Toshiyuki Shiraki

Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage

We introduce cap analysis gene expression (CAGE), which is based on preparation and sequencing of concatamers of DNA tags deriving from the initial 20 nucleotides from 5′ end mRNAs. CAGE allows high-throughout gene expression analysis and the profiling of transcriptional start points (TSP), including promoter usage analysis. By analyzing four libraries (brain, cortex, hippocampus, and cerebellum), we redefined more accurately the TSPs of 11-27% of the analyzed transcriptional units that were hit. The frequency of CAGE tags correlates well with results from other analyses, such as serial analysis of gene expression, and furthermore maps the TSPs more accurately, including in tissue-specific cases. The high-throughput nature of this technology paves the way for understanding gene networks via correlation of promoter usage and gene transcriptional factor expression.

A molecular neuroethological approach for identifying and characterizing a cascade of behaviorally regulated genes

Songbirds have one of the most accessible neural systems for the study of brain mechanisms of behavior. However, neuroethological studies in songbirds have been limited by the lack of high-throughput molecular resources and gene-manipulation tools. To overcome these limitations, we constructed 21 regular, normalized, and subtracted full-length cDNA libraries from brains of zebra finches in 57 developmental and behavioral conditions in an attempt to clone as much of the brain transcriptome as possible. From these libraries, ≈14,000 transcripts were isolated, representing an estimated 4,738 genes. With the cDNAs, we created a hierarchically organized transcriptome database and a large-scale songbird brain cDNA microarray. We used the arrays to reveal a set of 33 genes that are regulated in forebrain vocal nuclei by singing behavior. These genes clustered into four anatomical and six temporal expression patterns. Their functions spanned a large range of cellular and molecular categories, from signal transduction, trafficking, and structural, to synaptically released molecules. With the full-length cDNAs and a lentiviral vector system, we were able to overexpress, in vocal nuclei, proteins of representative singing-regulated genes in the absence of singing. This publicly accessible resource http://songbirdtranscriptome.net can now be used to study molecular neuroethological mechanisms of behavior.

Libraries enriched for alternatively spliced exons reveal splicing patterns in melanocytes and melanomas

It is becoming increasingly clear that alternative splicing enables the complex development and homeostasis of higher organisms. To gain a better understanding of how splicing contributes to regulatory pathways, we have developed an alternative splicing library approach for the identification of alternatively spliced exons and their flanking regions by alternative splicing sequence enriched tags sequencing. Here, we have applied our approach to mouse melan-c melanocyte and B16-F10Y melanoma cell lines, in which 5,401 genes were found to be alternatively spliced. These genes include those encoding important regulatory factors such as cyclin D2, Ilk, MAPK12, MAPK14, RAB4, melastatin 1 and previously unidentified splicing events for 436 genes. Real-time PCR further identified cell line–specific exons for Tmc6, Abi1, Sorbs1, Ndel1 and Snx16. Thus, the ASL approach proved effective in identifying splicing events, which suggest that alternative splicing is important in melanoma development.

Genome sequence and analysis of the Japanese morning glory Ipomoea nil.

Ipomoea is the largest genus in the family Convolvulaceae. Ipomoea nil (Japanese morning glory) has been utilized as a model plant to study the genetic basis of floricultural traits, with over 1,500 mutant lines. In the present study, we have utilized second- and third-generation-sequencing platforms, and have reported a draft genome of I. nil with a scaffold N50 of 2.88 Mb (contig N50 of 1.87 Mb), covering 98% of the 750 Mb genome. Scaffolds covering 91.42% of the assembly are anchored to 15 pseudo-chromosomes. The draft genome has enabled the identification and cataloguing of the Tpn1 family transposons, known as the major mutagen of I. nil, and analysing the dwarf gene, CONTRACTED, located on the genetic map published in 1956. Comparative genomics has suggested that a whole genome duplication in Convolvulaceae, distinct from the recent Solanaceae event, has occurred after the divergence of the two sister families.

Absolute expression values for mouse transcripts: re-annotation of the READ expression database by the use of CAGE and EST sequence tags

The RIKEN expression array database (READ) provides comprehensive gene expression data for the mouse, which were obtained as relative values from microarray double‐staining experiments with E17.5 mRNA as common reference. To assign absolute expression values for mouse transcripts within READ, we applied the E17.5 reference sample to CAGE (cap analysis of gene expression) and expressed sequence tag (EST) high‐throughput tag sequencing. Newly assigned values within the READ database were validated by comparison to expression data from serial analysis of gene expression, CAGE and EST experiments. These experiments confirmed the great significance of the absolute expression values within the improved READ database. The new Absolute READ database on absolute expression data is available under http://genome.gsc.riken.jp/absolute.