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.
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| Jiang, Peihua; Ji, Qingzhou; Liu, Zhan et al. (2004) The cysteine-rich region of T1R3 determines responses to intensely sweet proteins. J Biol Chem 279:45068-75 |
