BIOMOLECULAR STUDIES OF UNIQUE RETINAL SPECIFIC PROTEINS

最近，通过应用高度敏感的免疫学方法， 在神经视网膜基因表达的研究中，我们确定了四个 免疫学上不同的蛋白条带（MW 23、33、55和69 kD）， 存在于人、牛、大鼠和小鼠的正常成年视网膜中;但 在成年突变小鼠的无杆视网膜中缺失， 视网膜变性（RD）基因。 此外，没有一个蛋白质条带的 在非视网膜眼睛部分、非神经组织或CNS中发现了感兴趣的， PNS神经组织。 从出生后发育研究的年龄匹配 正常和突变同类小鼠，时间出现和消失 4条视网膜特异性蛋白带中有1条与形态学特征一致， 感光细胞群的成熟和退化。 本研究计划的主要目标是产生适当的 cDNA探针，将被应用到表达的详细分析， 一个或多个视网膜特异性蛋白质的基因在正常情况下， 和遗传性的退化状况 从23号开始 kD蛋白，并使用单特异性抗体和视网膜cDNA表达 从本实验室的文库中，23 kD的cDNA将被克隆。 中 平行的方法，23 kD的蛋白质将从视网膜中纯化，其 确定氨基酸序列，并将特异性寡核苷酸探针 制备并用于鉴定相应的cDNA克隆。 克隆的 将表征cDNA，并确定编码的蛋白质的身份。 确认 分别使用抗体和/或cDNA探针， 可以确定视网膜特异性蛋白质是否 光感受器特异性蛋白质或这些蛋白质中的任何一种或多种 其他视网膜细胞在响应于 感光细胞的“诱导调节”影响。 与这些 互补探针还可以定量， 表征和分离这些视网膜的信使RNA的编码， 特定蛋白质 预计所获得的结果和探针 将提供基因表达的详细检查， 在正常视网膜病变过程中编码所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， 遗传性失明小鼠的感光细胞。

Recently, and by applying a highly sensitive immunological approach to the study of gene expression in the neural retina, we identified four immunologically distinct protein bands (MW 23, 33, 55, and 69 kD) which are present in the normal adult retinas of human, cow, rat and mouse; but are missing from the rodless retinas of adult mutant mice homozygous for the retinal degeneration (rd) gene. Moreover, none of the protein bands of interest is found in non-retinal eye parts, non-neural tissues or CNS and PNS neural tissues. From postnatal developmental studies of age-matched normal and mutant congenic mice, the temporal appearance and disappearance of the four retinal specific protein bands coincides with the morphological maturation and degeneration of the photoreceptor cell population. It is the major objective of this research plan to generate the appropriate cDNA probes that will be applied to the detailed analysis of the expression of one or more of the genes for the retinal specific proteins under normal and genetically programmed degenerating conditions. Beginning with the 23 kD protein, and using monospecific antibodies and retinal cDNA expression libraries from this laboratory, the 23 kD cDNA will be cloned. In a parallel approach, the 23 kD protein will be purified from retina, its amino acid sequence determined and specific oligonucleotide probes will be made and used for identifying the corresponding cDNA clones. The cloned cDNA's will be characterized and the identity of the protein coded for confirmed. Using the antibody and/or cDNA probes respectively, it will be possible to determine whether the retinal specific proteins are photoreceptor specific proteins or whether any one or more of these proteins is differentially expressed by other retinal cells in response to an "inductive regulatory" influence of the photoreceptor cells. With these complementary probes it will be further possible to quantitate, characterize and isolate the messenger RNA"s coding for these retinal specific proteins. It is anticipated that the results and probes obtained will provide for the detailed examination 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 blindness.