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Cloning of genes involved in double strand break rejoining

Objective


To examine the molecular events involved in the cellular defence pathways that counteract ionizing radiation induced damage, X-ray sensitive mutants have been isolated and characterized. Amongst 9 mutants, isolated in our laboratory from Chinese hamster cells, 3 complementation groups have been identified: 2 groups defective in deoxyribonucleic acid (DNA) double strand break (DSB) repair and one group of mutants which closely resemble Ataxia-telangiectasia (A-T) cells. A-T is a human progressive neuroimmunologic recessive disorder characterized by sensitivity to ionizing radiation and proneness to cancer, and about 97% of tested A-T families link the disease to chromosome 11. The AT-like hamster mutants, despite having similar A-T cellular characteristics, are not homologous to AT-A, AT-C nor AT-D, as they are not localized on human chromosome 11. The only slightly increased X-ray induced mutability in the AT-like hamster cell mutants, indicates that the lesions which are not repaired in these mutants affect mainly chromosomal stability.

Cloning of genes involved in double srand break (dsb) rejoining.
The xrs complementation group of hamster mutants are all highly sensitive to ionising radiation and to radiomimetic drugs, and they have a defect in double strand break rejoining. In this study we aim to use XR-V15B, a member of this complementatlon group of mutants, to clone a complementing human repair gene. This V79B derived cell line appears to be genetically stable in contrast to the tendency of other members of this
complementation group to revert. The cloning procedure will
involve co-transfection of human DNA with a dominant selectable marker. Radiation resistant transfectants will be analysed for the presence of common human sequences and attempts made to
rescue the repair gene from such sequences. As an aid to
cloning we will attempt to assign the chromosomal location of the xrs gene using the technique of microcell mediated
chromosome transfer. We also aim to identify further mutants defective in dsb rejoining. Any mutants obtained will be
characterised and, in particular, examined for potential use in cloning additional genes involved in dsb rejoining.

Coordinator

MRC Cell Mutation Unit
Address
University Of Sussex Falmer
BN1 9RR Brighton
United Kingdom

Participants (1)

Rijksuniversiteit Leiden
Netherlands
Address
72,Wassenaarseweg
2333 AL Leiden