In situ accessibility of Escherichia coli 23S rRNA to fluorescently labeled oligonucleotide probes
In situ accessibility of Escherichia coli 23S rRNA to fluorescently labeled oligonucleotide probes
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DOI：
10.1128/aem.67.2.961-968.2001
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发表时间：
2001-02-01
影响因子：
4.4
通讯作者：
Amann, R
中科院分区：
文献类型：
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作者：
Fuchs, BM;Syutsubo, K;Amann, R
除了低细胞核糖体含量和细胞壁的不渗透性之外，用rRNA靶向寡核苷酸进行荧光原位杂交失败的主要原因之一是由于核糖体的高级结构导致探针靶位点不可接近。Wallner，W，Beisker，I，Schwippl，W，Ludwig和R，Amann，Appl，Environ，Microbiol，64：4973-4982，1998），用一组184个CY 3标记的寡核苷酸探针详细研究了大肠杆菌DSM 30083（T）的23 S rRNA的可及性。用流式细胞术定量探针赋予的荧光。最亮的信号来自探针23 S-2018，与位置2018至2035互补，探针赋予的细胞荧光分布在六个任意设置的亮度等级中（I至VI类，分别为23 S-2018的亮度的100至81%、80至61%、60至41%、40至21%、20至6%和5至0%）如下：I类，3%; II类，21%; III类，35%; IV类，18%; V类，16%;和VI类，7%。对所选区域的精细分辨率分析证实了寡核苷酸探针对23 S RNA的可及性的急剧变化。这与16 S rRNA的情况相似。事实上，在寡核苷酸探针与16 S和23 S rRNA的杂交之间没有发现显著差异。有趣的是，获得了与探针偶联的荧光染料类型对原位可及性的影响的指示。将结果转化为E.大肠杆菌，这可能会外推到其他细菌。因此，它可能有助于更好地开发23 S rRNA用于未来细菌鉴定的高潜力，
One of the main causes of failure of fluorescence in situ hybridization with rRNA-targeted oligonucleotides, besides low cellular ribosome content and impermeability of cell walls, is the inaccessibility of probe target sites due to higher-order structure of the ribosome, Analogous to a study on the 16S rRNA (B, M, Fuchs, G. Wallner, W, Beisker, I, Schwippl, W, Ludwig, and R, Amann, Appl, Environ, Microbiol, 64:4973-4982, 1998), the accessibility of the 23S rRNA of Escherichia coli DSM 30083(T) was studied in detail with a set of 184 CY3-labeled oligonucleotide probes. The probe-conferred fluorescence was quantified flow cytometrically. The brightest signal resulted from probe 23S-2018, complementary to positions 2018 to 2035, The distribution of probe-conferred cell fluorescence in six arbitrarily set brightness classes (classes I to VI, 100 to 81%, 80 to 61%, 60 to 41%, 40 to 21%, 20 to 6%, and 5 to 0% of the brightness of 23S-2018, respectively) was as follows: class I, 3%; class II, 21%; class III, 35%; class IV, 18%; class V, 16%; and class VI, 7%, A fine-resolution analysis of Selected areas confirmed steep changes in accessibility on the 23S RNA to oligonucleotide probes. This is similar to the situation for the 16S rRNA, Indeed, no significant differences were found between the hybridization of oligonucleotide probes to 16S and 23S rRNA, Interestingly, indications were obtained of an effect of the type of fluorescent dye coupled to a probe on in situ accessibility. The results were translated into an accessibility map for the 23S rRNA of E. coli, which may be extrapolated to other bacteria. Thereby, it may contribute to a better exploitation of the high potential of the 23S rRNA for identification of bacteria in the future,