MECHANISM OF CHROMOSOME EXCHANGE FORMATION IN HUMAN FIBROBLASTS - INSIGHTS FROM CHROMOSOME PAINTING

We have used the techniques of premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH) with a library for huma n chromosome 4 to analyze the rate of rejoining of chromosome breaks a nd development of exchange aberrations in AG1522 human fibroblasts. AG 1522 cells were irradiated in plateau phase with 10 Gy and fused with mitotic HeLa cells either immediately after irradiation or at interval s up to eight days later. The slides were then hybridized with the chr omosome 4 library and unrejoined breaks and exchange events (visualize d as bicolor chromosomes) scored in these cells. At the earliest time point after irradiation, the number of exchange events in the irradiat ed cells was low, but increased with kinetics similar to that of the j oining of the breaks. Furthermore, when we analyzed those cells which had exchange events for their distribution, almost all of the cells in itially contained one exchange event (1 bicolor chromosome). As time p rogressed, the number of cells containing exchanges with two exchange events per cell increased as the number with one exchange event per ce ll decreased. Extrapolation of the number of exchange events to zero t ime (with an estimate of 20 min for the fusion and condensation times) gave a value consistent with zero exchanges at zero time after irradi ation. In a separate experiment, we also scored AG 1522 cells at the f irst metaphase after a dose of 6 Gy and were able to show that as many as 50% of the complete exchanges were non-reciprocal in nature, that is, the two broken ends of a single break in chromosome 4 joined to tw o different chromosomes. These data support the classical breakage-and -reunion model rather than the Revell Exchange Theory of exchange form ation. (C) 1993 Wiley-Liss, Inc.