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首页> 外文期刊>Journal of bacteriology >DNA sequence divergence among derivatives of Escherichia coli K-12 detected by arbitrary primer PCR (random amplified polymorphic DNA) fingerprinting.
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DNA sequence divergence among derivatives of Escherichia coli K-12 detected by arbitrary primer PCR (random amplified polymorphic DNA) fingerprinting.

机译：通过任意引物PCR（随机扩增多态性DNA）指纹图谱检测到的大肠杆菌K-12衍生物之间的DNA序列差异。

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Derivatives of Escherichia coli K-12 of known ancestry were characterized by random amplified polymorphic DNA (RAPD) fingerprinting to better understand genome evolution in this family of closely related strains. This sensitive method entails PCR amplification with arbitrary primers at low stringency and yields arrays of anonymous DNA fragments that are strain specific. Among 150 fragments scored, eight were polymorphic in that they were produced from some but not all strains. Seven polymorphic bands were chromosomal, and one was from the F-factor plasmid. Five of the six mapped polymorphic chromosomal bands came from just 7% of the genome, a 340-kb segment that includes the terminus of replication. Two of these were from the cryptic Rac prophage, and the inability to amplify them from strains was attributable to deletion (excision) or to rearrangement of Rac. Two other terminus-region segments that resulted in polymorphic bands appeared to have sustained point mutations that affected the ability to amplify them. Control experiments showed that RAPD bands from the 340-kb terminus-region segment and also from two plasmids (P1 and F) were represented in approximate proportion to their size. Optimization experiments showed that the concentration of thermostable polymerase strongly affected the arrays of RAPD products obtained. Comparison of RAPD polymorphisms and positions of strains exhibiting them in the pedigree suggests that many sequence changes occurred in these historic E. coli strains during their storage. We propose that the clustering of such mutations near the terminus reflects errors during completion of chromosome replication, possibly during slow growth in the stab cultures that were often used to store E. coli strains in the early years of bacterial genetics.
机译：已知血统的大肠杆菌K-12衍生物通过随机扩增多态性DNA（RAPD）指纹图谱进行表征，以更好地了解这一密切相关菌株家族的基因组进化。这种灵敏的方法需要在低严格性下使用任意引物进行PCR扩增，并产生特定菌株的匿名DNA片段阵列。在所评分的150个片段中，有8个是多态性的，因为它们是由某些而非全部菌株产生的。 7条多态性条带是染色体，一条来自F因子质粒。六个映射的多态性染色体条带中的五个仅来自基因组的7％，这是一个340kb的片段，其中包括复制末端。其中两个来自神秘的Rac噬菌体，无法将它们从毒株中扩增出来的原因是Rac的缺失（切除）或重排。导致多态性条带的其他两个末端区域片段似乎具有持续的点突变，这影响了扩增它们的能力。对照实验表明，来自340-kb末端区域片段的RAPD条带以及来自两个质粒（P1和F）的RAPD条带的大小均与其比例成正比。优化实验表明，热稳定聚合酶的浓度强烈影响所获得的RAPD产物的排列。比较RAPD多态性和在谱系中表现出它们的菌株的位置表明，这些历史大肠杆菌菌株在储存过程中发生了许多序列变化。我们建议这种突变的聚集在末端附近反映了染色体复制完成过程中的错误，可能是在细菌遗传学的早期常用于存储大肠杆菌菌株的刺培养物中缓慢生长的过程中。

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