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

• Published in: Radiation research Vol. 135; no. 2; pp. 212 - 221
• Main Authors: Okinaka, Richard T., Anzick, Sarah L., Oller, Adriana, Thilly, William G.
• Format: Journal Article
• Language: English
• Published: Oak Brook, Il Academic Press, Inc 01-08-1993; Radiation Research Society
• Subjects: Base Sequence; Biological and medical sciences; Biological effects of radiation; Cell Line; Cell lines; Cultured cells; DNA; DNA, Single-Stranded; Electrophoresis, Polyacrylamide Gel - methods; Exons; Fundamental and applied biological sciences. Psychology; Gels; Gene Amplification; Genetic mutation; Humans; Hypoxanthine Phosphoribosyltransferase - genetics; Ionizing radiation; Ionizing radiations; Irradiation; Lymphocytes - radiation effects; Male; Molecular Sequence Data; Mutation; Polymerase Chain Reaction; Sequencing; Space life sciences; Tissues, organs and organisms biophysics; Ionizing irradiation; Cell line; Radiobiology; Gel electrophoresis; Animal; Denaturation; Radiation injury; Biophysics; Mutation; X ray irradiation
• ISSN: 0033-7587; 1938-5404
• DOI: 10.2307/3578297

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.