Structure of DNA formed in the first step of CAD gene amplification.

Thirty-three independent mutant cell lines were selected in single steps for resistance to low concentrations of N-(phosphonacetyl)-L-aspartate and the structure of their amplified DNA was probed, using a set of recombinant phage and cosmids containing a total of 380 kb of amplified DNA. In all 33 cell lines, the selected CAD gene and at least 65 kb of flanking DNA were amplified, an average of 2.6-fold. Six other regions of DNA were co-amplified in all 33 mutants, but sometimes to a different extent than CAD. Novel joints, marking recombinations which link amplified regions to each other, were found surprisingly rarely. There were only three within the 380 kb of DNA sequence examined in the total of 33 cell lines. Each novel joint was present in only one copy per cell, was found in a different cell line and was homologous to a different probe. The low frequency of novel joints is consistent either with very large amplified regions in the single-step mutants, possibly 10,000 kb of co-amplified DNA for each copy of the CAD gene, or with a strong bias against recombination in the cloned sequences used as probes. Our previous finding that CAD probes hybridize in situ to unusually large chromosome arms in several single-step mutants is most consistent with the first possibility.