老鼠生長休止基因蛋白八與其交互作用蛋白之生物功能探討

生長休止基因 8 (Gas8) 是由處於細胞靜止期的小鼠纖維母細胞株-NIH3T3中以基因捕捉法被選殖出來的，Gas8 主要表現在老鼠的睪丸中，而且其表現在後減數分裂時期是高度被調控的。此外，Gas8會積聚在round spermatids的細胞質還有精子的尾巴中。然而，Gas8在生理上的功能仍然不清楚，因此此論文的目標在於藉由分離和研究Gas8的結合蛋白(binding proteins)來探討Gas8的功能。我們藉由yeast two-hybrid 的系統，利用Gas8當作餌(bait)、mouse testis cDNA library當作魚(fish)來調出Gas8的結合蛋白。總共有三個Gas8的結合蛋白被分離出來，Gas8結合蛋白一(Gas8-BP1)稱作Axin，Gas8結合蛋白二(Gas8-BP2)稱作Meiosis Nuclear structural protein 1，Gas8結合蛋白三(Gas8-BP3)為一個新的蛋白稱作Mus musculus hypothetical protein 4921501M07。在這裡，我們描繪了Gas8結合蛋白三(Gas8-BP3)這個新的蛋白。結果顯示，在GST pull-down assay 還有co-immunoprecipitation的實驗中我們證明Gas8能和Gas8-BP3有交互作用，而且Gas8-BP3的C端部分可能和Gas8作結合。利用老鼠多種組織的北方點墨法(Northern blot)分析，我們證實了Gas8-BP3會高度表現在成年的老鼠睪丸中，而且它的表現在發育的睪丸中是被調控的。為了進一步了解Gas8-BP3在老鼠睪丸中表現的型態，我們自己製造了Gas8-BP3的抗體(antibody)並且利用此抗體作了免疫組織化學染色(immunohistochemical staining)的實驗。結果顯示Gas8-BP3的蛋白主要表現在round spermatids，spermatocytes而且也表現在spermatogonia中。此外，我們發現 Gas8 也能和微質管(microtubule)有物理上的交互作用。總結來說，Gas8 和Gas8-BP3兩者都會表現在老鼠的睪丸中並且在睪丸的發育中都是被調控的，顯示出它們兩者在睪丸發育的過程中扮演一些角色。

關鍵字

細胞休止基因；老鼠睪丸發育；老鼠精子

並列摘要

Gas8, originally identified in growth arrested NIH3T3 cells by Molony Murine leukemia virus gene-trapping is predominantly expressed in mouse testis, with its expression highly regulated during post meiotic sperm development. In addition, Gas8 accumulates in the tails of sperm, the cytoplasm of round spermatids and also in sperm flagella. However, Gas8 function remains unclear. This thesis investigated Gas8 function in mice by isolating and studying its binding proteins. We isolated Gas8 binding proteins by yeast two-hybrid assays using Gas8 as bait and a mouse testis cDNA library as the fish. Three Gas8 binding proteins were isolated. Gas8-BP1 is Axin, Gas8-BP2 is Meiosis Nuclear structural protein1, and Gas8-BP3 is Mus musculus hypothetical protein 4921501M07. In this study, the novel protein--Gas8-BP3 was further characterized. The results showed that Gas8 could interact with Gas8-BP3 in GST pull-down assays as well as in co-immunoprecipitation experiments, while the C-terminal portion of Gas8-BP3 may serve as the Gas8 interacting region. Using multi-tissue Northern blot analysis I confirmed that Gas8-BP3 was highly expressed in adult mouse testis and this expression was regulated developmentally. In order to understand the expression pattern of Gas8-BP3 in mouse testis, we generated Gas8-BP3 antibody and preformed the mouse testis immunohistochemical staining. The result suggested that Gas8-BP3 protein was predominantly expressed in round spermatids and spermatocytes but also expressed in spermatogonia. In addition, Gas8 physically interacted with microtubules. In summary, Gas8 and Gas8-BP3 are both expressed in testes and regulated developmentally, suggesting that they may play roles in spermatogenesis.

並列關鍵字

Growth arrest specific gene ； Gas8 ； microtubule ； spermatogenesis

參考文獻

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老鼠生長休止基因蛋白八與其交互作用蛋白之生物功能探討

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