| Cellular Translational Reporter Assay
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Author:
Date:
2014-03-20
[Abstract] The method described here allows measuring the effect of exogenously introduced modifications to in vitro-transcribed mRNA on the translation in cells. Using cells derived from knockout mice and control littermates, this method enables to compare the results in the presence or absence of specific gene products. In our lab, we used this protocol to check whether the exogenous addition of 5’ capping and 2’-O methylation to in vitro-mRNA affects the translational efficiency. Here we describe the details of our experiments.
[摘要] 本文所述的方法允许测量外部引入的修饰对体外转录的mRNA对细胞翻译的影响。 使用来自敲除小鼠和对照同窝出生的细胞，该方法能够比较存在或不存在特定基因产物的结果。 在我们的实验室中，我们使用该协议来检查外部添加5'帽化和2'-O甲基化在体外 -mRNA是否影响翻译效率。 这里我们描述我们的实验的细节。
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| Biochemical Assays for MTase Activity
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Author:
Date:
2014-01-20
[Abstract] Methyltransferase (MTase) transfers a methyl group (-CH3) from the donor S-adenosyl-L-methionine (AdoMet or SAM) to biologically active molecules such as hormones, neurotransmitters, lipids, proteins and nucleic acids. The addition of a methyl group causes a change in the physicochemical properties of the molecules. The mRNA cap structure is essential for cell and virus. Guanine-N7-methyltransferase (N7-MTase) methylates the GpppN cap at the N7 position of guanine, resulting in cap-0 structure (m7GpppN), and Ribose 2'-O-MTase further methylates the first nucleotide of higher eukaryotic ...
[摘要] 甲基转移酶（MTase）将甲基（-CH 3）从供体S-腺苷-L-甲硫氨酸（AdoMet或SAM）转移到生物活性分子例如激素，神经递质，脂质，蛋白质和核酸。 加入甲基引起分子的物理化学性质的改变。 mRNA帽结构对于细胞和病毒是必需的。 鸟嘌呤-N7-甲基转移酶（N7-MTase）甲基化在鸟嘌呤的N7位置的GpppN帽，导致cap-0结构（m7GpppN），核糖2'-O-MTase进一步甲基化高等真核细胞和病毒的第一个核苷酸 mRNA在核糖2'-OH位置形成cap-1（m7GpppNm）结构。 在这里，我们描述一种生物化学测定检测mRNA上限MTases的活动。
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