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Jin-Yong Jeong(정진용),Eun Sil Kim(김은실),Sam Woong Kim(김삼웅),Ho Young Kang(강호영),Jeong Dong Bahk(박정동) 한국생명과학회 2009 생명과학회지 Vol.19 No.3
플라스미드 pGKV21에는 229-nt single-strand DNA initiation (ssi) signal이 존재한다. Asymmetric PCR 기법으로 합성된 229-nt ssDNA 단편을 이용하여 실제로 RNA polymerase에 의한 priming ability와 protein interaction을 확인하였다. in vitro primer RNA 합성 실험 결과, 229-nt ssDNA 단편은 filamentous M13 phage의 주형 DNA에서와 비슷한 효율로 시발체 RNA를 합성하였으며, 이 반응은 strand-specific하게 이루어졌다. DNase I footprinting과 gel retardation 실험 결과, RNA polymerase와 SSB 단백질은 229-nt ssDNA 단편에 stable interaction을 하며, 시발체 RNA를 합성하였다. 또한, in vivo 조건 하에서 RNA polymerase의 저해제인 rifampicin을 처리하여 세포 내에 ssDNA 중간체가 집적되는 정도를 비교하여 본 결과, 플라스미드 pGKV21은 rifampicin-sensitive RNA polymerase가 상보가닥 합성에 관여 함을 보여 주었다. Plasmid pGKV21 contains a 229-nucleotide (nt) single-strand DNA initiation (ssi) signal. Using asymmetric PCR, we prepared a small single-stranded (ss) DNA fragment of the ssi signal and, using the 229-nt ssDNA fragment, determined the requirements of RNA polymerase for priming and DNA-protein interaction. The ssi fragment prepared was able to generate primer RNAs with almost the same efficiency as the M13Δlac182/229 phage DNA. However, the cssi (complementary strand of the ssi signal) fragment could not synthesize primer RNAs. This result suggests that the 229-nt ssi signal functions in a strand specific manner. Gel retardation and DNase I footprinting demonstrated that the synthesized ssi fragment could interact with both E. coli RNA polymerase and SSB protein to synthesize primer RNA. In Escherichia coli [pWVAp], an addition of rifampicin resulted in an accumulation of ssDNA, indicating that the host-encoded RNA polymerase is involved in the conversion of ssDNA to double-stranded plasmid DNA.
Jin Yong Jeong,Hak Soo Seo,Ho Yeon Kim,Moo Je Cho,Jeong Dong Bahk 생화학분자생물학회 1995 BMB Reports Vol.28 No.4
Using a mutant M13 phage derivative lacking a great part of the complementary strand synthesis origin, we identified six single-strand initiation (ssi) signals for DNA replication in pACYC184, pLG214, pGKV 21, and pDPT270 plasmids, and named them ssiA_(YC), ssiA_(LG, ssiB_(LG), ssiA_(KV), ssiA_(PT), and ssiB_(PT), respectively. Two of them were from pDPT270, one from downstream the on of pACYC184, two from pLG214, one from upstream the plus origin of pGKV21. Introduction of these ssi signals into the deleted ori_c site of a mutant filamentous M13 phage (M1301ac182) resulted in the restoration of growth activity of this phage. These ssi signals were classified into a number of groups on the basis of sequence similarity. ssiA_(YC) and ssiA_(LG) show extensive sequence homology to the n`-site (primosome assembly sites) of ColE1, whereas ssiB_(PT) is homologous to the n`-site of ΦX174. ssiA_(PT) belongs to G4-type ssi signals which require only dnaG primase and SSB protein for the priming of replication. In addition, possible: biological roles of these ssi signals are discussed.
Jin, Zheng-Lu,Hong, Joon-Ki,Yun, Dae-Jin,Lee, Sang-Yeol,Choi, Young-Ju,Bahk, Jeong-Dong,Roger N. Beachy,Cho, Moo-Je,Lim, Chae-Oh Plant molecular biology and biotechnology research 2002 Plant molecular biology and biotechnology research Vol.2002 No.-
Nicotiana benthamiana plants were transformed with the movement protein (MP) gene of tobacco mosaic virus (TMV), using Agrobacterium-mediated transformation. Plants regenerated from the transformed cells accumulated 30-kDa MP and complemented the activity of TMV MP when infected with chimeric TMVs containing defective MP. These transgenic plants displayed stunting, pale-green leaves, and starch accumulations, indicating that TMV MP altered the carbon partitioning for leaves involved in TMV cell-to-cell movement.