Singh A K, Kumar G, Shinohara T, Shichi H
Department of Ophthalmology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Exp Eye Res. 1996 Mar;62(3):299-308. doi: 10.1006/exer.1996.0036.
cDNA clones encoding S-antigen (arrestin) were isolated from the expression library constructed from porcine retina and sequenced. The 1490 base pair fragment contained a 1215 base pair open reading frame. From the nucleotide sequence, an amino acid sequence consisting of 405 residues was deduced and a molecular mass of 45,102 daltons was calculated. In order to determine whether the S-antigen mRNA transcript was expressed in anterior eye tissues, mRNA from ciliary non-pigmented epithelial cells and pigmented epithelial cells and iris was analysed by the reverse transcription polymerase chain reaction (PCR) using primers taken from sequences flanking the coding and non-coding regions of retinal S-antigen. Sequence analysis of the expected 611 base pairs in the 5' region and 672 base pairs in the 3' region of DNA fragments indicated that an identical mRNA for S-antigen was expressed in the anterior tissues examined. To investigate the in situ expression of S-antigen mRNA, 35S-labeled sense and antisense RNA probes were synthesized from the cDNA to label frozen sections of retina, ciliary body and iris and the treated sections were examined by autoradiography. The antisense probe labeled the layer between retinal pigmented epithelium and the outer nuclear layer of the retina, ciliary epithelium, and iris epithelium. From the results of sequencing PCR products and in situ hybridization, we concluded that, in porcine eye, the mRNA for S-antigen is expressed not only in the retina but also in the anterior tissues such as the ciliary epithelium and iris epithelium.
从猪视网膜构建的表达文库中分离出编码S抗原（抑制蛋白）的cDNA克隆并进行测序。1490个碱基对的片段包含一个1215个碱基对的开放阅读框。根据核苷酸序列推导了由405个残基组成的氨基酸序列，并计算出分子量为45102道尔顿。为了确定S抗原mRNA转录本是否在前眼部组织中表达，使用从视网膜S抗原编码区和非编码区侧翼序列获取的引物，通过逆转录聚合酶链反应（PCR）分析了睫状体无色素上皮细胞、色素上皮细胞和虹膜的mRNA。对DNA片段5'区域预期的611个碱基对和3'区域672个碱基对的序列分析表明，在所检测的前部组织中表达了相同的S抗原mRNA。为了研究S抗原mRNA的原位表达，从cDNA合成了35S标记的正义和反义RNA探针，用于标记视网膜、睫状体和虹膜的冰冻切片，并通过放射自显影检查处理后的切片。反义探针标记了视网膜色素上皮与视网膜外核层之间的层、睫状体上皮和虹膜上皮。根据PCR产物测序和原位杂交结果，我们得出结论，在猪眼中，S抗原mRNA不仅在视网膜中表达，而且在睫状体上皮和虹膜上皮等前部组织中也表达。
