Chromosomal mupping of a gene responsible for Bloom syndrome via microcell-mediated chromosome Transfer

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In order to identify the human chromosome which carries a mutated gene in cells from patients with the hereditary disorder, Bloom syndrome (BS), we performed chromosome transfer experimets via microcell fusion. Mouse A9 cells containing a single copy of pSV2neo-tagged chromosome 15 derived from normal human fibroblasts served as donor cells for transfer of human chromosome. Purified A9 microcells were fused with SV40-transformed cell line (GM8505) and spontaneously transformed cell line (GM1492E) derived from BS fibroblasts.Cell from BS is chracterized by an extremely high frequency of sister chromatid exchange (SCE). Thus, we examined the restoration of SCEs frequency in the microcells with the transfer of chromosome. The SCEs incidence in the both cell lines (GM8505, GM1492E) were restored by the transfer of chromosome 15. Chromosome analyzes revealed that these clones contained the intact chromosome 15, rearranged chromosome 15 or none. Thus, further analyzes with RFLP are needed for detailed mapping of the BS gene.Chromosome Transfer and SCE analyzesMouse A9 cells containing a single human chromosaome 15 were treated with colcemid to induce micronuclei and were enucleated by centrifugation. The microcells were fused to 2 BS cell lines. After incubation for 24 hr, cells were plated into dishes with medium containing G418. G418 resistant clones were isolated.SCEs were examined 24-27 h after the initiation of 5-bromo-2-deoxyuridine (BrdU) treatment ; BrdU (10ug/ml) was added at the same times as the chemical treatment. Slides were stained by FPG treatment and SCEs were scored.