人類同源箱基因TSX1之功能性探討

Functional Characterization of Human TSX1 Homeobox Gene

張雅津(Ya-Chin Chang)

指導教授 ： 謝秀梅

臺北醫學大學/醫學院/醫學科學研究所/碩士(2002年)

摘要

同源箱基因(homeobox gene)是一群能夠主導動物胚胎發育的基因，在越複雜的生物體中，同源箱基因的種類就越具多元性。這些基因的產物皆為基因調控蛋白，在生物發育過程中扮演著轉錄調節因子(transcription factor)的角色，來調節發育的過程;它們皆包含一段演化上相當保守的同源區(homeodomain)，是一段由60個氨基酸所組成並可以與DNA相結合的功能區。同源區立體結構上形成helix-turn-helix motif功能區，此結構在人類以及原核生物抑制因子 (repressor)上的DNA結合區 (DNA binding domain) 幾乎相同。同源箱基因已被證明在胚胎的發育上扮演極重要的角色，但相較之下，在幼年或成人器官上的研究較少。我們的研究著注於睪丸的發育與功能，至目前為止，文獻上探討睪丸與同源箱基因的關係，只知確實有一些同源箱基因表現在睪丸中，但對於這些基因在睪丸中的功能則是所知有限。我們過去曾利用同源區中高保留氨基酸序列設計退化引子(degenerate primer)，利用PCR的方法，從人類睪丸cDNA library中進行分子選殖，找到一個在睪丸專一表現的人類的同源箱基因-TSX1 (testis-specific homeobox gene)，長度為1244 bp，可轉譯出287個氨基酸的序列，其中包括一個類似paired-type的同源區序列。我們實驗室過去也曾利用反轉錄PCR (RT-PCR)及北方轉漬分析(Northern blot analysis)幾項分析，發現TSX1在睪丸具有高度的專一性。在比對基因庫的基因組序列時，我們發現TSX1位在染色體Xq23.1之位置。基因剔除鼷鼠( knockout mice )是目前探討基因在動物體內功能之最直接方法。但我們幾次篩選鼷鼠基因庫並沒有找到鼷鼠Tsx1同源基因，再經由Zoo blot實驗探討十二個物種的基因後，我們發現TSX1只存在人類基因組中，這樣的結果使TSX1基因剔除鼷鼠無法進行，因此我們另外建立了TSX1轉殖基因鼷鼠以進一步確認TSX1大量表現時，在動物體內可能造成之影響。目前以西方轉漬分析結果發現，在轉殖公鼠睪丸及TG29第二代轉殖母鼠之輸卵管看到有TSX1的表現，此外也在幾個不同的轉殖母鼠中看到一些有趣的表型(phenotype)，目前TSX1基因對轉殖鼷鼠所造成有關生殖方面的影響還無法確定。蛋白質方面的分析，我們已完成TSX1蛋白質體構築，將進一步產生抗體並計畫進行免疫組織化學染色、DNA結合位置確認試驗及免疫沈澱分析，藉由in vivo及in vitro的實驗做進一步分析以了解TSX1在精子成熟過程中可能扮演之角色。

關鍵字

同源箱基因

並列摘要

Several lines of evidence suggest that homeobox genes encode transcription factors to regulate developmental events in philologically diverse organisms. To identify novel homeobox genes involved in testis development, we employed a PCR strategy using homeodomain degenerate primers to screen human testis cDNA libraries. We have isolated a human testis specific homeobox gene, TSX1 (testis-specific homeobox gene). In situ hybridization analysis revealed that TSX1 gene is expressed in spermatocytes and spermatids during spermatogenesis. Analysis of the phenotypes of TSX1 knockout mice is a straight-forward approach to understand the in vivo function of a gene. We have not been able to isolate mouse Tsx1 ortholog after several times screening of mouse testis cDNA libraries with human TSX1 cDNA as probes. We employed Southern blot analysis by using a 12-species-DNA Zoo blot membrane, and found TSX1 is only present in human genome. Since there is no mouse TSX1 gene for making knockout construct, we therefore generated transgenic mice to further study the effect of TSX1 overexpression in the animal. TSX1 expressions were identified in testis, oviduct and uterus in some of the transgenic lines by Western analysis. We have also found some interesting phenotypes in some of the transgenic mice. Whether these phenotypes are correlated to the TSX1 expression or phenotypes related to reproduction are induced by TSX1 overexpression will need further analysis. In the study of protein level, we constructed expression plasmids to generate TSX1-fusion protein from the bacteria. TSX1-fusion protein will be used to raise antibody, identify the target-binding site and immunoprecipitate the TSX1-interacting proteins. Results from both in vitro and in vivo studies will further imply the function of TSX1 during spermatogenesis.

並列關鍵字

homeobox gene

參考文獻

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