BIOMOLECULAR STUDIES OF UNIQUE RETINAL SPECIFIC PROTEINS

本研究计划的长期目标是确定和 描述参与的遗传和环境因素， 光感受器的细胞和亚细胞浓度的调节 细胞特异性基因产物在发育和维持过程中 视野 更直接的是，小鼠基因的功能和表达 将分析视网膜钙传感器recoverin。的 特异性恢复肽在与视紫红质结合中的重要性 将使用偶联的体外转录/翻译来评估激酶 系统与通过以下方式修饰的恢复分子组合 表达/诱变技术。 时间和空间格局 recoverin在视杆细胞、视锥细胞、一些双极细胞和一种罕见的 神经节细胞群在参与神经细胞增殖的蛋白质中是独特的。 光传导 细胞外信号、顺式作用元件和 参与这种表达调节的转录因子是 未知拟议研究的一个目标是确定遗传 负责恢复素基因表达的调控元件 并评估其转录应答元件的功能状态 通过双脱氧核苷酸序列分析检测。鼠标的5 '端 将对基因进行测序，鉴定特异性DNA蛋白结合基序， 它们的开/关状态将通过免疫细胞化学和Western blot进行评估。 视网膜细胞中recoverin蛋白的分析 添加有激素或生长因子的化学成分确定的培养基。 将通过体外转染进一步分析recoverin启动子 Y 79视网膜母细胞瘤细胞与真核细胞载体， recoverin基因5 '端的确定的基因组序列， 细菌B-半乳糖苷酶报告基因。 成功的条件 还将寻求原代细胞培养物的转染。转基因 小鼠将用于确认转染数据并表征 recoverin基因的能力，支配时间，组织和细胞， 报告基因的特异性表达。 根据为该项目制定的初步战略， 55 kDa的感光细胞特异性蛋白，最初被鉴定为 recoverin和recoverducin的克隆、测序及表达 表征了 将确定其基因的内含子/外显子边界 以及它的染色体定位 这些研究的结果 将进一步加深我们对光传导、视觉、遗传 失明和失明是癌症的间接影响。 等 知识可能导致干预方法，以保护和/或恢复 视网膜中的视觉，否则为感光细胞编程 退化

The long term objective of this research plan is to identify and characterize the genetic and environmental factors involved in the regulation of cellular and subcellular concentrations of photoreceptor cell-specific gene products during the development and maintenance of vision. More immediately, the function and expression of the mouse gene for the retinal calcium sensor, recoverin, will be analyzed. The importance of specific recoverin peptides in the binding to rhodopsin kinase will be assessed using a coupled in vitro transcription/translation system in combination with recoverin molecules modified by expression/mutagenesis techniques. The temporal and spatial pattern of expression of recoverin in rods, cones, some bipolar cells and a rare population of ganglion cells is unique among the proteins participating in phototransduction. The extracellular signals, cis-acting elements and transcription factors involved in the regulation of this expression are unknown. An objective of the proposed research is to identify the genetic regulatory elements responsible for the expression of the recoverin gene and to assess the functional status of its transcription response elements detected by dideoxy nucleotide sequence analysis. The 5'end of the mouse gene will be sequenced, specific DNA protein binding motifs identified and their on/off status will be evaluated by immunocytochemical and Western analysis of the recoverin protein in postnatal retinal cells grown in a chemically defined medium to which the hormone or growth factor is added. The recoverin promoter will be further analyzed by in vitro transfection of Y79 retinoblastoma cells with a eukaryotic cell vector containing defined genomic sequences of the 5'end of the recoverin gene coupled to the bacterial B-galactosidase reporter gene. Conditions for successful transfection of the primary cell cultures will also be sought. Transgenic mice will be used to confirm the transfection data and to characterize the recoverin gene's ability to dictate the temporal, tissue- and cell- specific expression of the reporter gene. Following through on the initial strategy developed for this project, the 55kDa photoreceptor cell-specific protein, originally identified with recoverin and phosducin, will be cloned, sequenced and its expression characterized. The intron/exon boundaries of its gene will be determined and its chromosomal location assigned. The results from these studies will further our understanding of phototransduction, vision, hereditary blindness and blindness produced as a remote effect of cancer. Such knowledge may lead to methods of intervention to preserve and/or restore vision in retinas otherwise programmed for photoreceptor cell degeneration.