Molecular cloning and characterization of a cDNA encoding a larval cuticle protein of Bombyx mori

Department of Biology, Tokyo Metropolitan University, Minamiosawa, Japan.
Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology (Impact Factor: 1.55). 02/1999; 122(1):105-9. DOI: 10.1016/S0305-0491(98)10151-7
Source: PubMed


Cuticle proteins termed LCPs are the major protein components of the larval integument of the silkworm, Bombyx mori. We purified an 18 kDa LCP (LCP18) from the guanidine hydrochloride extract of the larval cuticle and identified an LCP18 cDNA clone. The deduced primary structure and mRNA expression pattern of LCP18 are similar to those of other Bombyx LCPs and to several cuticle proteins identified in other insect species. RNA blot analysis demonstrated that the biosynthesis of LCP18 is regulated in a stage-dependent manner at the level of mRNA in epidermal cells. An in vivo study using a juvenile hormone analogue suggested that juvenile hormone positively regulates expression of LCP18 mRNA during larval intermolt stages.

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    • "BmorCPR4 is also expressed abundantly in epV3 (30 clones; 12.9% of the epV3 cuticular protein transcripts), which is consistent with the former observation that this gene is also expressed in the intermolt stage [30]. BmorCPR4, the main component of the larval cuticle, is suggested to be orthologous to Hyalophora cecropia HCCP12 and Manduca sexta MSCP14.6 [30]. Similarly, BmorCPR38 and BmorCPR3 (5.7% and 3.1% of the epM cuticular protein transcripts, respectively) are abundantly expressed not only in epM but also in epV3 (5.6% and 8.6% of the epV3 cuticular protein transcripts, respectively; Figure 3). "
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    ABSTRACT: The insect cuticle is composed of various proteins and formed during the molt under hormonal regulation, although its precise composition and formation mechanism are largely unknown. The exhaustive catalogue of genes expressed in epidermis at the molt constitutes a massive amount of information from which to draw a complete picture of the molt and cuticle formation in insects. Therefore, we have catalogued a library of full-length cDNAs (designated epM) from epidermal cells during the last larval molt of Bombyx mori. Of the 10,368 sequences in the library, we isolated 6,653 usable expressed sequence tags (ESTs), which were categorized into 1,451 nonredundant gene clusters. Seventy-one clusters were considered to be isoforms or premature forms of other clusters. Therefore, we have identified 1,380 putative genes. Of the 6,653 expressed sequences, 48% were derived from 92 cuticular protein genes (RR-1, 24; RR-2, 17; glycine-rich, 29; other classes, 22). A comparison of epM with another epidermal EST data set, epV3 (feeding stage: fifth instar, day 3), showed marked differences in cuticular protein gene. Various types of cuticular proteins are expressed in epM but virtually only RR-1 proteins were expressed in epV3. Cuticular protein genes expressed specifically in epidermis, with several types of expression patterns during the molt, suggest different types of responses to the ecdysteroid pulse. Compared with other Bombyx EST libraries, 13 genes were preferentially included in epM data set. We isolated 290 genes for proteins other than cuticular proteins, whose amino acid sequences retain putative signal peptides, suggesting that they play some role in cuticle formation or in other molting events. Several gene groups were also included in this data set: hormone metabolism, P450, modifier of cuticular protein structure, small-ligand-binding protein, transcription factor, and pigmentation genes. We have identified 1,380 genes in epM data set and 13 preferentially expressed genes in epidermis at the molt. The comparison of the epM and other EST libraries clarified the totally different gene expression patterns in epidermis between the molting and feeding stages and many novel tissue- and stage-specifically expressed epidermal genes. These data should further our understanding of cuticle formation and the insect molt.
    BMC Genomics 09/2008; 9(1):396. DOI:10.1186/1471-2164-9-396 · 3.99 Impact Factor
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    • "The conventional polyA addition site (AATAAA) was found in 84% of the CPR genes within the first 500 nucleotides after the stop codon. Alternative sites (AATACA, AATATA, AATTAA) have been reported from some Bombyx cuticular protein genes [26,27]. We found the first two types in 14 additional genes. "
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    ABSTRACT: The most abundant family of insect cuticular proteins, the CPR family, is recognized by the R&R Consensus, a domain of about 64 amino acids that binds to chitin and is present throughout arthropods. Several species have now been shown to have more than 100 CPR genes, inviting speculation as to the functional importance of this large number and diversity. We have identified 156 genes in Anopheles gambiae that code for putative cuticular proteins in this CPR family, over 1% of the total number of predicted genes in this species. Annotation was verified using several criteria including identification of TATA boxes, INRs, and DPEs plus support from proteomic and gene expression analyses. Two previously recognized CPR classes, RR-1 and RR-2, form separate, well-supported clades with the exception of a small set of genes with long branches whose relationships are poorly resolved. Several of these outliers have clear orthologs in other species. Although both clades are under purifying selection, the RR-1 variant of the R&R Consensus is evolving at twice the rate of the RR-2 variant and is structurally more labile. In contrast, the regions flanking the R&R Consensus have diversified in amino-acid composition to a much greater extent in RR-2 genes compared with RR-1 genes. Many genes are found in compact tandem arrays that may include similar or dissimilar genes but always include just one of the two classes. Tandem arrays of RR-2 genes frequently contain subsets of genes coding for highly similar proteins (sequence clusters). Properties of the proteins indicated that each cluster may serve a distinct function in the cuticle. The complete annotation of this large gene family provides insight on the mechanisms of gene family evolution and clues about the need for so many CPR genes. These data also should assist annotation of other Anopheles genes.
    BMC Genomics 02/2008; 9(1):22. DOI:10.1186/1471-2164-9-22 · 3.99 Impact Factor
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    ABSTRACT: Juvenile hormone esterase (JHE) from hemolymph of the silkworm moth Bombyx mori was characterized for substrate specificity and inhibitor sensitivity. B. mori JHE hydrolyzed the juvenile hormone surrogate substrate methyl n-heptylthioacetothioate (HEPTAT) more efficiently than p-nitrophenyl acetate and 1-naphthyl acetate substrates widely used to assay total carboxylesterase activity. B. mori JHE was sensitive to 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP), which was developed as a selective inhibitor for lepidopteran JHE, and relatively insensitive to diisopropyl fluorophosphate (DFP), an inhibitor of serine esterases but not of all JHEs. Affinity purification with a trifluoromethyl ketone ligand was more efficient for purification of B. mori JHE than DEAE ion exchange chromatography.
    Bioscience Biotechnology and Biochemistry 09/2000; 64(8):1681-7. DOI:10.1271/bbb.64.1681 · 1.06 Impact Factor
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