Evidence for unequal crossing-over as the mechanism for amplification of some homogeneously staining regions.
The mechanism of DNA amplification in homogeneously staining regions (HSR) was studied in the human melanoma cell line MeWo. Three karyotypically distinguishable cell types within this cell line contain HSR on four different chromosomes, but all HSR contain the same amplified sequences derived from the short arm of chromosome #15. We examined metaphases of MeWo cells from different passages for changes in the length and location of the HSR. In addition, we examined the replication patterns of the HSR sequences and the organization of repeated sequences within these structures. We found that variation in the lengths of the HSR was due to fewer or more copies of a unit that consisted of a later-replicating, distamycin A/DAPI-positive block and active nucleolar organizing regions (NOR). Lateral asymmetry studies suggested that the satellite DNA sequences that are present within the HSR are organized in large inverted repeats. This organization would account for the pairing in both orientations with exchanges resulting in the types of derivative chromosomes observed. The frequency of sister chromatid exchanges (SCE) within the HSR was increased over other chromosomal regions and four examples of unequal SCE within the HSR, with prominent looping out of the longer chromatids, were seen. These results support a model of unequal SCE, rather than saltatory replication for the amplification of DNA sequences in these HSR.
D002460:Cell Line; D002871:Chromosome Banding; D003434:Crossing Over, Genetic; D004247:DNA / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics; D005784:Gene Amplification; D005819:Genetic Markers; D006801:Humans; D007621:Karyotyping; D008545:Melanoma / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D012854:Sister Chromatid Exchange; D013194:Staining and Labeling
D004273:DNA, Neoplasm; D005819:Genetic Markers; D004247:DNA