Two gap junction channel (innexin) genes of the Bombyx mori and their expression

Gap junctions are clusters of intercellular channels that are associated with embryonic development and neural signaling. Innexins, invertebrate gap junction proteins, have been identified in Drosophila and Caenorhabditis. Here, we report the isolation and characterization of two novel members of the insect innexin family, Bm inx2 and Bm inx4, from embryos of the silkworm, Bombyx mori, during the germ-band formation stage. Bm inx2 is a single copy gene with one exon, while Bm inx4 is a single copy gene with four exons and three introns. The predicted proteins show structural similarities with other innexin family members, including four transmembrane (TM) domains, two extracellular loops (ELs), one cytoplasmic loop (CL), and typical conserved amino acids. Bm inx2 is phylogenetically orthologous to the other insect inx2 genes, but Bm inx4 is not orthologous to any known innexin including Dm inx4. Interestingly, Northern blotting and in situ hybridization showed that Bm inx2 was variously expressed across all developmental stages and in various tissues, with high expression seen in the nervous system at the time of embryogenesis. In contrast, Bm inx4 was transiently expressed at the germ-band formation stage of embryogenesis, and was specifically expressed in the ovary and testis during the larval and pupal stages. The isolation and characterization of these novel genes should form the basis for further study of the functional events that occur during development and neuronal communication in B. mori.     

Molecular evolution of ankyrin: gain of function in vertebrates by acquisition of an obscurin/titin-binding-related domain

Ankyrins form a family of modular adaptor proteins that link between integral membrane proteins and the cytoskeleton. They evolved within the Metazoa as an adaptation for organizing membrane microstructure and directing membrane traffic. Molecular cloning has identified one Caenorhabditis elegans (unc-44), two Drosophila (Dank1, Dank2), and three mammalian (Ank1, Ank2, Ank3) genes. We have previously identified a 76-amino acid (aa) alternatively spliced sequence that is present in muscle polypeptides encoded by the rat Ank3 gene. A closely related sequence in a muscle Ank1 product binds the cytoskeletal muscle proteins obscurin and titin. This obscurin/titin-binding-related domain (OTBD) contains repeated modules of 18 aa: three are encoded by Ank1 and Ank2, two by Ank3; this pattern is conserved throughout vertebrate ankyrin genes. The C. elegans ankyrin, UNC-44, contains one 18-aa module as does the ankyrin gene in the urochordate Ciona intestinalis, but the insect ankyrins contain none. Our data indicate that an ancestral ankyrin acquired an 18-aa module which was preserved in the Ecdysozoa/deuterostome divide, but it was subsequently lost from arthropods. Successive duplications of the module led to a gain of function in vertebrates as it acquired obscurin/titin-binding activity. We suggest that the OTBD represents an adaptation of the cytoskeleton that confers muscle cells with resilience to the forces associated with vertebrate life.     

BmEts upregulates promoter activity of lebocin in Bombyx mori

The Ets family protein BmEts is assumed to be implicated in determination of diapause in the embryogenesis of Bombyx mori. In this study, we found that expression of BmEts was increased in the fat body and other tissues of the 5th instar larvae in response to Escherichia coli injection. Cotransfection experiments using a silkworm cell line revealed that overexpression of BmEts significantly elevated the activity of lebocin promoter but not of cecropin B1, cecropin D, attacin, and moricin promoters. Activation of the lebocin promoter by BmEts was dependent on at least two κB elements and the most proximal GGAA/T motif located on the 5'-upstream region. BmEts further synergistically enhanced E. coli or BmRelish1-d2 (active form)-stimulated lebocin promoter activation. Two κB elements were also found to be involved in promoter activation by BmRelish1-d2 and in synergistic promoter activation by BmEts and BmRelish1-d2 in the silkworm cells. Specific binding of recombinant BmEts to the proximal κB element and the most proximal GGAA/T motif and interaction between BmEts and BmRelish1 were also observed. To our knowledge, this is the first report of an Ets family protein directly regulating immune-related genes in invertebrates.     

The few virus-like genes of Cotesia congregata bracovirus

The origin of the symbiotic association between parasitoid wasps and bracoviruses is still unknown. From phylogenetic analyses, bracovirus-associated wasp species constitute a monophyletic group, the microgastroid complex. Thus all wasp-bracovirus associations could have originated from the integration of an ancestral virus in the genome of the ancestor of the microgastroids. In an effort to identify a set of virus genes that would give clues on the nature of the ancestral virus, we have recently performed the complete sequencing of the genome of CcBV, the bracovirus of the wasp Cotesia congregata. We describe here the putative proteins encoded by CcBV genome having significant similarities with sequences from known viruses and mobile elements. The analysis of CcBV gene content does not lend support to the hypothesis that bracoviruses originated from a baculovirus. Moreover, no consistent homology was found between CcBV genes and any set of genes constituting the core genome of a known free-living virus. We discuss the significance of the scarce homology found between proteins from CcBV and other viruses or mobile elements.     

Isolation of an imaginal disc growth factor homologue from Pieris rapae and its expression following parasitization by Cotesia rubecula

Endoparasitoid insects introduce maternal factors into the body of their host at oviposition to suppress cellular defences for the protection of the developing parasitoid. We have shown that transient expression of polydnavirus genes from a hymenopteran parasitoid Cotesia rubecula (CrPDV) is responsible for the inactivation of hemocytes from the lepidopteran host Pieris rapae. Since the observed downregulation of CrPDV genes in infected host tissues is not due to cis-regulatory elements at the CrV1 gene locus, we speculated that the termination of CrPDV gene expression may be due to cellular inactivation caused by the CrV1-mediated immune suppression of infected tissues. To test this assumption, we isolated an imaginal disc growth factor (IDGF) that is expressed in fat body and hemocytes, the target of viral infection and expression of CrPDV genes. Time-course experiments showed that the level of P. rapae IDGF is not affected by parasitization and polydnavirus infection. However, the amount of highly expressed genes, such as storage proteins, arylphorin and lipophorin, are significantly reduced following parasitization.     

BmICE-2 is a novel pro-apoptotic caspase involved in apoptosis in the silkworm, Bombyx mori

In this study we identified a potential pro-apoptotic caspase gene, Bombyx mori(B. mori)ICE-2 (BmICE-2) which encoded a polypeptide of 284 amino acid residues, including a ¹⁶⁹QACRG¹⁷³ sequence which surrounded the catalytic site and contained a p20 and a p10 domain. BmICE-2 expressed in Escherichia coli (E. coli) exhibited high proteolytic activity for the synthetic human initiator caspase-9 substrates Ac-LEHD-pNA, but little activity towards the effector caspase-3 substrates Ac-DEVD-pNA. When BmICE-2 was transiently expressed in BmN-SWU1 silkworm B. mori cells, we found that the high proteolytic activity for Ac-LEHD-pNA triggered caspase-3-like protease activity resulting in spontaneous cleavage and apoptosis in these cells. This effect was not replicated in Spodoptera frugiperda 9 cells. In addition, spontaneous cleavage of endogenous BmICE-2 in BmN-SWU1 cells could be induced by actinomycin D. These results suggest that BmICE-2 may be a novel pro-apoptotic gene with caspase-9 activity which is involved apoptotic processes in BmN-SWU1 silkworm B. mori cells.     

Distinct consequences of sterol sensor mutations in Drosophila and mouse patched homologs

The membrane protein Patched (Ptc) is a critical regulator of Hedgehog signaling. Ptc is among a family of proteins that contain a sterol sensor motif. The function of this domain is poorly understood, but some proteins that contain sterol sensors are involved in cholesterol homeostasis. In the SREBP cleavage-activating protein (SCAP), sterols inhibit the protein's activity through this domain. Mutations in two highly conserved residues in the SCAP sterol sensor have been identified that confer resistance to sterol regulation. We introduced the analogous mutations in the sterol sensor motif of fly Ptc and mouse Ptc1 and examined their effect on protein activity. In contrast to SCAP, the sterol sensor mutations had different affects on Drosophila Ptc; Ptc Y442C retained function, while Ptc D584N conferred dominant negative activity. In the wing imaginal disc, Ptc D584N overexpression induced Hedgehog targets by stabilizing Cubitus interruptus and inducing decapentaplegic. However, Ptc D584N did not induce collier, a gene that requires high levels of Hedgehog signaling. In mouse Ptc1, the Y438C and D585N mutations did not stimulate signaling in Shh-responsive cell lines but did complement murine ptc1(-/-) cells. The results suggest that mutations in sterol sensor motifs alter function differently between sterol sensor family members.     

A Rho GDP-dissociation inhibitor is involved in cytokinesis of Dictyostelium

Myotubularin-related genes define a novel highly conserved family of eukaryotic proteins of at least 11 human members. The hMTM1 gene that codes for myotubularin is mutated in X-linked myotubular myopathy, a severe congenital disease. Recently, we and others have characterized myotubularin as a potent and specific phosphatidylinositol 3-phosphate 3-phosphatase. In the present study we investigated the lipid phosphatase activity and the subcellular localization of two other members of the family, hMTMR2 protein that is mutated in the demyelinating neuropathy Charcot-Marie-Tooth type 4B and the FYVE-finger containing hMTMR3 protein. Our results show that both proteins are potent phosphatidylinositol 3-phosphate 3-phosphatases either in vitro or in yeast where they interfered with vesicular trafficking. Their localization is mainly cytoplasmic, with however strong labeling of Rac-inducible plasma membrane ruffles. The fact that the ubiquitously expressed hMTM1 and hMTMR2 genes are involved in different pathologies indicates that despite their shared enzymatic activity, they are not functionally redundant, at least in certain cell types. This might be explained by subtle differences in expression and/or in recruitment and regulation at their specific site of action.     

Characterization of Aquilegia Polycomb Repressive Complex 2 homologs reveals absence of imprinting

Epigenetic regulation is important for maintaining gene expression patterns in multicellular organisms. The Polycomb Group (PcG) proteins form several complexes with important and deeply conserved epigenetic functions in both the plant and animal kingdoms. The plant Polycomb Repressive Complex 2 (PRC2) contains four core proteins, Enhancer of Zeste (E(z)), Suppressor of Zeste 12 (Su(z)12), Extra Sex Combs (ESC), and Multicopy Suppressor of IRA 1 (MSI1), and functions in many developmental transitions. In some plant species, including rice and Arabidopsis, duplications in the core PRC2 proteins allow the formation of PRC2s with distinct developmental functions. In addition, members of the plant specific VEL PHD family have been shown to associate with the PRC2 complex in Arabidopsis and may play a role in targeting the PRC2 to specific loci. Here we examine the evolution and expression of the PRC2 and VEL PHD families in Aquilegia, a member of the lower eudicot order Ranunculales and an emerging model for the investigation of plant ecology, evolution and developmental genetics. We find that Aquilegia has a relatively simple PRC2 with only one homolog of Su(z)12, ESC and MSI1 and two ancient copies of E(z), AqSWN and AqCLF. Aquilegia has four members of the VEL PHD family, three of which appear to be closely related to Arabidopsis proteins known to associate with the PRC2. The PRC2 and VEL PHD family proteins are expressed at a relatively constant level throughout Aquilegia vulgaris development, with the VEL PHD family and MSI1 expressed at higher levels during and after vernalization and in the inflorescence. Both AqSWN and AqCLF are expressed in Aquilegia endosperm but neither copy is imprinted.     

Phylogenetic and evolutionary analysis of the septin protein family in metazoan

Septins, a conserved family of cytoskeletal GTP-binding proteins, were presented in diverse eukaryotes. Here, a comprehensive phylogenetic and evolutionary analysis for septin proteins in metazoan was carried out. First, we demonstrated that all septin proteins in metazoan could be clustered into four subgroups, and the representative homologue of every subgroup was presented in the non-vertebrate chordate Ciona intestinalis, indicating that the emergence of the four septin subgroups should have occurred prior to divergence of vertebrates and invertebrates, and the expansion of the septin gene number in vertebrates was mainly by the duplication of pre-existing genes rather than by the appearance of new septin subgroup. Second, the direct orthologues of most human septins existed in zebrafish, which suggested that human septin gene repertoire was mainly formed by as far as before the split between fishes and land vertebrates. Third, we found that the evolutionary rate within septin family in mammalian lineage varies significantly, human SEPT1, SEPT 10, SEPT 12, and SEPT 14 displayed a relative elevated evolutionary rate compared with other septin members. Our data will provide new insights for the further function study of this protein family.     

Cytochrome P450 CYP307A1/Spook: a regulator for ecdysone synthesis in insects

The prothoracic gland (PG) has essential roles in synthesizing and secreting a steroid hormone called ecdysone that is critical for molting and metamorphosis of insects. However, little is known about the genes controlling ecdysteroidogenesis in the PG. To identify genes functioning in the PG of the silkworm, Bombyx mori, we used differential display PCR and focused on a cytochrome P450 gene designated Cyp307a1. Its expression level positively correlates with a change in the hemolymph ecdysteroid titer. In addition, Drosophila Cyp307a1 is encoded in the spook locus, one of the Halloween mutant family members showing a low ecdysone titer in vivo, suggesting that Cyp307a1 is involved in ecdysone synthesis. While Drosophila Cyp307a1 is expressed in the early embryos and adult ovaries, the expression is not observed in the PGs of embryos or third instar larvae. These results suggest a difference in the ecdysone synthesis pathways during larval development in these insects.     

Bestrophin genes are expressed in Xenopus development

The Bestrophin-1/VMD2 gene has been implicated in Best disease, a juvenile-onset vitelliform macular dystrophy. The Bestrophin proteins have anion channel activity, and the four mammalian members share sequence homologies in multiple transmembrane domains and an RFP-tripeptide motif. The expression patterns and functions of the Bestrophin genes in retinal pigment epithelium have been studied intensively, whereas little is known about their roles in vertebrate embryogenesis. This study examined the roles of four Xenopus tropicalis homologs of BEST genes. The xtBest genes showed spatially and temporally distinct expression. xtBest-2 was the only maternally expressed Best gene, and both xtBest-2 and the Xenopus laevis Best-2 gene were expressed at the edge of the blastopore lip including the organizer. Ectopic expression of xBest-2 caused defects in dorsal axis formation and in mesodermal gene expression during gastrulation. These results suggest a new role of the Bestrophin family genes in early vertebrate embryogenesis.