DNA-SEQUENCE DIVERGENCE AMONG DERIVATIVES OF ESCHERICHIA-COLI K-12 DETECTED BY ARBITRARY PRIMER PCR (RANDOM AMPLIFIED POLYMORPHIC DNA) FINGERPRINTING
DNA-SEQUENCE DIVERGENCE AMONG DERIVATIVES OF ESCHERICHIA-COLI K-12 DETECTED BY ARBITRARY PRIMER PCR (RANDOM AMPLIFIED POLYMORPHIC DNA) FINGERPRINTING
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DOI：
10.1128/jb.176.6.1673-1682.1994
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发表时间：
1994-03-01
影响因子：
3.2
通讯作者：
BERG, DE
中科院分区：
文献类型：
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作者：
BRIKUN, I;SUZIEDELIS, K;BERG, DE
已知祖先的大肠杆菌K-12的衍生物的特征在于随机扩增多态性DNA（RAPD）指纹图谱，以更好地了解这个家族的密切相关的菌株的基因组进化。这种敏感的方法需要在低严格条件下用任意引物进行PCR扩增，并产生菌株特异性的匿名DNA片段阵列。在150个片段中，有8个是多态的，因为它们是从一些菌株产生的，但不是所有菌株。其中7条为染色体多态性带，1条为F因子质粒多态性带。六个多态性染色体带中的五个来自7%的基因组，一个340 kb的片段，包括复制的末端。其中两个是从隐藏的Rac原噬菌体，不能扩增它们从一些菌株是由于删除（切除）或重排的Rac。另外两个导致多态性条带的末端区域片段似乎具有持续的点突变，影响了扩增它们的能力。对照实验表明，从340-kb的末端区域片段，也从两个质粒（P1和F）的RAPD带表示在其大小的近似比例。优化实验表明，耐热聚合酶的浓度对RAPD扩增产物的排列有很大影响。RAPD多态性的比较和菌株在系谱中的位置表明，这些历史性的E。大肠杆菌菌株在其储存期间。我们认为，这种突变在末端附近的聚集反映了染色体复制完成过程中的错误，可能是在通常用于储存E.在细菌遗传学的早期，
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 some 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 shelved 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.