http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71 Sat, 11 Apr 2026 18:26:34 GMT 2026-04-11T18:26:34Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43916 標題: 鹿角蝴蝶蘭 Phalaenopsis cornu-cervi 含 (GA)n 微衛星體序列之選殖與定位; Isolation and physical mapping of (GA)n microsatellites in Phalaenopsis cornu-cervi 作者: Ling-Chiao Wu; 吳淩巧 摘要: 微衛星體序列在真核生物基因組中含量豐富。以(GA)11為引子進行單引子聚合酶連鎖反應，從鹿角蝴蝶蘭Phalaenopsis cornu-cervi分離出11個富含(GA)n之微衛星體序列，序列長度在120~860 bp之間，序列間平均相似度為54.45%，TIGR資料庫序列比對結果與許多植物之重複性序列相類似。5個選殖體W8-4、W8-5、W8-18、W8-35與W8-41、朵麗蘭（Phalaenopsis pulcherrima）之DpGA2和Phalaenopsis stuartiana 分離的W40-19利用螢光原位雜交實質定位的結果，皆位於鹿角蝴蝶蘭所有染色體的中節，且訊號強度相似。過去DpGA2定位結果，只位於朵麗蘭一對染色體的中節，有明顯差異。W8-18訊號亦位於 P.stuartiana、Phalaenopsis mannii 與朵麗蘭染色體的中節，且以P.stuartiana染色體中節的訊號最強，此與過去利用人工合成(GA)11定位的結果相吻合。; Microsatellites are highly abundant in eukaryotic genomes. Eleven (GA)n microsatellite-containing clones were isolated from Phalaenopsis cornu-cervi by single-primer PCR using (GA)11 as a primer. The size of sequences varied from 120 to 860 bp and the average sequence identity for those was 54.45%. Sequence homology searches of the TIGR database revealed similarities with repetitive sequences in many plants. Seven clones, W8-4, W8-5, W8-18, W8-35, and W8-41 from P. cornu-cervi, DpGA2 from P. pulcherrima, and W40-19 from P. stuartiana, were all mapped in the metaphase chromosomes of P. cornu-cervi by fluorescence in situ hybridization (FISH). All signals were similar in intensity and clustered in the centromeric regions of all chromosomes. It was found a quite exceptional distribution of DpGA2 probe which was only located in the centromeric regions of one chromosome pair of P. pulcherrima. The hybridization signals of the clone W8-18 were also clustered in the centromeric regions of P. mannii, P. pulcherrima, and P. stuartiana chromosomes. In addition, signals were much stronger in P. stuartiana than in other species. These results were consistent with the physical mapping of synthetic oligonucleotides (GA)11 in the previous study. Thu, 01 Jan 2009 00:00:00 GMT http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43916 2009-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32023 標題: 鯉魚硫胱胺酸蛋白酶cathepsin Z在卵子成熟過程中的研究; Study of Carp Cathepsin Z during Oocyte Maturation 作者: Chi-Min Kao; 高啟明 摘要: 此研究從鯉魚卵巢cDNA基因庫中選殖出一個新型的酵素，此酵素從未在鯉魚身上發表，也缺乏功能性的研究，經過比對，發現它屬於papain family家族的一員，與哺乳類的cathepsin Z相類似。cathepsin Z在分類上屬於cysteine protease。經過比對，此酵素有關於活性調控與形成三級結構的重要區域都與其它cysteine proteases類似。 觀察此酵素在鯉魚卵子最後成熟過程中的變化，發現cathepsin Z的mRNA在鯉魚注射GnRH-analogue 的8-12小時後表現量最多，稍後在排卵時又降到未注射前的水準，推測與卵細胞內卵黃的分解有關，也與先前的研究認定卵細胞內蛋白質分解在成熟過程中8-16小時達到最高峰相吻合。 此外，cathepsin Z存在於成熟大卵的表層顆粒與卵黃細胞質附近。在卵子發生表層反應時，會釋放到圍卵腔中，進而形成受精膜的一部分。最後，實驗表現的重組蛋白具有活性，可以分解gelatin，並且可以切割鯉魚的卵黃前質素(vitellogenin)，證明cathepsin Z具有內切酶的特性。; A cDNA encoding cathepsin Z (CTPZ) was cloned from a carp ovarian cDNA library. It is homologous to mammalian CTPZ. The amino acid residues important for protein folding and enzymatic activity of cysteine proteases are conserved in carp CTPZ. During oocyte maturation, cathepsin Z transcripts increased and reached peak at 8 h post-treatment of GnRH analogue by 9-folds compared to that of untreated oocytes, then declined near to the level of untreated oocytes in ovulated eggs. Cathepsin Z was discharged from cortical granules to perivitelline space after cortical reaction and became new component of fertilization envelope. Inside the oocyte, cathepsin Z was stained in the cytoplasm surrounding the yolk granules. The recombinant cathepsin Z has an enzymatic activity toward vitellogenin, and inhibited by PMSF or E-64, a specific inhibitor of thiol protease. The potential roles of cathepsin Z in carp eggs are discussed. Sun, 01 Jan 2006 00:00:00 GMT http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32023 2006-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/38575 標題: 鯉魚減數分裂基因c-Mos功能與活性調控之探討; Function and Regulation of Carp c-Mos 作者: Kuan-Cheng Lee; 李冠徵 摘要: 細胞進行有絲分裂時，細胞核當中的遺傳物質-染色體需進行一次複製、濃縮、經由紡錘體分離平均分配到兩個子細胞。進行減數分裂的生殖細胞染色體也需要經過以上等過程，但不同的是減數分裂中染色體只複製一次，而後卻有連續兩次的分離，形成單套染色體配子。顯然有絲分裂的機制在細胞行減數分裂時受到了修飾與特化。在大部分的脊椎動物裡，未成熟的卵需進行減數分裂，爾後在第二次減數分裂作停留等待受精，此一過程稱之為卵的成熟作用。 Ran系統在有絲分裂進程中參與染色體以及紡錘體狀態的調控。而Mos是減數分裂時的特有激酶，影響減數分裂時紡錘體形成、維持、變動以及染色體組態。為了探討此二者共同作用於M phase時，彼此之間交互作用狀況為何，因此選殖了鯉魚卵巢內的Mos基因異位表現於COS-1細胞，並觀察Mos與Ran是否有直接的結合。實驗結果發現，在此狀況下，針對Mos的共免疫沈澱複合物當中含有Hsp70/90等協助Mos構型的蛋白存在，而此共免疫複合沈澱物亦具有酶激活性，但卻無法偵測到Ran存在於其中。; Abstract In most vertebrate animals, the development of the immature oocyte into a fertilizable gamete, a process known as oocyte maturation, involves an arrest in the meiotic cell cycle while awaiting fertilization. Mos, a serine/threonine kinase, is specifically expressed during meiotic maturation of vertebrate oocytes. After germinal vesicle breakdown (GVBD), Mos is involved in the regulation of meiotic spindle assembly and chromatin organization. The activation of this kinase is also essential for the maintenance of metaphase Ⅱ arrest. Ran GTPase, which belongs to the Ras superfamily of small GTPase, and the protein that regulate its GTP binding and hydrolysis has a well-defined role in nuclear transport. Recent studies indicate that Ran has a central role in spindle assembly and nuclear envelope reformation. It seems that Ran system and Mos pathway coordinate in some events in M phase. To understand the inter-relationship of them, I ectopically express Carp Mos gene in COS-1 cell and investigate Mos-interacting proteins. Heat Shock Protein-Hsp70 and Hsp90 are detected in Mos co-immunoprecipitation complex with or without kinase activity, but Ran dose not exist in the complex. Sat, 01 Jan 2005 00:00:00 GMT http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/38575 2005-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/36370 標題: 鯉魚Septin7之選殖及其與精子專一蛋白質Msap交互作用之探討; Cloning of Carp Septin7 and Its Interaction with sperm specific protein, Msap 作者: Yi-Tsun Lee; 李依純 摘要: Septin是一種保守性相當高的細胞骨架(cytoskeleton)蛋白質，除了植物之外幾乎所有的真核生物中都含有Septin。septin基因最早在酵母菌（Saccharomyces cerevisiae）中發現，其在胞質分裂中佔有重要的地位，缺失septin的酵母菌在出芽生殖時會產生細胞形狀異常的現象。最近的研究顯示，Septin家族成員也參與許多其他的細胞內反應，如細胞極性、計畫性細胞死亡、細胞週期調控和細胞形狀形成等。在胺基酸序列方面，Septin家族主要有三個共同的結構：N端的basic domain、GTPase domain和C端的coiled-coil domain。在哺乳類，Septin的basic domain負責phosphoinositides之結合，而coiled-coil domain則和Septin家族蛋白質間的結合有關。 本實驗室在之前進行和精子發生相關基因Msap的二維電泳分析時，發現其和Septin家族蛋白質中的Septin7之間可能有結合的情形，且Septin7在鯉魚精子中主要分佈在basal body的位置，和近期的研究結果相似。於是本論文利用SMART RACE的系統選殖鯉魚睪丸中septin7的cDNA，結果顯示可能有兩種長度不同的septin7 transcripts存在於鯉魚的睪丸組織。利用特定引子進行PCR發現兩種transcripts都廣泛存在於各組織中，但彼此之間有表現量差異的情形。免疫組織化學染色可以看出在鯉魚睪丸組織中，Septin7蛋白質並沒有特殊的分佈位置，而是呈現散佈在組織中的情形。而利用Septin7重組蛋白質抗體進行各組織萃取液的西方轉印，結果也顯示Septin7在各個組織中廣泛存在，但以在睪丸的表現量為最多，而且50kDa為主要的表現形式。為了確認Msap和Septin7蛋白質之間的結合情形，利用二維電泳的方法分析pull down和免疫共沉的產物，發現Septin7和Msap之間的確有結合的關係。; Septin is a highly conserved group of cytoskeleton protein that have been found in almost all eukaryotes but not in protozoa or plants. The septin genes were originally identified in Saccharomyces cerevisiae, and have an important role during cytokinesis. The budding yeast mutants that exhibited defects in septins will produce abnormal morphology during the budding process. Recent researches implicate that different Septins have diverse cellular roles including cell polarity, programmed cell death, cell cycle regulation and cell morphogenesis. The septin family of genes possess three common structures: an N-terminal basic domain, GTPase domain and a C-terminal coiled-coil domain. In mammal cells, the function of the N-terminal basic region is possibly responsible for the interaction with phosphoinositides. Some data point that the C-terminal coiled-coil domain is also responsible for intermolecular interaction upon Septin complex formation. Previous studies about the spermatogenesis associated gene, msap, of our laboratory by using two dimensional electrophoresis discovered the possible interaction between Septin7 and Msap. The studies also revealed that Septins are localized in the basal body of carp sperm, which is consistent with other research. In this study, the septin7 cDNA is cloned by SMART RACE system and two variants with different length are obtained. By using PCR, we found that both two transcripts exist in all kind of tissue, but the relative expression is different. The result of immunohistochemistry revealed that the Septin7 is ubiquitously expressed present in all testicular tissue. Western blot by Septin7 antibody showed the Septin7 is expressed as 50 kDa as a major form in all kind of tissue, althouth enriched in testis. To confirm the interaction between Septin7 and Msap, pull down and co-immunoprecipitation were used. The results favored the possibility that Septin7 interacts with Msap. Sat, 01 Jan 2005 00:00:00 GMT http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/36370 2005-01-01T00:00:00Z