Nature © Macmillan Publishers Ltd 1997 letters to nature 624 NATURE | VOL 387 | 5 JUNE 1997 with RsaI before cDNA subtraction. cDNA prepared from RNA isolated on day 3 of induction was subtracted using cDNA prepared from undifferentiated cell RNA using a polymerase chain reaction (PCR)-select cDNA subtraction kit (Clonetech). The remaining cDNAs were randomly subcloned into a T-vector (Promega). Sixty-six clones were sequenced, and their sequences were com- pared with those in the GenBank/EMBL/DDBJ database. One clone (5m-1) was found to encode the 3'-UTR of a previously isolated seven-span orphan receptor 9 . The cDNA corresponding to the ORF of the orphan receptor was amplified by PCR from 1 g of human genomic DNA. The primers used for PCR were 5'-CGGGATCCCGATGGCGTCAGGAAACCCTTG-3' (sense), and 5'-GGAATTCCTAGTTCAGTTCGTTTAACTTG-3' (antisense). The PCR con- ditions were as follows: denaturation at 96 °C for 1 min, annealing at 55 °C for 1 min, elongation at 72 °C for 3 min; 30 cycles. The amplified fragment was randomly labelled with [ 32 P]dCTP, and was used to screen an HL-60 cDNA library, which was constructed in  Zap-II (Stratagene) from 5 g poly(A) þ RNA of HL-60 cells differentiated by exposure to 1 M retinoic acid for 3 days. 5  10 5 independent clones were screened and five clones (HL-1 to HL-5) were isolated by high-stringency washing. DNA sequencing revealed that HL-1 and HL-5 contain identical full-length ORFs. The ORF of HL-5 was subcloned in the mammalian expression vector pcDNA3 (Invitrogen), and the resulting plasmid designated pLTBR. Northern blot analysis. Poly(A) þ RNA (3 g) from HL-60 and U-937 cells was electrophoresed in a 1% agarose gel, and transferred to a Hybond-N nylon membrane (Amersham). Human multiple tissue northern blot filters I and II were purchased from Clonetech. The filters were hybridized with [ 32 P]dCTP- labelled ORF of the HL-5 clone or a human glutaraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA (Clonetech) in hybridization buffer contain- ing 4  SSC, 5  Denhardt 0 s solution, 0.2% SDS, 200 g ml -1 salmon sperm DNA, 50% formamide at 42 °C for 24 h. The filters were washed in 0:1  SSC, 0.1% SDS at 65 °C and subjected to autoradiography. Expression in mammalian cells and characterization. Cos-7, HEK-293 and C6-15 cells were cultured in DMEM, and CHO cells in F12 medium. Both media contained 10% fetal calf serum. DNA transfection was performed by lipofection using Transfectam (Gibco BRL) for Cos-7, HEK-293, and CHO cells 28 , or a calcium phosphate method for C6-15 glioma cells 27 . The membrane fractions were prepared as described 14 . Stable transformants were selected with 1 g l -1 Geneticin (Gibco BRL), and cloned by limiting dilution. Clones that showed increases in intracellular calcium following exposure to 100 nM LTB 4 were selected for further analysis. LTB 4 binding assay and measurements of cAMP, InsP 3 , and intracellular calcium were carried out using established protocols 19,28 . Chemotaxis assay. Polycarbonate filters with 8-m pores (Neuroprobe) were coated with 13.3 g ml -1 fibronectin (Sigma) in PBS for 60 min 29 . A dry coated filter was placed on a 96-blind well chamber (Neuroprobe) containing the indicated amounts of LTB 4 , and the CHO cells (200 l, 8  10 4 per well) were added to the top wells. The ligand solution and cell suspension were prepared in the same buffer (F-12 medium containing 0.1% BSA). After incubation at 37 °C in 5% CO 2 for 4 h, the filter was disassembled. The cells on the filter were fixed with methanol and stained with a Diff-Quick staining kit (International Reagents Corp.). The upper side of the filter was then scraped free of cells. 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T.Y. is a research fellow of the Japan Society for the Promotion of Science. Correspondence and requests for materials should be addressed to T.S. (e-mail: tshimizu@m.u-tokyo. ac.jp). DNA and amino-acid sequences will appear in the GenBank/EMBL/DDBJ databases under accession numbers D89078 and D89079. The three-dimensional structure of aquaporin-1 Thomas Walz*†, Teruhisa Hirai‡, Kazuyoshi Murata‡, J. Bernard Heymann*, Kaoru Mitsuoka‡, Yoshinori Fujiyoshi§, Barbara L. Smith¶, Peter Agre¶ & Andreas Engel* * M. E. Mu ¨ller-Institute for Microscopic Structural Biology at the Biozentrum, University of Basel, Basel CH-4056, Switzerland ‡ International Institute for Advanced Research, Matsushita Electric Industrial Co., Ltd., 3-4 Hikaridai, Seika 619-02, Japan § Department of Biophysics, Faculty of Science, Kyoto University, Kitashirakawa, Sakyo-Ku, Kyoto, 606-01, Japan ¶ Departments of Biological Chemistry and Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, USA ......................................................................................................................... The entry and exit of water from cells is a fundamental process of life. Recognition of the high water permeability of red blood cells led to the proposal that specialized water pores exist in the plasma membrane 1 . Expression in Xenopus oocytes and functional studies † Present address: Krebs Institute for Biomolecular Biology, Department of Molecular Biology and Biotechnology, University of Sheffield, PO Box 594, Sheffield S10 2UH, UK.