Radiation fusion hybrids for human chromosomes 3 and X generated at various irradiation doses

Abstract

We have used a gamma-irradiation (2.5-25 krads) cell fusion procedure to generate human-hamster somatic cell hybrids (IHB, irradiated human fragments in B14-150 cells), retaining small fragments derived from human chromosomes 3 and X. By using Alu-element mediated PCR amplification and dot-blot hybridization with human alphoid or total human DNA as probes, 86 positive hybrids were identified and selected for further analysis. Nonisotopic fluorescence in situ hybridization (FISH) with human DNA in a set of eight hybrids demonstrated the presence of from one to eight human fragments per cell independent of irradiation dose. In contrast, a significant dose-dependent variation of fragment sizes was shown in the analysis of the 86 hybrids with markers previously mapped to 3p (seven markers) and to Xq (21 markers). Using the Xq27-28 region as a model, 40% of the hybrids generated at 5 krads or less were found to have retained fragments in the range of 3-30 Mb, 10% retained the whole chromosome arm, and the remaining 50% retained fragments of less than 2-3 Mb. The proportion of fragments of 3 Mb or larger decreased rapidly at higher irradiation doses and was very low (less than 6%) in hybrids generated at 25 krads. Upon further characterization, the 86 hybrids analyzed here will provide a mapping panel for the entire chromosomes 3 and X with an estimated resolution in the range of 1-2 Mb on average, a size range amenable to PFGE and YAC contig mapping.