The new mouse OR-Z6 gene encodes a typical OR protein. A, Top, Partial restriction map of the 3 kb XbaI fragment carrying the complete OR-Z6 coding region (black box) and sequences 1.2 kb upstream and 1 kb downstream. Location of the gene-specific primers N and C used in RT-PCR analyses (C) is indicated by arrows. Restriction enzymes are the following: B,BamHI; E, EcoRI;S, SphI; X,XbaI. Bottom, Comparison of the deduced amino acid sequences of the coding regions of mouseOR-Z6 (mOR-Z6) and a humanOR-Z6 ortholog (hOR-Z6) inone-letter code. For hOR-Z6, only differences from the mOR-Z6 sequence are shown.Gray shading depicts the predicted seven transmembrane domains TM-1 to TM-7. The amino acid sequence domains of the two OR-Z6 proteins that match closely to the motifs conserved (indicated by a horizontal line) among OR proteins are PMYFFL (TM-2), MAVDRYVAVC (TM-3), SY (TM-5), K(A/S)FSTCASH (TM-6), and PFLNPF (TM-7). Both OR-Z6 proteins share 85% similarity over a total of 314 amino acids and exhibit cysteine residues in conserved positions (97, 179, stars) and a putative N-terminal receptor glycosylation site (arrowhead), as well as several serine and threonine phosphorylation sites in the third intracellular loop (IL-3). B, Genomic Southern blot hybridization for OR-Z6. Restriction digestion of mouse liver DNA (10 μg/lane) from wild-type (wt) and H-lacZ6 mice was as indicated (H,HindIII; S, SphI;X, XbaI). The Southern blot was hybridized with the digoxigenin-labeled OR-Z6 coding region. The DNA probe hybridized to a single fragment perlane, and the sizes of the hybridizing fragments for both wild-type and H-lacZ6 genomic DNA were identical (H, 23 kb; S, 1.72 kb; X, 3 kb). HindIII-digested λ DNA (M) is the length standard in kilobase pairs indicated by the numbers on theleft. C, RT-PCR performed with RNA from three different tissues demonstrating that OR-Z6 mRNA is expressed in olfactory tissues (OE, OB). The photograph shows a gel electrophoresis of the products derived from RT-PCR using the OR-Z6 specific primers N and C. The 899 bp amplicon was only evident in samples with preceding reverse transcription (+RT). No products were obtained from RT-PCR using liver mRNA (L) or from reactions omitting reverse transcriptase (−RT). The lower intensity of the fragment obtained from OB mRNA versus that from OE mRNA coincides with reports demonstrating that OR mRNA is much less abundant in axon terminals compared with their cell bodies (Ressler et al., 1994).