REGIONAL GENE-MAPPING USING MIXED RADIATION HYBRIDS AND REVERSE CHROMOSOME PAINTING

We describe a new approach for low-resolution physical mapping using p ooled DNA probe from mixed (non-clonal) populations of human-CHO cell hybrids and reverse chromosome painting. This mapping method is based on a process in which the human chromosome fragments bearing a complem enting gene were selectively retained in a large non-clonal population of CHO-human hybrid cells during a series of 12- to 15-Gy gamma irrad iations each followed by continuous growth selection. The location of the gene could then be identified by reverse chromosome painting on no rmal human metaphase spreads using biotinylated DNA from this populati on of ''enriched'' hybrid cells. We tested the validity of this method by correctly mapping the complementing human HPRT gene, whose locatio n is well established. We then demonstrated the method's usefulness by mapping the chromosome location of a human gene which complemented th e defect responsible for the hypersensitivity to ionizing radiation in CHO irs-20 cells. This method represents an efficient alternative to conventional concordance analysis in somatic cell hybrids where detail ed chromosome analysis of numerous hybrid clones is necessary. Using t his approach, it is possible to localize a gene for which there is no prior sequence or linkage information to a subchromosomal region, thus facilitating association with known mapping landmarks (e.g. RFLP, YAC or STS contigs) for higher-resolution mapping. (C) 1997 by Radiation Research Society.