鯉魚卵細胞中SKP1基因的選殖及表現

Abstract

細胞週期控制機制的基本架構建立了S期及M期交替出現的順序關係。環環相扣的CDK系統與ubiquitin蛋白質分解系統所構成的迴路形成一個以事件的發生為里程碑的細胞週期進程。一般體細胞的細胞週期進程為G1→S→G2→M，然而在進行減數分裂的細胞中則為G1→S→G2→MI→MII。減數分裂中的MI-MII明顯違背了有絲分裂中S-M交替的基本原則，因此減數分裂細胞中應存在一調控系統以修飾細胞週期的調控迴路。瞭解細胞週期迴路系統在減數分裂的狀態是瞭解減數分裂細胞週期本質的先決條件，而分析這個迴路中的元件則是重要的第一步。控制S期及M期蛋白質分解的ubiquitin蛋白質分解系統分別是SCF系統及APC系統。Skp1及Fzr分別是兩大系統中的重要元件。為調查減數分裂中ubiquitin蛋白質分解系統的狀態，本研究選殖了skp1 cDNA的全長及fzr cDNA的片段。將鯉魚skp1 cDNA的DNA序列翻譯成胺基酸序列後與其他生物中的skp1同源基因胺基酸序列比對，發現鯉魚Skp1與人類及豚鼠的Skp1胺基酸序列幾乎完全相同。以原位雜合反應偵測c-mos、fzr及skp1等細胞週期調控基因mRNA於卵巢中的表現狀態，發現c-mos及fzr的訊號均勻分佈於卵黃未堆積前的小卵細胞質中，而skp1的訊號則僅分佈於小卵外圍。以細菌表現及純化Skp1重組蛋白並以之製作抗血清，並以Skp1的抗血清進行免疫組織化學法以偵測卵巢中Skp1蛋白的表現，結果並無法測得任何預期訊號。

Cyclin-dependent kinases (CDKs) and the ubiquitin-dependent proteolysis systems are the principle regulators in the cell cycle. Tight connected cell cycle regulatory mechanisms order the mitotic cell cycle in the succession of G1 → S → G2 → M. However, the cell cycle progression in meiosis is shown to be G1 → S → G2 → MI → MII. The omitting of the S phase between the MI and MII apparently violates the rule set by the cell cycle regulatory mechanisms. During meiosis, a modifying machinery must functions to alter the cell cycle regulatory mechanisms. The ubiquitin-dependent proteolysis systems are the possible targets of the modification mechinery. There are two ubiquitin-dependent proteolysis systems involved in the cell cycle control. The SCF system majorly governs the onset of the S phase and the APC system directs the progression of the M phase. Skp1 protein is an essential part of the SCF complex. Fzr seems to control the substrate specificity in the APC system. To investigate the status of the ubiquitin-dependent proteolysis system in the meiotic cell cycle, the cDNA of SKP1 gene from the carp ovary was cloned and the partial DNA sequence of fzr gene with the same origin was also cloned. The deduced amino sequence of carp SKP1 gene shows extremely high homology to its counterparts in other vertebrates. In situ hybridization was performed to detect the distribution of c-mos, fzr and SKP-1 mRNAs in the carp ovary. It was found that c-mos and fzr mRNA disperses in the cytoplasm of the smaller oocytes before the stage of vitellogenesis while skp1 mRNA distributes peripherally. It is notable that I was never able to detect any signal within oocytes with thick yolk. A bacterial expressed recombinant Skp1 protein was produced to induce antiserum against Skp1 protein. However, Immunohistochemistry and immunoblot failed to detect the expression of endogenous Skp1 protein in fish oocytes.