Batni S, Scalzetti L, Moody S A, Knox B E
Department of Biochemistry and Molecular Biology, State University of New York Health Science Center, Syracuse, New York 13210, USA.
J Biol Chem. 1996 Feb 9;271(6):3179-86. doi: 10.1074/jbc.271.6.3179.
The abundant Xenopus rhodopsin gene and cDNA have been cloned and characterized. The gene is composed of five exons spanning 3.5 kilobase pairs of genomic DNA and codes for a protein 82% identical to the bovine rhodopsin. The cDNA was expressed in COS1 cells and regenerated with 11-cis-retinal, forming a light-sensitive pigment with maximal absorbance at 500 nm. Both Southern blots and polymerase chain reaction amplification of intron 1 revealed multiple products, indicating more than one allele for the rhodopsin gene. Comparisons with other vertebrate rhodopsin 5 upstream sequences showed significant nucleotide homologies in the 200 nucleotides proximal to the transcription initiation site. This homology included the TATA box region, Ret 1/PCE1 core sequence (CCAATTA), and surrounding nucleotides. To functionally characterize the rhodopsin promoter, transient embryo transfections were used to assay transcriptional control elements in the 5 upstream region using a luciferase reporter. DNA sequences encompassing -5500 to +41 were able to direct luciferase expression in embryo heads. Reporter gene expression was also observed in embryos microinjected with reporter plasmids during early blastomere stages. These results locate transcriptional control elements upstream of the Xenopus rhodopsin gene and show the feasibility of embryo transfections for promoter analysis of rod-specific genes.
非洲爪蟾视紫红质基因及互补DNA（cDNA）已被克隆并进行了特性分析。该基因由5个外显子组成，跨越3.5千碱基对的基因组DNA，编码一种与牛视紫红质有82%同源性的蛋白质。该cDNA在COS1细胞中表达，并与11-顺式视黄醛再生，形成一种在500纳米处具有最大吸光度的光敏感色素。Southern印迹法和对内含子1的聚合酶链反应扩增均显示有多种产物，表明视紫红质基因存在不止一个等位基因。与其他脊椎动物视紫红质5上游序列的比较显示，在转录起始位点近端的200个核苷酸中存在显著的核苷酸同源性。这种同源性包括TATA盒区域、Ret 1/PCE1核心序列（CCAATTA）及周围核苷酸。为了对视紫红质启动子进行功能特性分析，利用荧光素酶报告基因通过瞬时胚胎转染来检测5上游区域的转录控制元件。包含-5500至+41的DNA序列能够在胚胎头部指导荧光素酶表达。在早期卵裂球阶段向胚胎显微注射报告质粒时，也观察到了报告基因的表达。这些结果确定了非洲爪蟾视紫红质基因上游的转录控制元件，并表明胚胎转染用于杆状细胞特异性基因启动子分析的可行性。
