Somatic Cell and Molecular Genetics, Vol. 14, No. 3, 1988, pp. 273-286 Association of High Rate of Recombination with Amplification of Dominant Selectable Gene in Human Cells John P. Murnane and Michael J. Yezzi Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, California 94143 Received 6 November 1987--Final 22 December 1987 Abstract--The human cell line LM205, transformed with the pLR309 plasmid, contains a stably integrated selectable gene marker (neo) without a transcriptional promoter. Sponta- neous tandem duplication at the integration site relocates a Simian virus 40 transcriptional promoter to a position 5' to the neo gene at a rate of 5 x 10 -s events/cell/generation, as measured by subsequent resistance of the cells to the toxic antibiotic G418. The heterogeneity in the site of recombination observed in various G418-resistant (G418-R) subclones indicates that the sequences involved have little or no homology. The rate of tandem duplication involving the neo gene was not affected by DNA-damaging agents or by inhibitors of DNA synthesis. Although these tandem duplications were relatively stable in most G418-R subclones, others underwent further amplification of the neo gene during cloning. In one such cell line, RS-4, subclones isolated without G418 demonstrated a high degree of heterogeneity in the neo gene copy number (2-20), indicating that amplification was associated with a high rate of homologous recombination. Because LM205 was the only clone out of the 30 original clones transformed with pLR309 that demonstrated spontaneous G418-R colonies, cell DNA sequences near the integrated neo gene may promote this recombination. Inclusion of this cell DNA in the initial tandem duplication might then explain the high rate of duplication and deletion observed in the region of the neo gene in the RS-4 subclone. INTRODUCTION Rearrangement of the mammalian cell genome has been observed in the form of translocations, transpositions, and gene am- plification. Much of what little is known about the mechanisms involved in these processes has been deduced from the structure of the rearranged DNA. In some instances chromo- some transtocations seem to arise from incor- rect selection of sequences recognized by a developmentally regulated recombinational mechanism (1). In other cases, a more com- plex series of events results in multiple rear- rangements at the chromosome breakpoint (2). Transpositions in mammalian cells have been identified by the presence of new sequences integrated in various locations in the genome (3-5). These mobile elements are composed of short or long interspersed repeti- tive sequences (SINEs and LINEs, respec- tively) that apparently use pathways involving RNA intermediates. Amplification has been observed in genes such as protooncogenes in cancer cells and various metabolic enzymes in cells under selective pressure (6-8). Gene amplification could occur by various mecha- nisms in somatic mammalian cells, including unequal sister chromatid exchange (9-11) and rereplication followed by reintegration (9, 273 0740-7750/88/0500-0273506.00/0 1988 Plenum Publishing Corporation