Bruce J L, Hubner R J, Cole E M, McDowell C I, Webster J A
Central Research and Development, E. I. du Pont de Nemours and Co., Wilmington, DE 19880, USA.
Proc Natl Acad Sci U S A. 1995 May 23;92(11):5229-33. doi: 10.1073/pnas.92.11.5229.
To classify Listeria monocytogenes using taxonomic characters derived from the rRNA operons and their flanking sequences, we studied a sample of 1346 strains within the taxon. DNA from each strain was digested with a restriction endonuclease, EcoRI. The fragments were separated by gel electrophoresis, immobilized on a membrane, and hybridized with a labeled rRNA operon from Escherichia coli. The pattern of bands, positions, and intensities of hybridized fragments were electronically captured. Software was used to normalize the band positions relative to standards, scale the signal intensity, and reduce the background so that each strain was reproducibly represented in a data base as a pattern. With these methods, L. monocytogenes was resolved into 50 pattern types differing in the length of at least one polymorphic fragment. Pattern types representing multiple strains were recorded as the mathematical average of the strain patterns. Pattern types were arranged by size polymorphisms of assigned rRNA regions into subsets, which revealed the branching genetic structure of the species. Subtracting the polymorphic variants of a specific assigned region from the pattern types and averaging the types within each subset resulted in reduced sets of conserved fragments that could be used to recognize strains of the species. Pattern types and reduced sets of conserved fragments were conserved among different strains of L. monocytogenes but were not observed in total among strains of other species.
为了利用源自rRNA操纵子及其侧翼序列的分类特征对单核细胞增生李斯特菌进行分类，我们研究了该分类单元内的1346株菌株样本。用限制性内切酶EcoRI消化每个菌株的DNA。片段通过凝胶电泳分离，固定在膜上，并与来自大肠杆菌的标记rRNA操纵子杂交。杂交片段的条带模式、位置和强度通过电子方式捕获。使用软件将条带位置相对于标准进行归一化，对信号强度进行缩放，并降低背景，以便每个菌株在数据库中以一种模式可重复地呈现。通过这些方法，单核细胞增生李斯特菌被分为50种模式类型，至少有一个多态性片段的长度不同。代表多个菌株的模式类型记录为菌株模式的数学平均值。模式类型根据指定rRNA区域的大小多态性排列成子集，揭示了该物种的分支遗传结构。从模式类型中减去特定指定区域的多态性变体，并对每个子集中的类型进行平均，得到可用于识别该物种菌株的保守片段的简化集合。模式类型和保守片段的简化集合在单核细胞增生李斯特菌的不同菌株中是保守的，但在其他物种的菌株中未全部观察到。
