초파리 PERIOD 단백질의 아미노산 926-977이 생체시계의 작동에 관여하는 분자적 기전
Role of Amino Acid 926-977 in Drosophila PERIOD Protein for Circadian Clockworks
- 주제(키워드) 초파리+생체시계+dPERIOD+dCLOCK+전사활성+인산화
- 발행기관 아주대학교
- 발행년도 2010
- 학위수여년월 2010. 8
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000010906
- 본문언어 한국어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
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.
more목차
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
