Proc. Natl. Acad. Sci. USA Vol. 78, No. 5, pp. 3128-3132, May 1981 Genetics

Origin of replication from Xenopus laevis mitochondrial DNA promotes high-frequency transformation of yeast (DNA replication/yeast /recombinant DNA) VIRGINIA A. ZAKIAN Hutchinson Cancer Research Center, Genetics Division, 1124 Columbia Street, Seattle, Washington 98104 Communicated by Herschel L. Roman, February 2, 1981 ABSTRACT A specific fraction of chromosomal DNA from DNA (12, 13). This frequency is similar to the spacing of initi- both yeast and a wide variety of other , but not from ation sites (once in 36 kb) detected in small molecules of chro- , promotes high-frequency transformation in mosomal DNA by electron microscopy (15). (3) Preliminary data yeast. The containing these sequences are maintained as suggest that an 800-base pair (bp) region of the 1.4-kb TRP1 extra-chromosomal molecules in transformed cells. These results DNA fragment, which confers the ability to transform at high suggest that similar or identical sequences are used for the initi- frequency (16), can also be used preferentially as a template for ation of DNA replication in eukaryotes. To test this hypothesis, DNA synthesis in vitro (17). several foreign eukaryotic DNAs implicated directly or indirectly In addition to yeast DNA, a fraction ofthe DNA from a wide in the initiation of DNA replication have been examined for their variety of eukaryotes including Neurospora crassa, Dictyoste- ability to promote autonomous, extrachromosomal replication in melano- yeast. Simian 40 DNA, amplified Xenopus laevis ribosomal lium discoideum, Caenhorhabditis elegans, Drosophila DNA, X. laevis 5S ribosomal DNA, X. laevis mtDNA, and five gaster, and Zea mays (18) also promote high-frequency trans- different members of the Alu I family of human middle repetitive formation of recombinant DNA plasmids in yeast. In contrast, DNAs were cloned into the vector YIp5 and used to transform no DNA fragment from the Escherichia coli , in- yeast. Of these DNAs, only Xenopus mtDNA promoted high-fre- cluding the origin ofreplication, was capable ofpromotinghigh- quency transformation and extrachromosomal maintenance of frequency transformation ofyeast (18). These data raise the pos- YIp5 DNA. A 2.2-kilobase EcoRI fragment from the 17.4-kdlobase sibility that initiation of eukaryotic DNA replication occurs at mtDNA molecule was responsible for these activities. This frag- specific sequences and that these sequences are similar or iden- ment contains the sequence used for the initiation of replication tical in all eukaryotes. We have tested this hypothesis by ex- in Xenopus mitochondria. amining the abilities of several heterologous DNAs to replicate autonomously in yeast. The DNAs tested were Simian virus 40 In many bacterial chromosomal, , and viral DNAs, ini- (SV40); amplified ribosomal DNA (rDNA), 5S DNA, and tiation of DNA replication occurs at a single unique site (1). mtDNA, all from Xenopus laevis; and five members of the Alu Replication of small extrachromosomal eukaryotic DNAs such I family of middle repetitive DNAs from humans. Of these as mtDNA (for examples, see ref. 2), ribosomal DNA from Tet- DNAs, only X. laevis mtDNA exhibited high-frequency trans- rahymena (3) and Physarum (4), and viral DNAs (5) also begins formation of yeast and maintenance as an unstable extrachro- at unique sites. In contrast, the eukaryotic chromosome is rep- mosomal plasmid in transformed cells. A fragment containing licated from multiple initiation sites per DNA molecule, and the origin ofreplication ofthe mtDNA was responsible for these it is not known whether these sites occur at fixed chromosomal properties. loci. METHODS Yeast can be transformed by using recombinant DNA plas- MATERIAL AND mids containing a selectable yeast (6, 7). Most plasmids Strains and DNAs. The yeast strain 689 (a leu 2-3, leu2-112, carrying fragments of yeast DNA transform at low frequencies ura 3-50, can 1-101) and the vector YIp5 were supplied (1-10 colonies per jig of DNA) and are found integrated in a by D. Botstein, YIp5 is a 5.4-kb plasmid comprised of PBR322 relatively stable manner into chromosomal DNA (8). However, and 1100 bp of yeast DNA containing the URA3 gene (ref, 18; some yeast sequences enable a plasmid to transform at a high see Fig. 1). cloning was carried out in E. coli strain RR1 frequency (0.5 X 103 to 2 X 104 colonies per ,ug; ref. 8); plasmids (F- pro, leu, thi, lacy, StreR, r-m-endolF). The following re- carrying such sequences are found as supercoiled extrachro- combinant DNA plasmids were also used: (i) YRp12; which con- mosomal circles in transformed cells and are highly unstable tains a 1.4-kb EcoRI fragment with the TRP1 gene of yeast (8-13). The properties of plasmids that transform at high fre- in YIp5 (18); (ii) pXlm32, which contains a 17.4-kb quencies are believed to result from the ability of these DNAs BamHI fragment with a unit-length copy of X. laevis mtDNA to replicate autonomously in transformed cells. Moreover, it is in PBR322 (19); (iii) pXlm2l; which contains the EcoRI A frag- hypothesized that the sequences that confer these properties ment (15.2 kb) from X. laevis mtDNA in PMB9 (20); (iv) are those normally used for initiation ofyeast DNA replication pXlrlO1; which contains a HindIII fragment with an 11.5-kb full- (8-13). This hypothesis is supported by the following observa- length repeat unit of X. laevis amplified rDNA in PMB9 (R. tions. (i) Plasmids containing a specific region of 2-,Lum DNA, Reeder, personal communication); (v) pXlrll; which contains an endogenous yeast plasmid, also transform at high frequencies a 4.6-kb EcoRI fragment from X. laevis amplified rDNA in (6, 8, 14). (ii) Sequences capable of high-frequency transfor- ColE1 (21); (vi) pXlrl2; which contains a 5.9-kb EcoRI fragment mation occur about once in 30-40 kilobases (kb) ofchromosomal from X. laevis amplified rDNA in ColEl (21); (vii) pXlo8; which contains four repeat units of the 5S rRNA and spacer re- The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: kb, kilobase(s); bp, base pair(s); SV40, Simian virus 40; ment" in accordance with 18 U. S. C. ยง1734 solely to indicate this fact. rDNA, ribosomal DNA. 3128 Downloaded by guest on September 26, 2021 Genetics: Zakian Proc. Natl. Acad. Sci. USA 78 (1981) 3129 gions from X. laevis in PMB9 (22); and (viii) BLURs 2, 6, 8, 11, and 19-each plasmid contains a 250- to 310-bp fragment with a copy of an Alu I family middle repetitive human DNA se- quence inserted with BamHI linkers into PBR322 (23). SV40 DNA was either from D. Galloway, who isolated it from cells infected at low multiplicity, or was purchased from Bethesda Research Laboratories (Rockville, MD). E. coli Cloning and Transformation. Plasmid DNAs were prepared as described (24) and cleaved with the appropriate restriction enzymes (Bethesda Research Laboratories), and the fragments of interest were isolated by agarose gel electropho- resis. Fragments were electroeluted from gel slices and the eluant purified by passage over a DEAE-52 column (Whatman, URA3 preswollen) (25) or were transferred by electrophoresis directly onto DEAE paper (Whatman DE 81) followed by elution with Ylp5 1.0 M NaCl (A. Larsen, unpublished results) for use in ligation mixtures. T4 ligase (Bethesda Research Laboratories) was used FIG. 1. Structure ofYIp5 plasmid DNA (18). Cloning sites used in as directed. E. coli transformation was as described (24). this work are as follows: E, EcoRI; B,BamHI; and H,HindIII. The 1100 Yeast Transformation, DNA Preparations, and Stability bp of yeast DNA are represented by circles and the PBR322 DNA is Studies. Yeast cells were grown in Y minimal medium (26)/2% wepresented by a smooth-line. glucose/histidine (20 mg/ml)/leucine (20mg/ml). They were transformed as described by Beggs (6) with the following mod- 10). In 10 experiments, its average transformation frequency ifications. Spheroplasts were prepared by using Zymolyase was 4260 transformants per ,g. The percentage ofcells surviv- 60,000 (Kirin Brewery, Takasuki, Japan) at 0.1 mg/ml, and 109 ing the transformation procedures was also determined in each spheroplasts were incubated with 5 Ag of plasmid DNA plus experiment by plating spheroplasts on plates containing uracil. 20 ,ug ofsalmon sperm DNA. Carrier DNA was added because Viability in 12 different experiments ranged from 0.2% to 11.4% it increases the frequency of transformants (27). with an average of5.2%. In this paper, the transformation fre- Preparations enriched in plasmid DNA were prepared from quency of a plasmid is given in number of transformants per transformed yeast cells grown to mid-log phase J. F. Scott, microgram ofplasmid DNA. To allow direct comparison ofdata personal communication). Spheroplasts were obtained by in- from different experiments, all transformation frequencies have cubating 109 cells per ml in (0.5 mg/ml) Zymolyase 60,000 been adjusted to the number of transformants that would have (26). Cells were lysed gently with 0.1 vol 10% NaDodSO4, ad- been obtained if viability in the experiment had been 5%. justed to 1.2 M KOAc, and kept at 40C overnight. Cell debris SV40. Two different sources of SV40 DNA were used for E. was removed by centrifugation, and the supernatant was ex- coli cloning, and a number of independent isolates from each tracted with an equal volume of phenol/chloroform (1:1). The E. coli transformation were used in yeast transformations. The aqueous layer was extracted twice with diethyl ether and SV40 origin ofreplication was cloned into YIp5 both by BamHI ethanol was added. Samples were treated if necessary for 1 hr cleavage, which produces a single full-length fragment from at 370C with pancreatic RNase at 100 ,ug/ml. SV40 (pSBY; Fig. 2A, lane 2), and by HindIII cleavage, which For stability studies, transformed yeast cells were grown in produces six fragments with the origin located on a 1101-bp frag- supplemented Y minimal medium with or without uracil at 20 ment (31). In the HindIII cloning, all six fragments were cloned ,ug/ml. At 24-hr intervals over a 3-day period, cells from both in YIp5 and used to transform E. coli. Ampicillin-resistant, tet- cultures were diluted and plated with orwithout uracil at 20 ,ug/ racycline-sensitive colonies were selected and pooled, plasmid ml. DNA was made from the pool, and this DNA (pSHY; Fig. 2A, Containment. Cloning in E. coli and yeast was carried out lane 6) was used to transform yeast. In three different experi- under P2 and EKI (or HV1) conditions. ments, no yeast transformants were detected with either pSBY Other Procedures. Conditions for agarose gel electropho- or pSHY DNA. Therefore, yeast does not use the SV40 origin resis have been described (26). DNA was transferred to nitro- ofreplication to support autonomous replication of YIp5 DNA. cellulose filters (Schleicher & Schuell, BA85), by the Southern Amplified Ribosomal DNA (rDNA) from X. laevis. During procedure (28) and hybridized to nick-translated DNA probes oogenesis in the frog X. laevis, the genes coding for 18S and 28S (29) as described by Stalder et al. (30). rRNAs are amplified approximately 1000-fold (for review, see ref. 32). This amplification occurs by a rolling circle mechanism RESULTS of DNA replication (32). All copies ofamplified rDNA in an oo- Yeast strain 689, which carries a mutation in the URA3 gene, cyte should be identical to the original template circles and was transformed with YIp5 or a YIp5-derivative DNA. Because should therefore contain an origin for rolling circle replication. YIp5 and its derivatives carry a wild-type copy ofthe URA3 gene The transforming ability of rolling circle origins was tested by (Fig. 1), 689 cells transformed by these plasmids can be iden- cloning amplified rDNA into YIp5. Three types of plasmids tified by their ability to grow in the absence ofuracil. YIp5 alone were made and generously supplied by R. H. Reeder (see Fig. produces no true transformants of strain 689, presumably be- 2B). (i) pXY101 contains an 11.5-kb HindIII fragment from cause recombination between the 1100 bp ofyeast DNA in YIp5 pXlrlOl with an entire repeat unit from amplified rDNA (Fig. and the URA3-50 locus of strain 689 is too low to be detected 2B, lane 4); (ii) pXY1l contains a 4.6-kb EcoRI fragment from by these procedures (fewer than one recombinant in 5 x 108 pXlrl1 that consists entirely of sequences that are transcribed cells). In each experiment, a positive control DNA (YRp12) was into the 40S rRNA precursor (21) (Fig. 2B, lane 3); (iii) pXY12 also tested. YRpl2 transforms strain 689 at high frequency, as contains a 5.8-kb EcoRI fragment from pXlrl2 that includes the expected from the fact that it contains a 1.4-kb EcoRI fragment entire nontranscribed spacer region from a repeat unit and most from yeast inserted into YIp5 bearing both the TRP1 gene and of the DNA coding for the 18S rRNA (21) (Fig. 2B, lane 5). In a sequence that promotes high-frequency transformation (8, one experiment, the transformation frequencies were 7 and 12 Downloaded by guest on September 26, 2021 3130 Genetics: Zakian Proc. Natl. Acad. Sci. USA 78 (1981)

A B X. laevis 5S DNA. RNA III is the polymerase in eukaryotes that is responsible for of small RNAs such as tRNA and 5S rRNA. It is also possible that RNA poly- merase III provides the RNA primers required for DNA rep- lication (34). HindIII fragments of --700 bp each ofwhich con- tained a 5S rRNA gene and spacer unit from X. laevis, were cloned into YIp5 and generously provided by R. Reeder (pXY08; Fig. 2B, lane 6). Three different isolates were used in two ex- periments to transform yeast. No transformants were obtained. Therefore, the RNA polymerase III promotor in X. laevis 5S DNA does not support autonomous replication ofyeast plasmids. Alu I Middle Human DNA. About 3% of the hu- C D Repetitive man is comprised of members of a family of middle repetitive DNA sequences called Alu I or ubiquitous repeated DNA (34). Each member ofthis family ofrelated sequences has a size of -300 bp. Because a 14-bp region near the center of the Alu I sequence is similar to sequences found near the origins of replication of SV40, polyoma, and BK , it has been hypothesized that the Alu I sequences might serve as origins of replication in mammalian cells (34). We have inserted five different Alu I sequences from human DNA into YIp5 (pBY 2, 6, 8, 11 and 19; see Fig. 2C) and measured their ability to trans- form yeast. No true transformants were obtained with any of these plasmids. Therefore, yeast can not use a copy of the Alu I family DNA to support autonomous replication of plasmid FIG. 2. Gel electrophoresis of SV40 DNA, X. kaevis ribosomal DNA. DNAs, Alu I DNAs, andX. kaevis mitochondrial DNAs cloned in Up5. in at (A) SV40: Lanes 1,2, and 3, YIp5, pSBY, and SV40 DNAs, respectively, X. laevis mtDNA. Replication mtDNA begins each cleaved with BamHI; lane 4, HindIII A; lanes 5 and 6, HindIl a specific site on the DNA molecule and proceeds unidirec- SV40 and HindIll pSHY DNAs, respectively. Electrophoresis was in tionally from that site. In many higher cells, displacement-loop 0.5% (lanes 1-4) or 1.5% (lanes 5 and 6) agarose gels. (B) X. laevis 5S or D-loop structures are common intermediates in mtDNA rep- and rDNAs: Lane 1, EcoRI A; lane 2, linear YIp5; lane 3,EcoRI pXY11; lication: one end of the D loop marks the site from which unidi- lane 4, HindMI pXY101; lane 5, EcoRI pXY12; lanes 6 and 7, HindHu rectional replication proceeds (35). The D loop in the 17.4 kb pXY08 andHae HI 4X174 DNAs, respectively. Electrophoresis was in X. laevis mtDNA is found in EcoRI fragment of 2.2 kb 0.7% (lanes 1-5) or 1.2% (lanes 6 and 7) agarose gels. (C) Five different (20). human Alu I DNAs: Lanes 1 and 10, Hae IHI X174; lanes 2,4, and 6, A BamHI fragment containing full-length X. laevis mtDNA BamHI cleaved DNA from BLURs 2, 6, and 8, respectively; lanes 3,5, (17.4 kb) was isolated from pXlm32 and cloned into YIp5 7, 8, and 9, BamHI-cleaved pBY 2, 6, 8, 11, and 19, respectively. Elec- (pXY32; Fig. 2D, lane 2). In one experiment, transformation trophoresis was in 4% acrylamide gels. (D) Xenopus mtDNA: Lane 1, frequenceis of two independent isolates (pXY32-1 and pXY32- BamHI A; lane 2, BamHI pXY32; lane 3, BamHI pXlm32; lane 4, 2) were 532 and 503, respectively, versus 694 for YRp12. In BamHI A; lane 5, EcoRI pXY21; lane 7, Sal I A. Electrophoresis was another experiment, pXY32-1 gave a frequency of722 compared in 0.5% agarose gels. with 2500 for YRpl2. Thus, pXY32 plasmids transform at fre- quencies that are 30-70% that ofYRp12. The transformed phe- transformants per ,ug, for two independent isolates of pXY101, notype was unstable in these cells. 8 transformants per ug for pXY11, and 1280 transformants per X. laevis mtDNA was subcloned in YIp5: pXEY26 (Fig. 3, ,ug for YRp12. In a second experiment, the transformation fre- lane 3) carries a 2.2-kb EcoRI fragment isolated from pXlm32 quencies expressed in transformants per ,g for the plasmids DNA, which contains the origin of DNA replication (21), and were 1.3 (pXY101), 2.5 (pXYll), 1.3 (pXY12), and 3400 (YRp12). pXY21 (Fig. 3D, lane 5) carries an EcoRI fragment with the re- The transformation frequencies for plasmids containing Xen- maining 15.2 kb ofthe Xenopus mitochondrial genome (isolated opus-amplified rDNA are much lower than those for DNAs that from pXlm2l). In three different experiments, pXY21 produced replicate autonomously in yeast but are similar to those for no transformants (the frequencies for YRpl2 in the experiments DNAs that transform by integration into the chromosome (1-10 were 1125, 2045, and 6106 transformants per /kg). However, transformants per ,tg; ref. 8). To verify that the colonies ob- in different experiments, pXEY26 transformed at the following tained by transformation with Xenopus rDNA are due to inte- frequencies: 9517, 11,250, 1775, and 4286. These values were gration and not replication of the plasmids, their stability was 1.1, 10.1, 1.1, and 0.7 times the transformation frequency of examined during growth in the presence of uracil. Cells trans- YRpl2 in the same experiments. Thus, the region of the mi- formed by plasmids containing Xenopus rDNA were still able tochondrial genome used for initiation of DNA synthesis in X. to grow in the absence of uracil, even after 3 days of growth in laevis is responsible for high-frequency transformation ofyeast. nonselective media. Therefore, the most likely explanation for Evidence for Extrachromosomal Maintenance of Plasmid the low number of transformants produced after transformation DNA. The high-frequency transformation by pXEY26 was by plasmids containing Xenopus-amplified rDNA is that they shown to be associated with maintenance of the plasmid in ex- arise by integration into yeast chromosomal DNA. Presumably trachromosomal form in three ways. First, DNA preparations integration occurs at the yeast rDNA locus, which has been made from yeast cells transformed by pXEY26 were shown to shown by sequence determination studies (33) to share some contain these sequences in plasmid form (Fig. 4). Preparations homology with Xenopus rDNA. Thus, if Xenopus-amplified enriched in low molecular weight DNA were run on agarose rDNA contains an origin for rolling circle replication, this origin gels, transferred to nitrocellulose paper, and hybridized to nick- is not used by yeast to promote high-frequency transformation translated pXEY26 DNA. These procedures showed that four and extra chromosomal maintenance of plasmid DNAs. of four independent transformants contained a plasmid equal Downloaded by guest on September 26, 2021 Genetics: Zakian Proc. Natl. Acad. Sci. USA 78 (1981) 3131

1 ') ' A IS 7 stability ofthe URA3 phenotype. In these experiments, a trans- formed cell culture growing without uracil was divided into two parts: half was grown with and half was grown without uracil. Three times, at 24-hr intervals, cells from both cultures were plated on plates both with and without uracil, and the number ofcolonies was compared. The average percentages ofcells from the four cell lines that contained plasmids when the cells were grown without uracil were 25%, 32%, 43% and 45%. In all four lines, the transformed phenotype was unstable in the absence of selection pressure: after 72 hr, the percentage of cells with plasmids in cells growing with uracil was 0.02, 0.35, 0.08 and 0.7%, respectively. For comparison, an average of 18% of the cells in a line transformed by YRp12 contained the plasmid FIG. 3. Gel electrophoresis of plasmids containing origin of repli- in cation from X. laevis mitochondrial DNA. Lane 1, linear YIp5; lane 2, when grown the absence of uracil. After 72 hr of growth in EcoRI pXlm32; lane 3, EcoRI pXEY26; lane 4, EcoRI plasmid 2; lane the prescence of uracil, 0.2% of the cells carried the plasmid. 5, EcoRI plasmid 7; lane 6, Hindu A; lane 7, EcoRI plasmid 8. The 2.2- kb EcoRI fragment, which contains the origin ofreplication from Xen- opus mtDNA, was isolated from EcoRI-cleaved pXIm32 DNA (lane 2) DISCUSSION and cloned into YIp5 (lane 1), and the resultingplasmid (pXEY26, lane Eukaryotic chromosomal DNA is replicated from multiple ini- 3) was used to transform yeast. Low molecular weight DNA was iso- lated from two independent yeast cell lines transformed by pXEY26 tiation sites per DNA molecule. It is unclear whether or not and used to transform E. coli to ampicillin resistance. Plasmid DNA these sites are fixed chromosomal loci specifically recognized was isolated from eight different transformed E. coli colonies and as origin regions by the replication apparatus. Suggestive evi- cleaved with EcoRI. Plasmid DNA from three different E. coli colonies dence for specificity comes from a number of sources. Cytolog- (plasmids 2, 7, and 8) representing both yeast transformed cell lines ical studies and density shift experiments with cells from a va- are shown in lanes 4, 5, and 7. All eight plasmids from E. coli were riety of indicate that regions of DNA (for example, indistinguishable from pXEY26. Electrophoresis was in 0.5% agarose ) tend to replicate within specific subintervals gels. of the (for review, see ref. 36). Mutation studies with yeast show that individual genes also tend to replicate in specific in size to that of pXEY26. Second, plasmid DNA was rescued subintervals of S phase (37, 38). Electron microscopic studies from two different yeast transformed cell lines by using low with Drosophila DNA show that initiation sites are spaced at molecular weight DNA extracted from these cells to transform regular intervals along the chromosome (39, 40). In contrast, E. coli to ampicillin resistance. EcoRI digestion of the plasmids replication ofcircular DNAs injected into X. laevis eggs occurs subsequently isolated from E. coli showed that they were in- whether or not the injected DNA carries a eukaryotic origin of distinguishable from the pXEY26 DNA used in the initial yeast DNA replication (41). Even iforigins are specific, there are like- transformation (see Fig. 3). Additional proof for extrachromo- ly to be different classes of these sites because not all origins somal maintenance ofpXEY26 in yeast comes from its instability are used in each S phase (39, 42). in transformed cells (see below). Recombinant DNA plasmids containing specific fragments Stability of URA3 Phenotype in Cells Transformed by from yeast DNA can transform cells at high frequencies and are pXEY26. Four independent yeast transformants were tested for maintained in these cells as extrachromosomal plasmids. About 450 such sequences are found in the yeast genome (13). The A B properities of plasmids containing these sequences strongly 1 2 34567 1 2 3 4 5 6 suggest that they replicate autonomously in transformed cells (for review, see ref. 43) and have led to the hypothesis that they do so because they contain sequences normally used for initi- ation of chromosomal DNA replication. In addition, a fraction of DNA from every tested is also capable of sup- porting high-frequency transformation of yeast (18). However, -RC no fragment from E. coli, whose DNA has a complexity ofabout one-fourth that of yeast, displays these properties (18). To determine whether initiation sites are similar or identical in different eukaryotes, we have examined the transformation properties of a number of specific eukaryotic DNAs. The ideal DNA for this experiment is a segment ofchromosomal DNA that - ~~~CC has been shown to function as a replication origin in the donor . As no such sequence has yet been identified in any it is to use either FIG. 4. Presence of pXEY26 DNA in transformed cells. Strain 689 eukaryote (including yeast), necessary an ex- was transformed with pXEY26 DNA. DNAs from four independent trachromosomal origin (e.g., from viral or mtDNAs) or a seg- transformed cell lines (A and B, lanes 1-4) and from untransformed ment of DNA whose role in replication is speculative (e.g., 5S 689 (A and B, lanes 5) were treated with RNAse prior to electropho- rDNA or Alu I DNA). Ofthe DNAs tested here, only Xenopus resis. pXEY26 DNA is present in lane 6 (A and B). Unrestricted DNA mtDNA was able to promote high-frequency transformation in was subjected to electrophoresis in a 0.5% agarose gel in the presence yeast. A 2.2-kb fragment carrying the is ofethidium bromide at 0.5 pAg/ml (A), transferred to nitrocellulose, and responsible for the high-frequency transformation properties hybridized to nick-translated pXEY26 DNA (B). No hybridization was detected to DNA from nontransformed cells (B, lane 5). All ofthe trans- of Xenopus mtDNA. Like other plasmids carrying sequences formed cell lines (B, lanes 1-4) contained covalently closed copies of that promote high-frequency transformation, plasmids contain- pXEY26 DNA (CC) as well as relaxed (RC) and multimer circular ing the origin from mtDNA are maintained in extrachromo- forms of the plasmid. HindiII A DNA is present in A, lane 7. somal form in transformed cells. Downloaded by guest on September 26, 2021 3132 Genetics: Zakian, Proc. Natl. Acad. Sci. USA 78 (1981)

The failure ofthe SV40 origin to function in yeast may reflect 6. Beggs, J. D. (1978) Nature (London) 275, 104-109. its dependence on T antigen (31). Although the T-antigen cod- 7. Hinnen, A., Hicks, J. B. & Fink, G. R. (1978) Proc. Natl. Acad. ing region, as well as the origin of replication, is contained in Sci. USA 75, 1929-1933. 8. Struhl, K., Stinchcomb, D. T., Scherer, S. & Davis, R. W. (1979) pSBY DNA, yeast is probably unable to use this information to Proc. Nati. Acad. Sci. USA 76, 1035-1039. produce a functional T antigen: neither rabbit 8-globin DNA 9. Hsaio, C. L. & Carbon, J. (1979) Proc. Nati. Acad. Sci. USA 76, (44) nor the thymidine kinase gene from Herpes simplex (un- 3829-3833. published results) are expressed in yeast. Ifamplified Xenopus 10. Kingsman, A. J., Clarke, L., Mortimer, R. K. & Carbon, J. (1979) rDNA contains an origin for replication, this origin may be ac- Gene 7, 141-152. tive in Xenopus only in germ cells engaged in amplification and 11. Szostak, J. W. & Wu, R. (1979) Plasmid 2, 536-554. 12. Beach, D., Piper, M. & Shall, S. (1980) Nature (London) 284, may have replication requirements specific to a rolling circle 185-187. mechanism of replication. Neither 5S rDNA nor Alu I DNA 13. Chan, C. S. M. & Tye, B. K. (1980) Proc. Natl. Acad. Sci. USA have been shown directly to have a role in DNA replication and 77, 6329-633. their inability to function in yeast may simply reflect their lack 14. Broach, J. R. & Hicks, J. B. (1980) Cell 21, 501-508. ofreplication functions in the organisms from which they came. 15. Newlon, C. S. & Burke, W. (1980) in Mechanistic Studies ofDNA G. B. Kiss and R. E. Pearlman (personal communication) Replication and Genetic Recombination, ICN-UCLA Symposia on Molecular and Cellular Biology, eds. Alberts, B. & Fox, C. have recently shown that the region containing the origin of (Academic, New York), Vol. 19, pp. 399-409. DNA replication from the extrachromosomal rDNA of Tetra- 16. Stinchcomb, D. T., Struhl, K. & Davis, R. W. (1979) Nature hymena thermophila can also support high-frequency transfor- (London) 289, 39-43. mation in yeast. This origin is very near or at the center of the 17. Scott, J. F. (1980) in Mechanistic Studies ofDNA Replication and nontranscribed spacer region of the 20-kb palindromic rDNA Genetic Recombination, ICN-UCLA Symposia on Molecular and molecule (3) and is very rich in adenosine and deoxyribosyl- Cellular Biology, edsi Alberts, B. & Fox, C. (Academic, New York), Vol. 19, pp. 379-388. thymine (45). The region containing the origin of Xenopus 18. Stinchcomb, D. T., Thomas, M., Kelly, J., Selker, E. & Davis, mtDNA is also the only section of the DNA molecule in which R. W. (1980) Proc. Natl. Acad. Sci. USA 77, 4559-4563. no RNA coding sequences have been detected (19). However, 19. Rastl, E. & Dawid, I. B. (1979) Cell 18, 501-510. electron microscopic studies do not indicate that this region is 20. Ramirez, J. L. & Dawid, I. B. (1978)J. Mol. Biol. 119, 113-146. rich in adenosine and deoxyribosylthymine (I. Dawid, personal 21. Dawid, I. & Wellauer, P. (1976) Cell 8, 443-448. communication). 22. Birkenmeir, E. H., Brown, D. D. & Jordan, E. (1978) Cell 15, 1077-1086. The data reported here and those of G. B. Kiss and R. E. 23. Rubin, C. M., Houck, C. M., Deininger, P. L., Friedmann, T. Pearlman (personal communication) suggest that at least two & Schmid, C. W. (1980) Nature (London) 284, 372-374. regions containing sequences used for initiation of DNA syn- 24. Goodman, H. M., Olson, M. V. & Hall, B. D. (1977) Proc. Natl. thesis in other eukaryotic organisms can function in yeast to Acad. Sci. USA 74, 5453-5457. promote high-frequency transformation and extrachromosomal 25. Smith, H. 0. & Birnstiel, M. L. (1976) Nucleic Acids Res. 3, maintenance of plasmid DNA. These data support the theory 2387-2398. 26. Zakian, V. A., Brewer, B. J. & Fangman, W. L. (1979) Cell 17, that initiation sites are specific and that their sequences are con- 923-934. served in eukaryotes. The failure of other eukaryotic initiation 27. Nasmyth, K. A. & Reed, S. J. (1980) Proc. Natl. Acad. Sci. USA sites such as that of SV40 to function in yeast may reflect their 77, 2119-2123. divergence from a prototype sequence(s) or their dependence 28. Southern, E. (1975) J. Mol. Biol. 93, 503-517. on specific factors for activation (orboth). Furthermore, the data 29. Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg, P.. (1977) from yeast (13, 16), as well as from other eukaryotes (39, 42), J. Mol. Biol. 113, 737-751. 30. Stalder, J., Groudine, M., Dodgson, J. B., Engel, J. D. & Wein- suggest that not all initiation sites even from the same organism traub, H. (1980) Cell 19, 973-980. are identical. Perhaps only a subset ofthe initiation sites in any 31. Griffin, B. E. (1980) in DNA Tumor Viruses: Molecular Biology organism have a sequence recognized as an origin ofreplication ofTumor Viruses, ed. Tooze, J. (Cold Spring Harbor Laboratory, in all or most eukaryotes. Cold Spring Harbor, NY), 2nd Ed., pp. 61-123. In contrast to the replication of chromosomal DNA, repli- 32. Long, E. 0. & Dawid, I. B. (1980) Annu. Rev. Biochem. 49, cation of animal mtDNA proceeds unidirectionally and asym- 727-764. 33. Moss, T., Boseley, P. G. & Birnstiel, M. (1980) Nucleic Acids metrically from a single site on the DNA molecule. Replication Res. 8, 467-485. ofyeast mtDNA is under different genetic and temporal controls 34. Jelinek, W. R., Toomey, T. P., Leinwand, L., Duncan, C. H., than yeast chromosomal DNA (for review, see ref. 43). It will Biro, P. A., Chovdary, P. V., Weissman, S. M., Rubin, C. M., be ofinterest to see whether plasmids with the Xenopus origin Houck, C. M., Deininger, P. L. & Schmid, C. W. (1980) Proc. ofmtDNA have the replication properties ofmtDNA or ofchro- Natl. Acad. Sci. USA 77, 1398-1402. 35. Kasamatsu, K. & Vinograd, J. (1974) Annu. Rev. Biochem. 43, mosomal DNA. 695-719. 36. Edenberg, H. J. & Huberman, J. A. (1975) Annu. Rev. Genet. 9, It is a pleasure to acknowledge the expert technical assistance of Ms. 245-284. Doris Kupfer. I thank Drs. D. Botstein, I. Dawid, P. Deininger, and 37. Burke, W. & Fangman, W. L. (1975) Cell5, 263-269. J. Scott for recombinant DNA plasmids and, especially, Dr. R. H. 38. Kee, S. G. & Haber, J. E. (1975) Proc. Natl. Acad. Sci. USA 72, Reeder, who constructed the Xenopus rDNA plasmids used in this 1179-1193. work. I also thank Drs; W. L. Fangman and R. H. Reeder for critical 39. Blumenthal, A. B., Kriegstein, H. J. & Hogness, D. S. (1974) reading ofthe manuscript. This work was supported by a grant from the Cold Spring Harbor Symp. Quant. Biol. 38, 205-223. National Institutes of Health. 40. Zakian, V. A. (1976) J. Mol. Biol. 108, 305-331 41. Harland, R. M. & Laskey, R. A. (1980) Cell 21, 761-771. 1. Kolter, R. & Helinski, D. R. (1979) Annu. Rev. Genet. 13, 42. Callan, H. G. (1972) Proc. R. Soc. London Ser. B 181, 19-41. 355-391. 43. Fangman, W. L. & Zakian, V. A. (1981) in The Molecular Biology 2. Goddard, J. & Wolstenholme, D. R. (1978) Proc. Natl. Acad. Sci; of the Yeast Saccharomyces cerevisiae, ed. Broach, J., Jones, E. USA 75, 3886-3890. & Strathern, J. (Cold Spring Harbor Laboratory,.Cold Spring 3. Truett, M. A. & Gall, J. G. (1977) Chromosoma 64, 295-303. Harbor, NY), in press. 4. Vogt, V. & Braun, D. (1976) J. Mol. Biol. 106, 567-587. 44. Beggs, J., Van den Berg, J., VanOoyen, A. & Weissmann, C. 5. DePamphilis, M. L. & Wassarman, P. M. (1980) Annu. Rev. (1980) Nature (London) 283, 835-8. Biochem. 49, 627-66. 45. Kiss, G. B. & Pearlman, R. E.-(1981) Gene, in press. Downloaded by guest on September 26, 2021