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Recent progress in biological clock research has facilitated genetic analysis of circadian rhythm sleep disorders, such as delayed sleep phase syndrome (DSPS) and non-24-h sleep-wake syndrome (N-24). We analyzed the human period3 (hPer3) gene, one of the human homologs of the Drosophila clock-gene period (Per), as a possible candidate for rhythm disorder susceptibility. All of the coding exons in the hPer3 gene were screened for polymorphisms by a PCR-based strategy using genomic DNA samples from sleep disorder patients and control subjects. We identified six sequence variations with amino acid changes, of which five were common and predicted four haplotypes of the hPer3 gene. One of the haplotypes was significantly associated with DSPS (Bonferroni's corrected P = 0.037; odds ratio = 7.79; 95% CI 1.59-38.3) in our study population. Our results suggest that structural polymorphisms in the hPer3 gene may be implicated in the pathogenesis of DSPS.

Using an expression cloning strategy, a highanit meltonin receptor cDNA has been isolated from Xenopus laei dermal melanophores. Transient expression of the cDNA in COS-7 cells resulted in h-affinity 2-[125I-odomneatonn binding (Kd = 6.3 ± 0.3 x 10-11 M). In addition, six ligands exhibited a rank order of inhibition of specific 2-uI has revealed the presence of high-affinity melatonin receptors (Kd < 2 x 10-10 M) (4-6). Receptor affinity is sensitive to guanine nucleotides and activation of the receptors consistently leads to inhibition of adenylyl cyclase through a pertussis toxin-sensitive mechanism (7-11). High-affinity melatonin receptors thus appear to belong to the superfamily of guanine nucleotide binding protein (G protein)-coupled receptors. To better understand how melatonin acts at a cellular and molecular level, it is important to determine melatonin receptor structure.One of the earliest described actions of melatonin is its ability to cause melanin aggregation in dermal melanophores of amphibians (for review, see ref. 12). This action is mediated through a high-affinity melatonin receptor that is coupled to inhibitory G protein (Gi) (13,14). Using a cDNA library constructed from an immortalized cell line ofXenopus dermal melanophores and a mammalian cell expression cloning strategy, we have succeeded in cloning a high-affinity melatonin receptor. The cDNA encodes a protein that is a newly discovered member of the G protein-coupled receptor family. 11 MATERIALS AND METHODS Expression Cloning. Xenopus laevis dermal melanophores were cultured as described (15). The melanophores were derived from a clonal line that was isolated from a primary culture. Total cellular RNA was isolated from melanophores by extraction with guanidinium thiocyanate and separation in cesium chloride. Melanosomes were removed as described before centrifugation on the cesium chloride gradient (16). Poly(A)+ RNA was isolated by established methods (17).A random-primed cDNA library was constructed with a kit from Pharmacia. Double-stranded cDNA was ligated with nonpalindromic BstXI/EcoRI adaptors (Invitrogen). The cDNA was size-fractionated on an agarose gel, and cDNA>2 2 kb was recovered by electroelution. The size-selected cDNA was ligated into the expression vector pcDNAI (Invitrogen) and introduced into Escherichia coli MC1061/P3 by electroporation.A total of 4 x 105 recombinants were obtained from 5 pg of poly(A)+ RNA and divided into 54 pools of "7400 clones each. Plasmid DNA was prepared from each pool by the alkaline lysis method and transfected into COS-7 cells by the DEAE-dextran method (18). Three days after transfection, cells were incubated with 90 pM 125I-Mel in 50 mM Tris-HCl (pH 7.4) containing 100 mM NaCI, 5 mM KCl, 2 mM CaCl2, and 5% Nu-Serum I (Collaborative Biomedical Products, Bedford, MA) for 2 hr at room temperature. Cells were washed, air dried, and exposed to x-ray film for 14 days. A pool of clones that showed positive signals was subdivided, and the transfection procedure was repeated. This ...

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