臺灣博碩士論文加值系統

當環境中缺乏可發酵的醣類及氮源時，二倍體的酵母菌細胞會進行減數分裂，並產生四個單倍體的孢子以度過逆境，而在酵母菌減數分裂過程中有一些熱休克蛋白會表現，例如Hsp26蛋白。先前研究酵母菌Hsp26蛋白在孢子產生過程所扮演的角色時，我們發現可能存在一個與Hsp26有關的紡錘體位置檢控點 (spindle position checkpoint)，可監控從第二次減數分裂到孢子形成的過程，子細胞核在母細胞中的位置或細胞內形成的紡錘體是否適合形成孢子。

為了瞭解此檢控點如何監控紡錘體的形成及排列，先前找出幾個和Hsp26 蛋白有交互作用且可能參與在此調控機制的蛋白質。其中一個為Tem1蛋白。Tem1蛋白是酵母菌有絲分裂中mitotic exit network (MEN) pathway的重要分子，它在此pathway中扮演類似感應器的角色，去感應紡錘體的排列方向是否正確，以確保複製後的染色體能均勻地分配到母細胞與子細胞中。因為TEM1是必需基因，酵母菌缺乏Tem1蛋白會使細胞無法進行有絲分裂並死亡，我們無法利用基因剔除 (Gene knock-out)的方式來研究Tem1蛋白在減數分裂中的角色及功能，所以我們改利用CLB2 promoter去調控Tem1蛋白的表現，讓Tem1蛋白在有絲分裂時可以表現，在減數分裂中則無法表現，藉此研究Tem1蛋白在減數分裂中的角色及功能。我們發現CLB2p-TEM1突變會提高產孢效能。從減數分裂時間過程的分析顯示CLB2p-TEM1突變提高了產孢效能是影響在孢子形成的過程，相似的結果也可在hsp26突變中發現。除此之外，以benomyl處理細胞後，結果顯示hsp26突變和CLB2p-TEM1突變其產孢效能皆沒有明顯受到影響，但孢子存活率則有明顯降低的現象。而hsp26 CLB2p-TEM1突變的表型跟hsp26突變和CLB2p-TEM1突變的表型相同，結果顯示Hsp26蛋白和Tem1蛋白在減數分裂中可能參與在同一條調控路徑上。

綜合結果，我們認為Tem1蛋白可能參與在Hsp26蛋白參與的紡錘體位置檢控點 (spindle position checkpoint)調控孢子形成的機制中。根據Tem1蛋白在有絲分裂中的功能，Tem1蛋白可能也是扮演類似感應器的角色，去感應子細胞核在母細胞中的位置或細胞內形成的紡錘體是否適合形成孢子，若不適合，此檢控點會阻止孢子形成，以確保形成的孢子都具有能力存活。

我們進一步研究在減數分裂中僅有一次細胞分裂的細胞內此Hsp26蛋白相關的紡錘體位置檢控點 (spindle position checkpoint)是否有功能。缺乏Spo13蛋白的細胞在減數分裂中僅進行一次細胞分裂，我們觀察hsp26 spo13突變的產孢效能，發現其產孢效能並沒有提高，跟hsp26突變的產孢效能不同。此外，以benomyl或cold-shock處理細胞後，spo13突變的產孢效能並沒有明顯受到影響。綜合結果顯示在減數分裂中僅有一次細胞分裂的細胞內此檢控點不具有效應。

In the yeast Saccharomyces cerevisiae, diploid cells enter meiosis and produce four haploid spores when fermentable sugar and nitrogen resources are limited. Several stress proteins are induced during sporulation, including Hsp26. Our laboratory has discovered that there is a Hsp26-dependent spindle checkpoint to monitor spindle formation or position, and to regulate spore formation in the budding yeast.

To explore the mechanism of the Hsp26-dependent spindle checkpoint in monitoring spindle positioning, we studied Tem1, one of the Hsp26-interacting proteins. Tem1 is a critical component in the mitotic exit network (MEN) pathway, it plays a sensor for proper spindle positioning between the mother cell and the daughter bud. Since the TEM1 gene is essential, we could not use a knock-out method to explore the function of Tem1 in sporulation. We put the TEM1 gene under the control of the mitosis-specific CLB2 promoter to shut down TEM1 expression in meiosis. Interestingly, sporulation frequency was increased in the CLB2p-TEM1 cells. Meiotic time course studies showed that the increase in sporulation occurs at the step of spore formation, and similar result was obtained in the hsp26 cells. In addition, the response to benomyl treatment of the CLB2p-TEM1 mutant was the same to that of the hsp26 mutant. The phenotype of the hsp26 CLB2p-TEM1 double mutant indicated that Tem1 and Hsp26 might be involved in the same pathway of the spindle checkpoint for spore formation.

Our results suggest that Tem1 may be involved in the Hsp26-dependent spindle checkpoint. According to the function of Tem1 in mitosis, Tem1 might act as a sensor in the checkpoint to monitor spindle position, and in turn control spore formation.

We also investigated whether the Hsp26-dependent spindle checkpoint is functional in the cells undergoing only a single meiotic division. Because cells lacking Spo13 undergo a single meiotic division, we generated hsp26 deletion strain in the spo13 background and examined its effect on sporulation. However, the sporulation frequency of the hsp26 spo13 mutant is not increased, unlike that of the hsp26 mutant. In addition, the spo13 mutant did not display a decrease in sporulation after benomyl treatment or cold-shock treatment. These observations indicated that the spindle position checkpoint has no effect in the cells undergoing only a single meiotic division.

中文摘要 ---------------------------------------------- i
ABSTRACT ------------------------------------------- iii
TABLE OF CONTENTS -------------------------------------v
LIST OF TABLES ----------------------------------- viii
LIST OF FIGURES ------------------------------------- ix
CHAPTER 1. INTRODUCTION ------------------------------ 1
Vegetative cell division ----------------------------- 1
Budding ---------------------------------------------- 1
The spindle position checkpoint ---------------------- 2
Tem1 is the key component in MEN pathway ------------- 3
Sporulation ------------------------------------------ 4
Meiosis ---------------------------------------------- 4
Spo13 is an important regulator of meiosis I --------- 5
Spore formation -------------------------------------- 6
Yeast small heat shock proteins ---------------------- 8
The roles of heat shock proteins --------------------- 8
Yeast Hsp26 protein ---------------------------------- 9
Specific aims --------------------------------------- 12
CHAPTER 2. MATERIALS AND METHODS -------------------- 13
Culture conditions -----------------------------------13
DNA preparation, transformation --------------------- 14
Plasmid constructions ------------------------------- 15
Yeast strain constructions -------------------------- 18
Time course analyses -------------------------------- 21
Meiotic time course analyses ------------------------ 21
Nuclear division analyses --------------------------- 22
Benomyl treatment ----------------------------------- 23
Spore viability analysis ---------------------------- 23
Reversible thermal treatment ------------------------ 24
Cold-shock treatment -------------------------------- 25
Proteinextraction and western blot analysis --------- 25
CHAPTER 3. RESULTS ---------------------------------- 28
Analysis of Tem1 function in sporulation ------------ 28
Regulation of Tem1 expression by CLB2 promoter can support mitosis ------------------------------------- 28
Tem1 is not required for sporulation and spore viability ------------------------------------------------------ 29 Tem1 may be involved in the Hsp26-dependent spindle checkpoint ------------------------------------------ 30
Sporulation frequency in the CLB2p-TEM1 mutant is similar to that in the hsp26 mutant ------------------------- 30
The increase in sporulation occurs at spore formation, but not the initiation of meiosis ------------------- 31
Benomyl treatment affects sporulation in the wild-type cells, but not in the CLB2p-TEM1 mutant cells as well as the hsp26 mutant cells ------------------------------ 32
Benomyl-treated CLB2p-TEM1 mutant cells display a decline in spore viability ----------------------------------- 33
Reversible thermal treatment affects sporulation in the wild type, but not in the hsp26 and CLB2p-TEM1 mutants ------------------------------------------------------- 34
The two proteins, Tem1 and Hsp26, participate in the same pathway --------------------------------------------- 36
Hsp26-dependent spindle position checkpoint has no effect in the cells undergoing only a single meiotic division ------------------------------------------------------- 37
Sporulation frequency in the hsp26 spo13 mutant is not increased ------------------------------------------- 37
Benomyl treatment may not affect sporulation in the spo13 cells ----------------------------------------------- 38
Cold-shock treatment affects sporulation in the wild-type cells, but not in the spo13 mutant cells ------------ 40
Reversible thermal treatment affects sporulation in the wild type, but not in the hsp26, spo13 and hsp26 spo13 mutants --------------------------------------------- 40
Detection of Tem1 expression in meiosis ------------- 41
Epitope tagging of Tem1 ----------------------------- 41
The expression of Tem1 is shut down by the CLB2 promoter during sporulation ------------------------------------43
CHAPTER 4. DISCUSSION ------------------------------- 45
Tem1 is involved in the Hsp26-dependent spindle position checkpoint ------------------------------------------ 45
The role of Tem1 during sporulaiton ----------------- 45
Both the hsp26 mutation and CLB2p-TEM1 mutation suppress the effects caused by benomyl ----------------------- 46
The hsp26 and CLB2p-TEM1 mutant are less affected by the reversible thermal treatment ------------------------ 46
Tem1 and Hsp26 participate in the same pathway ------ 47
Hsp26-dependent spindle position checkpoint has no effect in the cells undergoing only a single meiotic division ------------------------------------------------------- 48
The expression of Tem1 in meiosis ------------------- 49
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