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 x 10(3) and 3.2 x 10(3) initial surviving 6-thioguanine-resistant (6-TGr) mutants in X-ray-treated and untreated cultures, respectively. The hprt exon 3 fragments were amplified from DNA extracted from these mixed 6-TGr 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.
Okinaka
R T
RT
Life Sciences Division, Los Alamos National Laboratory, New Mexico 87545.
Anzick
S L
SL
Oller
A
A
Thilly
W G
WG
D001483:Base Sequence; D002460:Cell Line; D004277:DNA, Single-Stranded; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005091:Exons; D005784:Gene Amplification; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008214:Lymphocytes / Q000528:radiation effects*; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction
D004277:DNA, Single-Stranded; D007041:Hypoxanthine Phosphoribosyltransferase