Linney E, Donerly S
Cell. 1983 Dec;35(3 Pt 2):693-9. doi: 10.1016/0092-8674(83)90102-2.
Restrictions fragments encompassing the DNA sequence changes, which allow polyoma mutants to productively infect embryonal carcinoma (EC) cells, have been coupled to the Herpes simplex virus thymidine kinase (HSV TK) gene and to the bacterial chloramphenicol acetyl transferase (CAT) gene. F9 TK(-) EC cells have been transfected with the TK constructions and transformation to TK(+) colonies has been determined. F9 EC cells, retinoic-acid-induced differentiating F9 cultures, mouse fibroblasts, and mouse myoblasts have been transfected with the CAT constructions and CAT enzyme activity has been measured from the transfected cells. The results suggest that the mutant changes function at the level of gene-expression enhancement, the mutant constructions increasing the TK transformation frequency and also yielding a higher level of CAT enzyme activity when compared with constructions having no polyoma fragment or the analogous wild-type polyoma restriction fragment. The sequence specificity for enhancement changes upon differentiation of F9 EC cells, with the CAT enzyme levels from the wild-type construction approaching the values obtained from a PyEC mutant construction having a single base-pair difference.
包含DNA序列变化的限制性片段，这些变化使多瘤病毒突变体能够有效地感染胚胎癌细胞，已被连接到单纯疱疹病毒胸苷激酶（HSV TK）基因和细菌氯霉素乙酰转移酶（CAT）基因上。F9 TK(-)胚胎癌细胞已用TK构建体进行转染，并确定了向TK(+)菌落的转化情况。F9胚胎癌细胞、视黄酸诱导分化的F9培养物、小鼠成纤维细胞和小鼠成肌细胞已用CAT构建体进行转染，并从转染细胞中测量了CAT酶活性。结果表明，突变变化在基因表达增强水平起作用，与没有多瘤病毒片段或类似野生型多瘤病毒限制性片段的构建体相比，突变构建体增加了TK转化频率，并且还产生了更高水平的CAT酶活性。F9胚胎癌细胞分化时增强变化的序列特异性，野生型构建体的CAT酶水平接近从具有单个碱基对差异的PyEC突变构建体获得的值。
