Molecular Genetics of a Lens Intrinsic Membrane Protein

描述(申请人提供)：人工晶状体是一种无血管的半固态器官，其功能是将入射光线折射到视网膜上。伴随这一功能的是镜片保持透明的必要性。晶状体透明度的丧失通常是永久性的，被称为白内障。细胞和器官的功能，如获得维持健康、透明的晶状体状态、处理细胞废物和维持膜的整体稳定性所需的营养物质，是通过晶状体细胞膜内的特定蛋白质或蛋白质组来实现的。适当地控制这些蛋白的表达及其随后的功能对于晶状体存活和维持透明度是至关重要的。调节晶状体中这些膜蛋白的机制目前尚不清楚。该项目的长期目标是了解晶状体纤维细胞固有膜蛋白MP19的功能以及该蛋白编码基因(LIM2)的调控。为了实现这一目标，我们将调查以下主题：a)。采用小鼠基因敲除技术，对LIM2基因进行消融，以研究MP19的功能。b)。利用哺乳动物表达载体，研究MP19的合成及其编码序列的各种突变。对表达的MP19蛋白进行截短，以研究MP19的膜拓扑结构。最后，我们将使用这些表达载体来研究MP19分子中可能存在的影响MP19向细胞膜转运的信号。c)。使用位点特异性RNA/DNA嵌合寡核苷酸(CO)研究LIM2基因点突变的功能后果。特定的一氧化碳将被注射到小鼠的原核中，然后移植到养母体内。将对含有成功突变的幼崽进行调查，以确定突变对晶状体发育和完整性的影响。 这些研究将使人们更好地了解MP19在维持晶状体透明度中的作用，以及它在白内障形成中的可能作用。

DESCRIPTION (provided by applicant): The ocular lens is an avascular, semisolid organ whose function is to refract incoming light upon the retina. Concomitant with this function is a necessity for the lens to remain transparent. A loss of transparency of the lens is usually permanent and is termed a cataract. Cell and organ functions, such as obtaining nutrients necessary for maintaining the healthy, transparent lens state, for handling of cell waste removal, and for maintenance of overall membrane stability are carried out through select and specific proteins or groupings of proteins within the lens cell membrane. Proper controls on expression of these proteins and their subsequent function is essential for lens survival, and maintenance of transparency. The mechanisms regulating these membrane proteins in the lens are presently unknown. The long term goals of this project are to understand the function of the lens fiber cell intrinsic membrane protein MP19 and the regulation of the gene coding for this protein (LIM2). To achieve this goal, we will investigate the following topics: a). Using knockout mouse technology, to ablate the Lim2 gene in order to study the function of MP19. b). Using mammalian expression vectors, the investigate the synthesis of MP19 and various mutations of this coding sequence. Truncations of the MP19 expressed protein will be made to investigate the membrane topology of MP19. Finally, we will use these expression vectors to investigate the putative signal residing in the MP19 molecule which effects transport of MP19 to the cell membrane. c). Investigate functional consequences of point mutations in the Lim2 gene using site-specific RNA/DNA chimeric oligonucleotides (CO). Specific CO will be injected into mouse pronuclei and subsequently implanted in foster mothers. Pups containing successful mutations will be investigated to determine the effect of the mutation on lens development and integrity. These studies will give a better understanding of the role of MP19 in the maintenance of lens transparency and its possible role in cataract formation.