鯉魚Mad2、Cdc20、Cdh1基因於減數分裂功能之探討

Abstract

有絲分裂細胞週期的設計原理建築於S phase與M phase相依且不相容的特性上，每一週期裡的S phase與M phase僅能進行一次且必須順序發生。但在減數分裂，DNA複製完成後卻進行了兩次分離，顯然違逆了有絲分裂的規範，而相繼兩個M phase也正是減數分裂最特殊的性狀。研究減數分裂的分子調控機制，可憑著有絲分裂的知識背景做為思考的架構，從中推敲減數分裂可能的特化形式。 APC為M phase之蛋白質分解系統，主宰了各調節因數活性消長的時程，而spindle assembly checkpoint功能在於檢查染色體與紡錘體之問的連繫狀態，能直接抑制APC系統的活性以延宕metaphase-anaphase transition啟動。檢視MI的染色體組態，包括chiasma構造、kinetochore與紡錘體之monopolar attachment 均為減數分裂所特有的，推規spindle assembly checkpoint檢查系統於此必受到修飾，而脊椎動物卵細胞受精前的metall arrest也隱約顯示與spindle assembly checkpoint有著密切的關連。基於上述理由，我選殖了spindle assembly checkpoint重要的成員-Mad2 以及APC活化因數-Cdc20與Cdhl，期盼以spindle assembly checkpoint與APC系統所形成的調控途徑為主軸，從中探索減數分裂M phase之分子運轉機制。 本實驗室以鯉魚為生物系統，從魚卵細胞裡選殖出Mad2、Cdc20與Cdhl基因後，利用細菌表現重組蛋白並製作多株抗體，其中Mad2抗體所進行的鯉魚卵巢組織之免疫偵測顯示，Mad2於第一次減數分裂的前期便已存在卵細胞核裡，但以Cdc20抗體所進行的免疫組織化學並無測得訊號。

The principle of cell cycle of mitosis is based on the characteristics of dependence and inconsistency between S phase and M phase. In each cycle, S phase and M phase can only proceed once and in order. But during the period of meiosis, DNA separates twice after its duplication, which obviously disobeys the rule of mitosis, and two consecutive M phases are exactly the most unique character of meiosis. We can make researches into molecular mechanism of meiosis by contemplating its possible specialized patterns with the background of mitosis. APC is the ubiquitin system of M phase, which is in charge of the sequential activity of each regulatory factor. Spindle assembly checkpoint functions on checking the connection status between chromosome and spindle, and it can inhibit APC system to delay the turn-on of metaphase-anaphase transition. Looking into MⅠchromosome, we know that chiasma structure, monopolar attachment between kinetochore and spindle only exist in meiosis. Hence we may conclude that the spindle assembly checkpoint must be modified at this key point. The meta Ⅱarrest of pre-fertilized vertebrate egg also implicitly shows its close relation with spindle assembly checkpoint. For the reasons above, I cloned Mad2, which is one important member of spindle assembly checkpoint, and Cdc20 and Cdh1, Which are APC activators, to explore the molecular mechanisms of M phase on the base of regulatory system consisted of spindle assembly checkpoint and APC. Carp is the biological experiment system of our laboratory. I cloned Mad2、Cdc20 and Cdh1 genes from carps’ oocytes, and the bacterial expressed recombinant proteins were produced to induce polyclonal antibody, Immunoncytochemistry was performed to detect the distribution of Mad2 protein in carp ovary and showed that Mad2 already exists in the nucleus of prophase I oocytes. However, immunoncytochemistry failed to detect the expression of endogenous Cdc20 protein in carp oocytes.