小鼠睪丸中新同源箱基因 Tex1 之分子特性探討
Molecular Characterization of A Novel Murine Testis Expressed Homeobox Gene, Tex1
中文摘要
同源箱基因在生物的發育過中扮演著轉錄調節因子的角色，並且在演化過程中具
有功能的保留性，此蛋白質的作用方式為藉由其同源箱區域和DNA 結合以達到
調控下游基因的表現，我們曾經利用degenerate oligonucleotides PCR 從小
鼠睪丸的cDNA library 中找到了這個基因，在本篇論文中經由對此基因的探
討，將其命名為Tex1 (testis expressed homeobox)，從 Tex1 的同源箱區
域發現在螺旋1 和螺旋 2 之間比大多數的同源箱基因多出 3 個氨基酸 AYP，再
經由與資料庫中基因比對後得知Tex1 其可歸屬於 TGIF (5＇TG3＇
interacting factor) 同源箱蛋白質的成員之一。本實驗經由北方墨染法和 RT-PCR 分析，證實了此基因在睪丸有高專一性的表現，且 Tex1 約是從小鼠出
生後的第21 天左右開始在睪丸中表現，利用原位雜交分析的結果發現 Tex1 只
專一性的表現在精細胞 (spermatid)，體細胞並沒有表現，基於以上的結果，
推論此基因可能在精子生成過程的型態變化階段中，扮演著一重要的角色。另一
方面，為了更進一步探討此基因在活體內的功能，目前正在篩選ES cell
clones，想了解 Tex1 基因功能的喪失，會對小鼠造成何種影響。此外，利用純 化的GST-Tex1 融合蛋白質進行 DNA 結合序列篩選的實驗，發現到 Tex1 會和 N (A/T) TG (A/T)6 的序列結合。
英文摘要
The homeobox gene superfamily is highly conserved during evolution. These genes act as transcription factors during several important developmental processes through their 60 amino acid homeodomains that recognize and bind to the regulatory region of their target genes. We isolated a novel homeobox gene from a mouse testis cDNA library by degenerate oligonucleotide polymerase chain reaction (PCR) screening.
Based on our studies, we designated the gene, Tex1 (testis expressed homeobox) . Three extra amino acid residues, AYP, are inserted between the first and second helices of the homeodomain, which makes Tex1 a TGIF subclass homeobox gene.
Tex1 was expressed specifically in the testis as seen by both Northern blot and
RT-PCR analysis. Tex1 expression is initiated at around day 20 of testes. Furthermore, the results of in situ hybridization analyses reveal that the expression of Tex1 in testis is restricted in the germ cells at spermatid stage. Based on these data, it is suggested that Tex1, a novel testis homeobox gene, may play a crucial role during
spermatogenesis. To further realize the in vivo biological function of this gene, we are
processing in knockout Tex1 through the ES homologous recombination system. To identify the Tex1 target genes, we have purified GST-Tex1 fusion protein and used it to find out the potential DNA-binding sequences of Tex1. A putative
consensus-binding site, N (A/T) TG (A/T)6, was identified. The phenotypes of the knockout mice and the downstream target gene identification will definitely let us more understand the function and molecular mechanism of Tex1 gene during spermatogenesis.
