Cloning of DNA from double minutes of Y1 mouse adrenocortical tumor cells: evidence for gene amplification

Abstract

We have isolated a metaphase chromosome fraction highly enriched in double minutes (dm) from a mouse adrenocortical tumor cell line (Y1-DM). We have cloned DNA from this dm-enriched fraction in the lambda vector Charon 4A, and have characterized two randomly chosen recombinant bacteriophage clones from this dm DNA library. When 32 P-labeled DNA from each recombinant was hybridized to Southern blots of restriction endonuclease-digested DNA from different mouse cell lines, large differences were seen in the intensity of the resulting autoradiographic images, depending on the source of the genomic DNA. A very strong signal was obtained with DNA from the Y1-DM cells and with DNA from a related Y1 subline that lacks dm but contains a marker chromosome bearing a large homogeneously staining region (HSR). Hybridization to DNA from parental inbred mice and from two unrelated mouse cell lines produced a significantly weaker signal than that obtained with DNA from the Y1 cells, but the DNA fragments from the sources were of similar size. Based on results from filter hybridization analysis, we estimate that sequences homologous to the cloned fragments are approximately 100- to 200-fold more abundant in the genome of the Y1-DM cells than in the parental mouse cells. The data are consistent with the hypothesis that dm and HSRs in these cells contain amplified genes.