (data not multimers are often produced by the recombination-proficient shown). All of the 27 bacterial plasmid DNA preparations tested yeast (25). Again, a correlation could be drawn between the could transform YNN27 to Ura+ at a high frequency, similar intensity of DNA hybridization and the growth rate of each to YRpl2. The nature of the insert DNA in nine of the hybrid transformant. The strains carrying Dd ars22, 23, and 24 had plasmids was further examined. Four ars+ plasmids presumably doubling times of 5.5, 5, and 26 hr, respectively. Dd ars22 and bearing yeast DNA inserts were labeled with 32P by nick- 23 hybridized almost as much 32P-labeled pBR322 as did arsl; translation and were hybridized to restriction endonuclease- Dd ars24 showed dramatically less hybridization. Likewise, the cleaved yeast DNA fixed to nitrocellulose sheets. In each case, the hybrid plasmid DNA annealed to the yeast DNA fragments in patterns consistent with each plasmid's structure (data not

_-->2S shown). Thus, all four hybrids contain intact, unique, yeast DNA fragments. Likewise, of five EcoRI-generated YIp5-D. melanogaster hybrids, four hybridized to the expected D. melanogaster EcoRI fragments. One hybrid annealed to several additional fragments, indicating that this ars-bearing DNA ( hr I P ),' fragment is associated with repetitive DNA sequences (data not 1)\ \ shown). Frequency of ars Loci. As mentioned above, eacheukaryotic DNA used to construct pools of YIpS hybrid molecules showed a characteristic frequency of transformation of YNN27 to Ura+. However, any calculation of the frequency of ars loci in a eukaryotic genome from the transformation frequency would require assumptions regarding the efficiency of the enzymatic _~~~~~~~~_ l ,s ;-'ii: processes used in constructing the hybrids, as well as the efficiency of transformation, the number of transformation com- petent cells, and the average number of DNA molecules taken up by each cell. To investigate the frequency of ars loci further, we trans- FIG. 3. Autonomously replicating YIp5-ars hybrids. Yeast DNA formed the bacterial strain BNN45 with a pool of YIp5-D. was purified from Ura+ transformants. Electrophoresis of the undi- melanogaster hybrid DNAs. Fifteen bacterial clones, each gested DNA was on 0.6% agarose in 40 mM Tris/20 mM acetic acid/2 containing one DNA hybrid, were isolated. The ability of each mM EDTA for 16 hr at 1 V/cm. The yeast chromosomal DNA and hybrid molecule to transform YNN27 was then tested. Thrge endogenous plasmid DNA migrated to the positions designated by of the 15 EcoRI-generated inserts allowed high-frequency the arrows. The gel was transferred to nitrocellulose and then hy- of YNN27 associated with autonomous bridized with approximately 5 X 106 cpm of 32P-labeled pBR322 DNA transformation repli- in 50% formamide/0.9 M NaCl/50 mM NaPO4, pH 7/5 mM EDTA/ cation of the hybrid molecules. The remaining hybrids did not 0.2% NaDodSO4 containing 100 ,ug of denatured salmon sperm DNA transform YNN27 to Ura+. The average D. melanogaster DNA per ml. The washed and dried nitrocellulose filter was used to expose fragment inserted by EcoRI digestion was 3 kb. Thus, we de- Kodak XR-5 x-ray film. Autoradiography was performed for a few tected approximately 1 ars locus per 15 kb of D. melanogaster days at -70°C using a DuPont Lightning Plus intensifying screen. DNA. Lanes Dd ars24, 23, and 22 contained DNA isolated from three independent yeast clones transformed by a pool of YIp5-D. discoideum hybrid DNA molecules. Lane Sc arsl contained yeast DNA purified DISCUSSION from a transformant carrying an arsl hybrid. Lanes Zm ars20, 19, and YIp5 alone has never been successfully used to transform a 18 contained DNA samples from three independent yeast clones ura3-52 strain to Ura+. Because the ura3-52 mutant contains transformed by a pool of YIp5-Z. mays DNA hybrids. All lanes contained approximately equal amounts of yeast chromosomal DNA and a small deletion, it is likely that the ura3 gene in YIp5 can not the endogenous yeast plasmid Scpl, as judged by ethidium bromide recombine with its genomic counterpart at a frequency suffi- fluorescence. cient to observe transformants (26). Transformation of a Downloaded by guest on September 30, 2021 Biochemistry: Stinchcomb et al. Proc. Nati. Acad. Sci. USA 77 (1980) 4563 ura3-52 strain does occur when DNA inserted into YIp5 allows 1. Matsubara, K. & Kaiser, A. D. (1968) Cold Spring Harbor Symp. the to into the or to itself Quant. Biol. 33,769-775. hybrid integrate genome propagate 2. Moore, D. D., Denniston-Thompson, K., Kruger, K. E., Furth, without integration. We have found no segment of E. coli DNA M. E., Williams, B. G., Daniels, D. L. & Blattner, F. R. (1978) that is capable of supporting autonomous replication in yeast. Cold Spring Harbor Symp. Quant. Biol. 43,155-163. Likewise, the yeast structural gene his3 and many other random 3. Hobom, G., Grosschedl, R., Lusky, M., Scherer, G., Schwarz, E. eukaryotic DNA segments, when inserted into YIp5, do not & Kossel, H. (1978) Cold Spring Harbor Symp. Quant. Biot. 43, allow high-frequency transformation of YNN27. 165-178. In contrast, YIp5 hybrids carrying arsi or certain DNA in- 4. Cohen, S. N. & Chang, A. C. Y. (1973) Proc. Nati. Acad. Sci. USA serts from all six eukaryotes tested will transform a ura3-52 70, 1293-1297. strain to transformed 5. Timmis, K., Cabello, F. & Cohen, S. N. (1975) Proc. Nati. Acad. Ura+. Invariably, the Ura+ yeast with Sci. USA 72,2242-2246. YIp5-eukaryotic DNA hybrids carried autonomously repli- 6. Lovett, M. & Helinski, D. (1976) J. Bacteriol. 127,982-987. cating molecules and demonstrated a phenotype qualitatively 7. Backman, K., Betlach, M., Boyer, H. & Yanofsky, S. (1978) Cold indistinguishable from that observed in arsl transformants. We Spring Harbor Symp. Quant. Biol. 43, 69-76. have shown that arsI fulfills the genetic criteria for an isolated 8. Kahn, M. L., Figurski, D., Ito, L. & Helinski, D. R. (1978) Cold chromosomal origin of replication (25). The DNA sequences Spring Harbor Symp. Quant. BMol. 43,99-104. responsible for autonomous replication of the YIp5 hybrid 9. Timmis, K. N., Andres, I., Solcombe, PR M. & Synenki, R. M. molecules are likely to be origins of replication in yeast. The (1978) Cold Spring Harbor Symp. Quant. Biol. 43, 105-110. 10. Crosa, J. H., Luttropp, L. K. & Falkow, S. (1978) Cold Spring foreign ars loci, however, may represent foreign chromosomal Harbor Symp. Quant. Biol. 43, 111-120. origins of replication or fortuitous sites at which yeast DNA 11. Zyskind, J. W., Deen, T. & Smith, D. W. (1979) Proc. Natl. Acad. replication is initiated. The frequency at which we detected Sci. USA 76,3097-3101. ars loci supports the former possibility. D. melanogaster 12. Hiraga, S. (1976) Proc. Natl. Acad. Sci. USA 73, 198-202. cleavage nuclei have an average origin-to-origin spacing of 7.9 13. Yasuda, S. & Hirota, Y. (1977) Proc. Natl. Acad. Sci. USA 74, kb as determined by electron microscopy. Cell culture nuclei 5458-5462. origins have an average spacing of 40 kb (37). Our finding of 14. Miki, T., Hiraga, S., Nagata, T. & Yura, S. (1978) Proc. Natl. 1 ars locus per 15 kb of D. DNA Acad. Sci. USA 75,5099-5103. approximately melanogaster 15. Sugimoto, K., Ota, A., Sugisaki, H., Takanami, M., Nishimura, falls within this range of origin-to-origin distances. A., Yasuda, S. & Hirota, Y. (1979) Proc. Natl. Acad. Sd. USA 76, Alternatively, the eukaryotic DNA inserts may activate a 575-579. previously silent ars locus in the vector (YIp5) sequences. Others 16. Meijer, M., Beck, E., Hansen, F. G., Bergens, H. E. N., Messer, have used different vectors carrying the yeast gene leu2 to W., von Meyerburg, K. & Schaller, H. (1979) Proc. Natl. Acad. isolate S. cerevtsiae arss (ref 38; S. M. Chan and B. K. Tye, Sci. USA 76,580-584. personal communication). Thus, ars function more likely is a 17. Struhl, K., Cameron, J. R. & Davis, R. W. (1976) Proc. Natl. Acad. characteristic of the inserted DNA than a property of the YIp5 Sci. USA 73, 1471-1475. vector. 18. Ratzkin, E. & Carbon, J. (1977) Proc. Natl. Acad. Sci. USA 74, 487-491. The three facets of the ars phenotype were qualitatively 19. Bach, M. L., Lacroute, F. & Botstein, D. (1979) Proc. Natl. Acad. interdependent. The transformed strains that grew more slowly Sci. USA 76,386-390. were also more unstable and contained fewer copies of the 20. Hinnen, A., Hicks, J. B. & Fink, G. R. (1978) Proc Natl. Acad. Sci. hybrid DNA. It is interesting to note that the YIp5-yeast and USA 75,1929-1933. YIp5-N. crassa ars hybrids all were similar to arsl in their 21. Beggs, J. D. (1978) Nature (London) 275,104-108. behavior. Some D. discoideum, C. elegans, D. melanogaster, 22. Hicks, J. B., Hinnen, A. & Fink, G. R. (1979) Cold Spring Harbor and Z. also as as ars 1. Symp. Quant. Biol. 43, 1305-1313. mays hybrids replicated well However, 23. Struhl, K., Stinchcomb, D. T., Scherer, S. & Davis, R. W. (1979) others were clearly less proficient. Comparison of several eu- Proc. Natl. Acad. Sci. USA 76,1035-1039. karyotic sequences that express the normal ars phenotype with 24. Hsiao, C.-L. & Carbon, J. (1979) Proc. Natl. Acad. Sci. USA 76, those that are less efficient arss should help to define the signals 3829-3833. responsible for the initiation of DNA replication in yeast. 25. Stinchcomb, D. T., Struhl, K. & Davis, R. W. (1979) Nature If the eukaryotic arss indeed contain chromosomal origins (London) 282, 39-43. of replication, they may be useful as templates for studies of in 26. Scherer, S. & Davis, R. W. (1979) Proc. Natl. Acad. Sci. USA 76, vitro DNA 4951-4955. replication in these eukaryotic systems. In addition, 27. Davis, R. W., Botstein, D. & Roth, J. (1980) Advanced Bacterial the eukaryotic arss may be capable of autonomous replication Genetics Laboratory Manual (Cold Spring Harbor Laboratory, in their homologous host cells as well as in yeast. Thus, an ars- Cold Spring Harbor, NY), in press. containing fragment linked to an appropriate selectable marker 28. Cameron, J. (1976) Dissertation (Stanford Univ., Stanford, CA). is a useful probe for studying transformation in these eukaryotic 29. Thomas, M. & Davis, R. W. (1975) J. Mol. Biol. 91,315-328. species. 30. McDonell, M. W., Simon, M. W. & Studier, F. (1977) J. Mol. Biol. 110, 119-146. 31. Southern, E. M. (1975) J. Mol. Biol. 98,503-517. 32. Rigby, P. W., Dieckmann, M., Rhodes, C. & Berg, P. (1977) J. Mol. Biol. 113,237-251. 33. Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C., We thank Stewart Scherer and Tom St. John for fruitful discussions Heyneker, H. L. & Boyer, H. W. (1977) Gene 2,95-113. and useful DNA. We are grateful to Seymour Fogel and Judith 34. Chevallier, M. R. & Aigle, M. (1979) FEBS Lett. 108, 179- Jaehning for constructive and critical readings of the manuscript. We 180. appreciate Bik Tye's liberal communication of results prior to publi- 35. Sheinin, R., Humbert, J. & Pearlman, R. E. (11978) Annu. Rev. cation. This work was supported in part by Grant GM21891 from the Biochem. 47, 277-316. National Institutes of Health, Grant 77-17859 from the National 36. Szostak, J. W. & Wu, R. (1979) Plasmid 2,536-554. Science Foundation, and Grant 7800503 from the U.S. Department 37. Blumenthal, A. B., Kriegstein, H. J. & Hogness, D. S. (1973) Cold of Agriculture. D.T.S. is a National Science Foundation Predoctoral Spring Harbor Symp. Quant. Biol. 38, 205-223. Fellow. J.K. and E.S. are supported by training grants from the Na- 38. Beach, D., Piper, M. & Shall, 5. (1980) Nature (London) 284, tional Institutes of Health. 185-187. Downloaded by guest on September 30, 2021