鯉魚c-mos基因表現蛋白性狀及功能之探討

Abstract

c-mos基因產物是個蛋白激?，在脊椎動物之卵成熟過程中調節各項重要事件。就蛙卵而言，Mos蛋白激?負責誘發GVBD（germinal vesicle breakdown），於第一次和第二次減數分裂過渡時抑制DNA合成，並維持成熟卵靜止於第二次減數分裂中期。Mos蛋白激?之作用機制與MPF（maturation-promoting factor）作用有著密不可分之關係，與Mos蛋白質交互作用之分子更結合成一複雜之交織。此多元化之分子性質，可能歸因於Mos蛋白質合成初期不穩定之性質與磷酸化狀態而達成。對於Mos蛋白之生理研究，多採融合蛋白進行研究。鯉魚卵成熟樣態與蛙卵系統有所不同，但鯉魚卵成熟之研究僅止於MPF，尚缺乏完整分子機制之構圖。本實驗室則用已選殖到的鯉魚c-mos基因，利用分子生物技術，促使細菌表現c-mos非融合蛋白，以極溫和之純化步驟獲取較近於內生性之Mos蛋白，並針對此蛋白作了一些基本性狀探討及與魚卵萃液之蛋白質之交互作用。發現到表現的Mos蛋白在純化後去鹽時有消失的現象。並且發現到細菌表現蛋白為—磷酸蛋白，且於表現之初便已呈磷酸化狀態。這些基本性狀及初步於卵萃液中之觀察對於未來c-mos蛋白在魚卵成熟分子機制的研究將會有所助益。

The Mos protein kinase, product of the c-mos proto-oncogene, is a key regulator of oocyte maturation in vertebrates. In Xenopus laevis, Mos protein kinase is involved in inducing germinal vesicle breakdown, inhibiting DNA replication during meiosis I/meiosis II transition and maintaining the metaphase arrest of meiosis II in mature eggs. Maturation promoting factor (MPF) activities during oocyte maturation are also regulated by Mos or Mos-mediated mechanisms. Mos also interacts with many molecules other than MPF. The multi-functional properties of Mos might be attributed to its various phosphorylation states and instable property. In spite of the apparent conformation problems, most researches in Xenopus oocyte system used Mos fusion protein as a tool. On the other hand, researches in fish oocyte maturation are in relatively slow progression without incorporating Mos into studies. We had cloned c-mos gene from the carp genomic library. In this study, the c-mos gene product of the carp was expressed as a non-fusion protein in E. coli. The expressed protein showed different characters from Mos fusion protein utilized in the study of Xenopus oocytes. Caracterization of the carp c-mos protein in vitro will help the investigation of Mos molecular mechanism in vivo in the future.