Amplified KpnL repetitive DNA sequences in homogeneously staining regions of a human melanoma cell line.
The human melanoma cell line MeWo was found to contain two populations of cells, one containing 83 chromosomes (hypotetraploid) and the other containing 43 chromosomes (hypodiploid). All of the hypodiploid cells, but none of the hypotetraploid cells, contained chromosomes with long homogeneously staining regions (HSR's) when examined with quinacrine fluorescence. These long HSR's on an X-chromosome and derivative chromosome 15, produced characteristic patterns of positive- and negative-staining areas along the HSR's with both conventional Giemsa (G) staining and C-banding. The C- and G-positive regions stained with distamycin A-DAPI, which is specific for the centromeric heterochromatin of chromosomes 1, 9, 15p, 16, and Y. DNA extracted from MeWo cells and digested with the restriction enzymes KpnL or Sau3A exhibited marked amplification of a 1.8-kilobase fragment. The amplified Sau3A fragment (D15Z1) was cloned, mapped, and partially sequenced. The sequenced region contained a five-base-pair repeat unit (adenine-adenine-thymine-guanine-guanine) that has undergone considerable divergence. Estimates of the size of the HSR's and the amount of amplified DNA suggested that each HSR contained at least 30,000 copies of the 1.8-kb KpnL fragment, representing about 50% of each HSR. The amplified sequence was identified as one member of the previously described KpnL family of repeated sequences. In situ hybridization of D15Z1 to MeWo metaphase chromosomes resulted in heavy labeling over both HSR's. These data suggested that centromeric heterochromatin and neighboring sequences on chromosome 15 have been amplified and some of this material translocated to the X-chromosome.
D004262:DNA Restriction Enzymes / Q000378:metabolism; D004273:DNA, Neoplasm / Q000302:isolation & purification / Q000378:metabolism; D005260:Female; D005784:Gene Amplification; D006801:Humans; D007621:Karyotyping; D008297:Male; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D010920:Placenta / Q000648:ultrastructure; D012091:Repetitive Sequences, Nucleic Acid; D013194:Staining and Labeling; D014178:Translocation, Genetic
D004273:DNA, Neoplasm; D004262:DNA Restriction Enzymes