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Follicular lymphoma (FL) of the gastrointestinal tract, particularly duodenal follicular lymphoma (DFL), is a rare variant of FL with indolent clinical behavior, and this disease is included in the 2008 World Health Organization classification system. In contrast to nodal follicular lymphoma (NFL), DFL occurs most frequently in the second part of the duodenum, lacks follicular dendritic cell meshworks and has memory B-cell characteristics. However, its molecular pathogenesis is still unclear. In the present study, we examined 10 DFL, 18 NFL and 10 gastric MALT lymphoma samples using gene expression analysis. Quantitative RT-PCR experiments and immunohistochemical analysis for 72 formalin-fixed, paraffin-embedded tissues from an independent series, including 32 DFL, 19 gastric MALT lymphoma and 27 NFL samples, were performed for validation of microarray data. Gene expression profiles of the three lymphoma types were compared using 2918 differentially expressed genes (DEG) and results suggested that DFL shares characteristics of MALT lymphoma. Among these DEG, CCL20 and MAdCAM-1 were upregulated in DFL and MALT but downregulated in NFL. In contrast, protocadherin gamma subfamily genes were upregulated in DFL and NFL. Quantitative RT-PCR and immunohistochemical studies demonstrated concordant results. Double immunofluorescence studies revealed that CCL20 and CCR6 were co-expressed in both DFL and MALT. We hypothesize that increased expression of CCL20 and MAdCAM-1 and co-expression of CCL20 and CCR6 may play an important role in tumorigenesis.

The Rice Annotation Project Database (RAP-DB) was created to provide the genome sequence assembly of the International Rice Genome Sequencing Project (IRGSP), manually curated annotation of the sequence, and other genomics information that could be useful for comprehensive understanding of the rice biology. Since the last publication of the RAP-DB, the IRGSP genome has been revised and reassembled. In addition, a large number of rice-expressed sequence tags have been released, and functional genomics resources have been produced worldwide. Thus, we have thoroughly updated our genome annotation by manual curation of all the functional descriptions of rice genes. The latest version of the RAP-DB contains a variety of annotation data as follows: clone positions, structures and functions of 31 439 genes validated by cDNAs, RNA genes detected by massively parallel signature sequencing (MPSS) technology and sequence similarity, flanking sequences of mutant lines, transposable elements, etc. Other annotation data such as Gnomon can be displayed along with those of RAP for comparison. We have also developed a new keyword search system to allow the user to access useful information. The RAP-DB is available at: http://rapdb.dna.affrc.go.jp/ and http://rapdb.lab.nig.ac.jp/.

We hypothesized that systemically circulating peripheral blood mononuclear cells (PBMCs) reflect the pathophysiology of type 2 diabetes. PBMCs were obtained from 18 patients with type 2 diabetes and 16 non-diabetic subjects. The expression of genes in the PBMCs was analyzed by using a DNA chip followed by statistical analysis for specific gene sets for biological categories. The only gene set coordinately up-regulated by the existence of diabetes and down-regulated by glycemic control consisted of 48 genes involved in the c-Jun N-terminal kinase (JNK) pathway. In contrast, the only gene set coordinately down-regulated by the existence of diabetes, but not altered by glycemic control consisted of 92 genes involved in the mitochondrial oxidative phosphorylation (OXPHOS) pathway. Our findings suggest that genes involved in the JNK and OXPHOS pathways of PBMCs may be surrogate transcriptional markers for hyperglycemia-induced oxidative stress and morbidity of type 2 diabetes, respectively.

Oocytes have unique characteristics compared with other cell types. In mouse and human oocytes, two X chromosomes are maintained in the active state. Previous microarray studies have shown that the balance of the expression state is maintained in haploid oocytes. Here, we investigated transcripts using RNA-sequence technology in mouse and human oocytes. The median expression ratio between X chromosome and autosomal genes (X:A) in immature mouse oocytes increased as the gene expression levels increased, reaching a value of 1. However, the ratio in mature oocytes was under 1 for all expression categories. Moreover, we observed a markedly low ratio resulting from the bimodal expression patterns of X–linked genes. The low X:A expression ratio in mature oocyte was independent of DNA methylation. While mature human oocytes exhibited a slightly low X:A expression ratio, this was the result of the skewed high frequency of lowly expressed X-linked genes rather than the bimodal state. We propose that this imbalance between the expression dosages of X-chromosome and autosomal genes is a feature of transcripts in mammalian oocytes lacking X-chromosome inactivation.

Transcriptome Auto-annotation Conducting Tool (TACT) is a newly developed web-based automated tool for conducting functional annotation of transcripts by the integration of sequence similarity searches and functional motif predictions. We developed the TACT system by integrating two kinds of similarity searches, FASTY and BLASTX, against protein sequence databases, UniProtKB (Swiss-Prot/TrEMBL) and RefSeq, and a unified motif prediction program, InterProScan, into the ORF-prediction pipeline originally designed for the ‘H-Invitational’ human transcriptome annotation project. This system successively applies these constituent programs to an mRNA sequence in order to predict the most plausible ORF and the function of the protein encoded. In this study, we applied the TACT system to 19 574 non-redundant human transcripts registered in H-InvDB and evaluated its predictive power by the degree of agreement with human-curated functional annotation in H-InvDB. As a result, the TACT system could assign functional description to 12 559 transcripts (64.2%), the remainder being hypothetical proteins. Furthermore, the overall agreement of functional annotation with H-InvDB, including those transcripts annotated as hypothetical proteins, was 83.9% (16 432/19 574). These results show that the TACT system is useful for functional annotation and that the prediction of ORFs and protein functions is highly accurate and close to the results of human curation. TACT is freely available at http://www.jbirc.aist.go.jp/tact/.

We identified a novel type of point mutation at the 22nd codon of the K-ras gene in a primary colon cancer. The mutation was C to A transversion substituting lysine (AAG) for normal glutamine (CAG) codon. Biological activity of this mutant K-ras gene was tested by expression of full-length cDNA clones in NIH3T3 cells. Most of the K-ras Lys22-transfected cells exhibited an increased saturation density, a lower serum requirement, and transformed morphology reminiscent to the typical K-ras Val12 transformants. However, the tumorigenicity of K-ras Lys22 transformants in nude mice was significantly less potent than that of K-ras Val12 transformants; only a high copy number transformant produced tumors. Even though the activation is incomplete, the finding that the majority of tumor cells in the specimen carried the K-ras Lys22 mutation suggests that this mutation might be advantageous for growth of tumor cells in vivo.

The unique antigen peptide pRL1 on BALB/c radiation-induced leukemia RL(male symbol)1 cells is derived from the normally untranslated 5' region of the mouse c-akt gene. Insertion of an endogenous long terminal repeat into the first coding exon of the gene resulted in the enhanced production of an altered akt protein, RL-akt, and creation of the tumor rejection antigen peptide pRL1. In this study, we constructed an RL-akt-expressing vector to investigate the transforming ability and anti-apoptotic activity of RK-akt in NIH/3T3 cells. RL-akt-expressing clones formed more colonies than did c-akt-expressing clones in soft agar and exhibited increased saturation density, a lower serum requirement for growth, and tumorigenicity on athymic nude mice. Immunoblot analysis of subcellular protein distribution showed that a considerable proportion of RL-akt was distributed in the membrane fraction. Thus, RL-akt expressed in NIH/3T3 cells appeared to behave like the v-akt oncoprotein. Furthermore, the RL-akt gene conferred resistance to the apoptosis induced by the calcium ionophore A23187 and by ultraviolet irradiation of NIH/3T3 cells. These findings indicate that the RL-akt gene is able to transform cells and exerts an anti-apoptotic effect on recipient cells, thereby implicating the gene in leukemogenesis of RL(male symbol)1 cells.

Introduction of biologics, such as infliximab, to the therapy of rheumatoid arthritis (RA) patients has revolutionized the treatment of this disease. However, biomarkers for predicting the efficacy of the drug at an early phase of treatment for selecting real responders have not been found. We here present predictive markers based on a thorough transcriptome analysis of white blood cells from RA patients. RNA from whole blood cells of consecutive 42 patients before the first infusion was analyzed with microarrays for training studies. Samples from the subsequent 26 consecutive patients were used for a prospective study. We categorized the results into no inflammation and residual inflammation groups using the serum C-reactive protein (CRP) level at 14 weeks after the first infusion. The accuracy of prediction in our study was 65.4%.

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 ∼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.

H-Invitational Database (H-InvDB; http://www.h-invitational.jp/) is a human transcriptome database, containing integrative annotation of 41,118 full-length cDNA clones originated from 21,037 loci. H-InvDB is a product of the H-Invitational project, an international collaboration to systematically and functionally validate human genes by analysis of a unique set of high quality full-length cDNA clones using automatic annotation and human curation under unified criteria. Here, 19,574 proteins encoded by these cDNAs were classified into 11,709 function-known and 7865 function-unknown hypothetical proteins by similarity with protein databases and motif prediction (InterProScan). The proportion of "hypothetical proteins" in H-InvDB was as high as 40.4%. In this study, we thus conducted data-mining in H-InvDB with the aim of assigning advanced functional annotations to those hypothetical proteins. First, by data-mining in the H-InvDB version of GTOP, we identified 337 SCOP domains within 7865 H-Inv hypothetical proteins. Second, by data-mining of predicted subcellular localization by SOSUI and TMHMM in H-InvDB, we found 1032 transmembrane proteins within H-Inv hypothetical proteins. These results clearly demonstrate that structural prediction is effective for functional annotation of proteins with unknown functions. All the data in H-InvDB are shown in two main views, the cDNA view and the Locus view, and five auxiliary databases with web-based viewers; DiseaseInfo Viewer, H-ANGEL, Clustering Viewer, G-integra and TOPO Viewer; the data also are provided as flat files and XML files. The data consists of descriptions of their gene structures, novel alternative splicing isoforms, functional RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein 3D structure, mapping of SNPs and microsatellite repeat motifs in relation with orphan diseases, gene expression profiling, and comparisons with mouse full-length cDNAs in the context of molecular evolution. This unique integrative platform for conducting in silico data-mining represents a substantial contribution to resources required for the exploration of human biology and pathology.

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology., An international team has systematically validated and annotated just over 21,000 human genes using full-length cDNA, thereby providing a valuable new resource for the human genetics community

Mutation of the Kirsten ras (K-ras) gene is one of most common alterations in solid tumors including lung and colorectal cancers. We developed new enriched PCR-RFLP assay to detect mutations of K-ras codon 61 at the 1st and 2nd letters and non-enriched PCR-RFLP assay to detect the 3rd letter mutation. One mutant allele among 10(3) wild-type alleles was detected by enriched PCR-RFLP assay, while one mutant in 10 wild-type alleles was detected by non-enriched PCR-RFLP assay for codon 61 3rd letter. We then examined K-ras codon 12, 13 and 61 mutations in lung and colorectal cancers using these assays. K-ras codon 12 mutation was detected in 10 of 109 (9%) lung cancer and 19 of 83 (23%) colorectal cancer cases. K-ras codon 13 mutation was detected in 2 of 83 (2%) colorectal and 0 of 109 NSCLC cases, respectively. There was no K-ras codon 61 mutation in either type of cancer. Our results demonstrate that enriched PCR-RFLP is a sensitive assay to detect K-ras codon 61 mutation, however, it was extremely rare in lung and colorectal cancers, suggesting organ-specific pathways in mutagenesis of the ras gene family.

E2F is a family of transcription factors implicated in the regulation of gene expression required for progression through the G(1)-S transition. We have previously detected tumor-specific mutations at a trinucleotide repeat coding sequence of E2F-4 gene in a subset of human sporadic colorectal cancers. The purpose of this study was to investigate the potential functional consequences of these E2F-4 mutations. We transfected NIH3T3 fibroblasts with expression constructs containing wild-type as well as mutant E2F-4 cDNA, and the effect of the E2F-4 mutations on proliferation was examined. Alteration in transactivation of the E2F consensus promoter sequence was also examined by transient cotransfection of a E2F-4 with a DP-2 construct into cultured human cells. Transfected cell clones overexpressing mutant E2F-4 grew more rapidly and showed higher proliferative activity by increased immunohistochemical staining for proliferating cell nuclear antigen (PCNA). All three mutant forms of E2F-4 showed elevated transactivation of the E2F consensus promoter sequence. Thus, expression of mutant E2F-4s confers a growth advantage in vivo, and this effect may be related to the acquisition of a neoplastic phenotype.

Genetic instability at microsatellites in some colorectal cancer (CRC) have been linked to the defects of human mismatch repair genes, but the targets of these defective genes have been largely unknown. We screened CRC specimens for alteration of E2F-4 gene by analyzing both cDNA and genomic sequences along with replication error (RER+) phenotype. Two out of 20 sporadic CRC patients showed RER+ phenotype. We found tumor-specific copy number alteration in 13 consecutive trinucleotide (CAG) repeats within the coding exon of E2F-4 exclusively in these 2 specimens. Thus, E2F-4 may be a clue of the target gene of defective repair genes in CRC with genetic instability in addition to the TGF-beta type II receptor gene.

The Human Anatomic Gene Expression Library (H-ANGEL) is a resource for information concerning the anatomical distribution and expression of human gene transcripts. The tool contains protein expression data from multiple platforms that has been associated with both manually annotated full-length cDNAs from H-InvDB and RefSeq sequences. Of the H-Inv predicted genes, 18 897 have associated expression data generated by at least one platform. H-ANGEL utilizes categorized mRNA expression data from both publicly available and proprietary sources. It incorporates data generated by three types of methods from seven different platforms. The data are provided to the user in the form of a web-based viewer with numerous query options. H-ANGEL is updated with each new release of cDNA and genome sequence build. In future editions, we will incorporate the capability for expression data updates from existing and new platforms. H-ANGEL is accessible at http://www.jbirc.aist.go.jp/hinv/h-angel/.