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, BmKrh1 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-loophelix 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 JHdependent 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 JHmediated 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. development | insecticide | steroid receptor coactivator

The Bombyx mori homolog of doublesex, Bmdsx, plays an essential role in silkworm sexual development. Exons 3 and 4 of Bmdsx pre-mRNA are specifically excluded in males. To explore how this occurs, we developed a novel in vivo sex-specific splicing assay system using sexually differentiated cultured cells. A series of mutation analyses using a Bmdsx minigene with this in vivo splicing assay system identified three distinct sequences (CE1, CE2, and CE3) positioned in exon 4 as exonic splicing silencers responsible for male-specific splicing. Gel shift analysis showed that CE1 binds to a nuclear protein from male cells but not that from female cells. Mutation of UAA repeats within CE1 inhibited the binding of the nuclear protein to the RNA and caused female-specific splicing in male cells. We have identified BmPSI, a Bombyx homolog of P-element somatic inhibitor (PSI), as the nuclear factor that specifically binds CE1. Down-regulation of endogenous BmPSI by RNA interference significantly increased female-specific splicing in male cells. This is the first report of a PSI homolog implicated in the regulated sex-specific splicing of dsx pre-mRNA.Sexual reproduction is the primary means to maintain variation for evolutionary survival. Sex determination cascades that drive the differentiation of dimorphic gonads, however, are some of the most rapidly evolved developmental events (28). Genetic and environmental cues direct the generation of two distinct sexual phenotypes via several key molecular signals. In Drosophila melanogaster and Caenorhabditis elegans, the ratio of X chromosomes to autosomes (X/A) is the primary signal for sex determination (19,23). In mammals, however, maleness is determined by the Y-linked gene SRY (17,41). Nevertheless, recent studies indicate some downstream sex determination genes are functionally similar in diverse species. Of these, DM domain (Doublesex/Mab-3 DNA-binding motif) genes have been shown in the past several years to regulate sexual development in multiple metazoan phyla, including arthropods, nematodes, and vertebrates, and thus this gene family may represent the first example of widespread conservation of sexual regulatory genes (20,46). The DM domain is a cysteine-rich DNA-binding motif first recognized in proteins encoded by the Drosophila sex determination gene doublesex (10, 48). As the name doublesex (dsx) suggests, this gene functions in both sexes: the primary transcript of dsx undergoes sex-specific alternative splicing, producing either a male-specific isoform, DSXM, or a female-specific isoform, DSXF (3, 6). Throughout most of the somatic tissues of the fruit fly, DSXM directs male development and DSXF directs female development.The mechanism of sex-specific dsx splicing has been well studied. Female-specific splicing of dsx requires TRA, TRA-2, and an exonic splicing enhancer (ESE) element located within the untranslated region of the fourth exon (29,34,35). Both TRA and TRA-2 contain Arg/Ser-rich (RS) domains, protein interaction domains characteristic of the Ser/Ar...

We identified a novel, 6,513-bp-long RNA, termed Bombyx mori macula-like latent virus (BmMLV) RNA, which abundantly expressed in B. mori cultured BmN cells. BmMLV RNA potentially encodes two proteins, putative RNA replicase and coat protein, which share structural features and sequence similarities with those of a plant RNA virus, the genus Maculavirus. Northern blot analysis showed that two transcripts were expressed in BmN cells: a 6.5-kb-long RNA, which contains both putative RNA replicase and coat protein genes, and a 1.2-kb-long RNA, which contains only a coat protein gene. Southern blot analysis showed that BmMLV RNA is not carried by the B. mori genome. RT-PCR analysis also revealed the presence of BmMLV RNA in several B. mori cell lines other than BmN cells, suggesting that BmMLV RNA latently exists in B. mori cultured cells. Infection studies showed that BmMLV virions were able to infect BmMLV-negative Spodoptera frugiperda Sf-9 cells and B. mori larvae. Electron microscopy and Northern blot analysis of a purified BmMLV revealed that isometric virions appear to be 28 to 30 nm in diameter and contain a 6.5-kb genomic RNA. These results showed that BmMLV is a novel macula-like virus infectious to and replicable in B. mori-derived cells.

Bmdsx is a sex-determining gene in the silkworm and is alternatively spliced in males and females. CE1 is a splicing silencer element responsible for the sex-specific splicing of Bmdsx. To identify sex-specific factors implicated in the sex-specific splicing of Bmdsx, we performed RNA affinity chromatography using CE1 RNA as a ligand. We have identified BmIMP, a Bombyx homolog of IGF-II mRNA binding protein (IMP), as a male-specific factor that specifically binds to CE1. The gene encoding BmIMP is localized on the Z chromosome and is male-specifically expressed in various tissues. Antisense inhibition of BmIMP expression increased female-specific splicing of Bmdsx pre-mRNA. Coimmunoprecipitation and glutathione S-transferase (GST) pulldown analyses demonstrated that BmIMP physically interacts with BmPSI, which has been identified as a factor implicated in the sex-specific splicing of Bmdsx, through the KH domains of BmIMP. The functional consequence of this interaction was examined using RNA mobility shift analysis. BmIMP increased BmPSI-CE1 RNA binding activity by decreasing the rate of BmPSI dissociation from CE1 RNA. Truncation analysis of BmIMP suggested that the KH domains are responsible for enhancing BmPSI-CE1 RNA binding activity. These results suggest that BmIMP may enhance the male-specific splicing of Bmdsx pre-mRNA by increasing RNA binding activity of BmPSI.Alternative splicing of pre-mRNA is an essential mechanism for differential gene expression that can produce functionally distinct proteins from a single gene according to the developmental or physiological state of cells in multicellular organisms (4,17). Recent studies estimate that about 70% of human genes are alternatively spliced (29), most strikingly, causing several thousands of different mRNA isoforms from a single gene. Although many examples describe how alternative splicing regulates gene expression, the mechanisms involved in the alternative splicing are less well understood (5,9,13,27,28,51). One of the best-studied systems in which the functions of splicing factors are known derives from the Drosophila melanogaster sex determination cascade. At the top of this cascade is the Sex-lethal (Sxl) gene. The functional protein of this master switch gene is produced early in development according to the primary sex determination signal, the X chromosome-to-autosome ratio (X:A ratio) (10,12,14,21). When the functional Sxl protein is produced, it directs the female-specific splicing of its downstream gene transformer (tra), giving rise to functional Tra protein (6). Tra, together with Tra2, binds to the cisregulatory element (dsxRE) within the female-specific exon of its downstream gene doublesex (dsx), activating a weak 3Ј splicing site preceding the female-specific exon to generate the female-type Dsx protein (Dsx F ), which regulates its downstream genes for female development. In males, the X:A ratio of 0.5 inhibits Sxl from being produced, causing male-specific splicing of tra, making its encoded product nonfunctional due to a premature ...

Pv11, a cell line derived from the anhydrobiotic insect, Polypedilum vanderplanki, was preserved in a dry form (only 6% residual moisture) at room temperature for up to 251 days and restarted proliferating after rehydration. A previous study already reported survival of Pv11 cells after desiccation, but without subsequent proliferation. Here, the protocol was improved to increase survival and achieve proliferation of Pv11 cells after dry storage. The method basically included preincubation, desiccation and rehydration processes and each step was investigated. So far, preincubation in a 600 mM trehalose solution for 48 h before dehydration was the most favourable preconditioning to achieve successful dry preservation of Pv11 cells, allowing about 16% of survival after rehydration and subsequent cell proliferation. Although the simple air-dry method established for Pv11 cells here was not applicable for successful dry-preservation of other insect cell lines, Pv11 is the first dry-preservable animal cell line and will surely contribute not only to basic but also applied sciences.

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