GENE EXPRESSION IN NORMAL AND CATARACTOUS LENS

透明的人类人工晶状体是分子和形态的 时间精确和空间定向的基因活性的缩影，它 从怀孕5/6周开始，一直持续到一生。 在最近的过去，我们实验室和其他调查人员的工作 帮助实现了对我们的 对α-晶体蛋白的理解，它是细胞的主要蛋白质 哺乳动物的晶状体。我们发现字母A和字母B都在 豆状核外组织，它们以独立的蛋白质形式存在。我们 提示这些蛋白质可能具有相似的、非结构的(非 晶体蛋白)在晶状体内和晶状体外都起作用。数据来自 其他实验室已经证明，像其他热量一样，αA和αB 休克蛋白(它们与之有初级序列同源性)，有 伴侣样活性，这些蛋白质的一个催化方面，可能 对他们在镜头中的作用来说是至关重要的。在本申请中，我们建议 研究αB-晶状体蛋白基因的转录调控。 镜头，特别强调热的作用的描绘 震撼因素。我们建议同时采取两种方法：(1)发展一种 大鼠晶状体的体外转录系统，这将使 晶状体特异性启动子元件的表征及鉴定 相互作用因子；以及(2)使用已知转录的探针 分离其同源物的因素(如HSF和C/EBP)进行研究 组织特异性异质性与基因表达的相关性 Alphab基因。这两种方法都将利用(A)高度敏感的 大鼠和人类αB基因的体内足迹，重点是 保守的DNA序列和(B)体外和体内的功能分析 瞬时表达系统。转录因子的特性 将涉及克隆、分离(从“正常”和正常的大鼠和 人晶状体c DNA文库)、序列分析及其在细菌中的表达 细胞。我们还建议对很可能发生的 甲型肝炎病毒基因自我调节的可能性。我们在概念上是 从技术上讲已经做好了充分的准备，以利用字母表 基因为阐明激活的机制方面提供了帮助 上皮/纤维细胞界面晶状体蛋白启动子的表达 与生俱来的遗传机制必须保持对我们的 对晶状体生长及其相关病理的理解， 白内障的发生。

The transparent human ocular lens is the molecular and morphological epitome of temporally precise and spatially directed gene activity, which commences in the 5/6th week of gestation and continues throughout life. In the recent past, work from our laboratory and other investigators has been instrumental in bringing about a remarkable revision of our understanding of alpha-crystallins, the predominant proteins of the mammalian lens. We showed that both alphaA and alphaB are expressed in extralenticular tissues and that they exist as independent proteins. We suggested that these proteins may perform similar, non-structural (non- crystallin) functions both inside and outside of the lens. Data from other laboratories have shown that alphaA and alphaB, like other heat shock proteins (with whom they share primary sequence homologies), have chaperone-like activities, a catalytic aspect of these proteins which may be fundamental to their role in the lens. In this application we propose to study the transcriptional regulation of alphaB-crystallin gene in the lens, with particular emphasis on the delineation of the role of heat shock factors. We suggest two simultaneous approaches: (1) Develop an in vitro transcription system from the rat lens, which will allow characterization of lens-specific promoter elements and identification of interacting factors; and (2) Use probes from known transcription factors (e.g., HSF and C/EBP) to isolate their homologues to investigate tissue-specific heterogeneity with relevance to the expression of the alphaB gene. Both these approaches will utilize (a) a highly sensitive in vivo footprinting of the rat and human alphaB genes with emphasis on conserved DNA sequences and (b) functional assays in in vitro and in transient expression systems. Characterization of transcription factors will involve cloning, isolation (from "normalized" and regular rat and human lens cDNA libraries), sequence analysis and expression in bacterial cells. We also propose to initiate investigations into the very likely possibility of autoregulation of the alphaB gene. We are conceptually and technically well poised to exploit the opportunity that the alphaB gene offers for elucidation of the mechanistic aspects of the activation of a crystallin promoter at the epithelium/fiber cell interface - an innate genetic mechanism which must remain fundamental to our understanding of lens growth and its attendant pathology, cataractogenesis.