Development and application of nano-subtraction technique

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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