睪丸鋅指蛋白基因TZF-376之分子特性探討

目前約有六分之一的夫婦可能面臨不孕症的困擾，而其中有一半問題是出自於男性。主要造成男性不孕的原因，又以在精子熟成（spermatogenesis）過程中發生障礙所造成的原因為最主要。已知有許多鋅指蛋白（zinc finger proteins）會在精子的發育過程表現，可能影響精子發育及其功能。鋅指蛋白是一種很常見的 DNA結合蛋白，一般是以扮演轉錄因子（transcription factor）的角色，調節許多其它基因之表現。為了尋找精子熟成過程中新的鋅指蛋白，我們利用網路上的基因庫進行人類表現序列標示（expressing sequence tags）（簡稱EST）clones的搜尋，輸入關鍵字testis及zinc finger，找到一些表現在睪丸的鋅指蛋白類EST clones，並針對這些EST clones之序列來設計PCR引子，起初進行分析的EST clones有AI376558、AI003931、AI014681、 AA417107四個，進行各個組織（例如：腦、肝、腎等組織）的反轉錄PCR（RT-PCR）實驗，以便瞭解此基因在各組織的表現，並製備riboprobes。結果發現AI376558在許多組織皆有表現，但是以睪丸表現量最高，此外在睪丸有兩個不同之PCR產物，我們猜測其有alternative splicing的情形，因此我們目前較專注於AI376558之進一步分析，並將其命名為TZF376。由定序結果發現這是一個具有四個C2H2鋅指區的鋅指蛋白，基因位在人類第一號染色體上。另外我們亦定序幾個相關的EST clones，結果顯示這個鋅指蛋白之alternative splicing相當地複雜，至少有四種不同的alternative splicing，其中有一個clone BE551217缺少一個20 bp之核酸片段，結果造成三個鋅指區之缺損，這個蛋白質有可能對具鋅指區之蛋白產生負調節作用。此外，我們根據第一號染色體上之基因組序列設計三組橫跨 TZF376 exons之primers，以檢測180例不孕症之男性是否有 TZF376基因之缺損情形。而更進一步原位雜交分析將有助於瞭解這個鋅指蛋白在睪丸中精細胞（germ cells）及體細胞（Sertoli and Leydig cells）的表現情形，如此可對基因之功能作一初步預測。

關鍵字

鋅指蛋白；表現序列標示；原位雜交法

並列摘要

About one-sixth of the couples have the problem with infertility nowadays, and half of the problems come from male. Most of male infertility results from the impairment of spermatogenesis. We searched EST Genebank and identified several testis zinc finger protein genes. We would like to know whether these genes could influence the development and function of testis. Zinc finger is an extremely common protein domain of DNA binding proteins, and zinc finger proteins usually regulate other gene expression via their roles as transcription factors. The sequences of these zinc finger EST clones was used to design the PCR primers. RT-PCR analysis indicated that one of the EST clones, AI376558 might have some alternative splicing processing. We now focus on this EST clone and name this gene TZF376. Sequence analysis of several related EST clones showed that the alternative splicing of TZF376 is complicated. We have found at least 5 different alternative spliced EST clones, BE551217, with a 20 nt deletion encodes a truncated protein without zinc finger domain. In situ hybridization analysis was performed to characterize the expression pattern of TZF376. The localization in germ cells, Sertoli or Leydig cells of testis will imply the function of the genes. Intensive PCR screening of the TZF376 coding region on human chromosome 1 were performed to see if any deletion occur in cellular DNA of 180 infertile patients.These results will imply the correlation between TZF376 gene infertility.

並列關鍵字

testis zinc finger ； EST ； in situ hybridization

參考文獻

Aapola, U., Kawasaki, K., Scott, H. S., Ollila, J., Vihinen, M., Heino, M., Shintani, A., Minoshima, S., Krohn, K., Antonarakis, S. E., Shimizu, N., Kudoh, J., and Peterson, P. (2000). Isolation and initial characterization of a novel zinc finger gene, DNMT3L, on 21q22.3, related to the cytosine-5-methyltransferase 3 gene family. Genomics 65, 293-8.

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Burke, P. S., and Wolgemuth, D. J. (1992). Zfp-37, a new murine zinc finger encoding gene, is expressed in a developmentally regulated pattern in the male germ line. Nucleic Acids Res. 20, 2827-34

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睪丸鋅指蛋白基因TZF-376之分子特性探討

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