Biomedical subjects

Katsuyuki Hashimoto

Publications and source records attributed to Katsuyuki Hashimoto.

17 recordsLinked to original sources

PubMed2007

Rate of evolution in brain-expressed genes in humans and other primates.

Brain-expressed genes are known to evolve slowly in mammals. Nevertheless, since brains of higher primates have evolved rapidly, one might expect acceleration in DNA sequence evolution in their brain-expressed genes. In this study, we carried out full-length cDNA sequencing on the brain transcriptome of an Old World monkey (OWM) and then conducted three-way comparisons among (i) mouse, OWM, and human, and (ii) OWM, chimpanzee, and human. Although brain-expressed genes indeed appear to evolve more rapidly in species with more advanced brains (apes > OWM > mouse), a similar lineage effect is observable for most other genes. The broad inclusion of genes in the reference set to represent the genomic average is therefore critical to this type of analysis. Calibrated against the genomic average, the rate of evolution among brain-expressed genes is probably lower (or at most equal) in humans than in chimpanzee and OWM. Interestingly, the trend of slow evolution in coding sequence is no less pronounced among brain-specific genes, vis-à-vis brain-expressed genes in general. The human brain may thus differ from those of our close relatives in two opposite directions: (i) faster evolution in gene expression, and (ii) a likely slowdown in the evolution of protein sequences. Possible explanations and hypotheses are discussed.

Animals↗

PubMed2005

Substitution rate and structural divergence of 5'UTR evolution: comparative analysis between human and cynomolgus monkey cDNAs.

The substitution rate and structural divergence in the 5'-untranslated region (UTR) were investigated by using human and cynomolgus monkey cDNA sequences. Due to the weaker functional constraint in the UTR than in the coding sequence, the divergence between humans and macaques would provide a good estimate of the nucleotide substitution rate and structural divergence in the 5'UTR. We found that the substitution rate in the 5'UTR (K5UTR) averaged approximately 10%-20% lower than the synonymous substitution rate (Ks). However, both the K5UTR and nonsynonymous substitution rate (Ka) were significantly higher in the testicular cDNAs than in the brain cDNAs, whereas the Ks did not differ. Further, an in silico analysis revealed that 27% (169/622) of macaque testicular cDNAs had an altered exon-intron structure in the 5'UTR compared with the human cDNAs. The fraction of cDNAs with an exon alteration was significantly higher in the testicular cDNAs than in the brain cDNAs. We confirmed by using reverse transcriptase-polymerase chain reaction that about one-third (6/16) of in silico "macaque-specific" exons in the 5'UTR were actually macaque specific in the testis. The results imply that positive selection increased K5UTR and structural alteration rate of a certain fraction of genes as well as Ka. We found that both positive and negative selection can act on the 5'UTR sequences.

Animals↗

PubMed2004

Integrative annotation of 21,037 human genes validated by full-length cDNA clones.

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology.

Alternative Splicing↗

PubMed2004

Novel missense mutation found in a Japanese patient with myeloperoxidase deficiency.

Myeloperoxidase (MPO; EC 1.11.1.7) plays an important role in the host defense mechanism against microbial diseases. The neutrophil disorder characterized by the lack of MPO activity, is speculated to be associated with a decreased level of immunity. A Japanese patient was identified with complete MPO deficiency through automated hematography. Neutrophil function analysis revealed that MPO activity was significantly diminished with slightly elevated superoxide production. Mutational analysis of the patient revealed a glycine to serine substitution (G501S) in the exon 9 region. This mutation was not detected in the 96 healthy controls analyzed. The amino acid substitution found may be responsible for the failure of mature MPO production in the patient. This is the first case of MPO deficiency of G501S missense mutation identified in a Japanese patient.

Adult↗

PubMed2003

Regulation of constitutive expression of mouse PTEN by the 5'-untranslated region.

PTEN tumor suppressor serves as a major negative regulator of survival signaling mediated by PI3 kinase/AKT/protein kinase B pathway, and is inactivated in various human tumors. Elucidation of mechanisms responsible for PTEN expression is important for providing insight into strategies to control the loss of PTEN expression in human cancers. Although recent studies suggested that p53 and Egr-1 can modulate induced PTEN expression, the mechanism responsible for ubiquitous constitutive expression of PTEN remains elusive. PTEN mRNA contains a highly conserved and GC-rich 5'-untranslated region (5'-UTR). Recently, it has been shown that the long 5'-UTR sequences of several growth-regulated mRNAs contain promoters that can generate mRNAs with shorter 5'-UTRs. In this paper, we tested whether the 5'-UTR sequence of mouse PTEN contains a promoter that is responsible for constitutive expression of PTEN. We found that the long 5'-UTR sequence of mouse PTEN severely inhibits translation of PTEN and a heterologous gene firefly luciferase. Deletion of the most 5'-UTR sequence would enhance translation efficiency 100-fold. We also showed that the 5'-UTR sequence of mouse PTEN does not have an internal ribosome entry site (IRES) that can mediate cap-independent initiation of translation. Instead, we found that the 5'-UTR sequence of mouse PTEN contains a strong promoter that drives the production of a transcript with shorter 5'-UTRs, which can be translated with higher efficiency. This promoter was mapped to the region between -551 and -220 bases upstream of the translation start codon. Cotransfection analysis using Drosophila SL2 cells showed that Sp1 is one of the major transcription factors that can constitutively activate this promoter. Two endogenous PTEN transcripts with 5'-UTRs of 193 and 109 bases were found in DU145 and H226 cell lines. Based on these observations, we conclude that the PTEN expression may be regulated at both transcriptional and translational levels, and that the 5'-UTR sequence of PTEN contains a promoter that is responsible for constitutive PTEN expression.

5' Untranslated Regions↗

PubMed2003

Molecular cloning, genomic characterization and over-expression of a novel gene, XRRA1, identified from human colorectal cancer cell HCT116Clone2_XRR and macaque testis.

BACKGROUND: As part of our investigation into the genetic basis of tumor cell radioresponse, we have isolated several clones with a wide range of responses to X-radiation (XR) from an unirradiated human colorectal tumor cell line, HCT116. Using human cDNA microarrays, we recently identified a novel gene that was down-regulated by two-fold in an XR-resistant cell clone, HCT116Clone2_XRR. We have named this gene as X-ray radiation resistance associated 1 (XRRA1) (GenBank BK000541). Here, we present the first report on the molecular cloning, genomic characterization and over-expression of the XRRA1 gene. RESULTS: We found that XRRA1 was expressed predominantly in testis of both human and macaque. cDNA microarray analysis showed three-fold higher expression of XRRA1 in macaque testis relative to other tissues. We further cloned the macaque XRRA1 cDNA (GenBank AB072776) and a human XRRA1 splice variant from HCT116Clone2_XRR (GenBank AY163836). In silico analysis revealed the full-length human XRRA1, mouse, rat and bovine Xrra1 cDNAs. The XRRA1 gene comprises 11 exons and spans 64 kb on chromosome 11q13.3. Human and macaque cDNAs share 96% homology. Human XRRA1 cDNA is 1987 nt long and encodes a protein of 559 aa. XRRA1 protein is highly conserved in human, macaque, mouse, rat, pig, and bovine. GFP-XRRA1 fusion protein was detected in both the nucleus and cytoplasm of HCT116 clones and COS-7 cells. Interestingly, we found evidence that COS-7 cells which over-expressed XRRA1 lacked Ku86 (Ku80, XRCC5), a non-homologous end joining (NHEJ) DNA repair molecule, in the nucleus. RT-PCR analysis showed differential expression of XRRA1 after XR in HCT116 clones manifesting significantly different XR responses. Further, we found that XRRA1 was expressed in most tumor cell types. Surprisingly, mouse Xrra1 was detected in mouse embryonic stem cells R1. CONCLUSIONS: Both XRRA1 cDNA and protein are highly conserved among mammals, suggesting that XRRA1 may have similar functions. Our results also suggest that the genetic modulation of XRRA1 may affect the XR responses of HCT116 clones and that XRRA1 may have a role in the response of human tumor and normal cells to XR. XRRA1 might be correlated with cancer development and might also be an early expressed gene.

Amino Acid Sequence↗

PubMed2003

A transposable element-mediated gene divergence that directly produces a novel type bovine Bcnt protein including the endonuclease domain of RTE-1.

Ruminant Bcnt protein with a molecular mass of 97 kDa (designated p97Bcnt) includes a region derived from the endonuclease domain of a retrotransposable element RTE-1. Human and mouse Bcnt proteins lack the corresponding region but have a highly conserved 82-amino acid region at the C-terminus that is not present in p97Bcnt. By screening a bovine BAC library, we found two more bcnt-related genes: human-type bcnt (h-type bcnt) and its processed pseudogene. Whereas the pseudogene is localized on chromosome 26, both bcntp97 and the h-type bcnt genes are found on bovine chromosome 18, a synteny region of human chromosome 16 on which human BCNT is localized. Complete nucleotide sequencing of the BAC clone reveals that the bcntp97 and h-type bcnt genes are located just 6 kb apart in a tandem manner. The two h-type bcnt and bcntp97genes are active at both the transcriptional level and the protein level. H-type bovine Bcnt is more like human BCNT than p97Bcnt, when compared at their N-terminal regions. However, phylogenetic analysis using the N-terminal region of the bcnt gene family revealed that the duplication of bovine genes occurred within the bovine lineage with significantly accelerated substitution in bcntp97. This acceleration was not ascribed definitely to positive selection. After duplication, one of the bovine bcnt genes recruited the endonuclease domain of an intronic RTE-1 repeat accompanied by the accelerated substitution at the 5'-ORF, resulting in creation of a novel type of Bcnt protein in bovine.

Amino Acid Sequence↗

PubMed2003

Gene expression profiling reveals the mechanism and pathophysiology of mouse liver regeneration.

Comprehensive analysis of the changes in gene expression during liver regeneration was carried out by using an in-house microarray composed of 2,304 distinct mouse liver cDNA clones. Mice were subjected to partial two-thirds hepatectomy, and changes in mRNA levels were monitored up to 48 h. Of the 2,304 genes analyzed, 496 genes showed expression levels measurable at all time points after the partial hepatectomy. 317 genes were up- or down-regulated 2-fold or more at least at one time point during liver regeneration and were classified into eight clusters based on their expression patterns. With a more stringent cut-off value of +/-2 S.D., 68 genes were listed and were classified into five clusters. In these two analyses with different clustering criteria, functionally categorized genes showed similar cluster distributions. Genes involved in protein synthesis and posttranslational processing were significantly enriched in the cluster characterized by rapid gene activation and subsequent persistence. This suggests the importance of modulating the efficiency of protein supply and/or altering the composition of protein population from the early phase of hepatocyte proliferation. Genes for two major liver functions, i.e. plasma protein secretion and intermediate metabolism were enriched in distinct clusters exhibiting the features of gradual gene activation and sustained repression, respectively. Therefore, these genes are differentially regulated during the regeneration, possibly leading to changes in the flow of amino acids and energy from enzyme proteins to plasma proteins in their synthesis. Thus, clustering analysis of expression patterns of functionally classified genes gave insights into mechanism and pathophysiology of liver regeneration.

Animals↗

PubMed2003

A vertebrate RNA-binding protein Fox-1 regulates tissue-specific splicing via the pentanucleotide GCAUG.

Alternative splicing is one of the central mechanisms that regulate eukaryotic gene expression. Here we report a tissue-specific RNA-binding protein, Fox-1, which regulates alternative splicing in vertebrates. Fox-1 bound specifically to a pentanucleotide GCAUG in vitro. In zebrafish and mouse, fox-1 is expressed in heart and skeletal muscles. As candidates for muscle-specific targets of Fox-1, we considered two genes, the human mitochondrial ATP synthase gamma-subunit gene (F1gamma) and the rat alpha-actinin gene, because their primary transcripts contain several copies of GCAUG. In transfection experiments, Fox-1 induced muscle-specific exon skipping of the F1gamma gene via binding to GCAUG sequences upstream of the regulated exon. Fox-1 also regulated mutually exclusive splicing of the alpha-actinin gene, antagonizing the repressive effect of polypyrimidine tract-binding protein (PTB). It has been reported that GCAUG is essential for the alternative splicing regulation of several genes including fibronectin. We found that Fox-1 promoted inclusion of the fibronectin EIIIB exon. Thus, we conclude that Fox-1 plays key roles in both positive and negative regulation of tissue-specific splicing via GCAUG.

Actinin↗

PubMed2003

Comparative DNA sequence analysis of mouse and human CC chemokine gene clusters.

The CC chemokines are a closely related subfamily of the chemokine superfamily. Most of the CC chemokine genes form a cluster on chromosome 11 in mice and chromosome 17 in humans. To date, 11 and 16 functional genes have been localized within the mouse and human clusters, respectively. Notably, some of the genes within these clusters appear to have no counterparts between the two species, and the orthologous relationships of some of the genes are difficult to establish solely on the basis of amino acid similarity. In this study, we have taken a comparative genomic approach to reveal some of the features that may be involved in the dynamic evolution of these gene clusters. We sequenced a 122-kb region containing five chemokine genes of the mouse CC cluster. This mouse sequence was combined with those determined by the Mouse Genome Sequencing Project, and the entire sequence of the mouse CC cluster was compared with that of the corresponding cluster in the human genome by percent identity plot and dot-plot analyses. Although no additional chemokine genes have been found in these clusters, our analysis has revealed that numerous gene rearrangements have occurred even after the diversification of rodents and primates, resulting in several species-specific chemokine genes and pseudogenes. In addition, phylogenetic analysis and comparison of the genomic sequences unambiguously identified the orthologous relationships of some of the chemokine genes in the mouse and human CC gene clusters.

Animals↗

PubMed2003

Analysis of 5'-end sequences of chimpanzee cDNAs.

We constructed full-length enriched cDNA libraries from chimpanzee brain, skin, and liver tissues by the oligo-capping method to establish a database of sequences of chimpanzee genes. Randomly selected clones from the libraries were subjected to one-pass sequencing from their 5'-ends. As a result, we collected 6813 chimpanzee cDNA sequences longer than 400 bp. Homology search against human mRNA sequences (RefSeq mRNAs) revealed that our collection included sequences of 1652 putative chimpanzee genes. In order to calculate the sequence identity between human and chimpanzee homologs, we constructed 5'-end consensus sequences of 226 chimpanzee genes by aligning at least three sequences for individual genes. Sequence identity was estimated by comparing these consensus sequences and the corresponding sequences of their human homologs. The average sequence identity of the 5'-end cDNAs was 99.30%. Those of the 5'-UTRs and CDSs were 98.79% and 99.42%, respectively. The results confirmed that human and chimpanzee genes are highly conserved at the nucleotide level. As for amino acids, the average sequence identity was 99.44%. The average synonymous (K(S)) and nonsynonymous (K(A)) divergences were estimated to be 1.33% and 0.28%, respectively.

5' Flanking Region↗

PubMed2002

Cynomolgus monkey testicular cDNAs for discovery of novel human genes in the human genome sequence.

BACKGROUND: In order to contribute to the establishment of a complete map of transcribed regions of the human genome, we constructed a testicular cDNA library for the cynomolgus monkey, and attempted to find novel transcripts for identification of their human homologues. RESULT: The full-insert sequences of 512 cDNA clones were determined. Ultimately we found 302 non-redundant cDNAs carrying open reading frames of 300 bp-length or longer. Among them, 89 cDNAs were found not to be annotated previously in the Ensembl human database. After searching against the Ensembl mouse database, we also found 69 putative coding sequences have no homologous cDNAs in the annotated human and mouse genome sequences in Ensembl. We subsequently designed a DNA microarray including 396 non-redundant cDNAs (with and without open reading frames) to examine the expression of the full-sequenced genes. With the testicular probe and a mixture of probes of 10 other tissues, 316 of 332 effective spots showed intense hybridized signals and 75 cDNAs were shown to be expressed very highly in the cynomolgus monkey testis, but not ubiquitously. CONCLUSIONS: In this report, we determined 302 full-insert sequences of cynomolgus monkey cDNAs with enough length of open reading frames to discover novel transcripts as human homologues. Among 302 cDNA sequences, human homologues of 89 cDNAs have not been predicted in the annotated human genome sequence in the Ensembl. Additionally, we identified 75 dominantly expressed genes in testis among the full-sequenced clones by using a DNA microarray. Our cDNA clones and analytical results will be valuable resources for future functional genomic studies.

Journal Article↗

PubMed2002

MacroH2A1.2 binds the nuclear protein Spop.

X-chromosome inactivation is a phenomenon by which one of the two X chromosomes in somatic cells of female mammals is inactivated for life. The inactivated X chromosomes are covered with Xist (X-inactive specific transcript) RNA, and also enriched with the histone H2A variant, macroH2A1.2. The N-terminal one-third of macroH2A1.2 is homologous to core histone H2A, but the function of the C-terminal two-thirds, which contains a basic, putative leucine zipper domain, remains unknown. In this study, we tried analyzing protein-protein interaction with a yeast two-hybrid system to interact with the nonhistone region of mouse macroH2A1.2. The results showed that macroH2A1.2 interacts with mouse nuclear speckled type protein Spop. The Spop protein has a unique composition: an N-terminal MATH, and a C-terminal BTB/POZ domain. Further binding domain mapping in a glutathione-S-transferase (GST) pull-down experiment revealed that macroH2A1.2 binds the MATH domain of Spop, which in turn binds to the putative leucine zipper domain of macroH2A1.2.

Animals↗

PubMed2002

A novel transactivating factor that regulates interferon-gamma-dependent gene expression.

We have previously identified a novel interferon (IFN)-stimulated cis-acting enhancer element, gamma-IFN-activated transcriptional element (GATE). GATE differs from the known IFN-stimulated elements in its primary sequence. Preliminary analysis has indicated that the GATE-dependent transcriptional response requires the binding of novel transacting factors. A cDNA expression library derived from an IFN-gamma-stimulated murine macrophage cell line was screened with a (32)P-labeled GATE probe to identify the potential GATE-binding factors. A cDNA coding for a novel transcription-activating factor was identified. Based on its discovery, we named it as GATE-binding factor-1 (GBF-1). GBF-1 homologs are present in mouse, human, monkey, and Drosophila. It activates transcription from reporter genes carrying GATE. It possesses a strong transactivating activity but has a weak DNA binding property. GBF-1 is expressed in most tissues with relatively higher steady-state levels in heart, liver, kidney, and brain. Its expression is induced by IFN-gamma treatment. GBF-1 is present in both cytosolic and nuclear compartments. These studies thus identify a novel transactivating factor in IFN signaling pathways.

Amino Acid Sequence↗

PubMed2002

Identification and characterization of a novel type of membrane-associated prostaglandin E synthase.

Membrane-associated prostaglandin E synthase (mPGE synthase) was previously purified to apparent homogeneity from the microsomal fraction of bovine heart (Watanabe, K., et al., Biochim. Biophys. Acta 1439, 406--414, 1999). The N-terminal 22-amino acid sequence of the purified enzyme was identical to that of the 88th to 109th amino acids deduced from the monkey (AB046026) or human (AK024100) cDNA that encodes a hypothetical protein with unknown function. The primary structure has the consensus region of glutaredoxin and of thioredoxin. We constructed an expression plasmid, using the vector (pTrc-HisA) and the monkey cDNA for the 290-amino-acid polypeptide. The recombinant protein with a M(r) of 33 kDa exhibited PGE synthase activity and was purified to apparent homogeneity by nickel-chelating column chromatography. The V(max) and K(m) values for PGH(2) of the purified recombinant mPGE synthase were about 3.3 mumol/min center dot mg of protein and 28 muM, respectively. The recombinant enzyme was activated by various SH-reducing reagents, i.e., dithiothreitol, glutathione (GSH), and beta-mercaptoethanol, in order of decreasing effectiveness. Moreover, the mRNA distribution was high in the heart and brain, but the mRNA was not expressed in the seminal vesicles. These results indicate that the recombinant mPGE synthase is identical to the enzyme purified from the microsomal fraction of bovine heart, and is a novel type of mPGE synthase based on the primary structure, a broad specificity of thiol requirement, and tissue distribution.

Amino Acid Sequence↗

PubMed2002

Search for genes positively selected during primate evolution by 5'-end-sequence screening of cynomolgus monkey cDNAs.

It is possible to assess positive selection by using the ratio of K(a) (nonsynonymous substitutions per plausible nonsynonymous sites) to K(s) (synonymous substitutions per plausible synonymous sites). We have searched candidate genes positively selected during primate evolution by using 5'-end sequences of 21,302 clones derived from cynomolgus monkey (Macaca fascicularis) brain cDNA libraries. Among these candidates, 10 genes that had not been shown by previous studies to undergo positive selection exhibited a K(a)/K(s) ratio > 1. Of the 10 candidate genes we found, 5 were included in the mitochondrial respiratory enzyme complexes, suggesting that these nuclear-encoded genes coevolved with mitochondrial-encoded genes, which have high mutation rates. The products of other candidate genes consisted of a cell-surface protein, a member of the lipocalin family, a nuclear transcription factor, and hypothetical proteins.

Animals↗

PubMed2001

Prediction of unidentified human genes on the basis of sequence similarity to novel cDNAs from cynomolgus monkey brain.

BACKGROUND: The complete assignment of the protein-coding regions of the human genome is a major challenge for genome biology today. We have already isolated many hitherto unknown full-length cDNAs as orthologs of unidentified human genes from cDNA libraries of the cynomolgus monkey (Macaca fascicularis) brain (parietal lobe and cerebellum). In this study, we used cDNA libraries of three other parts of the brain (frontal lobe, temporal lobe and medulla oblongata) to isolate novel full-length cDNAs. RESULTS: The entire sequences of novel cDNAs of the cynomolgus monkey were determined, and the orthologous human cDNA sequences were predicted from the human genome sequence. We predicted 29 novel human genes with putative coding regions sharing an open reading frame with the cynomolgus monkey, and we confirmed the expression of 21 pairs of genes by the reverse transcription-coupled polymerase chain reaction method. The hypothetical proteins were also functionally annotated by computer analysis. CONCLUSIONS: The 29 new genes had not been discovered in recent explorations for novel genes in humans, and the ab initio method failed to predict all exons. Thus, monkey cDNA is a valuable resource for the preparation of a complete human gene catalog, which will facilitate post-genomic studies.

Animals↗