Hidenori Kiyosawa

University of Tsukuba, Tsukuba, Ibaraki, Japan

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Publications (28)227.89 Total impact

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    ABSTRACT: To investigate the feasibility of developing a method for detection of gene doping in power-athletes, we devised an experimental model system. Myostatin is a potent negative regulator of skeletal muscle development and growth, and myostatin-knockout mice exhibit a double-muscle phenotype. To achieve knockdown, we constructed plasmids expressing short hairpin interfering RNAs (shRNAs) against myostatin. These shRNAs were transfected into C2C12 cultured cells or injected into the tibialis anterior (TA) muscle of adult mice. By performing in vitro and in vivo experiments, we found that some shRNAs effectively reduced the expression of myostatin, and that the TA muscle showed hypertrophy of up to 27.9%. Then, using real-time PCR, we tried to detect the shRNA plasmid in the serum or muscles of mice into which it had been injected. Although we were unable to detect the plasmid in serum samples, it was detectable in the treated muscle at least four weeks after induction. We were also able to detect the plasmid in muscle in the vicinity of the TA. This gene doping model system will be useful for further studies aimed at doping control.
  • Koji Numata, Hidenori Kiyosawa
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    ABSTRACT: Recent transcriptomic studies revealed that extensive proportions of genomes are transcribed, despite the limited fraction of protein-coding gene loci in the whole genome. Most transcripts are considered to be 'cryptic' output of the genome because of the lack of functional evidence; however, recent progress in molecular analyses has revealed that some of these transcripts at least have functional significance. This review article examines evidence of the functional significance of endogenous cis-antisense transcripts, which are the transcriptional output from the opposite strand of annotated genes. These transcripts are one of the most common types of transcripts that do not correspond to any protein-coding loci. Historical molecular studies revealed the existence of antisense transcripts associated with dozens of gene loci, whereas more recent genome-wide studies have shown that many genes have an antisense counterpart thus stimulating investigations into the functional significance of endogenous antisense transcripts. Here, we summarize the recent progress in the genome-wide characterization of the antisense transcriptome, and discuss the biological mechanisms that underlie the regulatory machinery of eukaryotic gene expression with respect to the potential roles of endogenous cis-antisense transcripts.
    Frontiers in Bioscience 01/2012; 17:300-15. DOI:10.2741/3928 · 4.25 Impact Factor
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    ABSTRACT: To investigate the feasibility of developing a method for detection of gene doping in power-athletes, we devised an experimental model system. Myostatin is a potent negative regulator of skeletal muscle development and growth, and myostatin-knockout mice exhibit a double-muscle phenotype. To achieve knockdown, we constructed plasmids expressing short hairpin interfering RNAs (shRNAs) against myostatin. These shRNAs were transfected into C2C12 cultured cells or injected into the tibialis anterior (TA) muscle of adult mice. By performing in vitro and in vivo experiments, we found that some shRNAs effectively reduced the expression of myostatin, and that the TA muscle showed hypertrophy of up to 27.9%. Then, using real-time PCR, we tried to detect the shRNA plasmid in the serum or muscles of mice into which it had been injected. Although we were unable to detect the plasmid in serum samples, it was detectable in the treated muscle at least four weeks after induction. We were also able to detect the plasmid in muscle in the vicinity of the TA. This gene doping model system will be useful for further studies aimed at doping control. Key pointsUsing a myostatin knockdown plasmid, we have succeeded in creating a model system for gene doping using mice that resulted in muscle hypertrophy greater than that reported previously.We confirmed that there was a limit of gene doping detection using real-time PCR, either from serum or muscle smple.This model experimental system can be utilized for examining indirect methods of gene doping detection such as immune responses to gene transfer or a profiling approach using DNA microarray.
    Journal of sports science & medicine 01/2012; 11(2):294-303. · 0.90 Impact Factor
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    ABSTRACT: Genomic imprinting is a phenomenon whereby monoallelic gene expression occurs in a parent-of-origin-specific manner. A subset of imprinted genes acquires a tissue-specific imprinted status during the course of tissue development, and this process can be analyzed by means of an in vitro differentiation system utilizing embryonic stem (ES) cells. In neurons, the gene Ube3a is expressed from the maternal allele only, and a paternally expressed non-coding, antisense RNA has been implicated in the imprinting process in mice and humans. Here, to study the genomic imprinting mechanism, we established F1 hybrid ES cells derived from two sub-species of Mus musculus and established an in vitro neuronal differentiation system in which neuron-specific imprinting of Ube3a was recapitulated. With this system, we revealed that the switch from biallelic expression to maternal, monoallelic expression of Ube3a occurs late in neuronal development, during the neurite outgrowth period, and that the expression of endogenous antisense transcript from the Ube3a locus is up-regulated several hundred-fold during the same period. Our results suggest that evaluation of the quality of ES cells by studying their differentiation in vitro should include evaluation of epigenetic aspects, such as a comparison with the genomic imprinting status found in tissues in vivo, in addition to the evaluation of differentiation gene markers and morphology. Our F1 hybrid ES cells and in vitro differentiation system will allow researchers to investigate complex end-points such as neuron-specific genomic imprinting, and our F1 hybrid ES cells are a useful resource for other tissue-specific genomic imprinting and epigenetic analyses.
    Human Molecular Genetics 12/2011; 21(6):1391-401. DOI:10.1093/hmg/ddr577 · 6.68 Impact Factor
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    ABSTRACT: Mammalian target of rapamycin (mTOR) pathway positively regulates the cell growth through ribosome biogenesis in many cell type. In general, myostatin is understood to repress skeletal muscle hypertrophy through inhibition of mTOR pathway and myogenesis. However, these relationships have not been clarified in skeletal muscle undergoing atrophy. Here, we observed a significant decrease of skeletal muscle mass at 2 weeks after denervation. Unexpectedly, however, mTOR pathway and the expression of genes related to myogenesis were markedly increased, and that of myostatin was decreased. However, de novo ribosomal RNA synthesis and the levels of ribosomal RNAs were dramatically decreased in denervated muscle. These results indicate that ribosome biogenesis is strongly controlled by factors other than the mTOR pathway in denervated atrophic muscle. Finally, we assessed rRNA transcription factors expression and observed that TAFIa was the only factor decreased. TAFIa might be a one of the limiting factor for rRNA synthesis in denervated muscle.
    Journal of Cellular Physiology 06/2011; 227(4):1569-76. DOI:10.1002/jcp.22871 · 3.87 Impact Factor
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    ABSTRACT: Recent studies have identified thousands of sense-antisense gene pairs across different genomes by computational mapping of cDNA sequences. These studies have shown that approximately 25% of all transcriptional units in the human and mouse genomes are involved in cis-sense-antisense pairs. However, the number of known sense-antisense pairs remains limited because currently available cDNA sequences represent only a fraction of the total number of transcripts comprising the transcriptome of each cell type. To discover novel antisense transcripts encoded in the antisense strand of important genes, such as cancer-related genes, we conducted expression analyses of antisense transcripts using our custom microarray platform along with 2376 probes designed specifically to detect the potential antisense transcripts of 501 well-known genes suitable for cancer research. Using colon cancer tissue and normal tissue surrounding the cancer tissue obtained from 6 patients, we found that antisense transcripts without poly(A) tails are expressed from approximately 80% of these well-known genes. This observation is consistent with our previous finding that many antisense transcripts expressed in a cell are poly(A)-. We also identified 101 and 71 antisense probes displaying a high level of expression specifically in normal and cancer tissues respectively. Our microarray analysis identified novel antisense transcripts with expression profiles specific to cancer tissue, some of which might play a role in the regulatory networks underlying oncogenesis and thus are potential targets for further experimental validation. Our microarray data are available at http://www.brc.riken.go.jp/ncrna2007/viewer-Saito-01/index.html.
    BMC Medical Genomics 05/2011; 4:42. DOI:10.1186/1755-8794-4-42 · 3.91 Impact Factor
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    ABSTRACT: The functionality of sense-antisense transcripts (SATs), although widespread throughout the mammalian genome, is largely unknown. Here, we analyzed the SATs expression and its associated promoter DNA methylation status by surveying 12 tissues of mice to gain insights into the relationship between expression and DNA methylation of SATs. We have found that sense and antisense expression positively correlate in most tissues. However, in some SATs with tissue-specific expression, the expression level of a transcript from a CpG island-bearing promoter is low when the promoter DNA methylation is present. In these circumstances, the expression level of its opposite-strand transcript, especially when it is poly(A)-negative was coincidentally higher. These observations suggest that, albeit the general tendency of sense-antisense simultaneous expression, some antisense transcripts have coordinated expression with its counterpart sense gene promoter methylation. This cross-strand relationship is not a privilege of imprinted genes but seems to occur widely in SATs.
    Genomics 12/2010; 96(6):333-41. DOI:10.1016/j.ygeno.2010.08.007 · 2.79 Impact Factor
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    Koji Numata, Chihiro Kohama, Kuniya Abe, Hidenori Kiyosawa
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    ABSTRACT: We investigated the allele- and strand-specific transcriptional landscape of a megabase-wide genomic region of mouse Ube3a (ubiquitin protein ligase E3A) by means of a highly parallel SNP genotyping platform. We have successfully identified maternal-specific expression of Ube3a and its antisense counterpart (Ube3a-ATS) in brain, but not in liver. Because of the use of inter-subspecies hybrid mice, this megabase-wide analysis provided high-resolution picture of the transcriptional patterns of this region. First, we showed that brain-specific maternal expression of Ube3a is restricted to the second half part of the locus, but is absent from the first half part. Balance of allelic expression is altered in the middle of the locus. Second, we showed that expression of the brain-specific Ube3a-ATS appeared to be terminated in the region upstream to the Ube3a transcription start site. The present study highlights the importance of locus-wide competition between sense and antisense transcripts.
    Nucleic Acids Research 12/2010; 39(7):2649-57. DOI:10.1093/nar/gkq1201 · 8.81 Impact Factor
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    ABSTRACT: Recent transcriptomic analyses in mammals have uncovered the widespread occurrence of endogenous antisense transcripts, termed natural antisense transcripts (NATs). NATs are transcribed from the opposite strand of the gene locus and are thought to control sense gene expression, but the mechanism of such regulation is as yet unknown. Although several thousand potential sense-antisense pairs have been identified in mammals, examples of functionally characterized NATs remain limited. To identify NAT candidates suitable for further functional analyses, we performed DNA microarray-based NAT screening using mouse adult normal tissues and mammary tumors to target not only the sense orientation but also the complementary strand of the annotated genes. First, we designed microarray probes to target the complementary strand of genes for which an antisense counterpart had been identified only in human public cDNA sources, but not in the mouse. We observed a prominent expression signal from 66.1% of 635 target genes, and 58 genes of these showed tissue-specific expression. Expression analyses of selected examples (Acaa1b and Aard) confirmed their dynamic transcription in vivo. Although interspecies conservation of NAT expression was previously investigated by the presence of cDNA sources in both species, our results suggest that there are more examples of human-mouse conserved NATs that could not be identified by cDNA sources. We also designed probes to target the complementary strand of well-characterized genes, including oncogenes, and compared the expression of these genes between mammary cancerous tissues and non-pathological tissues. We found that antisense expression of 95 genes of 404 well-annotated genes was markedly altered in tumor tissue compared with that in normal tissue and that 19 of these genes also exhibited changes in sense gene expression. These results highlight the importance of NAT expression in the regulation of cellular events and in pathological conditions. Our microarray platform targeting the complementary strand of annotated genes successfully identified novel NATs that could not be identified by publically available cDNA data, and as such could not be detected by the usual "sense-targeting" microarray approach. Differentially expressed NATs monitored by this platform may provide candidates for investigations of gene function. An advantage of our microarray platform is that it can be applied to any genes and target samples of interest.
    BMC Genomics 09/2009; 10:392. DOI:10.1186/1471-2164-10-392 · 4.04 Impact Factor
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    ABSTRACT: PTEN-induced kinase 1 (PINK1), which is identified as the gene transactivated by the tumor suppressor PTEN, has been found to be one of the causative genes in Parkinson's disease (PD). In order to understand PD, rodent models containing affected Pink1 such as loss-of-function mutations have been exploited. Recently, natural antisense RNA of PINK1 has been demonstrated to be involved in the regulation of the PINK1 locus. However, no antisense RNAs of Pink1 except for human have been reported so far. Therefore, in the present study, while searching for the Pink1 antisense RNAs in mouse, we found that the antisense RNAs are transcribed from a mouse genomic region corresponding to the human region from which the antisense RNAs are produced. Further, we investigated the localization of the antisense RNAs in mouse brain using in situ hybridization; this demonstrated that the antisense RNAs were localized in the regions of brain where the Pink1 mRNA was found. In addition, the mRNA and antisense RNAs were found more densely in the hippocampus than in the other brain regions in newborn and 1-week-old mice, while those RNAs were found uniformly in the mouse brain regions of embryo day (E) 14, E17, and 8-weeks-old.
    Cytogenetic and Genome Research 01/2009; 126(3):259-70. DOI:10.1159/000251963 · 1.84 Impact Factor
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    ABSTRACT: Increasing numbers of sense-antisense transcripts (SATs), which are transcribed from the same chromosomal location but in opposite directions, have been identified in various eukaryotic species, but the biological meanings of most SATs remain unclear. To improve understanding of natural sense-antisense transcription, we performed comparative expression profiling of SATs conserved among humans and mice. Using custom oligo-arrays loaded with probes that represented SATs with both protein-coding and non-protein-coding transcripts, we showed that 33% of the 291 conserved SATs displayed identical expression patterns in the two species. Among these SATs, expressional balance inversion of sense-antisense genes was mostly observed in testis at a tissue-specific manner. Northern analyses of the individual conserved SAT loci revealed that: (i) a smeary hybridization pattern was present in mice, but not in humans, and (2) small RNAs (about 60 to 80 nt) were detected from the exon-overlapping regions of SAT loci. In addition, further analyses showed marked alteration of sense-antisense expression balance throughout spermatogenesis in testis. These results suggest that conserved SAT loci are rich in potential regulatory roles that will help us understand this new class of transcripts underlying the mammalian genome.
    Human Molecular Genetics 07/2008; 17(11):1631-40. DOI:10.1093/hmg/ddn051 · 6.68 Impact Factor
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    ABSTRACT: Recently, it has been reported that antisense RNAs are transcribed from a large number of genes in various species including human and mouse. The Prdx2 gene, which is indicated to be involved in signal transduction related to platelet-derived growth factor as well as to protection from oxidizing agents, has been shown to produce sense and antisense transcripts. To obtain clues for possible roles of Prdx2 antisense transcripts, we have performed Northern blot analysis and in situ hybridization on tissues of 8-week-old C57BL/6J mice. The Northern blot analysis revealed that major parts of sense and antisense transcripts were poly(A-)-RNA. The analysis of the fractionated RNA of fibroblasts indicated that the poly(A-)-RNA would be localized in the cytoplasm of cells. The in situ hybridization demonstrated that the sense and antisense transcripts were localized in almost the same limited areas of brain, testis, and spleen. It also revealed that the sense and antisense transcripts coexisted in Purkinje cells. In thymus and stomach, the antisense transcripts were detected, but sense transcripts were not. When tissues of BALB/c mice were examined by in situ hybridization, the observations were essentially the same as those of C57BL/6J except that it appeared that the amounts of sense and antisense transcripts in testis of BALB/c were greater than those in C57BL/6J, and that the amounts of antisense transcripts in stomach of BALB/c were much smaller than those in C57BL/6J.
    Cytogenetic and Genome Research 02/2008; 121(3-4):222-31. DOI:10.1159/000138889 · 1.84 Impact Factor
  • Hidenori Kiyosawa
    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 06/2007; 52(5):441-8.
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    ABSTRACT: Recent large-scale transcriptomic analyses have identified numerous endogenously encoded cis-antisense RNAs that are thought to play important roles in diverse cellular processes although comprehensive comparative studies among multiple species have yet to be performed. To investigate conserved genomic features across various species that may be related to sense-antisense regulation, we performed comparative analysis of approximately 1000-2000 cis-encoded antisense RNA pairs from five model eukaryotes (Homo sapiens, Mus musculus, Drosophila melanogaster, Arabidopsis thaliana, and Oryza sativa). Analysis of overlapping patterns relative to the exon-intron structure revealed that the number of pairs sharing the 3' part of the transcripts was larger than that of the 5'-sharing pairs except in rice. Moreover, most of the well-conserved sense-antisense pairs between human and mouse exhibited 3'-overlaps, suggesting that regulatory mechanisms involving these regions may be important in sense-antisense transcription. Functional classification using Gene Ontology revealed that genes related to catalytic activity, nucleotide binding, DNA metabolism, and mitochondria were preferentially distributed within the set of exon-overlapping sense-antisense genes compared to the non-exon-overlapping group in animals. Despite the numerous sense-antisense pairs identified in human and mouse individually, the number of conserved pairs was extremely small (6.6% of the entire set). Whereas both genes of most of the conserved sense-antisense pairs had protein-coding potential, nearly half of the non-conserved pairs included a non-coding RNA, suggesting that non-coding sense-antisense RNAs may function in species-specific regulatory pathways.
    Gene 06/2007; 392(1-2):134-41. DOI:10.1016/j.gene.2006.12.005 · 2.08 Impact Factor
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    ABSTRACT: Genome-wide in silico analysis identified thousands of natural sense-antisense transcript (SAT) pairs in the mouse transcriptome. We investigated their expression using strand-specific oligo-microarray that distinguishes expression of sense and antisense RNA from 1947 SAT pairs. The majority of the predicted SATs are expressed at various steady-state levels in various tissues, and cluster analysis of the array data demonstrated that the ratio of sense and antisense expression for some of the SATs fluctuated markedly among these tissues, while the rest was unchanged. Surprisingly, further analyses indicated that vast amounts of multiple-sized transcripts are expressed from the SAT loci, which tended to be poly(A) negative, and nuclear localized. The tendency that the SATs are often not polyadenylated is conserved, even in the randomly chosen SAT genes in the plant Arabidopsis thaliana. Such common characteristics imply general roles of the SATs in regulation of gene expression.
    Genome Research 05/2005; 15(4):463-74. DOI:10.1101/gr.3155905 · 13.85 Impact Factor
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    ABSTRACT: Recent large-scale genome projects afford a unique opportunity to identify many novel disease genes and thereby better understand the genetic basis of human disease. Functional Annotation of Mouse (FANTOM) 2, the largest mouse transcriptome project yet, provides a wealth of data on novel genes, splice variants and non-coding RNA, and provides a unique opportunity to identify novel human disease genes. To demonstrate the power of combining the FANTOM 2 cDNA dataset with a positional candidate approach and bioinformatics analysis to identify genes underlying human genetic disease. By mapping all FANTOM 2 cDNA to the human genome, we were able to identify mouse clones that co-localised on the human genome with mapped but uncloned human disease loci. By this method we identified mouse and corresponding human genes mapping within the loci of 100 different human genetic diseases (mapped interval of <5 cM). Of particular interest was the elucidation through FANTOM 2 novel mouse gene data of candidate human genes for the following: (i) developmental -disorders: neural tube defect, Meckel syndrome, Wolf--Hirschhorn syndrome and keratosis follicularis spinulosa decalvans cum ophiasi; (ii) neurological disorders: benign familial infantile convulsions 3, early-onset cerebellar ataxia with retained tendon reflexes, infantile-onset spinocerebellar ataxia and vacuolar neuro-myopathy and (iii) cancer-related syndromes: tylosis with oesophageal cancer and low-grade B-cell chronic lymphatic leukaemia. The FANTOM 2 data will dramatically accelerate efforts to identify genes underlying human disease. It will also facilitate the creation of transgenic mouse models to help elucidate the function of potential human disease genes.
    Internal Medicine Journal 03/2004; 34(3):79-90. DOI:10.1111/j.1444-0903.2004.00581.x · 1.70 Impact Factor
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    ABSTRACT: We have used the FANTOM2 mouse cDNA set (60,770 clones), public mRNA data, and mouse genome sequence data to identify 2481 pairs of sense-antisense transcripts and 899 further pairs of nonantisense bidirectional transcription based upon genomic mapping. The analysis greatly expands the number of known examples of sense-antisense transcript and nonantisense bidirectional transcription pairs in mammals. The FANTOM2 cDNA set appears to contain substantially large numbers of noncoding transcripts suitable for antisense transcript analysis. The average proportion of loci encoding sense-antisense transcript and nonantisense bidirectional transcription pairs on autosomes was 15.1 and 5.4%, respectively. Those on the X chromosome were 6.3 and 4.2%, respectively. Sense-antisense transcript pairs, rather than nonantisense bidirectional transcription pairs, may be less prevalent on the X chromosome, possibly due to X chromosome inactivation. Sense and antisense transcripts tended to be isolated from the same libraries, where nonantisense bidirectional transcription pairs were not apparently coregulated. The existence of large numbers of natural antisense transcripts implies that the regulation of gene expression by antisense transcripts is more common that previously recognized. The viewer showing mapping patterns of sense-antisense transcript pairs and nonantisense bidirectional transcription pairs on the genome and other related statistical data is available on our Web site.
    Genome Research 07/2003; 13(6B):1324-34. DOI:10.1101/gr.982903 · 13.85 Impact Factor
  • Genome Research 06/2003; 13(6B). DOI:10.1101/gr.1462103 · 13.85 Impact Factor
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    ABSTRACT: Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.
    Nature 01/2003; 420(6915):563-73. DOI:10.1038/nature01266 · 42.35 Impact Factor
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    ABSTRACT: Non-insulin-dependent diabetes mellitus (NIDDM) is characterized by a breakdown of glucose homeostasis and is responsible for serious complications in various organs and vessels. Most of the genetic factors of NIDDM are yet unknown. Here, we identified two types of genetic factors that regulate homeostasis of blood glucose by measuring various pharmacokinetic parameters, some of which are used in the non-compartment analysis of drug metabolism in 340 F(2) progeny from the NIDDM model KK-A(y)/Ta Jcl mouse strain, and in non-diabetic PWK strain. We define "static glucokinesis" as the regulation of homeostasis that occurs during glucose deprivation, and "dynamic glucokinesis" as that during glucose stress; for instance, glucose tolerance test. Quantitative trait locus analysis revealed eight loci involved in the regulation of glucose homeostasis on chromosomes 7 ( Nidd1k), 2 ( Nidd2k), 1 ( Nidd3k, Nidd4k, and Nidd5k), 11 ( Nidd6k), 5 ( Nidd7k) (named Nidd1k through Nidd7k), and 4 ( Bwt1k). Nidd1k, Nidd4k, and Nidd7k were novel loci associated with NIDDM in mice. Nidd1k, Nidd2k, Nidd3k, and Nidd4k had linkage to factors characteristic of both static and dynamic glucokinesis. Nidd5k and Nidd6k showed linkage specific to markers of dynamic glucokinesis, and Nidd7k had linkage specific to static glucokinesis. Bwt1k was linked to obesity. Thus, the genetic factors for static glucokinesis and those for dynamic glucokinesis partially overlapped.
    Mammalian Genome 07/2002; 13(6):293-8. DOI:10.1007/s00335-001-2134-7 · 2.88 Impact Factor

Publication Stats

2k Citations
227.89 Total Impact Points

Institutions

  • 2005–2012
    • University of Tsukuba
      Tsukuba, Ibaraki, Japan
  • 2011
    • The University of Tokyo
      • Department of Animal Resource Sciences
      Edo, Tōkyō, Japan
  • 2002–2003
    • RIKEN
      • Biometal Science Laboratory
      Вако, Saitama, Japan