Abstract
麩胺酸(glutamate)在哺乳類動物的中樞神經系統中是一種主要的興奮性傳導物質。研究指出,麩胺酸受器(glutamate receptor)與腦部的學習、記憶功能有關。實驗室已從吳郭魚魚腦的 cDNA library 選殖出多種glutamate receptor subunits 的 cDNA。由推衍出的胺基酸序列和大鼠的比較下,以 fGluR2α、fGluR2β 和大鼠的相似性最高約 85% 。為了建立 fGluR2β 的基因結構, 分別利用 fGluR2β 的 cDNA 片段為探針, 進行 genomic library 的篩選,或是利用 chromosome walking 的方式,得到帶有 genomic DNA 的重組噬菌體。並且將 fGluR2β cDNA 利用限制酵素截切成適當片段當成探針,分別與不同限制酵素切過的重組噬菌體 DNA進行 Southern blot 的分析,顯示出 exon 所在的 DNA 片段。 並且藉由比對 cDNA 與 genomic DNA 的序列,決定出exon-intron junctions。同時亦進行5'-RACE(rapid amplification of cDNA ends)及 3'-RACE 以便得到完整長度的 fGluR2β cDNA。未完成的 fGluR2β 基因長度已大於55 kb,至少含有17個 exons, exon-intron junctions 符合標準splice donor and acceptor consensus sequences。與小鼠 GluR2 基因相較,fGluR2β與 GluR2 基因二者在 5'- 端皆有一個非常大的 intron存在於同源位置。在3'-端, fGluR2β 與 GluR2 基因皆可藉由3'-alternative splicing,產生 C-terminal variants。在小鼠 GluR2基因的 5'-UTR 是位在一個 exon 上,但是 fGluR2β 5'-UTR 所在的exons,至少有一個 intron 的存在。Glutamate plays an important role in the excitatorytransmission system in the mammalian brain. Glutamatereceptors may participate in basic functions of the brain suchas learning and memory. Two Oreochromis cDNA molecules, fGluR2α and fGluR2β, containing open reading frames that share 85%amino acid sequence identities to the rat glutamate receptorsubunit, Glur2. To characterize the gene structure of thefGluR2β gene, various restriction endonuclease-digestedfragments of the fGluR2β cDNA were used as probes to identifygenomic DNA fragments carrying exons of fGluR2β. The exon-intron junctions were determined by comparing cDNA and genomicDNA sequences. To obtain a full length fGluR2β cDNA, 5'-RACE(rapid amplification of cDNA ends) and 3'- RACE were employed.The incomplete fGluR2βgene spans more than 55 kb long andcontains at least 17 exons with splice junction donors andacceptors conforming to the consensus sequences. Theextraordinarily large size of the mouse intron 2 is conservedin the fGluR2β gene. 3'-alternative splicing is found in theC-terminal coding region of the fGluR2βgene transcripts. Thegene structures of the fGluR2β and mouse GluR2 are differentin that the fGluR2β gene contains at least one extra intronpresent in the region corresponding to the 5'-UTR of the fGluR2β.