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研究生：	陳奕興 Chen, Yi-Hsing
論文名稱：	黏菌發育必需基因之鑑別 Identification of Gene Required for Development in Dictyostelium
指導教授：	張文粲 Chang, Wen-Tsan
學位類別：	碩士 Master
系所名稱：	醫學院 - 生物化學研究所 Department of Biochemistry
論文出版年：	2002
畢業學年度：	90
語文別：	英文
論文頁數：	77
中文關鍵詞：	過氧化體、限制酶插入突變法、黏菌、檸檬酸合成酶
外文關鍵詞：	Restriction Enzyme-Mediated Integration, citrate synthase, Dictyostelium, peroxisome
相關次數：	點閱：99 下載：2
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檸檬酸合成酶普遍存在各生物體系，扮演催化各種營養物代謝的最終產物乙醯輔酶A進入檸檬酸循環的角色。檸檬酸循環同時具有分解產生能量及合成胞內物質的角色，其運轉順暢與否和細胞的生存有直接關係。有些物種擁有單一檸檬酸合成酶，有些則有兩個甚至三個。因物種不同，其主要存在於粒腺體，但也有些位於過氧化體（peroxisome），過氧化體具有代謝及分解有毒物質的功能，其數量，大小及蛋白質組成會因細胞的不同而不同。以限制酶插入突變法所產生的突變株WTC127，菌落大小明顯小於野生型菌落，且結構稀疏無法發育成成熟的子實體(fruiting body)，具有細胞生長缺陷的現象。被破壞的基因會轉譯成檸檬酸合成酶同源蛋白質(Citrate synthase homology protein, CshA)，分子量約55 KDa，而基因表現在細胞生長期至發育中期。在蛋白質胺端序列上具有進入過氧化體的訊息序列PTS2，因此CshA應該是屬於過氧化體間質蛋白。突變株在細菌的吞噬作用(phagocytosis)方面較野生型差。同樣的，細胞生長數量缺陷也在震盪培養下被證實，但在細胞分裂(cytokinesis)方面並沒有任何異常。最後，突變株細胞與細菌混合在飢餓的環境下進行發育，我們觀察到與細菌混合的狀態下可以重現在菌落中發育嚴重受到干擾的表現型，開始聚集發育的時間也比野生株延遲約20小時，證實了發育嚴重缺失的表現型與細菌的存在有直接的關係。這到底是細菌的代謝產物所致，亦或者是吞噬細菌後對突變株發育造成抑制，甚至是突變株無法分泌出某些物質所致?我們目前並不清楚這其中的關連。由於在飢餓的狀態下WTC127發育正常，因此我們對另外一個黏菌檸檬酸合成酶GltA(glutamate auxotroph)的基因表現感到興趣，是否當CshA缺失時GltA可能扮演替代品的角色。我們證實了黏菌中同源性檸檬酸合成酶的存在，同時我們找到部分gltA基因也知道其基因在黏菌的發育生活史的各個時期中持續表現，我們猜想當CshA缺失時GltA可能扮演替代品的角色，以致在飢餓時突變株發育正常。在另外一個以限制酶插入突變法所產生的突變株WTC180方面，被破壞的基因是未知基因，其突變株會進行正常聚集發育，但形成頂尖(apical tip)的時間約延遲6小時，最終會形成極為短小的結構，同時我們也以同源重組(homologous recombination)的方式證實了這個未知基因的被破壞，的確造成了這種特異的表現型。

Citrate synthase is an almost ubiquitous enzyme. Its role is a catalyst of the entry point reaction for entry of two-carbon units into the citric acid cycle. It is an essential step in the biosynthesis of amino acids. Some organisms have a single citrate synthase, while a few, have as many as three. Peroxisomes show a remarkable metabolic plasticity. Their size, numbers, protein composition and biochemical functions vary that depending on the organism, cell type and/or environmental condition. By screening developmentally morphological mutants generated by the restriction enzyme-mediated integration (REMI) mutagenesis, we have found two mutants WTC127 and WTC180. The plaques of WTC127 cells have sparse structures, and cannot form the terminal fruiting bodies, and the sizes of plaques are strikingly smaller than wild type cells on the bacterial plate. The disrupted gene of mutant strain WTC127 encodes a citrate synthase homology protein, CshA, contained a conserved peroxisomal targeting signal PTS2 nonapeptide sequence at the N terminus. Phagocytosis of cshA- cells is slightly worse than wild type and the results of flow cytometry are the same. The growth of cshA- cells have a strikingly defect in axenic medium, but mitosis of cshA- cells is normal. Finally, mixing cshA- cells with K. aerogenes and developing on KK2 agar, the aberrant developmental phenotype of cshA- mutant can be observed at 72 hr, and the aggregation of cshA- cells delayed about 20 hours. The presence of bacteria interferes the multicellular development of cshA- cells, but it is still unclear why cshA- cells cannot form mature fruiting bodies. Because the developmental program of WTC127 is normal on KK2 agar, we are interested in the role of another citrate synthase gene, gltA (glutamate auxotroph). We determined the existence of other citrate synthase homology protein, and GltA was considered the potential another citrate synthase in Dictyostelium. Analyzing the 3’-terminal of cDNA fragment, there is not any signal peptide including in C-terminal of GltA. RT-PCR analysis indicated that gltA mRNA is expressed throughout the development. We think that gltA is a substitute for cshA- cell during development. The WTC180 cells formed distinctly smaller structures than wild type on a bacterial plate. The disrupted gene of mutant strain WTC180 is a novel gene. Mutant cells were able to form normal aggregation streams upon starvation, but the formation of tip mound delayed about 6 hours. The terminal structures, very small size of fruiting bodies, completed at 36 hr.

ABSTRACT                                                                  I
中文摘要                                                       Ⅲ
誌謝                                                           V
CONTENT													   VI
FIGURE CONTENT	    									       VII
INTRODUCTION											          1
MATERIALS and METHODS									   15
RESULT                                                                24
DISCUSSION                                                                   31
REFERENCES                                                     35
FIGURES                                                              41
自述                                                                       77


FIGURE CONTENT
Figure 1    Possible integration of anaplerotic and bypass pathways with the TCA cycle                   41
Table   1     Model organisms used for the study of peroxisomes                                42
Figure 2     Targeting signals used by peroxisomal proteins                                  43
Figure 3      Life cycle of Dictyostelium                                                        44
Figure 4       Restriction enzyme-mediated integration in Dictyostelium                          45
Figure 5       REMI vectors                                                         46
Figure 6       Developmental phenotypes of the wild type and WTC127 
                     mutant on bacterial plates                                                              47
Figure 7         Southern blot analysis of the WTC127 mutant                                           48
Figure 8          Developmental morphology of the wild type and WTC127 mutant                                                                   49
Figure 9           Southern blot analysis of original and recreated mutants                                                                                                                                                                        50
Figure 10         Western blot analysis of the CshA protein                                                                                                                                                                                        51
Figure 11           SDS-PAGE analysis of purified CshA protein                                                                                                                                                                                             52
Figure 12   Amino acid sequence analysis of DdCshA                                                                                                                                                                                                                                               53
Figure 13  Sequence alignment of DdCshA and CmGCS                                                                 54
Figure 14  Southern blot analysis of the cshA copy number                                                                            55
Figure 15  Northern blot analysis of the cshA gene expression during 
                                                                                                      Dictyostelium development                                                                               56
Figure 16  Cellular location of the CshA protein                                                                                            57
Figure 17  N-terminal sequence alignment of citrate synthase proteins                                             58
Figure 18  Growth rate of REMI mutant WTC127 on the SM-agar bacterial plate                                                                       59
Table 2          Growth rate of REMI mutant WTC127 on the SM-agar bacterial plate                             60
Figure 19  Phagocytosis of the wild type and WTC127 mutant                                                                           61
Figure 20  Phagocytosis of the wild type and WTC127 mutant                                                                                                             62
Figure 21  Cell growth curve of the wild type and WTC127                                                                    63
Figure 22  DAPI staining of the cshA- cell                                                              64
Figure 23  Developmental morphology of the wild type and 
                                                                                                           WTC127 mutant on bacterial KK2 agar                                                                                 65
Figure 24  Southern blot analysis of cshA gene in Dictyostelium
                                                                                                      at lower hybridization stringency                                                         66
Figure 25  Partial nucleotide and deduced amino acid sequences of the gltA gene                                                                                                 67
Figure 26  Sequence alignment of CshA and GltA                                                                                      68
Figure 27  Sequence alignment of DdGltA and AtPCS                                             69
Figure 28  Comparison of Southern blot analyses of the gltA copy number and cshA 
                                                                                                                  gene in Dictyostelium at lower hybridization stringency (45oC)                                                       70
Figure 29  Expression of gltA during development                                                                       71
Figure 30  Southern blot analysis of the WTC180 mutant                                                             72
Figure 31  Morphology of the wild type and WTC180 mutant on bacterial agar                                    73
Figure 32  Developmental morphology of the wild type and WTC180 mutant                                         74
Figure 33        Southern blot analysis of original and recreated mutants                                                                       75
Figure 34  Partial nucleotide sequence of the WTC180                                                              76

                                    

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