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

使用人HPRT（次黄嘌呤磷酸核糖转移酶）的互补DNA作为检测突变的靶DNA。将该cDNA插入到逆转录病毒穿梭载体pZIP-NeoSV（X）1中。构建了能表达HPRT和NEO的表达载体pZIP（X）HPRT（新霉素抗性）基因，通过以下三种方法转染到源自小鼠Balb/c 3 T3细胞的2 TGOR细胞中;（a）磷酸钙转染，（B）与辅助病毒DNA共转染（pMOV <psi^->，其在病毒RNA的包装信号中有缺陷）和（c）感染从通过方法（B）构建的细胞获得的缺陷病毒。通过含G418（新霉素衍生物）的HAT培养基选择转化的细胞系。其中，6个细胞系显示自发突变为<6TG^R>的低频率。6个细胞系中的3个显示出由甲磺酸乙酯处理诱导的突变性。通过与COS细胞的细胞融合，从转化细胞中回收整合到宿主细胞染色体中的载体DNA。它们中的许多表现出缺失和重排，并且很少被恢复为完整的载体。然而，通过方法（c）获得的细胞系vHPRT-12显示了完整载体DNA的有效回收。用合成的引物和双脱氧法对HPRTcDNA进行碱基序列分析。HPRTcDNA编码区654个碱基对与文献报道的HPRTcDNA碱基序列一致。因此，细胞系vHPRT-12似乎适用于本项目。获得自发和X射线诱导的<6TG^R>突变体，并从这些突变体回收完整的载体DNA用于碱基序列分析。

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.