REGULATION OF CORNEAL EPITHELIAL RNA POLYMERASE ACTIVITY

了解调控RNA合成的分子机制 角膜上皮细胞的生长和分化是重要的 开发更有效的处理缺陷的方法， 角膜损伤或疾病。 正在进行的研究代表了 第一次深入的努力，以表征核酶的 在哺乳动物角膜上皮中的转录， 调节其活动的机制。 我们以前的 研究提供了环核苷酸介导的证据， 毒蕈碱胆碱能调节RNA聚合酶活性 兔角膜上皮细胞 建议的具体目标 主要研究内容如下：（1）RNA聚合酶I、II的表征 兔角膜上皮细胞（包括电泳 以及进一步的生化/动力学表征 色谱纯化的酶）。 (2)确定监管 胆碱能激动剂氨甲酰胆碱 增强兔角膜上皮RNA聚合酶活性。 有待检验的关于所观察到的 氨甲酰胆碱增强RNA聚合酶活性包括 药物的以下潜在作用，其中每一种都是 这与我们对一种或多种RNA的动力学数据暂时一致 来自分离的纯化的细胞核的粗酶的聚合酶研究： (a)对RNA聚合酶翻译后磷酸化的影响 环核苷酸依赖性蛋白激酶 （“靶向假设”）;（B）对其他蛋白磷酸化的影响 染色质相关蛋白（组蛋白，HMG非组蛋白） 影响DNA模板对RNA聚合酶的可及性;（c） 对RNA聚合酶合成的影响;（d）对相对生物量的影响 运动学和代谢学上不同的RNA的比例 具有不同底物亲和力的聚合酶同工酶。 实验方法包括电泳分析 磷酸化和核蛋白的合成 和对照细胞，结合色谱纯化 和RNA聚合酶I、II和 III、组蛋白和HMG蛋白。 长期目标 研究包括（1）阐明分子调控机制 胆碱能药物可能通过这种方式影响基因表达， 在角膜上皮细胞生长过程中的转录水平， 分化和（2）确定胆碱能药物的适用性 单用或与其他药物联用的管理药物 各种类型的损伤和眼表疾病。

Understanding molecular mechanisms regulating RNA synthesis during growth and differentiation of corneal epithelial cells is important in developing more effective treatment of defects arising from corneal injury or disease. The research in progress represents the first in-depth effort to characterize nuclear enzymes of transcription in mammalian corneal epithelium and to determine mechanisms by which their activity is regulated. Our previous studies have provided evidence for cyclic nucleotide-mediated, muscarinic cholinergic regulation of RNA polymerase activity in rabbit corneal epithelial cells. The specific aims of the proposed research are as follows: (1) To characterize RNA polymerase I, II and III of rabbit corneal epithelium (including electrophoretic and further biochemical/kinetic characterization of chromatographically purified enzymes). (2) To determine regulatory mechanisms by which the cholinergic agonist carbamylcholine enhances activity of RNA polymerase in rabbit corneal epithelium. Hypotheses to be tested concerning mechanisms mediating observed enhancement of RNA polymerase activity by carbamylcholine include the following potential effects of the drug, each of which is tentatively consistent with our kinetic data for one or more RNA polymerase studies of crude enzymes from isolated, purified nuclei: (a) Effects on posttranslational phosphorylation of RNA polymerase by cyclic nucleotide-dependent (cGMP-dependent) protein kinases ("targeted hypothesis"); (b) Effects on phosphorylation of other chromatin associated proteins (histones, HMG nonhistone proteins) influencing accessibility of DNA template to RNA polymerase; (c) Effects on RNA polymerase synthesis; (d) Effects on relative proportions of kinetically and electrophoretically distinct RNA polymerase isozymes with different substrate affinities. Experimental approaches include electrophoretic analysis of phosphorylation and synthesis of nuclear proteins in drug-treated and control cells, in conjunction with chromatographic purification and electrophoretic characterization of RNA polymerase I, II and III, histones and HMG proteins. Long-range objectives of this research include (1) elucidating molecular regulatory mechanisms by which cholinergic agents may influence gene expression at the transcriptional level during corneal epithelial cell growth and differentiation and (2) determining the suitability of cholinergic agents, alone or in combination with other drugs, for management of various types of injury and disease of the ocular surface.