A total of 4940 random sequence tags of the dimorphic yeast Yarrowia lipolytica, totalling 4.9 Mb, were analyzed. BLASTX comparisons revealed at least 1229 novel Y. lipolytica genes 1083 genes having homology with Saccharomyces cerevisiae genes and 146 with genes from various other genomes. This confirms the rapid sequence evolution assumed for Y. lipolytica. Functional analysis of newly discovered genes revealed that several enzymatic activities were increased compared to S. cerevisiae, in particular, transport activities, ion homeostasis, and various metabolism pathways. Most of the mitochondrial genes were identified in contigs spanning more than 47 kb. Matches to retrotransposons were observed, including a S. cerevisiae Ty3 and a LINE element. The sequences have been deposited with EMBL under the accession numbers AL409956^AL414895. ß

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 produced electrophoretic karyotypes of the reference strain E150 and of seven other isolates from different geographical origins to study the genomic organization of the dimorphic yeast Yarrowia lipolytica. These karyotypes differed in the number and size of the chromosomal bands. The karyotype of the reference stain E150 consisted of five bands of between 2.6 and 4.9 Mb in size. This strain contained at least five rDNA clusters, from 190 to 620 kb in size, which were scattered over most of the chromosomes. The assignment of 43 markers, including rRNA genes and three centromeres, to the E150 bands defined five linkage groups. Hybridization to the karyotypes of other isolates with pools of markers of each linkage group showed that linkage groups I, II, IV and V were conserved in the strains tested whereas group III was not and was split between at least two chromosomes in most strains. Use of a meganuclease I-SceI site targeted to one locus of E150 linkage group III showed that two chromosomes actually comigrated in band III of this strain. Our results are compatible with six chromosomes defining the haploid complement of strains of Y. lipolytica and that, despite an unprecedented chromosome length polymorphism, the overall structure of the genome is conserved in different isolates.