BIOMOLECULAR STUDIES OF UNIQUE RETINAL SPECIFIC PROTEINS

独特的视网膜特异性蛋白质的生物分子研究

• 批准号: 2159749
• 负责人: JAMES Francis MCGINNIS
• 金额: $ 7.34万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2159749
• 关键词: chromatography; complementary DNA; congenital eye disorder; cyclic GMP; electrophoresis; gene expression; genetic library; genetic manipulation; genotype; immunocytochemistry; in situ hybridization; laboratory mouse; laboratory rabbit; messenger RNA; molecular cloning; monoclonal antibody; nucleic acid probes; nucleic acid sequence; phosphodiesterases; protein sequence; protein transport; radioassay; recombinant DNA; retina; retina degeneration; visual photoreceptor; western blottings

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.

本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- 感光细胞发育过程中的特定基因产物 正常小鼠和遗传性视网膜病变小鼠的细胞 退化 更直接的目标包括： 重组DNA探针对应于三个感光细胞 特定蛋白质 它们是：环GMP的α亚基 磷酸二酯酶（PDE）;（2）33 k，一种结合磷酸二酯酶的磷蛋白。 转导素β，γ复合物，并且是光感受器所特有的 （3）23 k，正常成人中存在的蛋白质 小鼠视网膜和缺席的光感受器的视网膜 成年rd小鼠。 将从小鼠视网膜中分离cDNA， 本实验室通过筛选产生的cDNA表达文库 用抗体cDNA和合成寡核苷酸 的 特异基因的定性和定量特征 在视网膜发育过程中， 正常小鼠和遗传性视网膜变性（RD）小鼠的视网膜病变 和rds），使用Western、北方、南方和核苷酸 序列分析、免疫细胞化学和原位杂交 技术. 这些基因的位置分配 蛋白质将使用限制性内切酶的Southern分析来制备， 体细胞杂种染色体组的原位观察 杂交和重组近交系小鼠。 的 33 k、37 k（β转导蛋白）和48 k的亚细胞定位具有 免疫细胞化学显示， 动物所处的光照环境。 的 光诱导特异性染色改变的机制 因为内外节之间的这些蛋白质 检查和表征相对于光， 动物的基因型，运动的速度和方向， 特定的蛋白质和生化要求。 位点特异性 将使用抗48 k的单克隆抗体来观察变化 表位可用性和/或实际蛋白质移动。 是 预计获得的结果和探测器将提供 对编码以下基因表达的详细检查 在正常视网膜病变过程中鉴定的视网膜特异性蛋白质 在发育和遗传程序性退化过程中， 光感受器细胞发生在老鼠遗传。