ORIGINAL PAPER K. Hiratsu á S. Mochizuki á H. Kinashi Cloning and analysis of the replication origin and the telomeres of the large linear plasmid pSLA2-L in Streptomyces rochei Received: 2 December 1999 /Accepted: 12 April 2000 Abstract The replication origin and both terminal seg- ments were cloned from the large linear plasmid pSLA2- L in Streptomyces rochei 7434AN4. The basic replicon consists of a 1.9-kb DNA fragment, which contains the genetic information required for autonomous replication in circular form. Sequence analysis revealed two ORFs, RepL1 and RepL2, with no similarity to any of the replication initiator proteins in the database. Deletion and mutational analysis showed that RepL1 is essential for replication and RepL2 has a subsidiary function. The origin of replication may be located 800 bp up- stream of repL1. Sequencing of the left and right ter- minal segments revealed the presence of 12 palindromes. The sequence of the ®rst 90 bp, including palin- dromes I±IV, shows great similarity to that of other Streptomyces linear chromosomes and plasmids. These results suggest that the internal replication origins of the linear replicons vary widely, in contrast to the high de- gree of conservation of their telomeres. Key words Streptomyces á Linear plasmid á Replication origin á Telomere Introduction Streptomyces rochei 7434AN4, a producer of two macrolide antibiotics, lankacidin Harada et al. 1969; Uramoto et al. 1969) and lankamycin Keller-Schierlein and Roncari 1964), carries three linear plasmids, pSLA2-L 206 kb), M 100 kb), and S 17 kb) Kinashi et al. 1994). The smallest plasmid, pSLA2-S, was the ®rst linear plasmid to be isolated from bacteria Ha- yakawa et al. 1979; Hirochika et al. 1985). Since then, many linear plasmids in a wide range of sizes 12 kb to 1.7 Mb) have been isolated from actinomycetes Kinashi 1994). In addition, recent studies have indicated that all the chromosomes in Streptomyces species are linear Lin et al. 1993; Lezhava et al. 1995; Leblond et al. 1996; Redenbach et al. 1996; Pandza et al. 1997), so this seems to be a characteristic of the genus. These linear chro- mosomes and plasmids have an internally located rep- lication origin Calcutt and Schmidt 1992; Kieser et al. 1992; Zakrzewska-Czerwinska and Schrempf 1992; Shiman and Cohen 1992; Chang and Cohen 1994), terminal inverted repeats TIRs) and covalently bound terminal proteins TPs). They replicate bi-directionally from a replication origin near the center of the molecule Chang and Cohen 1994; Musialowski et al. 1994). Replication in the 3¢ direction is completed as far as the 3¢ ends, but that in the 5¢ direction does not reach the 5¢ ends. The recessed ends of the lagging strands may be ®lled in by protein-primed DNA synthesis Chang and Cohen 1994). Functional oriC regions have been isolated from the chromosomes of S. coelicolor A32) Musia- lowski et al. 1994) and S. lividans Zakrzewska-Czer- winska and Schrempf 1992), and appear to be conserved in several Streptomyces species Jakimowicz et al. 1998). The functional replication origin of linear Strepto- myces plasmids has been identi®ed only in pSLA2-S Chang et al. 1996), and comprises a series of direct repeats iterons) and two essential genes rep1 and rep2) similar to those of temperate bacteriophages of En- terobacteriaceae and Bacillus. The previously sequenced 12-kb linear plasmid, pSCL1, of S.clavuligerus Wu and Roy 1993) includes counterparts of the rep1, rep2, and iterons of pSLA2-S Chang et al. 1996). In contrast to the dierences in the replication origins between the linear chromosomes and plasmids, their telomeres Mol Gen Genet 2000) 263: 1015±1021 Ó Springer-Verlag 2000 Communicated by W. Goebel K. Hiratsu &) Department of Fermentation Technology, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8527, Japan E-mail: hiratsu@ipc.hiroshima-u.ac.jp Tel.: +81-824-24-7767; Fax: +81-824-24-7759 S. Mochizuki á H. Kinashi Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8527, Japan