Nübel U, Engelen B, Felske A, Snaidr J, Wieshuber A, Amann R I, Ludwig W, Backhaus H
Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Biochemie und Pflanzenvirologie, Braunschweig, Germany.
J Bacteriol. 1996 Oct;178(19):5636-43. doi: 10.1128/jb.178.19.5636-5643.1996.
Sequence heterogeneities in 16S rRNA genes from individual strains of Paenibacillus polymyxa were detected by sequence-dependent separation of PCR products by temperature gradient gel electrophoresis (TGGE). A fragment of the 16S rRNA genes, comprising variable regions V6 to V8, was used as a target sequence for amplifications. PCR products from P. polymyxa (type strain) emerged as a well-defined pattern of bands in the gradient gel. Six plasmids with different inserts, individually demonstrating the migration characteristics of single bands of the pattern, were obtained by cloning the PCR products. Their sequences were analyzed as a representative sample of the total heterogeneity. An amount of 10 variant nucleotide positions in the fragment of 347 bp was observed, with all substitutions conserving the relevant secondary structures of the V6 and V8 regions in the RNA molecules. Hybridizations with specifically designed probes demonstrated different chromosomal locations of the respective rRNA genes. Amplifications of reverse-transcribed rRNA from ribosome preparations, as well as whole-cell hybridizations, revealed a predominant representation of particular sequences in ribosomes of exponentially growing laboratory cultures. Different strains of P. polymyxa showed not only remarkably differing patterns of PCR products in TGGE analysis but also discriminative whole-cell labeling with the designed oligonucleotide probes, indicating the different representation of individual sequences in active ribosomes. Our results demonstrate the usefulness of TGGE for the structural analysis of heterogeneous rRNA genes together with their expression, stress problems of the generation of meaningful data for 16S rRNA sequences and probe designs, and might have consequences for evolutionary concepts.
通过温度梯度凝胶电泳（TGGE）对PCR产物进行序列依赖性分离，检测了多粘芽孢杆菌各菌株16S rRNA基因中的序列异质性。包含可变区V6至V8的16S rRNA基因片段用作扩增的靶序列。来自多粘芽孢杆菌（模式菌株）的PCR产物在梯度凝胶中呈现出清晰的条带模式。通过克隆PCR产物获得了六个具有不同插入片段的质粒，每个质粒分别展示了该模式单一条带的迁移特征。对它们的序列进行了分析，作为总异质性的代表性样本。在347 bp的片段中观察到10个可变核苷酸位置，所有取代都保留了RNA分子中V6和V8区域的相关二级结构。用专门设计的探针进行杂交显示了各个rRNA基因的不同染色体位置。从核糖体制剂中逆转录rRNA的扩增以及全细胞杂交表明，在指数生长的实验室培养物的核糖体中特定序列占主导地位。不同的多粘芽孢杆菌菌株在TGGE分析中不仅显示出明显不同的PCR产物模式，而且在用设计的寡核苷酸探针进行全细胞标记时也有区别，这表明活性核糖体中各个序列的不同表现。我们的结果证明了TGGE在异质rRNA基因结构分析及其表达方面的有用性，强调了为16S rRNA序列和探针设计生成有意义数据的压力问题，并且可能对进化概念产生影响。
