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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

我们设计了一种名为chum-RNA的小RNA分子，使我们能够在不使用PCR扩增的情况下，经过四轮基于T7的线性扩增后制备单细胞cDNA文库。Chum-RNA仅以0.49飞图的mRNA （730 mRNA分子）为底物合成cDNA，这一数量相当于单个哺乳动物细胞中少量的mRNA分子。对该文库独立cDNA克隆（6.6×10^5 cfu）的分析表明，在每一轮扩增过程中都发生了30倍的RNA扩增。单细胞cDNA文库中mrna的大小分布和表达与百万细胞cDNA文库保持了相似性。使用chum-RNA也可能促进涉及其他DNA/RNA修饰酶的反应，这些酶的米切里斯常数（K_m）值约为1mM，允许它们仅在少量底物存在时被激活。为了证明逐步减法对基础研究的有用性，我们对通过逐步减法技术分离的新基因进行了功能分析。例如，Lats2（大肿瘤抑制因子2）激酶（Yabuta等人，Genomics, 2000； Fujii等人，EMBO Rep, 2002）及其关联伙伴（Lats1， Aurora A等）定位于中心体并调节细胞周期（Toji等人，Genes Cells, 2004）。miRNA-372和-373两种mirna通过抑制LATS2的表达，在睾丸生殖细胞肿瘤中作为潜在的新型致癌基因发挥作用，这表明LATS2是一种重要的肿瘤抑制因子（Voorhoeve等，cell, 2006）。由于哺乳动物Lats1和Lats2在物种间高度保守，我们也研究了裂变酵母S. pombe的Lats2同源基因（Sid2和Mug27）。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在G2/M细胞中快速选择性上调Lats2的表达。这种正反馈回路构成了一种新的检查点途径，在维持适当的染色体数目中起着关键作用（Aylon等人，Gene Dev., 2006）。我们还对cyclin Gs所起的作用进行了功能分析。CyG1和CyG2招募携带B'α亚基的蛋白磷酸酶2A （PP2A）到它的靶蛋白上。Mdm2)。我们发现，细胞周期蛋白Gs的关联伙伴GAK与PP2A B’α亚基结合并磷酸化，从而调控PP2A B’α活性。连接蛋白26（C×26）在癌细胞自发转移中的作用分析，以及C×26-targeting有效阻断自发转移的药物的开发。我们报道了PP2A B'α亚基的截断形式在高转移性和C×26-overexpressing BL6小鼠黑色素瘤细胞中也过表达。CyG1和CyG2招募携带B'α亚基的蛋白磷酸酶2A （PP2A）到其靶蛋白（如Mdm2）。我们发现，细胞周期蛋白Gs的结合伙伴GAK与PP2A B'α亚基结合并磷酸化，从而调节PP2A B'α的活性。分析连接蛋白26（Cx26）在癌细胞自发转移中的作用，并开发靶向Cx26的有效阻断自发转移的药物。我们报道了PP2A B'α亚基的截断在高转移性和过表达cx26的BL6小鼠黑色素瘤细胞中也过表达。我们还对分裂酵母S. pombe的减数分裂特异性基因进行了功能分析，这有助于我们更好地理解减数分裂和有丝分裂细胞周期背后的分子机制。少