Biomedical subjects

Kenichi Matsubara

Publications and source records attributed to Kenichi Matsubara.

7 recordsLinked to original sources

PubMed2003

Collection, mapping, and annotation of over 28,000 cDNA clones from japonica rice.

We collected and completely sequenced 28,469 full-length complementary DNA clones from Oryza sativa L. ssp. japonica cv. Nipponbare. Through homology searches of publicly available sequence data, we assigned tentative protein functions to 21,596 clones (75.86%). Mapping of the cDNA clones to genomic DNA revealed that there are 19,000 to 20,500 transcription units in the rice genome. Protein informatics analysis against the InterPro database revealed the existence of proteins presented in rice but not in Arabidopsis. Sixty-four percent of our cDNAs are homologous to Arabidopsis proteins.

Alternative Splicing↗

PubMed2003

Impairment of the DNA binding activity of the TATA-binding protein renders the transcriptional function of Rvb2p/Tih2p, the yeast RuvB-like protein, essential for cell growth.

In Saccharomyces cerevisiae, two highly conserved proteins, Rvb1p/Tih1p and Rvb2p/Tih2p, have been demonstrated to be major components of the chromatin-remodeling INO80 complex. The mammalian orthologues of these two proteins have been shown to physically associate with the TATA-binding protein (TBP) in vitro but not clearly in vivo. Here we show that yeast proteins interact with TBP under both conditions. To assess the functional importance of these interactions, we examined the effect of mutating both TIH2/RVB2 and SPT15, which encodes TBP, on yeast cell growth. Intriguingly, only those spt15 mutations that affected the ability of TBP to bind to the TATA box caused synthetic growth defects in a tih2-ts160 background. This suggests that Tih2p might be important in recruiting TBP to the promoter. A DNA microarray technique was used to identify genes differentially expressed in the tih2-ts160 strain grown at the restrictive temperature. Only 34 genes were significantly and reproducibly affected; some up-regulated and others down-regulated. We compared the transcription of several of these Tih2p target genes in both wild type and various mutant backgrounds. We found that the transcription of some genes depends on functions possessed by both Tih2p and TBP and that these functions are substantially impaired in the spt15/tih2-ts160 double mutants that confer synthetic growth defects.

Adenosine Triphosphatases↗

PubMed2003

DNA alterations during multi-step development of human hepatocellular carcinomas revealed by laser capture microdissection.

In order to clarify early molecular events involved in liver carcinogenesis, we analyzed 53 liver-cirrhosis nodules (LCNs) from five patients and 13 micro-hepatocellular carcinoma (HCC) nodules from one patient and looked for alterations of microsatellites in genomic DNA after carefully preparing the tissue samples by laser-capture microdissection (LCM). Allelotyping was done with 20 markers corresponding to anonymous microsatellites and 13 corresponding to tumor suppressor genes (TSGs) that had shown significant alterations in HCCs. We detected both loss of heterozygosity (LOH) and microsatellite shifts (MS). Overall, 24 of 53 (47%) of LCNs showed LOH with any of the informative markers used in the study, reflecting that proportion of LCNs with clonal growth. The fractional allelic loss (FAL) index, an indication of total genomic complexity, was not significantly different between LCN and micro-HCC nodules, but their profiles of alteration were different. These profiles were classified into three groups: (1) LCN profile-allelic loss at chromosomal arms 1q and 14q, TBP and BRCA1; (2) HCC profile-LOH at 4q, 6q, 7q, 17p, NF1, IGFIIr and p53 in micro-HCC nodules; these changes in early lesions were identical to those seen in mature HCCs; (3) Common profile-LOH at NF1 and 6q, including IGFIIr, common to both LCN and HCC. No LCN showed LOH at p53 and Rb, loci that are generally altered in HCCs. However, 12 intra-tumoral nodules examined had lost p53 in all informative cases, although the loss of Rb was a late event. These results suggest that early genomic profiles confined to LCNs, and additional profiles that can be observed when liver tissue undergoes malignant transformation, support a model of multi-step development of HCC.

Journal Article↗

PubMed2002

Modulation of polyglutamine-induced cell death by genes identified by expression profiling.

The majority of triplet-repeat diseases are caused by mutated genes with an extended polyglutamine tract, exemplified by Huntington's disease (HD). In order to model HD pathogenesis in a controlled system, we developed stable PC12 cell lines that express exon 1 fragments of the huntingtin gene with 23 or 74 polyglutamines driven by an inducible doxycycline (dox)-sensitive promoter (HD-23Q or HD-74Q). We aimed to identify early perturbations induced by the mutation by studying expression levels of 1824 genes at 0, 5, 10 and 18 hours after induction, using adaptor-tagged competitive PCR (ATAC-PCR). At these time points, the cells show no appreciable death or mitochondrial impairment and very low inclusion levels. A total of 126 genes, including 69 known genes, exhibited statistically significant alterations in the HD-74Q cell lines but no changes in the HD-23Q lines. We tested 11 of these genes for their abilities to modulate polyglutamine-induced cell death in transiently transfected cell models. Five genes [glucose transporter 1 (Glut1), phosphofructokinase muscle isozyme (Pfkm), prostate glutathione-S -transferase 2 (Gstm2), RNA-binding motif protein 3 (Rbm3) and KRAB-A interacting protein 1 (Krip-1)] significantly suppressed cell death in both neuronal precursor and non-neuronal cell lines, suggesting that these transcriptional changes were relevant to polyglutamine pathology. The efficient recovery of functionally relevant genes supports the utility of gene expression profiling for discovering pathways related to pathogenesis, and the importance of analyzing molecular events in the early stages of disease.

Animals↗

PubMed2002

Comparison of gene expression profiling during postnatal development of mouse dentate gyrus and cerebellum.

Both the dentate gyrus of the hippocampus and the cerebellar cortex consist mainly of granule cells and develop postnatally. The granule cells in both tissues are presumed to be similar. Changes in gene expression were analyzed during the postnatal development of the dentate gyrus. Altogether, expression patterns of 1,937 genes were determined by adaptor-tagged competitive PCR. More than 90% of the genes belong to groups characterized by elevated expression either at earlier or later stages of development. A majority of the genes expressed showed marked changes during the developmental process, but there was little correlation between gene function and expression, unlike that observed during mouse postnatal cerebellar development. Despite anatomical and physiological similarities between these two processes, the gene expression profiles are completely different.

Animals↗

PubMed2002

Molecular classification of primary breast tumors possessing distinct prognostic properties.

The natural progression of breast cancer differs greatly between patients; the precise prediction of this disease course will improve the efficacy of therapeutics. Gene expression profiling may elucidate the undiscovered biological variations between seemingly similar cancers, leading to a new cancer classification system valuable in accurate diagnosis. The expression levels of 2412 genes, derived from 98 cancer samples, were precisely recorded by a high throughput RT-PCR technique, adapter-tagged competitive PCR. Subsequent cluster analysis revealed a molecular profile, correlating with estrogen receptor levels and the presence of lymph node metastases. We analyzed 301 cancer samples for the expression patterns of 21 genes critical in this categorization. The classification of the samples into three major groups was verified utilizing principal component analysis. This molecular classification system correlated significantly with early recurrence, independent of lymph node status. This malignant potential is associated with the expression levels of a group of genes, which comprise a set of candidates potentially useful in diagnostic prediction. These genes and the associated control mechanisms may also be effective therapeutic targets.

Breast Neoplasms↗

PubMed2002

A microarray-based method for detecting methylated loci.

CpG island DNA methylation plays an important role in regulating gene expression in development and carcinogenesis. We developed a new microarray-based method called methylation amplification DNA chip (MAD) for detecting differences in methylation. In this method, only methylated CpG islands from the two samples that we wanted to compare were amplified and used for hybridization. The resource material for the microarray was derived from the methylated DNA library of the sample in which we wanted to detect hypermethylation. Choosing the methylated DNA library as the resource material of the microarray increased the percentage of DNA fragments derived from hypermethylated loci on the microarray.

Animals↗