臺灣博碩士論文加值系統

在減數分裂過程中，染色體配對、聯會以及重組發生異常時，粗絲期檢控點會阻止細胞進入下一時期，直到缺失修復為止。在酵母菌(Saccharomyces cerevisiae)中，Ndt80是一個受到粗絲期檢控點調控的轉錄因子(transcription activator)，它能促使減數分裂中後期有關細胞核分裂與孢子生成基因的表現，缺少Ndt80的細胞會停留在粗絲期，因此在細胞從粗絲期進入第一次減數分裂的過程中，Ndt80扮演重要的角色。
之前本實驗室發現一個特殊的NDT80突變株，NDT80-bc。它可以使細胞不受粗絲期檢控點的調控，在染色體聯會發生異常的情況下(例如zip1突變株)完成減數分裂並產生孢子。有趣的是，zip1 NDT80/NDT80-bc細胞進入核分裂以及孢子生成的時間有延遲現象，推測是失去活性的Ndt80存在並干擾Ndt80-bc的能力。為了探討粗絲期檢控點調控Ndt80活性的機制，我們利用不同的抗原決定基分別標定Ndt80以及Ndt80-bc，經過初步產孢效能與蛋白質分析，我們決定利用NDT80-6XHA/NDT80-bc-6XMYC來同時觀察Ndt80及Ndt80-bc在野生型與zip1突變株中的表現。在zip1 NDT80-6XHA/NDT80-bc-6XMYC細胞中，核分裂與孢子生成仍然有延遲的現象。在蛋白質表現上，Ndt80與Ndt80-bc蛋白開始表現的時間相似，另一方面，Ndt80與Ndt80-bc表現量沒有明顯的差異。透過免疫螢光染色，我們發現在zip1 NDT80-6XHA/NDT80-bc-6XMYC細胞中，Ndt80與Ndt80-bc蛋白細進入細胞核的效率有明顯的差異，推測Ndt80蛋白進入細胞核的過程受到粗絲期檢控點的監控，進而調節Ndt80的活性。

Mutants that confer defects in meiotic recombination (e.g., zip1) will trigger the checkpoint mechanism to arrest cells at the pachytene stage of meiosis. One of the control targets of the pachytene checkpoint is Ndt80. Ndt80 is a meiosis-specific protein that activates transcription of the middle sporulation genes, including genes required for nuclear divisions and spore formation. The Ndt80 activity is regulated by the pachytene checkpoint. Therefore, NDT80 is a critical factor which controls the progression from the pachytene into meiosis I.
Previous studies have isolated NDT80-bc, a dominant NDT80 mutant, which is not regulated by the pachytene checkpoint. Interestingly, the zip1 NDT80/NDT80-bc cells displayed a delay in nuclear division and spore formation, suggesting that inactive Ndt80 might exist and interfere with the activity of Ndt80-bc. In order to explore the regulatory mechanism of the pachytene checkpoint on Ndt80, we tried to use different epitopes to label Ndt80 and Ndt80-bc, respectively. Among all of these tagged Ndt80 and Ndt80-bc, NDT80-6XHA/NDT80-bc-6XMYC heterozygous in wild-type and zip1 were used to monitor the difference between Ndt80 and Ndt80-bc simultaneously. The zip1 NDT80-6XHA/NDT80-bc-6XMYC cells also displayed a delay in the kinetics of nuclear division and spore formation. We found that there is no significant difference in kinetics of protein expression between Ndt80 and Ndt80-bc in the zip1 NDT80-6XHA/NDT80-bc-6XMYC cells. Cytological analyses showed that the kinetics of nuclear import between Ndt80 and Ndt80-bc were different in zip1 NDT80-6XHA/NDT80-bc-6XMYC. Our results suggest that the Ndt80 activity is probably controlled by the pachytene checkpoint at the step of nuclear import.

ABSTRACT .................................................i
中文摘要 ................................................ii
TABLE OF CONTENTS ......................................iii
LIST OF TABLES .......................................vi
LIST OF FIGURES ......................................vii

CHAPTER 1. INTRODUCTION ...............................1
Meiosis Overview ........................................1
Meiotic Checkpoint ...............................2
The Pachytene Checkpoint ...............................3
NDT80 .................................................4
NDT80-bc .................................................6

CHAPTER 2. MATERIALS AND METHODS ......................8
Media and Genetic Methods ...............................8
DNA Preparation, Transformation, and Molecular Biology Methods .................................................8
Plasmids Construction .....................................9
Yeast Strains ............................................11
Kinetics of the Meiotic Cells ............................12
Protein Extraction and Western Blot Analysis ............12
Immunofluorescence Staining of Yeast Cells ............13
Quantification of Subcellular Localization ............14

CHAPTER 3. RESULTS ..............................16
The Effects of Epitopes on Ndt80 and Ndt80-bc ............16
Epitope-tagged version of Ndt80 and Ndt80-bc ............16
The N-terminal 3XMYC affects the function of Ndt80-bc.....16
The effect of C-terminal epitopes on Ndt80 ............18
The Regulation of Ndt80 by the Pachytene Checkpoint.......19
Construction of NDT80-6XHA/NDT80-bc-6XMYC ............19
Time course analysis of zip1 NDT80-6XHA/NDT80-bc-6XMYC ...20
There is no significant difference in protein expression between Ndt80 and Ndt80-bc in zip1 .......................20
Nuclear import of Ndt80 and Ndt80-bc are different .......22

CHAPTER 4. DISCUSSION ....................................25
The Regulation of Ndt80 Activity by the Pachytene Checkpoint ...............................................25
The expression of Ndt80 is similar to Ndt80-bc ...........25
The phosphorylation of Ndt80 is delayed in
zip1 NDT80-6XHA/NDT80-bc-6XMYC ...........................25
The nuclear import of Ndt80 might be
regulated by the pachytene checkpoint ....................26
The phosphorylation and nuclear import of Ndt80 ..........28
The Effects of Epitopes on Ndt80 and Ndt80-bc ............29
The positional effect of 3XMYC on the mobility of Ndt80 ..29
General discussion for 3XMYC-Ndt80-bc ....................30
The effect of C-terminal epitopes on Ndt80 ...............31
REFERENCES ...............................................33
APPENDIX ................................................59

Bailis, J.M., and Roeder, G.S. (1998). Synaptonemal complex morphogenesis and sister-chromatid cohesion require Mek1-dependent phosphorylation of a meiotic chromosomal protein. Genes Dev. 12, 3551-3563.
Bailis, J.M., and Roeder, G.S. (2000). The pachytene checkpoint. Trends Genet. 16, 359-403.
Benjamin, K.R., Zhang, C., Shokat, K.M., and Herskowitz, I. (2003). Control of landmark events in meiosis by the CDK Cdc28 and the meiosis-specific kinase Ime2. Genes Dev. 17, 1524-1539.
Birnboim, H.C., and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513-1523.
Bishop, D.K., Park, D., Xu, L., and Kleckner, N. (1992). DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell 69, 439-456.
Chu, S., and Herskowitz, I. (1998). Gametogenesis in yeast is regulated by a transcriptional cascade dependent on Ndt80. Mol. Cell 1, 685-696.
Chu, S., DeRisi, J., Eisen, M., Mulholland, J., Botstein, D., Brown, P.O., and Herskowitz, I. (1998). The transcriptional program of sporulation in budding yeast. Science 282, 699-705.
Dahmann, C., and Futcher, B. (1995). Specialization of B-type cyclins for mitosis or meiosis in S. cerevisiae. Genetics 140, 957-963.
de los Santos, T., and Hollingsworth, N.M. (1999). Red1p, a MEK1-dependent phosphoprotein that physically interacts with Hop1p during meiosis in yeast. J. Biol. Chem. 274, 1783-1790.
Fingerman, I.M., Sutphen, K., Montano, S.P., Georgiadis, M.M., and Vershon, A.K. (2004). Characterization of critical interactions between Ndt80 and MSE DNA defining a novel family of Ig-fold transcription factors. Nucleic Acids Res. 32, 2947-2956.
Hartwell, L.H., and Weinert, T.A. (1989). Checkpoints: controls that ensure the order of cell cycle events. Science 246, 629-634.
Hepworth, S.R., Friesen, H., and Segall, J. (1998). NDT80 and the meiotic recombination checkpoint regulate expression of middle sporulation-specific genes in Saccharomyces cerevisiae. Mol. Cell Biol. 18, 5750-5761.
Hong, E.L., Shinohara, A., and Bishop, D.K. (2001). Saccharomyces cerevisiae Dmc1 protein promotes renaturation of single-strand DNA (ssDNA) and assimilation of ssDNA into homologous super-coiled duplex DNA. J. Biol. Chem. 276, 41906-41912.
Ito, H., Fukada, Y., Murata, K., and Kimura, A. (1983). Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163-168.
Keeney, S., Giroux, C.N., and Kleckner, N. (1997). Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell 88, 375-384.
Komeili A., and O''Shea E.K. (1999). Roles of phosphorylation sites in regulating activity of the transcription factor Pho4. Science 284, 977-980.
Komeili A., and O''Shea E.K. (2000). Nuclear transport and transcription. Curr Opin Cell Biol. 12, 355-360.
Lamoureux, J.S., Stuart, D., Tsang, R., Wu, C., and Glover, J.N. (2002). Structure of the sporulation-specific transcription factor Ndt80 bound to DNA. EMBO J. 21, 5721-5732.
Lindgren, A., Bungard, D., Pierce, M., Xie, J., Vershon, A., and Winter, E. (2000). The pachytene checkpoint in Saccharomyces cerevisiae requires the Sum1 transcriptional repressor. EMBO J. 19, 6489-6497.
Leu, J.Y., Chua, P.R., and Roeder, G.S. (1998). The meiosis-specific Hop2 protein of S. cerevisiae ensures synapsis between homologous chromosomes. Cell 94, 375-386.
Leu, J.Y., and Roeder, G.S. (1999). The pachytene checkpoint in S. cerevisiae depends on Swe1-mediated phosphorylation of the cyclin-dependent kinase Cdc28. Mol. Cell 4, 805-814.
Mitchell, A.P. (1994). Control of meiotic gene expression in Saccharomyces cerevisiae. Microbiol. Rev. 58, 56-70.
Montano, S.P., Cote, M.L., Fingerman, I., Pierce, M., Vershon, A.K., and Georgiadis, M.M. (2002a). Crystal structure of the DNA-binding domain from Ndt80, a transcriptional activator required for meiosis in yeast. Proc. Natl. Acad. Sci. USA 99, 14041-14046.
Montano, S.P., Pierce, M., Cote, M.L., Vershon, A.K., and Georgiadis, M.M. (2002b). Crystallographic studies of a novel DNA-binding domain from the yeast transcriptional activator Ndt80. Acta Crystallogr. D Biol. Crystallogr. 58, 2127-2130.
Murakami, H., and Nurse, P. (1999). Meiotic DNA replication checkpoint control in fission yeast. Genes Dev. 13, 2581-2593.
Murakami, H., and Nurse, P. (2000). DNA replication and damage checkpoints and meiotic cell cycle controls in the fission and budding yeasts. Biochem. J. 349, 1-12.
O''Neill, E.M., Kaffman, A., Jolly, E.R., and O''Shea, E.K. (1996). Regulation of PHO4 nuclear localization by the PHO80-PHO85 cyclin-CDK complex. Science 271, 209-212.
Pak, J., and Segall, J. (2002a). Regulation of the premiddle and middle phases of expression of the NDT80 gene during sporulation of Saccharomyces cerevisiae. Mol. Cell Biol. 22, 6417-6429.
Pak, J., and Segall, J. (2002b). Role of Ndt80, Sum1, and Swe1 as targets of the meiotic recombination checkpoint that control exit from pachytene and spore formation in Saccharomyces cerevisiae. Mol. Cell Biol. 22, 6430-6440.
Rockmill, B., Sym, M., Scherthan, H., and Roeder, G.S. (1995). Roles for two RecA homologs in promoting meiotic chromosome synapsis. Genes Dev. 9, 2684-2695.
Roeder, G.S. (1997). Meiotic chromosomes: it takes two to tango. Genes Dev. 11, 2600-2621.
Sambrook, J., and Russell, D.W. (2001). Molecular cloning. 3rd ed. Cold Spring Harbor Laboratory, New York.
Shonn, M.A., McCarroll, R., and Murray, A.W. (2000). Requirement of the spindle checkpoint for proper chromosome segregation in budding yeast meiosis. Science 289, 300-303.
Shubassi, G., Luca, N., Pak, J., and Segall, J. (2003). Activity of phosphoforms and truncated versions of Ndt80, a checkpoint-regulated sporulation-specific transcription factor of Saccharomyces cerevisiae. Mol Genet Genomics. 270, 324-336.
Shuster, E.O., and Byers, B. (1989). Pachytene arrest and other meiotic effects of the start mutations in Saccharomyces cerevisiae. Genetics 123, 29-43.
Smith, A.V., and Roeder, G.S. (1997). The yeast Red1 protein localizes to the cores of meiotic chromosomes. J. Cell Biol. 136, 957-967.
Sopko, R., Raithatha, S., and Stuart, D. (2002). Phosphorylation and maximal activity of Saccharomyces cerevisiae meiosis-specific transcription factor Ndt80 is dependent on Ime2. Mol. Cell Biol. 22, 7024-7040.
Stuart, D., and Wittenberg, C. (1998). CLB5 and CLB6 are required for premeiotic DNA replication and activation of the meiotic S/M checkpoint. Genes Dev. 12, 2698-2710.
Sym, M., Engebrecht, J.A., and Roeder, G.S. (1993). ZIP1 is synaptonemal complex protein required for meiotic chromosome synapsis. Cell 72, 365-378.
Tung, K.S., Hong, E.J., and Roeder, G.S. (2000). The pachytene checkpoint prevents accumulation and phosphorylation of the meiosis-specific transcription factor Ndt80. Proc. Natl. Acad. Sci. USA 97, 12187-12192.
Wang, Y. (2003). Analysis of yeast meiosis-specific transcription factor mutant, NDT80-bc. Master Thesis. National Taiwan University. Taiwan.
Wu, J.F. (2001). Relationship between Ndt80 phosphorylation and its function. Master Thesis. National Taiwan University. Taiwan.
Xie, J., Pierce, M., Gailus-Durner, V., Wagner, M., Winter, E., and Vershon, A.K. (1999). Sum1 and Hst1 repress middle sporulation-specific gene expression during mitosis in Saccharomyces cerevisiae. EMBO J. 18, 6448-6454.
Xu, L., Ajimura, M., Padmore, R., Klein, C., and Kleckner, N. (1995). NDT80, a meiosis-specific gene required for exit from pachytene in Saccharomyces cerevisiae. Mol. Cell Biol. 15, 6572-6581.