FIG. 2.

All three S. pombe RFBs are active in autonomously replicating plasmids with the same polarity as in their chromosomal context. (A) Detail of the rDNA NTS. The horizontal line represents the 1.6-kb XhoI-HindIII restriction fragment analyzed below, which includes both 17-bp reb1p binding sequences (striped boxes). (B) Diagram of plasmids pIRT1.6(+) and pIRT1.6(−) obtained by inserting the XhoI-HindIII fragment (internal arc) in both orientations into pIRT2. The ars1-containing fragment (large striped box) and the site where replication initiates (black rhomb) (11) are included. (C) 2D gel analyzing replication of the PvuII-EcoRV fragment of pIRT1.6(+) containing the rDNA insert. As in Fig. 1B, arrowheads labeled 1, 2, and 3 point to the spots of accumulated replication intermediates induced by RFB1, RFB2, and RFB3, respectively. (C') A longer exposure of the autoradiogram shown in panel C in which the signals corresponding to bubble-containing intermediates (open arrowhead) and double Ys (arrow) are visible. (D) 2D gel analyzing replication of the same fragment as in panel C but from pIRT1.6(−), in which no accumulated intermediates were detected. The probe used in both autoradiograms was the PvuII-EcoRV fragment from vector pIRT2.