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Development of a novel DNA-binding domain derived from Escherichia coli lac repressor and its application to artificial eukaryotic transcription factors

Other Titles
 Escherichia coli lac repressor에서 유래된 새 
Issue Date
2003
Description
Dept. of Medical Science/석사
Abstract
[한글] E.coli lac repressor DNA-binding domain (DBD)와 yeast의 GAL4 dimerization domain (DD)을 결합시켜 corepressor와 무관하게 항시 lac operator에 결합능력이 있는 새로운 DBD인 LacHG를 만들었다. 이 DBD에 SREBP-1a activation domain(AD)를 연결하여 제조한 인공 전사 인자 (LacAD) 는 유핵 세포내에서 표적 유전자의 발현을 증가시켰다. LacHG에서 GAL4 DD의 linker region을 제거하였을 때 전사의 활성은 크게 증가하였고, GAL4 DD대신 leucine zipper의 DD를 사용하였을 경우에는 LacAD와 비슷한 정도로 전사 활성을 나타내었다. PR 또는 ER의 ligand binding domain (LBD)과 AD를 연결하여 만든 인공핵 수용체 (LacAPR, LacAER)는 유핵 세포에서 외부 신호에 반응하여 target 유전자의 발현을 효과적으로 조절하였다. 또한 lac operator의 대칭성 서열 [SymL(-1)]이 원래의 lac operator 서열보다 LacAD에 훨씬 잘 반응하였다. 이상의 실험을 통해 무핵 세포에서 유래한 lac repressor의 DBD를 이용하여 유핵 세포 내에서 corepressor의 존재 유무에 관계없이 결합할 수 있고 유전자의 조절을 효과적으로 조절할 수 있는 전사활성 인자와 인공핵 수용체를 만들었다.
[영문]Novel DNA-binding domain (DBD), LacHG, was designed by joining the lac repressor DBD to GAL4 dimerization domain (DD), which could constitutively bind to lac operator. To assay the DNA-binding activities of LacHG, it was fused to the activation domain (AD) of SREBP-1a, resulting in artificial transcription activator, LacAD. The deletion of linker region (42-49 a. a.) of GAL4 DD, significantly enhanced the transcription activities of LacAD, while the linker deletion in PurHG, derived from purine repressor (PurR), decreased the transcription activities of PurAD. In reporter construct, the symmetric lac operator sequence [SymL(-1)] responded to LacAD much more efficiently than wild lac operator sequence. The LacHZ was generated by replacement of GAL4 DD in LacHG with GCN4 leucine zipper, which is well known as dimer forming motif. The LacHZ-AD activates the receptor expression as much as LacAD. The addition of nuclear localization signal (NLS) of SV 40 large T antigen to LacHZ-AD did not increase transcription activities. We made artificial nuclear receptor by insertion of ligand-binding domain (LBD) of progesterone receptor (PR, 645-891 a. a.) or estrogen receptor (ER, 279-554 a. a.) between DBD and AD for ligand-dependent activation of reporter gene. The mLacAPR, containing PR LBD, induced the reporter expression to about 6 folds in respond to RU486, even if this was much less than 12 fold induction in PurAPR containing PurHG instead of LacHG. The mLacAER, containing ER LBD, markedly increased the transcription of luciferase gene in respond to β-estradiol to about 63 folds comparable to PurAER. In present study, lac repressor DBD was first modified to constitutive DBD which specifically bind to lac operator sequence. Transcription factors, containing chimeric DBD derived lac repressor DBD, works well in eukaryotes as in forms of transcription activator or nuclear receptor.
URI
http://ir.ymlib.yonsei.ac.kr/handle/22282913/128441
Appears in Collections:
2. 학위논문 > 1. College of Medicine (의과대학) > 석사
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