Previous work has focussed on the analysis of mutagenic specificity at the natural adenine phosphoribosyltransferase (APRT) gene in Chinese Hamster ovary cells. These studies included the isolation of over 50 mutant genes and their subsequent DNA sequence determination. Although the determination of mutagenic specificity in mammalian cells appears feasible now, it still remains very laborious, requiring the construction of genomic libraries for each mutant gene. To facilitate the characterization of chromosomal mutations, an aprt cDNA gene (540 bp coding region) has been constructed and has been stably integrated in the genome of several mammalian cell lines using an infectious retrovirus shuttle vector. Cell lines carrying a single aprt provirus are useful for isolating mutations in the cDNA gene. A number of other sequences are present in the shuttle vector in order to allow rapid recovery of mutant sequences and subsequent DNA sequence analysis. These additional sequences include (1) the replication origin from Simian virus 40 (SV40 ori), which permits extensive in vivo amplification of the gene upon fusion of the mutant cells to cells with constitutive expression of the SV40 T-antigen; (2) the pBR322 plasmid replication ori, to propagate the amplified sequence in E. coli; (3) the tn5 neo gene, to select the recovered shuttle vector in E. coli; and (4) the bacteriophage M13 replication ori and packaging sequences, in order to produce transducing M13 particles carrying a single-stranded mutant gene, thus allowing rapid DNA sequencing. The aprt shuttle vector has been used to correct the APRT- phenotype of several mammalian cells, including human fibrosarcoma cells, human lymphoblasts, murine L cells.
