Qian X, Malchow R P, O'Brien J, al-Ubaidi M R
Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, USA.
Mol Vis. 1998 Mar 6;4:6.
The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is believed to play a crucial role in the processing of information within the vertebrate retina. Extracellular concentrations of GABA are thought to be tightly regulated by carrier-mediated transport proteins in neurons and glial cells. The purpose of this work was to isolate the gene that encodes one of these transport proteins in the skate retina.
cDNA clones were isolated from a skate retinal cDNA library using a mouse retinal GABA transporter (GAT1) cDNA as a probe. The PCR technique was used to fill sequence gaps, and 5' and 3' RACE were employed to amplify the 5' and 3' untranslated regions. The amplified fragments were subcloned into a T-vector. Blots containing RNA from 10 different tissues were probed to determine the size of the transcript and the tissue distribution.
Sequence analysis revealed that the skate retinal GABA transporter cDNA shared 72% identity with the mouse GABA transporter-1 at the DNA level and 80% identity at the amino acid level. Multiple sequence alignments showed that our sequence is closest to the Torpedo GABA transporter-1. Two transcripts, 4.5 and 7 kb, were detected in retina and possibly brain by RNA blot analysis. Fourteen introns were detected in the skate GABA transporter gene.
We successfully isolated a full length GABA transporter cDNA from the retina of the skate. The size of the full length sequence of the skate retinal GABA transporter is in agreement with the size of the smaller transcript detected on RNA blots. The larger transcript observed on the RNA blot may be the result of either alternative splicing or utilization of a downstream poly A signal.
抑制性神经递质γ-氨基丁酸（GABA）被认为在脊椎动物视网膜的信息处理过程中起关键作用。GABA的细胞外浓度被认为受到神经元和神经胶质细胞中载体介导的转运蛋白的严格调控。这项工作的目的是分离编码鳐鱼视网膜中这些转运蛋白之一的基因。
使用小鼠视网膜GABA转运体（GAT1）cDNA作为探针，从鳐鱼视网膜cDNA文库中分离cDNA克隆。采用PCR技术填补序列缺口，并利用5'和3' RACE扩增5'和3'非翻译区。将扩增片段亚克隆到T载体中。用来自10种不同组织的RNA印迹进行检测，以确定转录本的大小和组织分布。
序列分析显示，鳐鱼视网膜GABA转运体cDNA在DNA水平上与小鼠GABA转运体-1有72%的同一性，在氨基酸水平上有80%的同一性。多序列比对表明，我们的序列与电鳐GABA转运体-1最接近。通过RNA印迹分析在视网膜以及可能在脑中检测到两种转录本，大小分别为4.5 kb和7 kb。在鳐鱼GABA转运体基因中检测到14个内含子。
我们成功地从鳐鱼视网膜中分离出全长GABA转运体cDNA。鳐鱼视网膜GABA转运体全长序列的大小与RNA印迹上检测到的较小转录本的大小一致。RNA印迹上观察到的较大转录本可能是可变剪接或利用下游多聚腺苷酸信号的结果。
