Abstract
麩胺酸受器(glutamate receptor)是中樞神經系統中一種傳遞興奮性訊號的蛋白質。就藥理學及電生理研究而言，哺乳動物腦中所發現之離子通道型麩胺酸受器可分為AMPA、KA及NMDA- preferring受器等三大類。至今由吳郭魚中分析出八種cDNA分子，它們所轉譯出之蛋白質的胺基酸序列以兩個為一組，分別與大白鼠的四種AMPA preferring receptor subunits有很高的相似性。為研究各脊椎動物麩胺酸受器AMPA次型的種類及基因數目，我們選擇在大白鼠及吳郭魚的G1uR1~G1uR4胺基酸序列中保留性最高的區域設計degenerate primers，以RT-PCR大量複製(amplify)鳥類、爬蟲類、兩棲類及軟骨魚類的AMPA receptor like subunits的cDNA。由實驗結果我們得知，除了硬骨魚中真骨魚類(吳郭魚、河豚)可以發現多於四種與哺乳動物GluR1~GluR4相似之cDNA分子外，在其它物種均只尋得四種cDNA分子。另外我們在大鼠NMDAR1 cDNA相對應於 transmembranedomain region設計一組primers去大量複製魚腦中之NMDAR1 cDNA，再以此PCR產物為探針去篩選吳郭魚魚腦之cDNA library，只得到一種胺基酸序列與大鼠NR1胺基酸序列有高度相同性的fNR1 cDNA。而吳郭魚魚腦中NMDAR2受器之研究，採用與AMPA receptor like subunits的分析方式相同的方法，設計degenerate primers去找尋魚腦中的NMDAR2 cDNA，目前已找到7種胺基酸序列與大鼠之NMDAR2A~NMDAR2D胺基酸序列有很高相同性之吳郭魚NMDAR2 cDNA。這些結果暗示硬骨魚的 AMPA receptor likesubunits與NMDAR2 subunits可能在演化過程中發生了gene duplication的現象。Glutamate receptors constitute the excitatory neurotransmittersystem in the central nervous system (CNS). Based on thepharmacological and electrophysiological data, the mammalianionotropic receptors are classified into three subtypes:{SYMBOL 97 \f "Symbol"}-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate (KA), and N-methyl-D- aspartate(NMDA)-preferring receptors. Eight cDNA clones encodingproteins with high amino acid sequence identities to the fourmammalian AMPA-preferring receptor subunits were identifiedfrom Oreochromis sp. To study the numbers of vertebrateionotropic glutamate receptor genes, degenerated primersderived from the most conserved regions of glutamate receptorswere designed and used to amplify avian, reptilian, amphibian,and chondrichthyes glutamate receptor cDNAs by reversetranscription-PCR (RT-PCR). With the exception of bony fish(Oreochromis sp. and Tetraodon fluviatilis), there were onlyfour types of cDNA molecules encoding AMPA receptor likesubunits found in the brains of representative vertebralanimals. PCR primers were designed according to the conservedamino acid sequence of the rat NMDAR1 transmembrane region,then were used to amplify fish brain cDNA encoding the NMDAreceptor subunit. The fNR1 cDNA encoding protein with highamino acid sequence identity to rat NMDAR1 was identified byscreening Oreochromis sp. brain cDNA library with the RT-PCRproduct as a probe. RT-PCR with designed degenerated primerswas also performed to study the transcripts of NMDAR2-homologin the brain of Oreochromis sp. A total of 7 different cDNAmolecules homologous to the rat NMDAR2 subfamily wasidnetified. These results imply that gene duplication of AMPAreceptor like subunits and NMDAR2 subunits occurred during theevolution in bony fish.