Involvement of unstable chromosomal regions containing C-heterochromatin and fragile sites in the integration of amplified dhfr domains

A 10(-3) M methotrexate (MTX)-resistant variant (H2), selected from the murine fibrosarcoma line B77-3T3/AA12, was characterized after 5 (H2 MTXRes I) and 9 (H2 MTXRes II) months of in vitro propagation in the presence of the drug. Southern blot hybridization of wild-type and H2 MTXRes DNAs confirmed amplification of the dhfr gene without apparent rearrangements in its structure. Cytogenetic analysis revealed that double minutes (DMs) predominated in H2 MTXRes I, whereas homogeneously staining regions (HSRs) were the main feature of H2 MTXRes II cells. HSRs, shown to contain dhfr sequences by in situ chromosome hybridization, were localized within two rearranged chromosomes, designated as m1 and m2 because of their derivation from the marker chromosome m of AA12 cells. This chromosome, characterized by two interstitial C bands adjacent to two nonstaining gaps, was no longer observed in H2 MTXRes II cells. A role for nonrandom involvement of chromosome m in the integration of amplified DNA is suggested by the finding of another HSR-chromosome, m3, derived from m, in an independent MTXRes clone (B1). Rearrangement in one of the unstable C-band/gap regions of chromosome m is proposed as the unifying mechanism that may account for the outcome of the three HSR chromosomes observed.