Shuttle vector system for the analysis of specificity in radiation-induced mutation

利用人次黄嘌呤磷酸核糖基转移酶（HPRT）的互补DNA作为检测突变的靶DNA。将该cDNA插入由逆转录病毒衍生的穿梭载体pZIP-NeoSV(X)1中。将构建的表达HPRT和NEO（新霉素耐药）基因的载体pZIP(X)HPRT，通过以下三种方法转染小鼠Balb/c 3T3细胞衍生的2TGOR细胞；(a)磷酸钙转染，(b)辅助病毒DNA （pMOV <psi^->，缺乏病毒RNA的包装信号）共转染，(c)从方法(b)构建的细胞中获得缺陷病毒感染。用含G418（新霉素衍生物）的HAT培养基筛选转化细胞系。其中6个细胞系自发性突变频率低至<6TG^R>。6株细胞系中有3株表现出甲磺酸乙酯处理诱导的突变。通过与COS细胞的融合，从转化细胞中获得整合到宿主细胞染色体上的载体dna。其中许多显示了缺失和重排，很少恢复为完整的载体。然而，通过方法(c)获得的细胞系vHPRT-12显示了完整载体dna的有效恢复。这些完整的载体dna通过合成引物和双脱氧法对HPRTcDNA的碱基序列进行分析。HPRTcDNA编码区有654个碱基对与已有文献报道的HPRTcDNA碱基序列一致。因此，细胞系vHPRT-12似乎适合这个项目。获得了自发和x射线诱导的<6TG^R>突变体，并从这些突变体中恢复完整的载体dna进行碱基序列分析。

Complementary DNA of human HPRT (hypoxanthine phosphoribosyl transferase) was used as the target DNA for the detection of mutation. This cDNA was inserted into the shuttle vector, pZIP-NeoSV(X)1, which was derived from retrovirus. The vector constructed, pZIP(X)HPRT, which can express HPRT and NEO (neomycin-resistance) genes, was transfected into 2TGOR cells derived from mouse Balb/c 3T3 cell by the following three methods; (a) Ca-phosphate transfection, (b) co-transfection with helper virus DNA (pMOV <psi^-> which is deficient in packaging signal of viral RNA) and (c) infection of defective viruses obtained from the cells constructed by method (b). Transformed cell lines were selected by HAT medium with G418 (derivative of neomycin). Among those, 6 cell lines showed the low frequency of spontaneous mutation to <6TG^R> . Three out of the 6 cell lines showed the mutability induced by ethyl methanesulfonate treatment. Vector DNAs integrated into host cell chromosomes were recovered from transformed cells by the cell fusion with COS cells. Many of them showed deletions and rearrangements, and few were recovered as intact vectors. However, efficient recovery of the intact vector DNAs were shown by the cell line vHPRT-12 which was obtained by the method (c). These intact vector DNAs were used for the analysis of base sequence in HPRTcDNA by synthesized primers and dideoxy method. Six hundred and fifty four base pairs of coding region of HPRTcDNA were coincident with the base sequence of HPRTcDNA which was already reported in papers. Thus the cell line, vHPRT-12 is appeared to be siutable for this project. The spontaneous and X-ray induced <6TG^R> mutants were obtained and intact vector DNAs were recovered from these mutants for the analysis of base sequence.