[Isolation and characterization of functional replication origin from Streptomyces avermitilis].
Academic Article
Overview
abstract
Most eubacteria contain highly conservative gene clusters in the adjacent regions of oriC. According to this principle, a 1.4 kb DNA fragment containing parts of dnaA and dnaN genes of Streptomyces avermitilis was cloned by degenerate PCR. Sequence analysis of this fragment indicated that it encoded two partial genes in the order dnaA (the putative initiator protein) and dnaN (the beta subunit of DNA polymerase III). The intergenic non-coding region between dnaA and dnaN was found to contain 19 putative DnaA boxes, i.e. 9 nt long DnaA protein recognition sequences. It was confirmed that the location, orientation and spacing of DnaA boxes in this intergenic region are conserved among Streptomyces. The consensus sequence of DnaA box identified is (T/C)(T/C)(G/A/C)TCCACA (preferred bases in italic). When this fragment was cloned into Escherichia coli plasmid pQC156, which is otherwise non-replicative in Streptomyces, it exhibited autonomous replication activity in Streptomyces lividans, a closely related Streptomyces strain. Different parts of the oriC contribute unequally to the stability and transformation efficiency. The 3' region of oriC may contain features that support stable autonomous replication. The implications of these results for the understanding of the S. avermitilis oriC replication initiation process and its future application are discussed.
