Bioinformatic and genetic identification of replication origins in H. hispanica. A. Seven replication origins, oriC1-cdc6A and oriC2-cdc6E in the main chromosome; oriC4-cdc6G, oriC5-cdc6H, oriC6-cdc6I and oriC7-cdc6J in the minichromosome; and oriP-cdc6K in the megaplasmid, were predicted by searching ORB motifs (indicated with small triangles) in the IRs located directly adjacent to orc/cdc6 genes (indicated with red arrowheads) using MEME software. Logo representations of ORB elements are presented on the right, and the spaces represent sequences that are not conserved. oriC3*: predicted deficient origin adjacent to cdc6D gene. B. Replication assay for plasmids containing the origins predicted in A. (Up) Southern blot analysis with a bla gene probe: lane T contains crude DNA extracted from the H. hispanica transformants, and lane P represents the purified plasmid as an input control; (down) summaries of the identification of origins in H. hispanica and the five origins with ARS activity (oriC 1, 2, 6, 7, P) are indicated with filled ovals and are bolded in A.