Most lepidopteran species are herbivores, and interaction with host plants affects their gene expression and behavior as well as their genome evolution. Gustatory receptors (Grs) are expected to mediate host plant selection, feeding, oviposition and courtship behavior. However, due to their high diversity, sequence divergence and extremely low level of expression it has been difficult to identify precisely a complete set of Grs in Lepidoptera. By manual annotation and BAC sequencing, we improved annotation of 43 gene sequences compared with previously reported Grs in the most studied lepidopteran model, the silkworm, Bombyx mori, and identified 7 new tandem copies of BmGr30 on chromosome 7, bringing the total number of BmGrs to 76. Among these, we mapped 68 genes to chromosomes in a newly constructed chromosome distribution map and 8 genes to scaffolds; we also found new evidence for large clusters of BmGrs, especially from the bitter receptor family. RNA-seq analysis of diverse BmGr expression patterns in chemosensory organs of larvae and adults enabled us to draw a precise organ specific map of BmGr expression. Interestingly, most of the clustered genes were expressed in the same tissues and more than half of the genes were expressed in larval maxillae, larval thoracic legs and adult legs. For example, BmGr63 showed high expression levels in all organs in both larval and adult stages. By contrast, some genes showed expression limited to specific developmental stages or organs and tissues. BmGr19 was highly expressed in larval chemosensory organs (especially antennae and thoracic legs), the single exon genes BmGr53 and BmGr67 were expressed exclusively in larval tissues, the BmGr27-BmGr31 gene cluster on chr7 displayed a high expression level limited to adult legs and the candidate CO receptor BmGr2 was highly expressed in adult antennae, where few other Grs were expressed. Transcriptional analysis of the Grs in B. mori provides a valuable new reference for finding genes involved in plant-insect interactions in Lepidoptera and establishing correlations between these genes and vital insect behaviors like host plant selection and courtship for mating.
The glycine tRNA genes in silkworm Bombyx mon contain two regulatory regions upstream of the transcription start site as identified by direct transcription of 5' deletion mutants, transcription competition, gel mobility shift assays, and footprinting. A positive regulatory region is present in the immediate 5' flanking sequences of the four tRNAGIY clones studied. This region is essential for cell-free transcription in homologous extracts. A negative regulatory region is present farther upstream, and transcription competition experiments indicate its presence in three of the four clones exmned.The posterior silk gland of the silkworm Bombyx mori is a highly specialized organ involved in the synthesis of enormous amounts of the silk protein fibroin. Fibroin is rich in glycine (43%), alanine (28%), serine (12%), and tyrosine (5%) (1). To gear the system for synthesis of large quantities of fibroin, there is a functional adaptation in the silk glands of the B. mori larvae (2). The tRNA population in the posterior silk gland becomes highly enriched for the tRNA species cognate to the predominant amino acids in fibroin (3-5). To understand how the posterior silk gland produces such high levels of defined tRNA species, we have undertaken the study of regulation of expression of glycine tRNA genes. No tRNAGlY species that are exclusive to the silk gland have been identified so far (6-8), unlike the silk gland-specific tRNAAla (9, 10). It was therefore important to compare the relative transcriptional efficiencies of more than one tRNAly gene. In this paper, we report the presence of two regulatory regions for these tRNA GlY genes. An upstream positive regulatory region, within 40 base pairs (bp) of the transcription start site, is required for cell-free transcription of the clones studied. A negative regulatory element is present further upstream in three of the four clones examined. MATERIALS AND METHODSConstruction of Deletion Mutants. The six tRNAGly genes used in this study were a gift from A. Fournier. These clones were isolated from a B. mori genomic library in the phage A vector Charon 4. We have subcloned one of these, pBma2 (8) as an EcoRI-Sau3Al fragment in pUC18, which was designated pR8. The deletion mutants were constructed from this subclone. The 5' deletion constructs include pX3 (-2 to +108), pSX (-40 to +108), and pKX (-150 to +108). The subclone pA3 has all of the upstream sequences of pR8 (-300) and the coding region up to the Sma I site (+53). These constructs are listed in Fig. 1B.Preparation of Nuclear Extracts and in Vitro Transcription. Nuclear extracts from posterior silk glands of the fifth instar larvae were prepared as described for HeLa cell extracts (11) with modifications. The glands were homogenized in a buffer containing 2 M sucrose, 10o (vol/vol) glycerol, 10 mM Hepes (pH 7.9), 15 mM KCI, 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride. The homogenate was layered on a 3-ml cushion of the same buffer and centrifuged at 26,000 rpm for 1 hr at 40C in a Beckman SW-4...
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