Short DNA fragments without sequence similarity are initiation sites for replication in the chromosome of the yeast Yarrowia lipolytica

We have previously shown that both a centromere (CEN) and a replication ori gin are necessary for plasmid maintenance in the yeast Yarrowia lipolytica (Vernis et nl., 1997). Because of this requirement, only a small number of centromere-proximal replication origins have been isolated from Yarrowia. W e used a CEN-based plasmid to obtain noncentromeric origins, and several ne w fragments, some unique and some repetitive sequences, were isolated. Some of them were analyzed by two-dimensional gel electrophoresis and correspon d to actual sites of initiation (ORI) on the chromosome. We observed that a 125-bp fragment is sufficient for a functional ORI on plasmid, and that ch romosomal origins moved to ectopic sites on the chromosome continue to act as initiation sites. These Yarrowia origins share an 8-bp motif, which is n ot essential for origin function on plasmids. The Yarrowin origins do not d isplay any obvious common structural features, like bent DNA or DNA unwindi ng elements, generally present at or near eukaryotic replication origins. Y . lipolytica origins thus share features of those in the unicellular Saccha romyces cerevisiae and in multicellular eukaryotes: they are discrete and s hort genetic elements without sequence similarity.