Eukaryotic DNA Segments Capable of Autonomous Replication in Yeast (Transformation/Eukaryotic Origins of Replication) DAN T
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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+ transfor- mants 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 transforma- tion conditions (labeled "Ca2+, PEG"). Transformation to Ura+ results in colo- nies 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 (di- agrammed at left). However, hybrids be- tween 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 transfor- mation 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.