Shigeharu Kinoshita

Draft Genome of the Pearl Oyster Pinctada fucata: A Platform for Understanding Bivalve Biology

The study of the pearl oyster Pinctada fucata is key to increasing our understanding of the molecular mechanisms involved in pearl biosynthesis and biology of bivalve molluscs. We sequenced ∼1150-Mb genome at ∼40-fold coverage using the Roche 454 GS-FLX and Illumina GAIIx sequencers. The sequences were assembled into contigs with N50 = 1.6 kb (total contig assembly reached to 1024 Mb) and scaffolds with N50 = 14.5 kb. The pearl oyster genome is AT-rich, with a GC content of 34%. DNA transposons, retrotransposons, and tandem repeat elements occupied 0.4, 1.5, and 7.9% of the genome, respectively (a total of 9.8%). Version 1.0 of the P. fucata draft genome contains 23 257 complete gene models, 70% of which are supported by the corresponding expressed sequence tags. The genes include those reported to have an association with bio-mineralization. Genes encoding transcription factors and signal transduction molecules are present in numbers comparable with genomes of other metazoans. Genome-wide molecular phylogeny suggests that the lophotrochozoan represents a distinct clade from ecdysozoans. Our draft genome of the pearl oyster thus provides a platform for the identification of selection markers and genes for calcification, knowledge of which will be important in the pearl industry.

Deep sequencing of ESTs from nacreous and prismatic layer producing tissues and a screen for novel shell formation-related genes in the pearl oyster.

Background Despite its economic importance, we have a limited understanding of the molecular mechanisms underlying shell formation in pearl oysters, wherein the calcium carbonate crystals, nacre and prism, are formed in a highly controlled manner. We constructed comprehensive expressed gene profiles in the shell-forming tissues of the pearl oyster Pinctada fucata and identified novel shell formation-related genes candidates. Principal Findings We employed the GS FLX 454 system and constructed transcriptome data sets from pallial mantle and pearl sac, which form the nacreous layer, and from the mantle edge, which forms the prismatic layer in P. fucata. We sequenced 260477 reads and obtained 29682 unique sequences. We also screened novel nacreous and prismatic gene candidates by a combined analysis of sequence and expression data sets, and identified various genes encoding lectin, protease, protease inhibitors, lysine-rich matrix protein, and secreting calcium-binding proteins. We also examined the expression of known nacreous and prismatic genes in our EST library and identified novel isoforms with tissue-specific expressions. Conclusions We constructed EST data sets from the nacre- and prism-producing tissues in P. fucata and found 29682 unique sequences containing novel gene candidates for nacreous and prismatic layer formation. This is the first report of deep sequencing of ESTs in the shell-forming tissues of P. fucata and our data provide a powerful tool for a comprehensive understanding of the molecular mechanisms of molluscan biomineralization.

The role of proteoglycan in the development of sea urchins. I. Abnormal development of sea urchin embryos caused by the disturbance of proteoglycan synthesis.

Abstract The development of the sea urchins, Hemicentrotus pulcherrimus and Clypeaster japonicus, was examined under conditions which inhibit the synthesis of proteoglycan in various ways. The changes in the developmental fate were compared with the degree of inhibition of proteoglycan synthesis as measured by incorporation of labeled precursors. The inhibition of proteoglycan synthesis was achieved by treating the embryos with (1) aryl-β- d -xyloside (inhibition of coupling between protein and polysaccharide moieties); (2) sodium selenate (inhibition of sulfation of polysaccharide); and (3) 2-deoxy- d -glucose (inhibition of polysaccharide chain elongation). It was found that the development of embryos ceased at the blastula stage in all cases tested, irrespective of the step of the inhibition. The cessation of development became apparent when the incorporation of the labeled precursors was decreased to a level below 50% compared with the normal level. From these facts, it appears that the synthesis of proteoglycan is necessary for the realization of post-gastrular development.

Heparin as a possible initiator of genomic RNA synthesis in early development of sea urchin embryos.

Abstract In the early development of sea urchins, the uptake of sulfate ions into the embryo becomes prominent concurrent with the beginning of gastrulation, and the incorporated sulfate is invariably found in the heparin fraction. RNA synthesis in isolated nuclei obtained from pregastrular embryo is found to be enhanced on addition of an appropriate amount of heparin, and this effect is highly specific for heparin. Such enhancement is no longer observed when nuclei from postgastrular embryos are used. It has also been found that isolated nuclei change their morphological characteristics on treatment with heparin, becoming optically homogeneous, when examined under the phasecontrast microscope. Presumably, this indicates that loosening of the condensed parts of chromatin is occurring in the nuclei. Such a change is never observed in nuclei when they are treated with chondroitin sulfate or hyaluronate. The possibility is suggested that sulfate ions are incorporated into heparin molecules at the end of the blastula stage, and that this newly synthesized, or sulfate-enriched, heparin induces a change in the nuclear structure, namely, loosening of the chromatin, which in turn initiates extensive transcriptional activity in the embryonic nuclei. Such a series of events may be involved in the machinery needed for the switch-over from maternal gene-directed development to embryonic genome-dependent differentiation.

Dual Roles of Notch in Regulation of Apically Restricted Mitosis and Apicobasal Polarity of Neuroepithelial Cells

How the mitosis of neuroepithelial stem cells is restricted to the apical ventricular area remains unclear. In zebrafish, the mosaic eyes(rw306) (moe/epb41l5(rw306)) mutation disrupts the interaction between the putative adaptor protein Moe and the apicobasal polarity regulator Crumbs (Crb), and impairs the maintenance of neuroepithelial apicobasal polarity. While Crb interacts directly with Notch and inhibits its activity, Moe reverses this inhibition. In the moe(rw306) hindbrain, Notch activity is significantly reduced, and the number of cells that proliferate basally away from the apical area is increased. Surprisingly, activation of Notch in the moe(rw306) mutant rescues not only the basally localized proliferation but also the aberrant neuroepithelial apicobasal polarity. We present evidence that the Crb⋅Moe complex and Notch play key roles in a positive feedback loop to maintain the apicobasal polarity and the apical-high basal-low gradient of Notch activity in neuroepithelial cells, both of which are essential for their apically restricted mitosis.

The occurrence of two types of hemopexin-like protein in medaka and differences in their affinity to heme.

SUMMARY Full-length cDNA clones encoding two types of hemopexin-like protein, mWap65-1 and mWap65-2, were isolated from the HNI inbred line of medaka Oryzias latipes. The deduced amino acid sequence of mWap65-2 resembled mammalian hemopexins more closely than that of mWap65-1. Histidine residues required for the high affinity of hemopexins for hemes were conserved in mWap65-2, but not in mWap65-1. Surprisingly, mWap65-1, but not mWap65-2, showed heme-binding ability as revealed by hemin–agarose affinity chromatography, even though mWap65-1 lacked the essential histidine residues. Furthermore, RT-PCR analysis of different tissues demonstrated that the transcripts of mWap65-2 were restricted to liver, whereas those of mWap65-1 were found in various tissues including liver, eye, heart and brain. Quantitative RT-PCR revealed that transcripts of mWap65-2 were expressed earlier than those of mWap65-1 during ontogeny. However, the accumulated mRNA levels of both mWap65-1 and mWap65-2 did not differ significantly in fish acclimated to either 10°C or 30°C for 5 weeks. These characteristics suggest that the two proteins have different physiological functions and that mWap65-2 is not a hemopexin.

Quantitative expression analysis of nacreous shell matrix protein genes in the process of pearl biogenesis

A cultured pearl is produced in a pearl sac which forms layers of cells differentiated from an allograft mantle. Previous investigations claimed that genomic DNAs from grafting tissues were persistent during pearl aquaculture. However, the specific living status of the genes regulating pearl formation has remained unknown. This study examined the expression profiles of six genes encoding nacreous shell matrix proteins (NSMPs) in the pearl sac of aquaculture pearl oyster Pinctada fucata by real-time PCR. The comparative analysis of NSMP gene expression in the pearl sac and reference mantle tissues revealed that only a few NSMP genes maintained high transcription levels in the pearl sac. The impaired transcription levels of the nacrein gene refreshed the previous hypothesis, suggesting that CaCO(3) required for pearl secretion was not from pearl sac cells. Among the examined genes, only the N19 gene attained high expression levels in the pearl sac. These results provide new insights into the molecular mechanisms involved in pearl biogenesis.

The Diversity of Shell Matrix Proteins: Genome-Wide Investigation of the Pearl Oyster, Pinctada fucata

In molluscs, shell matrix proteins are associated with biomineralization, a biologically controlled process that involves nucleation and growth of calcium carbonate crystals. Identification and characterization of shell matrix proteins are important for better understanding of the adaptive radiation of a large variety of molluscs. We searched the draft genome sequence of the pearl oyster Pinctada fucata and annotated 30 different kinds of shell matrix proteins. Of these, we could identified Perlucin, ependymin-related protein and SPARC as common genes shared by bivalves and gastropods; however, most gastropod shell matrix proteins were not found in the P. fucata genome. Glycinerich proteins were conserved in the genus Pinctada. Another important finding with regard to these annotated genes was that numerous shell matrix proteins are encoded by more than one gene; e.g., three ACCBP-like proteins, three CaLPs, five chitin synthase-like proteins, two N16 proteins (pearlins), 10 N19 proteins, two nacreins, four Pifs, nine shematrins, two prismalin-14 proteins, and 21 tyrosinases. This diversity of shell matrix proteins may be implicated in the morphological diversity of mollusc shells. The annotated genes reported here can be searched in P. fucata gene models version 1.1 and genome assembly version 1.0 (http://marinegenomics.oist.jp/pinctada_fucata). These genes should provide a useful resource for studies of the genetic basis of biomineralization and evaluation of the role of shell matrix proteins as an evolutionary toolkit among the molluscs.

The behaviour and localization of intracellular relaxing system during cleavage in the sea urchin egg

Abstract A glycerinated model of cleaving sea urchin eggs was prepared using a new technique. The localization of the relaxing system in these eggs was determined by means of fluorescein-labeled antibodies. Some kind of cytoplasmic structure which reacts with anti-“relaxing granule”-serum was found to exist and show a directed migration during the cleavage in the sea urchin egg. As a result of directed movement of the “relaxing system” within the cell towards the centers of the mitotic apparatus during metaphase and anaphase, the presumptive furrowing region became temporarily poor in the relaxing system as compared with the polar regions. It was suggested that this relative deficiency of the relaxing system at the presumptive furrowing region will be the cause of the initiation of cytokinesis.

Molecular characterization of Mn-superoxide dismutase and gene expression studies in dietary restricted Brachionus plicatilis rotifers

Superoxide dismutases (SODs) promote a conversion of harmful reactive oxygen species (ROS) to relatively moderate forms, resulting in the extension of lifespan in the nematode Caenorhabditis elegans under caloric restriction. The lifespan of the rotifer Brachionus plicatilis is also markedly extended by caloric restriction. We, therefore, cloned cDNA encoding SOD activated with Mn (Mn SOD) from B. plicatilis and examined its expression pattern in rotifers raised with energy restricted diet. The full length deduced amino acid sequence of the rotifer Mn SOD showed 61% identity with the C. elegans ortholog. Four amino acid residues that are essential to the binding of this enzyme to Mn were conserved in the rotifer Mn SOD. Subsequently we examined the mRNA expression patterns of Mn SOD using highly sensitive quantitative real-time PCR for various rotifer populations that are likely to differ in their lifespans in experiments on calorie restricted diets. The accumulated mRNA levels of Mn SOD were found to increase in supposedly long-lived rotifers. These results suggest that Mn SOD is possibly related to the aging of B. plicatilis.