Identification and sequencing of the gene encoding DNA methyltransferase 3 (DNMT3) from sea cucumber, Apostichopus japonicus.

The sea cucumber Apostichopus japonicus is well known as a traditional tonic food and as a commercially important cultured aquatic species. This species produces saponins, and has a high potential to cope with environmental stress, such as aestivation, organ regeneration, and wound healing. Recently, several studies have shown that cellular reprogramming and the physiological responses of the sea cucumber to environmental changes, including aestivation, are potentially mediated by epigenetic DNA methylation. The DNA methyltransferase (DNMT)1 and DNMT3 genes are independent participants in the maintenance and de novo methylation of specific sequences. Sea urchin (Strongylocentrotus purpuratus) and starfish (Asterina pectinifera), which belong to the same phylum as A. japonicus, have both DNMT1 and DNMT3 genes. However, it was previously reported that DNMT1 is present, but DNMT3 is absent, in A. japonicus. In the present study, we sequenced the full-length cDNA of the A. japonicus DNMT3 gene. The newly sequenced DNMT3 gene comprises three major conserved domains (Pro-Trp-Trp-Pro (PWWP), plant homeodomain (PHD), and S-adenosylmethionine-dependent methyltransferase (AdoMet-MTase)), indicating that the DNMT3 possibly has de novo DNA methylation catalytic activity. Gene structure and phylogenetic analysis showed that sea cucumber DNMT3 is evolutionarily conserved in the Echinodermata. Next, we demonstrated the conservation of DNMT3 gene expression in sea cucumber and starfish belong to same phylum, echinoderm. Using reverse transcription-polymerase chain reaction, sea cucumber DNMT3 mRNA was detected in testis tissue, but not in other tissues tested, including the respiratory tree, muscle, tentacle, intestine, and ovary. This is inconsistent with previous reports, which showed the expression of DNMT3 in ovary, but not in testis of the starfish A. pectinifera, indicating the tissue- and species-specific expression of DNMT3 gene. Although further studies are needed to clarify the epigenetic regulatory mechanisms of DNMT3 and its application to the aquaculture industry, our findings may provide insights into the sea cucumber biology.

State-of-the-art housekeeping proteins for quantitative western blotting: Revisiting the first draft of the human proteome.

Western blotting (WB) analysis is the most popular and widely used methodology for protein detection and characterization over recent decades. In accordance with the advancement of the technologies for the acquisition of WB signals, a quantitative value is used to present the abundance of target proteins in a complex sample, thereby requiring the use of specific proteins as internal references that represent total proteins. Heretofore, proteins encoded by housekeeping genes such as GAPDH, ß-tubulin and ß-actin have been commonly used as loading controls without any hesitation because their mRNA expression levels tend to be high and constant in many different cells and tissues. Experimentally, however, some of the housekeeping reference proteins are often displayed with inconsistent expression levels in both homogeneous and heterogeneous tissues, and, in terms of mRNA levels, they have a weak correlation to the abundance of proteins. To estimate accurate, reliable, and reproducible protein quantifications, it is crucial to define appropriate reference controls. For this paper, we explored the recently released large-scale, human proteomic database ProteomicsDB including 16 857 liquid chromatography tandem-mass-spectrometry data from 27 human tissues, and suggest 20 ubiquitously- and constitutively-expressed, putative internal-reference controls for the quantification of differential protein expressions. Intriguingly, the most commonly used, known housekeeping genes were entirely excluded in our newly defined candidates. Although the applications of the candidates under many different biological conditions and in other organisms are yet to be empirically verified, we propose reliable, potential loading controls for a WB analysis in this paper.

Physiochemical Quality and Sensory Characteristics of koji Made with Soybean, Rice, and Wheat for Commercial doenjang Production.

The purpose of this study was to compare the physiochemical quality characteristics and sensory profiles of three types of koji: soybean, rice, and wheat koji. Koji is made by inoculating Aspergillus oryzae following the standard method of manufacturing. The physiochemical characteristic and sensory profiles were performed after fermenting samples of koji for a 72 h period. The physiochemical quality characteristics that were tested include pH, moisture content, color, acidity, TA, amino-type nitrogen content, reducing- and total-sugar content, and alcohol content; the enzymatic activities that were tested include amylase (α- and ß-) and protease (neutral and acidic) activities. A descriptive sensory analysis was conducted on three types of koji with a highly trained sensory panel (n = 7) using the SpectrumTM Method. Differences in physiochemical and sensory profiles were observed on three koji samples (p < 0.05). Soybean koji had higher values in acid and TA, while rice koji had the highest values in reducing and total sugar, at 90.3 mg/g and 107.5 mg/g respectively. Wheat koji had the lowest values in protease activities. The flavor profile of soybean koji was characterized by bean sprout, cracker, and cheonggukjang aromatics; that of rice koji was characterized by overripe banana, solvent, syrup, and parboiled rice aromatics; and that of wheat koji was characterized by woody and roasted aromatics.

Genome Sequences of Two Shewanella spp. Isolated from the Gut of the Sea Cucumber Apostichopus japonicus (Selenka, 1867).

In this study, we sequenced the genomes of two Shewanella spp., newly isolated from the gut of the sea cucumber Apostichopus japonicus (Selenka, 1867). The whole-genome sequences reported here will expand the repertoire of genomic information for the members of the genus Shewanella and will provide important insights into their roles within microbial communities.

Draft genome of the sea cucumber Apostichopus japonicus and genetic polymorphism among color variants.

The Japanese sea cucumber (Apostichopus japonicus Selenka 1867) is an economically important species as a source of seafood and ingredient in traditional medicine. It is mainly found off the coasts of northeast Asia. Recently, substantial exploitation and widespread biotic diseases in A. japonicus have generated increasing conservation concern. However, the genomic knowledge base and resources available for researchers to use in managing this natural resource and to establish genetically based breeding systems for sea cucumber aquaculture are still in a nascent stage. A total of 312 Gb of raw sequences were generated using the Illumina HiSeq 2000 platform and assembled to a final size of 0.66 Gb, which is about 80.5% of the estimated genome size (0.82 Gb). We observed nucleotide-level heterozygosity within the assembled genome to be 0.986%. The resulting draft genome assembly comprising 132 607 scaffolds with an N50 value of 10.5 kb contains a total of 21 771 predicted protein-coding genes. We identified 6.6-14.5 million heterozygous single nucleotide polymorphisms in the assembled genome of the three natural color variants (green, red, and black), resulting in an estimated nucleotide diversity of 0.00146. We report the first draft genome of A. japonicus and provide a general overview of the genetic variation in the three major color variants of A. japonicus. These data will help provide a comprehensive view of the genetic, physiological, and evolutionary relationships among color variants in A. japonicus, and will be invaluable resources for sea cucumber genomic research.