請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76098
標題: | 小鼠核分裂蛋白的基因選殖與組織特異性表現之研究 Isolation and Tissue Specific Expression of Various NuMA Isoforms in Mouse |
作者: | 施皜青 |
出版年 : | 1995 |
學位: | 碩士 |
摘要: | 早期在本實驗室中,牛食道上皮(bovine esopheal epithelium)底層小細胞之萃取物被用作免疫原(immunogen)以產生一系列的單株抗體(monoclonal antibodies)。其中一株mAb Wl所染上的抗原(antigen)在 interphase時均勻分佈於細胞核中,在metaphase時則呈新月形集中於紡錘極(spindle pole)附近。其後W1抗原被發現與在1980年首度被發現的NuMA(nuclear mitotic apparatus)為同一蛋白質。NuMA蛋白質的分子量約為240kDa ,結構為兩球狀區域(globular domains)夾一alpha-coiled coil domain。研究發現NuMA蛋白質為細胞進行有絲分裂不可或缺的要素,並與中心體(centrosome)、紡錘絲(spindle)、以及cdc2 kinase有密切關係。由於 alternative RNA splicing的結果,目前已發現至少有T33、U4、U6三種NuMA蛋白質的異型體(isforms)。此外,另因一段42bp序列之有無,同時可區分出二種分別具備或缺乏此一序列(+/- 42bp)的NuMA mRNA。本論文由小鼠 NuMA cDNA library選殖出可涵蓋3'端isoforms岐異區域的cDNA clones,先進行定序分析(sequencing),並與人類 NuMA相對位置之序列進行比對後,設計適當引子(Primer sets),以rtPCR (reverse transcriptase-polymerase chain reaction)技術偵測不同isoform在不同器官中之特殊分佈。結果顯示,小鼠與人類的NuMA T33 isoform無論在cDNA序列或蛋白質方面,皆有很高的相似性。T33與U4 mRNA在所有檢測的器官中皆有所分佈,而對於U6 mRNA卻無法偵測到其之存在。此外,在腎臟(kidney)與睾丸(testis)之中,同時存在著 +/- 42bp mRNA,然而在包括腦(brain)、心臟(heart)、小腸(intestine)、肝臟(liver)、肺(lung)、脾臟(spleen)以及卵巢(ovary)的器官中,卻只有+42bp mRNA的存在。 Nuclear extracts isolated from the small cells of the basal layer of bovine esophageal epithelium were used as immunogen for generation of monoclonal antibody (mAb) W1. W1 target antigen was found to be located at the nuclei of all cells examined. In interphase cells, the target antigen of mAb W1 was diffusely distributed in the nucleus. During metaphase, however, the W1 antigen formed prominent crescents at the poles of the mitotic spindle. This W1 antigen was later found to be identical to the NuMA (nuclear mitotic apparatus) protein which was first discovered by Lydersen in 1980. NuMA is a 240-kDa protein with a long alpha coiled-coil domain and two globular domains. Many reports have shown a closely relationship among the NuMA protein, the centrosome, the spindle, and cdc2 kinase. Recently, it has been reported that there are at least three different isoforms of NuMA (T33, U4, and U6) which were generated by alternative RNA splicing with diversity in their carboxy-terminus. In additional, two other types of NuMA mRNAs with or without the presence of the 42-bp sequence were also found in cells. In this study, mouse NuMA cDNA clones that span the entire 3'-end coding region were isolated, sequenced, and were compared to its human counterpart. My results showed that the mouse T33 isoform had high identity to that of human form. However, high degree of diversity were also observed between mouse and human NuMA U4 isoforms. rtPCR was then applied to detect the tissue specific distribution of various NuMA isoforms. In this respect, T33 and U4 mRNAs were present in all organs examined, however, no U6 mRNA was detected. In contrast, +/- 42bp mRNA coexisted in the kidney and the testis while +42bp mRNA appeared to be the predominant form found in most organs, including brain, heart, intestine, liver, lung, spleen, and ovary. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76098 |
全文授權: | 未授權 |
顯示於系所單位: | 動物學研究所 |
文件中的檔案:
沒有與此文件相關的檔案。
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。