| Summary: | 碩士 === 國立中興大學 === 生命科學院碩士在職專班 === 98 === ABSTRACT
Plasmid pSN (2272 bp) from Haloterrigena sp. H13 was sequenced. The deduced amino acid sequence of the largest ORF (330 a.a.) individually shows 68 %, 67% and 60 % similarity to the putative replication proteins (Rep) of pGRB1, pHSB and pHGN1 from Halobacterium sp. and contains three sequence motifs that conserved in the Rep proteins of rolling circle replication (RCR) mechanism. Based on the homology of double strain origin and Rep protein sequence (http://www.essex.ac.uk/bs/staff/osborn/DPR/DPR_RCRdata. htm), pSN belongs to the Group XII of RCR. Plasmid pYH (3820 bp) from Methanocalculus taiwanensis strain P2F9704a was sequenced. The deduced amino acid sequence of the largest ORF (562 a.a.) individually shows 28 % similarity to the putative replication protein of pHEN7, helicase-like protein of pRN2 from Sulfolobus islandicus and 27 % similarity to primase protein of pDL10 from Acidianus ambivalens. Based on the homology of Rep protein sequence, pYH belongs to the pRN family. The single stranded intermediates form of pSN and pYH were detected by southern blotting and PCR, which confirm that pSN and pYH replicate through RCR mechanism. DNA encoding ORF330 was cloned into Escherichia coli and a expected polypeptide was detected. Western blotting with anti-His antibody confirm that this overexpressed protein is the translational product of orf330. Phylogenetic analysis based on the amino acid sequences of archaeal Rep proteins indicated that RepSN of pSN is clustered to the plasmids of the aerobic extremely halophilic Euryarchaeota. And RepYH of pYH is clustered to the plasmids of the acidophilic and thermophilic Crenarchaeota.
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