Development and application of nano-subtraction technique

纳米减影技术的发展及应用

基本信息

批准号：
15101006
负责人：
NOJIMA Hiroshi
金额：
$ 68.97万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (S)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2007
项目状态：
已结题

项目摘要

We have designed a small RNA molecule called chum-RNA that has enabled us to prepare a single-cell cDNA library after four rounds of T7 based linear amplification, without using PCR amplification. Chum-RNA drove cDNA synthesis from only 0.49 femtograms of mRNA (730 mRNA molecules) as a substrate, a quantity that corresponds to a minor population of mRNA molecules in a single mammalian cell. Analysis of the independent cDNA clone of this library (6.6×10^5 cfu) suggests that 30-fold RNA amplification occurred in each round of the amplification process. The size distribution and representation of mRNAs in the resulting one-cell cDNA library retained its similarity to that of the million-cell cDNA library. The use of chum-RNA might also facilitate reactions involving other DNA/RNA modifying enzymes whose Michaelis constant (K_m) values are around 1mM, allowing them to be activated in the presence of only small quantities of substrate.To show the usefulness of stepwise-subtraction for basic … More research, we have performed functional analysis of novel genes isolated by the stepwise-subtraction technique. For example, Lats2 (large tumor suppressor 2) kinase (Yabuta, et. al., Genomics, 2000; Fujii, et. al., EMBO Rep., 2002) and its association partners (Lats1, Aurora A etc.), localize at the centrosome and regulate the cell cycle(Toji, et. al., Genes Cells, 2004). Two miRNAs, miRNA-372 and -373, function as potential novel oncogenes in testicular germ cell tumors by inhibiting LATS2 expression, which suggests that Lats2 is an important tumor suppressor (Voorhoeve, et. al., Cell, 2006). Since mammalian Lats1 and Lats2 are highly conserved across species, we have also studied the Lats2 homologs of the fission yeast S. pombe (Sid2 and Mug27). Of particular interest is that Lats2 binds Mdm2, thereby inhibiting its E3 ligase activity and activating p53, while p53 rapidly and selectively upregulates Lats2 expression in G2/M cells. This positive feedback loop constitutes a novel checkpoint pathway that plays a critical role in the maintenance of proper chromosome numbers (Aylon, et. al., Gene Dev., 2006).We also performed functional analysis of the roles played by cyclin Gs. CyG1 and CyG2 recruit protein phosphatase 2A (PP2A) bearing the B'α subunit to its target proteins(e.g. Mdm2). We found that GAK, an association partner of cyclin Gs, associates and phosphorylates the PP2A B'α subunit, thus regulating PP2A B'α activity Analysis of the role connexin 26(C×26) plays in the spontaneous metastasis of cancer cells, and the development of C×26-targeting drugs that potently block spontaneous metastasis. We reported that a truncated form of the PP2A B'α subunit is also overexpressed in the highly metastatic and C×26-overexpressing BL6 mouse melanoma cells. CyG1 and CyG2 recruit protein phosphatase 2A (PP2A) bearing the B'α subunit to its target proteins (e.g. Mdm2). We found that GAK, an association partner of cyclin Gs, associates and phosphorylates the PP2A B'α subunit, thus regulating PP2A B'α activity. Analysis of the role connexin 26(Cx26) plays in the spontaneous metastasis of cancer cells, and the development of Cx26-targeting drugs that potently block spontaneous metastasis. We reported that a truncated from of the PP2A B'α subunit is also overexpressed in the highly metastatic and Cx26-overexpressing BL6 mouse melanoma cells. We also performed functional analyses of the meiosis-specific genes of fission yeast S. pombe, which help us to better understand the molecular mechanisms behind both the meiotic and mitotic cell cycles. Less

我们设计了一个称为Chum-RNA的小RNA分子，它使我们能够在四轮基于T7的线性扩增后准备一个单细胞cDNA库，而无需使用PCR扩增。 CHUM-RNA仅从0.49个mRNA（730个mRNA分子）作为底物中驱动cDNA合成，该量与单个哺乳动物细胞中的少量mRNA分子相对应。对该文库的独立cDNA克隆的分析（6.6×10^5 CFU）表明，在每个回合过程中，都会发生30倍RNA扩增。由此产生的单细胞cDNA文库中mRNA的大小分布和表示保留了与百万细胞cDNA库的相似性。 Chum-RNA的使用还可能促进涉及其他DNA/RNA修饰酶的反应，其Michaelis Constance（K_M）值约为1mm，从而使它们仅在仅少量底物的情况下激活。为了显示逐步获得的逐步研究的实用性……对基本的研究进行了更多的研究，我们已经对新的基因进行了隔离的分析，从而通过逐步进行了逐步分析。例如，LATS2（大型肿瘤2）激酶（Yabuta等人，Genomics，2000; Fujii等，等，Embo Rep。，2002）及其关联伴侣（Lats1，Aurora a等），位于中心体并调节细胞周期（Toji Cycle（Toji）（TOJI）（toji等）。通过抑制LATS2表达，两个miRNA MiRNA -372和-373在验证生殖细胞肿瘤中起潜在的新型癌基因，这表明LATS2是重要的肿瘤抑制因子（Voorhoeve等，等，Cell，Cell，2006）。由于哺乳动物LATS1和LATS2在各种物种之间都高度保守，因此我们还研究了裂变酵母菌S. pombe（SID2和MUG27）的LATS2同源物。特别令人感兴趣的是LATS2结合MDM2，从而抑制其E3连接酶活性并激活p53，而p53迅速，有选择地上调G2/M细胞中的LATS2表达。这种积极的反馈回路构成了一种新颖的检查点途径，在维持适当的染色体数字中起着至关重要的作用（Aylon等人，Gene Dev。，2006）。我们还对Cyclin Gs起的作用进行了功能分析。 CYG1和CYG2募集蛋白质磷酸酶2a（PP2A），其B'α亚基与其靶蛋白（例如MDM2）。我们发现，Cyclin GS的关联合作伙伴GAK会促成PP2AB'α亚基，因此对Connexin 26（C×26）作用的PP2AB'α活性分析进行了调节，在癌细胞的赞助转移中发挥了C×26范围的药物的发展。我们报告说，在高度转移和C×26过表达的BL6小鼠黑色素瘤细胞中，PP2AB'α亚基的截短形式也过表达。 CYG1和CYG2募集蛋白质磷酸酶2a（PP2A），其B'α亚基与其靶蛋白（例如MDM2）。我们发现Cyclin GS的关联合作伙伴GAK会促成PP2AB'α亚基，从而控制了PP2AB'α活性。对癌细胞的发起人转移以及CX26靶向药物的发展，对连接蛋白26（CX26）作用的分析均在潜在地阻断发起人的转移。我们报告说，在高度转移和CX26过表达的BL6小鼠黑色素瘤细胞中，从PP2AB'α亚基的截短也过表达。我们还对裂变酵母菌的减数分裂特异性基因进行了功能分析，该基因有助于我们更好地了解减数分裂和有丝分裂细胞周期背后的分子机制。较少的