小鼠睪丸之同源箱基因Tse1之分子選殖與分析

Molecular Cloning and Analysis of a Testis — Specific Expression Homeobox Gene , Tse1

孫璧玉(Pi-Yu Sun)

指導教授 ： 謝秀梅 博士

臺北醫學大學/醫學院/醫學科學研究所/碩士(2001年)

摘要

中 文 摘 要 同源箱基因群（homeobox gene superfamily）在生物的發育過程中，扮演著重要的調控角色。同源區蛋白中的同源區 ( homeodomain ) 包含60個高度保留性的氨基酸。同源區蛋白即藉由此同源區去辨認下游的目標基因的調控區，並與之結合，以調節該基因的表現。近年來發現某些同源箱基因在動物出生後，對器官的成熟與在胚胎發育過程中亦具有調控的作用。本實驗即針對此方面的基因作研究，利用已知的同源區蛋白最保守的序列，設計退化性引子(degenerate primer) ，以PCR方式對小鼠testis cDNA基因庫進行新同源箱基因的分子選殖，再將此方式所得的新同源箱基因片段作為探針，去篩選小鼠的testis cDNA library中更長片段的cDNA clone，結果我們得到一段長度為839 bp的新同源箱基因Tse1片段。由小鼠各不同器官之總RNA作多組織北方墨點法 (northern blot) 分析，結果顯示Tse1的RNA主要表現在睪丸，全長約為1.2Kb。進一步再取各時期小鼠睪丸的總RNA作北方墨點法，由結果得知Tse1在20天的睪丸表現量最高。由另一個小鼠testis cDNA library之篩選，得到Tse1的full length cDNA總長為1117 bp。經由原位雜交 (in situ hybridization) 實驗顯示Tse1表現在小鼠睪丸的精細胞產生過程以及萊氏細胞 (Leydig cells)，由此推測Tse1可能參與精子成熟的過程並可能參與雄性賀爾蒙的分泌調節。在Genomic DNA 方面由Genomic DNA library 篩選及南方墨點法(southern blot)得到兩個包含Tse1 cDNA片段的Genomic DNA 菌株， 經過核酸定序分析並配合由美國國家衛生院所建立的NCBI網站搜尋genomic 的資訊結果，目前已得到部分的3’端延伸片段，未來將進一步釐清Tse1 Genomic DNA的詳細序列，以便進行Tse1基因剔除鼠的建立。日後可藉由基因剔除鼠，以了解Tse1之基因功能並深入探討此基因與賀爾蒙之間的關係。

關鍵字

同源箱基因

並列摘要

Abstract The homeobox genes play a central role in animal development. These genes encode homeodomain proteins that act as transcription factors which regulate expression of gene cascades. The homeodomain of these homeobox genes, consisting of a conserved 60 amino-acid region, recognizes and binds to regulatory sites of their target genes, and then initiates gene cascades and cell type specificities. Tse1 is a novel mouse homeobox gene identified by degenerated-oligonucleotide PCR screening from a mouse testis cDNA library. A 839 bp cDNA fragment of Tse1 was used as a probe to screen testis cDNA library and obtained a 1117 bp full—length cDNA clone which encoded 293 a.a. with a Prd-like homeodomain. Analysis of tissue and stage expression patterns of Tse1 gene by Northern hybridization, we found that Tse1 transcript was about 1.2kb and mainly expressed in testis with the highest level at postnatal day 20. Tse1 transcript was also detected in c-kit mutant (w/ wv and wv/ wv) testes in which germ cells were depleted. These results suggest that Tse1 gene is possible expressed in somatic cells of testes. In situ hybridization further confirmed that Tse1 expressions were in spermatids and leydig cells and implied that Tse1 may play a role during spermatogenesis and may be involved in hormonal reguation during the puberty. Gene structure of Tse1 was identified by sequence comparison between cDNA and genomic DNA. Information obtained from these genomic DNA sequences will be used to construct the Tse1 knock-out construct and establish the Tse1 knock-out mice to elucidat the in vivo function of Tse1 in spermatogenesis.

並列關鍵字

Homeobox Gene

參考文獻

Billeter, M. (1996). Homeodomain-typeDNA recognition Prog. Biophys. Mol. Biol. 66: 211-225.

Boettger, T. and Kessel, T. (1997). The chick Nkx2-8gene A novel member of the NK-2 family. Dev. Genes Evol 207: 65-72.

Branford, W., Zhao, G., Valerius, M., Weinstein, M., Birkenmeier, E., Rowe, L., Potter, S. (1997). Spx, anovel X-l inked homeobox gene expressed during spermatogenesis. Mech-Dev. 65: 87-98.

Burglin, T. R., Finney, M., Coulson, A. andRuvkun, G. (1989). Caenorhabditis elegans has scores of homeobox- containing genes. Nature (London). 341: 239-243.

Capel, B., Albrecht, K. H., Washburn, L L, Eicher, E. M. (1999). Migration of mesonephric cells into the mammalian gonad depends on Sry. Mechanisms of Development. 84:127-131.