Kre6 (yeast 1,6-ß-transglycosylase) homolog, PhTGS, is essential for ß-glucan synthesis in the haptophyte Pleurochrysis haptonemofera.

Haptophytes synthesize unique ß-glucans containing more ß-1,6-linkages than ß-1,3 linkages, as a storage polysaccharide. To understand the mechanism of the synthesis, we investigated the roles of Kre6 (yeast 1,6-ß-transglycosylase) homologs, PhTGS, in the haptophyte Pleurochrysis haptonemofera. RNAi of PhTGS repressed ß-glucan accumulation and simultaneously induced lipid production, suggesting that PhTGS is involved in ß-glucan synthesis and that the knockdown leads to the alteration of the carbon metabolic flow. PhTGS was expressed more in light, where ß-glucan was actively produced by photosynthesis, than in the dark. The crude extract of E. coli expressing PhKre6 demonstrated its activity to incorporate 14C-UDP-glucose into ß-glucan of P. haptonemofera. These findings suggest that PhTGS functions in storage ß-glucan synthesis specifically in light, probably by producing the ß-1,6-branch.

Overexpression of Tisochrysis lutea Akd1 identifies a key cold-induced alkenone desaturase enzyme.

Alkenones are unusual long-chain neutral lipids that were first identified in oceanic sediments. Currently they are regarded as reliable palaeothermometers, since their unsaturation status changes depending on temperature. These molecules are synthesised by specific haptophyte algae and are stored in the lipid body as the main energy storage molecules. However, the molecular mechanisms that regulate the alkenone biosynthetic pathway, especially the low temperature-dependent desaturation reaction, have not been elucidated. Here, using an alkenone-producing haptophyte alga, Tisochrysis lutea, we show that the alkenone desaturation reaction is catalysed by a newly identified desaturase. We first isolated two candidate desaturase genes and found that one of these genes was drastically upregulated in response to cold stress. Gas chromatographic analysis revealed that the overexpression of this gene, named as Akd1 finally, increased the conversion of di-unsaturated C37-alkenone to tri-unsaturated molecule by alkenone desaturation, even at a high temperature when endogenous desaturation is efficiently suppressed. We anticipate that the Akd1 gene will be of great help for elucidating more detailed mechanisms of temperature response of alkenone desaturation, and identification of active species contributing alkenone production in metagenomic and/or metatranscriptomic studies in the field of oceanic biogeochemistry.

Stable Nuclear Transformation System for the Coccolithophorid Alga Pleurochrysis carterae.

Of the three dominant marine microalgal groups, dinoflagellates and diatoms can undergo genetic transformation; however, no transformation method has been established for haptophytes to date. Here, we report the first stable genetic transformation of a coccolithophore, Pleurochrysis carterae, by means of polyethylene glycol (PEG)-mediated transfer of a bacterial hygromycin B-resistance gene. Together with the novel transient green fluorescent protein (GFP) expression system, this approach should facilitate further molecular-based research in this phylum.

Bivalve-specific gene expansion in the pearl oyster genome: implications of adaptation to a sessile lifestyle.

INTRODUCTION: Bivalve molluscs have flourished in marine environments, and many species constitute important aquatic resources. Recently, whole genome sequences from two bivalves, the pearl oyster, Pinctada fucata, and the Pacific oyster, Crassostrea gigas, have been decoded, making it possible to compare genomic sequences among molluscs, and to explore general and lineage-specific genetic features and trends in bivalves. In order to improve the quality of sequence data for these purposes, we have updated the entire P. fucata genome assembly. RESULTS: We present a new genome assembly of the pearl oyster, Pinctada fucata (version 2.0). To update the assembly, we conducted additional sequencing, obtaining accumulated sequence data amounting to 193× the P. fucata genome. Sequence redundancy in contigs that was caused by heterozygosity was removed in silico, which significantly improved subsequent scaffolding. Gene model version 2.0 was generated with the aid of manual gene annotations supplied by the P. fucata research community. Comparison of mollusc and other bilaterian genomes shows that gene arrangements of Hox, ParaHox, and Wnt clusters in the P. fucata genome are similar to those of other molluscs. Like the Pacific oyster, P. fucata possesses many genes involved in environmental responses and in immune defense. Phylogenetic analyses of heat shock protein70 and C1q domain-containing protein families indicate that extensive expansion of genes occurred independently in each lineage. Several gene duplication events prior to the split between the pearl oyster and the Pacific oyster are also evident. In addition, a number of tandem duplications of genes that encode shell matrix proteins are also well characterized in the P. fucata genome. CONCLUSIONS: Both the Pinctada and Crassostrea lineages have expanded specific gene families in a lineage-specific manner. Frequent duplication of genes responsible for shell formation in the P. fucata genome explains the diversity of mollusc shell structures. These duplications reveal dynamic genome evolution to forge the complex physiology that enables bivalves to employ a sessile lifestyle in the intertidal zone.

Identification and characterization of a matrix protein (PPP-10) in the periostracum of the pearl oyster, Pinctada fucata.

The periostracum is a layered structure that is formed as a mollusk shell grows. The shell is covered by the periostracum, which consists of organic matrices that prevent decalcification of the shell. In the present study, we discovered the presence of chitin in the periostracum and identified a novel matrix protein, Pinctada fucata periostracum protein named PPP-10. It was purified from the sodium dodecyl sulfate/dithiothreitol-soluble fraction of the periostracum of the Japanese pearl oyster, P. fucata. The deduced amino acid sequence was determined by a combination of amino acid sequence analysis and cDNA cloning. The open reading frame encoded a precursor protein of 112 amino acid residues including a 21-residue signal peptide. The 91 residues following the signal peptide contained abundant Cys and Tyr residues. PPP-10 was expressed on the outer side of the outer fold in the mantle, indicating that PPP-10 was present in the second or third layer of the periostracum. We also determined that the recombinant PPP-10 had chitin-binding activity and could incorporate chitin into the scaffolds of the periostracum. These results shed light on the early steps in mollusk shell formation.

Initiating the mollusk genomics annotation community: toward creating the complete curated gene-set of the Japanese Pearl Oyster, Pinctada fucata.

The genome sequence of the Japanese pearl oyster, the first draft genome from a mollusk, was published in February 2012. In order to curate the draft genome assemblies and annotate the predicted gene models, two annotation Jamborees were held in Okinawa and Tokyo. To date, 761 genes have been surveyed and curated. A preparatory meeting and a debriefing were held at the Misaki Marine Biological Station before and after the Jamborees. These four events, in conjunction with the sequence-decoding project, have facilitated the first series of gene annotations. Genome annotators among the Jamboree participants added 22 functional categories to the annotation system to date. Of these, 17 are included in Generic Gene Ontology. The other five categories are specific to molluskan biology, such as "Byssus Formation" and "Shell Formation", including Biomineralization and Acidic Proteins. A total of 731 genes from our latest version of gene models are annotated and classified into these 22 categories. The resulting data will serve as a useful reference for future genomic analyses of this species as well as comparative analyses among mollusks.

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.

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.

Structural features and gene-expression profiles of actin homologs in Porphyra yezoensis (Rhodophyta).

The marine red alga Porphyra yezoensis contains an actin gene family consisting of at least four isoforms (PyACT1, 2, 3 and 4). The amino acid identity between isoforms exceeds 83%, and each contains a putative nuclear export signal (NES). We scanned the sequences for amino acids in regions homologous to the intermonomeric interface of actin filaments. Few residues expected to engage in cross-linking were conserved between the four isoforms. The results of the sequence analyses suggest that PyACT2 probably functions in the nucleus as a monomer (G-actin) or in other unconventional forms. In addition, the distribution and position of the introns were different from those in florideophycean actin genes. The expression level of PyACT3 in matured gametophytes was significantly higher than in those in a vegetative state, although the mRNA was detected at similar levels in both apical and basal parts of thalli. The expression levels of PyACT2 and 4, on the other hand, did not change significantly between the matured and vegetative gametophytes. The PyACT3 may serve as a molecular marker for monitoring thallus maturation in this species.

IDENTIFICATION OF GENES PREFERENTIALLY EXPRESSED DURING ASEXUAL SPORULATION IN PORPHYRA YEZOENSIS GAMETOPHYTES (BANGIALES, RHODOPHYTA)(1).

Asexual reproduction via archeospores in Porphyra yezoensis Ueda gametophytes is a very valuable character to nori farming; however, there is little information available on the molecular basis of the developmental process. To identify genes involved in the Porphyra asexual sporulation, we compared the gene expression profiles derived from four developmental stages of the life cycle (three from gametophytes; one from sporophytes) using cDNA macroarray, which includes 4,896 nonredundant expressed sequence tag (EST) groups. Candidate genes were screened by two different macroarray data analyses combined with reverse transcription-PCR (RT-PCR) analysis or Northern analysis. RT-PCR analysis revealed that nine genes (one: similarity to 5'-3' exoribonuclease; the other eight: no sequence similarity to known proteins) were expressed with a gametophyte (G)-specific manner, and two genes (named ASPO2608, ASPO1527) were expressed only in gametophytes that formed archeospores. The deduced amino acid sequences for the latter two genes are predicted to contain signal peptides for secretion at their N-termini. Northern analysis revealed that expression levels of Calvin cycle genes in the gametophytic stage that formed archeospores (G-A stage) were higher than those of the gametophyte blade with no archeospores (G-NA stage). In the macroarray analysis based on the rank data of G-preferentially expressed genes, which were detected in the previous P. yezoensis EST analysis, one gene encoding the cyclase associated protein (CAP) exhibited a change upwardly in the G-A stage >1,000 ranks to the G-NA stage. We propose that ASPO2608 and CAP may function in a signaling pathway of asexual sporulation.

IDENTIFICATION OF CROSS-FERTILIZED CONCHOCELIS USING CLEAVED AMPLIFIED POLYMORPHIC SEQUENCE MARKERS IN CROSS-EXPERIMENTS OF PORPHYRA YEZOENSIS (BANGIALES, RHODOPHYTA)(1).

As a part of the construction of a Porphyra yezoensis Ueda genetic linkage map, we conducted intraspecific cross-experiments and subsequent screening of cross-fertilized conchocelis by cleaved amplified polymorphic sequence (CAPS) analysis. The cross-experiments were carried out between males of the wildtype (KGJ) and females of the recessive green mutant (TU-2) using two methods, controlled and random crosses. A total of 42 and 186 wildtype-colored conchocelis colonies were obtained from the former and latter experiments, respectively. Among those, 49 DNA samples (14% and 23% obtained from the former and latter crosses, respectively) showed biparental CAPS patterns in the two gene regions (EF-1α open reading frame [ORF] region and V-ATPase). This study represents the first report in which the cross-fertilized conchocelis of P. yezoensis has been directly confirmed by molecular marker. The combination of the simple DNA extraction and CAPS analysis may be applicable in genetic studies of other macroalgae that are monoecious and/or grow slowly in laboratory culture.

Identification of symbiotically expressed coral mRNAs using a model infection system.

Hermatypic (or reef-building) corals live in obligatory mutualistic symbiosis with the symbiotic dinoflagellates Symbiodinium spp. (generally known as zooxanthellae). In an attempt to establish a model symbiosis system consisting of a coral host and a monoclonal population of zooxanthellae, infectivity of five cultured Symbiodinium cell lines was tested on naturally aposymbiotic juveniles of Acropora tenuis. A clade A3 strain (PL-TS-1) infected the juveniles at high density and promoted growth of the host. To identify host genes involved in the establishment or maintenance of symbiosis, mRNA expression patterns were compared between aposymbiotic and PL-TS-1-infected juvenile polyps using the suppression subtractive hybridization technique. Two mRNAs, the expression levels of which were augmented more than twofold by the presence of the symbionts, were thereby identified. One of the mRNAs, AtSym-02, encodes a novel protein of 322 amino acids which is predicted to be a glycosylated trans-membrane protein.

Hemocyanin in the exoskeleton of crustaceans: enzymatic properties and immunolocalization.

We investigated the enzymatic properties and immunohistochemical localization of cuticular hemocyanin, a known oxygen transporter in the prawn Penaeus japonicus. The molecular weight of hemocyanin purified from the cuticle was estimated to be 67-77 k using SDS-PAGE, and the purified protein was effectively converted into a phenoloxidase-like enzyme by an SDS-treatment. The activated enzyme catalyzed the o-hydroxylation of monophenols and the oxidation of o-diphenols and was inhibited by typical inhibitors of phenoloxidase. These characteristics were nearly identical to the enzymatic properties of hemolymph hemocyanin. Immunological detection showed a diffuse distribution of hemocyanin over the exocuticle and endocuticle, and a higher signal level was observed in the latter. Based on these results, roles of hemocyanin in various physiological processes such as immune response and sclerotization of the cuticle were discussed.

A crustacean Ca2+-binding protein with a glutamate-rich sequence promotes CaCO3 crystallization.

The DD4 mRNA of the penaeid prawn Penaeus japonicus was shown previously to be expressed in the epidermis adjacent to the exoskeleton specifically during the post-moult period, when calcification of the exoskeleton took place. The encoded protein possessed a Ca2+-binding site, suggesting its involvement in the calcification of the exoskeleton. In the present study, an additional ORF (open reading frame) of 289 amino acids was identified at the 5' end of the previous ORF. The newly identified part of the encoded protein included a region of approx. 120 amino acids that was highly rich in glutamate residues, and contained one or more Ca2+-binding sites. In an immunohistochemical study, signals were detected within calcified regions in the endocuticular layer of the exoskeleton. Bacterially expressed partial segments of the protein induced CaCO3 crystallization in vitro. Finally, a reverse transcription-PCR study showed that the expression was limited to an early part of the post-moult period, preceding significant calcification of the exoskeleton. These observations argue for the possibility that the encoded protein, renamed crustocalcin (CCN), promotes formation of CaCO3 crystals in the exoskeleton by inducing nucleation.