E . coli cryptic miniplasmid p 15A 에서 유래하는 플라스미드 pACYC 184 의 ssi 시그날에 관한 연구

박정동,Sakai Hiroshi,Tohru Komano ( Jeong Dong Bahk,Hiroshi Sakai,Tohru Komano ) 생화학분자생물학회 1990 BMB Reports Vol.23 No.1

Single-strand DNA initiation (ssi) signals are DNA requirements for initiation of DNA synthesis on the single-strand DNA templates. For primary screening of ssi signal function, we used plaque morphology method. Plasmid pACYCl84 derived from E. coli minicryptic plasmid p15A had only one ssi signal which consists of 119-nt stretch. This segment played critical roles in ss DNA phage growth activity and performed primase-dependent replication instead of RNA polymerase-dependent one. Three kinds of energetically stable stem and loop structures were expected. Especially, the orientation and location of this 119-nt stretch, when compared with those of the origin of p15A, favor that this ssi signal might be taken part in the lagging strand replication of plasmid pACYCl84.

Primer RNA Synthesis by E. coli RNA Polymerase on the SSB-coated 229-nt ssi Signal of Lactococcal Plasmid pGKV21

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.

초고온 archaeon인 Thermococcus profundus에서 P93 복합체의 분리 및 구조적 특성

이미홍,김숙경,윤영근,박성철,박정동,정강원,Lee, Mi-Hong,Kim, Suk-Kyoung,Yun, Young-Gun,Park, Seong-Cheol,Bahk, Jeong-Dong,Cheong, Gang-Won 한국현미경학회 2000 Applied microscopy Vol.30 No.2

초고온 archaeon인 Thermococcus profundus에서 매우 거대한 단백질 복합체를 분리 및 구조를 규명하였다. 거대 복합체는 93kDa단백질(P93 complex)로 구성된 homomultimer이며, 강한 내열성을 보여주고 있다. 순수 분리한 P93 complex를 SDS(최종 농도 1%)와 $85^{\circ}C$에서 12시간 항온시킨 후, SDS-PAGE와 전자현미경에서 구조적 변화를 관찰할 수 없었다. 음착색된 P93 complex의 전자현미경 사진에서 하나의 형태만을 보여주고 있으며, 구조의 규명을 위해 image processing을 하였다. 이의 구조는 3대칭 중심에 core(혹은 hole)이 뚜렷이 존재하며 이를 중심으로 단백질이 모여 있는 형태를 보여 주고 있다. 또한 P93 complex는 가장자리에서 뚜렷한 형태를 보여주지 않은 부분, 즉 flexible부분을 포함하고 있다. Gel filtration과 2차원 구조를 기초로 P93 complex는 24 homomultimer로 되어 있음을 추정하였다. An unusually large protein complex was found in the cytosol of the hyperthmophilic archaeon. Thermococcus profundus. The purified protein was shown to be a homomultimer of 93 kDa subunit (P93 complex). The complex is extremely heat stable. During 12 hrs incubation with SDS (final concentration 1%) at $85^{\circ}C$, no changed structure could be observed. Electron image analysis of negatively stained showed that the complex has a single, stable characteristic view and a well-preserved core with threefold rotational symmetry. The periphery of the assembly is composed of a nebulose, possibly flexible, component. Based on the projected structure suggest the P93 complex from T. profundus is composed 24 homomultimer.

E . coli cryptic miniplasmid p 15A 에서 유래하는 플라스미드 pACYC 184 의 ssi 시그날에 관한 연구

박정동,Sakai, Hiroshi,Komano, Tohru 생화학분자생물학회 1993 BMB Reports Vol.17 No.3

Single-strand DNA initiation (ssi) signals are DNA requirements for initiation of DNA synthesis on the single-strand DNA templates. For primary screening of ssi signal function, we used plaque morphology method. Plasmid pACYCl84 derived from E. coli minicryptic plasmid p15A had only one ssi signal which consists of 119-nt stretch. This segment played critical roles in ss DNA phage growth activity and performed primase-dependent replication instead of RNA polymerase-dependent one. Three kinds of energetically stable stem and loop structures were expected. Especially, the orientation and location of this 119-nt stretch, when compared with those of the origin of p15A, favor that this ssi signal might be taken part in the lagging strand replication of plasmid pACYCl84.

Reconstitution of Artificially Designed inaz Gene and Its Expression in vivo

BAHK, JEONG DONG,CHO, MOO JE,LEE, DAE SIL 경상대학교 유전공학연구소 1992 遺傳工學硏究所報 Vol.11 No.-

In order to express the artifically designed inaz gene, which was deduced from the InaZ protein of Pseudomonas syringae, in Escherichia coli, a fused protein was planned. One unit of the fused protein consists of N-term. 50 amino acids of LacZ protein of E. coli and one unit of the repeated 48 amino acids from the core domain of InaZ protein of Ps. syringae. Each of them consists of eight synthetic DNA fragments. All of them were phosphorylated, block-ligated and reconstituted. Theresfter, the reconstituted DNA stretah was inserted into the plasmid vector pASI containing a P_L promoter of bacteriophage lambda. The recombinant plasmid pASLF was transformed to an expression host, E. coli N4839-1. After performing expression under a permissible or impermissible temperature, the cultures were applied to the buffer gradient gel and ckecked. At the near of 12-13 kD region a clear thick band was observed to be the fused INA protein.

Identification of nucleotide sequences for initiating single-stranded phage DNA replication from plasmid pACYC184

Jeong, Jin Yong,Cho, Moo Je,Bahk, Jeong Dong 慶尙大學校 기초과학연구소 1992 基礎科學硏究所報 Vol.8 No.-

대장균 플라스미드 p15A 유도체인 플라스미드 pACYC184의 복제 개시점 하류에 위치 한 159-nt 영역에서 하나의 단일 가닥 복제 개시(ssi)signal이 발견 되었다. 이 ssi signal은 돌연변이 M13파아지(M13Δlac182)를 이용한 plaque morphology assay를 이용하여 선별하였다. M13Δlac182는 상보 가닥 DNA를 합성하는 복제 개시 부위(ori_c)의 많은 부분이 결실 되었기 때문에 작고 탁한 plaque을 형성한다. 159-nt의 ssi 단편을 가지고 있는 재조합된 phage(M13Δlac182/pACYC184ssi)는 야생형 M13mp18 phage와 거의 같은 효율로 생육 하였다. 그러므로 159-nt 단편은 M13Δlac182의 결실된 복제 개시점으로 부터 18-nt 아래에 위치한다. 플라스미드pACY184에서 발견된 ssi signal은 다른 여러가지 플라스미드에서 발견된 ssi signal들의 DNA 염기배열과 높은 homology를 보여 주었다. 159-nt 단편 중에서 가능한 2차 구조를 그려볼 수 있었으며 이 2차 구조에서 몇몇 conserved sequences(n'단백질 인식 부위; dnaB, dnaC, 그리고 dnaG 의존형 복재 개시 signal ; primer RNA 개시 부위)가 발견되었다. A single-strand initiation(ssi) signal for phage DNA synthesis was identified in the 159-bp region of plasmid pACYC184, a derivative of plasmid p15A of Escherichia coli. The ssi signal was identified by using a plaque morphology assay with a mutant M13 phage(M13Δlac182) which forms small turbid plaques because it lacks the greater part of the origin of complementary DNA strand synthesis(ori_c). The recombinant phage (M13Δlac182/pACYC184ssi) carrying the 159-bp insertion grew as efficiently as wild-type M13mp 18 phage and thus this 159-bp DNA segment can recover the defect in phage replication of M13Δlac182. This region is located 18-nt downstream from p15A origin of DNA replication. The direction of chain elongation in DNA synthesis is opposite to that of the leading strand. The ssi signal of plasmid pACYC184 shows sequence homology to the ssi signals of some other plasmids. In this region, we found a potential stem and loop structure. Its stem region contains the consensus sequence, 5'-CGCTCGCCGCAT-3', known as dnaB, dnaC and dnaG protein-dependent initiation signal and 5'-GAAGCGG-3', known as an n' protein recognition site. Furthermore, trinucleotide sequence, 5'-CTG-3', known as a primer RNA initiation site, was also maintained.