Colocalization of centromeric and replicative functions on autonomously replicating sequences isolated from the yeast Yarrowia lipolytica.

Abstract

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...

An Origin of Replication and a Centromere Are Both Needed To Establish a Replicative Plasmid in the YeastYarrowia lipolytica

“…On the basis of the genetic behavior in Y. lipolytica of plasmids bearing the ARS18 and ARS68 elements, we had concluded that a centromere was present in each of these sequences (26). We decided to investigate if this was also the case for ARS2, another ARS element described for this yeast (57).…”

“…These two ARS plasmids displayed a behavior different from that of traditional ARS elements in S. cerevisiae: low copy number, higher mitotic stability, and scarcity in a genomic DNA library. These unusual properties were later attributed to the presence of a centromere within each of the two ARS elements (26). At the same time another group also described the isolation of two ARS sequences from this yeast (57).…”

“…2; the NotI site belongs to a polylinker located a few base pairs from the left BamHI site) into the plasmid pINA731 digested by NotI and NheI (NheI and NotI are two vector sites, NotI being located close to the BglII site of ARS68, which is cloned in the reverse direction in this plasmid [see reference 26]). Transformation of yeast INAG33122 was achieved with NotIlinearized pINA888 by selecting for Leu ϩ clones.…”

“…Y. lipolytica transformation has been described elsewhere (3). Chromosome separation was achieved according to published protocols (26,81). The preparation of replication intermediates and 2D gel analyses followed methods already described by Huberman (43).…”

Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production

“…Yet, the genetic tractability of Y. lipolytica [18][19][20][21][22][23][24] coupled with its modest, innate de novo lipogenesis (B10-15% lipid content in wild type 13,14,[25][26][27] ) make it a potential candidate as a platform organism for superior lipid production.…”