Kyushu University Academic Staff Educational and Research Activities Database
List of Reports
Fumihito Miura Last modified date:2022.06.02

Associate Professor / Bioregulation / Department of Basic Medicine / Faculty of Medical Sciences

1. Study on the elucidation of the mechanism for cisplatin-resistance in ovarian cancer using Comparative Genomic Hybridization and Laser Scanning Cytometry.
2. C. Uematsu, J. Nishida, K. Okano, F. Miura, T. Ito, Y. Sakaki, H. Kambara, Multiplex polymerase chain reaction (PCR) with color-tagged module-shuffling primers for comparing gene expression levels in various cells, Nucleic acids research, 10.1093/nar/29.16.e84, Vol.29, No.16, p.84, 2001.08, A method based on the multiplex polymerase chain reaction (PCR) and gel electrophoresis for the comparative analysis of gene expression levels was developed. Using the method many cDNA fragments from different sources can be compared simultaneously. Competitive PCR amplification of expressed genes from different sources was performed by using 'module-shuffling primers' (MPSs). The MPSs (labeled with different fluorophores) consist of sequence modules of 3 or 4 nt. The modules are arranged in different orders in each primer; therefore, the base sequences of the primers are different but their melting temperatures are identical. The genes expressed in different sources are ligated with tags complementary with the MPSs. Tag-ligated fragments are mixed in one tube and amplified at the same amplification efficiency by the MPSs. Amplified fragments are detected separately by multiple-color gel electrophoresis. This method can detect different amounts of each expressed gene, up to a difference in amounts of 30%, and its detection limit is 0.1 amol per assay..
3. Fumihito Miura, Tetsushi Yada, Kenta Nakai, Yoshiyuki Sakaki, Takashi Ito, Differential display analysis of mutants for the transcription factor Pdr1p regulating multidrug resistance in the budding yeast, FEBS Letters, 10.1016/S0014-5793(01)02792-2, Vol.505, No.1, pp.103-108, 2001.09, The transcription factor Pdr1p recognizes Pdr1p/Pdr3p-response element (PDRE) to activate genes involved in multidrug resistance of the budding yeast. To identify novel targets of Pdr1p, we compared transcriptomes among the yeast cells bearing wild, disrupted and gain-of-function alleles of PDR1 using a high-throughput fluorescent differential display PCR. Consequently, we identified 20 transcripts apparently regulated by Pdr1p, which are derived from well-known target genes as well as those that have never been described in the context of drug resistance. Intriguingly, among the latter, a previously unrecognized gene bearing a small putative open reading frame preceded by a functional PDRE was found. © 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies..
4. Yoichi Yamada, Hidemi Watanabe, Fumihito Miura, Hidenobu Soejima, Michiko Uchiyama, Tsuyoshi Iwasaka, Tsunehiro Mukai, Yoshiyuki Sakaki, Takashi Ito, A comprehensive analysis of allelic methylation status of CpG islands on human chromosome 21q, Genome Research, 10.1101/gr.1351604, Vol.14, No.2, pp.247-266, 2004.02, Approximately half of all human genes have CpG islands (CGIs) around their promoter regions. Although CGIs usually escape methylation, those on Chromosome X in females and those in the vicinity of imprinted genes are exceptions: They have both methylated and unmethylated alleles to display a "composite" pattern in methylation analysis. In addition, aberrant methylation of CGIs is known to often occur in cancer cells. Here we developed a simple Hpall-McrBC PCR method for discrimination of full, null, incomplete, and composite methylation patterns, and applied it to all computationally identified CGIs on human Chromosome 21q. This comprehensive analysis revealed that, although most CGIs (103 out of 149) escape methylation, a sizable fraction (31 out of 149) are fully methylated even in normal peripheral blood cells. Furthermore, we identified seven CGIs showing the composite methylation, and demonstrated that three of them are indeed methylated monoallelically. Further analyses using informative pedigrees revealed that two of the three are subject to maternal allele-specific methylation. Intriguingly, the other CGI is methylated in an allele-specific but parental-origin-independent manner. Thus, the cell seems to have a broader repertoire of methylating CGIs than previously thought, and our approach may contribute to uncover novel modes of allelic methylation..
5. 出芽酵母の系統的転写開始点解析:完全長cDNAライブラリシーケンシングと系統的RACE法によるアプローチ.
6. マウス始原生殖細胞におけるDNAメチローム解析
【目的】DNAメチル化は哺乳類生殖細胞分化の過程でダイナミックに変化するエピゲノム修飾の一つであり、各配偶子におけるゲノムインプリント確立・外来性リピート配列の抑制などの重要な役割を担う。また、生殖細胞の最終形態である精子・卵子ではまったく異なるメチル化パターンを示す。しかし、そのようなメチル化の性差が生じる各領域のメチル化・脱メチル化のターゲティング機構は明らかにされていない。我々は雌雄始原生殖細胞の包括的なDNAメチル化マップ(DNAメチローム)を作製し、生殖細胞形成過程におけるDNAメチロームの性差を詳細にプロファイリングした。【方法】胎齢10.5-16.5日齢Oct4-GFPマウス胚からFACS法によるソーティングによりマウス始原生殖細胞を雌雄それぞれ2000-4000細胞回収した。微量サンプルからの調整が可能となるPost-Bisulfite Adaptor Tagging(PBAT)法により、DNAメチローム解析用のDNAライブラリーを作製し、HiSeq2000(Illumina)を用いて高速シークエンスを行った。【結果】解析した胎齢を通して雌雄生殖細胞間の全体的なCpGメチル化の性差がみられた。胎齢10.5日から13.5日にかけて、X染色体上のCpGアイランドならびにインプリント制御領域(ICR)のメチル化の消去とともにゲノムワイドな脱メチル化がみられる一方、CpG-richな一部のレトロトランスポゾンにおいて高度なメチル化が残っていることが明らかとなった。また、16.5日齢において父方ICRのメチル化の上昇がみられるが、母方ICRの一部も、雌雄始原生殖細胞間のメチル化差異領域として同定することができた。本発表では生殖細胞におけるDNAメチル化の性差についてより詳細に解説し、機能的性差との関わりについて議論する。.
7. 体細胞クローンマウス精子のDNAメチレーションエラー.
8. 新井 恵吏, 三浦 史仁, 十時 泰, 山下 聡, 田 迎, 後藤 政広, 尾島 英知, 中村 浩実, 濱 奈津子, 鈴木 穣, 伊藤 隆司, 柴田 龍弘, 金井 弥栄, 国際ヒトエピゲノムコンソーシアム(IHEC)における日本人正常肝細胞の標準エピゲノムプロファイル解析, 日本癌学会総会記事, Vol.74回, pp.E-1157, 2015.10.
9. 体細胞クローンマウス精子のDNAメチル化異常の検出.