2026-09-22T09:29:04Zhttps://repository.ajou.ac.kr/oai/request

oai:repository.ajou.ac.kr:201003/22682024-10-11T02:17:38Zcom_201003_14372com_201003_14368col_201003_14373

Role of Amino Acid 926-977 in Drosophila PERIOD Protein for Circadian Clockworks 초파리 PERIOD 단백질의 아미노산 926-977이 생체시계의 작동에 관여하는 분자적 기전 선, 우철 김, 은영 대학원 의생명과학과 200824177 선, 우철 초파리 생체시계 dPERIOD dCLOCK 전사활성 인산화 생체시계의 분자적 기전은 transcriptional-translational feedback loop이다. 초파리 feedback loop의 첫 번째 loop에서는 bHLH/PAS 도메인을 함유한 전사인자인 dCLOCK (dCLK) 과 CYCLE (CYC) 가 dperiod (dper)와 timeless (tim) 의 발현을 촉진하고 합성된 dPER 단백질과 TIM 단백질이 이합체로 결합하여 dCLK/CYC의 전사활성을 억제하여 dper 와 tim 의 발현이 하루를 주기로 진동케 한다. 여기에는 dPER에 의한 dCLK/CYC 전사활성의 억제가 필수적이다. 본 연구에서는 dPER에서 dCLK/CYC의 전사활성을 억제하는데 필요한 도메인으로 알려진 dCLK CYC inhibition domain (CCID) 에서 특히 종간 보존성이 매우 높은 아미노산 926-977 부분의 역할을 탐구하였다. dPER로부터 아미노산 926-977 을 제거한 변이체인 dPER(Δ926-977) 은 초파리 세포라인 Schneider 2 (S2) 세포에서 dCLK/CYC의 전사활성을 억제하지 못하였다. dPER(Δ926-977) 는 dCLK과 결합하지 못하였는데, 이로부터 아미노산 926-977이 dCLK의 binding domain 으로 작용한다는 사실을 밝혀내었다. 더욱이 아미노산 949-977만을 내부적으로 제거한 dPER(Δ949-977) 가 S2 세포에서 dCLK/CYC의 전사활성을 억제하지 못하고 dCLK과의 결합능력이 떨어지는 것으로 보아 아미노산 949-977이 dCLK과의 결합에 필요한 최소 도메인인 것을 확인하였다. 아미노산 926-977의 in vivo 역할을 탐색하기 위하여 dPER(Δ926-977)를 발현시키는 초파리를 제작하였고, 이 초파리의 생체리듬을 분석하여 본 결과 주기가 길어지기는 하였으나 정상적인 생체리듬을 갖는 것을 알 수 있었다. dCLK의 결합에 필수적인 아미노산 926-977이 없는 dPER가 in vivo 에서 생체시계 유전자의 발현이 진동하게 하는 feedback loop작동에 정상적인 기능을 한다는 사실은 매우 흥미로우며 이에 대한 후속 연구가 진행되어야 할 것으로 보인다. "국문요약 ····················································································································· ⅰ 차례 ····························································································································· ⅲ 그림 차례 ··················································································································· ⅴ 표 차례 ······················································································································· ⅶ Ⅰ. 서론 ························································································································ 1 Ⅱ. 재료 및 방법 ········································································································ 8 A. Plasmid ················································································································ 8 1. Site-direct mutagenesis를 이용하여 만든 plasmid들 ································· 8 2. Rapid and efficient PCR-based mutagenesis를 이용하여 만든 plasmid들 (Ko 와 Ma, 2005) ···························································································· 8 3. Cloning으로 만든 plasmid들 ········································································ 8 4. 참고문헌을 통해 이미 사용되어 인용된 plasmid들 ······························· 8 B. Site-direct mutagenesis ························································································ 9 1. QuickChange site-directed mutagenesis kit ···················································· 9 2. Site-directed mutagenesis를 위한 primer ···················································· 10 3. Rapid and efficient PCR-based mutagenesis method (Ko 와 Ma, 2005) ······ 11 4. PCR-based Rapid site directed mutagenesis를 위한 Primer들 ··················· 13 C. Drosophila melanogaster Schneider line 2 (S2) 세포의 배양과transfection ······························································································································· 15 iv D. 형질전환 초파리 (Transgenic Drosophila melanogaster) ···························· 16 E. Locomotor activity assays ·················································································· 17 F. Immunoblot analysis ·························································································· 18 G. Immunoprecipitation (IP) ·················································································· 21 H. Luciferase Reporter Assay 와 β-Galactosidase Enzyme Assay System ········· 23 Ⅲ. 결과 ······················································································································ 25 A. S2 세포에서 dPERΔ926-977은 dCLK의 전사활성을 억제하지 못한다 ····························································································································· 25 B. S2 cells에서 dPERΔ926-977은 dCLK과 결합하지 못한다 ········ 28 C. dPERΔ926-977은 DBT에 의한 인산화에 결함을 나타내었다 ····· 32 D. 아미노산 949-977이 dCLK과의 결합에 필요한 최소 도메인이다 ····· 34 E. dPER의 아미노산949-977를 Ser과 Thr을 모두 Ala으로 바꾸어도 DBT에 의한 인산화가 일어난다 ································································ 38 F. dPERΔ926-977을 발현하는 형질전환 초파리는 주기가 길어지기는 하였으나 정상적인 생체리듬을 가졌다 ···················································· 43 Ⅳ. 고찰 ······················································································································ 46 Ⅴ. 결론 ······················································································································ 48 참고문헌 ····················································································································· 50 ABSTRACT ················································································································ 53" Master 2011-04-13T05:33:35Z 2011-04-13T05:33:35Z 2010 2010 Thesis Theses http://repository.ajou.ac.kr/handle/201003/2268 http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000010906 000000010906 ko