Development of a chub mackerel with less-aggressive fry stage by genome editing of arginine vasotocin receptor V1a2.

Genome editing is a technology that can remarkably accelerate crop and animal breeding via artificial induction of desired traits with high accuracy. This study aimed to develop a chub mackerel variety with reduced aggression using an experimental system that enables efficient egg collection and genome editing. Sexual maturation and control of spawning season and time were technologically facilitated by controlling the photoperiod and water temperature of the rearing tank. In addition, appropriate low-temperature treatment conditions for delaying cleavage, shape of the glass capillary, and injection site were examined in detail in order to develop an efficient and robust microinjection system for the study. An arginine vasotocin receptor V1a2 (V1a2) knockout (KO) strain of chub mackerel was developed in order to reduce the frequency of cannibalistic behavior at the fry stage. Video data analysis using bioimage informatics quantified the frequency of aggressive behavior, indicating a significant 46% reduction (P = 0.0229) in the frequency of cannibalistic behavior than in wild type. Furthermore, in the V1a2 KO strain, the frequency of collisions with the wall and oxygen consumption also decreased. Overall, the manageable and calm phenotype reported here can potentially contribute to the development of a stable and sustainable marine product.

LsSpt23p is a regulator of triacylglycerol synthesis in the oleaginous yeast Lipomyces starkeyi.

The oleaginous yeast Lipomyces starkeyi has considerable potential in industrial application, since it can accumulate a large amount of triacylglycerol (TAG), which is produced from sugars under nitrogen limitation condition. However, the regulation of lipogenesis in L. starkeyi has not been investigated in depth. In this study, we compared the genome sequences of wild-type and mutants with increased TAG productivity, and identified a regulatory protein, LsSpt23p, which contributes to the regulation of TAG synthesis in L. starkeyi. L. starkeyi mutants overexpressing LsSPT23 had increased TAG productivity compared with the wild-type strain. Quantitative real-time PCR analysis showed that LsSpt23p upregulated the expression of GPD1, which encodes glycerol 3-phosphate dehydrogenase; the Kennedy pathway genes SCT1, SLC1, PAH1, DGA1, and DGA2; the citrate-mediated acyl-CoA synthesis pathway-related genes ACL1, ACL2, ACC1, FAS1, and FAS2; and OLE1, which encodes ∆9 fatty acid desaturase. Chromatin immunoprecipitation-quantitative PCR assays indicated that LsSpt23p acts as a direct regulator of SLC1 and PAH1, all the citrate-mediated acyl-CoA synthesis pathway-related genes, and OLE1. These results indicate that LsSpt23p regulates TAG synthesis. Phosphatidic acid is a common substrate of phosphatidic acid phosphohydrolase, which is used for TAG synthesis, and phosphatidate cytidylyltransferase 1 for phospholipid synthesis in the Kennedy pathway. LsSpt23p directly regulated PAH1 but did not affect the expression of CDS1, suggesting that the preferred route of carbon is the Pah1p-mediated TAG synthesis pathway under nitrogen limitation condition. The present study contributes to understanding the regulation of TAG synthesis, and will be valuable in future improvement of TAG productivity in oleaginous yeasts. KEY POINTS: LsSpt23p was identified as a positive regulator of TAG biosynthesis LsSPT23 overexpression enhanced TAG biosynthesis gene expression and TAG production LsSPT23M1108T overexpression mutant showed fivefold higher TAG production than control.

Prdm12 regulates inhibitory neuron differentiation in mouse embryonal carcinoma cells.

The epigenetic regulatory system significant influences the fate determination of cells during developmental processes. Prdm12 is a transcriptional regulator that modulates gene expression epigenetically. The Prdm12 gene has been shown to be expressed in neural tissues, specifically during development, but its detailed function is not fully understood. This study investigated the function of the Prdm12 gene in P19 mouse embryonic tumor cells as a model for neural differentiation. A decrease in the expression of neuron-specific genes and the alterations of dendrites and axons morphology was confirmed in Prdm12-knockout P19 cells. In addition, almost no astrocytes were generated in Prdm12-knockout P19 cells. Comprehensive gene expression analysis revealed that there was a reduction in the expression of the inhibitory neuron-specific genes Gad1/2 and Glyt2, but not the excitatory neuron-specific gene VGLUT2, in Prdm12-knockout P19 cells. Furthermore, the expression of inhibitory neuron-related factors, Ptf1a, Dbx1, and Gsx1/2, decreased in Prdm12-knockout P19 cells. Gene expression analysis also revealed that the Ptf1a, Hic1, and Foxa1 genes were candidate targets of Prdm12 during neurogenesis. These results suggest that Prdm12 regulates the differentiation of inhibitory neurons and astrocytes by controlling the expression of these genes during the neural differentiation of P19 cells.

Analysis of the light regulatory mechanism in carotenoid production in Rhodosporidium toruloides NBRC 10032.

Light stimulates carotenoid production in an oleaginous yeast Rhodosporidium toruloides NBRC 10032 by promoting carotenoid biosynthesis genes. These genes undergo two-step transcriptional activation. The potential light regulator, Cryptochrome DASH (CRY1), has been suggested to contribute to this mechanism. In this study, based on KU70 (a component of nonhomologous end joining (NHEJ)) disrupting background, CRY1 disruptant was constructed to clarify CRY1 function. From analysis of CRY1 disruptant, it was suggested that CRY1 has the activation role of the carotenogenic gene expression. To obtain further insights into the light response, mutants varying carotenoid production were generated. Through analysis of mutants, the existence of the control two-step gene activation was proposed. In addition, our data analysis showed the strong possibility that R. toruloides NBRC 10032 is a homo-diploid strain.

Effect of light on carotenoid and lipid production in the oleaginous yeast Rhodosporidium toruloides.

The oleaginous yeast Rhodosporodium toruloides is receiving widespread attention as an alternative energy source for biofuels due to its unicellular nature, high growth rate and because it can be fermented on a large-scale. In this study, R. toruloides was cultured under both light and dark conditions in order to understand the light response involved in lipid and carotenoid biosynthesis. Our results from phenotype and gene expression analysis showed that R. toruloides responded to light by producing darker pigmentation with an associated increase in carotenoid production. Whilst there was no observable difference in lipid production, slight changes in the fatty acid composition were recorded. Furthermore, a two-step response was found in three genes (GGPSI, CAR1, and CAR2) under light conditions and the expression of the gene encoding the photoreceptor CRY1 was similarly affected.

Association Between Serum ß-Alanine and Risk of Dementia.

We examined the association between serum concentrations of ß-alanine, a metabolite of carnosine and anserine, and the risk of dementia in a general population of elderly Japanese persons. In 2007, 1,475 residents of Hisayama, Japan, aged 60-79 years and without dementia were divided into 4 groups according to quartiles of serum ß-alanine concentrations (quartile 1, lowest; quartile 4, highest) and followed for a median of 5.3 years. During follow-up, 117 subjects developed all-cause dementia (Alzheimer in 77 cases and vascular dementia in 31). The risk of all-cause dementia decreased with increasing serum ß-alanine levels after adjustment for potential confounding factors (quartile 2, hazard ratio (HR) = 0.73 (95% confidence interval (CI): 0.45, 1.18); quartile 3, HR = 0.50 (95% CI: 0.28, 0.89); quartile 4, HR = 0.50 (95% CI: 0.27, 0.92); P = 0.01 for trend). A similar inverse association was observed for Alzheimer disease (quartile 2, HR = 0.78 (95% CI: 0.44, 1.38); quartile 3, HR = 0.53 (95% CI: 0.26, 1.06); quartile 4, HR = 0.53 (95% CI: 0.25, 1.10); P = 0.04 for trend) but not for vascular dementia. We found that higher serum ß-alanine levels were significantly associated with lower risks of all-cause dementia and Alzheimer disease. Because serum ß-alanine levels reflect intakes of carnosine/anserine, higher intakes of carnosine/anserine might be beneficial for the prevention of dementia.

Cellulase productivity of Trichoderma reesei mutants developed in Japan varies with varying pH conditions.

The ascomycete Trichoderma reesei is known to produce a variety of cellulases and hemicellulases and the hyper-cellulolytic mutants of this fungus are useful as industrial cellulase producers. In Japan, PC-3-7, derived from the early mutant QM9414, is well-known as a cellulase hyperproducing mutant. In addition to the productivity of enzymes, the composition of secreted enzymes greatly influences biomass saccharification. Therefore, we evaluated the cellulase productivity of T. reesei mutants in Japan at different pH as a factor influencing enzyme production. At higher pH values, QM9414 exhibited reduced cellulase productivity whereas PC-3-7 maintained high cellulase productivity and gene expression at the transcriptional level. The gene encoding the pH-responsive transcription factor PACI did not mutate in PC-3-7, and its expression pattern against different pH conditions was similar between QM9414 and PC-3-7. Furthermore, the deletion of pac1 encoding PACI caused different expression patterns of cellulase genes between QM9414 and PC-3-7. Therefore, we suggest that T. reesei possesses a pH-responsive cellulase production mechanism that is different from the PACI-related mechanism. Finally, we identified that N-25, a strain developed at an early stage of mutant development acquired cellulase productivity at a higher pH. In this investigation, we also found and tested candidate genes possibly affecting pH response using comparative genome analysis.

The habu genome reveals accelerated evolution of venom protein genes.

Evolution of novel traits is a challenging subject in biological research. Several snake lineages developed elaborate venom systems to deliver complex protein mixtures for prey capture. To understand mechanisms involved in snake venom evolution, we decoded here the ~1.4-Gb genome of a habu, Protobothrops flavoviridis. We identified 60 snake venom protein genes (SV) and 224 non-venom paralogs (NV), belonging to 18 gene families. Molecular phylogeny reveals early divergence of SV and NV genes, suggesting that one of the four copies generated through two rounds of whole-genome duplication was modified for use as a toxin. Among them, both SV and NV genes in four major components were extensively duplicated after their diversification, but accelerated evolution is evident exclusively in the SV genes. Both venom-related SV and NV genes are significantly enriched in microchromosomes. The present study thus provides a genetic background for evolution of snake venom composition.

Pig testis extract augments adiponectin expression and secretion through the peroxisome proliferator-activated receptor signaling pathway in 3T3-L1 adipocytes.

Adiponectin is a key molecule whose upregulation may ameliorate symptoms of type 2 diabetes mellitus and disorders of lipid metabolism. Several plant-derived components have been shown to enhance adiponectin secretion; however, there have been no reports on the effects of animal-derived products. Therefore, in the current study, we investigated whether hot-water extracts of specific livestock by-products induce the expression of adiponectin in mouse 3T3-L1 adipocytes. Out of the 11 extracts tested, pig testis extract (PTE) was found to enhance adiponectin expression and secretion by 3T3-L1 cells. Furthermore, simultaneous treatment with PTE and daidzein, a soy phytoestrogen, synergistically enhanced adiponectin secretion. Moreover, pretreatment with an estrogen receptor ß antagonist (PHTPP) diminished adiponectin secretion from daidzein-treated cells but not from PTE-treated cells. Transcriptome analyses revealed that both daidzein and PTE regulate the peroxisome proliferator-activated receptor signaling pathway, although differences in the regulation of gene expression were observed between PTE- and daidzein-treated cells. These results suggest that PTE ameliorates lipid metabolic dysfunction by promoting adipocyte differentiation and enhancing adiponectin secretion via a mechanism different from that of daidzein.

Genome-wide identification of pistil-specific genes expressed during fruit set initiation in tomato (Solanum lycopersicum).

Fruit set involves the developmental transition of an unfertilized quiescent ovary in the pistil into a fruit. While fruit set is known to involve the activation of signals (including various plant hormones) in the ovary, many biological aspects of this process remain elusive. To further expand our understanding of this process, we identified genes that are specifically expressed in tomato (Solanum lycopersicum L.) pistils during fruit set through comprehensive RNA-seq-based transcriptome analysis using 17 different tissues including pistils at six different developmental stages. First, we identified 532 candidate genes that are preferentially expressed in the pistil based on their tissue-specific expression profiles. Next, we compared our RNA-seq data with publically available transcriptome data, further refining the candidate genes that are specifically expressed within the pistil. As a result, 108 pistil-specific genes were identified, including several transcription factor genes that function in reproductive development. We also identified genes encoding hormone-like peptides with a secretion signal and cysteine-rich residues that are conserved among some Solanaceae species, suggesting that peptide hormones may function as signaling molecules during fruit set initiation. This study provides important information about pistil-specific genes, which may play specific roles in regulating pistil development in relation to fruit set.

Duplication and Loss of Function of Genes Encoding RNA Polymerase III Subunit C4 Causes Hybrid Incompatibility in Rice.

Reproductive barriers are commonly observed in both animals and plants, in which they maintain species integrity and contribute to speciation. This report shows that a combination of loss-of-function alleles at two duplicated loci, DUPLICATED GAMETOPHYTIC STERILITY 1 (DGS1) on chromosome 4 and DGS2 on chromosome 7, causes pollen sterility in hybrid progeny derived from an interspecific cross between cultivated rice, Oryza sativa, and an Asian annual wild rice, O. nivara Male gametes carrying the DGS1 allele from O. nivara (DGS1-nivaras ) and the DGS2 allele from O. sativa (DGS2-T65s ) were sterile, but female gametes carrying the same genotype were fertile. We isolated the causal gene, which encodes a protein homologous to DNA-dependent RNA polymerase (RNAP) III subunit C4 (RPC4). RPC4 facilitates the transcription of 5S rRNAs and tRNAs. The loss-of-function alleles at DGS1-nivaras and DGS2-T65s were caused by weak or nonexpression of RPC4 and an absence of RPC4, respectively. Phylogenetic analysis demonstrated that gene duplication of RPC4 at DGS1 and DGS2 was a recent event that occurred after divergence of the ancestral population of Oryza from other Poaceae or during diversification of AA-genome species.

Elucidation of the sequential transcriptional activity in Escherichia coli using time-series RNA-seq data.

Functional genomics and gene regulation inference has readily expanded our knowledge and understanding of gene interactions with regards to expression regulation. With the advancement of transcriptome sequencing in time-series comes the ability to study the sequential changes of the transcriptome. Here, we present a new method to augment regulation networks accumulated in literature with transcriptome data gathered from time-series experiments to construct a sequential representation of transcription factor activity. We apply our method on a time-series RNA-Seq data set of Escherichia coli as it transitions from growth to stationary phase over five hours and investigate the various activity in gene regulation process by taking advantage of the correlation between regulatory gene pairs to examine their activity on a dynamic network. We analyse the changes in metabolic activity of the pagP gene and associated transcription factors during phase transition, and visualize the sequential transcriptional activity to describe the change in metabolic pathway activity originating from the pagP transcription factor, phoP. We observe a shift from amino acid and nucleic acid metabolism, to energy metabolism during the transition to stationary phase in E. coli.

Identification of RAN1 orthologue associated with sex determination through whole genome sequencing analysis in fig (Ficus carica L.).

With the aim of identifying sex determinants of fig, we generated the first draft genome sequence of fig and conducted the subsequent analyses. Linkage analysis with a high-density genetic map established by a restriction-site associated sequencing technique, and genome-wide association study followed by whole-genome resequencing analysis identified two missense mutations in RESPONSIVE-TO-ANTAGONIST1 (RAN1) orthologue encoding copper-transporting ATPase completely associated with sex phenotypes of investigated figs. This result suggests that RAN1 is a possible sex determinant candidate in the fig genome. The genomic resources and genetic findings obtained in this study can contribute to general understanding of Ficus species and provide an insight into fig's and plant's sex determination system.

Regulation of matrix metalloproteinase-1 and alpha-smooth muscle actin expression by interleukin-1 alpha and tumour necrosis factor alpha in hepatic stellate cells.

Hepatic stellate cells (HSCs) are key players in liver fibrosis and regeneration via collagen degradation and synthesis. These phenomena involve inflammatory cytokines released from non-parenchymal liver cells such as Kupffer cells. Although the effects of individual cytokines on many cell types have been investigated in various conditions, such as inflammation and tissue fibrosis, investigating the effect of combined cytokines would further our understanding of the regulatory mechanisms in tissue fibrosis. Here, we report the effect of multiple cytokine combinations on primary HSCs. We first examined the effect of individual cytokines and then the simultaneous exposure of different cytokines, including interleukin-6 (IL-6), IL-1 alpha (IL-1α), platelet-derived growth factor (PDGF), tumour necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-ß), on matrix metalloproteinase-1 (MMP1) gene expression in primary HSCs. We observed that the combination of all five cytokines induced higher levels of MMP1 gene expression. Of these cytokines, TNF-α and IL-1α were found to be the key cytokines for not only inducing MMP1 expression, but also increasing α-smooth muscle actin gene expression. In conclusion, the combined treatment of TNF-α and IL-1α on HSCs had an enhanced effect on the expression of the fibrotic genes, MMP1 and α-smooth muscle actin, so appears to be an important regulator for tissue regeneration. This finding suggests that stimulation with combined anti-fibrotic cytokines is a potential approach in the development of a novel therapy for the recovery of liver fibrosis.

Development and evaluation of rice giant embryo mutants for high oil content originated from a high-yielding cultivar 'Mizuhochikara'.

Rice bran oil is a byproduct of the milling of rice (Oryza sativa L.). It offers various health benefits and has a beneficial fatty acid composition. To increase the amount of rice bran as a sink for triacylglycerol (TAG), we developed and characterized new breeding materials with giant embryos. To induce mutants, we treated fertilized egg cells of the high-yielding cultivar 'Mizuhochikara' with N-methyl-N-nitrosourea (MNU). By screening M2 seeds, we isolated four giant embryo mutant lines. Genetic analysis revealed that the causative loci in lines MGE12 and MGE13 were allelic to giant embryo (ge) on chromosome 7, and had base changes in the causal gene Os07g0603700. On the other hand, the causative loci in lines MGE8 and MGE14 were not allelic to ge, and both were newly mapped on chromosome 3. The TAG contents of all four mutant lines increased relative to their wild type, 'Mizuhochikara'. MGE13 was agronomically similar to 'Mizuhochikara' and would be useful for breeding for improved oil content.

Draft Genome Sequence of Bacillus clausii AKU0647, a Strain That Produces Endo-ß-N-Acetylglucosaminidase A.

To comprehensively identify glycosyl hydrolase genes in the genome of Bacillus clausii strain AKU0647, which produces endo-ß-N-acetylglucosaminidase A (Endo-A), we conducted whole-genome shotgun sequencing. We identified several other putative glycosyl hydrolase genes apart from the Endo-A gene, and report these findings here.

Transcriptomic features associated with energy production in the muscles of Pacific bluefin tuna and Pacific cod.

Bluefin tuna are high-performance swimmers and top predators in the open ocean. Their swimming is grounded by unique features including an exceptional glycolytic potential in white muscle, which is supported by high enzymatic activities. Here we performed high-throughput RNA sequencing (RNA-Seq) in muscles of the Pacific bluefin tuna (Thunnus orientalis) and Pacific cod (Gadus macrocephalus) and conducted a comparative transcriptomic analysis of genes related to energy production. We found that the total expression of glycolytic genes was much higher in the white muscle of tuna than in the other muscles, and that the expression of only six genes for glycolytic enzymes accounted for 83.4% of the total. These expression patterns were in good agreement with the patterns of enzyme activity previously reported. The findings suggest that the mRNA expression of glycolytic genes may contribute directly to the enzymatic activities in the muscles of tuna.

Physiological Properties and Genome Structure of the Hyperthermophilic Filamentous Phage φOH3 Which Infects Thermus thermophilus HB8.

A filamentous bacteriophage, φOH3, was isolated from hot spring sediment in Obama hot spring in Japan with the hyperthermophilic bacterium Thermus thermophilus HB8 as its host. Phage φOH3, which was classified into the Inoviridae family, consists of a flexible filamentous particle 830 nm long and 8 nm wide. φOH3 was stable at temperatures ranging from 70 to 90°C and at pHs ranging from 6 to 9. A one-step growth curve of the phage showed a 60-min latent period beginning immediately postinfection, followed by intracellular virus particle production during the subsequent 40 min. The released virion number of φOH3 was 109. During the latent period, both single stranded DNA (ssDNA) and the replicative form (RF) of phage DNA were multiplied from min 40 onward. During the release period, the copy numbers of both ssDNA and RF DNA increased sharply. The size of the φOH3 genome is 5688 bp, and eight putative open reading frames (ORFs) were annotated. These ORFs were encoded on the plus strand of RF DNA and showed no significant homology with any known phage genes, except ORF 5, which showed 60% identity with the gene VIII product of the Thermus filamentous phage PH75. All the ORFs were similar to predicted genes annotated in the Thermus aquaticus Y51MC23 and Meiothermus timidus DSM 17022 genomes at the amino acid sequence level. This is the first report of the whole genome structure and DNA multiplication of a filamentous T. thermophilus phage within its host cell.

Draft Genome Sequence of the Fish Pathogen Mycobacterium pseudoshottsii Strain JCM15466, a Species Closely Related to M. marinum.

Mycobacterium pseudoshottsii is a slowly growing photochromogenic mycobacterium and fish pathogen isolated from wild marine fishes. M. pseudoshottsii closely resembles M. marinum, which is a human and animal pathogen. Here, we report the draft genome sequence of M. pseudoshottsii strain JCM15466, originally isolated from striped bass, Morone saxatilis.