Toru Togawa

Transcriptional regulation of juvenile hormone-mediated induction of Krüppel homolog 1, a repressor of insect metamorphosis

The Krüppel homolog 1 gene (Kr-h1) has been proposed to play a key role in the repression of insect metamorphosis. Kr-h1 is assumed to be induced by juvenile hormone (JH) via a JH receptor, methoprene-tolerant (Met), but the mechanism of induction is unclear. To elucidate the molecular mechanism of Kr-h1 induction, we first cloned cDNAs encoding Kr-h1 (BmKr-h1) and Met (BmMet1 and BmMet2) homologs from Bombyx mori. In a B. mori cell line, BmKr-h1 was rapidly induced by subnanomolar levels of natural JHs. Reporter assays identified a JH response element (kJHRE), comprising 141 nucleotides, located ∼2 kb upstream from the BmKr-h1 transcription start site. The core region of kJHRE (GGCCTCCACGTG) contains a canonical E-box sequence to which Met, a basic helix–loop–helix Per-ARNT-Sim (bHLH–PAS) transcription factor, is likely to bind. In mammalian HEK293 cells, which lack an intrinsic JH receptor, ectopic expression of BmMet2 fused with Gal4DBD induced JH-dependent activity of an upstream activation sequence reporter. Meanwhile, the kJHRE reporter was activated JH-dependently in HEK293 cells only when cotransfected with BmMet2 and BmSRC, another bHLH–PAS family member, suggesting that BmMet2 and BmSRC jointly interact with kJHRE. We also found that the interaction between BmMet2 and BmSRC is dependent on JH. Therefore, we propose the following hypothesis for the mechanism of JH-mediated induction of BmKr-h1: BmMet2 accepts JH as a ligand, JH-liganded BmMet2 interacts with BmSRC, and the JH/BmMet2/BmSRC complex activates BmKr-h1 by interacting with kJHRE.

Annotation and analysis of a large cuticular protein family with the R&R Consensus in Anopheles gambiae

BackgroundThe 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.ResultsWe 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.ConclusionThe 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.

Developmental expression patterns of cuticular protein genes with the R&R Consensus from Anopheles gambiae

CPR proteins are the largest cuticular protein family in arthropods. The whole genome sequence of Anopheles gambiae revealed 156 genes that code for proteins with the R&R Consensus and named CPRs. This protein family can be divided into RR-1 and RR-2 subgroups, postulated to contribute to different regions of the cuticle. We determined the temporal expression patterns of these genes throughout post-embryonic development by means of real-time qRT-PCR. Based on expression profiles, these genes were grouped into 21 clusters. Most of the genes were expressed with sharp peaks at single or multiple periods associated with molting. Genes coding for RR-1 and RR-2 proteins were found together in several co-expression clusters. Twenty-five genes were expressed exclusively at one metamorphic stage. Five out of six X-linked genes showed equal expression in males and females, supporting the presence of a gene dosage compensation system in A. gambiae. Many RR-2 genes are organized into sequence clusters whose members are extremely similar to each other and generally closely associated on a chromosome. Most genes in each sequence cluster are expressed with the same temporal expression pattern and at the same level, suggesting a shared mechanism to regulate their expression.

Purification and cDNA cloning of evolutionally conserved larval cuticle proteins of the silkworm, Bombyx mori

A specific set of structural proteins termed larval cuticle proteins (LCPs) accumulates in integuments during larval development of the silkworm, Bombyx mori. Two major larval cuticle proteins, LCP17 and LCP22, were purified from the guanidine hydrochloride extract of the larval cuticle, and specific antibodies were raised against these proteins. Immunoblot analysis revealed that both LCPs are actively synthesized during larval intermolt stages, whereas the LCP17 epitope is also slightly but significantly detectable in pupal integuments. cDNA clones for LCPs were isolated by immunoscreening of the cDNA expression library constructed from larval epidermal mRNA. Predicted amino acid sequences of LCP17 and LCP22 are homologous to cuticle proteins from other insect species, including Manduca sexta, Drosophila melanogaster and Locusta migratoria. This fact suggests that these cuticle protein genes originated from a common ancestral gene and have been conserved during evolution. Northern blot hybridization demonstrated that the expression of LCP17 as well as LCP22 mRNA is controlled in a stage-specific manner in the epidermis of the final instar larvae, suggesting a common regulatory mechanism for transcription of these two intermolt genes.

Overexpression of grappa encoding a histone methyltransferase enhances stress resistance in Drosophila

Histone deacetylases, such as silent information regulator 2 (Sir2) and Rpd3 are involved in chromatin silencing and implicated in lifespan determination in several organisms. The yeast Dot1 gene encoding a histone methyltransferase affects localization of silencing proteins including Sir2, and plays an essential role in the repair of damaged DNA. However, it is not known whether an alteration of a histone methyltransferase activity influences lifespan or stress resistance, which is often associated with extended lifespan. Here we investigated whether the Drosophilagrappa (gpp) gene, a Dot1 homolog influences lifespan and stress resistance using transgenic flies overexpressing gpp and those bearing a partial loss-of-function mutation. Overexpression of gpp throughout the adult stage did not extend the lifespan, but significantly enhanced resistances when they were kept on medium containing 1% H(2)O(2), or those with poor nutrients. As well, gpp-overexpressing flies were behaviourally more active than control flies. We investigated whether gpp overexpression induced anti-oxidant genes, Catalase, Sod, Sod2, GstD2, dhd, TrxT and Trx-2. However, none of these genes was induced. A partial loss-of-function mutations in gpp dramatically reduced the lifespan under oxidative and caloric stresses. Taken together, these results demonstrated that gpp is required for normal lifespan and stress resistance, and that its overexpression increases stress resistance in Drosophila, without obvious induction of representative anti-oxidant genes.

Efficient measurement of H2O2 resistance in Drosophila using an activity monitor

We have established a method for the efficient measurement of oxidative stress resistance inDrosophila melanogaster, using a commercially available activity monitor. Conditions under which flies in glass tubes placed in the monitor can survive over one month at 25 °C were optimized. The active periods of flies were reduced by administration of H2O2 into the media in a dose-dependent manner. Although we used only eight flies per assay, far fewer individuals than in conventional methods, it was possible to detect the effects of H2O2 at a statistically significant level. Increased levels of H2O2 resistance were confirmed in transgenic flies overexpressing antioxidant enzymes, catalase or Cu/Zn superoxide dismutase. We applied the method to determine oxidative stress resistance in fly lines bearing insertions of a gene misexpression vector. H2O2 resistance in these flies varied considerably depending on the insertion, and positively correlated with previously determined longevity. We identified one insertion that conferred a significantly higher level of resistance to H2O2compared to controls. Molecular analysis of the insertion revealed that a misexpressed transcript matched an expressed sequence tag, and suggested that its full-length product was overproduced upon GAL4 activation. Our method should be applicable to the systematic screening for genes involved in the antioxidant mechanism in Drosophila.

Molecular cloning and characterization of a cDNA encoding a novel cuticle protein in the silkworm, Bombyx mori

We have cloned the full length of a novel cDNA named Bombyx mori cuticle protein that contains an AlaAlaProAla/Val-repeat (BMCPA) from a cDNA library of integument in the larval silkworm. Both a typical tandem repeat (A-A-P-A/V) for cuticle protein and a unique tandem repeat with Ser, Ala, Gly, Pro, Val, Tyr and Thr were observed in the predicted amino acid sequence of the cDNA encoding BMCPA. Approximately 80% of the amino acids in BMCPA were composed of Ser, Ala, Gly, Pro, Val and Tyr. Northern-hybridization analysis indicated that BMCPA mRNA is expressed only in the larval epidermis and that the expression pattern of the BMCPA gene in the developmental stage was observed mainly at the larval stage. We propose BMCPA may be a novel component of cuticle, and may play an important role in the integument of the larval silkworm.

Developmental Changes in the Localization of Protein Kinase CK2 in Non-Diapause and Diapause Eggs of the Silkworm, Bombyx mori

To analyze the role of protein kinase CK2 (CK2) during early embryogenesis in non-diapause and diapause of the silkworm, the distribution and localization of Bombyx mori CK2 (BmCK2) were investigated by an immunohistochemical technique using antibodies against the &agr;- and &bgr;-subunits of BmCK2. Both were localized in blastoderm cells of non-diapause and diapause eggs until 24 h after oviposition. More than 24 h after oviposition, however, the distribution of BmCK2 was different in non-diapause and diapause eggs. In non-diapause eggs, BmCK2 was mainly localized in yolk cells. In contrast, in diapause eggs, the localization was mainly observed in germ-band cells. Furthermore, we confirmed that the RNA helicase-like protein that was localized together with BmCK2 in non-diapause eggs was phosphorylated by BmCK2 in vitro. These data suggest that the role of BmCK2 is different in non-diapause and diapause eggs.