Figure 2.

Activation of the dormant chromosomal ARS301 and ARS302/303. (A) Detection of RIs from ARS301. α factor-arrested cells were released from the G₁ block into medium containing HU. At the indicated times replication intermediates that originated from ARS301 were studied using the alkaline gel electrophoresis method as described (Santocanale and Diffley 1998). The relevant genotype is indicated. A 207-bp HindIII–BamHI DNA fragment containing the ARS301 sequence prepared from plasmid pCS1 (Santocanale and Diffley 1996) was used as a probe. (B) Characterization of RIs from ARS301. DNAs prepared from α factor-arrested cells (α) or cells released into HU containing medium for 90 min (HU 90′) were analyzed as described in Materials and Methods. (C) Detection of replication intermediates from ARS302. Cells were grown and RIs analyzed as in A. A 1-kb DNA fragment of chromosome III (14,000–15,000 nucleotides) containing the ARS302 sequence was used as a probe for hybridization (lanes 1–6). In lanes 7–12 a 0.8-kb fragment of chromosome III (21,200–22,000 nucleotides) that does not contain origin sequences and lies between ARS303 and ARS304 was used as a probe.