Development and application of nano-subtraction technique

我们设计了一种称为chum-RNA的小RNA分子，使我们能够在四轮基于T7的线性扩增后制备单细胞cDNA文库，而不使用PCR扩增。Chum-RNA仅以0.49毫微克mRNA（730个mRNA分子）为底物驱动cDNA合成，该数量对应于单个哺乳动物细胞中的少量mRNA分子。对该文库的独立cDNA克隆（6.6 × 10^5 cfu）的分析表明，在每轮扩增过程中发生了30倍的RNA扩增。在所得到的单细胞cDNA文库中的mRNA的大小分布和代表性保留其相似性的百万细胞cDNA文库。chum-RNA的使用也可能促进涉及其他DNA/RNA修饰酶的反应，这些酶的米氏常数（K_m）值约为1mM，允许它们在仅少量底物存在下被激活。 ...更多信息 研究中，我们已经进行了功能分析的新基因分离的逐步减法技术。例如，Lats2（大肿瘤抑制因子2）激酶（Yabuta，et.例如，Genomics，2000; Fujii，et.例如，EMBO代表，2002年）及其协会合作伙伴（Lats1，Aurora A等），定位于中心体并调节细胞周期（Toji，et.例如，Genes Cells，2004）。两种miRNA，miRNA-372和-373，通过抑制LATS 2表达在睾丸生殖细胞肿瘤中作为潜在的新癌基因起作用，这表明LATS 2是一种重要的肿瘤抑制因子（Voorhoeve，et.例如，Cell，2006）。由于哺乳动物Lats1和Lats2在物种间高度保守，我们还研究了裂殖酵母S. pombe（Sid2和Mug27）。特别令人感兴趣的是，Lats2结合Mdm 2，从而抑制其E3连接酶活性并激活p53，而p53快速并选择性地上调G2/M细胞中的Lats2表达。这种正反馈环构成了一种新的检查点途径，其在维持适当的染色体数目中起关键作用（Aylon，et.例如，吉恩·德夫，2006）。我们还对细胞周期蛋白Gs所起的作用进行了功能分析。CyG1和CyG2将携带B'α亚基的蛋白磷酸酶2A（PP2A）募集至其靶蛋白（例如Mdm 2）。我们发现GAK是细胞周期蛋白Gs的结合伙伴，它结合并磷酸化PP2A B 'α亚基，从而调节PP2A B' α活性。分析连接蛋白26（C × 26）在癌细胞自发转移中的作用，并开发有效阻断自发转移的C × 26靶向药物。我们报道了PP2A B 'α亚基的截短形式在高转移性和C × 26过表达的BL 6小鼠黑素瘤细胞中也过表达。CyG1和CyG2将携带B'α亚基的蛋白磷酸酶2A（PP2A）募集至其靶蛋白（例如Mdm 2）。我们发现GAK是cyclin Gs的结合伙伴，它结合并磷酸化PP2AB 'α亚基，从而调节PP2AB' α活性。分析连接蛋白26（Cx26）在癌细胞自发转移中的作用，并开发有效阻断自发转移的Cx26靶向药物。我们报道了PP2A B 'α亚基的截短形式在高转移性和Cx26过表达的BL 6小鼠黑素瘤细胞中也过表达。我们还对裂殖酵母S.粟酒裂殖酵母，这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。少

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