Overexpression of Rev1 promotes the development of carcinogen-induced intestinal adenomas via accumulation of point mutation and suppression of apoptosis proportionally to the Rev1 expression level.

Cancer development often involves mutagenic replication of damaged DNA by the error-prone translesion synthesis (TLS) pathway. Aberrant activation of this pathway plays a role in tumorigenesis by promoting genetic mutations. Rev1 controls the function of the TLS pathway, and Rev1 expression levels are associated with DNA damage induced cytotoxicity and mutagenicity. However, it remains unclear whether deregulated Rev1 expression triggers or promotes tumorigenesis in vivo. In this study, we generated a novel Rev1-overexpressing transgenic (Tg) mouse and characterized its susceptibility to tumorigenesis. Using a small intestinal tumor model induced by N-methyl-N-nitrosourea (MNU), we found that transgenic expression of Rev1 accelerated intestinal adenoma development in proportion to the Rev1 expression level; however, overexpression of Rev1 alone did not cause spontaneous development of intestinal adenomas. In Rev1 Tg mice, MNU-induced mutagenesis was elevated, whereas apoptosis was suppressed. The effects of hREV1 expression levels on the cytotoxicity and mutagenicity of MNU were confirmed in the human cancer cell line HT1080. These data indicate that dysregulation of cellular Rev1 levels leads to the accumulation of mutations and suppression of cell death, which accelerates the tumorigenic activities of DNA-damaging agents.

Generation and evaluation of an anti-REV1 monoclonal antibody.

Continuous exposure of cells to exogenous and endogenous agents produces many types of DNA damage during normal cell cycles. Post-replication repair, consisting of error-free and error-prone sub-pathways, is required for tolerance of such DNA damage. REV1 plays a crucial role in regulation of the error-prone pathway. To facilitate analysis of its cellular functions, we here generated a mouse Rev1 monoclonal antibody, called D6, which also recognizes human REV1. The epitope for the antibody could be mapped between 860-877 amino acid residues of human REV1, which was located outside of the conserved catalytic domain. Although the antibody unfortunately could not specifically detect endogenous mouse and human REV1 by western blotting and immunohistochemistry, we established a method to identify endogenous human REV1 by immunoprecipitation-western blotting analysis combining D6 and separately generated polyclonal antibodies.

The H-Invitational Database (H-InvDB), a comprehensive annotation resource for human genes and transcripts.

Here we report the new features and improvements in our latest release of the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/), a comprehensive annotation resource for human genes and transcripts. H-InvDB, originally developed as an integrated database of the human transcriptome based on extensive annotation of large sets of full-length cDNA (FLcDNA) clones, now provides annotation for 120 558 human mRNAs extracted from the International Nucleotide Sequence Databases (INSD), in addition to 54 978 human FLcDNAs, in the latest release H-InvDB_4.6. We mapped those human transcripts onto the human genome sequences (NCBI build 36.1) and determined 34 699 human gene clusters, which could define 34 057 (98.1%) protein-coding and 642 (1.9%) non-protein-coding loci; 858 (2.5%) transcribed loci overlapped with predicted pseudogenes. For all these transcripts and genes, we provide comprehensive annotation including gene structures, gene functions, alternative splicing variants, functional non-protein-coding RNAs, functional domains, predicted sub cellular localizations, metabolic pathways, predictions of protein 3D structure, mapping of SNPs and microsatellite repeat motifs, co-localization with orphan diseases, gene expression profiles, orthologous genes, protein-protein interactions (PPI) and annotation for gene families. The current H-InvDB annotation resources consist of two main views: Transcript view and Locus view and eight sub-databases: the DiseaseInfo Viewer, H-ANGEL, the Clustering Viewer, G-integra, the TOPO Viewer, Evola, the PPI view and the Gene family/group.

Curated genome annotation of Oryza sativa ssp. japonica and comparative genome analysis with Arabidopsis thaliana.

We present here the annotation of the complete genome of rice Oryza sativa L. ssp. japonica cultivar Nipponbare. All functional annotations for proteins and non-protein-coding RNA (npRNA) candidates were manually curated. Functions were identified or inferred in 19,969 (70%) of the proteins, and 131 possible npRNAs (including 58 antisense transcripts) were found. Almost 5000 annotated protein-coding genes were found to be disrupted in insertional mutant lines, which will accelerate future experimental validation of the annotations. The rice loci were determined by using cDNA sequences obtained from rice and other representative cereals. Our conservative estimate based on these loci and an extrapolation suggested that the gene number of rice is approximately 32,000, which is smaller than previous estimates. We conducted comparative analyses between rice and Arabidopsis thaliana and found that both genomes possessed several lineage-specific genes, which might account for the observed differences between these species, while they had similar sets of predicted functional domains among the protein sequences. A system to control translational efficiency seems to be conserved across large evolutionary distances. Moreover, the evolutionary process of protein-coding genes was examined. Our results suggest that natural selection may have played a role for duplicated genes in both species, so that duplication was suppressed or favored in a manner that depended on the function of a gene.

Retionic acid and its receptors are required for expression of aryl hydrocarbon receptor mRNA and embryonic development of blood vessel and bone in the medaka fish, Oryzias latipes.

Retinoic acid (RA), the active derivative of vitamin A, is essential for normal embryonic development of vertebrates because both the lack and excess of RA result in developmental malformations. We previously reported that aryl hydrocarbon receptor (AHR) is also required for vascular and bone formation by regulating cytochrome P450 expression. However, little is known about the roles of retinoic acid receptors (RAR) and retinoid X receptors (RXR) in the embryonic development of blood vessels and molecular cross-talk between RAR/RXR and AHR. We report for the first time that RA and RAR/RXR are required for expression of AHR mRNA and the embryonic development of blood vessel and bone. The embryonic organogenesis of medaka fish was specifically inhibited by an inhibitor of RA synthesis (diethylaminobenzaldehyde), antagonists of RAR (Ro41-5253) and RXR (Ro71-4595), agonist (beta-naphthoflavone) and antagonist (alpha-naphthoflavone) of AHR, and excess RA. These reagents are useful for future studies to elucidate molecular mechanisms for vascular and bone formation in the medaka embryogenesis. Our results also show that medaka embryos may be useful for screening inhibitors of vascular formation for anti-cancer drugs.

No effects of estrogen receptor overexpression on gonadal sex differentiation and reversal in medaka fish.

In order to elucidate a possible role of estrogen receptor in the gonadal sex differentiation and the sex reversal with sex steroids, we examined for the formation of testis or ovary in transgenic medaka fish overexpressing the medaka estrogen receptor under the constitutive medaka beta-actin promoter. The transgenic fish underwent the genetically determined gonadal differentiation and showed the same sex-reversal rates as those of wild-type non-transgenic fish after treatments with estrogen and androgen. These results present invaluable data to reconsider the role of estrogen receptor in the gonadal sex determination.

Aryl hydrocarbon receptor is required for prevention of blood clotting and for the development of vasculature and bone in the embryos of medaka fish, Oryzias latipes.

The aryl hydrocarbon receptor (AHR) is a member of ligand-activated transcription factors and conserved among vertebrates. To investigate the role of AHR in fish development, medaka embryos were treated with agonist (2,3,7,8-tetrachlorodibenzo-p-dioxin), antagonists (alpha-naphthoflavone and resveratrol), and inhibitor (piperonyl butoxide) of cytochromes (Cyts) P450 encoded by a battery of target genes. These embryos were found to have similar abnormal phenotypes. Among the most consistent phenotypes were blood clotting and malformation of bone that were associated with vascular damages. These results thus indicate that control of AHR is important for proper development of fish embryos. AHR may control levels of Cyts P450 that are responsible for synthesis and metabolism of a toxic compound that caused the abnormal phenotypes. Complementary DNA fragments encoding AHR homologs were cloned from medaka embryos. AHR-specific mRNA was ubiquitously expressed in embryos and adult tissues.

Estrogen inhibits development of yolk veins and causes blood clotting in transgenic medaka fish overexpressing estrogen receptor.

We established three transgenic medaka fish lines overexpressing the medaka estrogen receptor under the constitutive medaka beta-actin promoter. The transgenic embryos became hypersensitive to estrogens (17 beta-estradiol and 17alpha-ethinylestradiol), and failed to develop yolk veins while blood clots formed in the blood island within 3 days after exposure to the estrogens. The embryos developed normally if exposed to estrogen after an early neurula stage, suggesting that the sensitive stage is before neurulation. The developmental defects were recovered by incubation with an anti-estrogen, tamoxifen. These results indicate that activation of estrogen receptor caused the estrogen-induced developmental defects. Our results show that the transgenic embryos can be used to assay the blood clotting activity of estrogenic compounds in vivo.