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Origin of Replication from Xenopus Laevis Mitochondrial DNA

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Origin of Replication from Xenopus Laevis Mitochondrial DNA 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 replicon/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 eukaryotes, but not from ation sites (once in 36 kb) detected in small molecules of chro- Escherichia coli, promotes high-frequency transformation in mosomal DNA by electron microscopy (15). (3) Preliminary data yeast. The plasmids 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 virus 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 chromosome, 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, plasmid, 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 gene (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 cloning 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). Bacteria 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 genes 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. §1734 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 cell 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.
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