ADENOVIRUS MEDIATED REGULATION OF PINEAL PHOTORESPONSES

描述(申请人摘要)：内源性生物钟允许生物体 适当地安排他们的行为和生理反应的时间 环境循环，例如每日的光循环和白天的季节变化 长度。脊椎动物时钟的一个重要输出是松果体激素。 褪黑激素。昼夜节律控制的褪黑素合成节律 被每日的光周期重置。褪黑激素的合成受到调节 独立于生物钟和光。因此，光可以调节 褪黑素的合成有两条途径。松果体细胞培养提供了一种有用的 系统来研究这两条途径；松果体细胞有节律性地释放 褪黑素在体外培养数周及其节律受 灯。药理学方法已被广泛用于体外研究。 松果体节律；然而，还没有对这个系统的遗传操作 这是因为转基因或“敲除”方法改变了基因的表达 特定的基因还不可能在鸡身上进行遗传操作 松果体细胞基因表达。复制缺陷的重组腺病毒 表达有义、突变、核糖体或反义松果体cDNA 将被用来感染松果体细胞。P-视蛋白，一种松果体特有的视蛋白，将被 在松果体细胞培养中表达并“敲除”。松果体-表达 G蛋白α杆状转导蛋白将被敲除，而GTPase缺陷杆状 长时间激活的转导蛋白α突变体将在 松果体细胞。CGMP PDE的野生型伽马亚基和两个突变体，一个 这是过度活跃的，而不活跃的，将在 松果体细胞。这些基因操作对褪黑素的影响 松果体细胞的生物合成、节律性和光敏性 用静止法、流通法和放射免疫测定法进行评估 褪黑激素。对这些影响做出了具体的预测 松果体对光反应的分子遗传学调控 雏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.