© Springer-Verlag 1983

Chromosomal DNA Sequences from Ustilago maydis Promote Autonomous Replication of Plasmids in Saccharomyces cerevisiae Geoffrey R. Banks National Institute for Medical Research, Mill Hill, London NW7 1AA, UK

Summary. U. maydis chromosomal DNA sequences which promote the autonomous replication of plasmid YIp5 in S. cerevisiae YNN27 have been isolated and three of them characterised in some detail. Their properties are idential to yeast ars sequences in that plasmids containing them are maintained extrachromosomally as circular double-stranded DNA molecules, are mitotically unstable in yeast transformants and transform yeast at high frequencies. There is no sequence homology between the three U. maydis sequences and they are not reiterated in the U. maydis genome.

Key words: ars sequences - Ustilago - Yeast

Introduction Electron microscopy and fibre autoradiography have revealed that the DNA of eukaryotic chromosomes is organised into multiple replicating units or replicons, in which bidirectional replication is initiated (for review see Edenberg and Huberman 1975). Moreover, initiation at such sites is both a spatially and temporally ordered process (Dawes and Carter 1974; Burke and Fangman 1975; Kee and Haber 1975; Braun and Willi 1969; Muldoon et al. 1971). As a result, a given region of the DNA appears to be replicated within a specific interval of the S phase in successive cell generations. Such specificity is not, however, immutable because the distance between replicon initiations in both Drosophila cleavage embryos and Triturus spermatocytes differs significantly from those in their somatic cell counterparts (Blumenthal et at. 1973; Callan 1972). Because the rates of DNA chain elongation are comparable for both cell types in each case, the overall rate of DNA synthesis can be adjusted to the particular requirements

of the cell type. In addition, DNA injected into Xenopus eggs is replicated in a controlled fashion even in the absence of a eukaryotic DNA replication origin (Harland and Laskey 1980). It has been discovered recently that specific DNA sequences can be isolated from the chromosomal DNA of S. cerevisiae by recombinant DNA techniques which, when incorporated into a plasmid containing a yeast gene encoding a selectable phenotype, can transform S. cerevisiae at high frequencies. Such plasmids are maintained within transformants extrachromosomally, replicate autonomously and are mitotically and meiotically rather unstable in contrast to plasmids lacking the specific sequences, which transform at low frequencies and integrate into chromosomal DNA (Struhl et al. 1979; Stinchcomb et al. 1979; Hsiao and Carbon 1979; Kingsman et al. 1979; Szostak and Wu 1979; Chan and Tye 1980; Beggs 1978; Hinnen et al. 1978). As a result of these observations, it has been suggested that the specific yeast sequences (ars's) are normally sites of initiation of chromosomal DNA replication, this activity being retained when they are incorporated into plasmids. Sequences which promote autonomous plasmid replication in yeast transformants have now been isolated from chromosomal and organelle DNA of a variety of eukaryotic organisms (Stinchcomb et al. 1980; Beach and Nurse 1981; Zakian 1981; Gorman et al. 1981; Hyman et al. 1982), but not from prokaryotic ones (Stinchcomb et al. 1980), although a Staphylococcus aureus plasmid is an exception to this role (Goursot et al. 1982). This paper describes the cloning and characterisation of sequences from chromosomal DNA of the basidiomycete fungus U. maydis with ars activity in yeast in order to complement and advance our studies of the biochemistry of DNA replication in this organism (Banks et al. 1976; Banks and Yarranton 1976; Yarran-

G.R. Banks: Ars-like Sequences from U. maydis

80 t o n a n d B a n k s 1 9 7 7 ; Jeggo a n d B a n k s 1 9 7 5 ; B a n k s a n d S p a n o s 1 9 7 5 ) a n d t o develop a n Ustilago t r a n s f o r m a t i o n s y s t e m to facilitate surrogate genetics o f D N A replicat i o n , repair a n d r e c o m b i n a t i o n genes (see a c c o m p a n y i n g paper).

Materials a n d M e t h o d s

Strains. E. coli SF8 (C600 thr6 leuB thi- hsr- hsm- lop11 recBC), plasmids YIp5, YRpl2 and S. cerevisiae YNN27 (atrpl289 ura3-52 gal2) were kindly provided by R. Davis and pBR322 by D. Sherratt. YRpl2 contains the yeast TRP1 gene (with an associated ars sequence) inserted into the EcoRI site of YIp5 (Scherer and Davis 1979). U. maydis 521 AB was from the collection of R. Holliday.

Enzymes. Restriction endonucleases were from Bethesda Research Laboratories, Inc. and were used as recommended by the supplier. PstI was a gift from G. T. Yarranton and bacteriophage T4 DNA ligase was isolated by a published procedure (Panet et al. 1973). E. coli DNA polymerase I was from Boehringer Mannheim, glusulase from Endo Labs., Inc.

Media. t?. coli was grown in L-broth (Miller 1972) or, for plasmid amplification, in M9 minimal medium (Miller 1972). S. cerevisiae was grown in YEPD (1% yeast extract, 2% Difco peptone and 2% glucose) or in YNB selective medium (0.67% difco yeast nitrogen base lacking amino acids and 2% glucose). U. maydis was grown in complete medium (Holliday 1974). The following were added as required (final concentrations) - L-tryptophan (20 /~g/ml), uracil (20 ~g/ml), chtoramphenieol (180/~g/ml), and ampicillin (50 ~g/ml).

DNA. Preparative and rapid plasmid DNA isolations from E. coli were by the methods of Guerry et al. (1973), followed by CsCl-ethidium bromide centrifugation, and Burke and IshHorowitz (1981), respectively. Rapid yeast DNA isolation was by the method of Nasmyth and Reed (1980). U. maydis chromosomal DNA was isolated as follows (unpublished methods of G. T. Yarranton). 20 g frozen U. maydis 521 was thawed by suspension in 60 ml cold 50 mM Tris-HC1 pH 7.5, 5 mM EDTA and 0.75 M sucrose. The suspension was passed twice through a French pressure cell at 18,000 p.s.i, and centrifuged at 1,000 rpm and 4 °C for 10 min in a Sorvail SS-34 rotor. The supernatant was carefully removed and the loose pellet re-extracted with 40 ml of the sucrose buffer. The nuclei in the combined supernatants were centrifuged at 8,000 rpm for 20 min and resuspended in 2 ml sucrose buffer. 2 ml 50 mM Tris-HC1 pH 7.5, 5 mM EDTA followed by 0.1 ml 10% sodium dodecyl sulphate were added to lyse the nuclei and the resulting suspension extracted with an equal volume of phenol-chloroform. After gentle mixing, the suspension was centrifuged followed by addition of 0.2 ml 5 M NaC1 and 8 ml ethanol to precipitate nucleic acids from the aqueous phase. The suspension was held at - 2 0 °C for 60 rain, centrifuged and the pellet dissolved in 4 mt TE buffer (10 mM Tris-HC1 pH 8.0, 1 mM EDTA). Preheated pancreatic RNAse was added to 50 t~g/ml, the solution incubated at 37 °C for 30 min, mixed with an equal volume of phenol and centrifuged. Two volumes of ethanol were added to the aqueous phase, which was then held at - 2 0 °C for 120 min and centrifuged. The pellet was dissolved in TE and chromosomal DNA further purified by CsCl-ethidium bromide centrifugation.

Electrophoresis. Horizontal submarine 0.8% agarose (BioRad) gels were run in a buffer containing 10.8 g Trizma base, 0.25 g Na 2 • EDTA. 2H20 and 5.5 g boric acid per 1, overnight at 20 mA. After staining in 2 t~g/ml ethidium bromide, DNA bands were detected over a Birchover Instruments transilluminator and photographed on Type 55 Polaroid film with a Polaroid MP-4 Land camera.

Transformation. E. eoli SF8 and S. cerevisiae YNN27 were transformed with plasmid DNAs by published procedures (Mandel and Higa 1970; Hinnen et al. 1978). Hybridization. DNA was transferred from 0.7% agarose gels to sheets of nitrocellulose (Schleicher and Schiill BA85) in 20 x SSC (SSC is 0.015 M sodium citrate, 0.15 M sodium chloride, pH 7.0 by the m e t h o d o f Southern (1975). Plasmid DNA hybridization probes, labelled with [a-32p]-dTTP by nick translation (Rigby et al. 1973), were hybridized to the filters in 5 x SSCPE (5 mM EDTA and 40 mM NaPO 4 in 5 x SSC) containing 0.2% SDS and 32 #g/ml denatured salmon sperm DNA at 65 °C for 16 h. The filters were washed in 5 x SSCPE at 65 °C, 2 x SSC at room temperature, dried and autoradiographed on Fuji RX X-ray film.

Cloning. 5 ~g U. maydis 521 chromosomal DNA and 5 ~g YIp5 DNA were separately digested with EcoRI enzyme, the two mixtures combined, extracted with phenol and then chloroform and the DNA ethanol precipitated. The drained precipitate was dissolved in T4 DNA ligase buffer (66 mM Tris-HC1 pH 7.6, 6.6 mM MgC12, 10 mM dithiothreitol and 1 mM ATP) and 15 units T4 DNA ligase added, volume 0.05 ml. After incubation at 12 °C for 60 min a further 20 units ligase and T4 ligase buffer was added to a final volume of 0.25 ml. Incubation was continued at 12 °C for 20 h, 12.5 #1 5 M NaC1 and two volumes of ethanol then added. The precipitated DNA was held in ethanol-solid CO 2 for 60 rain, centrifuged, washed with 70% ethanol, drained and dissolved in 22 #1 TE buffer and used for the transformation of S. cerevisiae YNN27. Cloning procedures were carried out under GMAG recommendations.

Results Cloning and Selection o f U. maydis DNA Sequences. T h e p l a s m i d Y I p 5 c o m p r i s e s the yeast U R A 3 gene i n s e r t e d i n t o t h e AvaI site o f p B R 3 2 2 (Fig. 1; S t r u h l e t al. 1979). It will t r a n s f o r m yeast strains w i t h p o i n t m u t a t i o n s in the h o m o l o g o u s c h r o m o s o m a l gene t o uracil i n d e p e n d e n c e b y c h r o m o s o m e i n t e g r a t i o n at l o w f r e q u e n c i e s ( B a c h et al. 1979). Strain Y N N 2 7 c o n t a i n s a d e l e t i o n - r e a r r a n g e m e n t in this gene (ura3-52) a n d uracil i n d e p e n d e n t t r a n s f o r m a n t s are n o t observed, p r e s u m a b l y b e c a u s e o f the l i m i t e d ura3 D N A s e q u e n c e h o m o l o g y ( S c h e r e r a n d Davis 1979). If, h o w e v e r , Y I p 5 c o n t a i n s a y e a s t ars s e q u e n c e or s e q u e n c e s f r o m o t h e r o r g a n i s m s w h i c h can f u n c t i o n in a n analogous fashion, uracil i n d e p e n d e n t t r a n s f o r m a n t s are isolated at h i g h f r e q u e n cies ( S t i n c h c o m b et al. 1 9 7 9 ; S t i n c h c o m b e t al. 1 9 8 0 ; Z a k i a n 1 9 8 1 ; H y m a n et al. 1982). S u c h p l a s m i d s are maintained and replicated autonomously within the t r a n s f o r m a n t s as closed, circular d o u b l e - s t r a n d e d D N A

81

G. R. Banks: Ars-like Sequences from U. maydis EH

B

A

Fig. 1. Plasmid YIp5. Restriction endonuclease sites are E EcoRI, B BamHI, H HindlII, A AvaI, S SalI and P PstI Table 1. Yeast transformant generation times Transformant containing plasmid

Generation time - h

YRp12 pURY1 pURY2 pURY19

4.0 4.5 6.4 7.9

lacking a centromere sequence) is unstable under selective growth conditions and dramatically so under nonselective ones because of plasmid loss, an observation diagnostic for autonomous plasmid replication (Stinchcomb et al. 1979; Stinchcomb et al. 1980). The three transformants were grown to 6 x 106 cells per ml in selective medium, and the culture diluted 1,000-fold into selective and non-selective media. After ten generation of growth in each, aliquots were plated onto both selective and non-selective agar plates to determine the percentages of uracil prototrophic cells in both cultures. Although large differences are apparent in the three percentages after growth in selective medium, all three dropped dramatically after ten generations of growth in the presence of uracil (Table 2), strongly suggesting that their plasmids were mitotically unstable and consistent with their autonomous replication.

Growth Rates. The generation times of the three transformants were determined in YNB medium. All were extended when compared to that for a Y R p l 2 transformant and the 2.5 h of YNN27 growing in uracil supplemented YNB (Table 1).

Identity o f Transformant DNAs. If all three transformants possessed autonomously replicating plasmids, they should be detectable in DNA extracted from each transformant by hybridization and by transformation of E. coli. Rapid DNA preparations from the three transformants were electrophoresed in an agarose gel, transferred to nitrocellulose and probed with [32p] pBR322 DNA. In each case a pattern of hybridization typical of a closed, circular plasmid DNA and its nicked derivatives was observed (Fig. 2). Each plasmid was larger than YIp5, no hybridization to chromosomal DNA was detected or to DNA isolated from non-transformed YNN27, even after over-exposure of the resulting autoradiogram (not shown). E. coli SF8 was transformed with DNA extracted from each of the yeast transformants and ampicillin resistant bacterial transformants selected. Colonies were randomly picked from each transformation, small cultures grown up and their plasmid DNA contents analysed by agarose gel electrophoresis (Fig. 3). All bacterial transformants resulting from the DNA of a given yeast transformant contained plasmids of the same size, whilst the plasmids derived from the three original yeast transformants differed in size from one another, were all larger than YIp5 and were identical to the sizes observed in the hybridization experiment described above. These experiments demonstrate that the three original yeast transformants possessed extrachromosomal, autonomously replicating plasmids of different sizes which can be transferred intact to E. coli by transformation. Thus they contain DNA of different sizes inserted into YIp5. No integration of pBR322 DNA sequences into the yeast chromosome was detected.

Mitotic Stability. The uracil independence of yeast strains transformed with ars containing YIp5 (but

Origin o f YIp5 DNA Inserts. If the DNAs inserted into YIp5 originated from U. maydis chromosomal DNA,

Table 2. Mitotic stability of yeast transformants Transformant containing plasmid

YRp12 pURY1 pURY2 pURY19

% ura3+ cells after 10 generations -uracil

+uracil

17 10 76 4

0.2 0.8 18.0 0.03

molecules and the system thus allows selection of DNA sequences from any organism with ars activity in yeast. YIp5 containing U. maydis chromosomal DNA was prepared as described above and used to transform S. cerevisiae YNN27 to uracil independence. Transformant colonies selected on YNB agar plates were colony purified twice to ensure that they were stable on selective medium, resulting in ten stable transformants per /~g DNA. Three faster growing ones (on selective agar), containing palsmids pURY1, pURY2 and pURY19, were selected for detailed characterisation.

82

G.R. Banks: Ars-like Sequences from U. maydis

Fig. 2. Autoradiogram of [32p]pBR322 DNA hybridizations. Lane 1, YIp5 DNA; lane 2, YRp12 yeast transformant DNA; lane 3, pURY1 yeast transformant DNA; lane 4, pURY2 yeast transformant DNA; lanes 5 and 6, pURY19 yeast transformant DNA (two concentrations); lane 7, pBR322 DNA; lane 8, YRpl2 DNA. All DNAs were electrophoresed on the same agarose gel. The open circular (o) linear (1) and supercoiled (s) DNA species of YIp5 and YRpl2 are labelled

Fig. 3. Agarose gel electrophoresis of plasmid DNAs. Lane 1, YRpl2 DNA; lane 2, YIp5 DNA; lanes 3-7, DNAs in five E. coli ampicillin resistant colonies resulting from transformation by the total DNA extracted from the yeast transformant containing pURY1; lanes 8-12, same as lanes 3-7 but the yeast transformant containing pURY2 was the DNA source; lanes 13-16, same as lanes 3 - 7 but the yeast transformant containing pURY19 was the DNA source

the plasmids containing them should hybridize to EcoRI fragments of U. maydis DNA of the same sizes as the inserts (note that no hybridization of YIp5 sequences alone to U. maydis DNA was detectable not shown). U. maydis chromosomal DNA was digested with EcoRI, electrophoresed on an agarose gel, transferred to nitrocellulose and probed separately with 3~p. labelled pURY1, pURY2 and pURY19 DNAs which had been purified from the E. coli transformants described above, pURYI hybridized to a U. maydis DNA fragment identical in size to the fragment inserted into YIp5 (Fig. 4A, lanes 3 and 2, respectively). A similar result obtained for pURY2 (Fig. 4B, lanes 3 and 2). pURY19 in fact contains two EcoRI fragments inserted into YIp5 (Fig. 4C, lane 2), which were identical in size to two U. maydis EcoRI fragments which hybridized to pURY19 (Fig. 4C, lane 3). In addition, there is hybridization to a fragment fractionally larger than linear YIp5 DNA. These results established that the DNAs inserted into YIp5 originated from U. maydis chromosomal DNA and that they represent unique EcoRI fragments in the U. maydis genome, except possibly for the

pURY19 insert. Included in each of the three hybridization experiments (but not shown in Fig. 4) were the other pURY plasmids. There was no detectable hybridization of one U. maydis insert to the other two and thus the three inserts share no sequence homologies.

Yeast Transformation by p UR Y1, pUR Y2 and PUR Y19. So eerevisiae YNN27 was transformed by the pURY1, pURY2 and pURY19 plasmids to uracil independence and transformation frequencies determined (Table 3). All three transformed YNN27 at high frequencies, comparable to YRpl2, but in contrast to YIp5. This result is expected for plasmids which can be replicated autonomously in yeast. Preliminary Mapping on the Inserted DNA. Plasmids pURY1, pURY2 and pURY19 were each digested separately or in combinations with EcoRI, BamHI, PstI, SalI and HindlII restriction endonucleases and the restriction sites mapped after agarose gel electrophoresis (Fig. 5). The sizes of the U. maydis DNA inserts were 1.1, 1.7 and 3.6 (comprising two EcoRI fragments of

G. R. Banks: Am-like Sequences from U. maydis

83

1.2 and 2.4) kilobase pairs, respectively. Each insert is different in sequence from the other two, in accordance with the conclusion derived from the hybridization experiments described above.

Discussion

Fig. 4A-C. Autoradiograms of [32p]pURY1 (A), pURY2 (B) and pURY19 (C) hybridizations. A lane 1, pURY1 DNA; lane 2, EcoRI cleaved pURY1 DNA; lane 3; EcoRI cleaved U. maydis DNA; B lane 1, pURY2 DNA; lane 2, EcoRI cleaved pURY2 DNA; lane 3, EcoRI cleaved U. maydis DNA; C lane 1, pURY19 DNA; lane 2, EcoRI cleaved pURY19 DNA; lane 3, EcoRI cleaved U, maydis DNA

Table 3. Transformation frequencies of S. cerevisiae YNN27 by plasmid DNAs Plasmid

Transformation per pg plasmid DNA

YIp5 YRp12 pURY1 pURY2 pURY19

0 3.6 3.4 1.9 1.9

x x x x

104 104 104 104

1.1 k b E

E

--1

I-- pURY2

1.7kb E 3"6kb

[ --

pURY 19

Fig. 5. Preliminary restriction map of cloned U. maydis ars fragements in YIp5. E EcoRI, B BamHI, H HindlII, S SalI. Sizes in kilobase pairs

Specific DNA sequences which promote the extrachromosomal maintainance and autonomous replication of plasmids in yeast transformants have been isolated from yeast chromosomal DNA at a frequency which might be expected from the estimated number of replicons per genome (Struhl et al. 1979; Hsiao and Carbon 1979; Kingsman et al. 1979; Szostak and Wu 1979; Chan and Tye 1980; Chanet al. 1981; Stinchcomb et al. 1981; Tschumper and Carbon 1981). The properties which they confer upon plasmids are consistent with a role in the initiation of the plasmid DNA replication and, by inference, in chromosomal replicon initiation in situ. Additional roles, such as in plasmid segregation at mitosis, have not been unequivocally excluded. These properties include autonomous replication and lack of detectable integration into chromosomes; mitotic and meiotic instability (plasmids also containing centromeric sequences (Chart et al. 1981; Stinchcomb et al. 1981; Tschumper and Carbon 1981) and the trp-1 circle (Scott and Brajkovich 1981) are exceptions), despite a relatively high average copy number; cis acting and transformation of yeast at high frequencies. The properties of the cloned U. maydis chromosomal sequences described above suggest that in yeast transformants they are functionally identical to the yeast ars sequences and in agreement with observations that chromosomal DNA from a wide variety of eukaryotes contains similar functional sequences (Stinchcomb et al. 1980; Beach and Nurse 1981; Zakian 1981; Gorman et al. 1981; Hyman et al. 1982). The hybridization and mapping experiments provide evidence that the three U. maydis ars sequences possess no large-scale homologies. Two classes of yeast ars sequences have been identified, unique and ones reiterated in the yeast genome (Chan and Tye 1980; Chan et al. 1981 ). The three sequences characterised in detail above appear to be on the unique class. The status of the third fragment hybridizing to pURY19 (Fig. 4C, lane 3) is equivocal because the insert comprises a double EcoRI fragment and it is not yet known which of the two subfragments possess ars activity, but it is clearly not extensively reiterated. With the development of a U. maydis transformation system (see accompanying paper) we intend to determine if the present unique ars sequences are also functional in U. maydis itself and to isolate further sequences to determine if a reiterated class exists. The plasmids characterised above may also

84 b e o f u t i l i t y as s u b s t r a t e s for U. maydis D N A replicat i o n p r o t e i n s to d e t e r m i n e i f r e p l i c a t i o n in vitro can b e i n i t i a t e d specifically at an ars s e q u e n c e , as appears to b e the case w i t h yeast ars p l a s m i d s (J. C a m p b e l l , J. Scott, p e r s o n a l c o m m u n i c a t i o n s ) .

References Bach M-L, Lacroute F, Botstein D (1979) Proc Natl Acad Sci USA 7 6 : 3 8 6 - 3 9 0 Banks GR, Holloman WK, Kairis MV, Spanos A, Yarranton GT (1976) Eur J Biochem 62:131-142 Banks GR, Spanos A (1975) J Mol Biol 9 3 : 6 3 - 7 7 Banks GR, Yarranton GT (1976) Eur J Biochem 6 2 : 1 4 3 - 1 5 0 Beach D, Nurse P (1981) Nature 290:140-142 Beggs JD (1978) Nature 2 7 5 : 1 0 4 - 1 0 9 Blumenthal AB, Kriegstein H J, Hogness DS (1974) Cold Spring Harbor Symposium Quant Biol 38:205-223 Braun R, Will H (1969) Biochim Biophys Acta 174:246-252 Burke JF, Ish-Horowitz D (1981) Nucleic Acids Res 9 : 2 9 8 9 2998 Burke W, Fangman WL (1975) Cell 5 : 2 6 3 - 2 6 9 Callan HG (1972) Proc Royal Soc London Series B 181:19-41 Chan CSM, Maine G, Tye B-K (1981) Functional components of the Saccharomyees cerevisiae chromosomes - replication origins and centromeric sequences. In: Ray DS (ed) The initiation of DNA replication. Academic Press, New York, p 455 Chan CSM, Tye B-K (1980) Proc Natl Acad Sci USA 7 7 : 6 3 2 9 6333 Dawes IW, Carter BLA (1974) Nature 250:709-712 Edenberg HJ, Huberman JA (1975) Annu Rev Gen 9 : 2 4 5 - 2 8 4 Gorman JA, Dove WF, Warran N (1981) Mol Gen Genet 183: 306-313 Goursot R, Goze A, Niaudet B, Ehrlich SD (1982) Nature 298: 488-490 Guerry P, LeBlanc DJ, Falkow S (1973) J Bacteriol 116:10641066 Harland RM, Laskey RA (1980) Cell 21:761-771 Hinnen A, Hicks JB, Fink GR (1978) Proc Natl Acad Sci USA 75:1929-1933 HoUiday R (1974) Ustilago maydis. In: King RC (ed) Handbook of Genetics, vol 1. Plenum Press, New York, p 575 Hsaio CI, Carbon J (1979) Proc Natl Acad Sci USA 7 6 : 3 8 2 9 3833

G.R. Banks: Ars-like Sequences from U. maydis Hyman BC, Cramer JH, Rownd RH (1982) Proc Natl Acad Sci USA 79:1578-1582 Jeggo PA, Banks GR (1975) Biochim Biophys Acta 1 4 2 : 2 0 9 224 Kee SG, Haber JE (1975) Proc Natl Acad Sci USA 7 2 : 1 1 7 9 1183 Kingsman AJ, Clarke L, Mortimer RK, Carbon J (1979) Gene 7:141-i52 Mandel M, Higa A (1970) J Mol Biol 53:159-162 Miller JH (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, New York Muldoon JJ, Evans TE, Nygaard OF, Evans HH (1971) Biochim Biophys Acta 247:310-321 Nasmythe K, Reed SI (1980) Proc Natl Acad Sei USA 7 7 : 2 1 1 9 2123 Panet A, van de Sande JH, Loewen PC, Khorana HG, Raae A J, Lillehaug JR, Kleppe K (1973) Biochemistry 12:50455050 Rigby PWJ, Dieckmann M, Rhodes C, Berg P (1977) J Mol Biol 113:737-751 Scherer S, Davis RW (1979) Proc Natl Acad Sci USA 7 6 : 4 9 5 1 4955 Scott JF, Brajkovich CM (1981) Replication properties of trplRl-circle: a high copy number yeast chromosomal DNA plasmid. In: Ray DS (ed) The initiation of DNA replication. Academic Press, New York, p 517 Southern EM (1975) J Mol Bio198:503-517 Stinchcomb DT, Mann C, Selker E, Davis RW (1981) DNA sequences that allow the replication and segragation of yeast chromosomes. In: Ray DS (ed) The initiation of DNA replication of DNA replication. Academic Press, New York, p. 473 Stinchcomb DT, Struhl K, Davis RW (1979) Nature 2 8 2 : 3 9 - 4 3 Stinchcomb DT, Thomas M, Kelly J, Selker E, Davis RW (1980) Proc Natl Acad Sci USA 77:4559-4563 Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) Proc Natl Acad Sci USA 7 6 : 1 0 3 5 - 1 0 3 9 Szostak JW, Wu R (1979) Plasmid 2 : 5 3 6 - 5 5 4 Tschumper G, Carbon J (1981) Sequencing and subcloning analysis of autonomously replicating sequences from yeast chromosomal DNA. In: Ray DS (ed) The initiation of DNA replication. Academic Press, New York, p 489 Yarranton GT, Banks GR (1977) Eur J Biochem 77:521-527 Zakian VA (1981) Proc Natl Acad Sci USA 7 8 : 3 1 2 8 - 3 1 3 2

C o m m u n i c a t e d b y B. S. C o x Received October 25, 1982