Relationship between amino acid composition and gene expression in the mouse genome.

Background: Codon bias is a phenomenon that refers to the differences in the frequencies of synonymous codons among different genes. In many organisms, natural selection is considered to be a cause of codon bias because codon usage in highly expressed genes is biased toward optimal codons. Methods have previously been developed to predict the expression level of genes from their nucleotide sequences, which is based on the observation that synonymous codon usage shows an overall bias toward a few codons called major codons.

GeneWaltz--A new method for reducing the false positives of gene finding.

Background: Identifying protein-coding regions in genomic sequences is an essential step in genome analysis. It is well known that the proportion of false positives among genes predicted by current methods is high, especially when the exons are short. These false positives are problematic because they waste time and resources of experimental studies.

The RNA-binding protein Mex3b has a fine-tuning system for mRNA regulation in early Xenopus development.

Post-transcriptional control by RNA-binding proteins is a precise way to assure appropriate levels of gene expression. Here, we identify a novel mRNA regulatory system involving Mex3b (RKHD3) and demonstrate its role in FGF signaling. mex3b mRNA has a 3' long conserved UTR, named 3'LCU, which contains multiple elements for both mRNA destabilization and translational enhancement.

Evaluation of the effect of CpG hypermutability on human codon substitution.

Understanding the cause underlying the changes in amino acid composition of proteins is essential for understanding protein evolution and function. Accurate models of DNA and protein evolution are essential for studying molecular evolution. Although many models have been developed, most models assume that each site evolves independently and that substitutions are time reversible.

The universal trend of amino acid gain-loss is caused by CpG hypermutability.

Understanding the cause of the changes in the amino acid composition of proteins is essential for understanding the evolution of protein functions. Since the early 1970s, it has been known that the frequency of some amino acids in protein sequences is increasing and that of others is decreasing. Recently, it was found that the trends of amino acid changes were similar in 15 taxa representing Bacteria, Archaea, and Eukaryota.

Exploration of human ORFeome: high-throughput preparation of ORF clones and efficient characterization of their protein products.

In this study, we established new systematic protocols for the preparation of cDNA clones, conventionally termed open reading frame (ORF) clones, suitable for characterization of their gene products by adopting a restriction-enzyme-assisted cloning method using the Flexi cloning system. The system has following advantages: (1) preparation of ORF clones and their transfer into other vectors can be achieved efficiently and at lower cost; (2) the system provides a seamless connection to the versatile HaloTag labeling system, in which a single fusion tag can be used for various proteomic analyses; and (3) the resultant ORF clones show higher expression levels both in vitro and in vivo. With this system, we prepared ORF clones encoding 1,929 human genes and characterized the HaloTag-fusion proteins of its subset that are expressed in vitro or in mammalian cells.

The H-Invitational Database (H-InvDB), a comprehensive annotation resource for human genes and transcripts.

Here we report the new features and improvements in our latest release of the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/), a comprehensive annotation resource for human genes and transcripts.

Preparation of a set of expression-ready clones of mammalian long cDNAs encoding large proteins by the ORF trap cloning method.

Although we have so far identified and sequenced >2000 human long cDNAs, known as KIAA cDNAs, half of them have yet to be functionally annotated. Expression-ready cDNA clones derived from these genes, where the open reading frame (ORF) of the gene of interest is placed under the control of an appropriate promoter, are critical for functional characterization of these gene products. In this study, we attempted to systematically convert original cDNA clones to expression-ready forms for native and fusion proteins.

Influence of the 3'-UTR-length of mKIAA cDNAs and their sequence features to the mRNA expression level in the brain.

We have previously described the sequence features of approximately 1500 mouse KIAA (mKIAA) genes in comparison with those of human KIAA genes (Okazaki, N., Kikuno, R., Inamoto, S.

CDNA library construction from a small amount of RNA: adaptor-ligation approach for two-round cRNA amplification using T7 and SP6 RNA polymerases.

In this study, we developed a method that allows cDNA library construction from a small amount of RNA without causing serious size bias in the resulting cDNA population. For this purpose, we adopted two-round cRNA amplification by T7 and SP6 RNA polymerases. The first-round cDNAs, flanked by the promoter sequences of T7 and SP6 RNA polymerases, were synthesized from 1 microg total RNA and then subjected to two rounds of cRNA amplification.

Alternative splice variants encoding unstable protein domains exist in the human brain.

Alternative splicing has been recognized as a major mechanism by which protein diversity is increased without significantly increasing genome size in animals and has crucial medical implications, as many alternative splice variants are known to cause diseases. Despite the importance of knowing what structural changes alternative splicing introduces to the encoded proteins for the consideration of its significance, the problem has not been adequately explored. Therefore, we systematically examined the structures of the proteins encoded by the alternative splice variants in the HUGE protein database derived from long (>4 kb) human brain cDNAs.

Prediction of the coding sequences of mouse homologues of FLJ genes: the complete nucleotide sequences of 110 mouse FLJ-homologous cDnas identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a mouse cDNA project to predict protein-coding sequences of mouse KIAA-homologous genes since 2001. As an extension of this project, we also started to accumulate mouse cDNA clones homologous to the human FLJ cDNA clones which are another long cDNA resource produced in our institute. We have isolated the cDNA clones from size-fractionated cDNA libraries derived from five different mouse tissues and natural killer T-cells.

The CAP-Gly domain of CYLD associates with the proline-rich sequence in NEMO/IKKgamma.

CYLD was originally identified as the human familial cylindromatosis tumor suppressor. Recently, it was reported that CYLD directly interacts with NEMO/IKKgamma and TRAF2 in the NF-kappaB signaling pathway. The two proteins bind to a region of CYLD that contains a Cys-box motif and the third cytoskeleton-associated protein-glycine conserved (CAP-Gly) domain.

Method for systematic targeted isolation of homologous cDNA fragments in a multiplex format.

In this study, a two-step method for systematic multiplex cloning of homologous cDNAs from related species was developed. The first step, called MUCH (multiplex cloning of homologous genes), is cloning of partial but authentic cDNA fragments of homologous cDNAs by hybridization to arrayed cRNA probes of specified genes on a nylon membrane, followed by PCR amplification of the hybridized fragments. The second step is PCR-based screening of a library that contains longer cDNA inserts based on the sequences obtained in the first step.

The Kazusa cDNA project for identification of unknown human transcripts.

The Kazusa cDNA project is unique by its focus on sequencing large human cDNAs (>4 kb). We describe an overview of the human cDNA sequence data accumulated during the first phase of the project on over 2000 cDNAs and its integration with the genome sequence. In the second phase of the project, which aims at bridging the human genome and proteome using the output of the first phase, we are very carefully evaluating our cDNA clones and, when necessary, experimentally revising them.

HUGE: a database for human KIAA proteins, a 2004 update integrating HUGEppi and ROUGE.

We have been developing a Human Unidentified Gene-Encoded (HUGE) protein database (http://www.kazusa.or.

Prediction of the coding sequences of mouse homologues of KIAA gene: III. the complete nucleotide sequences of 500 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have conducted a human cDNA project to predict protein-coding sequences (CDSs) in large cDNAs (> 4 kb) since 1994, and the number of newly identified genes, known as KIAA genes, already exceeds 2000. The ultimate goal of this project is to clarify the physiological functions of the proteins encoded by KIAA genes. To this end, the project has recently been expanded to include isolation and characterization of mouse KIAA-counterpart genes.

A new heat shock gene, AgsA, which encodes a small chaperone involved in suppressing protein aggregation in Salmonella enterica serovar typhimurium.

We discovered a novel small heat shock protein (sHsp) named AgsA (aggregation-suppressing protein) in the thermally aggregated fraction from a Salmonella enterica serovar Typhimurium dnaK-null strain. The -10 and -35 regions upstream of the transcriptional start site of the agsA gene are characteristic of sigma(32)- and sigma(72)-dependent promoters. AgsA was strongly induced by high temperatures.

Genome-wide expression analysis of mouse liver reveals CLOCK-regulated circadian output genes.

CLOCK is a positive component of a transcription/translation-based negative feedback loop of the central circadian oscillator in the suprachiasmatic nucleus in mammals. To examine CLOCK-regulated circadian transcription in peripheral tissues, we performed microarray analyses using liver RNA isolated from Clock mutant mice. We also compared expression profiles with those of Cryptochromes (Cry1 and Cry2) double knockout mice.

Characterization of long cDNA clones from human adult spleen. II. The complete sequences of 81 cDNA clones.

To accumulate information on the coding sequences (CDSs) of unidentified genes, we have conducted a sequencing project of human long cDNA clones. Both the end sequences of approximately 10,000 cDNA clones from two size-fractionated human spleen cDNA libraries (average sizes of 4.5 kb and 5.

Prediction of the coding sequences of mouse homologues of KIAA gene: II. The complete nucleotide sequences of 400 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have accumulated information of the coding sequences of uncharacterized human genes, which are known as KIAA genes, and the number of these genes exceeds 2000 at present. As an extension of this sequencing project, we recently have begun to accumulate mouse KIAA-homologous cDNAs, because it would be useful to prepare a set of human and mouse homologous cDNA pairs for further functional analysis of the KIAA genes. We herein present the entire sequences of 400 mouse KIAA cDNA clones and 4 novel cDNA clones which were incidentally identified during this project.

Prediction of the coding sequences of mouse homologues of KIAA gene: I. The complete nucleotide sequences of 100 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a human cDNA project to predict protein-coding sequences in long cDNAs (> 4 kb) since 1994. The number of these newly identified human genes exceeds 2000 and these genes are known as KIAA genes. As an extension of this project, we herein report characterization of cDNAs derived from mouse KIAA-homologous genes.

Protein-protein interactions between large proteins: two-hybrid screening using a functionally classified library composed of long cDNAs.

Large proteins have multiple domains that are potentially capable of binding many kinds of partners. It is conceivable, therefore, that such proteins could function as an intricate framework of assembly protein complexes. To comprehensively study protein-protein interactions between large KIAA proteins, we have constructed a library composed of 1087 KIAA cDNA clones based on prior functional classifications done in silico.