Yasuhiro Kasai

The medaka draft genome and insights into vertebrate genome evolution

Teleosts comprise more than half of all vertebrate species and have adapted to a variety of marine and freshwater habitats. Their genome evolution and diversification are important subjects for the understanding of vertebrate evolution. Although draft genome sequences of two pufferfishes have been published, 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 and developmental genetics. 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 5′-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.

5′-end SAGE for the analysis of transcriptional start sites

Identification of the mRNA start site is essential in establishing the full-length cDNA sequence of a gene and analyzing its promoter region, which regulates gene expression. Here we describe the development of a 5′-end serial analysis of gene expression (5′ SAGE) that can be used to globally identify transcriptional start sites and the frequency of individual mRNAs. Of the 25,684 5′ SAGE tags in the HEK293 human cell library, 19,893 matched to the human genome. Among 15,448 tags in one locus of the genome, 85.8%–96.1% of the 5′ SAGE tags were assigned within −500 to +200 nt of mRNA start sites using the RefSeq, UniGene and DBTSS databases. This technique should facilitate 5′-end transcriptome analysis in a variety of cells and tissues.

5'SAGE: 5'-end Serial Analysis of Gene Expression database

To comprehensively identify transcription start sites and the frequencies of individual mRNAs in human cell libraries, a method of 5′ end Serial Analysis of Gene Expression (SAGE) was developed recently, which makes it possible to collect a large amount of start site information, and subsequently, we have established a related database server called 5′SAGE. This database displays the observed frequencies of individual 5′ end SAGE tags and previously unknown transcription start sites in the promoter regions, introns and intergenic regions of known genes. 5′SAGE will be useful for analyzing promoter regions and start site variation in different tissues, and is freely available at http://5sage.gi.k.u-tokyo.ac.jp/.

Divalent cation binding to the well-conserved tandem G-A pairs and flanking C-G pair in hammerhead ribozyme as revealed by heteronuclear NMR spectroscopy

Hammerhead ribozymes have catalytically important tandem G-A pairs (G12-A9 and G8-A13 pairs) and flanking C11.1-G10.1 pair [so called A9-G10.1 motif ] in the core region, and the A9-G10.1 motif captures the divalent cation.1−3 In this study, we measured 31P-, 1H-, and 13C-NMR spectroscopy of the RNA oligomer, GA10: r(GGACGAGUCC)2, to examine whether this motif by itself (in the absence of other catalytic loops) might be sufficient to capture structurally and catalytically important metal ions in solution. GA10 forms a self-associated duplex, and contains tandem GS-A6∗ pairs and flanking C4-G7∗ pairs which mimics an A9-G10.1 motif of hammerhead ribozymes. (The residues with “*” belongs to the opposite strand of the duplex.)4 Titrations were performed using MgCl2, CdCl2, NaClO4, and Co(NH3)6Cl3. Typical acquisition parameters for 1D 31P-NMR spectra were 313 K, a spectral width of 10000 Hz digitized into 16384 points (0.61 Hz/point and 0.0020 ppm/point), and 512 scans were averaged. For the accurate assignment of 31P-resonances, 1H-31P HMQC NOESY spectra4 were measured at several points during the titration. Other spectra were recorded as described before.4 We deduced that the A9-G10.1 motif was able to capture a Mg(II) and Cd(II) ions in solution in the absence of any other part of a hammerhead ribozyme since the chemical shift values of the phosphorus atom of A6