BIOMOLECULAR STUDIES OF UNIQUE RETINAL SPECIFIC PROTEINS

这项研究计划的长期目标是确定和 描述涉及的遗传和环境因素 光感受器细胞和亚细胞浓度的调节 细胞特异性基因产物在细胞发育和维持过程中的作用 幻象。更直接的是，小鼠基因的功能和表达 对于视网膜钙传感器，恢复素，将被分析。这个 恢复素特异性多肽在视紫红质结合中的重要性 将使用体外转录/翻译耦合的方法来评估激酶 结合经以下修饰的恢复素分子的系统 表达/突变技术。时间和空间格局 恢复素在视杆细胞、视锥细胞、一些双极细胞和一种罕见的 神经节细胞群是参与蛋白质中独特的 光转导。细胞外信号、顺式作用元件和 参与这一表达调控的转录因子有 未知。这项拟议的研究的一个目标是识别基因 恢复素基因表达的调控元件 并评估其转录反应元件的功能状态 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，确定特定的DNA蛋白结合基序，并 他们的开/关状态将通过免疫细胞化学和Western 培养的新生视网膜细胞恢复素蛋白的分析 添加荷尔蒙或生长因子的化学定义的介质。 恢复素启动子将通过体外转染法进一步分析。 Y79视网膜母细胞瘤细胞表达载体的构建 Recoverin基因5‘端的已定义基因组序列与 细菌B-半乳糖苷酶报告基因。成功的条件 还将寻求对原代细胞培养物进行转基因。转基因 小鼠将被用来确认转基因数据并表征 恢复素基因决定时间、组织和细胞的能力- 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光感受器细胞特异性蛋白，最初鉴定为 Recoverin和Phosducin基因的克隆、测序及其表达 特色化的。其基因的内含子/外显子边界将被确定 并指定了它的染色体位置。这些研究的结果 将进一步加深我们对光传导、视觉、遗传的理解 由于癌症的远程影响而产生的失明和失明。是这样的 知识可能导致干预方法来保存和/或恢复 视网膜中的视觉，否则为光感受器细胞编程 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.