豬卵母細胞特異性連接組蛋白基因表現調控及其功能之探討

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Title:	豬卵母細胞特異性連接組蛋白基因表現調控及其功能之探討
Other Titles:	Regulation of Gene Expression and Functional Analysis of Oocyte Specific H1 Linker Histone in Porcine Embryo
Authors:	鄭登貴,歐柏榮
Contributors:	行政院國家科學委員會東海大學畜產與生物科技學系
Date:	2010
Issue Date:	2012-04-27T03:05:05Z (UTC)
Abstract:	組蛋白(histone)為真核生物體細胞染色質中的基本結構蛋白質，依其所含鹼性胺基酸相對比例可區分為5 種類型組蛋白(H1、H2A、H2B、H3 和H4)，其中組蛋白H1 又稱連接組蛋白(linker histone)，在哺乳動物細胞中存在11 種亞單元，於發育不同時期能組成不同的結合模式，並在特定階段相互更換。卵母細胞特異性連接組蛋白(oocyte-specific linker histone H1, H1Foo)首先於小鼠中發現，已知職司細胞核內染色質結構之維持，並可能涉及卵母細胞的成熟、受精後精子染色質重構(chromatin remodeling)、胚原性與母源性基因轉換調控，與體細胞核移植時對體細胞核的再程序化(reprogramming)；惟豬卵母細胞之該基因全長核?酸序列、蛋白質分子特性，及其可能涉及之生理作用機制，則迄未有明。鑑於豬隻繁殖過程中，豬胚存活率決定豬隻繁殖力，且早期胚存活之關鍵為4-至8-細胞胚期階段，即母源性基因停止表現，胚原性基因開始轉錄或轉譯之際，在此過程中，除特定轉錄因子的數量、活性外，染色質結構成分的變化也扮演關鍵性的角色。本研究室已針對源自生發泡(germinal vesicle, GV)階段之豬卵母細胞之總RNA 樣品，透過RNA 連接?仲介(RNA ligase–mediate, RLM)策略，成功獲得兩種不同長度 (1170 bp 及1215 bp)之豬H1Foo(pH1Foo)完整cDNA，經基因結構分析與定序結果，證明該二不同長度之pH1Foo cDNA 產物，乃基因轉錄後不同選擇性剪接(alternative splicing)所使然；前述事實暗示，pH1Foo 將因此產生不同之開放讀架(open reading frame)，其結果是否涉及調控豬胚早期發育潛能，誠待進一步詳加釐清之。職是之故，本三年期研究計畫，擬延續前述之試驗結果，先行構築重組蛋白表現載體，並產製多株抗體後，進而探討不同選擇性剪接之pH1Foo 於豬胚早期發育各不同階段之表現情形，並透過5’調節區報導基因的分析及染色質免疫沈澱法(chromatin immunoprecitation, ChIP)的研究探討pH1Foo 基因表現之調控，最後利用siRNA 或專一性抗體基因抑制策略，及大量表現H1foo 蛋白質的方法探討H1Foo 對於早期胚發育時所扮演的角色，最後冀能建立發育相關基因的系統研究體系，為深入探討早期胚發育關鍵因子之調節機制奠定基礎。 Eukaryotic histone proteins are mainly responsible for the nucleosome structure of the chromosomal fiber through package and order the DNA into nucleosome to provide the first level of chromatin organization. Among them, core histones (histones H2A, H2B, H3 and H4) play an essential role in constraining DNA wrapped around the histone octamer to produce a nucleosome core particle, while linker histones (histone H1 family) locate the exterior of the nucleosome core and associate with linker DNA to keep the chromatin in a helical and compact state to regulate gene expression. The oocyte-specific linker histone H1 (H1Foo) is first found in the mouse oocyte and participates in various steps of embryogenesis including oocyte maturation, sperm chromatin remodeling after fertilization and donor cell reprogramming after somatic nuclear transfer. The sequence of full-length H1Foo cDNA has been identified in mice, human, bovine and other non-mammalian species, but not in porcine. In our previously study, we had identified an alternative splicing form of the pH1Foo gene exists in porcine GV-stage oocyte. The DNA sequence analysis showed that these two mRNAs (1170- and 1215-bp, respectively) were made from the alternative splicing of exon 4 of pH1Foo gene. Toward a better understanding of chromatin remodeling in pig oocyte, identification and characterization of the porcine specific H1Foo gene would be an important prerequisite for explication of the maternal control of germline development following fertilization. In this 3-year of research project, polyclonal antibodies against pH1Foo will be firstly generated. Further studies will be focused on identification and characterization of pH1Foo differentially expressed during early embryo development. Promoter deletion analysis and chromatin immunoprecipitation technique will be used to study the mechanism of H1Foo gne expression. Final attempts will be addressed on the siRNA-based and overexpression of H1Foo protein techniques to study the role of pH1Foo protein in early embryo development. In addition, DNA microarray will be used to investigate the effect of H1Foo protein on expression of other genes. The understanding of molecular mechanisms of pH1Foo governing the development of the oocyte and early embryo will help us to better understand the regulation of the timing and extent of switching between somatic H1s and H1Foo.
Relation:	研究編號：NSC99-2628-B029-001-MY3 研究期間：2010-08~ 2011-07
Appears in Collections:	[畜產與生物科技學系所] 國科會研究報告

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