Pan Yi, Comiskey Daniel F, Kelly Lisa E, Chandler Dawn S, El-Hodiri Heithem M
Center for Molecular and Human Genetics, The Research Institute at Nationwide Children's Hospital, Columbus, OH.
Graduate Program in Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, OH; Center for Childhood Cancer, The Research Institute at Nationwide Children's Hospital, Columbus, OH; Center for RNA Biology, Wexner Medical Center, The Ohio State University, Columbus, OH.
Mol Vis. 2016 Dec 14;22:1421-1428. eCollection 2016.
The photoreceptor conserved element-1 (PCE-1) sequence is found in the transcriptional regulatory regions of many genes expressed in photoreceptors. The gene product functions by binding to PCE-1 sites. However, other transcriptional regulators have also been reported to bind to PCE-1. One of these, , is expressed in retinal progenitor and bipolar cells. The purpose of this study is to identify gene products and characterize gene product expression and function with respect to the PCE-1 site.
gene products were amplified with PCR. Expression patterns were determined with in situ hybridization using whole or sectioned embryos and digoxigenin- or fluorescein-labeled antisense riboprobes. DNA binding characteristics of the gene products were analyzed with electrophoretic mobility shift assays (EMSAs) using in vitro translated proteins and radiolabeled oligonucleotide probes. Gene transactivation assays were performed using luciferase-based reporters and in vitro transcribed effector gene products, injected into embryos.
We identified one and two gene products. The two gene products are generated by alternate mRNA splicing. We verified that these gene products are expressed in the developing retina and that expression resolves into distinct cell types in the mature retina. Finally, we found that gene products can bind the PCE-1 site in vitro and that the two isoforms have different gene transactivation activities.
gene products are expressed in the developing and mature neural retina. gene products can bind the PCE-1 site in vitro and influence the expression of a rhodopsin promoter-luciferase reporter gene. The two isoforms of have different gene transactivation activities in this reporter gene system.
光感受器保守元件1（PCE-1）序列存在于许多在光感受器中表达的基因的转录调控区域。该基因产物通过与PCE-1位点结合发挥作用。然而，也有报道称其他转录调节因子也能与PCE-1结合。其中之一， 在视网膜祖细胞和双极细胞中表达。本研究的目的是鉴定 基因产物，并就PCE-1位点对 基因产物的表达和功能进行表征。
用聚合酶链反应（PCR）扩增 基因产物。使用完整或切片的 胚胎以及地高辛或荧光素标记的反义核糖探针，通过原位杂交确定表达模式。使用体外翻译的蛋白质和放射性标记的寡核苷酸探针，通过电泳迁移率变动分析（EMSA）分析 基因产物的DNA结合特性。使用基于荧光素酶的报告基因和体外转录的效应基因产物进行基因反式激活分析，将其注射到 胚胎中。
我们鉴定出一种 和两种 基因产物。这两种 基因产物是由交替的mRNA剪接产生的。我们证实这些基因产物在发育中的视网膜中表达，并且在成熟视网膜中表达分化为不同的细胞类型。最后，我们发现 基因产物在体外可以结合PCE-1位点，并且两种 异构体具有不同的基因反式激活活性。
基因产物在发育中和成熟的神经视网膜中表达。 基因产物在体外可以结合PCE-1位点，并影响视紫红质启动子-荧光素酶报告基因的表达。在该报告基因系统中， 的两种异构体具有不同的基因反式激活活性。
