De Togni P, Fox H B, Morrissey S, Tansey L R, Levy S B, Babior B M
Department of Basic and Clinical Research, Research Institute of Scripps Clinic, La Jolla, CA 92037.
Blood. 1988 Feb;71(2):463-6.
The plasmid pUC18 contains a lacZ alpha-complementation gene that codes for a small peptide that can complement the delta M15 mutation of the Escherichia coli lacZ (beta-galactosidase) gene, converting bacteria carrying that mutated gene from the lacZ- to the lacZ+ phenotype. This plasmid was used in experiments designed to study mutagenesis by human neutrophils. E coli carrying pUC18 were incubated with neutrophils under conditions in which little ingestion of the bacteria took place; the plasmid was then isolated and transformed into an E coli strain (BOZO) that carries the lacZ delta M15 mutation. Of these transformants, 11 of 205,000 were lacZ, suggesting that in these 11, alpha-complementation had been lost through a mutation. No lac- colonies were detected among several hundred thousand BOZO transformed with plasmid isolated from incubations in which phagocytosis could take place, nor from incubations from which neutrophils were omitted. Despite the lac- phenotype of these 11 transformants, plasmids reisolated from nine of them showed normal alpha-complementing ability when transformed into fresh BOZO. These findings indicated that in these nine, the mutations were located in the chromosomes of the transformed BOZO. It thus appears that on exposure to activated neutrophils, a plasmid may acquire a lesion (? mutation) that can somehow be transferred to the genome of a recipient microorganism, resulting in repair of the damaged plasmid accompanied by mutation of the recipient's chromosome. Restriction mapping of the DNA from four of these nine chromosomal mutants suggested that the mutations did not represent major insertions or deletions in the portion of the bacterial chromosome corresponding to the pUC18 lac operon insert, nor in the remainder of the lacZ delta M15 gene. These results confirm previous work showing that exposure to activated neutrophils can induce mutations in biological systems, and provides an experimental model in which the mechanism of neutrophil-mediated mutagenesis may be examined.
质粒pUC18含有一个乳糖Zα-互补基因，该基因编码一种小肽，可互补大肠杆菌乳糖Z（β-半乳糖苷酶）基因的ΔM15突变，使携带该突变基因的细菌从乳糖Z-表型转变为乳糖Z+表型。该质粒用于旨在研究人类中性粒细胞诱变作用的实验。携带pUC18的大肠杆菌在几乎不发生细菌摄取的条件下与中性粒细胞一起孵育；然后分离质粒并将其转化到携带乳糖ZΔM15突变的大肠杆菌菌株（BOZO）中。在这些转化体中，205,000个中有11个是乳糖Z，这表明在这11个中，α-互补通过突变而丧失。在用可发生吞噬作用的孵育物中分离的质粒转化的几十万BOZO中，以及在省略中性粒细胞的孵育物中，均未检测到乳糖-菌落。尽管这11个转化体具有乳糖-表型，但从其中9个重新分离的质粒在转化到新鲜的BOZO中时显示出正常的α-互补能力。这些发现表明，在这9个中，突变位于转化的BOZO的染色体中。因此，似乎在暴露于活化的中性粒细胞时，质粒可能获得一个损伤（？突变），该损伤可以以某种方式转移到受体微生物的基因组中，导致受损质粒的修复并伴随受体染色体的突变。对这9个染色体突变体中的4个的DNA进行限制性图谱分析表明，这些突变在与pUC18乳糖操纵子插入片段相对应的细菌染色体部分中，以及在乳糖ZΔM15基因的其余部分中，均不代表主要的插入或缺失。这些结果证实了先前的工作，即暴露于活化的中性粒细胞可诱导生物系统中的突变，并提供了一个实验模型，可在其中研究中性粒细胞介导的诱变机制。
