Li Chibo, Ding Xi-Qin, O'Brien John, Al-Ubaidi Muayyad R, Naash Muna I
Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, College of Medicine, Chicago, Illinois, USA.
Invest Ophthalmol Vis Sci. 2003 Jun;44(6):2433-41. doi: 10.1167/iovs.02-1152.
A great deal of information about functionally significant domains of a protein may be obtained by comparison of primary sequences of gene homologues over a broad phylogenetic base. This study was designed to identify evolutionarily conserved domains of the photoreceptor disc membrane protein peripherin/rds by analysis of the homologue in a primitive vertebrate, the skate.
A skate retinal cDNA library was screened using a mouse peripherin/rds clone. The 5' and 3' untranslated regions of the skate peripherin/rds (srds) cDNA were isolated by the rapid amplification of cDNA ends (RACE) approach. The gene structure was characterized by PCR amplification and sequencing of genomic fragments. Northern and Western blot analyses were used to identify srds transcript and protein, respectively.
A new homologue of peripherin/rds was identified from the skate retinal cDNA library. SRDS is a glycoprotein with a predicted molecular mass of 40.2 kDa. The srds gene consists of two exons and one small intron and transcribes into a single 6-kb message. Phylogenetic analysis places SRDS at the base of peripherin/rds family and near the division of that group and the branch leading to rds-like and rom-1 genes. SRDS protein is 54.5% identical with peripherin/rds across species. Identity is significantly higher (73%) in the intradiscal domains. Sequence comparison revealed the conservation of all residues that have been shown, on mutation, to associate with retinitis pigmentosa and showed conservation of most residues associated with macular dystrophies. Comparison with ROM-1 and other rds-like proteins revealed the presence of a highly conserved domain in the large intradiscal loop.
Srds represents the skate orthologue of mammalian peripherin/rds genes. Conservation of most of the residues associated with human retinal diseases indicates that these residues serve important functional roles. The high degree of conservation of a short stretch within the large intradiscal loop also suggests an important function for this domain.
通过在广泛的系统发育基础上比较基因同源物的一级序列，可以获得大量有关蛋白质功能重要结构域的信息。本研究旨在通过分析原始脊椎动物鳐鱼中的同源物，鉴定光感受器盘膜蛋白外周蛋白/视网膜变性慢（peripherin/rds）的进化保守结构域。
使用小鼠外周蛋白/视网膜变性慢克隆筛选鳐鱼视网膜cDNA文库。通过cDNA末端快速扩增（RACE）方法分离鳐鱼外周蛋白/视网膜变性慢（srds）cDNA的5'和3'非翻译区。通过PCR扩增和基因组片段测序对基因结构进行表征。分别使用Northern和Western印迹分析来鉴定srds转录本和蛋白质。
从鳐鱼视网膜cDNA文库中鉴定出一种新的外周蛋白/视网膜变性慢同源物。SRDS是一种糖蛋白，预测分子量为40.2 kDa。srds基因由两个外显子和一个小内含子组成，并转录成一条单一的6 kb信息。系统发育分析将SRDS置于外周蛋白/视网膜变性慢家族的基部，靠近该组与导致rds样和rom-1基因的分支的划分处。SRDS蛋白在不同物种间与外周蛋白/视网膜变性慢的同一性为54.5%。在盘内结构域中的同一性显著更高（73%）。序列比较显示，所有已证明在突变时与色素性视网膜炎相关的残基都具有保守性，并且大多数与黄斑营养不良相关的残基也具有保守性。与ROM-1和其他rds样蛋白的比较显示，在大的盘内环中有一个高度保守的结构域。
Srds代表哺乳动物外周蛋白/视网膜变性慢基因的鳐鱼直系同源物。与人类视网膜疾病相关的大多数残基的保守性表明这些残基发挥着重要的功能作用。大的盘内环中一小段序列的高度保守也表明该结构域具有重要功能。
