Sex pheromones released by female moths are detected with high specificity and sensitivity in the olfactory sensilla of antennae of conspecific males. Bombykol in the silkmoth Bombyx mori was the first sex pheromone to be identified. Here we identify a male-specific G protein-coupled olfactory receptor gene, B. mori olfactory receptor 1 ( BmOR-1 ), that appears to encode a bombykol receptor. The BmOR-1 gene is located on the Z sex chromosome, has an eight-exon/seven-intron structure, and exhibits male-specific expression in the pheromone receptor neurons of male moth antenna during late pupal and adult stages. Bombykol stimulation of Xenopus laevis oocytes expressing BmOR-1 and BmGαq elicited robust dose-dependent inward currents on two-electrode voltage clamp recordings, demonstrating that the binding of bombykol to BmOR-1 leads to the activation of a BmGαq-mediated signaling cascade. Antennae of female moths infected with BmOR-1 -recombinant baculovirus showed electrophysiological responses to bombykol but not to bombykal. These results provide evidence that BmOR-1 is a G protein-coupled sex pheromone receptor that recognizes bombykol.

The transcription factor Bach1 heterodimerizes with small Maf proteins to repress Maf recognition element (MARE)-dependent gene expression. The repressor activity of Bach1 is inhibited by the direct binding of heme. To investigate the involvement of Bach1 in the heme-dependent regulation of the expression of the β-globin gene, mouse erythroleukemia (MEL) cells were cultured with succinylacetone (SA), a specific inhibitor of heme biosynthesis, and the level of β-globin mRNA was examined. A marked decrease of β-globin mRNA in SA-treated cells was observed, and this decrease was reversed by the addition of hemin. An iron chelator, desferrioxamine, also lowered the level of β-globin mRNA. The heme-dependent expression of β-globin is a transcriptional event since the expression of the human β-globin gene promoter-reporter gene containing the microlocus control region (μLCR) was inhibited when human erythroleukemia K562 cells and MEL cells were cultured with SA. Hemin treatment restored the decrease in promoter activity caused by SA. The control of the μLCR-β-globin promoter reporter gene by heme was dependent on DNase I-hypersensitive site 2 (HS2), which contains MARE. The MARE binding activity of Bach1 in K562 and MEL cells increased upon SA treatment, and the increase was diminished by the treatment with hemin. Transient expression of Bach1 suppressed the μLCR activity, and this repressor activity was cancelled by treatment with hemin. The expression of a mutated Bach1 lacking heme-binding sites led to a loss in the heme responsiveness of the μLCR. Furthermore, chromatin immunoprecipitation experiments revealed that Bach1 bound to the MARE of HS2 increased by the treatment of MEL cells with SA, and this was cancelled by hemin. We propose that heme positively regulates the β-globin gene expression by blocking the interaction of Bach1 with the MARE in the LCR.

The Bombyx mori fibroin light (L)-chain gene was cloned and the green fluorescent protein (GFP) gene inserted into exon 7. The chimeric L-chain–GFP gene was used to replace the polyhedrin gene of Autographa californicanucleopolyhedrovirus (AcNPV). This recombinant virus was used to target the L-chain–GFP gene to the L-chain region of the silkworm genome. Female moths were infected with the recombinant virus and then mated with normal male moths. Genomic DNA from their progenies was screened for the desired targeting event. This analysis showed that the chimeric gene had integrated into the L-chain gene on the genome by homologous recombination and was stably transmitted through generations. The chimeric gene was expressed in the posterior silk gland, and the gene product was spun into the cocoon layer.