title: The use of integrating DNA vectors to analyse the molecular defects in ionising radiation-sensitive mutants of mammalian cells including ataxia telangiectasia.
abstract: Integrating DNA vectors, encoding selectable recombinant genes, were used to assess rejoining and recombination in wild-type mammalian cells and their ionising radiation-sensitive mutants. To provide a simple model of an important radiation-induced lesion - the DNA double-strand break - the vectors were cut with restriction endonucleases at specific single sites. If these breaks were made in the coding sequence of a selectable gene, the fidelity of the rejoin/recombination process could be measured by survival of vector-transformed cells in selective medium. Rejoining was assessed using vectors without internal homologies, while recombination was measured using pairs of fragments or deletion vectors carrying homologous regions. Initial experiments were made with vectors carrying a single selectable gene but, to overcome potential artefacts, 2-gene vectors were then constructed where one gene acts as a linked marker and (unbroken) control for the other (broken) gene. Available data are reviewed to show that, compared to their respective wild-type counterparts: (1) an ataxia telangiectasia (A-T) cell line and the hamster irs1 mutant show a consistent reduction in the fidelity of rejoining double-strand breaks (while the hamster mutants irs2, irs3, xrs series, and EM9 show wild-type fidelity); (2) the hamster EM9 mutant shows a reduction in ability to recombine homologous vector fragments (while the A-T line and probably the xrs mutants show show wild-type abilities); and (3) the xrs mutants show a reduction in overall transformation frequency with vector DNA, whether broken or not, while the other mutants tested show approximately wild-type frequencies. A critical account of the techniques and data is given, together with speculations on the molecular nature of the processes which are defective in these mutants, leading to radiosensitivity.
authors.affiliation: Cell and Molecular Biology, Division, MRC Radiobiology Unit, Didcot, Oxon, Great Britian.
authors.forename: J
authors.initials: J
authors.lastname: Thacker
mesh_terms: D001260:Ataxia Telangiectasia / Q000235:genetics*; D002460:Cell Line; D004260:DNA Repair; D004262:DNA Restriction Enzymes / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D009154:Mutation; D011839:Radiation, Ionizing; D011995:Recombination, Genetic; D013539:Simian virus 40 / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D004262:DNA Restriction Enzymes