Segregation and rearrangement of coamplified genes in different lineages of mutant cells that overproduce adenylate deaminase.

Abstract

Four genes encoding proteins designated as W, X, Yl, and Y2 were found previously to be amplified at different levels in a Chinese hamster fibroblast mutant line selected for overproduction of adenylate deaminase. To gain information on the molecular mechanisms responsible, we studied the levels of amplification and the structures of these four genes in several lineages of mutant cells with comparable activities of adenylate deaminase, the selected enzyme. Only the W gene was amplified in all the lines. In one line, the X, Y1, and Y2 genes were coamplified, while in others either the Yl gene or the pair X and Y2 were coamplified. The results were consistent with linkage of all the genes-in a particular order-in an amplifiable sequence with variable endpoints. Novel joints with a nonrandom distribution were observed. We frequently detected rearranged copies of the W gene, but very few novel joints were present in the other three genes in the six highly amplified lines examined. Some of the novel joints in gene W were highly amplified; they were generated by reamplification of a rearrangement that appeared at an early selection step. In some lines, reamplification was accompanied by deletion or mass correction of preexisting units. We discuss mechanisms which might account for these observations.We have shown previously (4) that unstable mutants overproducing adenylate deaminase (AMPD) can be isolated from Chinese hamster fibroblast line GMA32 by supplementing the growth medium with adenine, azaserine, and the AMPD inhibitor coformycin. Stepwise increases in the concentration of coformycin allow the recovery of mutants which accumulate increasing amounts of the target enzyme. Proteins other than AMPD also accumulate during these selections, and their levels return to those of wild-type cells in revertants with wild-type levels of AMPD activity. Plasmid cDNA probes specific for genes encoding four such proteins-designated W, X, Y1, and Y2-were isolated and used to show that the four genes are amplified at different levels in the AMPD-overproducing mutants studied in the previous experiments (5). We report here an analysis of the abundance, structure, and expression of these four genes in a variety of clones with comparable levels of AMPD activity, derived independently through multiple steps of selection. We again observed that the four genes were not coamplified to the same extent within the same line. Moreover, the pattern of amplification differed from clone to clone. In some cases, striking differences between the amplification and transcription levels of a gene were detected; they were shown to result from genetic rearrangements. The rearrangements were not randomly distributed along the amplified units, as judged from their preferential location in one of these genes. We discuss mechanisms able to generate the coamplification pattern observed in these clones. MATERIALS AND METHODSCell lines and mutant selection. The GMA32 line of Chinese hamster fibroblasts, conditions for its growth, and the details of stepw...

Sister chromatid fusion initiates amplification of the dihydrofolate reductase gene in Chinese hamster cells.

“…6Corresponding author. Debatisse et al 1986;Ma et al 1988]; (3} the multiple amplicons are arrayed in tandem, either in the body of chromosomes as abnormally banding regions (ABRs; Biedler and Spengler 1975) or as acentromeric, autonomously replicating episomal elements (termed double minutes; Kaufman et al 1979;Barker 1982).…”

Preferential amplification of rearranged sequences near amplified adenylate deaminase genes.

“…On the contrary, the highly amplified units within each line are relatively homogeneous. Virtually all the units in each amplified array can be derived from a single rearranged structure that is likely to be formed early in the amplification process (5,9,22,32). For the AMPD system, we demonstrate here that, in highly resistant mutants of families 5 and 6, the amplified DNA constituted relatively homogeneous units formed preferentially from a single initial unit.…”

“…In these cases, most of the amplified units seem to be derived from a single rearranged structure, formed early in * Corresponding author. the amplification process (5,9,32). In the present work, we show that rearranged structures formed at single copy number during the first step of selection are preferentially amplified.…”

“…We used cosmids covering the whole region to probe Northern (RNA) blots of poly(A)+ mRNA from the wild-type line and from line 422 (Fig. 1A), which, in contrast to the other highly amplified mutants, accumulates large amounts of both W and Y1 mRNAs (9 and 56/Y1/6 detected W and Y1 mRNAs, respectively (Fig. 6).…”

“…Thus, during the amplification process, pieces of DNA from different parts of the genome are joined by recombination to form novel structures and novel joints (new restriction fragments). In some highly resistant cell lines, novel structures are amplified enough to be cloned by differential screening or to be detected by direct visualization or in gel renaturation, and highly redundant novel joints have been detected (2,9,22,32,37). In these cases, most of the amplified units seem to be derived from a single rearranged structure, formed early in * Corresponding author.…”