GENE EXPRESSION IN THE NORMAL AND CATARACTOUS LENS

各种病因的白内障，包括遗传性白内障， 与γ-晶体蛋白水平改变有关 我们现在成功了 在克隆不同的γ-晶体蛋白cDNA序列，我们已经确定 牛γ-晶状体蛋白mRNA的完整核苷酸序列。 γ-晶体蛋白的特征是在终末合成， 区分透镜纤维细胞，因此，可以用作方便的 差异基因表达的标志物。 本研究将试图 阐明参与发展的分子遗传机制， 调节空间和时间控制的异质性， 正常情况下，小牛和人透镜中的γ-晶体蛋白 分化和疾病状态。 为了实现这一目标，使用克隆的 我们的实验室里已经有了探针，我们将分离出主要的人类 γ-晶体蛋白基因，并通过全序列对其进行表征 分析。 使用重组DNA技术，DNA序列将被删除， 来自或添加到分离的人和/或牛基因。 表达 这些基因将在无细胞系统和体内监测， 将它们显微注射到LESN上皮细胞的细胞核中， 维持在原代培养物中（γ-晶体蛋白在 这些细胞）。 分化的透镜纤维的转录调控 和上皮细胞将在体外和体内进行研究， 研究γ-晶体蛋白的核转录物的加工过程 mRNA。 利用限制性内切酶和特定的人和牛克隆 DNA探针，我们将试图映射序列多态性，突变或 疑似患者的γ-晶状体蛋白基因序列存在缺陷 遗传性白内障的几率 本研究将探讨 基因表达的调节可能参与了 维持眼睛的透明度和发展中的白内障 眼镜.

Cataracts of varied etiologis, including heriditary cataracts, are associated with altered levels of gamma-crystallins. We now have succeeded in cloning different gamma-crystallin cDNA sequences and we have determined the complete nucleotide sequence of a bovine gamma-crystallin mRNA. Gamma-crystallins are characteristically synthesised in the terminally differentiating lens fibre cells and therefore, can be used as convenient markers of differential gene expression. The present study will attempt to elucidate the molecular genetic mechanisms involved in the development and regulation of the spatially and temporally controlled heterogeneity of gamma-crystallins in the calf and the human lens, during normal differentiation and diseased states. Toward this goal, using the cloned probes already available in our laboratory, we will isolate the major human gamma-crystallin gene(s) and characterize them by complete sequence analyses. Using recombinant DNA techniques, DNA sequences will be deleted from or added to the isolated human and/or bovine gene(s). The expression of these genes will be monitored in cell-free systems and in vivo, by microinjecting them into the nuclei of the lesn epithelial cells, maintained in primary cultures (gamma-crystallins are not synthesised in these cells). Transcriptional regulation in the differentiated lens fibres and epithelial cells will be investigated both in vitro and in vivo by studying the processing of the nuclear transcripts of the gamma-crystallin mRNA. Utilizing restriction enzymes and specific human and bovine cloned DNA probes, we will attempt to map sequence polymorphisms, mutations or defects in the gamma-crystallin gene sequences of the individuals suspected of having cataracts of genetic origin. This study will investigate the possible involvement of the regulation of gene expression in the maintenance of ocular transparency and in the development of cataractous lenses.