Quinto I, Scala G, Mallardo M, Arcucci A, Ruocco M R, De Lorenzo F
Dipartimento di Biochimica e Biotecnologie Mediche, II Facoltà di Medicina e Chirurgia, Università degli Studi di Napoli, Italy.
Carcinogenesis. 1992 Mar;13(3):439-45. doi: 10.1093/carcin/13.3.439.
A promoterless neo gene was stably transfected in rodent fibroblasts to act as a reporter gene for rearrangements resulting in its expression at the different genomic integration sites. Nine clones were isolated which had integrated a varying copy number of neo at one or more genomic sites but still displayed a Neo- phenotype (G418-sensitive). These clones were analyzed for their ability to become Neo+ (G418-resistant) either spontaneously or after mutagen treatment. They were all able to generate G418-resistant subclones spontaneously at frequencies ranging from 2 x 10(-8) to 6 x 10(-5). The acquired G418-resistance was always associated with amplification and enhanced transcription of neo. No correlation was observed between the frequency of occurrence of G418-resistance and the number of copies or integration sites of neo. When treated with the mutagens mitomycin C or methylmethane sulfonate, only one clone, RH15, produced G418-resistant subclones in a dose-related fashion. In this mutagen-inducible clone, DNA lesions of a different nature (monoadducts or cross-links) were equally efficient in the induction of G418-resistance. Amplification and enhanced transcription of the neo gene were observed in both the spontaneous and mutagen-induced G418-resistant subclones of RH15 cell line. These findings indicate that the exogenous neo gene integrated at different genomic sites was acting as a reporter gene for amplification. Interestingly, while all nine integration sites were observed to amplify spontaneously, only one could be induced to amplify by mutagens. This suggests that different genomic regions display differing susceptibilities to mutagen-mediated amplification. This may be important in view of the major role played by mutagen-mediated gene amplification in carcinogenesis.
将无启动子的新霉素基因稳定转染至啮齿动物成纤维细胞中，作为一种报告基因，用于检测重排情况，该重排会导致其在不同基因组整合位点表达。分离出九个克隆，这些克隆在一个或多个基因组位点整合了不同拷贝数的新霉素基因，但仍表现出Neo-表型（对G418敏感）。分析这些克隆自发或经诱变处理后变为Neo+（对G418耐药）的能力。它们都能够自发产生对G418耐药的亚克隆，频率范围为2×10(-8)至6×10(-5)。获得的G418耐药性总是与新霉素基因的扩增和转录增强相关。未观察到G418耐药性的发生频率与新霉素基因的拷贝数或整合位点之间存在相关性。当用诱变剂丝裂霉素C或甲基磺酸甲酯处理时，只有一个克隆RH15以剂量相关的方式产生了对G418耐药的亚克隆。在这个诱变可诱导的克隆中，不同性质的DNA损伤（单加合物或交联）在诱导G418耐药性方面同样有效。在RH15细胞系的自发和诱变诱导的对G418耐药的亚克隆中均观察到新霉素基因的扩增和转录增强。这些发现表明，整合在不同基因组位点的外源新霉素基因作为扩增的报告基因。有趣的是，虽然观察到所有九个整合位点都能自发扩增，但只有一个能被诱变剂诱导扩增。这表明不同的基因组区域对诱变介导的扩增具有不同的敏感性。鉴于诱变介导的基因扩增在致癌过程中起主要作用，这可能很重要。
