Eukaryotic DNA segments capable of autonomous ... - Europe PMC

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 sequences capable of replicating autonomously in the yeast Saccharomyces cerevisiae. YIp5, a vector that contains the yeast gene ura3, does not transform a ura3 deletion mutant to Ura+. Hybrid YIp5-Escherichia coli DNA molecules also fail to produce transformants. However, collections of molecular hybrids between YIpS and DNA from any of six eukaryotes tested (S. cerevisiae, Neurospora crassa, Dictyostelium discoideum, Ceanhorabditis elegans, Drosophila melanogaster, and Zea mays) do transform the deletion mutant. The Ura+ transformants grow slowly, are unstable under nonselective conditions, and carry the transforming DNA as autonomously replicating, supercoiled circular molecules. Such a phenotype is qualitatively identical to that of strains transformed by molecules containing a yeast chromosomal origin of replication. Thus, these DNA hybrid molecules may contain eukaryotic origins of replication. Ile isolated sequences may be useful in determining the signals controlling DNA replication in yeast and in studying both DNA replication and transformation in other eukaryotic organisms.

The ability of extrachromosomal DNA molecules to replicate autonomously has been utilized to isolate prokaryotic origins of replication. Typically, DNA is introduced into bacteria via phage infection, conjugation, or Ca2+-mediated transformation. A given DNA molecule will replicate independently of integration into the host genome only if it contains an initiation site recognized by the essential replication enzymes and factors. Propagation of such extrachromosomal DNA molecules can be ensured by selecting for the expression of a linked marker-e.g., a gene encoding drug resistance or a gene capable of complementing a host lesion. This rationale has been used to isolate and define the origins of replication of X (1-3), F and R factor plasmids (4-10), and the Salmonella typhimurium (11) and Escherichia 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 molecules capable of complementing corresponding E. coli auxotrophs (17-19). Hinnen et al. (20) used chimeric molecules containing one of these yeast markers (leu2) to demonstrate transformation; auxotrophic yeast mutants were complemented at low frequency (1-10 colonies per ,g of DNA) and the transforming DNA was found to be integrated into the host genome. Other hybrid molecules containing segments of a yeast plasmid (21-23) or other segments of chromosomal DNA (23-25) were found to transform yeast at high frequencies (5000-50,000 colonies per ug of DNA). One such chromosomal segment (termed arsl for autonomously replicating sequence) was shown to behave as an origin of replication, capable of autoThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

nomous replication in the absence of recombination with the yeast genome (25). Thus, when transformed into yeast, a chimeric molecule carrying an origin of replication has a readily selectable property: high-frequency transformation. Here we report the isolation of additional DNA sequences from S. cerevssiae, Neurospora crassa, Dictyostelium discoideum, Caenorhabditis elegans, Drosophila nelanogaster, and Zea mays that allow autonomous replication in yeast cells. By analogy, such sequences may contain other eukaryotic origins of repli-

cation. MATERIALS AND METHODS Bacterial and Yeast Strains. The strains used in this work are shown in Table 1. YNN27 is a ura3-52 strain that is transformed by YRp12 (see Fig. 2) at a particularly high frequency (2000-10,000 colonies per gg of DNA). It was obtained by crossing YNN6 and YNN34 and assessing the transformation ability of strains grown from individual spores. Growth and storage conditions used for all strains have been described (27). DNA. Bacterial plasmid DNA was purified by repeated isopycnic centrifugation in CsCl (27). Chromosomal yeast DNA was prepared by the method of Cameron (28). N. crassa DNA was purified from conidia (unpublished method). E. coil, D. melanogaster, D. discoideum, C. elegans, and Z. mays DNAs were generous gifts of Lee Rowan, Louise Prestidge, Alan Jacobsen, David Hirsh, and Irwin Rubenstein, respectively. pSY317, a kanamycin-resistant plasmid carrying the E. coli origin of replication, was provided by Seiichi Yasuda. Enzymes and Reagents. EcoRI endonuclease was purified by the published procedure (29). T4 DNA ligase and DNA polymerase I were generously provided by Stewart Scherer. All other enzymes and reagents were purchased from commercial suppliers and were used as described (27). Construction of Hybrid DNA Molecules. Random DNA fragments were inserted into YIp5 to produce pools of hybrid molecules. After digestion with the appropriate restriction endonuclease(s) (EcoRI, HindIII, BamHI, or codigestion with EcoRI and HindIII), the YIp5 and chromosomal DNAs (each at 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, pH 7.4/10 mM MgSO4/1 mM ATP/10 mM dithiothreitol at 40C for 1-24 hr. This ligation mixture was directly used to transform yeast cells. Hybrids were constructed between YIp5 and the E. coil origin of replication, oriC, by mixing and ligating EcoRI-digested pSY317 and YIp5 DNAs (as described above). Two fragments of 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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Proc. Natl. Acad. Sci. USA 77 (1980)

Biochemistry: Stinchcomb et al. Table 1. Strains used Strain

Synonym

Bacteria: SF8 BNN20 BNN45 LE392 BNN70 Esrm recA Bacteria containing plasmids: PNN33 trpC9830(YRp12) PNN36 MB1000(YIp5) PNN52 BNN70(YIp5-Ec3l7a) PNN53 BNN70(YIp5-Ec3l7b) Yeast: YNN6 D13-1A SX1-2 YNN34 M1-2B YNN27

C600 rK-mK- recBC- lopll lig+ C600 rK-mK+ rec+ supE44 supF thy F+ asn recA strr

F. Schachat L. Enquist S. Yasuda

Trp+ tetr ampr trp lac- Pyr+tetr ampr Asn+ tetr ampr Asn+ tetr ampr

26 23 This study This study

a his3-532 trp1-289gal2 a trpl-289 ura3-52 gal2 gallO a trpl-289 ura3-52 gal2

23 26 This study

DNA was mixed with 108 yeast spheroplasts and embedded in

linked gene encoding asparagine synthetase asn (15); the other fragment encodes kanamycin resistance (6). The asn bacterial strain, BNN70, was then transformed to tetracycline resistance with the ligated DNA. Clones carrying YIp5-oriC hybrids were Asn+, tetracycline resistant, ampicillin resistant, and kanamycin sensitive. Two plasmid DNAs, YIp5-Ec317a and YIp5-Ec317b, were purified, and were demonstrated to contain the oriC fragment in each orientation as assessed by restriction endonuclease cleavage and agarose gel electrophoresis (30). Yeast Transformations. Transformation of yeast strains was performed as described (23). Approximately 0.5 ,ug of YIp5

E. co/i

agar on a 9-cm plate.

Analysis of Transformants. Growth rates of yeast transformants in the standard yeast minimal medium were measured by using a Klett-Summerson colorimeter. To assess the stability of the transformed phenotype, cultures grown under selective conditions were diluted 1:1000 into rich medium and were grown until saturated. The percentage of cells that remained Ura+ was then determined by duplicate platings onto selective and nonselective agar plates. E. coil transformations, rapid DNA preparations, agarose gel electrophoresis, transfer

Eukaryotic DNA

DNA

YI p5

I EcoRI

+ EcoRI Eco RI

00% R____ =

I-as

R _

(

~~~~~~~.

+t

s

R

ura3~~+

No Ura+ colonies

Source or ref.

Genotype

No Ura+ colonies

Slow Growing Ura+ colonies

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 of fragments (second vertical arrow marked "ligase"). The pools of hybrid 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 vector YIp5 fails to transform the ura3-52 yeast mutant (diagrammed in the middle of the of figure). 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 endonucleases EcoRI, HindIII, BamHI, and Sal I, respectively.

Biochemistry: Stinchcomb et al. to nitrocellulose paper, and hybridization with 32P-labeled pBR322 DNA were carried out with minor modifications of the published procedures (23, 27, 31, 32). RESULTS Selective System for ars. The rationale for isolating DNA sequences that allow autonomous replication in yeast (which we term ars for autonomously replicating sequence) is shown in Fig. 1. The yeast/E. colh vector YIp5 [a hybrid of pBR322 DNA (33) and the yeast gene ura3] has not been observed to transform YNN27 or any other yeast strain carrying the ura3-52 allele (26). In contrast, YRp12, a YIpS hybrid containing the previously identified arsl locus (Fig. 2), will transform YNN27 at high frequency (2000-10,000 transformants per jug of DNA). The resulting transformants demonstrate the complete ars phenotype: they grow slowly, they are mitotically unstable (upon dilution and growth in rich medium the transformants rapidly lose the Ura+ phenotype and, concurrently, the transforming DNA), and they bear the hybrid DNA as extrachromosomal supercoiled molecules (25). The different behaviors of YIpS and YRpi2 provide the basis for isolating other DNA fragments that permit autonomous replication in yeast. When such sequences are inserted into YIpS, the hybrid DNA should transform a ura3-52 strain at high frequency and the transformants thereby produced should show the ars phenotype. Search for E. coli arss. Because the signals that control yeast and E. colh gene expression are sufficiently similar to allow the expression of yeast genes in E. coil (17-19) as well as E. coil genes in yeast (34), we asked whether E. col sequences could direct autonomous replication in yeast. This was answered in two ways. A pool of hybrid molecules was constructed consisting of random EcoRI-generated E. col DNA fragments inserted into YIp5. We used two different preparations of E. colh DNA; both had been used previously to isolate functional genes. The transformation regimen was followed with YNN27 cells and the collection of chimeric molecules. As shown in Fig. 1, no transformants were obtained with E. coil DNA (4 Mg) but other DNA preparations yielded Ura+ colonies at the expected frequencies. This result precludes the existence of several E. colh sequences capable of autonomous replication in yeast. However, trp I

.sI

se

YRpl2

Ylp5-E

c 317a

Ylp5&E c 317b

FIG. 2. Structure of YIp5-origin hybrids. YRp12 is a 7.0-kb plasmid carrying pBR322 sequences (33), the yeast genes, ura3 and trpl, and a chromosomal origin of replication. YIp5-Ec317a and YIp5-Ec3l7b are hybrids containing the ura3 gene, the E. coli chromosomal origin of replication, oriC, and the asparagine synthetase gene asn. The two hybrids differ only in the orientation of the E. coli DNA as determined by the HindIll endonuclease cleavage site. All symbols areas in Fig. 1.

Proc. Natl. Acad. Sci. USA 77 (1980)

4561

if only one such sequence exists, it may have escaped detection. The most likely candidate is the E. col origin of replication. To test its ability to direct autonomous replication upon transformation of yeast, we inserted an EcoRI-generated fragment carrying oriC into YIp5 in both orientations [YIpS-Ec317a and YIp5-Ec317b (Fig. 2)]. When transformation of YNN27 with either YIp5-Ec317a or YIp5-Ec317b was attempted, no transformants were generated, indicating that the E. coli chromosomal origin of replication does not support autonomous replication of hybrid molecules in yeast. Isolation of Eukaryotic arss. Eukaryotic DNA seemed a potentially fertile source of arss; the signals controlling chromosomal replication may be similar to those regulating autonomous replication in yeast (25, 35). Furthermore, arss may be abundant in eukaryotic DNAs because eukaryotic genomes are divided into replication units that are, on the average, 10- to 100-fold smaller than the E. col chromosome (35). Several pools of hybrid molecules were made by inserting restriction endonuclease-generated segments of different eukaryotic chromosomal DNAs into YIp5. EcoRI was used to fragment N. crassa, D. discoideum, C. elegans, D. melanogaster, and Z. mays DNA. D. melanogaster DNA was also cleaved with HindIII and EcoRI simultaneously. Both the endogenous yeast plasmid Scpl and the yeast ribosomal gene cluster are known to transform yeast with a high frequency (21-23, 36). We wished to exclude these sequences from our search for new yeast ars loci. Neither Scpl nor the rDNA repeat contains a cleavage site for BamHI. Therefore, we constructed YIp5-yeast hybrids by ligating DNAs cleaved with BamHI. Under these conditions, no YIp5-rDNA or YIp5-Scpl hybrid molecules should form. YNN27 was transformed with each separate pool of YIp5eukaryotic DNA hybrids. As diagrammed in Fig. 1, all eukaryotic pools generated Ura+ yeast colonies. The frequency at which YNN27 was transformed to Ura+ varied from approximately 50 colonies per Mg of YIp5-N. crassa or YIp5-C. elegans hybrids to 2000 colonies per Mug for the pool of D. discoideum hybrids (all values represent Ura+ transformants per mass of YIp5 DNA present in a hybrid pool and are corrected for the different YRp12 transformation efficiencies observed in each different experiment). Two separate pools of YIp5-D. melanogaster hybrids constructed by EcoRI cleavage of different DNA preparations yielded 800 and 1000 Ura+ transformants per ug of DNA. Moreover, YIp5-D. melanogaster hybrids constructed by using HindIII generated 600 Ura+ colonies per Mug of hybrid DNA upon transformation of YNN-27. The similarity 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 transformation 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 YIp5yeast DNA hybrids showed generation times of 4-8.5 hr. Surprisingly, 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. discoideum, C. elegans, D. melanogaster, and Z. mays hybrids varied from 4.5 to 62 hr. The distribution of doubling times was highly skewed with a cluster around 4-10 hr and with isolated hybrids requiring days to double their cell number. All of the Ura+ transformants were unstable. After growing approximately 10 generations under nonselective conditions, 95% or more of each transformed strain (transformed by YRp12 DNA or a YIp5-eukaryotic hybrid DNA) lost the Ura+ char-

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Proc. Natl. Acad. Sci. USA 77 (1980)

acter. There seemed to be a rough correlation between relative instability and growth rate. Those transformants with longer doubling times lost the Ura+ phenotype more quickly in rich media (see below). The state of the DNA responsible for the Ura+ phenotype of the transformants was determined. Yeast DNA was isolated from each transformed strain. The circular, extrachromosomal DNA was separated from the linear, high molecular weight, chromosomal DNA by agarose gel electrophoresis. After transfer to nitrocellulose paper, the yeast DNA was probed for the YIp5 chimeric sequences by hybridization with 32P-labeled pBR322 DNA. Fig. 3 shows such an analysis of seven Ura+ transformants: three transformed by YIp5-D. discoideum hybrid DNA, one by YRp12, and three resulting from transformation by a pool of YIp5-Z. mays hybrids. In each instance (the 7 transformants shown as well as 62 others representing all sources of hybrid DNAs), the transforming hybrid DNA molecules migrated in unique positions, distinct from both the yeast chromosomal DNA and the endogenous yeast plasmid. The multiple bands of hybridizing DNA are most simply explained as supercoiled and nicked circles of monomer, dimer, and (in some cases) trimer forms of the transforming DNA. Such multimers are often produced by the recombination-proficient yeast (25). Again, a correlation could be drawn between the intensity of DNA hybridization and the growth rate of each transformant. The strains carrying Dd ars22, 23, and 24 had doubling times of 5.5, 5, and 26 hr, respectively. Dd ars22 and 23 hybridized almost as much 32P-labeled pBR322 as did arsl; Dd ars24 showed dramatically less hybridization. Likewise, the -->2S

_

(

P ),'

hr I

1)\ \

_~~~~~~~~ _ ,s

l

;-'ii:

FIG. 3. Autonomously replicating YIp5-ars hybrids. Yeast DNA from Ura+ transformants. Electrophoresis of the undigested DNA was on 0.6% agarose in 40 mM Tris/20 mM acetic acid/2 mM EDTA for 16 hr at 1 V/cm. The yeast chromosomal DNA and endogenous plasmid DNA migrated to the positions designated by the arrows. The gel was transferred to nitrocellulose and then hybridized with approximately 5 X 106 cpm of 32P-labeled pBR322 DNA in 50% formamide/0.9 M NaCl/50 mM NaPO4, pH 7/5 mM EDTA/ 0.2% NaDodSO4 containing 100 ,ug of denatured salmon sperm DNA per ml. The washed and dried nitrocellulose filter was used to expose Kodak XR-5 x-ray film. Autoradiography was performed for a few days at -70°C using a DuPont Lightning Plus intensifying screen. 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 from a transformant carrying an arsl hybrid. Lanes Zm ars20, 19, and 18 contained DNA samples from three independent yeast clones transformed by a pool of YIp5-Z. mays DNA hybrids. All lanes contained approximately equal amounts of yeast chromosomal DNA and the endogenous yeast plasmid Scpl, as judged by ethidium bromide fluorescence. was purified

Z. mays hybrids Zm arsl8 and 19 doubled in 7.5 and 8.5 hr and showed less hybridization than Zm ars20, which doubled in 4.5 hr. Because approximately equal amounts of yeast chromosomal and yeast plasmid (Scpl) DNA were applied to each well, those strains whose DNA annealed to more 32P-labeled pBR322 carried more copies of the foreign hybrid sequences. If the bacterial plasmid sequences encoding drug resistance and replication functions are being propagated without alteration in the Ura+ transformants, it should be possible to transform 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 transformants. Bacterial clones were not obtained with DNA preparations from 15 of the slowest growing Ura+ yeast transformants. This is presumably due to the low transformation efficiency of the crude DNA preparations and the small amount of YIp5 hybrid DNA present. The drug-resistant bacterial clones carried YIp5-eukaryotic DNA hybrids as demonstrated by restriction endonuclease digestion and agarose gel electrophoresis of rapid plasmid DNA preparations. Each hybrid contained intact YIp5 sequences with one or two insert DNA fragments varying from 2 to 9 kb in total length (data not shown). All of the 27 bacterial plasmid DNA preparations tested could transform YNN27 to Ura+ at a high frequency, similar to YRpl2. The nature of the insert DNA in nine of the hybrid plasmids was further examined. Four ars+ plasmids presumably bearing yeast DNA inserts were labeled with 32P by nicktranslation and were hybridized to restriction endonucleasecleaved 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 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 fragment is associated with repetitive DNA sequences (data not 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 processes used in constructing the hybrids, as well as the efficiency of transformation, the number of transformation competent cells, and the average number of DNA molecules taken up by each cell. To investigate the frequency of ars loci further, we transformed the bacterial strain BNN45 with a pool of YIp5-D. melanogaster hybrid DNAs. Fifteen bacterial clones, each containing one DNA hybrid, were isolated. The ability of each hybrid molecule to transform YNN27 was then tested. Thrge of the 15 EcoRI-generated inserts allowed high-frequency transformation of YNN27 associated with autonomous replication of the hybrid molecules. The remaining hybrids did not transform YNN27 to Ura+. The average D. melanogaster DNA fragment inserted by EcoRI digestion was 3 kb. Thus, we detected approximately 1 ars locus per 15 kb of D. melanogaster DNA.

DISCUSSION YIp5 alone has never been successfully used to transform a ura3-52 strain to Ura+. Because the ura3-52 mutant contains a small deletion, it is likely that the ura3 gene in YIp5 can not recombine with its genomic counterpart at a frequency sufficient to observe transformants (26). Transformation of a

Biochemistry: Stinchcomb et al. ura3-52 strain does occur when DNA inserted into YIp5 allows the hybrid to integrate into the genome or to propagate itself without integration. We have found no segment of E. coli DNA that is capable of supporting autonomous replication in yeast. Likewise, the yeast structural gene his3 and many other random eukaryotic DNA segments, when inserted into YIp5, do not allow high-frequency transformation of YNN27. In contrast, YIp5 hybrids carrying arsi or certain DNA inserts from all six eukaryotes tested will transform a ura3-52 strain to Ura+. Invariably, the Ura+ yeast transformed with YIp5-eukaryotic DNA hybrids carried autonomously replicating molecules and demonstrated a phenotype qualitatively indistinguishable from that observed in arsl transformants. We have shown that arsI fulfills the genetic criteria for an isolated chromosomal origin of replication (25). The DNA sequences responsible for autonomous replication of the YIp5 hybrid molecules are likely to be origins of replication in yeast. The foreign ars loci, however, may represent foreign chromosomal origins of replication or fortuitous sites at which yeast DNA replication is initiated. The frequency at which we detected ars loci supports the former possibility. D. melanogaster cleavage nuclei have an average origin-to-origin spacing of 7.9 kb as determined by electron microscopy. Cell culture nuclei origins have an average spacing of 40 kb (37). Our finding of approximately 1 ars locus per 15 kb of D. melanogaster DNA falls within this range of origin-to-origin distances. Alternatively, the eukaryotic DNA inserts may activate a previously silent ars locus in the vector (YIp5) sequences. Others have used different vectors carrying the yeast gene leu2 to isolate S. cerevtsiae arss (ref 38; S. M. Chan and B. K. Tye, personal communication). Thus, ars function more likely is a characteristic of the inserted DNA than a property of the YIp5 vector.

The three facets of the ars phenotype were qualitatively interdependent. The transformed strains that grew more slowly were also more unstable and contained fewer copies of the hybrid DNA. It is interesting to note that the YIp5-yeast and YIp5-N. crassa ars hybrids all were similar to arsl in their behavior. 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 eukaryotic sequences that express the normal ars phenotype with those that are less efficient arss should help to define the signals responsible for the initiation of DNA replication in yeast. If the eukaryotic arss indeed contain chromosomal origins of replication, they may be useful as templates for studies of in vitro DNA replication in these eukaryotic systems. In addition, the eukaryotic arss may be capable of autonomous replication in their homologous host cells as well as in yeast. Thus, an arscontaining fragment linked to an appropriate selectable marker is a useful probe for studying transformation in these eukaryotic species.

We thank Stewart Scherer and Tom St. John for fruitful discussions and useful DNA. We are grateful to Seymour Fogel and Judith Jaehning for constructive and critical readings of the manuscript. We appreciate Bik Tye's liberal communication of results prior to publication. This work was supported in part by Grant GM21891 from the National Institutes of Health, Grant 77-17859 from the National Science Foundation, and Grant 7800503 from the U.S. Department of Agriculture. D.T.S. is a National Science Foundation Predoctoral Fellow. J.K. and E.S. are supported by training grants from the National Institutes of Health.

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