在環境變化之下，增生中的細胞延緩細胞週期以防止進一步的損傷積累。酵母菌的Cip1蛋白已知與Cdk1和Cln2具有交互作用；然而Cip1的功能和調控目前還仍有許多的未知。在本篇論文中，Cip1的表現由細胞週期調控因子Mcm1和壓力調控因子Msn2/4所共同調節。過表現Cip1透過抑制G1時期的Cdk1-G1細胞週期蛋白複合物，且在滲透壓壓力刺激下的Cip1蛋白T65、T69和T73磷酸化可以增強Cip1蛋白和Cdk1-G1細胞週期蛋白複合物的相互作用，從而抑制細胞週期。Cip1蛋白的主要抑制目標為Cdk1-Cln3複合物，從而阻止Whi5蛋白的磷酸化並抑制早期G1時期的進行。在滲透壓力下，Cip1的表現引發暫時性的G1時期延遲，而這與另一種被高滲透壓活化的CKI，Sic1蛋白在功能上是冗餘的。這些研究結果顯示，Cip1蛋白的功能類似於哺乳動物p21蛋白，皆作為壓力誘導的CDK抑制物，藉由抑制G1時期來減緩細胞週期，以利細胞應對外在環境的壓力。

Upon environmental changes, proliferating cells delay cell cycle to prevent further damage accumulation. Yeast Cip1 is a Cdk1 and Cln2 associated protein. However, the function and regulation of Cip1 are still poorly understood. Here we report that Cip1 expression is co-regulated by the cell cycle-mediated factor Mcm1 and the stress-mediated factors Msn2/4. Overexpression of Cip1 arrests cell cycle through inhibition of Cdk1-G1 cyclin complexes at G1 stage and the stress-activated protein kinase-dependent Cip1 T65, T69, and T73 phosphorylation may strengthen the Cip1 and Cdk1-G1 cyclin interaction. Cip1 accumulation mainly targets Cdk1-Cln3 complex to prevent Whi5 phosphorylation and inhibit early G1 progression. Under osmotic stress, Cip1 expression triggers transient G1 delay which plays a functionally redundant role with another hyperosmolar activated CKI, Sic1. These findings indicate that Cip1 functions similar to mammalian p21 as a stress induced CDK inhibitor to decelerate cell cycle through G1 cyclins to cope with environmental stresses.

CONTENTS i
口試委員會審定書 vi
致謝 vii
中文摘要 viii
ABSTRACT ix
INTRODUCTION 1
Environmental stress and cellular response 1
Yeast G1 cell cycle regulation 1
Cyclin-dependent kinase inhibitor in yeast 2
The regulation of Mcm1 3
The stress-responsive transcription factors, Msn2/4 4
The new G1 CKI in yeast 4
RESULTS 6
Overexpression of Cip1 induces G1 arrest 6
Cip1 is a CKI of all G1 cyclins 7
CIP1 is expressed by Mcm1 with peak expression at G1 7
CIP1 expression is induced upon stress 9
Stress-driven CIP1 expression depends on Msn2/4 10
Cip1 is phosphorylated under osmotic stress 12
Hog1 phosphorylates Cip1 T65 and T73 under osmotic stress 13
Cip1 contributes to osmostress-induced transitory G1 delay 15
Cip1 phosphorylation may enhance the binding to Cdk1-Clns 18
Cip1 impedes G1 through inhibiting Cdk1-Cln3 activity 18
Cdk1-Cln3 interaction prevents Cip1-Cln3 binding 20
Cip1 localizes to the nucleus upon osmotic stress 20
Human p21 expression stimulates G1 arrest in yeast cells 21
DISCUSSION 22
MATERIALS & METHODS 28
Yeast strains and plasmids 28
RNA preparation and Northern blotting 29
Cell growth media and condition for stress response 30
EMSA 30
RNA purification and quantitative reverse transcription PCR 31
ChIP analysis 31
α-factor/nocodazole trap assay 32
Budding index under osmotic stress 33
Isolation of newborn cells by elutriation 33
Co-immunoprecipitation 33
MS analysis 34
Phosphor-specific antibody preparation 35
Dot blot analysis 36
Western blot analysis 36
In vitro kinase assay 37
Fluorescence microscopy 38
FIGURES AND FIGURE LEGENDS 39
Figure 1. Overexpression of CIP1 causes cell cycle arrest at G1 through inhibition of all Cdk1-G1 cyclin complexes. 39
Figure 2. CIP1 expression at M/G1 is mediated by Mcm1 through ECB elements on CIP1 promoter. 41
Figure 3. Mcm1 binds to the CIP1 promoter at late M phase. 43
Figure 4. CIP1 expression is induced under several stresses. 44
Figure 5. CIP1 expression is induced under several stresses. 46
Figure 6. CIP1 single deleted cells are not sensitive to stresses. 48
Figure 7. Deletion of Msn2/4 upstream suppressors slightly increases CIP1 expression in the absence of stress. 49
Figure 8. Msn2/4 regulate CIP1 expression through two STRE binding sites. 51
Figure 9. Mass analysis of the phosphorylation on Cip1. 53
Figure 10. Cip1 is phosphorylated by Hog1 kinase directly under osmotic stress. 55
Figure 11. Characterization of the phosphor-specific antibodies against Cip1 T65 and T73. 57
Figure 12. The phosphorylation of Cip1 is responsible for hyperosmotic stress induced transitory G1 delay. 59
Figure 13. Cell cycle progression in Cip1 overexpressed or deleted cells. 61
Figure 14. Cip1 regulates G1/S progression by inhibiting Cdk1-Cln3 complex activity. 62
Figure 15. Cip1 localizes to the nucleus after osmotic stress. 65
Figure 16. Human p21 expression stimulates G1 arrest in yeast cells. 66
Figure 17. Mcm1 and Mbf1 drive CIP1 transcription at different cell cycle stages. 67
Figure 18. A model of Cip1-involved regulations. 69
TABLES 72
Table 1. Strains Used in This Study. 72
Table 2. Constructs Used in This Study. 74
Table 3. Primers Used in This Study 76
REFERENCES 79
APPENDIXES 85
Appendix I. Contribution Table 85
Appendix II. Instability of Succinate Dehydrogenase in SDHD Polymorphism Connects Reactive Oxygen Species Production to Nuclear and Mitochondrial Genomic Mutations in Yeast 86

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