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Androgen receptor (AR) is a critical transcription factor that regulates various target genes and contributes to the pathophysiology of prostate cancer hormone dependently. Here, we identify amyloid precursor protein (APP) as a primary androgen target through chromatin immunoprecipitation (ChIP) combined with genome tiling array analysis (ChIP-chip). ChIP-treated DNA were obtained from prostate cancer LNCaP cells with R1881 or vehicle treatment using AR or acetylated histone H3 antibodies. Ligand-dependent AR binding was further enriched by PCR subtraction. Using chromosome 21/22 arrays, we identified APP as one of the androgen-regulated genes with adjacent functional AR binding sites. APP expression is androgen-inducible in LNCaP cells and APP immunoreactivity was correlated with poor prognosis in patients with prostate cancer. Gain-of-function and loss-of-function studies revealed that APP promotes the tumor growth of prostate cancer. The present study reveals a novel APP-mediated pathway responsible for the androgen-dependent growth of prostate cancer. Our findings will indicate that APP could be a potential molecular target for the diagnosis and treatment of prostate cancer. [Cancer Res 2009;69(1):137–42]

The Human Genome Project (HGP) is one of the most important international achievements in life sciences, to which Japanese scientists made remarkable contributions. In the early 1980s, Akiyoshi Wada pioneered the first project for the automation of DNA sequencing technology. Ken-ichi Matsubara exhibited exceptional leadership to launch the comprehensive human genome program in Japan. Hideki Kambara made a major contribution by developing a key device for high-speed DNA sequencers, which enabled scientists to construct human genome draft sequences. The RIKEN team led by Yoshiyuki Sakaki (the author) played remarkable roles in the draft sequencing and completion of chromosomes 21, 18, and 11. Additionally, the Keio University team led by Nobuyoshi Shimizu made noteworthy contributions to the completion of chromosomes 22, 21, and 8. In April 2003, the Japanese team joined the international consortium in declaring the completion of the human genome sequence. Consistent with the HGP mandate, Japan has successfully developed a wide range of ambitious genomic sciences.

The disease model of familial amyloidotic polyneuropathy-7.2-hMet30 mice-manifests amyloid deposition that consists of a human amyloidogenic mutant transthyretin (TTR) (TTR V30M). Our previous study found amyloid deposits in 14 of 27 7.2-hMet30 mice at 21-24 months of age. In addition, non-fibrillar TTR deposits were found in amyloid-negative 7.2hMet30 mice. These results suggested that TTR amyloidogenesis required not only mutant TTR but also an additional factor (or factors) as an etiologic molecule. To determine the differences in serum proteome in amyloid-positive and amyloid-negative mice in the 7.2-hMet30 model, we used proteomic analyses and studied serum samples obtained from these mice. Hemopexin (HPX) and transferrin (Tf) were detected in the serum samples from amyloid-positive mice and were also found in amyloid deposits via immunohistochemistry, but serum samples from amyloid-negative mice did not contain HPX and Tf. These two proteins were also not detected in non-fibrillar TTR deposits. In addition, in silico analyses suggested that HPX and Tf facilitate destabilization of TTR secondary structures and misfolding of TTR. These results suggest that HPX and Tf may be associated with TTR amyloidogenesis after fibrillogenesis in vivo.

Protobothrops flavoviridis (habu) (Crotalinae, Viperidae) is a Japanese venomous snake, and its venom contains the enzymes with a variety of physiological activities. The phospholipases A2 (PLA2s) are the major components and exert various toxic effects. They are expressed abundantly in the venom gland. It is thought that the venom gland-specific transcription factors play a key role for activation of PLA2 genes specifically expressed in the venom gland. Thus, the full-length cDNA library for P. flavoviridis venom gland after milking of the venom was made to explore the transcription factors therein. As a result, three cDNAs encoding epithelium-specific ETS transcription factors (ESE)-1, -2, and -3 were obtained. Among them, ESE-3 was specifically expressed in the venom gland and activated the proximal promoters of venom PLA2 genes, which are possibly regarded as the representatives of the venom gland-specific protein genes in P. flavoviridis. Interestingly, the binding specificity of ESE-3 to the ETS binding motif located near TATA box is well correlated with transcriptional activities for the venom PLA2 genes. This is the first report that venom gland-specific transcription factor could actually activate the promoters of the venom protein genes.

Objective Previous studies have indicated that type-1 and type-2 interleukin-1 (IL-1) receptors (IL-1R1 and IL-1R2) play important roles in periodontitis progression. We investigated the association between periodontitis and polymorphisms in the IL-1R1 and IL-1R2 genes ( IL1R1 and IL1R2 ). Design We searched for genetic variants in IL1R1 and IL1R2 in 24 Japanese patients with aggressive periodontitis (AgP) and 24 periodontally healthy controls. Thirty-eight single nucleotide polymorphisms (SNPs) were identified within genomic regions containing all exons and relevant exon-intron boundaries in IL1R1 and IL1R2 . Possible associations of each gene locus with AgP were investigated in 119 AgP patients and 102 periodontally healthy controls using allelotypes, genotypes, and haplotypes. Results Significant differences were noted in the frequencies of 3 SNPs in IL1R2 (rs3819370, rs3218974 and rs3218977) for AgPs and controls ( p = 0.012, p = 0.008, and p = 0.038, respectively), after adjustment for gender and smoking status in the additive model ( p = 0.016, p = 0.007, and p = 0.027, respectively) and 2 haplotypes ( p = 0.010 and p = 0.011, respectively) constructed from 2 SNPs (rs3819370 and rs3218974) that showed the lowest p -values after adjustment of covariates in additive models. Conclusion A genetic susceptibility locus for AgP may lie within or close to the IL1R2 locus. Further studies in other populations are necessary to confirm these results.

The first studies that identified leptin and its receptor (LepR) in mammals were based on mutant animals that displayed dramatic changes in body-weight and regulation of energy homeostasis. Subsequent studies have shown that a deficiency of leptin or LepR in homoeothermic mammals results in hyperphagia, obesity, infertility and a number of other abnormalities. The physiological roles of leptin-mediated signaling in ectothermic teleosts are still being explored. Here, we produced medaka with homozygous LepR gene mutation using the targeting induced local lesions in a genome method. This knockout mutant had a point mutation of cysteine for stop codon at the 357th amino acid just before the leptin-binding domain. The evidence for loss of function of leptin-mediated signaling in the mutant is based on a lack of response to feeding in the expression of key appetite-related neuropeptides in the diencephalon. The mutant lepr−/− medaka expressed constant up-regulated levels of mRNA for the orexigenic neuropeptide Ya and agouti-related protein and a suppressed level of anorexigenic proopiomelanocortin 1 in the diencephalon independent of feeding, which suggests that the mutant did not possess functional LepR. Phenotypes of the LepR-mutant medaka were analyzed in order to understand the effects on food intake, growth, and fat accumulation in the tissues. The food intake of the mutant medaka was higher in post-juveniles and adult stages than that of wild-type (WT) fish. The hyperphagia led to a high growth rate at the post-juvenile stage, but did not to significant alterations in final adult body size. There was no additional deposition of fat in the liver and muscle in the post-juvenile and adult mutants, or in the blood plasma in the adult mutant. However, adult LepR mutants possessed large deposits of visceral fat, unlike in the WT fish, in which there were none. Our analysis confirms that LepR in medaka exert a powerful influence on the control on food intake. Further analyses using the mutant will contribute to a better understanding of the role of leptin in fish. This is the first study to produce fish with leptin receptor deficiency.

Summary Several thermophilic hydrogenotrophic methanogens naturally aggregate in their habitats in association with hydrogen-producing bacteria for efficient transfer of the methane fermentation intermediates to produce methane. However, physiology of aggregation and the identity of aggregation-specific genes remain to be elucidated. Here, we isolated and characterized a hydrogen and formate-utilizing Methanothermobacter sp. CaT2 that is capable of self-aggregation and utilizing formate. CaT2 produced methane from propionate oxidation in association with a syntrophic propionate-oxidizing bacterium faster than other methanogens, including Methanothermobacter thermautotrophicus ΔH and Methanothermobacter thermautotrophicus Z-245. CaT2 also aggregated throughout the culture period and was coated with polysaccharides, which was not found on the ΔH and Z-245 cells. Sugar content (particularly of rhamnose and mannose) was also higher in the CaT2 cells than the ΔH and Z-245 cells. Comparative genomic analysis of CaT2 indicated that four candidate genes, all of which encode glycosyltransferase, were involved in aggregation of CaT2. Transcriptional analysis showed that one glycosyltransferase gene was expressed at relatively high levels under normal growth conditions. The polysaccharide layer on the CaT2 cell surface, which is probably assembled by these glycosyltransferases, may be involved in cell aggregation.

Plants belonging to the Brassicaceae family exhibit species-specific profiles of glucosinolates (GSLs), a class of defence compounds against pathogens and insects. GSLs also exhibit various human health-promoting properties. Among them, glucoraphanin (aliphatic 4-methylsulphinylbutyl GSL) has attracted the most attention because it hydrolyses to form a potent anticancer compound. Increased interest in developing commercial varieties of Brassicaceae crops with desirable GSL profiles has led to attempts to identify genes that are potentially valuable for controlling GSL biosynthesis. However, little attention has been focused on genes of kale (Brassica oleracea var. acephala). In this study, we established full-length kale cDNA libraries containing 59 904 clones, which were used to generate an expressed sequence tag (EST) data set with 119 204 entries. The EST data set clarified genes related to the GSL biosynthesis pathway in kale. We specifically focused on BoMYB29, a homolog of Arabidopsis MYB29/PMG2/HAG3, not only to characterize its function but also to demonstrate its usability as a biological resource. BoMYB29 overexpression in wild-type Arabidopsis enhanced the expression of aliphatic GSL biosynthetic genes and the accumulation of aliphatic GSLs. When expressed in the myb28myb29 mutant, which exhibited no detectable aliphatic GSLs, BoMYB29 restored the expression of biosynthetic genes and aliphatic GSL accumulation. Interestingly, the ratio of methylsulphinyl GSL content, including glucoraphanin, to that of methylthio GSLs was greatly increased, indicating the suitability of BoMYB29 as a regulator for increasing methylsulphinyl GSL content. Our results indicate that these biological resources can facilitate further identification of genes useful for modifications of GSL profiles and accumulation in kale.

The endoplasmic reticulum (ER) membrane-bound transcription factors ATF6α and ATF6β mediate adjustment of chaperone levels to increased demands in the ER, which is essential for development of the notochord; the latter synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis before formation of the vertebra., ATF6α and ATF6β are membrane-bound transcription factors activated by regulated intramembrane proteolysis in response to endoplasmic reticulum (ER) stress to induce various ER quality control proteins. ATF6α- and ATF6β single-knockout mice develop normally, but ATF6α/β double knockout causes embryonic lethality, the reason for which is unknown. Here we show in medaka fish that ATF6α is primarily responsible for transcriptional induction of the major ER chaperone BiP and that ATF6α/β double knockout, but not ATF6α- or ATF6β single knockout, causes embryonic lethality, as in mice. Analyses of ER stress reporters reveal that ER stress occurs physiologically during medaka early embryonic development, particularly in the brain, otic vesicle, and notochord, resulting in ATF6α- and ATF6β-mediated induction of BiP, and that knockdown of the α1 chain of type VIII collagen reduces such ER stress. The absence of transcriptional induction of several ER chaperones in ATF6α/β double knockout causes more profound ER stress and impaired notochord development, which is partially rescued by overexpression of BiP. Thus ATF6α/β-mediated adjustment of chaperone levels to increased demands in the ER is essential for development of the notochord, which synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis before formation of the vertebra.

The common marmoset is a new world monkey, which has become a valuable experimental animal for biomedical research. This study developed cDNA libraries for the common marmoset from five different tissues. A total of 290 426 high-quality EST sequences were obtained, where 251 587 sequences (86.5%) had homology (1E(-100)) with the Refseqs of six different primate species, including human and marmoset. In parallel, 270 673 sequences (93.2%) were aligned to the human genome. When 247 090 sequences were assembled into 17 232 contigs, most of the sequences (218 857 or 15 089 contigs) were located in exonic regions, indicating that these genes are expressed in human and marmoset. The other 5578 sequences (or 808 contigs) mapping to the human genome were not located in exonic regions, suggesting that they are not expressed in human. Furthermore, a different set of 118 potential coding sequences were not similar to any Refseqs in any species, and, thus, may represent unknown genes. The cDNA libraries developed in this study are available through RIKEN Bio Resource Center. A Web server for the marmoset cDNAs is available at http://marmoset.nig.ac.jp/index.html, where each marmoset EST sequence has been annotated by reference to the human genome. These new libraries will be a useful genetic resource to facilitate research in the common marmoset.

Roberts syndrome and SC phocomelia (RBS/SC) are genetic autosomal recessive syndromes caused by establishment of cohesion 1 homolog 2 ( ESCO 2) mutation. RBS/SC appear to have a variety of clinical features, even with the same mutation of the ESCO2 gene. Here, we established and genetically characterized a medaka model of RBS/SC by reverse genetics. The RBS/SC model was screened from a mutant medaka library produced by the Targeting Induced Local Lesions in Genomes method. The medaka mutant carrying the homozygous mutation at R80S in the conserved region of ESCO2 exhibited clinical variety (i.e. developmental arrest with craniofacial and chromosomal abnormalities and embryonic lethality) as characterized in RBS/SC. Moreover, widespread apoptosis and downregulation of some gene expression, including notch1a, were detected in the R80S mutant. The R80S mutant is the animal model for RBS/SC and a valuable resource that provides the opportunity to extend knowledge of ESCO2. Downregulation of some gene expression in the R80S mutant is an important clue explaining non-correlation between genotype and phenotype in RBS/SC.

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis.

Genome sequencing of Streptomyces species has highlighted numerous potential genes of secondary metabolite biosynthesis. The mining of cryptic genes is important for exploring chemical diversity. Here we report the metabolite-guided genome mining and functional characterization of a cryptic gene by biochemical studies. Based on systematic purification of metabolites from Streptomyces sp. SN-593, we isolated a novel compound, 6-dimethylallylindole (DMAI)-3-carbaldehyde. Although many 6-DMAI compounds have been isolated from a variety of organisms, an enzyme catalyzing the transfer of a dimethylallyl group to the C-6 indole ring has not been reported so far. A homology search using known prenyltransferase sequences against the draft sequence of the Streptomyces sp. SN-593 genome revealed the iptA gene. The IptA protein showed 27% amino acid identity to cyanobacterial LtxC, which catalyzes the transfer of a geranyl group to (−)-indolactam V. A BLAST search against IptA revealed much-more-similar homologs at the amino acid level than LtxC, namely, SAML0654 (60%) from Streptomyces ambofaciens ATCC 23877 and SCO7467 (58%) from S. coelicolor A3(2). Phylogenetic analysis showed that IptA was distinct from bacterial aromatic prenyltransferases and fungal indole prenyltransferases. Detailed kinetic analyses of IptA showed the highest catalytic efficiency (6.13 min −1 μM −1 ) for l -Trp in the presence of dimethylallyl pyrophosphate (DMAPP), suggesting that the enzyme is a 6-dimethylallyl- l -Trp synthase (6-DMATS). Substrate specificity analyses of IptA revealed promiscuity for indole derivatives, and its reaction products were identified as novel 6-DMAI compounds. Moreover, Δ iptA mutants abolished the production of 6-DMAI-3-carbaldehyde as well as 6-dimethylallyl- l -Trp, suggesting that the iptA gene is involved in the production of 6-DMAI-3-carbaldehyde.

Parkinson's disease is a neurodegenerative disease associated with the degeneration of dopaminergic neurons in the substantia nigra. The PTEN-induced kinase 1 gene (PINK1) is responsible for recessive inherited familial Parkinson's disease (PARK6). Neither the function of PINK1 nor its role in the prevention of Parkinson's disease is fully understood. Gene disruption of PINK1 causes remarkably different phenotypes in animal models such as Drosophila melanogaster, zebrafish, and mouse, none of which recapitulate Parkinson's-disease-like symptoms. We established PINK1-gene-disrupted medaka fish. These mutant fish grew normally at first, then developed significant decrease in the frequency of spontaneous swimming movements in the late-adult stage. Although the mutants did not show any dopaminergic cell loss, the amount of 3,4-dihydroxyphenylacetic acid, a major metabolite of dopamine, decreased. Thus, PINK1 contributes to the maintenance of dopamine metabolism, even before the selective death of dopaminergic neurons. Our animal model is therefore a valuable tool to detect pathogenesis in Parkinson's patients in the early stages.

Patterns of Early Migration In order to gain insight into various migrations that must have happened during movement of early humans into Asia and the subsequent populating of the largest continent on Earth, the HUGO Pan-Asian SNP Consortium (p. 1541 ) analyzed genetic variation in almost 2000 individuals representing 73 Asian and two non-Asian populations. The results suggest that there may have been a single major migration of people into Asia and a subsequent south-to-north migration across the continent. While most populations from the same linguistic group tend to cluster together in terms of relatedness, several do not, clustering instead with their geographic neighbors, suggesting either substantial recent mixing among the populations or language replacement. Furthermore, data from indigenous Taiwanese populations appear to be inconsistent with the idea of a Taiwan homeland for Austronesian populations.

Alternative splicing (AS) is a mechanism by which multiple types of mature mRNAs are generated from a single pre-mature mRNA. In this study, we completely sequenced 1800 full-length cDNAs from Arabidopsis thaliana, which had 5' and/or 3' sequences that were previously found to have AS events or alternative transcription start sites. Unexpectedly, these sequences gave us further evidence of AS, as 601 out of 1800 transcripts showed novel AS events. We focused on the combination patterns of multiple AS events within individual genes. Interestingly, some specific AS event combination patterns tended to appear more frequently than expected. The two most common patterns were: (i) alternative donor-0 approximately 12 times of exon skips-alternative acceptor and (ii) several times ( approximately 8) of retained introns. We also found that multiple AS events in a transcript tend to have the same effects concerning the length of the mature mRNA. Our current results are consistent with our previous observations, which showed changes in AS profiles under different conditions, and suggest the involvement of hypothetical cis- and trans-acting factors in the regulation of AS events.

We have described a new DNA sequencing platform based on the Sanger chemistry, in which the large-scale microfabricated channel plates and electrophoretic system result in higher-throughput DNA sequencing. Three hundred and eighty-four channels are arranged in a fan-like shape on a 25x47 cm glass plate, on which 384 oval sample holes are connected to each channel coupled to the opposite anode access holes. Two microfabricated plates are set on the sequencing apparatus, in which sequencing electrophoresis is conducted on one plate and the preparation process is on another plate. Each sample hole is loaded with 2.3 microL volume of sample and injected into separation channels electrokinetically. High-quality sequencing data were acquired using the pUC18 template, achieving an average read-length of 1001 bases with 99% accuracy and a throughput of 5 Mbases per day per instrument. To assess the performance in actual sequencing field, the bacterial artificial chromosome shotgun library of the Pseudorca crassidens genome was sequenced, using 1/80 of the quantity of Sanger reagent (0.1 microL). We believe that this is the first demonstration of the useful performance of DNA sequencing using monolithic microfabricated devices with walk-away operation.

A large collection of full-length cDNAs is essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. We obtained a total of 39,936 soybean cDNA clones (GMFL01 and GMFL02 clone sets) in a full-length-enriched cDNA library which was constructed from soybean plants that were grown under various developmental and environmental conditions. Sequencing from 5' and 3' ends of the clones generated 68 661 expressed sequence tags (ESTs). The EST sequences were clustered into 22,674 scaffolds involving 2580 full-length sequences. In addition, we sequenced 4712 full-length cDNAs. After removing overlaps, we obtained 6570 new full-length sequences of soybean cDNAs so far. Our data indicated that 87.7% of the soybean cDNA clones contain complete coding sequences in addition to 5'- and 3'-untranslated regions. All of the obtained data confirmed that our collection of soybean full-length cDNAs covers a wide variety of genes. Comparative analysis between the derived sequences from soybean and Arabidopsis, rice or other legumes data revealed that some specific genes were involved in our collection and a large part of them could be annotated to unknown functions. A large set of soybean full-length cDNA clones reported in this study will serve as a useful resource for gene discovery from soybean and will also aid a precise annotation of the soybean genome.

RNA interference (RNAi) is a mechanism by which double-stranded RNAs (dsRNAs) suppress specific transcripts in a sequence-dependent manner. dsRNAs are processed by Dicer to 21-24-nucleotide small interfering RNAs (siRNAs) and then incorporated into the argonaute (Ago) proteins. Gene regulation by endogenous siRNAs has been observed only in organisms possessing RNA-dependent RNA polymerase (RdRP). In mammals, where no RdRP activity has been found, biogenesis and function of endogenous siRNAs remain largely unknown. Here we show, using mouse oocytes, that endogenous siRNAs are derived from naturally occurring dsRNAs and have roles in the regulation of gene expression. By means of deep sequencing, we identify a large number of both approximately 25-27-nucleotide Piwi-interacting RNAs (piRNAs) and approximately 21-nucleotide siRNAs corresponding to messenger RNAs or retrotransposons in growing oocytes. piRNAs are bound to Mili and have a role in the regulation of retrotransposons. siRNAs are exclusively mapped to retrotransposons or other genomic regions that produce transcripts capable of forming dsRNA structures. Inverted repeat structures, bidirectional transcription and antisense transcripts from various loci are sources of the dsRNAs. Some precursor transcripts of siRNAs are derived from expressed pseudogenes, indicating that one role of pseudogenes is to adjust the level of the founding source mRNA through RNAi. Loss of Dicer or Ago2 results in decreased levels of siRNAs and increased levels of retrotransposon and protein-coding transcripts complementary to the siRNAs. Thus, the RNAi pathway regulates both protein-coding transcripts and retrotransposons in mouse oocytes. Our results reveal a role for endogenous siRNAs in mammalian oocytes and show that organisms lacking RdRP activity can produce functional endogenous siRNAs from naturally occurring dsRNAs.

We constructed and characterized arrayed bacterial artificial chromosome (BAC) libraries of five Drosophila species (D. melanogaster, D. simulans, D. sechellia, D. auraria, and D. ananassae), which are genetically well characterized in the studies of meiosis, evolution, population genetics, and developmental biology. The BAC libraries comprise 8,000 to 12,500 clones for each species, estimated to cover the most of the genomes. We sequenced both ends of most of these BAC clones with a success rate of 91%. Of these, 53,701 clones consisting of non-repetitive BAC end sequences (BESs) were mapped with reference of the public D. melanogaster genome sequences. The BES mapping estimated that the BAC libraries of D. auraria and D. ananassae covered 47% and 57% of the D. melanogaster genome, respectively, and those of D. melanogaster, D. sechellia, and D. simulans covered 94-97%. The low coverage by BESs of D. auraria and D. ananassae may be due to the high sequence divergence with D. melanogaster. From the comparative BES mapping, 111 possible breakpoints of chromosomal rearrangements were identified in these four species. The breakpoints of the major chromosome rearrangement between D. simulans and D. melanogaster on the third chromosome were determined within 20 kb in 84E and 30 kb in 93E/F. Corresponding breakpoints were also identified in D. sechellia. The BAC clones described here will be an important addition to the Drosophila genomic resources.

The laboratory rat is one of the most extensively studied model organisms. Inbred laboratory rat strains originated from limited Rattus norvegicus founder populations, and the inherited genetic variation provides an excellent resource for the correlation of genotype to phenotype. Here, we report a survey of genetic variation based on almost 3 million newly identified SNPs. We obtained accurate and complete genotypes for a subset of 20,238 SNPs across 167 distinct inbred rat strains, two rat recombinant inbred panels and an F2 intercross. Using 81% of these SNPs, we constructed high-density genetic maps, creating a large dataset of fully characterized SNPs for disease gene mapping. Our data characterize the population structure and illustrate the degree of linkage disequilibrium. We provide a detailed SNP map and demonstrate its utility for mapping of quantitative trait loci. This community resource is openly available and augments the genetic tools for this workhorse of physiological studies. This work was supported by European Union grants LSGH-2004-005235 and LSHG-CT-2005-019015. D.G. is supported by a Wellcome Trust Senior Fellowship in Basic Biomedical Science (057733/Z/99/A). M.-T.B. and D.G. acknowledge support from the Wellcome Cardiovascular Functional Genomics Initiative (066780/Z/01/Z)

Evidence that some types of epilepsies show strong genetic predisposition has been well documented. AP3M2 is considered to be an epileptogenic gene because AP3M2 knockout mice exhibit symptoms of spontaneous epileptic seizures. In order to investigate whether the AP3M2 gene causes susceptibility to epilepsy, we performed mutation screening of the genomic DNA of 190 patients with six epilepsy types; this screening involved all the 9 exons and the relevant exon-intron boundaries of AP3M2. Although neither missense nor nonsense mutations were detected, we identified 21 sequence variations, of which 16 variations were novel. Of the 21 variations, 11 were detected in 5' and 3' UTRs, while the remaining variations were detected in introns. Although the present study failed to identify the possible AP3M2 mutations that may cause epilepsy, our results suggest that some AP3M2 mutations still remain candidates for unmapped disorders including epilepsy, febrile seizure, and other neuronal developmental disorders associated with functional abnormalities of GABAergic transmission.

Opalin is a transmembrane protein detected specifically in mammalian oligodendrocytes. Opalin homologs are found only in mammals and not in the genome sequences of other animal classes. We first determined the nucleotide sequences of Opalin orthologs and their flanking regions derived from four prosimians, a group of primitive primates. A global comparison revealed that an evolutionarily conserved region exists in the first intron of Opalin. When the conserved domain was assayed for its enhancer activity in transgenic mice, oligodendrocyte-directed expression was observed. In an oligodendroglial cell line, Oli-neu, the conserved domain showed oligodendrocyte-directed expression. The conserved domain is composed of eight subdomains, some of which contain binding sites for Myt1 and cAMP-response element binding protein (CREB). Deletion analysis and cotransfection experiments revealed that the subdomains have critical roles in Opalin gene expression. Over-expression of Myt1, treatment of the cell with leukemia inhibitory factor (LIF), and cAMP analog (CREB activator) enhanced the expression of endogenous Opalin in Oli-neu cells and activated the oligodendrocyte enhancer. These results suggest that LIF, cAMP signaling cascades and Myt1 play significant roles in the differentiation of oligodendrocytes through their action on the Opalin oligodendrocyte enhancer.

The mammalian molecular clock is composed of feedback loops to keep circadian 24-h rhythms. Although much focus has been on transcriptional regulation, it is clear that posttranscriptional controls also play important roles in molecular circadian clocks. In this study, we found that mouse LARK (mLARK), an RNA binding protein, activates the posttranscriptional expression of the mouse Period1 (m Per1 ) mRNA. A strong circadian cycling of the mLARK protein is observed in the suprachiasmatic nuclei with a phase similar to that of mPER1, although the level of the Lark transcripts are not rhythmic. We demonstrate that LARK causes increased mPER1 protein levels, most likely through translational regulation and that the LARK1 protein binds directly to a cis element in the 3′ UTR of the m Per1 mRNA. Alterations of m Lark expression in cycling cells caused significant changes in circadian period, with m Lark knockdown by siRNA resulting in a shorter circadian period, and the overexpression of mLARK1 resulting in a lengthened period. These data indicate that mLARKs are novel posttranscriptional regulators of mammalian circadian clocks.

Peroxisomes play an important role in beta-oxidation of fatty acids. All peroxisomal matrix proteins are synthesized in the cytosol and post-translationally sorted to the organelle. Two distinct peroxisomal signal targeting sequences (PTSs), the C-terminal PTS1 and the N-terminal PTS2, have been defined. Import of precursor PTS2 proteins into the peroxisomes is accompanied by a proteolytic removal of the N-terminal targeting sequence. Although the PTS1 signal is preserved upon translocation, many PTS1 proteins undergo a highly selective and limited cleavage. Here, we demonstrate that Tysnd1, a previously uncharacterized protein, is responsible both for the removal of the leader peptide from PTS2 proteins and for the specific processing of PTS1 proteins. All of the identified Tysnd1 substrates catalyze peroxisomal beta-oxidation. Tysnd1 itself undergoes processing through the removal of the presumably inhibitory N-terminal fragment. Tysnd1 expression is induced by the proliferator-activated receptor alpha agonist bezafibrate, along with the increase in its substrates. A model is proposed where the Tysnd1-mediated processing of the peroxisomal enzymes promotes their assembly into a supramolecular complex to enhance the rate of beta-oxidation.

Numerous microbes inhabit the human intestine, many of which are uncharacterized or uncultivable. They form a complex microbial community that deeply affects human physiology. To identify the genomic features common to all human gut microbiomes as well as those variable among them, we performed a large-scale comparative metagenomic analysis of fecal samples from 13 healthy individuals of various ages, including unweaned infants. We found that, while the gut microbiota from unweaned infants were simple and showed a high inter-individual variation in taxonomic and gene composition, those from adults and weaned children were more complex but showed a high functional uniformity regardless of age or sex. In searching for the genes over-represented in gut microbiomes, we identified 237 gene families commonly enriched in adult-type and 136 families in infant-type microbiomes, with a small overlap. An analysis of their predicted functions revealed various strategies employed by each type of microbiota to adapt to its intestinal environment, suggesting that these gene sets encode the core functions of adult and infant-type gut microbiota. By analysing the orphan genes, 647 new gene families were identified to be exclusively present in human intestinal microbiomes. In addition, we discovered a conjugative transposon family explosively amplified in human gut microbiomes, which strongly suggests that the intestine is a ‘hot spot’ for horizontal gene transfer between microbes.

Aggressive periodontitis (AgP) is characterized by the early onset of the rapid and progressive destruction of the alveolar bone. We investigated the correlation of single nucleotide polymorphisms (SNPs) in candidate genes with AgP in the Japanese population in order to determine the genetic risk factors for this complex disease. Among 11 genes related to bone formation and resorption, 43 known SNPs were tested in 98 case and 88 control samples for association with AgP by using SNP genotyping techniques. Among these, three polymorphisms located in the colony stimulating factor 1 (CSF1) gene showed a positive association with AgP. This is the first case of an association between a CSF1 polymorphism and a human disease.

Background: Recent studies have demonstrated a significant association between mutations in genes involved in the GHR/IGF1 signaling pathway and extension of the lifespan of model organisms. Exon 3 insertion or deletion is one common polymorphism in the growth hormone receptor (GHR) of humans. The exon 3 deletion allele is reported to have stronger signaling in the GH/GHR pathway, which may correlate to short lifespan. Methods: We investigated the common polymorphic variation in 119 Japanese semisuper centenarians (SSC; older than 105) compared with 104 healthy younger controls via the polymorphism-based polymerase chain reaction method. Results: The frequency of exon 3 deletion variation of GHR in SSC was found to be higher than controls, although this was not significant statistically. Also, the single nucleotide polymorphism genotype frequency and allele frequency exhibited no differences between SSC and controls. Conclusions: These results show that SSC in Japan do not tend to have the allele of GHR, which has a lower signaling capacity.

Recent advances in DNA microarray technology have enabled the identification of small alterations throughout the genome. We used standard karyotype analysis, followed by DNA microarray analysis and PCR to precisely map the chromosomal 4p deletion and determine the deletion breakpoints in the genome of an epileptic patient. The karyotype of the patient was 46,XY,del(4)(p15.2p15.3) as determined by G-banding analysis. We used a high-density oligonucleotide genotyping array to estimate the size of the deletion (4.5 Mb) and to locate the breakpoints within a 9-kb region on one side of the deletion and a 100-kb region on the other side. We amplified by PCR and sequenced the genomic region encompassing the breakpoints, and mapped the deletion to regions extending from 21648457 to 26164287 and from 26164505 to 26167493, respectively (chromosome 4 of NCBI Homo sapiens Genome Build 35.1). The deletion involves 18 genes, one of which (CCKAR) is partially deleted.

Three mammalian Period ( Per ) genes, termed Per1 , Per2 , and Per3 , have been identified as structural homologues of the Drosophila circadian clock gene, period ( per ). The three Per genes are rhythmically expressed in the suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals. The phases of peak mRNA levels for the three Per genes in the SCN are slightly different. Light sequentially induces the transcripts of Per1 and Per2 but not of Per3 in mice. These data and others suggest that each Per gene has a different but partially redundant function in mammals. To elucidate the function of Per1 in the circadian system in vivo , we generated two transgenic rat lines in which the mouse Per1 ( mPer1 ) transcript was constitutively expressed under the control of either the human elongation factor-1α ( EF-1 α) or the rat neuron-specific enolase ( NSE ) promoter. The transgenic rats exhibited an ≈0.6–1.0-h longer circadian period than their wild-type siblings in both activity and body temperature rhythms. Entrainment in response to light cycles was dramatically impaired in the transgenic rats. Molecular analysis revealed that the amplitudes of oscillation in the rat Per1 ( rPer1 ) and rat Per2 ( rPer2 ) mRNAs were significantly attenuated in the SCN and eyes of the transgenic rats. These results indicate that either the level of Per1 , which is raised by overexpression, or its rhythmic expression, which is damped or abolished in over expressing animals, is critical for normal entrainment of behavior and molecular oscillation of other clock genes.

Recent progress in the human genome project allows us to study the genetic basis of complex traits such as atherosclerosis and diabetes. Among various approaches, a genetic approach such as affected sib-paired analysis and case-control study has successfully identified genes for susceptibility to Alzheimer's disease and diabetes. Several points to be considered are discussed for their successful application to athroscelorosis. In addition, functional genomic approaches based on powerful technology such as cDNA microarray and knockout mice allow us to identify genes and proteins involved in the complex biological (disease) process. As a successful example, a transgenic mice study of circadian rythm in transgenic mice is described.

The Impact is an evolutionarily conserved gene subjected to genomic imprinting in mouse but not in human. A characteristic tandem repeat similar to those found in many other imprinted genes and an elevated expression level, both observed only for the mouse gene, are implicated in the evolution of imprinting, to which the repeat might have contributed via enhancement of the expression. To pursue the possibility further, we examined the correlation among the repeat, expression level, and imprinting of Impact in various mammals ranging from rodents, lagomorphs, carnivores, artiodactyls to primates. Intriguingly, rabbit Impact is abundantly expressed and imprinted like those of rodents, but is missing the repeat from its first intron like those of other mammals that express both alleles weakly. It thus seems that lineage-specific enhancement of gene expression rather than the tandem repeat per se played a critical role in the evolution of imprinting of Impact.

Chromosome 18 appears to have the lowest gene density of any human chromosome and is one of only three chromosomes for which trisomic individuals survive to term. There are also a number of genetic disorders stemming from chromosome 18 trisomy and aneuploidy. Here we report the finished sequence and gene annotation of human chromosome 18, which will allow a better understanding of the normal and disease biology of this chromosome. Despite the low density of protein-coding genes on chromosome 18, we find that the proportion of non-protein-coding sequences evolutionarily conserved among mammals is close to the genome-wide average. Extending this analysis to the entire human genome, we find that the density of conserved non-protein-coding sequences is largely uncorrelated with gene density. This has important implications for the nature and roles of non-protein-coding sequence elements.

Previous studies revealed thatIgf2andMpr/Igf2rare imprinted in eutherian mammals and marsupials but not in monotremes or birds.Igf2lies in a large imprinted cluster in eutherians, and its imprinting is regulated by long-range mechanisms. As a step to understand how the imprinted cluster evolved, we have determined a 490-kb chicken sequence containing the orthologs of mammalianAscl2/Mash2, Ins2andIgf2. We found that most of the genes in this region are conserved between chickens and mammals, maintaining the same transcriptional polarities and exon–intron structures. However,H19, an imprinted noncoding transcript, was absent from the chicken sequence. ChickenASCL2/CASH4andINS, the orthologs of the imprinted mammalian genes, showed biallelic expression, further supporting the notion that imprinting evolved after the divergence of mammals and birds. TheH19imprinting center and many of the local regulatory elements identified in mammals were not found in chickens. Also, a large segment of tandem repeats and retroelements identified between the two imprinted subdomains in mice was not found in chickens. Our findings show that the imprinted genes were clustered before the emergence of imprinting and that the elements associated with imprinting probably evolved after the divergence of mammals and birds.

MSM/Ms is an inbred strain derived from the Japanese wild mouse, Mus musculus molossinus. It is believed that subspecies molossinus has contributed substantially to the genome constitution of common laboratory strains of mice, although the majority of their genome is derived from the west European M. m. domesticus. Information on the molossinus genome is thus essential not only for genetic studies involving molossinus but also for characterization of common laboratory strains. Here, we report the construction of an arrayed bacterial artificial chromosome (BAC) library from male MSM/Ms genomic DNA, covering ∼1× genome equivalent. Both ends of 176,256 BAC clone inserts were sequenced, and 62,988 BAC-end sequence (BES) pairs were mapped onto the C57BL/6J genome (NCBI mouse Build 30), covering 2,228,164 kbp or 89% of the total genome. Taking advantage of the BES map data, we established a computer-based clone screening system. Comparison of the MSM/Ms and C57BL/6J sequences revealed 489,200 candidate single nucleotide polymorphisms (SNPs) in 51,137,941 bp sequenced. The overall nucleotide substitution rate was as high as 0.0096. The distribution of SNPs along the C57BL/6J genome was not uniform: The majority of the genome showed a high SNP rate, and only 5.2% of the genome showed an extremely low SNP rate (percentage identity = 0.9997); these sequences are likely derived from the molossinus genome.

The SAMP8 mouse is a suitable animal model to examine the decline in learning ability with senescence. We tried to identify genes related to brain senescence by using fluorescent differential display analysis. Total RNAs were extracted from hippocampus and cerebrum of groups of five male SAMP8 and SAMR1 (control) mice, 8 months of age. A cDNA fragment of 165 bp encoding a part of EST was cloned. An mRNA sequence of 2487 bp that consists of five exons and contains ORF (684 bp for 228 amino acids) could be linked to chromosome 15 by using EST clusters in the National Center for Biotechnology Information (NCBI) database. Expression of 2.5-kb cDNA was confirmed in hippocampus and cerebrum by RT-PCR, and sequenced. This was a novel gene, named as ankyrin small ( ank-s ), the protein of which contained 4 ankyrin-repeats in N-terminal side and membrane-binding site in C-terminus side. Northern blot showed that expression of ank-s was the highest in brain, especially in hippocampus where its expression doubled after birth and in 8-month-old SAMP8 was twice that in SAMR1 mice. These results suggest that ank-s is related to neuronal degeneration in SAMP8 hippocampus.

The mammalian SCN contains a biological clock that drives remarkably precise circadian rhythms in vivo and in vitro. This study asks whether the cycle-to-cycle variability of behavioral rhythms in mice can be attributed to precision of individual circadian pacemakers within the SCN or their interactions. The authors measured the standard deviation of the cycle-to-cycle period from 7-day recordings of running wheel activity, Period1 gene expression in cultured SCN explants, and firing rate patterns of dispersed SCN neurons. Period variability of the intact tissue and animal was lower than single neurons. The median variability of running wheel and Period1 rhythms was less than 40 min per cycle compared to 2.1 h in firing rate rhythms of dispersed SCN neurons. The most precise SCN neuron, with a period deviation of 1.1 h, was 10 times noisier than the most accurate SCN explant (0.1 h) or mouse (0.1 h) but comparable to the least stable explant (2.1 h) and mouse (1.1 h). This variability correlated with intrinsic period in mice and SCN explants but not with single cells. Precision was unrelated to the amplitude of rhythms and did not change significantly with age up to 1 year after birth. Analysis of the serial correlation of cycle-to-cycle period revealed that approximately half of this variability is attributable to noise outside the pacemaker. These results indicate that cell-cell interactions within the SCN reduce pacemaker noise to determine the precision of circadian rhythms in the tissue and in behavior.

Mammalian genes subject to genomic imprinting often form clusters and are regulated by long-range mechanisms. The distal imprinted domain of mouse chromosome 7 is orthologous to the Beckwith-Wiedemann syndrome domain in human chromosome 11p15.5 and contains at least 13 imprinted genes. This domain consists of two subdomains, which are respectively regulated by an imprinting center. We here report the finished-quality sequence of a 0.6-Mb region encompassing the more centromeric subdomain. The sequence contains four imprinted genes (Ascl2/Mash2, Ins2, Igf2 and H19) and reveals previously unidentified CpG islands and tandem repeats, which may be features of imprinted genes. Most interestingly, a unique 210-kb segment consisting almost exclusively of tandem repeats and retroelements is identified. This segment, located between Th and Ins2, has features of heterochromatin-forming DNA and is highly methylated at CpG sites. The segment exhibits asynchronous replication on the parental chromosomes, a feature of the imprinted domains. We propose that this repeat segment could serve either as a boundary between the two subdomains or as a target for epigenetic chromatin modifications that regulate imprinting.

Approximately half of all human genes have CpG islands (CGIs)around their promoter regions. Although CGIs usually escape methylation, those on Chromosome X in females and those in the vicinity of imprinted genes are exceptions: They have both methylated and unmethylated alleles to display a “composite” pattern in methylation analysis. In addition, aberrant methylation of CGIs is known to often occur in cancer cells. Here we developed a simple HpaII-McrBC PCR method for discrimination of full, null, incomplete, and composite methylation patterns, and applied it to all computationally identified CGIs on human Chromosome 21q. This comprehensive analysis revealed that, although most CGIs (103 out of 149)escape methylation, a sizable fraction (31 out of 149)are fully methylated even in normal peripheral blood cells. Furthermore, we identified seven CGIs showing the composite methylation, and demonstrated that three of them are indeed methylated monoallelically. Further analyses using informative pedigrees revealed that two of the three are subject to maternal allele-specific methylation. Intriguingly, the other CGI is methylated in an allele-specific but parental-origin-independent manner. Thus, the cell seems to have a broader repertoire of methylating CGIs than previously thought, and our approach may contribute to uncover novel modes of allelic methylation.

The recurrent translocation t(1;3)(p36;q21) is associated with myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML) characterized by trilineage dysplasia, especially dysmegakaryopoiesis and a poor prognosis. Recently, the two genes involved in this translocation have been identified: the MEL1 gene at 1p36.3, and the RPN1 gene at 3q21. The breakpoint in RPN1 is centromeric to the breakpoint cluster region of the inv(3) abnormality. Because the MEL1 transcript is detected only in leukemic cells with t(1;3)(p36;q21), ectopic expression of MEL1 driven by RPN1 at 3q21 is thought to contribute to the pathogenesis of t(1;3)(p36;q21) leukemia. However, the precise breakpoint in the patients has not yet been identified. With fluorescence in situ hybridization analysis by use of BAC/PAC probes, we identified the breakpoint at 1p36.3 in three MDS/AML patients with t(1;3)(p36;q21): within the first intron of the MEL1 gene (one patient) or within a 29-kb region located in the 5′ region of MEL1 (two other patients). We detected several sizes of MEL1 transcript in two patients including the first patient, although we have not yet clarified whether MEL1 transcripts were different among the patients and whether a truncated MEL1 transcript was expressed in the first patient. This patient showed an unusual clinical profile, repeating progression to overt leukemia and conversion to MDS three times during the 29-month survival period, which might be related to a different molecular mechanism in this patient. © 2003 Wiley-Liss, Inc.

Gene regulatory networks elucidated from strategic, genome-wide experimental data can aid in the discovery of novel gene function information and expression regulation events from observation of transcriptional regulation among genes of known and unknown biological function. To create a reliable and comprehensive data set for the elucidation of transcription regulation networks, we conducted systematic genome-wide disruption expression experiments of yeast on 118 genes with known involvement in transcription regulation. We report several novel regulatory relationships between known transcription factors and other genes with previously unknown biological function discovered with this expression library. Here we report the downstream regulatory subnetworks for UME6 and MET28. The elucidated network topology among these genes demonstrates MET28's role as a nodal point between genes involved in cell division and those involved in DNA repair mechanisms.

The clinical significance of osteonectin in human stomach cancer was examined immunohistochemically and molecular biologically in 31 differentiated and eight undifferentiated stomach adenocarcinomas and 19 non-cancer stomach tissues. Osteonectin-mAb-stained cells were observed in stroma of 90% differentiated and 63% undifferentiated adenocarcinomas, and of 26% non-cancer stomach tissues. Competitive reverse transcriptase polymerase chain reaction results generally coincided with immunohistochemical data. The present results suggest that osteonectin is highly expressed in reactive stroma associated with invasive differentiated adenocarcinomas and that it may serve as a useful clinical diagnostic marker for stomach cancer.

We report a familial case with a proximal interstitial deletion of chromosome 21q [del(21q)]. Although the mother in the family was phenotypically normal, her first child was affected with both sensorineural hearing loss and moderate mental retardation, and the second affected child had mild mental retardation but not sensorineural hearing loss. We determined breakpoints of the del(21q) in the mother and her two affected children by fluorescence in situ hybridization analysis using 45 DNA clones and the molecular analysis using 21 DNA markers. The proximal breakpoint of the del(21q) was located at a region between 0.33 Mb and 0.46 Mb distal to the centromere, and the distal breakpoint was at a region between 14.6 Mb and 14.9 Mb. The finding indicates that the three individuals had an approximate 14-Mb deletion within 21q11.2-q21.3. Molecular analysis showed that both affected children shared the same maternal haplotype of their del(21q), but a crossover was detected in the paternally inherited normal chromosome 21. These data suggest that unmasking of deleterious genes on the paternally derived chromosome 21 of the two children as a result of the deletion may affect the extent of their mental retardation and/or sensorineural hearing loss. Usher syndrome 1E may be a candidate disease locus related to the sensorineural hearing loss of the first child.

The present study was designed to define molecular alterations in the initiation stage of rat stomach carcinogenesis. Groups of male Lewis rats, 6 weeks old, were given drinking water with or without N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; 100 mg/liter). Total RNA was isolated from the stomach pyloric mucosa, and fluorescent differential display analysis was performed. A cDNA fragment of 125 bp encoding an extracellular matrix-associated matricellular glycoprotein, osteonectin, was identified after 14 days of MNNG exposure. A severalfold increase in expression was observed after 14 and 27 days of MNNG exposure, as determined by northern blot and RT-PCR. Immunohistochemistry revealed that osteonectin-mAb-stained fibroblastic cells appeared in interstitial tissue of pyloric mucosa. Additionally the gene expression of other extracellular matrix proteins, viz., collagen type III, fibronectin, osteopontin, proteoglycan NG2, laminin gamma1 and S-laminin, was also markedly increased, as determined by competitive RT-PCR after 14 days of MNNG exposure. The gene expression of osteonectin and the six other extracellular matrix proteins was elevated in twelve stomach adenocarcinomas and adenomas induced by MNNG in Lewis and WKY rats. Osteonectin-mAb-stained fibroblastic cells were evident in interstitial tissue of stomach tumor. These results suggest that osteonectin-expressing fibroblastic cells appear in the interstitial tissue of pyloric mucosa from the early initiation stage of rat stomach chemical carcinogenesis, and that this phenomenon probably plays a role in cancer development.

Dendritic cell appearance and differentiation during early and late stages of rat stomach carcinogenesis were studied in the pyloric mucosa. Young male rats were given drinking water with or without N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; 100 mg/liter) for 14 days. Use of competitive RT-PCR and northern blotting showed that MNNG exposure induced 3- to 4-fold greater expression of the genes for integrin beta7 and integrin alphaE2 (identical with antigen OX-62, a dendritic cell marker), as well as three cytokines, IL-4, GM-CSF and TNFalpha, in the stomach pyloric mucosa of resistant Buffalo rats compared to sensitive ACI rats. These genes were minimally expressed in control animals. The results confirm the appearance of dendritic cells in the target pyloric mucosa and suggest the possibility that dendritic cell differentiation and maturation are induced by various cytokines, at least in Buffalo rats. Competitive RT-PCR showed expression of integrin alphaE2 and beta7, MHC class II-associated invariant chain (Ii), MHC class II, B7-1, CD28, GM-CSF and TNFalpha genes in all 12 examined stomach adenocarcinomas and adenomas induced in male Lewis and WKY rats with 30 weeks' MNNG exposure, suggesting the presence of dendritic cells in tumors. OX-62 staining and western blotting for OX-62 also confirmed the presence of dendritic cells in tumors. However, the population of dendritic cells in tumors was less than that in the pyloric mucosa after 14 days' MNNG exposure. The present results suggest that immune defense involving dendritic cells is marshaled from the very early initiation stage during rat stomach cancer development, but is downgraded in developed tumors.