. 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 ...

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.

Wild-type strains of Saccharomycopsis lipolytica are able to use lysine as a carbon or a nitrogen source, but not as a unique source for both. Mutants were selected that could not use lysine either as a nitrogen or as a carbon source. Some of them, however, utilized N-6-acetyllysine or 5-aminovaleric acid. Many of the mutants appeared to be blocked in both utilizations, suggesting a unique pathway for lysine degradation (either as a carbon or as a nitrogen source). Genetic characterization of these mutants was achieved by complementation and recombination tests.

Fourteen lipase low-producing (Lip-) mutants of Yarrowia lipolytica were obtained by UV mutagenesis. Complementation studies showed that ten of these mutants belonged to one complementation group. Two other mutants, Lip-132 and 223, complemented those of the first group. Lip-1123 complemented mutants of the first and second groups. Lip-1351 did not complement Lip-742, 417, 529 and 1133, which belonged to the first group. Genetic recombination analysis of the Lip-mutants by random ascospore analysis showed that mutants of the first and second groups have lesions in the same gene, which is proposed to be the lipase structural gene. Inter-allelic complementation occurs between certain mutants of these groups. Lip-1123 has a lesion in a different gene.

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