• 한국미생물·생명공학회지
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• 제20권2호
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• pp.122-128
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• 1992

Runaway replciation plasmid pSY35AT를 이용하여 이 plasmid가 가지고 있는 $cI_{857}$ repressor를 숙주 MC1065를 이용하여 $30^{\circ}C$에서 $37^{\circ}C$로 온도를 올려 shift-up 법으로 생산하였다. $cI_{857}$은 wild type cI repressor 정제법을 수정하여 사용하였으며 정제된 $cI_{857}$ repressor 농도는 0.11mg/ml이었다. 그리고 정제된 $cI_{857}$ repressor와 $^3H-CTP$로 labelinf한 PrOr과의 결합활성은 cell 파쇄액보다 약 23배 높았으며, 온도가 올라갈수록 결합활성은 떨어졌다.

• BMB Reports
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• 제37권2호
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• pp.254-259
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• 2004

The wild-type and temperature-sensitive (ts) repressor genes were cloned from the temperate mycobacteriophage L1 and its mutant L1cIts391, respectively. A sequencing analysis revealed that the $131^{st}$ proline residue of the wild-type repressor was changed to leucine in the ts mutant repressor. The 100% identity that was discovered between the two DNA regions of phages L1 and L5, carrying the same sets of genes including their repressor genes, strengthened the speculation that L1 is a minor variant of phage L5 or vice versa. A comparative analysis of the repressor proteins of different mycobacteriophages suggests that the mycobacteriophage-specific repressor proteins constitute a new family of repressors, which were possibly evolved from a common ancestor. Alignment of the mycobacteriophage-specific repressor proteins showed at least 7 blocks (designated I-VII) that carried 3-8 identical amino acid residues. The amino acid residues of blocks V, VI, and some residues downstream to block VI are crucial for the function of the L1 (or L5) repressor. Blocks I and II possibly form the turn and helix 2 regions of the HTH motif of the repressor. Block IV in the L1 repressor is part of the most charged region encompassing amino acid residues 72-92, which flanks the putative N-terminal basic (residues 1-71) and C-terminal acidic (residues 93-183) domains of L1 repressor.

• BMB Reports
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• 제40권5호
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• pp.740-748
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• 2007

To gain insight into the structure and function of repressor proteins of bacteriophages of gram-positive bacteria, repressor of temperate Staphylococcus aureus phage ${\phi}11$ was undertaken as a model system here and purified as an N-terminal histidine-tagged variant (His-CI) by affinity chromatography. A ~19 kDa protein copurified with intact His-CI (~ 30 kDa) at low level was resulted most possibly due to partial cleavage at its Ala-Gly site. At ~10 nM and higher concentrations, His-CI forms significant amount of dimers in solution. There are two repressor binding sites in ${\phi}11$ cI-cro intergenic region and binding to two sites occurs possibly by a cooperative manner. Two sites dissected by HincII digestion were designated operators $O_L$ and $O_R$, respectively. Equilibrium binding studies indicate that His-CI binds to $O_R$ with a little more strongly than $O_L$ and binding species is probably dimeric in nature. Interestingly His-CI binding affinity reduces drastically at elevated temperatures ($32-42^{\circ}C$). Both $O_L$ and $O_R$ harbor a nearly identical inverted repeat and studies show that ${\phi}11$ repressor binds to each repeat efficiently. Additional analyses indicate that ${\phi}11$ repressor, like $\lambda$ repressor, harbors an N-terminal domain and a C-terminal domain which are separated by a hinge region. Secondary structure of ${\phi}11$ CI even nearly resembles to that of $\lambda$ phage repressor though they differ at sequence level. The putative N-terminal HTH (helix-turn-helix) motif of ${\phi}11$ repressor belongs to the HTH -XRE-family of proteins and shows significant identity to the HTH motifs of some proteins of evolutionary distant organisms but not to HTH motifs of most S. aureus phage repressors.

• BMB Reports
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• 제37권6호
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• pp.709-714
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• 2004

The wild-type repressor CI of temperate mycobacteriophage L1 and the temperature-sensitive (ts) repressor CIts391 of a mutant L1 phage, L1cIts391, have been separately overexpressed in E. coli. Both these repressors were observed to specifically bind with the same cognate operator DNA. The operator-binding activity of CIts391 was shown to differ significantly than that of the CI at 32 to $42^{\circ}C$. While 40-95% operator-binding activity was shown to be retained at 35 to $42^{\circ}C$ in CI, more than 75% operator-binding activity was lost in CIts391 at 35 to $38^{\circ}C$, although the latter showed only 10% less binding compared to that of the former at $32^{\circ}C$. The CIts391 showed almost no binding at $42^{\circ}C$. An in vivo study showed that the CI repressor inhibited the growth of a clear plaque former mutant of the L1 phage more strongly than that of the CIts391 repressor at both 32 and $42^{\circ}C$. The half-life of the CIts391-operator complex was found to be about 8 times less than that of the CI-operator complex at $32^{\circ}C$. Interestingly, the repressor-operator complexes preformed at $0^{\circ}C$ have shown varying degrees of resistance to dissociation at the temperatures which inhibit the formation of these complexes are inhibited. The CI repressor, but not that of CIts391, regains most of the DNA-binding activity on cooling to $32^{\circ}C$ after preincubation at 42 to $52^{\circ}C$. All these data suggest that the 131st proline residue at the C-terminal half of CI, which changed to leucine in the CIts391, plays a crucial role in binding the L1 repressor to the cognate operator DNA, although the helix-turn-helix DNA-binding motif of the L1 repressor is located at its N-terminal end.

• 한국미생물·생명공학회지
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• 제19권1호
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• pp.45-51
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• 1991

$cI_{857}$ repressor 단백질을 대량으로 얻기위해 tac promoter 하류에 $cI_{857}$ 구조 유전자를 삽입하는 것을 검토하였다. $cI_{857}$ 유전자를 포함하는 DNA 단편을 plasmid PUC12를 이용하여 대량생산후, HphI으로 부분 분해하여 $cI_{857}$ 구조 유전자만을 취하고, tac promoter 하류에 삽입시켰다. 그리고 $\lambda$ phage $cI_{90}$에 의해 $30^{\circ}C$에서는 용원성을, $42^{\circ}C$에서는 용균성을 보이는 균주를 선택함으로 tac promoter 하류에 cI857 구조 유전자가 삽입된 pDR540-$cI_{857}$을 선택할 수가 있었다. 이 plasmid는 $lacI^q$ JM103 뿐만 아니라 각종 E.coli에서 $cI_{857}$-repressor 단백질을 균체 단백질당 약 17까지 생산하였다.

• BMB Reports
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• 제42권3호
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• pp.154-159
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• 2009

JARID1B (jumonji AT rich interactive domain 1B) is a large nuclear protein that is highly expressed in breast cancers and is proposed to function as a repressor of gene expression. In this paper, a phage display screen using the N-terminus of JARID1B as bait identified one of the JARID1B interacting proteins, namely PcG protein (Polycomb group) hPc2. We demonstrated that the C-terminal region, including the COOH box, was required for the interaction with the N-terminus of JARID1B. In a reporter assay system, co-expression of JARID1B with hPc2 significantly enhanced the transcriptional repression. These results support a role for hPc2 acting as a transcriptional co-repressor.

• 생명과학회지
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• 제28권12호
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• pp.1432-1437
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• 2018

클라라 세포에 의해 생산되는 클라라 세포 분비 단백질(CCSP)은 폐를 염증으로부터 보호하는데 중요한 역할을 한다. 이 연구는 CCSP 유전자 발현에 관여하는 프로모터 부위에서 repressor에 결합할 수 있는 cis-element를 밝히는데 있다. DNaseI footprinting법을 사용하여 mCCSP 프로모터의 -812에서 -768 bp (45 bp) 사이에서 3 개의 보호된 motif를 찾았고, 그 중 하나인 D3 모티프(GCCTGGGAA)는 다른 3 가지 동물들과 염기서열이 100% 일치하였다. 45 bp를 사용한 EMSA 분석에서 D3 모티프(GGCCTGGGAA)는 45 bp에 높은 경쟁을 보였으나, 변이된 D3 모티프가 ($G{\underline{AA}}TG{\underline{TT}}AA$)를 사용되었을 때, 경쟁은 상당히 감소되었다. 이는 mCCSP 프로모터의 45 bp의 D3 모티프가 단백질과 DNA 상호 작용을 위한 중요한 element임을 시사한다. -756-Luc과 -812-Luc을 이용한 transient transfection 분석 결과, -756-Luc은 -812-Luc보다 CCSP의 발현이 현저하게 감소되었다. 이는 mCCSP 프로모터의 45 bp부위가 repressor의 결합 부위로서 기능을 할 수 있음을 의미한다. -812-Luc에 KLF4를 co-transfection 한 결과, KLF4는 CCSP 발현을 현저하게 저해(repression)함을 밝혔다. 그러나 -768-Luc이 사용되었을 때 KLF4에 의한 repression은 관찰되지 않았다. 이것은 KLF4가 CCSP 유전자의45 bp에 결합할 수 있고, 전사 억제자 역할을 하여 mCCSP 발현을 억제 할 수 있음을 명확히 보여 준다. 또한 이는 45 bp 중, D3 모티프가 KLF4의 결합에 강하게 관여 함을 시사한다. 이 반응에 KLF4에 대한 항체가 첨가되었을 때는 super-shifted 밴드가 관찰되었으나, SP1에 대한 항체가 사용되었을 때는 관찰되지 않았다. 이는 KLF4가 CCSP 프로모터의 45 bp 영역에 결합하여 repressor기능 할 수 있고, D3 모티프가 KLF4의 특이적 결합에 관여 할 수 있음을 시사한다.

• Archives of Pharmacal Research
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• 제21권3호
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• pp.320-325
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• 1998

To elucidate of role(s) of tTA as a repressor in the tTA-mediated gene repression system, we introduced mutations into the acidic domain of VP16 and examined the effects of such various mutations. In the transient repression experiment, a region containing 34 amino acids of the activation domain of VP16 (412-456) which interacts with TFIIB was found to be necessary and sufficient for the tTA-mediated repression of gene expression. However, in the experiment to investigate the fact that tTA-regulated repression is related to the activation function of VP16, we found that the repression abilities of tTA derivatives did not correlate exactly with their activation abilities. Therefore, we conclude that increased mass of VP16 in tTA might be also important for efficient repression in addition to functional domain of VP16.

• 한국가축번식학회지
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• 제19권4호
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• pp.283-291
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• 1996

Transgenic animal을 응용할 수 있는 분야에서는 이식유전자의 기능을 정확하게 규명하고 이를 바탕으로 실질적인 유전적인 개량을 이루기 위해서 이식유전자의 발현을 조절할 수 있는 정교한 system이 필요하다. 유전자의 미세주입법에 의해 transgenic animal을 생산할 수 있는데 이용되고 있는 tissue-specific promoter에 의한 이식유전자의 발현조절은 필요로 하는 시기나 양 등을 인위적으로 조절하고자 하는데 한계점을 갖고 있다. 이러한 이식유전자 발현의 문제점을 극복하기 위해 효모의 recombinase나 미생물의 repressor 단백질과 이들의 binding site인 operator sequence를 이용하여 인위적으로 이식유전자의 발현을 조절할 수 있는 system이 개발되고 있다. Cre/loxP system은 site-specific recombination에 의해 DNA sequence를 제거함으로서 이식유전자의 발현을 조절할 수 있다. 이식유전자 발현의 장소와 양을 조절하기 위해서는 미생물이 이용하고 있는 repressor와 이들의 operator sequence를 적용하여 ligand binary system이 개발되었다. Lac repressor system에서는 isopropyl-$\beta$-D-thiogalactoside (IPTG)가 이식유전자 발현을 조절할 수 있는 positive regulator로서 작용하고, tetracycline-VP16 system에서는 tetracycline이나 유사물질들이 negative regulator로서 이용할 수 있다. 이러한 binary system은 transgenic animal에서 이식유전자 발현의 장소와 시기 또한 양을 효과적으로 조절하는데 적용할 수 있는 것으로 나타났다. 따라서 기존의 binary system과 함께 새로운 regulatory system의 장점을 이용하여 보다 완벽한 이식유전자의 인위적인 조절 system을 이룩함으로서 transgenic animal technology의 실용화를 앞당길 것으로 기대된다.

• BMB Reports
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• 제42권5호
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• pp.293-298
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• 2009

Temperate mycobacteriophage L1 encodes an unusual repressor (CI) for regulating its lytic-lysogenic switching and, in contrast to the repressors of most temperate phages, it binds to multiple asymmetric operator DNAs. Here, ions like $Na^+$, $Cl^-$, and $acetate^-$ ions were demonstrated to facilitate the optimal binding of CI to cognate operator DNA, whereas $K^+$, $Li^+$, ${NH_4}^+$, $Mg^{2+}$, $carbonate^{2-}$, and $citrate^{3-}$ ions significantly affected its operator binding activity. Of these ions, $Mg^{2+}$ unfolded CI most severely at room temperature and, compared to $Mg^{2+}$, $Na^+$ provided improved thermal stability to CI. Furthermore, the intrinsic tryptophan fluorescence of CI was changed notably upon replacing $Na^+$ with $Mg^{2+}$ and these opposing effects of $Mg^{2+}$ and $Na^+$ were also noticed in their actions on the C-terminal fragment (CTD) of CI. Taken together, $Na^+$ appeared to be more appropriate than $Mg^{2+}$ for maintaining the biologically active conformation of CI needed for its optimal binding to operator DNA.