Analysis of Large X-Ray-Induced Mutant Populations by Denaturing Gradient Gel Electrophoresis

To increase the precision by which predominant point mutations can be observed, hypoxanthine guanine phosphoribosyl transferase (HPRT)-deficient mutants selected en masse from large X-irradiated cultures of human lymphoblastoid cells (line TK6) were analyzed by denaturing gradient gel electrophoresis (DGGE). Four independent experiments yielded approximately$7\times 10^{3}$ and 3.2× 10³ initial surviving 6-thioguanine-resistant$(6\text{-}{\rm TG}^{{\rm r}})$ mutants in X-ray-treated and untreated cultures, respectively. The hprt exon 3 fragments were amplified from DNA extracted from these mixed$6\text{-}{\rm TG}^{{\rm r}}$ cell populations by employing the polymerase chain reaction using modified T7 DNA polymerase. DGGE was used to separate the mutant sequences from the wild-type as mutant/wild-type heteroduplexes. The X-irradiated populations contained several mutant bands in the 104-bp low-melting region of exon 3 that were not observed in the untreated cultures. Two exon 3 specific mutations were observed in more than one treated culture and various tests for potential biases suggested that these were radiation-specific mutational hotspots. These two recurring mutations were specific 1-bp deletions in either a run of four T:A's (bp 294-297) or a run of 3 A:T's (bp 247-249). Several other "sporadic" signals observed in X-irradiated cultures were caused by small deletions ranging from 2 to 25 bp in length.