GENE EXPRESSION IN THE NORMAL AND CATARACTOUS LENS

各种病因的白内障，包括遗传性白内障， 与伽马晶体蛋白水平的变化有关。我们现在已经成功了 在克隆不同的伽马晶体蛋白cDNAs序列时，我们已经确定 牛的伽马晶体蛋白基因的完整核苷酸序列。 伽马晶体蛋白的特征是在末端合成 因此，可以方便地使用区分晶状体纤维细胞 差异基因表达的标记。本研究将试图 阐明与发育和发育有关的分子遗传学机制 空间和时间控制的非均质性的调节 正常情况下，小腿和人类晶状体中的伽马晶体蛋白 分化与病态。为了实现这一目标，使用克隆的 我们实验室里已经有的探头，我们将分离出主要的人类 γ-晶体蛋白基因(S)及其全序列分析 分析。使用重组DNA技术，DNA序列将被删除 从分离的人和/或牛基因中分离或添加到分离的人和/或牛基因中(S)。表达方式 这些基因中的一部分将在无细胞系统和体内进行监测，通过 将它们微量注射到LESN上皮细胞的细胞核中， 保持在原代培养中(伽马晶体蛋白不是在 这些细胞)。分化的晶状体纤维中的转录调控 上皮细胞将在体外和体内通过以下方式进行研究 γ-晶体蛋白核转录产物的处理研究 MRNA.利用限制性内切酶克隆人和牛的特异性 DNA探针，我们将尝试绘制序列多态、突变或 疑似个体的γ-晶体蛋白基因序列存在缺陷 患有遗传性白内障。这项研究将调查 基因表达调控可能参与了 眼部透明度的维持与白内障的发生 隐形眼镜。

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.