.1 Rare Actinomycetes Genes In Vivo Biological Research. Chain Of Fungal Dna Fragment Of Genetic Operating System Initially Established

There are two parts in this thesis, one is studies on genetic elements in rareactionmycetes. 7 circular plasmids, 2 linear plasmids and one prophage wereisolated from 200 rare actinomycetes strains. Most of these strains are linearchromosomes.A 1339bp DNA fragment of the putative oriC region from the rifamycin SVproducer Amycolatopsis mediterranei U32 was cloned by PCR amplificationemploying primers designed based on the conserved flanking genes of dnaA anddnaN. The 884bp sequence of the intergenic region between dnaA and dnaN genesconsists of 19 DnaA-boxes and two 13-mer AT-rich sequences, which is similar tothe oriC structure of Streptomyces lividans. A mini-chromosome constructed bycloning the putative U32 oriC DNA fragment into an E. coli plasmid was able toreplicate autonomously, but unstable, in A. mediterranei U32 with estimated copynumber of 2 per cell. Although efficient replication of the mini-chromosome inU32 requires the complete set of the DnaA-boxes and AT-rich regions, only one ofthe AT-rich sequences together with part of the DnaA-boxes are sufficient,suggesting the presence of combinatorial alternatives for a functional oriC regionof A. mediterranei U32. Phylogenetic analysis based on definite oriC sequencesamong eubacteria reflect well the relationship between these species. A 6.8kb circular plasmid, pSR119 with 8-10 copies per cell, was isolatedfrom Streptosporangium SC119. Sequence analysis of pSR119 show there are 12ORFs, including genes for conjugation, transposase and transcriptional regulator.Phage NC174 from Nocardia SC174 have been isolated by PFGE electrophoresis,its sequence contain all feature of phage, for example genes of capsid protein,terminase, tail protein and integrase.The other part is construction of genetic manipulation system for large DNAfragment in Streptomyces. A series of cloning vector have been developed by usingtelomere of Streptomyces chromosome, replicon of linear plasmid pSLA2 andSLP2. We tried to clone large DNA fragment of Streptomyces avermilitischromosome. Meanwhile, we developed a method for cloning large DNA fragmentof telomere region of Streptomyces chromosome used the recombination system invivo and conjugative ability of linear plasmid. A 10kb DNA fragment of telomereregion was cloned successfully.