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DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 董桂書 | |
dc.contributor.author | Tzu-Ming Ho | en |
dc.contributor.author | 何子明 | zh_TW |
dc.date.accessioned | 2021-06-08T05:22:20Z | - |
dc.date.copyright | 2005-07-28 | |
dc.date.issued | 2005 | |
dc.date.submitted | 2005-07-25 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24335 | - |
dc.description.abstract | 在酵母菌減數分裂過程中有一些熱休克蛋白的表現,而這些熱休克蛋白在減數分裂中的功能目前尚未清楚。本論文的目標在於研究酵母菌Hsp26蛋白質在孢子產生時期的功能。Hsp26屬於small heat shock protein superfamily,它除了會受到熱休克而表現,在diauxic shift以及產孢過程中也會表現。
我們發現hsp26突變並不會降低酵母菌的產孢效能,反而會提高了產孢效能。在hsp26突變株細胞中可以發現有一些不正常的孢子產生。從減數分裂時間過程的分析顯示hsp26突變提高了產孢效能是影響在孢子形成的過程。我們假設Hsp26蛋白可能扮演一個紡垂體位置檢控點 (spindle position checkpoint) 的角色,來偵測已完成核分裂的子細胞在母細胞中的位置是否有平均分配而決定孢子形成的過程是否會繼續進行。為了驗證這個假設,我們使用了一些方法來干擾孢子形成的過程。發現當野生型細胞受到高溫處理一段時間後所產生的孢子中,dyads所佔的比例有明顯提高,而在hsp26突變株中則沒有受到影響。我們更進一步的使用benomyl處理細胞,專一性的使減數分裂細胞的紡垂體 (spindle) 發生缺失。結果顯示hsp26突變株並沒有明顯受到影響而野生型的細胞其產孢效能則有明顯降低的現象。另外,我們使用兩個會影響有絲分裂紡垂體方位(spindle orientation)的突變株kar9和dyn1來影響減數分裂時期細胞紡垂體的位置。這個結果也顯示hsp26突變可以隱抑kar9和dyn1所造成的產孢效能缺陷。最後,Hsp26蛋白與一個參與在有絲分裂紡垂體位置檢控點 (mitotic spindle position checkpoint) 的蛋白Tem1有明顯的交互作用,這個結果也支持了我們的假設。綜合所有的結果,我們認為在酵母菌的產孢過程中有一個Hsp26蛋白所參與的紡垂體位置檢控點(spindle position checkpoint),可監控從第二次減數分裂到孢子形成的過程,而確保產生正常的孢子。 | zh_TW |
dc.description.abstract | In the budding yeast, diploid cells enter meiosis under nutrient starvation. Several heat shock proteins are induced during sporulation. The function of these heat shock proteins is unclear. In this study, we explore the role of Hsp26 in sporulation. Hsp26 is a member of the small heat shock protein superfamily. It is induced by heat shock and accumulates after diauxic shift and during the course of sporulation.
The hsp26 mutant does not decrease sporulation; instead, the sporulation frequency in the hsp26 mutant is ~20% higher than that in wild-type cells at 30℃ and ~50% higher at 32.5℃. Meiotic time course studies showed that the increase in sporulation occurs at the step of spore formation. Based on these results, we hypothesize that Hsp26 may play a spindle position checkpoint role in monitoring the distribution of daughter nuclei within the mother cells and in determining whether spore formation should proceed normally. Consistent with this hypothesis, an increase of abnormal asci, such as linear triads, is in the hsp26 mutants. In addition, when wild-type sporulating cells subjected to the reversible thermal treatment yield dyads, however, the hsp26 mutants still produced tetrads mostly. Furthermore, we have used benomyl to specifically cause spindles defects in meiotic cells. The hsp26 mutants are not affected, while the wild-type cells display a defect in sporulation. Additionally, two mutants, kar9 and dyn1, which cause misorientation of mitotic spindles were used to disturb the spindle positioning during meiosis. The results show that the sporulation defects caused by the kar9 and dyn1 mutants could be suppressed by the hsp26 mutation. Finally, the interaction between Hsp26 and Tem1 further suggests that the spindle position checkpoint is involved in our proposed checkpoint machinery. These results demonstrate that there is a novel Hsp26-dependent spindle position checkpoint to control the transition from meiosis II to spore formation. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T05:22:20Z (GMT). No. of bitstreams: 1 ntu-94-R92b43005-1.pdf: 1462440 bytes, checksum: ae0b51fb8feda783c0220c3b728e37e2 (MD5) Previous issue date: 2005 | en |
dc.description.tableofcontents | ABSTRACT...............................................i
中文摘要..............................................ii TABLE OF CONTENTS....................................iii LIST OF TABLE.........................................vi LIST OF FIGURES......................................vii CHAPTER 1. INTRODUCTION ...............................1 The Importance of Meiosis..............................1 Checkpoint and Meiotic Cell Cycle Control..............2 The Roles of Heat-Shock Proteins in Meiosis............4 Mouse HSP70-2 Protein..................................4 Yeast Ssa3 Protein ....................................5 Yeast Hsp26 Protein ...................................6 Specific Aim ..........................................7 CHAPTER 2. MATERIALS AND METHODS .....................8 Strains, Media, and Growth Conditions .................8 DNA Preparation, Transformation, and Plasmid Constructions..........................................9 General methods .......................................9 Plasmid Constructions .................................9 Yeast Constructions .................................13 Constructions of yeast null mutants ..................13 Epitope tagging Hsp26.................................15 Protein Extraction and Western Blot Analysis..........15 Time Course Analyses..................................16 Meiotic time course analyses..........................16 Nuclear division analyses.............................17 Cytology Analyses ....................................18 Whole cell staining and immunofluorescence with yeast cells...........................................18 Chromosome spreading and immunostaining ..............19 Image analysis ......................................20 Yeast Two-Hybrid Assay ...............................21 Reversible Thermal Treatment .........................21 Benomyl Treatment.....................................22 Spore Viability Analyses .............................23 CHAPTER 3. RESULTS ...................................24 Analysis of hsp26 Mutant in Sporulation .............24 Sporulation frequency in the hsp26 mutant is increased.............................................24 Abnormal asci are observed in the hsp26 mutants.......25 The increase in sporulation occurs at the step of spore formation,but not the initiation of meiosis.....25 Hsp26 and the Transition from Meiosis to Spore Formation ............................................27 Reversible thermal treatment affects sporulation in the wild-type cells, but not in the hsp26 mutants.....27 Benomyl, a microtubule inhibitor, affects sporulation in the wild-type cells, but not in the hsp26 mutant cells.................................................28 Benomyl-treated cells have a delay in the process of asci maturation.......................................29 Benomyl-treated hsp26 mutant cells display a decline in spore viability....................................30 Hsp26 and the Spindle Positioning.....................31 The sporulation defects caused by the kar9 and dyn1 mutants could be suppressed by the hsp26 mutation.....32 Hsp26-Interacting Protein.............................36 Hsp26 protein interacts with Tem1 and Ady3............36 Hsp26 protein has the transcription activation ability...............................................37 Detection of the Hsp26 Protein Expression.............38 Epitope tagging of Hsp26..............................38 Hsp26 is expressed during sporulation.................38 Subcellular Localization of the Hsp26 Protein.........40 Hsp26 localizes in nuclei during sporulation .........40 Hsp26 localizes to meiotic chromosomes after pachytene ............................................40 CHAPTER 4. DISCUSSION.................................42 Hsp26 Functions in the Transition from Meiosis II to Spore Formation ......................................42 The hsp26 mutant is less affected by the reversible thermal treatment ....................................42 The hsp26 mutation suppresses the effects caused by benomyl ..............................................43 The defects in sporulation caused by the kar9 and dyn1 could be suppressed by the hsp26 mutation .......43 Hsp26 is localized to chromosomes after pachytene.....44 Hsp26 Interacts with Tem1.............................45 The Spindle Position Checkpoint in Meiosis............45 The possible target of the Hsp26-dependent spindle position checkpoint ..................................45 The spindle position checkpoint in mitosis............47 The possibility for the meiotic spindle position checkpoint............................................47 REFERENCES ..........................................49 APPENDIX ............................................92 | |
dc.language.iso | en | |
dc.title | 酵母菌Hsp26蛋白在孢子產生時的作用 | zh_TW |
dc.title | The role of yeast Hsp26 in sporulation | en |
dc.type | Thesis | |
dc.date.schoolyear | 93-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳蕾惠,陳枝乾 | |
dc.subject.keyword | 酵母菌,熱休克蛋白26,減數分裂, | zh_TW |
dc.subject.keyword | Budding yeast,Hsp26,Meiosis,Sporulation, | en |
dc.relation.page | 93 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2005-07-26 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
顯示於系所單位: | 分子與細胞生物學研究所 |
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