|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1993 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1992 : ¥1,000,000 (Direct Cost : ¥1,000,000)
1. The 2287bp plasmid, pMAl.and the 4993bp cryptic plasmid, pMA2, both from Microcystis aeruginosa f.aeruginosa Kutzing, a unicellular cyanobacterium originally derived from Kasumigaura lake, were completely sequenced and analyzed. The predicted amino acid sequence (253 residues) of an open reading frame had identities of 47%, 48%, and 53% with replication-associated protein of Bacillus amyloliquefaciens's plasmid, pFTB14, B.subtillis BAA1's pBAA1, and B.coagulans's pBC1, respectively, when conservative aminoacid substitutions were included Such high-level identities were also shown with rep proteins and oriregions in a group of Gram-positive bacterial plasmids such as Lactococci and Staphylococci that are known to replicate via single-strand intermediates. The pMA1 does hybridize with a plasmid with a plasmid, pUS1-3, derived from another unicellular cyanobacterium, Synechocystis sp. PCC 6803.
2. Single-stranded plasmid DNA of the pPM1 that was specifically degraded by S1 nuclease was detected by Southern hybridization. This observation suggest that such small cryptic plasmid as pMA1 replicate through a rolling circle mechanism in the living cells, probably by the replication protein encoded on the pMA1.
3.pMA1(2287bp)にはグラム陽性菌であるBacillus sp.Lactobacillus sp.のプラスミド類が有するreplication proteinと相同性の高い領域、Ori領域及び複製開始点が存在していた。この検索結果に基づいてpMA1の複製機構を調べた。pMA1を菌体より抽出後、S1 nuclease処理、Agarose gel電気泳動、Nylontransfer membrane移送及びそれぞれのプラスミドプローブによるHybridization実験の結果からpMA1は明らかにRolling Circle Replication機構に従って複製される事が証明された。現在このreplication proteinの証明を進めている。