Two sequences (ARS18 and ARS68) displaying autonomous replication activity were previously cloned in the yeast Yarrowia lipolytica. (4,5). The copy number ofARS plasmids in Y. lipolytica has been reported to be about 3 per plasmid-containing cell, which is also very different from the 50-100 copies for S. cerevisiae ARS plasmids (6) but closer to the 1-2 copies per cell described for centromeric plasmids (3).We wondered whether ARS18 and ARS68 are ARS sequences associated with Y. lipolytica centromeres or with some kind of stabilizing sequence like that described for Schizosaccharomyces pombe (7). The structure of S. cerevisiae centromeres has been extensively investigated (8): they contain two conserved sequences (CDEI and CDEIII) separated by an A+T-rich region (CDEII), and a functional centromere is contained within a 125-bp sequence (9). In contrast, the three centromeres of the fission yeast Sch. pombe cover very large regions (35, 55, and 110 kb) displaying a complex pattern of repetitive DNA sequences (10, 11). Thus the two types of centromeres from Sch. pombe and S. cerevisiae are very different (12). The Sch. pombe centromeres have an organization similar to those of higher eukaryotes, and the S. cerevisiae centromeres could be more representative of those from other yeasts, such as Kluyver-The 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. omyces marxianus var. lactis (hereafter, K. lactis). Indeed DNA fragments probably corresponding to K. lactis centromeres (13) are very similar in sequence to the S. cerevisiae centromeres, except for a larger CDEII region (14). However, they are not recognized as centromeres in S. cerevisiae. Our analysis of Y. lipolytica ARS18 and ARS68 shows that each carries functional centromeres within a 1-kb DNA fragment and that they do not share any significant sequence similarity with known S. cerevisiae, K. lactis, or Sch. pombe sequences. MATERIALS AND METHODSStrains and Plasmids. The following Y. lipolytica strains were used: INAG 33122 (MatB, leu2-35, xpr2, lys2-5), E122 (MatA,, 21805-9 (MatA, leu2-35, ura3-18), and 22301-3 (MatB, ura3-302, leu2-270, his-i). Mutations leu2-270 and ura3-302 are in vitro-generated deletions ofLEU2 and URA3 (gift of E. Fabre, Institut National de la Recherche Agronomique). The Y. lipolytica URA3 gene was obtained from L. Davidow (Pfizer). The ARS18-URA3 plasmid pINA311 was given to us by E. Fabre.Genetic and Molecular Biology Techniques. ARS68 was cloned on a 2.3-kb BamHI-Bgl II fragment (see Fig. 2) into the BamHI site of the pBluescript vector (Stratagene) in both orientations. Both plasmids were sequentially digested with Kpn I and HindIII and then treated with exonuclease III by using the Erase-a-Base kit (Promega). The DNA was religated, giving a series of plasmids with various deletions of the ARS68 region. Several ARS18 restriction fragments were cloned in pBluescrip...

. We have used the integration properties of centromeric sequences to show that all Y. lipolytica ARS elements so far isolated are composed of both a replication origin and a centromere. The sequence and the distance between the origin and centromere do not seem to play a critical role, and many origins can function in association with one given centromere. A centromeric plasmid can therefore be used to clone putative chromosomal origins coming from several genomic locations, which confer the replicative property on the plasmid. The DNA sequences responsible for initiation in plasmids are short (several hundred base pairs) stretches which map close to or at replication initiation sites in the chromosome. Their chromosomal deletion abolishes initiation, but changing their chromosomal environment does not. Although the nature of higher eukaryotic replication originsis not yet clear (12), that of origins in the yeast Saccharomyces cerevisiae is relatively well understood. It has been possible to clone S. cerevisiae genomic sequences that confer on a plasmid the ability to replicate extrachromosomally in this yeast. These sequences are called ARS (autonomously replicating sequence) elements. Most ARS elements are active as origins in their chromosomal context, as shown by two-dimensional (2D) gel replicon mapping studies (8,44,62). ARS elements are usually smaller than 150 bp, and they contain an essential motif that matches the 11-bp ARS consensus sequence (ACS; WTT TAYRTTTW) in at least 9 of 11 positions (61, 72). The ACS is essential for initiation of replication, both on plasmids and in the chromosome (17). Also essential is domain B, located at the 3Ј end of the ACS (54,55,76). Depending on the ARS element, domain B can be subdivided into two or three subdomains with variable sequences but conserved roles (42, 66). Finally, a DNA unwinding element is frequently present in ARS elements and is important for replication of plasmids (60) and chromosomes (41). Initiation at ARS elements is under strict cell cycle control (reviewed in reference 18).In contrast to the defined origin sequences of S. cerevisiae, the degree to which specific sequences are employed as origins in the differentiated cells of higher organisms is not yet clear. Most studies employing labeling techniques suggest that initiation takes place at specific sites or in very small (a few kilobases or less) initiation zones (15,19,35). In contrast, all studies employing 2D gel replicon mapping techniques-and some studies using labeling techniques (31, 71)-suggest that initiation can take place at any of numerous locations within large initiation zones of 50 kb or more (reviewed in reference 36). Furthermore, in contrast to the discrete ARS elements of S. cerevisiae, most human DNA pieces larger than ϳ10 kb permit extrachromosomal plasmid replication (38,51). In this case, initiation does not start at a preferred locus, a result which is also observed in plasmid transformation in Xenopus eggs (46,53). Demonstrations that the choice of initiation sites ...

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Previous attempts is isolate autonomously replicating sequences (ars) from the dimorphic yeast Yarrowia lipolytica have been unsuccessful. We isolated a Fil- mutant unable to produce hyphae and growing only in a yeast form to facilitate ars isolation. This mutant was transformed with a Y. lipolytica DNA bank and several unstable clones were obtained. Extrachromosomal plasmids were evidenced in yeast, recovered in Escherichia coli and characterized by restriction mapping. They were able to retransform Fil- and Fil+ yeast strains at high frequency and transformants displayed a slightly unstable phenotype. The detailed analysis of the plasmids showed that only two different ars sequences had been isolated, each of them corresponding to a unique sequence in the Y. lipolytica genome. We concluded that functional ars sequences that can be cloned on plasmids are rare in this yeast.