Veigl M L, Donover S P, Anderson R D, Akst L, Sedwick C E, Sedwick W D
Department of Medicine, Case Western Reserve University, Ireland Cancer Center of University Hospitals, Cleveland, OH 44106, USA.
Environ Mol Mutagen. 1995;26(1):16-25. doi: 10.1002/em.2850260104.
Previous studies of doxorubicin-induced mutations employing F' lacl/lacO as an endogenous gene target have focused on properties of large deletions with 3' endpoints residing in the lacO region of the target gene. This study considers the influence of Lac repressor binding on the distribution of these deletions. Results of the DNA sequence level analysis of spontaneous and doxorubicin-induced i-d and lacO mutations in Escherichia coli uvrB- are reported for mutants isolated under conditions where Lac repression is relieved by isopropyl-beta-D-thiogalactopyranosid (IPTG; an inducer that prevents repressor binding to lacO). The location of deletions isolated from doxorubicin-treated cultures in the presence and absence of IPTG suggests that doxorubicin preferentially focuses deletion endpoints adjacent to its binding sites in lacO and that the distribution of these deletion endpoints is not modulated by Lac repressor binding. In contrast, spontaneous deletion endpoints are preferentially clustered in the loop away from the palindromic sequences under conditions of repression. However, when the Lac repressor/lacO binding complex is dissociated by IPTG, the spontaneous 3'-deletion endpoints distribute proportionally between the putative stem and loop of the lacO palindrome. The single most striking effect of IPTG induction of the Lac operon was elimination of a "hot spot" for T:A-->C:G transitions at position +6 in lacO. This base substitution "hot spot," which accounted for 17.6% of total doxorubicin-induced mutants and 16.4% of spontaneous mutants in repressed bacterial cultures, accounted for approximately 1% of total mutations in similar experiments carried out in the presence of IPTG. A large number of mutations at the +6 position are induced only by doxorubicin in the absence of IPTG, however, suggesting that both doxorubicin-induced and spontaneous mutation at this transition "hot spot" are mediated by Lac repressor binding to lacO.
以往利用F'lacl/lacO作为内源性基因靶点对阿霉素诱导突变进行的研究，主要集中在3'端位于靶基因lacO区域的大缺失的特性上。本研究考虑了Lac阻遏物结合对这些缺失分布的影响。报告了在异丙基-β-D-硫代半乳糖苷（IPTG；一种阻止阻遏物与lacO结合的诱导剂）解除Lac阻遏的条件下分离得到的大肠杆菌uvrB-中自发和阿霉素诱导的i-d和lacO突变的DNA序列水平分析结果。在有和没有IPTG的情况下，从阿霉素处理的培养物中分离出的缺失位置表明，阿霉素优先将缺失端点集中在其在lacO中的结合位点附近，并且这些缺失端点的分布不受Lac阻遏物结合的调节。相比之下，在阻遏条件下，自发缺失端点优先聚集在远离回文序列的环中。然而，当Lac阻遏物/lacO结合复合物被IPTG解离时，自发的3'-缺失端点在lacO回文的假定茎和环之间按比例分布。IPTG诱导Lac操纵子最显著的单一效应是消除了lacO中+6位T:A→C:G转换的“热点”。这种碱基替换“热点”在受抑制的细菌培养物中占阿霉素诱导突变总数的17.6%和自发突变总数的16.4%，在IPTG存在下进行的类似实验中约占总突变数的1%。然而，只有在没有IPTG的情况下，阿霉素才会在+6位诱导大量突变，这表明在这个转换“热点”处，阿霉素诱导的突变和自发突变都是由Lac阻遏物与lacO的结合介导的。
