| Trypanosomatid, fluorescence-based in vitro U-insertion/U-deletion RNA-editing (FIDE)
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Author:
Date:
2021-03-05
[Abstract] Gene expression within the mitochondria of African trypanosomes and other protozoan organisms relies on a nucleotide-specific RNA-editing reaction. In the process exclusively uridine (U)-nucleotides are site-specifically inserted into and deleted from sequence-deficient primary transcripts to convert them into translatable mRNAs. The reaction is catalyzed by a 0.8 MDa multiprotein complex termed the editosome. Here we describe an improved in vitro test to quantitatively explore the catalytic activity of the editosome. The assay uses synthetic, fluorophore-derivatized oligoribonucleotides as editing substrates, which enable the automated electrophoretic separation of the reaction products by capillary electrophoresis (CE) coupled to laser-induced fluorescence (LIF) detection systems. The ...
[摘要] [摘要]非洲锥虫和其他原生动物生物线粒体内的基因表达依赖于核苷酸特异性的RNA编辑反应。在该过程中,仅将尿苷(U)-核苷酸位点特异性插入序列不足的初级转录物中,并从中缺失,以将其转化为可翻译的mRNA。该反应由0.8 MDa的多蛋白复合物催化,该复合物被称为编辑体。在这里我们描述了一种改进的体外试验,以定量探索Editosome的催化活性。该测定使用合成的,荧光团衍生的寡核糖核苷酸 作为编辑底物,可通过耦合到激光诱导荧光(LIF)检测系统的毛细管电泳(CE)自动分离反应产物。该测定法功能强大,只需要纳克级的材料,并且通过使用多毛细管CE / LIF仪器,可以高度平行的方式进行测定。进一步的改进包括使用硫代磷酸酯修饰的,因此具有RNase耐性的底物RNA,以及用于同时监测U插入和U缺失反应的多重型荧光团标记策略。该测定方法对于研究酶体的机理和酶学是有用的。ħ H但是,它也可以在高通量执行以筛选RNA编辑特异性抑制剂。
图形摘要:
基于荧光的体外U插入/ U缺失RNA编辑(FIDE)分析的特征
[背景]中的RNA编辑反应动质体原生动物如非洲锥虫和利什曼原虫表示一个信使的最显着的转录后修饰(米)的RNA(综述Göringer ...
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| Antimicrobial Sensitivity Assay for Bdellovibrio bacteriovorus
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Author:
Date:
2020-12-20
[Abstract] Bdellovibrio bacteriovorus, an obligate predatory bacterium [i.e., bacteria that kill and feed on other bacteria (prey)], has the potential to be used as a probiotic for the disinfection of surfaces or for the treatment of bacterial infections. One option is to use this organism in combination with antimicrobials to potentiate the effectiveness of treatments. In order to make this approach feasible more has to be known about the ability of B. bacteriovorus to resist antibiotics itself. Standard assays to determine the minimum inhibitory concentration (MIC) are not suitable for B. bacteriovorus, since the small size of this bacterium (0.25-0.35 by 0.5-2 μm) prevents scattering at OD600. Since these predatory bacteria require larger prey bacteria for growth (e.g., E. coli dimensions are ...
[摘要] [摘要]蛭弧菌弧菌,一种专性捕食细菌[即,细菌杀灭和进料的其它细菌(猎物)] ,有可能使被用作表面的消毒益生菌或细菌感染的治疗。一种选择是将这种生物体与抗菌素联合使用以增强治疗效果。为了使该方法可行,必须进一步了解细菌噬菌芽孢杆菌自身抵抗抗生素的能力。标准测定法来确定最小抑制浓度(MIC)是不适合B.弧菌,由于 的小尺寸该细菌(0.25-0.35由0.5-2的微米)防止在OD散射600 。由于这些细菌掠夺需要用于生长较大的捕食的细菌(例如,大肠杆菌(E.coli)尺寸为1由1-2微米),第这里描述的用于抗微生物敏感性测定方法E基础是OD的降低600期间引起的猎物裂解增长。以前关于掠食性细菌对抗菌素耐药性的研究采用的方法无法直接将抗菌素耐药性水平与其他细菌种类进行比较。在这里,我们描述了一种确定细菌对芽孢杆菌的抗药性的方法,该方法可与被测试的参照生物体尽可能接近相同的实验条件进行比较。简要地说,最低抑菌浓度(MIC)值的B.弧菌通过在存在和不存在不同浓度的抗微生物剂的混合捕食者/猎物培养物的600nm处测量的吸光度的减少来确定。值得注意的是,可以使用不同的条件,猎物细菌和/或抗菌剂来修改此方法以获得其他掠食性细菌的抗菌素MIC值。
[背景]噬菌蛭弧菌是一种革兰氏阴性细菌掠夺是“早”在其他革兰氏阴性细菌的物种,生长在他们的牺牲,并最终杀死它们。细菌噬菌芽孢杆菌以及其他掠食性细菌有潜力用作益生菌以消毒表面或治疗细菌感染(Tyson和Elizabeth ...
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