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<正>Ever since the first RNA nucleoside modification was characterized in 1957~[1],over 100 distinct chemical modifications have been identified in RNA to date~[2].Most of these modifications were characterized in non-coding RNAs(nc RNAs),including t RNA,r RNA,and small nuclear RNA(sn RNA)~[3].Studies in the past few decades have located various modifications in these nc RNAs and revealed their functional roles~[3].For instance,N1-methyladenosine(m~1A),which is typically 相似文献
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Artificial microRNA (amiRNA) technology is used for gene silencing in Arabidopsis. We describe a method for constructing amiRNA vectors that requires only one PCR and one ligation reaction. Vectors produced by this method are the same as those from the method of Schwab et al. (Plant Cell 2006, 18:1121-1133). Transgenic plants created by this method can therefore be tested in the same way or compared with existing transgenic material without the risk of alteration to the amiRNA skeleton. With optimized parameters, 36-42 % colonies had the insertion in the expected orientation and 85-95 % of these had the correct sequence. Using this method, a transient gene knock-down analysis in Arabidopsis could be completed in 4-5 days. 相似文献
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The human obesity susceptibility gene, FTO, encodes a protein that is homologous to the DNA repair AlkB protein. The AlkB family proteins utilize iron(II), alpha-ketoglutarate (alpha-KG) and dioxygen to perform oxidative repair of alkylated nucleobases in DNA and RNA. We demonstrate here the oxidative demethylation of 3-methylthymine (3-meT) in single-stranded DNA (ssDNA) and 3-methyluracil (3-meU) in single-stranded RNA (ssRNA) by recombinant human FTO protein in vitro. Both human and mouse FTO proteins preferentially repair 3-meT in ssDNA over other base lesions tested. They showed negligible activities against 3-meT in double-stranded DNA (dsDNA). In addition, these two proteins can catalyze the demethylation of 3-meU in ssRNA with a slightly higher efficiency over that of 3-meT in ssDNA, suggesting that methylated RNAs are the preferred substrates for FTO. 相似文献
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Histone acetylation plays important roles for the regulation of many fundamental cellular processes. Saccharomyces cerevisiae Rtt109 is an important class of histone acetyltransferases (HATs), which promote genome stability by directly acetylating newly synthesized histone H3 lysine 56 (H3-K56) through an unknown mechanism. Here, we report the crystal structures of Rtt109 at 2.2 A and Rtt109/Acetyl-CoA binary complex at 1.9 A. The structure displays a vise-like topology with mixed three-layered alpha/beta module forming the central module, whose core region resembles the structure of GCN5 HAT domain and P300/CBP HAT domain. Using structural and biochemical analyses, we have discovered the catalytic active site and have identified Asp288 as the deprotonation residue and Lys290 as the autoacetylation residue. We have further proposed the unique H3-K56 anchoring pocket and the potential H3alphaN binding groove. Our work has provided structural insights to understand the acetylation mechanism of H3-K56 by Rtt109. 相似文献
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Xuming Wang Yong Yang Chulang Yu Jie Zhou Ye Cheng Chengqi Yan Jianping Chen 《Molecular biotechnology》2010,46(3):211-218
Artificial microRNA (amiRNA) has become a powerful tool for gene silencing in plants. A new method for easy and rapid construction
of rice artificial miRNA vector is described. The procedure involved modification of the pCAMBIA1300-UR vector by insertion
of a ‘vector modification fragment’. This was prepared from the precursor of Os-amiR528 by eliminating the central miRNA-containing region while simultaneously creating an AfeI restriction site. The fragment was then introduced to the destination vector to produce a multipurpose ‘Highly Efficient
gene Silencing Compatible vector’ (HESC vector). AfeI was used to produce linearized HESC vectors, and a blunt end PCR product that included amiRNA sequence was cloned into this
site by a single ligation reaction to create the completed amiRNA vector. Tests showed that the method was highly efficient,
and greatly reduced the time needed for vector construction and resulted in a DNA sequence identical to that of the current
method, making it particularly suitable for use in a systems biology approach to functional genomic research. 相似文献