BIOMOLECULAR STUDIES OF UNIQUE RETINAL SPECIFIC PROTEINS

这项研究计划的长期目标是确定 参与调节的遗传和环境因素 感光细胞的细胞和亚细胞浓度- 光感受器发育过程中的特异基因产物 正常小鼠和遗传性视网膜小鼠的细胞 退化。更直接的目标包括一代人 与三个光感受器相对应的重组DNA探针 特定的蛋白质。它们是：环状GMP的阿尔法亚单位 磷酸二酯酶(PDE)；(2)33K，一种结合 转导蛋白β，伽马复合体，是光感受器所特有的 细胞；和(3)23K，一种存在于正常成人中的蛋白质 小鼠的视网膜和无光感受器的视网膜 成年RD小鼠。将从小鼠视网膜中分离出cDNAs 筛选本实验室构建的表达文库 抗体、cDNAs和人工合成的寡核苷酸。这个 特定基因的质量和数量特征 产品将在视网膜发育过程中确定 正常小鼠和遗传性视网膜变性(RD)小鼠 和rds)，使用Western、Northern、Southern和核苷酸 序列分析、免疫细胞化学和原位杂交 技巧。这些基因的位置分配 蛋白质将使用限制性内切酶的Southern分析 体细胞杂交染色体组的原位消化 小鼠的杂交和重组近交系。这个 33k、37k(β转导蛋白)和48k的亚细胞定位 已被免疫细胞化学证明是暂时的和依赖于 动物所处的照明环境。这个 光诱导特定染色发生移位的机制 因为这些位于内部和外部片段之间的蛋白质 与光有关的检查和特征， 动物的基因，运动的速度和方向， 特定的蛋白质和生化要求。站点特定 抗48k的单抗将被用于观察变化 在表位可用性和/或实际的蛋白质运动中。它是 预计所获得的结果和探测将提供 编码基因表达的详细检查 正常视网膜中已鉴定的视网膜特异性蛋白 发育和在遗传程序性退化过程中 发生在遗传性小鼠身上的感光细胞。

The long term objective os this research plan is to identify the genetic and environmental factors involved in the regulation of cellular and subcellular concentrations of photoreceptor cell- specific gene products during development of the photoreceptor cells in normal mice and in mice with inherited retinal degeneration. The more immediate objectives include the generation of recombinant DNA probes corresponding to three photoreceptor specific proteins. They are: The alpha subunit of cyclic GMP phosphodiesterase (PDE); (2) 33k, a phosphoprotein which binds the transducin beta, gamma complex and is unique to photoreceptor cells; and (3) 23k, a protein which is presence in normal adult mouse retina and absent from the photoreceptorless retina of the adult rd mouse. The cDNA's will be isolated from mouse retinal cDNA expression libraries generated in this laboratory by screening with antibodies, cDNA's and synthetic oligonucleotides. The qualitative and quantitative characteristic of the specific gene products will be determined during the development of the retinas of normal mice and of those with inherited retinal degeneration (rd and rds), using Western, Northern, Southern, and nucleotide sequence analysis; immunocytochemistry and in situ hybridization techniques. The positional assignment of the genes for these proteins will be made using Southern analysis of restriction digests of somatic cell hybrid chromosome panels, in situ hybridization and recombinant inbred lines of mice. The subcellular localization of 33k, 37k (beta transducin) and 48k has been shown by immunocytochemistry to be transient and dependent on the lighting environment to which the animal is exposed. The mechanism by which light induces a shift in the specific staining for these proteins between the inner and the outer segments will be examined and characterized with respect to the light, the genotype of the animal, the rate and direction of movement, the specific protein, and the biochemical requirements. Site specific monoclonal antibodies against 48k will be used to visualize changes in epitope availability and/or actual protein movement. It is anticipated that the results and probes obtained will provide for the detailed examinations of the expression of the genes coding for the identified retinal specific proteins during normal retinal development and during the genetically programmed degeneration of photoreceptor cells that occurs in mice with hereditary.