Evolution and stability of chromosomal DNA coamplified with the CAD gene.

Abstract

We have compared clones of Syrian hamster cells selected for the first amplification of the CAD gene with clones selected for further amplification. The large domain amplified initially was not reamplified as an intact unit. Instead, subregions were reamplified preferentially, and parts of the initial array were often lost. These events reduced the average amount of coamplified DNA accompanying each copy of the selected gene. The degree of amplification was small in the first step (about three extra copies of CAD per cell), but second-step amplifications to a high copy number (up to 60 extra copies per cell) occurred frequently. After several separate steps of amplification, highly condensed arrays that brought many CAD genes close together were formed. In striking contrast to the stability of these highly amplified arrays, the low-copy chromosomal arrays formed early were quite unstable and were often lost completely within 1 or 2 months of growth without selection. The results suggest that different mechanisms may be involved in the first step of amplification and in the later evolution of an already amplified array.

Molecular structure and evolution of double-minute chromosomes in methotrexate-resistant cultured mouse cells.

“…The NotI digestion patterns indicate that DMs do not undergo any detectable rearrangements during this process despite the months of growth in the presence of toxic levels of the DNA-damaging agent MTX. This finding is quite different from the observation in drug-resistant hamster cells containing HSRs that the DNA undergoes complex rearrangements (17,24). Fig.…”

A central role for chromosome breakage in gene amplification, deletion formation, and amplicon integration.

“…A third mechanism, recombination within a replication intermediate (see Wahl 1989), does not directly predict chromosome breakage, heterogeneous deletions from a single parental cell, or vast differences in amplicon sizes. Finally, it has been proposed that the initial amplification intermediates are very large, intrachromosomal, and highly unstable, and they decrease in size over time (Saito et al 1989). This model requires that multiple, typically rare recombination events would have to occur every few cell divisions to account for the observed copy number heterogeneity.…”

“…Previous studies have suggested that chromosomal amplicons might be unstable and capable of generating copy number heterogeneity (e.g., see Saito et al 1989). We tested this possibility by isolating six subclones from P75-20 (referred to as P75-20S5 to P75-20S10), which contained the major classes of chromosomally amplified DHFR genes.…”

“…In most cases, the instability derives from acentric extrachromosomal molecules ranging in size from -100 kb to >2000 kb [e.g., episomes and double minute chromosomes (DMs); for references, see Cowell 1982;Wahl 1989]. However, in one instance, unstable early stage amplification products have been postulated to be chromosomal (Saito et al 1989). (2) In the late phase, the amplified sequences are typically genetically stable, and they are detected in cells subjected to multiple step selection or extensive cell culture.…”

Chromosomal destabilization during gene amplification.

“…These are paired acentric circular structures called (double) minute chromosomes (DMs) and expanded chromosomal regions (ECRs). Several recent studies indicate that DMs can originate from submicroscopic circular elements called episomes (5,6,37,40,50); however, in some cases, DMs may be generated without such precursors by a process which involves chromosome fragmentation (44). Studies with various genes in different cell lines suggest, and in one case directly show, that DMs integrate into a chromosome to form an ECR (5,7,28,47,49,50; this study).…”