Teleosts comprise more than half of all vertebrate species and have adapted to a variety of marine and freshwater habitats 1 . Their genome evolution and diversification are important subjects for the understanding of vertebrate evolution. Although draft genome sequences of two pufferfishes have been published 2,3 , analysis of more fish genomes is desirable. Here we report a high-quality draft genome sequence of a small egg-laying freshwater teleost, medaka (Oryzias latipes). Medaka is native to East Asia and an excellent model system for a wide range of biology, including ecotoxicology, carcinogenesis, sex determination 4-6 and developmental genetics 7 . In the assembled medaka genome (700 megabases), which is less than half of the zebrafish genome, we predicted 20,141 genes, including 2,900 new genes, using 59-end serial analysis of gene expression tag information. We found single nucleotide polymorphisms (SNPs) at an average rate of 3.42% between the two inbred strains derived from two regional populations; this is the highest SNP rate seen in any vertebrate species. Analyses based on the dense SNP information show a strict genetic separation of 4 million years (Myr) between the two populations, and suggest that differential selective pressures acted on specific gene categories. Four-way comparisons with the human, pufferfish (Tetraodon), zebrafish and medaka genomes revealed that eight major interchromosomal rearrangements took place in a remarkably short period of 50 Myr after the whole-genome duplication event in the teleost ancestor and afterwards, intriguingly, the medaka genome preserved its ancestral karyotype for more than 300 Myr.We applied the whole-genome shotgun approach to an inbred strain, , derived from the southern Japanese population, as the main target. A total of 13.8 million reads amounting to approximately 10.6-fold genome coverage were obtained from the shotgun plasmid, fosmid and bacterial artificial chromosome (BAC) libraries. A newly developed RAMEN assembler was used to process the shotgun reads to generate contigs and scaffolds. The N50 values (50% of nucleotides in an assembly are in scaffolds-or contigs-longer than or equal to the N50 value) are ,1.41 megabases (Mb) for scaffolds and ,9.8 kilobases (Kb) for contigs. The total length of the contigs reached 700.4 Mb, which, from now on, we refer to as the medaka genome size.To construct ultracontigs, the scaffolds were integrated with the medaka genetic map by using SNP markers. For this purpose, we further obtained about 2.8-fold coverage of shotgun reads from another inbred strain HNI (refs 9, 10), which is derived from the northern Japanese population. The reads were assembled by RAMEN to scaffolds covering 648 Mb. Aligning the HNI contigs with the HdrR genome using BLASTZ 11 , we identified 16.4 million SNPs as well as 1.40 million insertions and 1.45 million deletions in non-repetitive regions (Supplementary Table 2). We selected 2,401 SNPs and genetically mapped them onto medaka chromosomes using a backcross panel between the...

We performed threefold shotgun sequencing of the silkworm (Bombyx mori) genome to obtain a draft sequence and establish a basic resource for comprehensive genome analysis. By using the newly developed RAMEN assembler, the sequence data derived from whole-genome shotgun (WGS) sequencing were assembled into 49,345 scaffolds that span a total length of 514 Mb including gaps and 387 Mb without gaps. Because the genome size of the silkworm is estimated to be 530 Mb, almost 97% of the genome has been organized in scaffolds, of which 75% has been sequenced. By carrying out a BLAST search for 50 characteristic Bombyx genes and 11,202 non-redundant expressed sequence tags (ESTs) in a Bombyx EST database against the WGS sequence data, we evaluated the validity of the sequence for elucidating the majority of silkworm genes. Analysis of the WGS data revealed that the silkworm genome contains many repetitive sequences with an average length of <500 bp. These repetitive sequences appear to have been derived from truncated transposons, which are interspersed at 2.5- to 3-kb intervals throughout the genome. This pattern suggests that silkworm may have an active mechanism that promotes removal of transposons from the genome. We also found evidence for insertions of mitochondrial DNA fragments at 9 sites. A search for Bombyx orthologs to Drosophila genes controlling sex determination in the WGS data revealed 11 Bombyx genes and suggested that the sex-determining systems differ profoundly between the two species.

Circulating tumor cells (CTC) in blood have attracted attention both as potential seeds for metastasis and as biomarkers. However, most CTC detection systems might miss epithelial-mesenchymal transition (EMT)-induced metastatic cells because detection is based on epithelial markers. First, to discover novel markers capable of detecting CTCs in which EMT has not been repressed, microarray analysis of 132 colorectal cancers (CRC) from Japanese patients was conducted, and 2,969 genes were detected that were overexpressed relative to normal colon mucosa. From the detected genes, we selected those that were overexpressed CRC with distant metastasis. Then, we analyzed the CRC metastasis-specific genes (n ¼ 22) to determine whether they were expressed in normal circulation. As a result, PLS3 was discovered as a CTC marker that was expressed in metastatic CRC cells but not in normal circulation. Using fluorescent immunocytochemistry, we validated that PLS3 was expressed in EMTinduced CTC in peripheral blood from patients with CRC with distant metastasis. PLS3-expressing cells were detected in the peripheral blood of approximately one-third of an independent set of 711 Japanese patients with CRC. Multivariate analysis showed that PLS3-positive CTC was independently associated with prognosis in the training set (n ¼ 381) and the validation set [n ¼ 330; HR ¼ 2.17; 95% confidence interval (CI) ¼ 1.38-3.40 and HR ¼ 3.92; 95% CI ¼ 2.27-6.85]. The association between PLS3-positive CTC and prognosis was particularly strong in patients with Dukes B (HR ¼ 4.07; 95% CI ¼ 1.50-11.57) and Dukes C (HR ¼ 2.57; 95% CI ¼ 1.42-4.63). PLS3 is a novel marker for metastatic CRC cells, and it possesses significant prognostic value. Cancer Res; 73(7); 2059-69. Ó2012 AACR.

Understanding intratumor heterogeneity is clinically important because it could cause therapeutic failure by fostering evolutionary adaptation. To this end, we profiled the genome and epigenome in multiple regions within each of nine colorectal tumors. Extensive intertumor heterogeneity is observed, from which we inferred the evolutionary history of the tumors. First, clonally shared alterations appeared, in which C>T transitions at CpG site and CpG island hypermethylation were relatively enriched. Correlation between mutation counts and patients’ ages suggests that the early-acquired alterations resulted from aging. In the late phase, a parental clone was branched into numerous subclones. Known driver alterations were observed frequently in the early-acquired alterations, but rarely in the late-acquired alterations. Consistently, our computational simulation of the branching evolution suggests that extensive intratumor heterogeneity could be generated by neutral evolution. Collectively, we propose a new model of colorectal cancer evolution, which is useful for understanding and confronting this heterogeneous disease.

The functions of many long non-coding RNAs (ncRNAs) in human cancers have not yet been elucidated. The long ncRNA HOTAIR is expressed from the developmental HOXC locus located on chromosome 12q13.13. Previous reports have demonstrated that HOTAIR associates with chromatin modifications in cooperation with the Polycomb complex PRC2, and promotes breast and colorectal cancer metastasis. In this study, we examined the clinical significance of HOTAIR expression in patients with hepatocellular carcinoma (HCC). HOTAIR expression was detected in primary HCCs in 13 out of 64 patients. Patients with HOTAIR expression had significantly poorer prognoses and a larger primary tumor size than those without HOTAIR expression, similar to studies in breast and colorectal cancers. Moreover, introduction of human HOTAIR into liver cancer cells revealed that HOTAIR promoted more rapid proliferation compared to control cells. Thus, although the clinical significance of HOTAIR expression in HCC may not be as pronounced as that in breast and colorectal cancers, the current study demonstrates that HOTAIR expression is associated with HCC progression, warranting further studies.

Lysophosphatidic acid (LPA) is a simple bioactive phospholipid with diverse effects on various cells, that interacts with three G protein-coupled transmembrane receptors, LPA1, LPA2, and LPA3. The expression pattern and functions of these LPA receptors in various tumors have not been fully examined, except in ovarian cancer. To evaluate the LPA receptor expression profile in human colorectal cancer and in normal mucosa, we used real-time reverse transcription-polymerase chain reaction (RT-PCR) and measured the expression levels of LPA1, LPA2, and LPA3 messenger RNA (mRNA) in 26 colorectal cancers and 16 corresponding normal tissue samples. Normal epithelium expressed both LPA1 and LPA2 mRNA at similar levels. In comparison, colorectal cancers expressed LPA1 mRNA at a significantly lower level (0.3-fold; Po0.05), and LPA2 mRNA at a significantly higher level (three-fold; Po0.05), as compared with normal tissues. Thus, the ratio of LPA2/LPA1 increased markedly during malignant transformation (18-fold increase). LPA3 mRNA was expressed at only a low level in both normal and cancer tissues. We also assessed LPA2 expression immunohistochemically using a rat anti-LPA2 monoclonal antibody, and confirmed high expression of LPA2 in colorectal cancer at the protein level. As for LPA1, we examined Western blot analysis for 16 matched normal and cancer tissues. It revealed a significant decrease in the expression of LPA1 protein in cancer tissues compared to normal mucosa in nine of 16 cases, and in the remaining seven cases the expression levels was much the same. These results suggested that alteration of LPA receptor expression might be an important event in the development of colorectal cancer, and therefore, LPA and its receptors could be a chemopreventive target against colorectal cancer. Keywords: aberrant expression of LPA2; carcinogenesis; colorectal cancer; LPA receptor; lysophosphatidic acid; phospholipid Lysophosphatidic acid (LPA, 1-or 2-acyl-sn-glycero-3-phosphate), the simplest glycerophospholipid, mediates a broad range of cellular responses, including smooth muscle cell contraction, platelet aggregation, neurite retraction/cell rounding, regulation of cell proliferation, protection from apoptosis, modulation of chemotaxis, and transcellular migration.

To clarify the molecular basis of human TLR9 (hTLR9) gene expression, the activity of the hTLR9 gene promoter was characterized using the human myeloma cell line RPMI 8226. Reporter gene analysis and EMSA demonstrated that hTLR9 gene transcription was regulated via four cis-acting elements, cAMP response element, 5′-PU box, 3′-PU box, and a C/EBP site, that interacted with the CREB1, Ets2, Elf1, Elk1, and C/EBPα transcription factors. Other members of the C/EBP family, such as C/EBPβ, C/EBPδ, and C/EBPε, were also important for TLR9 gene transcription. CpG DNA-mediated suppression of TLR9 gene transcription led to decreased binding of the trans-acting factors to their corresponding cis-acting elements. It appeared that suppression was mediated via c-Jun and NF-κB p65 and that cooperation among CREB1, Ets2, Elf1, Elk1, and C/EBPα culminated in maximal transcription of the TLR9 gene. These findings will help to elucidate the mechanism of TLR9 gene regulation and to provide insight into the process by which TLR9 evolved in the mammalian immune system.

The rostral and caudal parts of the ventrolateral medulla play a major role in the control of blood pressure. Both regions contain a high density of receptor binding sites for angiotensin II, and it has been shown previously that microinjection of angiotensin II into the rostral ventrolateral medulla causes a rise in blood pressure. The aims of this study were to determine the cardiovascular effects of microinjection of angiotensin II and its specific antagonist T here is considerable evidence that angiotensin II (Ang II) in the brain may play a role in hypertension. For example, it is well established that Ang II can influence fluid and electrolyte balance and arterial blood pressure via an action on circumventricular organs and the nucleus tractus solitarius (for review, see Reference 1). Recently, it has also been demonstrated that microinjection of Ang II into sites within the rostral part of the ventrolateral medulla (VLM) or application of the peptide to the nearby ventral surface results in an increase of blood pressure. -3 Furthermore, the sites at which Ang II microinjection produces the largest pressor responses correspond very closely to the location of Ang II receptor binding sites.2 These sites are confined to a highly localized region referred to as the subretrofacial nucleus, which contains sym-