No abstract

Previous analyses by fluorescence in situ hybridization of structures present 20-30 cell generations after the primary events of mali gene amplification have shown that tens of megabases of DNA separate each copy of the selected gene in chromosomal arrays that contain up to 15 copies. Since these structures are very unstable, it is neceary to study amplified DNA as soon as possible after it has been formed to relate the structures observed to the primary mechanisms that generated them. Previously, new amplifications of the CAD gene were analyzed in colonies of 105 N-(phosphonoacetyl)-L-aspartate-resistant Syrian hamster BHK cells. CAD is on the p arm of chromosome B9 and the amplified genes were usually found in large extensions of B9p, with one copy in its normal position. We now report that dividing drug-resistant cells have been physically separated from static drug-sensitive cells, to allow the amplified structures to be observed only a few cell generations after they have been formed. The most informative results are that about one-third of the newly formed chromosomes carrying amplified CAD genes are dicentric and that about halfof these carry two B9q arms. These observations reveal that recombination between the p telomeric regions of two B9 sister chromatids is an important primary event of amplification in this system. The resulting dicentric chromosomes can then enter bridge-breakage-fusion cycles that provide the means to increase the number of CAD genes per cell in successive generations by an asymmetric distribution at each cell division.Several recent papers in which fluorescence in situ hybridization has been used to look at structures formed early in gene amplification have shed light on this process and have dramatically changed our ideas about the mechanisms involved. Trask and Hamlin (1) studied methotrexate-resistant populations of Chinese hamster cells derived from a single cell by serial selection with increasing concentrations of drug over a period of about 8 weeks. The amplified dihydrofolate reductase genes were chromosomal and spaced tens of megabases apart. They were located on extended structures derived from the same chromosome arm that carries the single gene in unselected cells. Trask and Hamlin (1) concluded that replicative mechanisms were unlikely to be responsible and favored recombinational mechanisms such as sister chromatid exchange.We studied (2) amplifications of the carbamoyl-P synthetase, aspartate transcarbamylase, dihydroorotase (CAD) gene in Syrian hamster cells resistant to N-(phosphonoacetyl)-L-aspartate (PALA) at a somewhat earlier stage, when colonies had reached about 105 cells. We also saw (2) chromosomally amplified genes tens of megabases apart, usually on the same chromosome arm that carried the CAD gene in unselected cells. Furthermore, there were two strong indications that the structures formed in the initial event had changed considerably by the 105-cell stage: individual cells within a single clone had quite different numbers of CAD genes and som...

Rodent cells resistant to N-phosphonacetyl-