蝾螈红色和蓝色视锥视觉色素的分子克隆。

Xu L, Hazard E S, Lockman D K, Crouch R K, Ma J

Department of Ophthalmology, Medical University of South Carolina, Charleston, SC 29425, USA.

Mol Vis. 1998 Jul 15;4:10.

PURPOSE

Salamander retinas are known to contain at least three cone pigments and two rod pigments. The purpose of this study was to clone and characterize the visual pigments from salamander cones.

METHODS

cDNA fragments of cone pigments were amplified from a salamander retina cDNA library by PCR using a pair of primers with consensus for visual pigments. These fragments were cloned and used as probes for library-screening. The full-length cDNAs were isolated from the retinal library using the cloned PCR products as probes. DNA sequences were determined by the dideoxynucleotide chain termination method.

RESULTS

Two pigment cDNAs were cloned and sequenced from the salamander library. The global GenBank search showed that they do not match any existing sequences but have significant sequence similarity to visual pigments. One of the pigment cDNAs showed a high sequence homology with red cone pigments from other species and thus, was designated as a red cone opsin. The other pigment was designated as a blue cone opsin as it is most homologous to the chicken and goldfish blue cone pigments. Both cDNAs contain a full-length coding region encoding 365 amino acids in the red and 363 amino acids in the blue cone pigment. Hydropathy analysis predicted that both pigments could form seven hydrophobic transmembrane helices. Both pigments retain the key amino acid residues critical for maintaining the structure and function of opsins and have similar G-protein interaction sequences which differ from that of rod opsin. Phylogenetic analysis indicates that the red opsin belongs to the L group and the blue opsin belongs to the M1 group of visual pigments.

CONCLUSIONS

The salamander red and blue cone pigments share high sequence homology with the cone pigments of other species.

目的

已知蝾螈视网膜含有至少三种视锥色素和两种视杆色素。本研究的目的是克隆并鉴定蝾螈视锥细胞中的视觉色素。

方法

使用一对对视觉色素具有共识的引物，通过PCR从蝾螈视网膜cDNA文库中扩增视锥色素的cDNA片段。将这些片段克隆并用作文库筛选的探针。使用克隆的PCR产物作为探针从视网膜文库中分离全长cDNA。通过双脱氧核苷酸链终止法测定DNA序列。

结果

从蝾螈文库中克隆并测序了两个色素cDNA。全球GenBank搜索表明，它们与任何现有序列均不匹配，但与视觉色素具有显著的序列相似性。其中一个色素cDNA与其他物种的红色视锥色素具有高度的序列同源性，因此被指定为红色视锥视蛋白。另一种色素被指定为蓝色视锥视蛋白，因为它与鸡和金鱼的蓝色视锥色素最同源。两个cDNA都包含一个全长编码区，红色视锥色素编码365个氨基酸，蓝色视锥色素编码363个氨基酸。亲水性分析预测，这两种色素都可以形成七个疏水跨膜螺旋。两种色素都保留了对视蛋白结构和功能至关重要的关键氨基酸残基，并且具有与视杆视蛋白不同的相似G蛋白相互作用序列。系统发育分析表明，红色视蛋白属于视觉色素的L组，蓝色视蛋白属于M1组。