Figure 1.

Cloning of Y. lipolytica origins with a centromeric plasmid. (A) The cloning vector pINA732-Δ contains the LEU2 gene (dotted area), the CEN1 centromere, and flanking genomic sequences. Y. lipolytica genomic DNA digested with Sau3A was ligated into the BamHI site of the vector. (B) Characteristics of the eight ORI elements analyzed in this article. Demonstration that five of them are active origins comes from Vernis et al. (1997) and from data presented below in this article. We therefore adopt the ORI designation for all of them. Two of these sequences, marked by a star (ori1068 and ori3018), were subjected to systematic size reduction to determine the minimal functional fragment (Figure 4). The chromosomal localization of the different fragments was determined by pulsed-field gel electrophoresis and Southern blotting using reference strain E150. Chromosomal bands are numbered according to Vernis et al. (1997). Chromosomal activity (active; nt, not tested) refers to the observation of replication initiation signals after 2D gel electrophoresis.