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P Carninci

Publications and source records attributed to P Carninci.

At least 19 recordsLinked to original sources
PubMed2002

A framework for integrating the songbird brain.

Biological systems by default involve complex components with complex relationships. To decipher how biological systems work, we assume that one needs to integrate information over multiple levels of complexity. The songbird vocal communication system is ideal for such integration due to many years of ethological investigation and a discreet dedicated brain network. Here we announce the beginnings of a songbird brain integrative project that involves high-throughput, molecular, anatomical, electrophysiological and behavioral levels of analysis. We first formed a rationale for inclusion of specific biological levels of analysis, then developed high-throughput molecular technologies on songbird brains, developed technologies for combined analysis of electrophysiological activity and gene regulation in awake behaving animals, and developed bioinformatic tools that predict causal interactions within and between biological levels of organization. This integrative brain project is fitting for the interdisciplinary approaches taken in the current songbird issue of the Journal of Comparative Physiology A and is expected to be conducive to deciphering how brains generate and perceive complex behaviors.

Animals↗
PubMed2002

On biased distribution of introns in various eukaryotes.

We conducted comprehensive analyses on intron positions in the Mus musculus genome by comparing genomic sequences in the GenBank database and cDNA sequences in the mouse cDNA library recently developed by Riken Genomic Sciences Center. Our results confirm that introns have a tendency to be located toward the 5' end of the gene. The same type of analysis was conducted in the coding region of seven eukaryotes (Saccharomyces cerevisiae, Plasmodium falciparum, Caenorhabditis elegans, Drosophila melanogaster, M. musculus, Homo sapiens, Arabidopsis thaliana). Introns in genes with a single intron have a locational bias toward the 5' end in all species except A. thaliana. We also measured the distance from the start codon to the position of the intron, and found that single introns prefer the location immediately after the start codon in S. cerevisiae and P. falciparum. We discuss three possible explanations for these findings: (1) they are the consequence of intron loss by reverse-transcriptase; (2) they are necessary to accommodate the function; and (3) they are concerned with the mechanism of pre-mRNA splicing.

Animals↗
PubMed2002

Comprehensive sequence analysis of translation termination sites in various eukaryotes.

Recent investigations into the translation termination sites of various organisms have revealed that not only stop codons but also sequences around stop codons have an effect on translation termination. To investigate the relationship between these sequence patterns and translation as well as its termination efficiency, we analysed the correlation between strength of consensus and translation efficiency, as predicted according to Codon Adaptation Index (CAI) value. We used RIKEN full-length mouse cDNA sequences and ten other eukaryotic UniGene datasets from NCBI for the analyses. First, we conducted sequence profile analyses following translation termination sites. We found base G and A at position +1 as a strong consensus for mouse cDNA. A similar consensus was found for other mammals, such as Homo sapiens, Rattus norvegicus and Bos taurus. However, some plants had different consensus sequences. We then analysed the correlation between the strength of consensus at each position and the codon biases of whole coding regions, using information content and CAI value. The results showed that in mouse cDNA, CAI value had a positive correlation with information content at positions +1. We also found that, for positions with strong consensus, the strength of the consensus is likely to have a positive correlation with CAI value in some other eukaryotes. Along with these observations, biological insights into the relationship between gene expression level, codon biases and consensus sequence around stop codons will be discussed.

3' Untranslated Regions↗
PubMed2002

Cytoplasmic RNA extraction from fresh and frozen mammalian tissues.

The quality of collections of expressed sequence tags andfull-length cDNAs is adversely affected by the presence of "junk" clones derivedfrom unspliced or partially spliced RNAs present in conventional total RNA preparations. One can overcome this problem by using intact cytoplasmic RNA to create cDNA libraries, but the methods in the literature that describe the preparation of RNA only work well for extracting cultured cells. Cell lines are not as diverse as one would like, and to clone comprehensive sets of human and model organism full-length cDNAs, libraries have to be prepared from tissue samples. Thus, we have developed a robust and inexpensive method that allows intact cytoplasmic RNA to be extracted from both fresh and frozen mammalian tissues. A mouse full-length, cap-trapped cDNA library prepared with RNA using this new procedure had excellent characteristics.

Animals↗
PubMed2001

Correlation between sequence conservation of the 5' untranslated region and codon usage bias in Mus musculus genes.

The codon adaptation index (CAI) values of all protein-coding sequences of the full-length cDNA libraries of Mus musculus were computed based on the RIKEN mouse full-length cDNA library. We have also computed the extent of consensus in flanking sequences of the initiator ATG codon based on the 'relative entropy' values of respective nucleotide positions (from -20 to +12 bp relative to the initiator ATG codon) for each group of genes classified by CAI values. With regard to the two nucleotides positions (-3 and +4) known to be highly conserved in Kozak's consensus sequence, a clear correlation between CAI values and relative entropy values was observed at position -3 but this was not significant at position +4, although a significant correlation was found at position -1 of the consensus sequence. Further, although no correlation was observed at any additional positions, relative entropy values were very high at positions -4, -6, and -8 in genes with high CAI values. These findings suggest that the extent of conservation in the flanking sequence of the initiator ATG codon including Kozak's consensus sequence was an important factor in modulation of the translation efficiency as well as synonymous codon usage bias particularly in highly expressed genes.

5' Untranslated Regions↗
PubMed2001

Delineating developmental and metabolic pathways in vivo by expression profiling using the RIKEN set of 18,816 full-length enriched mouse cDNA arrays.

We have systematically characterized gene expression patterns in 49 adult and embryonic mouse tissues by using cDNA microarrays with 18,816 mouse cDNAs. Cluster analysis defined sets of genes that were expressed ubiquitously or in similar groups of tissues such as digestive organs and muscle. Clustering of expression profiles was observed in embryonic brain, postnatal cerebellum, and adult olfactory bulb, reflecting similarities in neurogenesis and remodeling. Finally, clustering genes coding for known enzymes into 78 metabolic pathways revealed a surprising coordination of expression within each pathway among different tissues. On the other hand, a more detailed examination of glycolysis revealed tissue-specific differences in profiles of key regulatory enzymes. Thus, by surveying global gene expression by using microarrays with a large number of elements, we provide insights into the commonality and diversity of pathways responsible for the development and maintenance of the mammalian body plan.

Animals↗
PubMed2001

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.

Animals↗
PubMed2001

Comparative evaluation of 5'-end-sequence quality of clones in CAP trapper and other full-length-cDNA libraries.

To enhance the usefulness of the laboratory mouse and to facilitate the rapid assay of gene functions we have been collecting the entire set of mouse full-length cDNA by one-pass sequencing. To collect full-length cDNA clones efficiently, it is critical to construct high-quality cDNA libraries. In recent years, we have been developing a way to construct full-length cDNA libraries by using biotinylation of the cap structure (the 'CAP-trapper' method) coupled with treatment to increase reverse transcriptase efficiency at high temperature by the addition of trehalose. In this paper we report our evaluation of the quality of CAP trapper and a number of other full-length cDNA libraries, including the results of 5' end analysis of clones in CAP trapper and the other libraries. We used a procedure that compared the 5'-ends of cDNA clones with those of genes in the public databases. Our analysis showed that 63% of cDNA clones in CAP trapper libraries had sequences that were either the same length as those of equivalent genes in the public database or 5'-extended, and that 90% of these clones maintained their coding sequences. These results indicate that the CAP trapper library is a promising tool for collecting full-length cDNA in large-scale projects. Comparison of the quality of CAP trapper with that of other full-length-cDNA libraries confirmed the value of these libraries.

Algorithms↗
PubMed2001

Balanced-size and long-size cloning of full-length, cap-trapped cDNAs into vectors of the novel lambda-FLC family allows enhanced gene discovery rate and functional analysis.

We have developed a new class of cloning vectors: lambda-full-length cDNA (lambda-FLC) cloning vectors. These vectors can be bulk-excised for preparing full-length cDNA libraries in which a high proportion of the plasmids carry large inserts that can be transferred into other (for example, functional) vectors. Unlike other cloning vectors, lambda-FLC vectors accommodate a broad range of sizes of eukaryotic cDNA inserts because they contain "size balancers." Further, the main protocol we use for direct bulk excision of plasmids is mediated by a Cre-lox system and is apparently free of size bias. The average size of the inserts from excised plasmid cDNA libraries was 2.9 kb for standard and 6.9 kb for size-selected cDNA. The average insert size of the full-length cDNA libraries was correlated to the rate of new gene discovery, suggesting that effectively cloning rarely expressed mRNAs requires vectors that can accommodate large inserts from a variety of sources. Part of the vectors are also suitable for bulk transfer of inserts into various functional vectors.

Animals↗
PubMed2001

Computer-based methods for the mouse full-length cDNA encyclopedia: real-time sequence clustering for construction of a nonredundant cDNA library.

We developed computer-based methods for constructing a nonredundant mouse full-length cDNA library. Our cDNA library construction process comprises assessment of library quality, sequencing the 3' ends of inserts and clustering, and completing a re-array to generate a nonredundant library from a redundant one. After the cDNA libraries are generated, we sequence the 5' ends of the inserts to check the quality of the library; then we determine the sequencing priority of each library. Selected libraries undergo large-scale sequencing of the 3' ends of the inserts and clustering of the tag sequences. After clustering, the nonredundant library is constructed from the original libraries, which have redundant clones. All libraries, plates, clones, sequences, and clusters are uniquely identified, and all information is saved in the database according to this identifier. At press time, our system has been in place for the past two years; we have clustered 939,725 3' end sequences into 127,385 groups from 227 cDNA libraries/sublibraries (see http://genome.gse.riken.go.jp/).

5' Untranslated Regions↗
PubMed2001

Monitoring the expression pattern of 1300 Arabidopsis genes under drought and cold stresses by using a full-length cDNA microarray.

Full-length cDNAs are essential for functional analysis of plant genes. Using the biotinylated CAP trapper method, we constructed full-length Arabidopsis cDNA libraries from plants in different conditions, such as drought-treated, cold-treated, or unstressed plants, and at various developmental stages from germination to mature seed. We prepared a cDNA microarray using approximately 1300 full-length Arabidopsis cDNAs to identify drought- and cold-inducible genes and target genes of DREB1A/CBF3, a transcription factor that controls stress-inducible gene expression. In total, 44 and 19 cDNAs for drought- and cold-inducible genes, respectively, were isolated, 30 and 10 of which were novel stress-inducible genes that have not been reported as drought- or cold-inducible genes previously. Twelve stress-inducible genes were identified as target stress-inducible genes of DREB1A, and six of them were novel. On the basis of RNA gel blot and microarray analyses, the six genes were identified as novel drought- and cold-inducible genes that are controlled by DREB1A. Eleven DREB1A target genes whose genomic sequences have been registered in the GenBank database contained the dehydration-responsive element (DRE) or DRE-related CCGAC core motif in their promoter regions. These results show that our full-length cDNA microarray is a useful material with which to analyze the expression pattern of Arabidopsis genes under drought and cold stresses, to identify target genes of stress-related transcription factors, and to identify potential cis-acting DNA elements by combining the expression data with the genomic sequence data.

Arabidopsis↗
PubMed2001

Cloning full-length, cap-trapper-selected cDNAs by using the single-strand linker ligation method.

We have developed the single-strand linker ligation method (SSLLM), which uses DNA ligase to add a dsDNA linker to single-stranded (ss) full-length cDNA. The linkers have random 6-bp (dN6 or dGN5) 3' overhangs that can ligate to any cDNA sequence, thereby facilitating the production of cDNA libraries with titers exceeding 1 x 10(6) independent clones. We confirmed that the 5' ends of cDNA inserts cloned by using SSLLM are full-length and include the 5' untranslated regions. The great advantage of our method is that the elimination of the GC tail simplifies the sequencing and protein translation of the full-length clones. Further, our method tags ss cDNAs more efficiently than does the traditional RNA ligase reaction.

5' Untranslated Regions↗
PubMed2001

Removal of polyA tails from full-length cDNA libraries for high-efficiency sequencing.

We have developed a method to overcome sequencing problems caused by the presence of homopolymer stretches, such as polyA/T, in cDNA libraries. PolyA tails are shortened by cleaving before cDNA cloning with type IIS restriction enzymes, such as GsuI, placed next to the oligo-dT used to prime the polyA tails of mRNAs. We constructed four rice Cap-Trapper-selected, full-length normalized cDNA libraries, of which the average residual polyA tail was 4 bases or shorter in most of the clones analyzed Because of the removal of homopolymeric stretches, libraries prepared with this method can be used for direct sequencing and transcriptional sequencing without the slippage observed for libraries prepared with currently available methods, thus improving sequencing accuracy, operations, and throughput.

Animals↗
PubMed2000

Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes.

In the effort to prepare the mouse full-length cDNA encyclopedia, we previously developed several techniques to prepare and select full-length cDNAs. To increase the number of different cDNAs, we introduce here a strategy to prepare normalized and subtracted cDNA libraries in a single step. The method is based on hybridization of the first-strand, full-length cDNA with several RNA drivers, including starting mRNA as the normalizing driver and run-off transcripts from minilibraries containing highly expressed genes, rearrayed clones, and previously sequenced cDNAs as subtracting drivers. Our method keeps the proportion of full-length cDNAs in the subtracted/normalized library high. Moreover, our method dramatically enhances the discovery of new genes as compared to results obtained by using standard, full-length cDNA libraries. This procedure can be extended to the preparation of full-length cDNA encyclopedias from other organisms.

Animals↗
PubMed2000

RIKEN integrated sequence analysis (RISA) system--384-format sequencing pipeline with 384 multicapillary sequencer.

The RIKEN high-throughput 384-format sequencing pipeline (RISA system) including a 384-multicapillary sequencer (the so-called RISA sequencer) was developed for the RIKEN mouse encyclopedia project. The RISA system consists of colony picking, template preparation, sequencing reaction, and the sequencing process. A novel high-throughput 384-format capillary sequencer system (RISA sequencer system) was developed for the sequencing process. This system consists of a 384-multicapillary auto sequencer (RISA sequencer), a 384-multicapillary array assembler (CAS), and a 384-multicapillary casting device. The RISA sequencer can simultaneously analyze 384 independent sequencing products. The optical system is a scanning system chosen after careful comparison with an image detection system for the simultaneous detection of the 384-capillary array. This scanning system can be used with any fluorescent-labeled sequencing reaction (chain termination reaction), including transcriptional sequencing based on RNA polymerase, which was originally developed by us, and cycle sequencing based on thermostable DNA polymerase. For long-read sequencing, 380 out of 384 sequences (99.2%) were successfully analyzed and the average read length, with more than 99% accuracy, was 654.4 bp. A single RISA sequencer can analyze 216 kb with >99% accuracy in 2.7 h (90 kb/h). For short-read sequencing to cluster the 3' end and 5' end sequencing by reading 350 bp, 384 samples can be analyzed in 1.5 h. We have also developed a RISA inoculator, RISA filtrator and densitometer, RISA plasmid preparator which can handle throughput of 40,000 samples in 17.5 h, and a high-throughput RISA thermal cycler which has four 384-well sites. The combination of these technologies allowed us to construct the RISA system consisting of 16 RISA sequencers, which can process 50,000 DNA samples per day. One haploid genome shotgun sequence of a higher organism, such as human, mouse, rat, domestic animals, and plants, can be revealed by seven RISA systems within one month.

Animals↗
PubMed1999

Increased specificity of reverse transcription priming by trehalose and oligo-blockers allows high-efficiency window separation of mRNA display.

We have developed a method for high-efficiency window separation of cDNA display by increasing the specificity of priming in reverse transcription. In the conventional method, two-base anchored oligo(dT) primers (5'dT16VN3', where N is any base and V is G, A or C) are used to make windows for the display of transcripts. However, reverse transcriptase often extends misprimed oligonucleotides. To avoid mispriming from dT16VN primers, we have developed two new technologies. One is higher temperature priming with reverse transcriptase thermoactivated by the disaccharide trehalose. The other is the use of competitive oligonucleotide blockers that hybridize to the non-selectively primed mRNAs, preventing the mispriming from the VN site. These methods were combined to improve restriction landmark cDNA scanning (RLCS), resulting in the elimination of the redundant signals that appear in different windows. This was achieved by the increased specificity of initiation of reverse trans-cription from the beginning of poly(A) sites. This method paves the way for the precise visualization of transcripts to allow expression profiles in individual tissues and at each developmental stage to be understood.

Animals↗