Characterization of the Xenopus rhodopsin gene
Characterization of the Xenopus rhodopsin gene
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DOI：
10.1074/jbc.271.6.3179
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发表时间：
1996-02-09
影响因子：
4.8
通讯作者：
Knox, BE
中科院分区：
文献类型：
--
作者：
Batni, S;Scalzetti, L;Knox, BE
已克隆并鉴定了非洲爪哇丰富的视紫红质基因和cDNA.该基因由5个外显子组成，跨越3.5kb的基因组DNA，编码一种蛋白质，与牛视紫质有82%的同源性。该基因在COS1细胞中表达，经11-顺式视网膜再生，在500 nm处形成最大吸光度的光敏色素。Southern杂交和聚合酶链式反应扩增内含子1都显示了多个产物，表明视紫红质基因有不止一个等位基因。与其他脊椎动物视紫红质5‘上游序列的比较显示，转录起始点附近的200个核苷酸具有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)和周围的核苷酸。为了确定视紫红质启动子的功能特性，利用瞬时胚胎转基因技术，利用荧光素酶报告基因检测了5‘上游区域的转录调控元件。-5500到+41之间的DNA序列能够直接在胚头中表达荧光素酶。在卵裂球早期显微注射报告基因的胚胎中也观察到了报告基因的表达。这些结果定位了非洲爪哇视紫红质基因上游的转录调控元件，并表明胚胎转染用于杆状特异基因启动子分析的可行性。
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.