Regional gene mapping using mixed radiation hybrids and reverse chromosome painting.
We describe a new approach for low-resolution physical mapping using pooled 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 complementing gene were selectively retained in a large non-clonal population of CHO-human hybrid cells during a series of 12- to 15-Gy gamma irradiations each followed by continuous growth selection. The location of the gene could then be identified by reverse chromosome painting on normal human metaphase spreads using biotinylated DNA from this population of "enriched" hybrid cells. We tested the validity of this method by correctly mapping the complementing human HPRT gene, whose location is well established. We then demonstrated the method's usefulness by mapping the chromosome location of a human gene which complemented the 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 detailed chromosome analysis of numerous hybrid clones is necessary. Using this 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.
Lin
J Y
JY
Department of Radiological Health Sciences, Colorado State University, Fort Collins 80523-1673, USA.
Bedford
J S
JS
D000818:Animals; D016466:CHO Cells; D002874:Chromosome Mapping / Q000379:methods*; D002875:Chromosomes / Q000528:radiation effects*; D006224:Cricetinae; D053938:DNA Fragmentation / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D005720:Gamma Rays; D005816:Genetic Complementation Test / Q000379:methods*; D006801:Humans; D006822:Hybrid Cells; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D012641:Selection, Genetic; D014965:X-Rays