초파리 생체리듬에 영향을 주는 프롤린 cis/trans 이성질화 효소의 규명
- 발행기관 아주대학교
- 지도교수 김은영
- 발행년도 2015
- 학위수여년월 2015. 2
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000019503
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Circadian rhythms are widespread in all life and are driven by cell-autonomous molecular oscillator. This cellular oscillator is based on the negative feedback loops comprised of core “clock” genes. Work in the last several years has explained the importance of post-transcriptional regulation, especially protein phosphorylation in the aspect of cellular oscillators. A common feature of animal circadian clocks is the progressive phosphorylation of PERIOD (PER) proteins, which is highly dependent on casein kinase Iδ/ε (CKIδ/ε; termed DOUBLETIME [DBT] in Drosophila) and ultimately leads to the rapid degradation of hyper-phosphorylated isoforms via a mechanism involving the F-box protein SLIMB. Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes that accelerate energetically unfavorable cis/trans isomerization. Such conformational changes can act as a molecular switch to determine the fate of proteins through several mechanisms including phosphorylation, ubiquitination, and protein interaction. In search for PPIases which might play crucial role in circadian clock function, we performed genome-wide in vivo RNAi screen via binary GAL4/UAS system. Among the 23 types of Drosophila PPIases, we identified 7 kinds of PPIase which showed a lengthened circadian period when their expression is down-regulated in timeless expressing cells: CG3511, CG5808, CG7747, CG7768, CG10907, CG11777, CG13892, CG17051. We focused on CG17051 (dod) and CG5808. DOD is a Drosophila homolog of mammalian PIN, especially targeting phospho-Ser/Thr-Pro. When DOD expression is down-regulated in timeless expressing cells, circadian period is lengthened about 1h (≅24.8 h). Also, in S2 cell based assay, when dod is overexpressed, degradation of hyper-phosphorylated isoforms of PER-V5 is delayed. We confirmed that DOD interacts with hyper-phosphorylated isoforms of PER in S2 cells. CG5808 is a family of Cyclophilin-like peptidyl-prolyl cis/trans isomerase. When CG5808 expression is down-regulated in timeless expressing cells, circadian period is most severely lengthened about 4.4 h (≅28.1 h). Also, in Drosophila clock neuron s-LNv, the level of PER is decreased during ZT22-ZT2 and the nuclear entry of TIM is delayed at ZT22. This study gives an insight that PPIase DOD and CG5808 are involved in Drosophila circadian rhythm regulation.
more목차
ABSTRACT············································································ⅰ
TABLE OF CONTENTS ·························································· ⅲ
LIST OF FIGURES ································································ ⅴ
LIST OF TABLES ································································· ⅵ
Ⅰ. INTRODUCTION ································································ 1
Ⅱ. MATERIALS AND METHODS ·············································· 5
A. PPIase genes in Drosophila melanogaster ···························· 5
B. Knockdown fly strains ····················································· 7
C. Fly behavioral assays ······················································ 7
D. Plasmids and methods for S2 cell-based assays ······················ 8
E. Immunoblotting ······························································ 8
F. Immunoprecipitation ··························································9
G. Immunohistochemistry······················································ 10
H. Quantitative real-time RT–PCR ········································ 11
Ⅲ. RESULTS ········································································ 12
A. PPIases alters Drosophila circadian activity rhythms ··············· 12
B. DOD ·········································································· 19
1. Daily cycle of core clock protein in dod knockdown flies········ 19
2. Degradatin of PER is attenuated when DOD is overexpressed in S2 cells ·································································· 22
3. DOD interacts with hyperphosphorylated PER in S2 cells ······ 28
C. CG5808 ······································································· 30
1. Daily cycle of core clock protein in CG5808 knockdown flies ·· 30
2. PER and TIM are altered in key brain pacemaker neurons when CG5808 is down-regulated ··········································· 34
Ⅳ. DISCCUTION ··································································· 37
Ⅴ. CONCLUTION ·································································· 40
REFERENCES ······································································· 41
국문요약 ·············································································· 46
