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GENETIC AND BIOCHEMICAL BASIS OF RADIATION SENSITIVITY IN CULTURED HUMAN AND OTHER MAMMALIAN CELLS

Objective

THE PRINCIPAL AIM OF THE FIRST PROJECT WILL BE THE ISOLATION AND CHARACTERIZATION OF THE HUMAN GENES CONTROLLING DNA REPAIR AND RADIOSENSITIVITY, WITH THE ULTIMATE AIM OF PROVIDING A COMPLETE MOLECULAR DESCRIPTION OF THE NATURE AND FUNCTION OF THESE GENES, AND THEREBY OBTAINING A FULLER UNDERSTANDING OF THE WAYS IN WHICH HUMAN CELLS RESPOND TO RADIATION.
The approach of this study has been to collect mutants represented in the human population, in patients with certain genetic disorders, and to characterise the nature of the molecular defect in these human mutants. Methods are also described which have increased sensitivity for detecting radiosensitivity at the cellular level and finally data are presented on the cloning of deoxyribonucleic acid (DNA) repair genes, making use of fission yeast as a stepping stone to cloning human DNA repair genes.
RADIATION SENSITIVE CELLS WILL BE CO-TRANSFECTED WITH DNA CONTAINING A CLONED SELECTABLE MARKER AND GENOMIC DNA FROM NORMAL CELLS, FOLLOWED BY SELECTION OF CELLS BOTH FOR THE MARKER AND FOR RADIATION-RESISTANCE. USING AN ATAXIA-TELANGIECTASIA CELL LINE AS THE RECIPIENT, ONE RADIATION RESISTANT DERIVATIVE HAS SO FAR BEEN ISOLATED AND CHARACTERISED. DNA FROM SUCH RESISTANT TRANSFECTANTS WILL BE CLONED INTO A BACTERIOPHAGE VECTOR TO PRODUCE A LIBRARY FROM WHICH IT SHOULD BE POSSIBLE TO IDENTIFY CLONES CONTAINING THE MARKER GENE, WHICH SHOULD BE CLOSELY LINKED TO THE GENE OF INTEREST.

WORK IN THIS LABORATORY OVER THE PAST TEN YEARS HAS PRINCIPALLY INVOLVED THE USE OF DIPLOID HUMAN FIBROBLASTS.UNFORTUNATELY BECAUSE OF THEIR LIMITED LIFESPAN IN CULTURE THESE CELLS ARE UNSUITABLE AS RECIPIENTS IN THE TRANSFECTION EXPERIMENTS OUTLINED ABOVE. SOME EFFORT WILL THEREFORE BE PUT INTO THE PRODUCTION OF IMMORTAL CELL LINES BOTH OF LYMPHOID AND FIBROBLAST ORIGIN.

APART FROM THE MOLECULAR CLONING WORK FURTHER CELL STRAINS FROM RADIATION-SENSITIVE INDIVIDUALS WILL BE ADDED TO THE LARGE COLLECTION OF FIBROBLAST STRAINS WHICH CONSTITUTED THE BASIS OF OUR PREVIOUS CONTRACT. AS THEY ARE FOUND SUCH STRAINS WILL BE CHARACTERISED.

WORK WILL CONTINUE ON CELLS HETEROZYGOUS FOR RADIOSENSITIVITY GENES, AND OTHERS WHICH APPEAR TO BE AT THE LOWER BOUNDARY OF THE NORMAL HUMAN RADIATION RESPONSE. INDIVIDUALS FROM THESE TWO CATEGORIES (WHICH PRESUMABLY OVERLAP) ARE CLEARLY NOT TOO UNCOMMON IN THE HUMAN POPULATION. CELLS FROM HETEROZYGOTES FOR ATAXIA-TELANGIECTASIA ARE, AS A GROUP, MORE RADIOSENSITIVE THAN NORMALS AND WORK TOWARDS A DIAGNOSTIC TEST FOR HETEROZYGOTES WILL CONTINUE. COLLABORATION WITH OTHER CONTRACTORS WILL ALSO BE DIRECTED TOWARDS THE COMPARISON OF DNA REPAIR GENES FROM DIFFERENT SPECIES. WHEREVER APPROPRIATE COLLABORATION WITH OTHER CONTRACTING LABORATORIES IN DEVELOPING AND PERFORMING COMPLEMENTATION ANALYSES AND GENE MAPPING STUDIES WILL BE PURSUED.

THE SECOND PROJECT IS CONCERNED WITH THE RELATION BETWEEN DNA REPAIR AND MUTAGENESIS IN HUMAN CELLS. STUDIES WILL CONTINUE ON THE MUTABILITY OF REPAIR-DEFICIENT CELLS, BUT USING MORE SOPHISTICATED MUTAGENESIS SYSTEMS INVOLVING RECOMBINANT DNA TECHNOLOGY. THESE ARE CURRENTLY UNDER DEVELOPMENT IN THIS LABORATORY. THEY INVOLVE (1) THE STABLE INTEGRATION OF BACTERIAL GENES INTO THE HUMAN CHROMOSOMES, FOLLOWED BY MUTAGENESIS, USING THESE GENES TO MEASURE THE MUTATION FREQUENCY. THE MUTATED GENES CAN THEN BE RECOVERED, REINTRODUCED INTO BACTERIA, AND THE NATURE OF THE MUTATION DETERMINED BY DNA SEQUENCING. (2) AN ALTERNATIVE APPROACH INVOLVES THE USE OF PLASMID SHUTTLE VECTORS WHICH CAN REPLICATE IN MAMMALIAN OR BACTERIAL CELLS. MUTATIONS PRODUCED IN THE MAMMALIAN CELL, CAN AGAIN BE ANALYSED IN BACTERIA. (3) SPECIFIC TYPES OF MUTATIONS (EG. AMBER OR FRAMESHIFTS) CAN BE CONSTRUCTED IN CLONED GENES BY SITE-SPECIFIC MUTAGENESIS. THESE MUTATED GENES CAN BE INTRODUCED INTO MAMMALIAN CELLS AND REVERSIONS MEASURED IN THE MAMMALIAN CELLS BY PROCEDURES (1) OR (2).

ATTENTION WILL BE GIVEN TO EVENTS SUCH AS TRANSPOSITION, SEQUENCE REARRANGEMENT AND GENE SWITCHING WHICH HAVE BEEN LITTLE STUDIED AFTER IRRADIATION. IT MAY ALSO PROVE FEASIBLE TO APPLY OUR EXPERTISE IN MUTAGENESIS TO AN INVESTIGATION OF THE INTERACTION BETWEEN IONIZING RADIATION AND OTHER MUTAGENS TO WHICH MAN MAY BE EXPOSED. RECOMBINANT DNA APPROACHES WILL FACILITATE THE STUDY OF THE RELATION BETWEEN SPECIFIC DNA LESIONS AND MUTAGENESIS AND THEY MAY ALSO YIELD VALUABLE INFORMATION ON THE IMPORTANCE OF UNTARGETED EVENTS.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Medical Research Council (MRC)
Address
20 Park Crescent
W1N 4AL London
United Kingdom