Cloning and identification of telomere and internal origin of the endophytic Streptomyces linear plasmid pYY8L from Allium fistulosum

Abstract:

Abstract: Strain 36R-2-1B was isolated from Allium fistulosum and identified as Streptomyces spp., harboring a ~280 kb linear plasmid designated pYY8L. [Objective] Cloning, sequencing and analysis of telomere and internal replication origin of pYY8L. [Methods] pYY8L telomere was cloned by a modified procedure ─“alkaline treatment and enzyme digestion in gel”. The internal replication origin of pYY8L was cloned by construction of a cosmid library and then sub-cloning. [Results] A ~280 kb DNA band (pYY8L) of strain 36R-2-1B was detected by pulsed-field gel electrophoresis. The 152-bp telomere, containing six small palindromes and potentially forming complicated secondary structure, was cloned and sequenced. The replication origin of pYY8L was initially cloned on a cosmid and then sub-cloned as a 4891-bp autonomous replication sequence. This sequence was predicted to contain six genes, two of them resembling replication genes of Streptomyces linear plasmid SCP1, but their adjacent iterons were different. [Conclusion] New telomere and internal replication origin of Streptomyces linear plasmid pYY8L were cloned and identified. This is the first example of report on telomere and replication genes of linear plasmid from endophytic Streptomyces.