ADENOVIRUS MEDIATED REGULATION OF PINEAL PHOTORESPONSES

描述（申请人摘要）：内源性生物钟允许生物体 来适当地安排他们的行为和生理反应， 环境周期，如每天的光照周期和一天中的季节变化 长度脊椎动物生物钟的一个重要输出是松果体激素 褪黑素生物钟控制的褪黑激素合成节律 被每日的光照周期重置褪黑激素的合成受到调节 独立于生物钟和光线。因此，光调节 褪黑激素的合成通过两条途径。松果体细胞培养提供了一种有用的 系统来研究这两种途径;松果体细胞有节奏地释放 褪黑激素在培养物中培养数周，它们在体外的节律被 光药理学方法已被广泛用于体外研究 松果体节律;然而，没有遗传操纵这个系统已经 这是因为通过转基因或“敲除”方法来改变 鸡的特定基因不可能被遗传操纵 松果体细胞基因表达复制缺陷型重组腺病毒 表达有义、突变、核糖体或反义形式的松果体cDNA 将用于感染松果体细胞。P-视蛋白，一种松果体特异性视蛋白， 在松果体细胞培养物中表达和“敲除”。松果体表达 G蛋白α杆转导蛋白将被敲除， 具有延长激活的转导素α突变体将在 松果体细胞cGMP PDE的野生型γ亚基和两种突变形式，一种是 一个是过度活跃的，一个是功能减退的，将被表达为 松果体细胞这些基因操作对褪黑激素的影响 生物合成和节律性以及松果体细胞的光敏性将 通过静态和流通培养以及放射免疫测定法进行评估 褪黑激素具体预测的影响，这些 松果体对光的反应的分子遗传操纵， 小鸡体内存在视网膜样光传导通路的假说 松果体细胞

DESCRIPTION (applicant's abstract): Endogenous circadian clocks allow organisms to appropriately time their behavioral and physiological responses to environmental cycles such as daily light cycles and seasonal changes in day length. A prominent output of the clock in vertebrates is the pineal hormone melatonin. The rhythm of melatonin synthesis controlled by the circadian clock is reset by the daily light cycle. Melatonin synthesis is regulated independently by the circadian clock and by light. Thus, light regulates melatonin synthesis via two pathways. Pinealocyte cultures provide a useful system to study both these pathways; pinealocytes rhythmically release melatonin for weeks in culture and their rhythms in vitro are entrained by light. Pharmacological methods have been extensively used to study in vitro pineal rhythms; however, no genetic manipulation of this system has been achieved because transgenic or "knock out" methods to alter expression of specific genes have not been possible in chickens to genetically manipulate pinealocyte gene expression. Replication-deficient, recombinant adenoviruses that express sense, mutant, ribosomal or antisense versions of pineal cDNAs will be used to infect pinealocytes. P-opsin, a pineal-specific opsin, will be expressed and "knocked out" in pinealocyte cultures. The pineal-expressed G-protein alpha rod transducin will be knocked out and a GTPase-deficient rod transducin alpha mutant with prolonged activation will be expressed in pinealocytes. The wild type gamma subunit of cGMP PDE and two mutant forms, one that is hyperactive and one that is hypoactive, will be expressed in pinealocytes. The effects of these genetic manipulations on melatonin biosynthesis and rhythmicity and the photosensitivity of the pinealocytes will be assessed by both static and flow through cultures and by radioimmunoassay for melatonin. Specific predictions are made for the effects of these molecular-genetic manipulations on the pineal's response to light based upon the hypothesis that a retina-like phototransduction pathway exists in the chick pinealocyte.