Harukazu Suzuki

Functional annotation of a full-length mouse cDNA collection

The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.

Update of the FANTOM web resource: From mammalian transcriptional landscape to its dynamic regulation

The international Functional Annotation Of the Mammalian Genomes 4 (FANTOM4) research collaboration set out to better understand the transcriptional network that regulates macrophage differentiation and to uncover novel components of the transcriptome employing a series of high-throughput experiments. The primary and unique technique is cap analysis of gene expression (CAGE), sequencing mRNA 5′-ends with a second-generation sequencer to quantify promoter activities even in the absence of gene annotation. Additional genome-wide experiments complement the setup including short RNA sequencing, microarray gene expression profiling on large-scale perturbation experiments and ChIP–chip for epigenetic marks and transcription factors. All the experiments are performed in a differentiation time course of the THP-1 human leukemic cell line. Furthermore, we performed a large-scale mammalian two-hybrid (M2H) assay between transcription factors and monitored their expression profile across human and mouse tissues with qRT-PCR to address combinatorial effects of regulation by transcription factors. These interdependent data have been analyzed individually and in combination with each other and are published in related but distinct papers. We provide all data together with systematic annotation in an integrated view as resource for the scientific community (http://fantom.gsc.riken.jp/4/). Additionally, we assembled a rich set of derived analysis results including published predicted and validated regulatory interactions. Here we introduce the resource and its update after the initial release.

RLCS, Restriction Landmark cDNA Scanning

It is important to identify and isolate differentially expressed genes whose expression underlies many biological processes such as development, differentiation, and cellular response to various stimuli, and also pathological changes that arise in diseases. Classically, two hybridization methods, differential and subtractive hybridization, have been used to analyze and isolate such genes [1–3]. However, differential hybridization is effective only for mRNAs expressed abundantly in one of the two types of cells. Subtractive hybridization is rather empirical and poor in reproducibility. Furthermore, both methods are time-consuming. Recently, Liang and Pardee have developed a novel method called differential display using an arbitrarily primed reverse transcription-coupled PCR and sequencing gels [4]. This has proved to be a powerful technique for detecting changes in eukaryotic gene expression; it is simple, sensitive, and time-saving. However, PCR-mediated amplification with arbitrary primers requires delicate control, depending on the primers used [5,6], and cDNA probes thus obtained often give false positive signals on Northern blot analysis [7,8]. Furthermore, the differential display seems less sensitive in detecting rare mRNA populations, since it has a strong bias toward higher abundance mRNAs [9].