The major goal of this project is to define the structure of the retinal/pineal gene, N-acetyltransferase (NAT) and to examine NAT gene expression in visual and neural transduction. The primary objective of this five year research proposal is to answer the question: Is the retinal/pineal NAT enzyme a key component of a circadian driving oscillator and is the retinal/pineal NAT enzyme controlled by environmental cues at the molecular level? This project will investigate one specific phase of this endogenous rhythm and the entrainment by the light:dark cycle of the rate=limiting enzyme that synthesizes melatonin. The pineal is an excellent system to study all steps in neurochemical transduction, including ligand-receptor interactions, second messenger regulation, and selective regulation of specific genes. Two characteristics for studying molecular mechanisms of pineal neurotransduction are (1) innervation by sympathetic fibers involving alpha- and beta-receptors; and (2) high amplitude circadian rhythms in biochemical activities, including some that are rapidly responsive to known molecular triggers. Although the retina shares similar proteins and biochemical pathways with the pineal, the role of melatonin in the retina is not understood. The experiments will characterize the retinal/pineal tissue-specific genes coding for NAT. Normal tissue-specific expression will be analyzed in retinas and pineals of rats and inbred mice. Primary pineal cell cultures will also be used to study the role adrenergic regulation has in gene expression. The project will have three specific aims: 1. Isolate full-length cDNAs and characterize cDNA probes encoding the retinal/pineal enzyme, NAT, by screening a rat pineal expression library. 2. Generate antisera for immunological probes against recombinant fusion proteins and synthesized peptides identified from the translated cDNAs encoding NAT. 3. Apply these recombinant and immunological probes to localize, characterize and measure changes in circadian regulation and environmental modulation of their mRNA and protein for NAT. The experiments described will employ immunological and recombinant DNA tools to examine the influence of environmental cues on the genes expressed in the pineal and retina. The pineal and retina have been extensively studied by physiological, pharmacological and biochemical approaches. These studies will extend these studies concerning the fundamental molecular nature of circadian rhythms in the nervous system. Biological circadian rhythms are important not only in our daily lives, but in maintaining our health throughout life. Knowledge of the normal structure of genes expressed in the pineal and retina may lead to a better understanding the involvement of the melatonin synthesizing enzyme in visual and neural transduction.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29NS028126-03
Application #
3477954
Study Section
Neurology C Study Section (NEUC)
Project Start
1990-01-01
Project End
1994-12-31
Budget Start
1992-01-01
Budget End
1992-12-31
Support Year
3
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
Zhan-Poe, X; Craft, C M (1999) Biochemical characterization of recombinant serotonin N-acetyltransferase. J Pineal Res 27:49-58
Craft, C M; Murage, J; Brown, B et al. (1999) Bovine arylalkylamine N-acetyltransferase activity correlated with mRNA expression in pineal and retina. Brain Res Mol Brain Res 65:44-51
Craft, C M; Xu, J; Slepak, V Z et al. (1998) PhLPs and PhLOPs in the phosducin family of G beta gamma binding proteins. Biochemistry 37:15758-72
Gauer, F; Craft, C M (1996) Circadian regulation of hydroxyindole-O-methyltransferase mRNA levels in rat pineal and retina. Brain Res 737:99-109
Craft, C M; Whitmore, D H (1995) The arrestin superfamily: cone arrestins are a fourth family. FEBS Lett 362:247-55
Gauer, F; Kedzierski, W; Craft, C M (1995) Identification of circadian gene expression in the rat pineal gland and retina by mRNA differential display. Neurosci Lett 187:69-73
Carcamo, B; Hurwitz, M Y; Craft, C M et al. (1995) The mammalian pineal expresses the cone but not the rod cyclic GMP phosphodiesterase. J Neurochem 65:1085-92
Craft, C M; Whitmore, D H; Wiechmann, A F (1994) Cone arrestin identified by targeting expression of a functional family. J Biol Chem 269:4613-9
Lolley, R N; Rong, H; Craft, C M (1994) Linkage of photoreceptor degeneration by apoptosis with inherited defect in phototransduction. Invest Ophthalmol Vis Sci 35:358-62
Wiechmann, A F; Craft, C M (1993) Localization of mRNA encoding the indolamine synthesizing enzyme, hydroxyindole-O-methyltransferase, in chicken pineal gland and retina by in situ hybridization. Neurosci Lett 150:207-11

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