Identification of the new odorant receptor geneOR-Z6 at the Hlz6 locus.A, Gel electrophoresis is shown of the products obtained after PCR of the four P1 clones 6386–6389 (lanes 5–8) using the two different primer sets primers p26 and p27 and primers ps2 and ps4. All P1 clones carry the 3′-flanking fragment as demonstrated by the 160 bp amplicon derived from primers ps2 and ps4 (lanes 5–8). The 390 bp PCR product (star) derived from the degenerate OR primers p26 and p27 is only evident for P1 clone 6386 (lane 5) and the positive control (+C, lane 4) using mouse genomic DNA. The thickness of the band in the positive control (lane 4) reflects the amplification of numerous genomic OR genes. The positive control for primers ps2 and ps4 (+C, lane 2; mouse genomic DNA) shows a 160 bp amplicon, whereas negative control reactions, omitting template DNA, yielded no products for primers ps2 and ps4 (−C,lane 1) or primers p26 and p27 (−C,lane 3). HaeIII-digested ΦX174 phage DNA (M) is the length standard (lane 9). Fragment sizes in base pairs are indicated by thenumbers at the right.B, Restriction digestion (left) followed by Southern blot hybridization (right) shows that the P1 clone 6386 carries a single OR gene. Eight hundred nanograms of each P1 6386 DNA digest (E, EcoRI;S, SacI; X,XbaI) were resolved on a 0.6% agarose gel, transferred onto a nylon membrane, and subjected to Southern blot hybridization. The probe was prepared by labeling the 390 bp PCR fragment obtained inA with digoxigenin-conjugated dUTP via PCR using primers p26 and p27. Hybridizing fragment sizes were 4 kb (EcoRI), 11 kb (SacI), and 3 kb (XbaI). The 3 kb hybridizing XbaI fragment (arrowhead) was subcloned and sequenced.HindIII-digested λ DNA (M) is the length standard in kilobase pairs indicated by the numbers at theleft.